Cancer cells are often exposed to a hypoxic environment due to indiscriminate proliferation and incomplete vasculature formation. Hypoxia-inducible factor-1 (HIF-1), which is induced by a hypoxic environment, promotes tumor malignancy by regulating cancer cell metabolism, metastasis, angiogenesis, and cancer stem cell formation. Previous studies have demonstrated increased activation of HIF-1-induced signaling pathways in triple-negative breast cancer (TNBC). Therefore, a hypoxic environment or hypoxia-induced genes are considered as attractive therapeutic targets for TNBC. We have previously identified CCN3 as a novel player in the cancer progression of TNBC. In this study, using public databases and cell lines with altered CCN3 expression, we found that CCN3 expression and hypoxia gene expression signatures were positively correlated, and that HIF1 alpha and HIF1 alpha target gene expression were regulated by CCN3. Furthermore, the induction of HIF1 alpha by CCN3 was regulated at the post-transcriptional level, mainly by IMP3-mediated modulation of mRNA stability and translation of HIF1 alpha. Additionally, we found that HIF1 alpha directly regulates expression of CCN3 by binding to the promoter region of CCN3. This CCN3-HIF1 alpha positive feedback loop may play an important role in HIF1 alpha-induced tumorigenesis in TNBC and be involved in the metastasis of TNBC cells.

CCN3/IMP3/HIF1α positive feedback loop enhances malignant progression of triple-negative breast cancer

Nidogen-1 (NID1) is a secreted glycoprotein widely distributed in basement membranes. NID1 interacts with extracellular matrix proteins such as collagen and laminin and has been implicated in the progression of various cancers. However, study on the role of NID1 in breast cancer is scarce and inconsistent. In this work, we found that the expression of NID1 is significantly lower in breast cancer tissue than in normal tissue. In addition, NID1 expression correlated negatively with a poor prognosis for breast cancer patients. Based on those findings, we speculated that NID1 might act as a cancer suppressor in breast cancer. To investigate the role of NID1 in breast cancer, we constructed NID1-overexpressing cell lines. NID1 overexpression decreased breast cancer cell proliferation, migration, and in vivo tumor growth. Moreover, glucose metabolism, which is known to enhance cancer cell proliferation and migration, was also decreased by NID1 overexpression. Mechanistically, NID1 overexpression downregulated hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression at the transcription level. Furthermore, we found that NID1 reduced integrin beta 1 stability and downregulated the transcription of HIF-1 alpha through the FAK/Src/NF-kappa B p65 signaling axis, which is downstream of integrin beta 1. Together, the results of this study demonstrate the tumor suppressive role of NID1 in triple-negative breast cancer.

Nidogen-1 suppresses cell proliferation, migration, and glycolysis via integrin β1-mediated HIF-1α downregulation in triple-negative breast cancer

This study aimed to investigate the characteristics of out-of-office blood pressure (BP) measurements in patients with apparent treatment-resistant hypertension (aRH) enrolled from 15 tertiary care centers in South Korea. aRH was defined as having uncontrolled office BP ≥ 130/80 mmHg despite receiving three classes of antihypertensive medication or any level of BP despite receiving ≥4 classes of antihypertensive medication. Patients with complete data for office BP, 24-h ambulatory BP monitoring (ABPM), and home BP measurements at baseline were included. BP control status between ABPM and home BP measurements was compared. Out of 1457 patients, 823 meeting the enrollment criteria were included (mean age: 59.9 ± 13.6 years; 57.5% male patients). Among them, 7.2% had controlled BP, 8.7% had whitecoat uncontrolled hypertension, 15.1% had masked uncontrolled hypertension, and 69% had sustained hypertension, as measured through baseline ABPM. Additionally, 43% of patients with controlled BP based on home BP measurement had nocturnal hypertension. Relying solely on home BP measurement may result in misclassifying 70% of patients as having either controlled BP or whitecoat uncontrolled BP. This study reaffirms the circadian pattern of resistant hypertension, characterized by a higher prevalence of non-dipping and rising patterns, even in patients with BP controlled based on ABPM. Considering the persistent difference between home BP measurement and ABPM, even at a lower home BP threshold, integrating both measurements into the management of aRH is advisable.

Discrepancies between home blood pressure and ambulatory blood pressure monitoring in apparent treatment-resistant hypertension: analysis from the Korean resistant hypertension cohort

Cysteine-rich angiogenic inducer 61 (CYR61) is a protein from the CCN family of matricellular proteins that play diverse regulatory roles in the extracellular matrix. CYR61 is involved in cell adhesion, migration, proliferation, differentiation, apoptosis, and senescence. Here, we show that CYR61 induces chemoresistance in triple-negative breast cancer (TNBC). We observed that CYR61 is overexpressed in TNBC patients, and CYR61 expression correlates negatively with the survival of patients who receive chemotherapy. CYR61 knockdown reduced cell migration, sphere formation and the cancer stem cell (CSC) population and increased the chemosensitivity ofTNBC cells. Mechanistically, CYR61 activated Wnt/β-catenin signaling and increased survivin expression, which are associated with chemoresistance, the epithelial–mesenchymal transition, and CSC-like phenotypes. Altogether, our study demonstrates a novel function of CYR61 in chemotherapy resistance in breast cancer.

CYR61 confers chemoresistance by upregulating survivin expression in triple-negative breast cancer

The Drosophila lymph gland houses blood progenitors that give rise to myeloid-like blood cells. Initially, blood progenitors proliferate, but later, they become quiescent to maintain multipotency before differentiation. Despite the identification of various factors involved in multipotency maintenance, the cellular mechanism controlling blood progenitor quiescence remains elusive. Here, we identify the expression of nitric oxide synthase in blood progenitors, generating nitric oxide for post-translational S-nitrosylation of protein cysteine residues. S-nitrosylation activates the Ire1-Xbp1-mediated unfolded protein response, leading to G2 cell-cycle arrest. Specifically, we identify the epidermal growth factor receptor as a target of S-nitrosylation, resulting in its retention within the endoplasmic reticulum and blockade of its receptor function. Overall, our findings highlight developmentally programmed S-nitrosylation as a critical mechanism that induces protein quality control in blood progenitors, maintaining their undifferentiated state by inhibiting cell-cycle progression and rendering them unresponsive to paracrine factors.

S-nitrosylation-triggered unfolded protein response maintains hematopoietic progenitors in Drosophila

Background: Although the representative treatment for colorectal cancer (CRC) is radiotherapy, cancer cells survive due to inherent radioresistance or resistance acquired after radiation treatment, accelerating tumor malignancy and causing local recurrence and metastasis. However, the detailed mechanisms of malignancy induced after radiotherapy are not well understood. To develop more effective and improved radiotherapy and diagnostic methods, it is necessary to clearly identify the mechanisms of radioresistance and discover related biomarkers. Methods: To analyze the expression pattern of miRNAs in radioresistant CRC, sequence analysis was performed in radioresistant HCT116 cells using Gene Expression Omnibus, and then miR-1226-5p, which had the highest expression in resistant cells compared to parental cells, was selected. To confirm the effect of miR-1226-5 on tumorigenicity, Western blot, qRT-PCR, transwell migration, and invasion assays were performed to confirm the expression of EMT factors, cell mobility and invasiveness. Additionally, the tumorigenic ability of miR-1226-5p was confirmed in organoids derived from colorectal cancer patients. In CRC cells, IRF1, a target gene of miR-1226-5p, and circSLC43A1, which acts as a sponge for miR-1226-5p, were discovered and the mechanism was analyzed by confirming the tumorigenic phenotype. To analyze the effect of tumor-derived miR-1226-5p on macrophages, the expression of M2 marker in co-cultured cells and CRC patient tissues were confirmed by qRT-PCR and immunohistochemical (IHC) staining analyses. Results: This study found that overexpressed miR-1226-5p in radioresistant CRC dramatically promoted epithelial-mesenchymal transition (EMT), migration, invasion, and tumor growth by suppressing the expression of its target gene, IRF1. Additionally, we discovered circSLC43A1, a factor that acts as a sponge for miR-1226-5p and suppresses its expression, and verified that EMT, migration, invasion, and tumor growth are suppressed by circSLC43A1 in radioresistant CRC cells. Resistant CRC cells-derived miR-1226-5p was transferred to macrophages and contributed to tumorigenicity by inducing M2 polarization and secretion of TGF-beta. Conclusions: This study showed that the circSLC43A1/miR-1226-5p/IRF1 axis is involved in radioresistance and cancer aggressiveness in CRC. It was suggested that the discovered signaling factors could be used as potential biomarkers for diagnosis and treatment of radioresistant CRC.

miR-1226-5p is involved in radioresistance of colorectal cancer by activating M2 macrophages through suppressing IRF1

Background: Resistance acquired after radiotherapy is directly related to the failure of various cancer treatments, including GBM. Because the mechanism for overcoming radioresistance has not yet been clearly identified, the development of diagnostic and therapeutic markers to treat radioresistance is necessary. Since increased expression of stemness- and EMT-related markers are reported to be closely correlated with radioresistance, research is underway to develop new drugs targeting these factors. Methods: To develop an anticancer drug that overcomes radioresistance, a library of drugs already approved by the FDA was used. After treating radioresistant GBM cells with each drug, the expression of stemness- and EMT-related markers was confirmed by qRT-PCR, and as a result, Atomoxetine (ATX) was selected. It was confirmed that radioresistance-induced cell migratory, invasive, sphere formation abilities, and tumor growth using a xenograft mouse model were suppressed upon ATX treatment. Using a miRNA prediction tool, we discovered miR-520d-5p, which targets Notch2 and Hey1, key factors in radioresistance, and discovered circATIC targeting this miRNA, revealing its relationship with ATX. We demonstrated the expression regulation mechanism and signaling mechanism between circATIC, miR-520d-5p, Notch2, and Hey1 factors using a luciferase reporter assay. In addition, the results at the cellular level were clinically verified by confirming the correlation between radiation, miR-520d-5p, and circATIC using patient plasma by qRT-PCR. Results: ATX showed potential as a treatment for radioresistance by suppressing the malignant phenotype by regulating the circATIC/miR-520d-5p/Notch2-Hey1 signaling mechanism in vitro and in vivo using radioresistant GBM cells. Conclusions: This study revealed that ATX suppresses radioresistance through the circATIC/miR-520d-5p/Notch2-Hey1 signaling pathway. These results showed the potential of ATX as a new drug that can overcome radioresistance, a major challenge in cancer treatment, and the signaling factors identified in this mechanism suggest the possibility of use as potential targets for the diagnosis and treatment of radioresistance.

Atomoxetine suppresses radioresistance in glioblastoma via circATIC/miR-520d-5p/Notch2-Hey1 axis

Triple-negative breast cancer (TNBC), the most aggressive subtype, presents a critical challenge due to the absence of approved targeted therapies. Hence, there is an urgent need to identify effective therapeutic targets for this condition. While epidermal growth factor receptor (EGFR) is prominently expressed in TNBC and recognized as a therapeutic target, anti-EGFR therapies have yet to gain approval for breast cancer treatment due to their associated side effects and limited efficacy. Here, we discovered that intercellular adhesion molecule-1 (ICAM-1) exhibits elevated expression levels in metastatic breast cancer and serves as a pivotal binding adaptor for EGFR activation, playing a crucial role in malignant progression. The activation of EGFR by tumor-expressed ICAM-1 initiates biased signaling within the JAK1/STAT3 pathway, consequently driving epithelial-to-mesenchymal transition and facilitating heightened metastasis without influencing tumor growth. Remarkably, ICAM-1-neutralizing antibody treatment significantly suppressed cancer metastasis in a breast cancer orthotopic xenograft mouse model. In conclusion, our identification of ICAM-1 as a novel tumor intrinsic regulator of EGFR activation offers valuable insights for the development of TNBC-specific anti-EGFR therapies.

Tumor-intrinsic role of ICAM-1 in driving metastatic progression of triple-negative breast cancer through direct interaction with EGFR

Background: Solid tumors promote tumor malignancy through interaction with the tumor microenvironment, resulting in difficulties in tumor treatment. Therefore, it is necessary to understand the communication between cells in the tumor and the surrounding microenvironment. Our previous study revealed the cancer malignancy mechanism of Bcl-w overexpressed in solid tumors, but no study was conducted on its relationship with immune cells in the tumor microenvironment. In this study, we sought to discover key factors in exosomes secreted from tumors overexpressing Bcl-w and analyze the interaction with the surrounding tumor microenvironment to identify the causes of tumor malignancy. Methods: To analyze factors affecting the tumor microenvironment, a miRNA array was performed using exosomes derived from cancer cells overexpressing Bcl-w. The discovered miRNA, miR-6794-5p, was overexpressed and the tumorigenicity mechanism was confirmed using qRT-PCR, Western blot, invasion, wound healing, and sphere formation ability analysis. In addition, luciferase activity and Ago2-RNA immunoprecipitation assays were used to study the mechanism between miR-6794-5p and its target gene SOCS1. To confirm the interaction between macrophages and tumor-derived miR-6794-5p, co-culture was performed using conditioned media. Additionally, immunohistochemical (IHC) staining and flow cytometry were performed to analyze macrophages in the tumor tissues of experimental animals. Results: MiR-6794-5p, which is highly expressed in exosomes secreted from Bcl-w-overexpressing cells, was selected, and it was shown that the overexpression of miR-6794-5p increased migratory ability, invasiveness, and stemness maintenance by suppressing the expression of the tumor suppressor SOCS1. Additionally, tumor-derived miR-6794-5p was delivered to THP-1-derived macrophages and induced M2 polarization by activating the JAK1/STAT3 pathway. Moreover, IL-10 secreted from M2 macrophages increased tumorigenicity by creating an immunosuppressive environment. The in vitro results were reconfirmed by confirming an increase in M2 macrophages and a decrease in M1 macrophages and CD8+ T cells when overexpressing miR-6794-5p in an animal model. Conclusions: In this study, we identified changes in the tumor microenvironment caused by miR-6794-5p. Our study indicates that tumor-derived miR-6794-5p promotes tumor aggressiveness by inducing an immunosuppressive environment through interaction with macrophage.

Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. TNBC patients typically exhibit unfavorable outcomes due to its rapid growth and metastatic potential. Here, we found overexpression of CCN3 in TNBC patients. We identified that CCN3 knockdown diminished cancer stem cell formation, metastasis, and tumor growth in vitro and in vivo. Mechanistically, ablation of CCN3 reduced activity of the EGFR/MAPK pathway. Transcriptome profiling revealed that CCN3 induces glycoprotein nonmetastatic melanoma protein B (GPNMB) expression, which in turn activates the EGFR pathway. An interrogation of the TCGA dataset further supported the transcriptional regulation of GPNMB by CCN3. Finally, we showed that CCN3 activates Wnt signaling through a ligand-dependent or -independent mechanism, which increases microphthalmia-associated transcription factor (MITF) protein, a transcription factor inducing GPNMB expression. Together, our findings demonstrate the oncogenic role of CCN3 in TNBC, and we propose CCN3 as a putative therapeutic target for TNBC.

CCN3/NOV promotes metastasis and tumor progression via GPNMB-induced EGFR activation in triple-negative breast cancer

Brain type of creatine kinase (CKB) regulates energy homeostasis by reversibly transferring phosphate groups between phosphocreatine and ATP at sites of high energy demand. Several types of cancer cells exhibit upregulated CKB expression, but the function of CKB in cancer cells remains unclear. In this study, we investigated the function of CKB in breast cancer by overexpressing CKB in MDA-MB-231 cells. The overexpression of CKB did not affect cell growth rate, cell cycle distribution, ATP level or key mediators of aerobic glycolysis and lactate dehydrogenase isoform levels. Meanwhile, CKB overexpression did increase resistance to doxorubicin. TGF-beta-induced Smad phosphorylation and Smad-dependent transcriptional activity were significantly up-regulated by CKB expression without changes in inhibitory Smad protein levels. Moreover, treatment with TGF-beta considerably enhanced cell viability during doxorubicin treatment and decreased doxorubicin-induced apoptosis in CKB-expressing MDA-MB-231 cells compared to control cells. These results suggest that CKB attenuates doxorubicin-induced apoptosis and potentiates resistance to doxorubicin by enhancing TGF-beta signaling in MDA-MB-231 cells.

Brain type of creatine kinase induces doxorubicin resistance via TGF-beta signaling in MDA-MB-231 breast cancer cells

About 70% of breast cancers overexpress estrogen receptor α (ERα, encoded by ESR1). Tamoxifen, a competitive inhibitor of estrogen that binds to ER, has been widely used as a treatment for ER-positive breast cancer. However, 20–30% of breast cancer is resistant to tamoxifen treatment. The mechanisms underlying tamoxifen resistance remain elusive. We found that Yes-associated protein (YAP; also known as YAP1), connective tissue growth factor (CTGF; also known as CCN2) and cysteine-rich angiogenic inducer 61 (Cyr61; also known as CCN1) are overexpressed, while ERα is downregulated in tamoxifen-resistant breast cancer. Inhibition of YAP, CTGF and Cyr61 restored ERα expression and increased sensitivity to tamoxifen. Overexpression of YAP, CTGF, and Cyr61 led to downregulation of ERα and conferred resistance to tamoxifen in ER-positive breast cancer cells. Mechanistically, CTGF and Cyr61 downregulated ERα expression at the transcriptional level by directly binding to the regulatory regions of the ERα-encoding gene, leading to increased tamoxifen resistance. Also, CTGF induced Glut3 (also known as SLC2A3) expression, leading to increased glycolysis, which enhanced cell proliferation and migration in tamoxifen-resistant cells. Together, these results demonstrate a novel role of YAP, CTGF and Cyr61 in tamoxifen resistance and provide a molecular basis for their function in tamoxifen-resistant breast cancer.

YAP, CTGF and Cyr61 are overexpressed in tamoxifen-resistant breast cancer and induce transcriptional repression of ERα

The objective of this study was to compare the diagnostic accuracy of office blood pressure (BP) threshold of 140/90 and 130/80 mmHg for correctly identifying uncontrolled out-of-office BP in apparent treatment-resistant hypertension (aTRH). We analyzed 468 subjects from a prospectively enrolled cohort of patients with resistant hypertension in South Korea (clinicaltrials.gov: NCT03540992). Resistant hypertension was defined as office BP >= 130/80 mmHg with three different classes of antihypertensive medications including thiazide-type/like diuretics, or treated hypertension with four or more different classes of antihypertensive medications. We conducted different types of BP measurements including office BP, automated office BP (AOBP), home BP, and ambulatory BP. We defined uncontrolled out-of-office BP as daytime BP >= 135/85 mmHg and/or home BP >= 135/85 mmHg. Among subjects with office BP < 140/90 mmHg and subjects with office BP < 130/80 mmHg, 66% and 55% had uncontrolled out-of-office BP, respectively. The prevalence of controlled and masked uncontrolled hypertension was lower, and the prevalence of white-coat and sustained uncontrolled hypertension was higher, with a threshold of 130/80 mmHg than of 140/90 mmHg, for both office BP and AOBP. The office BP threshold of 130/80 mmHg was better able to diagnose uncontrolled out-of-office BP than 140/90 mmHg, and the net reclassification improvement (NRI) was 0.255. The AOBP threshold of 130/80 mmHg also revealed better diagnostic accuracy than 140/90 mmHg, with NRI of 0.543. The office BP threshold of 130/80 mmHg showed better than 140/90 mmHg in terms of the correspondence to out-of-office BP in subjects with aTRH.

Office blood pressure threshold of 130/80 mmHg better predicts uncontrolled out-of-office blood pressure in apparent treatment-resistant hypertension

Understanding the interactions between nanoparticles (NPs) and human immune cells is necessary for justifying their utilization in consumer products and biomedical applications. However, conventional assays may be insufficient in describing the complexity and heterogeneity of cell-NP interactions. Herein, mass cytometry and single-cell RNA-sequencing (scRNA-seq) are complementarily used to investigate the heterogeneous interactions between silver nanoparticles (AgNPs) and primary immune cells. Mass cytometry reveals the heterogeneous biodistribution of the positively charged polyethylenimine-coated AgNPs in various cell types and finds that monocytes and B cells have higher association with the AgNPs than other populations. scRNA-seq data of these two cell types demonstrate that each type has distinct responses to AgNP treatment: NRF2-mediated oxidative stress is confined to B cells, whereas monocytes show Fc gamma-mediated phagocytosis. Besides the between-population heterogeneity, analysis of single-cell dose-response relationships further reveals within-population diversity for the B cells and naive CD4(+) T cells. Distinct subsets having different levels of cellular responses with respect to their cellular AgNP doses are found. This study demonstrates that the complementary use of mass cytometry and scRNA-seq is helpful for gaining in-depth knowledge on the heterogeneous interactions between immune cells and NPs and can be incorporated into future toxicity assessments of nanomaterials.

Mass Cytometry and Single-Cell RNA-seq Profiling of the Heterogeneity in Human Peripheral Blood Mononuclear Cells Interacting with Silver Nanoparticles

Cluster of differentiation 133 (CD133) is a transmembrane glycoprotein that has been reported as a marker of cancer stem cells or cancer-initiating cells in various cancers. However, its contribution to tumorigenesis and differentiation remains to be elucidated. To determine the role of CD133 in colon cancer, we silenced CD133 in human colon cancer cells. Silencing of CD133 results in decreased cell proliferation, survival, migration, invasion, and glucose transport. These effects are mediated by downregulation of the human epidermal growth factor receptor 3 (HER3)/Akt/mTOR signaling pathway, culminating in reduced expression of the glucose transporter GLUT1. We also confirm that the cellular phenotypes of CD133-silenced cells are mediated by GLUT1 downregulation. We conclude that CD133 is a potential tumor initiator that positively regulates GLUT1 expression through modulation of HER3/Akt/mTOR signaling.

Silencing of CD133 inhibits GLUT1-mediated glucose transport through downregulation of the HER3/Akt/mTOR pathway in colon cancer

Phthalates are mainly used as binders and plasticizers in various industrial products including detergents, surfactants, waxes, paints, pharmaceuticals, food products, and cosmetics. However, they have been reported to be endocrine disruptors, which are chemicals that can mimic or disturb endocrines, causing interference to the endocrine system. Recently, there have been numerous reports showing that phthalates have negative health impacts such as asthma, breast cancer, obesity, type II diabetes, and male infertility. Due to these effects, there is an urgent need for phthalate alternatives. In this study, the potential cytotoxicity of phthalates and their substitutes were screened in HaCaT cells, a human keratinocyte cell line, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) thiazolyl blue assay, immunocytochemistry, flow cytometric analysis, and western blotting. We confirmed that common phthalates such as butyl benzyl phthalate (BBP), di-n-butyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEHP) have genotoxic effects, leading to cell death. Among the known phthalate substitutes, tributyl O-acetylcitrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylcitrate (ATHC) were tested for cytotoxicity. As a result, ATEC showed similar levels of cytotoxicity with the phthalates whereas ATBC and ATHC did not show significant cytotoxicity even in high doses (5 mg/ml).

Toxicological assessment of phthalates and their alternatives using human keratinocytes

Titanium dioxide nanoparticles, due to their smaller size and increased surface area comparted to the bulk form, are known to be bioreactive and have unexpected toxicological outcomes. Previous studies have shown that nanoscale titanium dioxide induces reactive oxygen species (ROS)-mediated cytotoxicity and genotoxicity. Although many reports have discussed the ROS-mediated cytotoxic effects of titanium dioxide nanoparticles (TiO2-NPs), their effects on the receptor-ligand association are unknown. In this study, the possibility that TiO2-NPs can interfere with the receptor-ligand binding was assessed by monitoring alterations in the phosphorylation status of proteins downstream of the epidermal growth factor receptor (EGFR) signaling cascade. TiO2-NPs blocked ligand-induced EGFR autophosphorylation, leading to the deactivation of EGFR downstream effectors such as Akt and extracellular signal-regulated kinase signaling, inducing cell death.

Titanium dioxide nanoparticles induce apoptosis by interfering with EGFR signaling in human breast cancer cells

In this paper, we investigate the tracking of the 3D moving target when multiple measurements are available. We propose a particle filter-based 3D target tracking algorithm with measurement of time difference of arrival (TDoA), frequency difference of arrival (FDoA), and angle of arrival (AoA). We analyze the performance of the proposed scheme by MATLAB simulations and show the efficiency of the particle filter for 3D moving target tracking with various types of measurements.

3D moving target tracking with measurement fusion of TDoA/FDoA/AoA

Correction to: ECM1 regulates cell proliferation and trastuzumab resistance through activation of EGF-signaling

Bisphenol-A (BPA) was first synthesized in the 1890s and has been used in many plastic products. However, BPA is known to act as an endocrine disruptor and has been found to be toxic to human health. Many alternative substances have been developed to replace BPA, but it is still widely used worldwide. In this study, we identified the potential cytotoxicity of BPA by evaluating toxicity using human keratinocytes. Also, we evaluated cytotoxicity of BPA substitutes to determine their suitability as an alternative to BPA. The proliferation assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and western blot analysis showed that BPA significantly affect cell viability, induction of apoptotic fraction and increased activation of DNA-damage marker protein. In addition, through the same experiments, the substitutes of BPA were shown to be significantly less toxic than BPA, and the least toxicity was observed with 1,4-cyclohexanedimethanol (CHDM) and terephthalic acid (TPA). In conclusion, this study suggests that cytotoxicity of BPA induces apoptosis of human keratinocytes, and that CHDM and TPA are the most suitable substitutes for BPA.

Cytotoxicity measurement of Bisphenol A (BPA) and its substitutes using human keratinocytes

The CCN protein family is composed of six matricellular proteins, which serve regulatory roles rather than structural roles in the extracellular matrix. First identified as secreted proteins which are induced by oncogenes, the acronym CCN came from the names of the first three members: CYR61, CTGF, and NOV. All six members of the CCN family consist of four cysteine-rich modular domains. CCN proteins are known to regulate cell adhesion, proliferation, differentiation, and apoptosis. In addition, CCN proteins are associated with cardiovascular and skeletal development, injury repair, inflammation, and cancer. They function either through binding to integrin receptors or by regulating the expression and activity of growth factors and cytokines. Given their diverse roles related to the pathology of certain diseases such as fibrosis, arthritis, atherosclerosis, diabetic nephropathy, retinopathy, and cancer, there are many emerging studies targeting CCN protein signaling pathways in attempts to elucidate their potentials as therapeutic targets.

Role of the CCN protein family in cancer

Nonylphenol polyethoxylates (NPEOs) are a major group of nonionic surfactants widely used in various detergents, cleaners, plastics, papers, and agro-chemical products. Nonylphenol (NP), which is a final degraded metabolite derived from NPEOs, has been reported as an endocrine disrupter, known to mimic or disturb reproductive hormone functions. Concern about the hazards of NP and NPEOs has generated legal restrictions and action plans worldwide. Considering the fact that NP and NPEOs are majorly used in the production of products such as detergents, shampoos, and cosmetics which frequently come into contact with the skin, we investigated the effects of NP and NPEOs on a human keratinocyte cell line (HaCaT). In this study, the toxicity of NP and NPEOs was screened in HaCaT cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue assay and Western blotting. The potential cytotoxicity of substitutes was assessed by dose-response assays, relative cell viability, and genotoxicity caused by specific alterations in DNA damage response proteins (including ataxia-telangiectasia mutated, p53, Chk1, Chk2, and Histone H2A.X). We demonstrated that NP and NPEOs are toxic to HaCaT cells, as revealed by the decreased cell viability after 24h treatment. NPs and NPEOs also induced apoptosis and DNA damage as shown by the activation of Poly(ADP-ribose) polymerase, Caspase-3, and Histone H2A.X.

Comparative toxicological evaluation of nonylphenol and nonylphenol polyethoxylates using human keratinocytes

Elevated glucose levels in cancer cells can be attributed to increased levels of glucose transporter (GLUT) proteins. Glut1 expression is increased in human malignant cells. To investigate alternative roles of Glut1 in breast cancer, we silenced Glut1 in triple-negative breast-cancer cell lines using a short hairpin RNA (shRNA) system. Glut1 silencing was verified by Western blotting and qRT-PCR. Knockdown of Glut1 resulted in decreased cell proliferation, glucose uptake, migration, and invasion through modulation of the EGFR/MAPK signaling pathway and integrin β1/Src/FAK signaling pathways. These results suggest that Glut1 not only plays a role as a glucose transporter, but also acts as a regulator of signaling cascades in the tumorigenesis of breast cancer.

Glut1 promotes cell proliferation, migration and invasion by regulating epidermal growth factor receptor and integrin signaling in triple-negative breast cancer cells

Galectin-1 is a beta-galactoside binding protein secreted by many types of aggressive cancer cells. Although many studies have focused on the role of galectin-1 in cancer progression, relatively little attention has been paid to galectin-1 as an extracellular therapeutic target. To elucidate the molecular mechanisms underlying galectin-1-mediated cancer progression, we established galectin-1 knock-down cells via retroviral delivery of short hairpin RNA (shRNA) against galectin-1 in two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and Hs578T. Ablation of galectin-1 expression decreased cell proliferation, migration, invasion, and doxorubicin resistance. We found that these effects were caused by decreased galectin-1-integrin 1 interactions and suppression of the downstream focal adhesion kinase (FAK)/cSrc pathway. We also found that silencing of galectin-1 inhibited extracellular signalregulated kinase (ERK)/signal transducer and activator of transcription 3 (STAT3) signaling, thereby down-regulating survivin expression. This finding implicates STAT3 as a transcription factor for survivin. Finally, rescue of endogenous galectin-1 knockdown and recombinant galectin-1 treatment both recovered signaling through the FAK/c-Src/ERK/STAT3/survivin pathway. Taken together, these results suggest that extracellular galectin-1 contributes to cancer progression and doxorubicin resistance in TNBC cells. These effects appear to be mediated by galectin-1-induced up-regulation of the integrin beta 1/FAK/c-Src/ERK/STAT3/survivin pathway. Our results imply that extracellular galectin-1 has potential as a therapeutic target for triple-negative breast cancer.

Binding of galectin-1 to integrin β1 potentiates drug resistance by promoting survivin expression in breast cancer cells

Cancer cells require increased aerobic glycolysis to support rapid cell proliferation. For their increased energy demands, cancer cells express glucose transporter (Glut) proteins at a high level. Glut1 is associated with basal-like breast cancer and is considered a potential therapeutic target. To investigate the possibility of Glut1 as a therapeutic target in breast cancer cells, we downregulated Glut1 in triple negative breast cancer (TNBC) cell lines using a short hairpin system. We determined whether Glut1 silencing might enhance anti-proliferative effect of chemotherapeutic agents. Contrary to our hypothesis, ablation of Glut1 attenuated apoptosis and increased drug resistance via upregulation of p-Akt/p-GSK-3 beta (Ser9)/beta-cateninisurvivin. These results indicated that the potential of Glut1 as a therapeutic target should be carefully reevaluated.

Silencing of Glut1 induces chemoresistance via modulation of Akt/GSK-3β/β-catenin/survivin signaling pathway in breast cancer cells

Background/Aim: Silica nanoparticles (nano-SiO2) are widely used in many industrial areas and there is much controversy surrounding cytotoxic effects of such nanoparticles. In order to determine the toxicity and possible molecular mechanisms involved, we conducted several tests with two breast cancer cell lines, MDA-MB-231 and Hs578T. Materials and Methods: After exposure to nano-SiO2, growth, apoptosis, motility of breast cancer cells were monitored. In addition, modulation of signal transduction induced by nano-SiO2 was detected through western blot analysis. Results: Treatment of nano-SiO2 repressed the growth of breast cancer cell lines. It also increased apoptosis and reduced cell motility. Moreover, exposure to nano-SiO2 significantly disturbed the dimerization of epidermal growth factor receptor (EGFR), followed by down-regulation of its downstream cellular sarcoma kinase (c-SRC) and signal transducer and activator of transcription 3 (STAT3) signaling cascades. Conclusion: Nano-SiO2 has a cytotoxic effect on MDA-MB-231 and Hs578T breast cancer cells via modulation of EGFR signaling cascades.

Cytotoxic Effect of Nano-SiO2 in Human Breast Cancer Cells via Modulation of EGFR Signaling Cascades

Cluster of differentiation 44 (CD44) is a transmembrane glycoprotein that has been identified as a cancer stem cell marker in various cancer cells. Although many studies have focused on CD44 as a cancer stem cell marker, its effect on cancer cell metabolism remains unclear. To investigate the role of CD44 on cancer cell metabolism, we established CD44 knock-down cells via retroviral delivery of shRNA against CD44 in human breast cancer cells. Silencing of CD44 decreased the glycolytic phenotype of cancer cells, affecting glucose uptake, ATP production, and lactate production. We also found that ablation of the CD44-induced lactate dehydrogenase (LDH) isoenzyme results in a shift to LDH1 due to LDHA down-regulation and LDHB up-regulation, implying the importance of LDH isoenzyme modulation on cancer metabolism. The expression of glycolysis- related proteins including hypoxia inducible factor-1 alpha (HIF-1 alpha) and LDHA was decreased by CD44 silencing. These effects were due to the up-regulation of liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) a activity by reduction in c-Src and Akt activity in CD44 knock-down cells. Finally, induction of LKB1/AMPK alpha activity blocked the expression of HIF-1 alpha and its target gene, LDHA. Inversely, LDHB expression was repressed by HIF-1 alpha. Collectively, these results indicate that the CD44 silencing-induced metabolic shift is mediated by the regulation of c-Src/Akt/LKB1/AMPK alpha/HIF-1 alpha signaling in human breast cancer cells.

Ablation of CD44 induces glycolysis-to-oxidative phosphorylation transition via modulation of the c-Src–Akt–LKB1–AMPKα pathway

Stanniocalcin-1 (STC-1), a secreted glycoprotein, is highly expressed in a variety of human malignancies. However, the role of STC-1 has not been fully elucidated in breast cancer cells. Here, we investigated whether STC-1 acts as a prognostic factor in triple-negative breast cancer (TNBC) patients, and we explored the cellular mechanism in breast cancer cells. The level of STC-1 expression was directly associated with the relapse-free and overall survival of basal-type breast cancer patients. Breast cancer patients with a high level of STC-1 had poor prognosis. In addition, our results showed that the level of STC-1 expression was markedly higher in TNBC than in non-TNBC cells. Invasiveness of the TNBC cells was also significantly increased in response to recombinant human STC-1 treatment. In contrast, the invaded cell numbers were completely decreased by STC-1 siRNA overexpression in the Hs578T and MDA-MB-231 TNBC cells. Our results showed that the phosphorylation of c-Jun N-terminal protein kinase (JNK) and c-Jun was increased after STC-1 treatment but not the phosphorylation of ERK and p38 MAPKs in the Hs578T and MDA-MB-231 TNBC cells. Furthermore, expression of one invasion-related gene MMP-9, was increased by STC-1 treatment. STC-1-induced MMP-9 expression was suppressed by SP600125 (a JNK inhibitor) in the Hs578T cells. STC-1-induced cell invasiveness was also inhibited by SP600125. Taken together, we demonstrated that aberrant STC-1 expression is associated with poor prognosis and stimulates the invasiveness of TNBC cells through the JNK/c-Jun-dependent signaling pathway.

Elevated STC‑1 augments the invasiveness of triple‑negative breast cancer cells through activation of the JNK/c‑Jun signaling pathway

CD44 was recently identified as a cancer initiation marker on the cell membrane. The cytoplasmic tail of CD44 is known to bind ERM (ezrin, radixin, moesin) proteins, cytoskeletal proteins like ankyrin, and the non-receptor tyrosine kinase c-Src. CD44 transmits its oncogenic signaling via c-Src and its downstream effectors. To investigate the role of CD44 in breast cancer cells, we generated CD44 knock-down cells via retroviral delivery of shRNA against CD44. We found that silencing of CD44 decreased the proliferation, migration, and invasion of breast cancer cells. The expression and activity of cell migration-related proteins, including c-Src, paxillin, and FAK were decreased by CD44 silencing. We also found that the c-Jun protein level was negatively regulated via induction of a GSK-3 beta-dependent degradation pathway in CD44 knock-down cells. The expression level of Sp1, a target gene product of c-Jun, was also decreased in these cells. Finally, CD44 knock-down suppressed both mRNA and protein levels of c-Sic and its downstream MAPK pathway as a result of down-regulation of Sp1 as a transcription factor for c-Src. Collectively, these results indicate that biological changes induced by CD44 silencing are mediated by cumulative down-regulation of c-Jun, Sp1, and c-Src in human breast cancer cells.

CD44 regulates cell proliferation, migration, and invasion via modulation of c-Src transcription in human breast cancer cells

Several groups have reported that TGF beta 1 regulates cellular responses to gamma-irradiation; however, the exact mechanism has not been fully elucidated. In the current study, the role of TGF beta 1 in cellular responses to gamma-irradiation was investigated in detail. The data indicate that TGF beta 1 pretreatment decreased the aftermath of ionizing radiation (IR)-induced DNA damage in a SMAD-dependent manner. To determine the underlying mechanism for these effects, the extent of IR-induced DNA repair activity in the presence or absence of TGF beta 1 was examined. Studies reveal that TGF beta 1 upregulated DNA ligase IV (Lig4), augmented IR-induced nuclear retention of the DNA ligase, and enhanced nonhomologous end-joining (NHEJ) repair activity. In addition, knockdown of Lig4 reduced the TGF beta 1-induced protection against IR. Overall, these data indicate that TGF beta 1 facilitates the NHEJ repair process upon gamma-irradiation and thereby enhances long-term survival.

TGF beta 1 Protects Cells from gamma-IR by Enhancing the Activity of the NHEJ Repair Pathway

The Warburg effect is an oncogenic metabolic switch that allows cancer cells to take up more glucose than normal cells and favors anaerobic glycolysis. Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein that is overexpressed in various types of carcinoma. Using two-dimensional digest-liquid chromatography-mass spectrometry (LC-MS)/MS, we showed that the expression of proteins associated with the Warburg effect was upregulated in trastuzumab-resistant BT-474 cells that overexpressed ECM1 compared to control cells. We further demonstrated that ECM1 induced the expression of genes that promote the Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and hypoxia-inducible factor 1 alpha (HIF-1 alpha). The phosphorylation status of pyruvate kinase M2 (PKM-2) at Ser37, which is responsible for the expression of genes that promote the Warburg effect, was affected by the modulation of ECM1 expression. Moreover, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting the Warburg effect mediated by PKM2.

ECM1 promotes the Warburg effect through EGF-mediated activation of PKM2

Fibronectin (FN), an extracellular matrix ligand, plays a pivotal role in cell adhesion, migration, and oncogenic transformation. Aberrant FN expression is associated with poor prognoses in various types of cancer, including breast cancer. In the current study, we investigated the relationship between FN induction and HER2 expression in breast cancer cells. Our results showed that the level of FN expression increased in response to HER family ligands, EGF and TGF-alpha in a time- and dose-dependent manner. On the other hand, EGF-induced FN expression decreased in response to trastuzumab, which is a HER2-targeted monoclonal antibody. However, EGF-induced FN expression was not affected by trastuzumab in JIMT-1 breast cancer cells, which are trastuzumab insensitive cells. Next, we introduced the HER2 gene into MDA-MB231 cells to verify the relationship between FN and HER2. The level of FN expression significantly increased in HER2-overexpressed MDA-MB231 cells. In contrast, the induction of FN by HER2 was significantly decreased in response to trastuzumab treatment. In addition, the induction of FN by HER2 was down-regulated by the MEK 1/2 specific inhibitor, U0126. Using conditioned culture media of vec- and HER2-overexpressed MDA-MB231 cells, we observed the cell morphology, adhesion, and invasion of MDA-MB231 cells. Interestingly, in conditioned culture media of HER2-overexpressed MDA-MB231 cells, the cell morphology was altered, and adhesion and invasion of MDA-MB231 cells significantly increased. In addition, our results showed that recombinant human FN augmented cell adhesion and invasion of MDA-MB231 cells while these inductions decreased in response to an FN inhibitor. Therefore, we demonstrated that the induction of FN by HER2 triggers cell adhesion and invasion capacities.

Induction of fibronectin by HER2 overexpression triggers adhesion and invasion of breast cancer cells

Members of the EGFR family are potent mediators of normal cell growth and development. HER2 possesses an active tyrosine kinase domain, but no direct ligand has been identified. To investigate the differential effect of HER2 in breast cell lines, HER2 was overexpressed in MCF-10A, MCF7 and MDA-MB-231 cells. HER2 overexpression promoted proliferation, survival and migration in MCF-10A and MCF-7 cells. No significant differences were seen in proliferation, survival or migration between MDA-MB-231 vec and HER2 cells. The activity of downstream HER2 proteins increased in MCF-10A HER2 and MCF-7 HER2 cells but not in MDA-MB-231 HER2 cells. Exogenously expressed HER2 failed to associate with EGFR or HER3 in MDA-MB-231 cells, while overexpression of HER2 enhanced HER family dimerization in MCF-10A and MCF-7 cells.

EGFR negates the proliferative effect of oncogenic HER2 in MDA-MB-231 cells

Introduction: Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein with putative functions in cell proliferation, angiogenesis and differentiation. Expression of ECM1 in several types of carcinoma suggests that it may promote tumor development. In this study, we investigated the role of ECM1 in oncogenic cell signaling in breast cancer, and potential mechanisms for its effects. Methods: In order to find out the functional role of ECM1, we used the recombinant human ECM1 and viral transduction systems which stably regulated the expression level of ECM1. We examined the effect of ECM1 on cell proliferation and cell signaling in vitro and in vivo. Moreover, tissues and sera of patients with breast cancer were used to confirm the effect of ECM1. Results: ECM1 protein was increased in trastuzumab resistant (TR) cells, in association with trastuzumab resistance and cell proliferation. Through physical interaction with epidermal growth factor receptor (EGFR), ECM1 potentiated the phosphorylation of EGFR and extracellular signal-regulated kinase upon EGF treatment. Moreover, ECM1-induced galectin-3 cleavage through upregulation of matrix metalloproteinase 9 not only improved mucin 1 expression, but also increased EGFR and human epidermal growth factor receptor 3 protein stability as a secondary signaling. Conclusions: ECM1 has important roles in both cancer development and trastuzumab resistance in breast cancer through activation of EGFR signaling.

Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

We report here that CD24 knockdown resulted in decreased expression of Notch1 in MCF-7 cells. CD24-downstream p38MAPK was shown to regulate Notch1 at the level of mRNA stability. We also found that CD24-mediated cell migration, invasion, mammosphere formation, and drug resistance was regulated by its downstream target Notch1. Together, our results indicate that CD24 may regulate the epithelial to mesenchymal transition and sternness through Notch1 signaling in breast cancer cells.

CD24 regulates stemness and the epithelial to mesenchymal transition through modulation of Notch1 mRNA stability by p38MAPK

S100A4, also known as the mts1 gene, has been reported as an invasive and metastatic marker for many types of cancers. S100A4 interacts with various target genes that affect tumor cell metastasis; however, little is known about cellular signaling pathways elicited by S100A4. In the current study, we demonstrate an inhibitory effect of S100A4 on beta-catenin signaling in breast cancer cells. By overexpressing S100A4 in MCF-7, MDA-MB-231 and MDA-MB-453 breast cancer cells, we observed the down-regulation of beta-catenin expression and beta-catenin-dependent TCF/LEF transcriptional activities. The activity of GSK3 beta, which phosphorylates beta-catenin and induces proteasomal degradation of beta-catenin, was increased in S100A4-overexpressing cell lines. Blocking Glycogen Synthase Kinase (GSK3 beta) activity by lithium chloride or Dvl gene overexpression restored beta-catenin expression. We also found that increased GSK3 beta activity was due to decrease in Ala activity resulting from Egr-1-induced phosphatase and tensin homolog (PTEN) expression. S100A4 induced Egr-1 nuclear localization by increasing the association between Egr-1 and importin-7 and this effect was reduced in S100A4 mutants that harbored a defect in nuclear localization signals. Collectively, we verify herein that S100A4 may act as a tumor suppressor in breast cancers by down-regulating the central signaling axis for tumor cell survival.

S100A4 negatively regulates beta-catenin by inducing the Egr-1-PTEN-Akt-GSK3 beta degradation pathway

Purpose: Keratin19 (KRT19) is the smallest known type I intermediate filament and is used as a marker for reverse transcriptase PCR-mediated detection of disseminated tumors. In this study, we investigated the functional analysis of KRT19 in human breast cancer. Experimental Design: Using a short hairpin RNA system, we silenced KRT19 in breast cancer cells. KRT19 silencing was verified by Western blot analysis and immunocytochemistry. We further examined the effect of KRT19 silencing on breast cancer cells by cell proliferation, migration, invasion, colony formation assay, cell-cycle analysis, immunocytochemistry, immunohistochemistry, and mouse xenograft assay. Results: Silencing of KRT19 resulted in increased cell proliferation, migration, invasion, and survival. These effects were mediated by upregulation of Akt signaling as a result of reduced PTEN mRNA expression. Silencing of KRT19 decreased the nuclear import of early growth response-1 (Egr1), a transcriptional factor for PTEN transcription, through reduced association between Egr1 and importin-7. We also confirmed that silencing of KRT19 increased tumor formation in a xenograft model. Conclusions: KRT19 is a potential tumor suppressor that negatively regulates Akt signaling through modulation of Egr1 nuclear localization.

Regulation of Cell Proliferation and Migration by Keratin19-Induced Nuclear Import of Early Growth Response-1 in Breast Cancer Cells

Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

In breast cancer, the HER2 (human epidermal growth factor receptor 2) receptor tyrosine kinase is associated with extremely poor prognosis and survival. Notch signalling has a key role in cell-fate decisions, especially in cancer-initiating cells. The Notch intracellular domain produced by Notch cleavage is translocated to the nucleus where it activates transcription of target genes. To determine the combinatory effect of HER2 and Notch signalling in breast cancer, we investigated the effect of HER2 on Notch-induced cellular phenomena. We found the down-regulation of Notch-dependent transcriptional activity by HER2 overexpression. Also, the HER2/ERK (extracellular-signal-regulated kinase) signal pathway down-regulated the activity of gamma-secretase. When we examined the protein level of Notch target genes in HER2-overexpressing cells, we observed that the level of survivin, downstream of Notch, increased in HER2 cells. We found that activation of ERK resulted in a decrease in XAF1 [XIAP (X-linked inhibitor of apoptosis)-associated factor 1] which reduced the formation of the XIAP-XAF1 E3 ligase complex to ubiquitinate survivin. In addition, Thr(34) of survivin was shown to be the most important residue in determining survivin stability upon phosphorylation after HER2/Akt/CDK1 (cyclin-dependent kinase 1)-cyclin B1 signalling The results of the present study show the combinatorial effects of HER2 and Notch during breast oncogenesis.

HER2 stabilizes survivin while concomitantly down-regulating survivin gene transcription by suppressing Notch cleavage

Apigenin induces apoptosis via extrinsic pathway, inducing p53 and inhibiting STAT3 and NF kappa B signaling in HER2-overexpressing breast cancer cells

To determine the role of CD24 in breast cancer cells, we knocked down CD24 in MCF-7 human breast cancer cells by retroviral delivery of shRNA. MCF-7 cells with knocked down CD24 (MCF-7 hCD24 shRNA) exhibited decreased cell proliferation and cell adhesion as compared to control MCF-7 mCD24 shRNA cells. Decreased proliferation of MCF-7 hCD24 shRNA cells resulted from the inhibition of cell cycle progression from G1 to S phase. The specific inhibition of MEK/ERK signaling by CD24 ablation might be responsible for the inhibition of cell proliferation. Phosphorylation of Src/FAK and TGF-beta 1 -mediated epithelial to mesenchymal transition was also down-regulated in MCF-7 hCD24 shRNA cells. Reduced Src/FAK activity was caused by a decrease in integrin beta 1 bound with CD24 and subsequent destabilization of integrin beta 1. Our results suggest that down-regulation of Raf/MEK/ERK signaling via Src/FAK may be dependent on integrin beta 1 function and that this mechanism is largely responsible for the CD24 ablation-induced decreases in cell proliferation and epithelial to mesenchymal transition.

CD24 regulates cell proliferation and transforming growth factor beta-induced epithelial to mesenchymal transition through modulation of integrin beta 1 stability

Primary breast sarcomas (PBSs) that arise from mammary stroma are very rare, highly aggressive and therapy-resistant tumors with a heterogeneous phenotype. In this study, we sought to identify tumor-initiating cells (TICs) in PBSs and to describe their features. We isolated long-term self-renewing sarcospheres (designated NDY-1) from primary breast carcinosarcoma tissue (sarcoma component >95%) using the anchorage-independent culture method. NDY-1 spheres expressed various mesenchymal cell markers, and their tumorigenic potential was markedly reduced in adherent culture conditions, compared to spheres. Screening for integrins revealed a marked decrease in CD49d expression in adherent culture conditions of NDY-1. The CD49d(+/high) subpopulation sorted from NDY-1 spheres displayed higher cell viability and sphere-forming ability than CD49d(-/low) population in vitro. Moreover, the CD49d(+/high) population displayed high tumor initiating ability in limiting dilution transplantation to NOD/SCID mice, and the xenotransplanted CD49d(+/high) population recapitulated the complexity of the original primary tumors. Greater doxorubicin resistance was exhibited by the CD49d(+/high) population, compared with the CD49d(-/low) population. Thus, our results collectively demonstrate that CD49d(+/high) cells from sarcospheres display enhanced sphere-forming, drug resistance and tumor-initiating abilities. To our knowledge, this is the first study to identify TICs from breast sarcoma.

The CD49d(+/high) subpopulation from isolated human breast sarcoma spheres possesses tumor-initiating ability

We identified two S/T-P-S/T domains (2122-2124, 2126-2128) inducing Notch intracellular domain (NICD) nuclear localization. The GFP-NICD (1963-2145) fusion protein deletion mutant without classical NLS was localized in the nucleus like the full length GFP-NICD. However, quadruple substitution mutant (T2122A T2124A S2126A T2128A) showed increased cytoplasmic localization. GSK-3 beta enhanced nuclear localization and transcriptional activity of WT NICD but not of quadruple substitution mutant. In vitro kinase assays revealed that GSK-3 beta phosphorylated S and T residues in NICD S/T-p-S/T domains. These results suggest that the novel SIT-P-SIT domain, phosphorylated by GSK-3 beta is also involved in the nuclear localization of NICD as well as classical NLS.

Glycogen synthase kinase 3-beta phosphorylates novel S/T-P-S/T domains in Notch1 intracellular domain and induces its nuclear localization

Phytoestrogens are known to prevent tumor induction. But their molecular mechanisms of action are still unknown. This study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with an increase of sub G(0)/G(1) apoptotic fractions. Overexpression of HER2 did not confer resistance to apigenin in MCF-7 cells. Apigenin-induced extrinsic apoptosis pathway up-regulating the levels of cleaved caspase-8, and inducing the cleavage of poly (ADP-ribose) polymerase, whereas apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential maintaining red fluorescence and did not affect the levels of B-cell lymphoma 2 (BCL2) and Bcl-2-associated X protein. Moreover, apigenin reduced the tyrosine phosphorylation of HER2 (phospho-HER2 level) in MCF-7 HER2 cells, and up-regulated the levels of p53, phospho-p53 and p21 in MCF-7 vec and MCF-7 HER2 cells. This suggests that apigenin induces apoptosis through p53-dependent pathway. Apigenin also reduced the expression of phospho-JAK1 and phospho-STAT3 and decreased STAT3-dependent luciferase reporter gene activity in MCF-7 vec and MCF-7 HER2 cells. Apigenin decreased the phosphorylation level of I kappa B alpha in the cytosol, and abrogated the nuclear translocation of p65 within the nucleus suggesting that it blocks the activation of NF kappa B signaling pathway in MCF-7 vec and MCF-7 HER2 cells. Our study indicates that apigenin could be a potential useful compound to prevent or treat HER2-overexpressing breast cancer.

Background: The clinical implication of Ras/Raf/ERK pathway activity in breast cancer tissue and its association with response to chemotherapy is controversial. We aimed to explore the value of p90RSK phosphorylation, a downstram molecule of the pathway, in predicting chemotherapy response in breast cancer. Methods: The expression of phosphorylated p90RSK (phospho-p90RSK) and chemotherapy response was measured in 11 breast cancer cell lines and 21 breast cancer tissues. The predictive value of phospho-p90RSK was validated in core needle biopsy specimens of 112 locally advanced breast cancer patients who received anthracycline and taxane-based neoadjuvant chemotherapy. Results: In 11 breast cancer cell lines, the relative expression of phospho-p90RSK was inversely correlated with cell survival after doxorubicin treatment (p = 0.021). Similar association was observed in fresh tissues from 21 breast cancer patients in terms of clinical response. In paraffin-embedded, formalin-fixed tissues from core needle biopsy tissues from 112 patients, positive phospho-p90RSK expression was associated with greater tumor shrinkage and smaller post-chemotherapy tumor size. The association between phospho-p90RSK expression and chemotherapy response was more evident in estrogen receptor(ER)-positive tumors. The expression of phosphor-p90RSK did not show a significant relationship with the incidence of pCR. P90RSK silencing using siRNA did not affect the cancer cell's response to doxorubicin, and the expression of phospho-p90RSK was highly correlated with other Ras/Raf/ERK pathway activation. Conclusion: Our results suggest that phospho-p90RSK expression, which reflects the tumor's Ras/Raf/ERK/p90RSK pathway activation can be a potential predictive marker for chemotherapy response in ER-positive breast cancer which needs further independent validation.

Phosphorylation of p90RSK is associated with increased response to neoadjuvant chemotherapy in ER-positive breast cancer

Protein kinase C delta (PKC delta) plays a significant role in the regulation of growth, apoptosis, and differentiation in a diversity of cell types. We investigated the effect of PKC delta on Notch1 intracellular domain (NICD)-mediated transcription with Notch transcription reporter constructs. The results indicate that co-expression of PKC delta down-regulated NICD-dependent transcription. Co-expression of a dominant negative PKC delta (K376R) variant lacking kinase activity was also able to downregulate NICD-dependent transcription, suggesting that PKC delta exerts its inhibitory effect via a kinase-independent mechanism(s). Interestingly, expression of PKC delta as well as K376R induced NICD up-regulation by inhibiting proteasome-mediated degradation of NICD, indicating that NICD protein quantity is not proportional to its transcriptional activity. When the subcellular distribution of NICD was investigated by both subcellular fractionation and immunocytochemistry, it was found that PKC delta and K376R effectively impaired proper nuclear localization of NICD, possibly via a physical association between NICD and PKC delta, which was confirmed by co-immunoprecipitation experiments. Chromatin immunoprecipitation assays revealed that both PKC delta and K376R inhibit the association of NICD with the promoter region of its target gene, Hes1. Furthermore, silencing of PKC delta resulted in increased NICD nuclear localization and NICD transcriptional activity in MCF-7 cells. PKC delta silencing-induced increase in anti-apoptotic survivin could not rescue apoptosis induced by doxorubicin. The data herein indicate that PKC delta can induce down-regulation of NICD transcriptional activity via a kinase-independent inhibition of NICD nuclear targeting and dissociation of NICD from target gene promoters.

Protein kinase C delta negatively regulates Notch1-dependent transcription via a kinase-independent mechanism in vitro

To investigate the role of p27 on estrogen receptor (ER)a-mediated transcription, we generated MCF-7 cells with knocked down p27 via retroviral delivery of p27 shRNA. Suppression of p27 expression in MCF-7 cells resulted in up-regulation of ER alpha-mediated transcription by estradiol compared to the levels in control MCF-7 cells. Accordingly, transient transfection studies in 293T cells revealed that overexpression of p27 reduced ER alpha-mediated transcription. The effect of p27 on ER alpha transcriptional activity was independent of cell cycle arrest by p27, as cell cycle arrest induced by serum starvation did not significantly affect ER alpha-mediated transcription. Further, we observed that p27 inhibited nuclear localization of ER alpha, and that p27 was associated with ER alpha in the cytoplasm. We also investigated the role of p27 in the modulation of ER alpha transcriptional activity in the nucleus. We found that p27 negatively modulated ERa transcriptional activity by inhibiting association of cyclin D1 with ERa and recruiting BRCA1 to ER alpha transcriptional complex. Taken together, these data suggest that p27 inhibits ER alpha transcriptional activity by two independent mechanisms, namely, physical nuclear exclusion of ER alpha, and modulation of the ERa transcriptional complex.

Down-regulation of estrogen receptor alpha (ER alpha) transcriptional activity by p27 is mediated by inhibition of ERa alpha nuclear localization and modulation of the ER alpha transcriptional complex

The biochemical mechanism by which the human tumorous imaginal disc1(S) (hTid-1(S)) interferes with actin cytoskeleton organization in keratinocytes of human skin epidermis was investigated. We found that hTid-1, specifically hTid-1(S), interacts with MK5, a p38-regulated/activated protein kinase, and inhibits the protein kinase activity of MK5 that phosphorylates heat shock protein HSP27 in cultured HeLa cells. Thus, hTid-1(S) expression inhibits the phosphorylation of HSP27 known to play important roles in F-actin polymerization and actin cytoskeleton organization. The interplay between MK5/HSP27 signaling and hTid-1(S) expression was supported by the inhibition of HSP27 phosphorylation and MK5 activity in HeLa cells in response to hypoxia during which hTid-1(S) expression was down-regulated. We also found that overexpression of hTid-1(S) results in the inhibition of HSP27 phosphorylation, F-actin polymerization, and actin cytoskeleton organization in transduced HaCaT keratinocytes. This study further proposes that the loss of hTid-1(S) expression in the basal layer of skin epidermis correlates with the enhanced HSP27 phosphorylation, keratinocyte hyperproliferation, and excess actin cytoskeleton organization in lesional psoriatic skin.

Absence of a Human DnaJ Protein hTid-1(S) Correlates with Aberrant Actin Cytoskeleton Organization in Lesional Psoriatic Skin

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin-beta 1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral-MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HAG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.

A functional comparison between the HER2(high)/HER3 and the HER2l(ow/)HER3 dimers on heregulin-beta 1-induced MMP-1 and MMP-9 expression in breast cancer cells

To dissect the isoform-specific roles of Akt in breast cancer cells, constitutively active Akt isoforms were introduced into MDA-MB-231 cells. Both Akt1 and Akt2 efficiently inhibited the growth of MDA-MB-231 cells. Overexpression of Akt1 down-regulated ERK activity inhibiting Ser 259 phosphorylation of c-Raf and subsequent downstream signaling. Akt2 overexpression up-regulated the cell cycle inhibitor p27. Cycloheximide decay assays showed that Akt2 increased the stability and nuclear localization of p27, thus inhibiting the cyclin E/CDK2 complex. These results suggest that the inhibition of cell proliferation by Akt1 and Akt2 is mediated by isoform-specific mechanisms.

Akt isoform-specific inhibition of MDA-MB-231 cell proliferation

In this study, we investigated the underlying mechanism by which phytoestrogens suppress the growth of normal (MCF-10A) and malignant (MDA-M B-231) estrogen receptor a (ER alpha) negative breast cells. We hypothesized that phytoestrogen inhibits the proliferation of ER alpha-negative breast cancer cells. We found that all tested phytoestrogens (genistein, apigenin, and quercetin) suppressed the growth of both MCF-10A and MDA-MB-231 cells, as revealed by proliferation assays. These results were accompanied by an increase in the sub-G0/G1 apoptotic fractions as well as an increase in the cell population in the G2/M phase in both cell types, as revealed by cell cycle analysis. When we assessed the effect of phytoestrogens on the level of intracellular signaling molecules by Western blot analysis, we found that phytoestrogens increased the level of active p53 (phospho-p53) without changing the p53 level in both MCF-10A and MDA-MB-231 cells. Phytoestrogens also induced an increase in p21, a p53 target gene, and a decrease in either Bcl-xL or cyclin B1 in both cell types. In contrast, the protein levels of phosphatase and tensin homolog, cyclin D1, cell division control protein 2 homolog, phospho-cell division control protein 2 homolog, and p27 were not changed after phytoestrogen treatment. Our data indicate that phytoestrogens induce apoptotic cell death of ER alpha-negative breast cancer cells via p53-dependent pathway and suggest that phytoestrogens may be promising agents in the treatment and prevention of ER alpha-negative breast cancer.

Induction of apoptotic cell death by phytoestrogens by up-regulating the levels of phospho-p53 and p21 in normal and malignant estrogen receptor alpha-negative breast cells

Cluster of differentiation 24 (CD24) is a small glycosylphosphatidylinositol-linked cell surface molecule that is expressed in a variety of human carcinomas, including breast cancer. To determine the role of CD24 in breast cancer cells, we expressed CD24 in CD24-negative/low and cluster of differentiation 44 (CD44)-positive MDA-MB-231 metastatic breast cancer cells. Forced expression of CD24 resulted in a decrease in c-Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase signaling and reduced cell proliferation. Apoptosis induced by DNA damage was greatly enhanced in MDA-MB-231 CD24 cells as compared with MDA-MB-231 vec cells. CD24 expression efficiently attenuated DNA damage-induced nuclear factor-kappaB (NF-kappa B) signaling in MDA-MB-231 cells. However, in CD24-positive and CD44-negative/low MCF-7 cells, knockdown of CD24 did not significantly affect DNA damage-induced apoptosis nor NF-kappa B signaling. Silencing of CD24 in CD24/CD44-double-positive MDA-MB-468 cells partially rescued DNA damage-induced apoptosis. Transient transfection studies with 293T cells also revealed that CD24 attenuated cell viability and NF-kappa B signaling only when CD44 was cotransfected. These data indicate that CD24 expression potentiated DNA-induced apoptosis by suppressing antiapoptotic NF-kappa B signaling in CD44-expressing cells.

CD24 enhances DNA damage-induced apoptosis by modulating NF-kappa B signaling in CD44-expressing breast cancer cells

Aim of the study: We performed this study to investigate the anti-cancer activity of Pharbitis nil (PN) ethanol extract which has been used for herbal medicinal treatment against diseases in East Asia. Materials and methods: We analyzed the effects of PN extract on proliferation of breast cancer cell lines, MCF-7 control vector (vec) and MCF-7 human epidermal growth factor receptor 2 (HER2) cells engineered to overexpress oncogenic HER2 via retroviral infection. We performed the proliferation assay to measure the growth rate of the cells. FACS analysis was used to analyze the cell cycle. Western blot analysis was used to investigate the effect of PN on the level and activation of intracellular molecules. Results: We found that PN extract inhibited the proliferation of both MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with the increase of sub G0/G1 apoptotic fractions. When we check the efficiency of PN on the level of intracellular signaling molecules, we found that PN extract induced the inhibition of phosphorylation of HER2 and its downstream effectors, Akt and extracellular signal-regulated kinases (ERK). Active forms of both Akt and ERK were gradually decreased in PN-treated MCF-7 vec and MCF-7 HER2 cells suggesting that the growth suppressive activity of PN is related to signaling pathway. The level of cyclin D also diminished in PN-treated both cells suggesting that PN may inhibit the growth of MCF-7 vec and MCF-7 HER2 cells by perturbing cell cycle progression. It should be noted that PN decreased the growth rate of both MCF-7 vec and MCF-7 HER2 cells without changing the level and activation of p53. Conclusion: PN extract suppressed the proliferation rate of HER-2 overexpressing MCF-7 breast cancer cells inducing apoptotic cell death in vitro. Our data demonstrates that PN extracts contain useful antitumor activity especially against HER2 overexpressing breast cancer.

Induction of apoptotic cell death by Pharbitis nil extract in HER2-overexpressing MCF-7 cells

To assess the effects of endocrine-disrupting chemicals on the expression and activity of aromatase in the gonads of Bombina orientalis, a common amphibian, we intraperitoneally injected nonylphenol or bisphenol-A and then examined aromatase mRNA levels by RT-PCR as well as aromatase enzymatic activity by tritiated water release assays. To design primers for the RT-PCR, we cloned the B. orientalis aromatase gene using RT-PCR and degenerate primers. The full-length cDNA was obtained by 5'- and 3'-RACE PCR. The complete sequence of the B. orientalis aromatase gene revealed an open reading frame of 1500 bp encoding a deduced protein of 500 amino acids. Semi-quantitative RT-PCR indicated that nonylphenol or bisphenol-A injection did not significantly affect the expression of B. orientalis aromatase mRNA. However, a 48-hr treatment with nonyphenol or bisphenol A reduced aromatase activity to 47% and 32% of the control, respectively. These results suggest that endocrine disruptors can effectively modulate the activity of B. orientalis aromatase without affecting its mRNA levels.

Effects of Endocrine Disruptors on Bombina orientalis P450 Aromatase Activity

MMTV-neu transgenic mice overexpressing NEU in their mammary glands develop tumor after 6 months of age. To find a novel protein biomarker using this mouse model, we identified and characterized the proteins that were differently expressed between primary mammary epithelial cells from 2 months old MMTV-neu heterozygote mice and wild type (WT) littermates using two-dimensional digest (ChemDigest (TM)/Trypsin)-LC-MS/MS. The differentially expressed proteins were selected and analyzed using DAVID Bioinformatics resource. The proteins involved in anti-apoptosis, purine metabolism, ribosome and proteasome functions were upregulated, whereas cell adhesion-related proteins were downregulated in PMECs from MMTV-neu mice when compared with WT PMECs. The results indicate that several functional units are coregulated by HER2/NEU. We hypothesize that these changes in the cellular proteome may be responsible for early onset of HER2/NEU-driven tumorigenesis.

Protein Expression Profiling of Primary Mammary Epithelial Cells Derived from MMTV-neu Mice Revealed that HER2/NEU-Driven Changes in Protein Expression Are Functionally Clustered

Danshen is widely used in traditional Chinese medicine, often in combination with other herbs. To check the effect of Danshen on the proliferation of breast cancer cells, Danshen extract was used to treat MCF-7 and MCF-7 HER2 cells, the latter of which overexpresses HER2. HER2 is a receptor tyrosine kinase, and is involved in signal transduction pathways leading to tumor cell proliferation. MTT and cell proliferation assails revealed that Danshen strongly inhibited the proliferation of both MCF-7 vec cells and MCF-7 HER2 cells. Flow cytometry analyses indicated that Danshen induced cell cycle delay in the G1 phase. HER2 expression was shown to confer resistance to Danshen-induced inhibition of proliferation and cell cycle delay, suggesting that HER2 is responsible for the resistance to Danshen. Danshen treatment induced the down-regulation of Akt phosphorylation and an increase in p27 in MCF-7 vec and MCF-7 HER2 cells. Nevertheless, MCF-7 HER2 cells were more resistant to the Danshen-induced inhibition of Akt phosphorylation and p27 up-regulation.

Danshen (Salvia miltiorrhiza) Extract Inhibits Proliferation of Breast Cancer Cells via Modulation of Akt Activity and p27 Level

In this article, we introduce a rapid and simple fabrication method to realize a 3-dimensional (3-D) microfluidic channel with a near-perfect circular cross section. This new concept of fabrication method is defined by metal wire removal process, where the metal wire such as a thin soldering wire for the 3-D circular shape is commercially available. For the microfluidic channel mold, PDMS (polydimethylsiloxane) was poured on several shapes such as 3-D circular, helix, and double helix shapes, of soldering wire and solidified. The soldering wire was then melted out by heating. With the two-step process, rapidly and simply fabricated 3-D circular microfluidic channels can be obtained. CPAE (endothelial cell line) cells were cultured inside the channel to evaluate the biocompatibility of the fabricated microfluidic channel. Our method will be very useful in making various circular shapes of 3-D microfluidic devices that need multi-depth and round corners inside the channel.

A rapid and simple fabrication method for 3-dimensional circular microfluidic channel using metal wire removal process

Phosphatase and tensin homolog (PTEN) is a tumor suppressor that inhibits PI3K/Akt signaling. To examine the effect of PTEN on breast cancer cell proliferation, we expressed PTEN in MDA-MB-468 cells (MDA-MB-468 PTEN) by retroviral infection and tested the cell proliferation rate. We found that the growth rate of MDA-MB-468 PTEN cells was significantly lower than that of MDA-MB-468 control vector cells (MDA-MB-468 vec). When the PI3K/Akt signaling inhibitor LY294002 and the MEK/Erk signaling inhibitor U0126 were applied, LY294002 reduced cell proliferation in MDA-MB-468 PTEN and MDA-MB-468 vec by 20%, while U0126 led to a >60% reduction in MDA-MB-468 PTEN and a 20% reduction in MDA-MB-468 vec cells. FACS analysis demonstrated that the subG0/G1 apoptotic fraction was significantly increased in MDA-MB-468 PTEN cells after U0126 treatment, while LY294002 treatment in both cell lines and U0126 treatment in MDA-MB-468 vec cells led to a modest increase in the apoptotic fraction. This phenomenon was accompanied by the down-regulation of p-Erk. p-Erk levels were significantly lower after U0126 treatment in MDA-MB-468 PTEN cells. Similar results were observed in MDA-MB-231 cells, which express endogenous PTEN. The growth of MDA-MB-231 cells was significantly inhibited after U0126 treatment, compared to LY294002, while PTEN-null ZR-75-1 cells did not show increased sensitivity to U0126 over LY294002. Taken together, these findings suggest that blockade of PI3K/Akt signaling by PTEN may render breast cancer cells more dependent on the MEK/Erk pathway for their proliferation and survival.

PTEN sensitizes MDA-MB-468 cells to inhibition of MEK/Erk signaling for the blockade of cell proliferation

We have previously reported that MMTR (MAT1-mediated transcriptional repressor) is a co-repressor that inhibits TFIIH-mediated transcriptional activity via interaction with MAT1 (Kang et al., 2007). Since MAT1 is a member of the CAK kinase complex that is crucial for cell cycle progression and that regulates CDK phosphorylation as well as the general transcription factor TFIIH, we investigated MMTR function in cell cycle progression. We found that MMTR over-expression delayed G1/S and G2/M transitions, whereas co-expression of MAT1 and MMTR rescued the cell growth and proliferation rate. Moreover, MMTR was required for inhibition of CAK kinase-mediated CDK1 phosphorylation. We also showed that the expression level of MMTR was modulated during cell cycle progression. Our data support the notion that MMTR is an intrinsic negative cell cycle regulator that modulates the CAK kinase activity via interaction with MAT1.

Corepressor MMTR/DMAP1 is an intrinsic negative regulator of CAK kinase to regulate cell cycle progression

TPA is a potent regulator of cell growth, including cell proliferation and differentiation. In this study, we determined the effect of silibinin on TPA-induced growth arrest in breast cancer cells. Silibinin increased growth arrest of the G2/M phase in a dose-dependent fashion. Silibinin decreased the basal level of cyclin B1 and cdc2 expression, which is involved in S phase and G2/M transition. In addition, TPA-induced G2/M phase arrest was increased by silibinin. Under the same conditions, TPA-induced down-regulation of cyclin B1 and cdc2 was decreased by silibinin. In contrast, TPA-induced p21 expression was further increased by silibinin. To determine the regulatory mechanism of TPA-induced growth arrest, we pre-treated cells with various inhibitors, such as UO126, SB203580, and LY294002. Interestingly, TPA-induced growth arrest was significantly increased by LY294002, but not by UO126 and SB203580. In addition, TPA-induced down-regulation of cyclin B1 was inhibited by LY294002; however, the basal level of p21 was increased by TPA and TPA-induced p21 expression was further increased by LY294002. Finally, adenoviral constitutively active-Akt (Ad-CA-Akt) overexpression regulated the up-regulation of cyclin B1 and the down-regulation of p21. Therefore, we have demonstrated that silibinin has an additive effect on TPA-induced growth arrest through the PI-3-kinase/Akt-dependent pathway.

12-O-Tetradecanoyl phorbol-13-acetate (TPA)-induced growth arrest is increased by silibinin by the down-regulation of cyclin B1 and cdc2 and the up-regulation of p21 expression in MDA-MB231 human breast cancer cells

Cyclooxygenase-2 (COX-2) has been reported to be elevated in many cancers, including breast and colorectal cancers, resulting in accumulation of prostaglandin E-2 in the cancer cell environment. In this study, we investigated the effect of pifithrin (PFT)-alpha, an inhibitor of p53 transactivation, on COX-2 expression in breast and fibrosarcoma cells. Our results showed that COX-2 expression was dose-dependently increased by PFT-alpha in MDA-MB231 breast cancer cells with mutant p53. In addition, the expression level of COX-2 was also increased by PFT-alpha in normal fibroblasts as well as in HT1080 fibrosarcoma cells with p53 wild-type cells. To verify the regulatory mechanism of COX-2 in response to PFT-alpha, we pre-treated cells with a mitogen-activated protein kinase (MAPK) kinase (MEK)1/2 inhibitor (UO126) and a phosphoinositide-3 (PI-3K) inhibitor (LY294002). PFT-alpha-induced COX-2 expression was significantly decreased by UO126 and LY294002 in MDA-MB231 cells. However, the phosphorylation of extracellular signal-regulated kinase (ERK) was increased by PFT-alpha, but not Akt phosphorylation. Finally, we confirmed the correlation of the MEK and PI-3K pathway and COX-2 expression using the constitutively active (CA)-MEK and myr-Akt adenovirus systems. COX-2 expression was increased by CA-MEK transfection, but not by myr-Akt. Taken together, we have demonstrated that PFT-alpha-induced COX-2 expression is regulated through a MEK/ERK pathway in MDA-MB231 human breast cancer cells.

Pifithrin-alpha an Inhibitor of p53 Transactivation, Up-Regulates COX-2 Expression through an MAPK-Dependent Pathway

p27 is a member of the Kip family of cyclin-dependent kinase inhibitors and its overexpression results in cell cycle arrest at G1 and/or apoptosis. In addition to its role as a regulator of cell cycle progression, p27 can also participate in cell motility, especially when it is mislocalized in the cytosol. To further elucidate the role of p27 in the motility of MDA-MB-231 breast cancer cells. we performed p27 knockdown in MDA-MB-231 cells by RNA interference. Infection of MDA-MB-231 cells with retroviruses harboring p27 short hairpin RNA (shRNA) designed from human p27 cDNA resulted in efficient inhibition of p27 expression, while p27 shRNA designed from mouse p27 cDNA did not affect p27 expression in MDA-MB-231 cells. MDA-MB-231 cells infected with human p27 shRNA (MDA-MB-231 hp27shRNA) showed increased proliferation compared to control MDA-MB-231 cells and MDA-MB-231 cells infected with mouse p27 shRNA (MDA-MB-231 mp27shRNA). Wound healing assays revealed that migration of MDA-MB-231 hpshRNA cells was markedly impaired compared to MDA-MB-231 mpshRNA cells, especially when cycloheximide was added to block protein synthesis. Immunostaining of p27 in MDA-MB-231 cells showed that p27 predominantly localized in the nuclei. These results suggest that both nuclear and cytosolic p27 call promote cancer cell motility.

Effect of p27 on motility of MDA-MB-231 breast cancer cells

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.

Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Nurr1 is a transcription factor specific for the development and maintenance of the midbrain dopamine (DA) neurons. Exogenous Nurr1 in neural precursor (NP) cells induces the differentiation of DA neurons in vitro that are capable of reversing motor dysfunctions in a rodent model for Parkinson disease. The promise of this therapeutic approach, however, is unclear due to poor cell survival and phenotype loss of DA cells after transplantation. We herein demonstrate that Nurr1 proteins undergo ubiquitin-proteasome-system-mediated degradation in differentiating NP cells. The degradation process is activated by a direct Akt-mediated phosphorylation of Nurr1 proteins and can be prevented by abolishing the Akt-target sequence in Nurr1 (Nurr1(Akt)). Overexpression of Nurr1(Akt) in NP cells yielded DA neurons in which Nurr1 protein levels were maintained for prolonged periods. The sustained Nurr1 expression endowed the Nurr1(Akt)-induced DA neurons with resistance to toxic stimuli, enhanced survival, and sustained DA phenotypes in vitro and in vivo after transplantation. STEM CELLS 2009; 27: 2238-2246

Generation of Dopamine Neurons with Improved Cell Survival and Phenotype Maintenance Using a Degradation-Resistant Nurr1 Mutant

The effect of Akt on Notch intracellular domain (NICD)-mediated transcription was investigated. Transfection experiments revealed that constitutively active Akt down-regulates NICD-dependent transcription. Kinase inactive dominant negative Akt did not affect NICD transcriptional activity, indicating that Akt kinase activity is responsible for the down-regulation. Studies using histone deacetylase (HDAC) and silencing mediator of retinoid and thyroid hormone receptor (SMRT) revealed that modulation of NICD transcriptional activity is not mediated by an HDAC-dependent mechanism or recruitment of the co-repressor SMRT. Akt inhibited proper nuclear localization of NICD, and phosphorylated NICD both in vitro and caused its hyperphosphorylation in vivo. These results may suggest possible regulation of NICD transcriptional activity by Akt-mediated phosphorylation and subsequent inhibition of proper nuclear localization of NICD.

Down-regulation of Notch-dependent transcription by Akt in vitro

We have investigated the effect of Akt on estrogen receptor (ER) a protein level and its transcriptional activity. Transient transfection studies revealed that constitutively active AM up-regulated ER alpha at the post-transcriptional level. Studies using Akt inhibitor and dominant-negative Akt1 showed that Akt1 kinase activity is required for the up-regulation of ERa. Cycloheximicle decay assays and studies with proteasome inhibitor indicated that Akt1-mediated up-regulation of ER alpha was maintained by inhibiting proteasome-mediated degradation of ERa. When Akt consensus phosphorylation site mutant, ER alpha S167A was tested for Akt1-mediated up-regulation, increase of ER alpha S167A by Akt1 was significantly impaired as compared to wild type ERa. In addition, dominant-negative glycogen synthase kinase (GSK) 3 beta and LiCl could also partially up-regulate ERa protein level, suggesting that concerted action of Akt1-mediated phosphorylation on S167 and kinase activity of Akt-downstream GSK3 beta could affect ERa protein level. Paradoxically, co-expression of Akt1 could down-regulate transcriptional activity of ER alpha. The inhibitory effect of Akt1 on ERa transcriptional activity was not attributable to changes in subcellular distribution of ERa. Transfection studies using increasing amount of Akt1 and ERa indicated that the transcriptional activity of ER alpha was negatively regulated by ERa protein quantities at higher ER alpha concentrations. Chromatin immunoprecipitation assays revealed that at Akt1 concentration high enough to induce up-regulation of ERa, association of ERa to promoter region of ERa target pS2 gene was impaired. Taken together, these data suggest that Akt1 could increase ER alpha protein level with simultaneous reduction in its transcriptional activity, possibly by modulating association of ERa to the target gene promoters.

Akt stabilizes estrogen receptor alpha with the concomitant reduction in its transcriptional activity

Background: The biological effects of CD24 (FL-80) cross-linking on breast cancer cells have not yet been established. We examined the impact of CD24 cross-linking on human breast cancer cell line MCF-7. Methods: MCF-7 and MDA-MB-231 cells were treated with anti-rabbit polyclonal IgG or anti-human CD24 rabbit polyclonal antibodies to induce cross-linking, and then growth was studied. Changes in cell characteristics such as cell cycle modulation, cell death, survival in three-dimensional cultures, adhesion, and migration ability were assayed after CD24 cross-linking in MCF-7. Results: Expression of CD24 was analyzed by flow cytometry in MDA-MB-231 and MCF-7 cells where 2% and 66% expression frequencies were observed, respectively. CD24 cross-linking resulted in time-dependent proliferation reduction in MCF-7 cells, but no reduction in MDA-MB-231 cells. MCF-7 cell survival was reduced by 15% in three-dimensional culture after CD24 cross-linking. Increased MCF-7 cell apoptosis was observed after CD24 cross-linking, but no cell cycle arrest was observed in that condition. The migration capacity of MCF-7 cells was diminished by 30% after CD24 cross-linking. Conclusion: Our results showed that CD24 cross-linking induced apoptosis and inhibited migration in MCF-7 breast cancer cells. We conclude that CD24 may be considered as a novel therapeutic target for breast cancer.

CD24 cross-linking induces apoptosis in, and inhibits migration of, MCF-7 breast cancer cells

The plant alkaloid berberine has many biological activities including the ability to induce cell cycle arrest and apoptosis, making it a potentially useful agent for targeting cancer cells. We have analyzed the effects of berberine oil MCF-7 breast cancer cells. Berberine was added to MCF-7 cells in Culture, and proliferation, side population (SP) cells and expression of ABCG2 were examined. Berberine caused a close-dependent reduction in Proliferation. Hoechst 33342 dye staining and FACS analysis revealed that berberine treatment caused a decrease in SP cells relative to untreated controls. In addition, berberine treatment was associated with a decrease in expression of ABCG2 relative to untreated controls. These results indicate that the growth inhibitory effects of berberine treatment on MCF-7 cells may be partly via effects oil SP and ABCG2 expression. Further work is warranted to explore whether berberine may be a novel therapeutic drug useful for targeting breast cancer stem cells.

Berberine Diminishes the Side Population and ABCG2 Transporter Expression in MCF-7 Breast Cancer Cells

Phosphorylation of the cytochrome P450 aromatase has been proposed as a switch to rapidly modulate enzymatic activity and estrogen biosynthesis. Herein, we demonstrate that aromatase serine-118 is a potential phosphoiylation site in mammalian cells. The amino acid context surrounding 5118 is highly conserved among diverse animal species and suggests that an AGC-like kinase may phosphorylate aromatase. Mutation of 5118 to Ala blocked phosphorylation. Mutation of 5118 to either Ala or Asp destabilized aromatase, indicating an important structural role for S118. The phosphomimetic S118D mutant showed decreased specific enzymatic activity, decreased Vmax, and increased Km, while the S118A phospho-inhibiting mutant showed opposite effects. Our findings suggest that phosphorylation of 5718 may decrease aromatase activity, presenting a mechanism whereby kinase signaling may modulate estrogen production and hormone balance.

Aromatase is phosphorylated in situ at serine-118

The effects of berberine on the behavior of breast tumors have not yet been established. To determine whether this compound is useful in the treatment of breast cancer, we analyzed the impact of berberine on the human breast cancer cell lines MCF-7 and MDA-MB-231 cells. Berberine was added to proliferating MCF-7 and MDA-MB-231 cells in culture. Following treatment, changes in cell growth characteristics such as proliferation, cell cycle duration, and the degree of apoptosis were assayed. Following berberine treatment, a time-dependent reduction in proliferation was observed in both cell lines at differing concentrations: 20 mu M for MCF-7 and 10 mu M for MDA-MB-231 cells. Annexin V staining showed an increase in apoptosis in both cell lines of 31 % in MCF-7and 12 % in MDA-MB-231 cells compared to their respective controls. In addition, 12 % of the MCF-7 cells were arrested at G0/G1, compared to 62 % of control cells. These results demonstrate that treatment with berberine inhibits growth in both MDA-MB-231 and MCF-7 cells. In addition, they show that this partly occurs through the induction of apoptosis in MDA-MB-231 cells, and through both cell cycle arrest and induction of apoptosis in MCF-7 cells. Thus, berberine may be a novel therapeutic drug for breast cancer.

Berberine inhibits growth of the breast cancer cell lines MCF-7 and MDA-MB-231

Neural precursor cells (NPCs) are regarded as a promising source of donor cells in transplantation-based therapies for neurodegenerative disorders. However, poor survival and limited neuronal differentiation of the transplanted NPCs remain critical limitations for developing therapeutic strategies. In this study, we investigated the effects of the proneural basic helix-loop-helix (bHLH) transcription factors Mash1 and Neurogenin 2 (Ngn2) in neuronal differentiation and survival of NPCs. Induction of Mash1 or Ngn2 expression strikingly enhanced neuronal differentiation of cultured NPCs in vitro. Ngn2-transduced NPCs underwent a rapid cell cycle arrest, which often accompanies differentiation. In contrast, cells continuously expanded upon Mash1 expression during NPC differentiation. Notably, sonic hedgehog (SHH) was upregulated by Mash1 and mediated the proliferative and survival effects of Mash1 on NPCs. Upon transplantation into adult rat brains, Mash1-expressing NPCs yielded large grafts enriched with neurons compared to control LacZ-transduced NPCs. Interestingly, enhancements in neuronal yield, as well as in donor cell survival, were also achieved by transplanting Ngn2-transduced NPCs. We show that a differentiation stage- and cell density-dependent survival effect of Ngn2 involves neurotrophin3 (NT3)/TrkC-mediated signaling. Together, these findings suggest potential benefits of bHLH gene manipulation to develop successful transplantation strategies for brain disorders.

Mash1 and Neurogenin 2 Enhance Survival and Differentiation of Neural Precursor Cells After Transplantation to Rat Brains via Distinct Modes of Action

CD24 has been reported to have a role in metastasis of several malignancies including breast cancer. We investigated the effect of CD24 expression on biologic features in human breast cancer cells, and found that CD24 expression is associated with more rapid growth and enhanced ability of adhesion and invasion in MCF-7. The proportion of cells was higher in synthetic phase and lower in arrestic phase for CD24(high) than for CD24(low/-) cells. Cyclin D1 and p27 had stronger expressions in CD24(low/-) cells than CD24(high) cells. These suggested one possible pathway for CD24 effect on proliferation.

Isolation of CD24(high) and CD24(low/-) cells from MCF-7: CD24 expression is positively related with proliferation, adhesion and invasion in MCF-7

A transcription corepressor, MAT1-mediated transcriptional repressor (MMTR), was found in mouse embryonic stem cell lines. MMTR orthologs (DMPA1) are found in a wide variety of life forms from yeasts to humans. MMTR down-regulation in differentiating mouse embryonic stem cells in vitro resulted in activation of many unrelated genes, suggesting its role as a general transcriptional repressor. In luciferase reporter assays, the transcriptional repression activity resided at amino acids 221 to 468. Histone deacetylase I (HDAC1) interacts with MMTR both in vitro and in vivo and also interacts with MMTR in the nucleus. Interestingly, MMTR activity was only partially rescued by competition with dominant-negative HDAC1(H141A) or by treatment with an HDAC inhibitor, trichostatin A (TSA). To identify the protein responsible for HDAC1-independent MMTR activity, we performed a yeast two-hybrid screen with the full-length MMTR coding sequence as bait and found MAT1. MAT1 is an assembly/targeting factor for cyclin-dependent kinase-activating kinase which constitutes a subcomplex of TFIIH. The coiled-coil domain in the middle of MAT1 was confirmed to interact with the C-terminal half of MMTR, and the MMTR-mediated transcriptional repression activity was completely restored by MAT1 in the presence of TSA. Moreover, intact MMTR was required to inhibit phosphorylation of the C-terminal domain in the RNA polymerase 11 largest subunit by TFIIH kinase in vitro. Taken together, these data strongly suggest that MMTR is part of the basic cellular machinery for a wide range of transcriptional regulation via interaction with TFIIH and HDAC.

Corepressor MMTR/DMAP1 is involved in both histone deacetylase 1-and TFIIH-mediated transcriptional repression

We investigated the effect of constitutive active Akt expression on anti-proliferative and apoptotic effect of tamoxifen in MCF-7 human breast cancer cells. Forced expression of Akt(DD) (T308D, S473D) resulted in increased phosphorylation of GSK3 beta, a physiological substrate of Akt. When estrogen receptor (ER) mediated transcription was determined by luciferase assays, there was more than 2-fold increase in estradiol-dependent transcription in MCF-7 cells overexpressing Akt(DD) (MCF-7 Akt(DD)) compared to vector control cells (MCF-7 vec). MCF-7 Akt(DD) cells showed increased proliferation in a medium containing charcoal stripped serum supplemented with estradiol. When the cell cycle profiles were examined, there was an increase in S-phase and a reduction in G1 phase in MCF-7 Akt(DD) cells as compared to MCF-7 vec cells. Overexpression of Akt(DD) also attenuated tamoxifen-mediated apoptosis. These results suggest that Akt could confer resistance to anti-estrogen mediated cell death and inhibition of proliferation.

Expression of active Akt protects against tamoxifen-induced apoptosis in MCF-7 cells

In HER2 (ErbB2)-overexpressing cells, transforming growth factor beta (TGF-beta), via activation of phosphoinositide-3 kinase (PI3K), recruits actin and actinin to HER2, which then colocalizes with Vav2, activated Rac1, and Pak1 at cell protrusions. This results in prolonged Rac1. activation, enhanced motility and invasiveness, Bad phosphorylation, uncoupling of Bad/Bcl-2, and enhanced cell survival. The recruitment of the HER2/Vav2/Rac1/Pak1/actin/actinin complex to lamellipodia was abrogated by actinin siRNAs, dominant-negative (dn) p85, gefitinib, and dn-Rac1 or dnPak1, suggesting that the reciprocal interplay of PI3K, HER2 kinase, and Rac GTPases with the actin cytoskeleton is necessary for TGF-beta action in oncogene-overexpressing cells. Thus, by recruiting the actin skeleton, TGF-beta "cross-links" this signaling complex at cell lamellipodia; this prolongs Rac1 activation and increases metastatic properties and survival of HER2-overexpressing cells.

HER2/Neu (ErbB2) signaling to Rac1-Pak1 is temporally and spatially modulated by transforming growth factor beta

Emerging evidence suggests that brain aquaporins (AQPs) play important roles in the dynamic regulation of brain water horneostasis and the production of cerebrospinal fluid (CSF) under normal, as well as pathological, conditions. To date, the spatiotemporal expression patterns of AQP1, 4, and 9 have been elucidated in brain tissues. However, the expression of AQP7, an aquaglyceroporin associated with brain development, has not been shown. In the present study, we examined expression of AQP7 during perinatal and adult brain development in the mouse. Throughout brain development, the immunoreactivity of AQP7 was largely found in the choroid plexus (CP). AQP7 immunoreactivity in ependyma (Ep), pia, and blood vessels (BV) was increased during perinatal to postnatal development. Cells in the different layers of cerebral cortex became a little positive for AQP7 immunoreactivity during postnatal development. Optimized semi-quantitative RT-PCR and Western blot analysis revealed that AQP7 mRNA and protein levels increased during perinatal development of brain. To our knowledge, this is the first report on the pattern of AQP7 expression in brain tissues. These results suggest that AQP7 is an important structural element in the choroid plexus and is possibly involved in the production of CSF during brain development in mice.

Aquaporin7 expression during perinatal development of mouse brain

We generated a p27(Kip1) mutant (p27 Delta NLS) that localized exclusively in cell cytosol. Expression of p27 Delta NLS in MCF7 breast cancer cells down-regulated RhoA and increased motility, survival, and Akt levels without an effect on cell cycle distribution. RNA interference of p27 in U87 glioma cells, which express p27 predominantly in the cytoplasm, inhibited motility and survival. Conversely, knockdown of p27 in COS7 cells, with > 95% nuclear p27 expression, accelerated proliferation but had no effect on motility or survival. U87 cells in which p27 had been eliminated by RNA interference exhibited lower Akt levels, shorter Akt turnover, and markedly impaired tumorigenicity in vivo. These xenografts were less invasive and exhibited increased apoptosis compared with p27-expressing tumors. Expression of cytosolic p27 in primary human breast carcinomas correlated linearly with Akt content as measured by immunohistochemistry. These data suggest that cytoplasmic p27 can exert oncogenic functions by modulating Akt stability, cell survival, and tumorigenicity.

Reduction of cytosolic p27(Kip1) inhibits cancer cell motility, survival, and tumorigenicity

As the LIF-induced Jak1/STAT3 pathway has been reported to play a crucial role in self-renewal of mESCs, we sought to determine if Jak2, which is also expressed in mESCs, might also be involved in the pathway. By employing an RNAi strategy, we established both Jak2 and Jak2/Tyk2 knockdown mESC clones. Both Jak2 and Jak2/Tyk2 knockdown clones maintained the undifferentiated state as wild-type controls, even in a very low concentration of LIF. However, we observed not only faster onset of differentiation but also differential expression of tissue-specific lineage genes for ectodermal and mesodermal, but not endodermal origins from embryoid bodies generated from both types of knockdown clones compared to the wild-type. Furthermore, the reduced level of Jak2 caused differentiation of mESCs in the presence of LIF when the Writ pathway was activated by LiCl treatment. Taken together, we demonstrated that Jak2 and Tyk2 are not involved in LIF-induced STAT3 pathway for self-renewal of mESCs, but play a role in early lineage decision of mESCs to various differentiated cell types.

Jak2 and Tyk2 are necessary for lineage-specific differentiation, but not for the maintenance of self-renewal of mouse embryonic stem cells

We have examined the effects of transforming growth factor-beta (TGF beta) signaling on mammary epithelial cell survival. Transgenic mice expressing an active mutant of Alk5 in the mammary gland (MMTV-Alk5(T204D)) exhibited reduced apoptosis in terminal endbuds and during postlactational involution. Transgene-expressing mammary cells contained lower Smad2/3 and higher c-myc levels than controls, high ligand-independent phosphatidylinositol-3 kinase (PI3K) and Akt activities, and were insensitive to TGF beta-mediated growth arrest. Treatment with a proteasome inhibitor increased Smad2/3 levels and ligand-independent Smad transcriptional reporter activity, as well as reduced both c-myc protein and basal cell proliferation. Treatment with an Alk5 kinase small-molecule inhibitor upregulated Smad2/3 levels, reduced PI3K activity, P-Akt, and c-myc, and inhibited cell survival. Although Alk5(T204D)-expressing mice did not develop mammary tumors, bigenic MMTV-Alk(T204D) x Neu mice developed cancers that were more metastatic than those occurring in MMTV-Neu transgenics. These data suggest that (1) TGF beta can signal to PI3K/Akt and enhance mammary epithelial cell survival in vivo before cytological or histological evidence of transformation, and (2) TGF beta signaling can provide epithelial cells with a 'gain-of-function' effect that synergizes with oncogene-induced transformation.

분자세포생물학연구실

교수님 소개

신인철

연구실 소개

연구실 키워드

알럼나이가 간 회사

연구실 지원 방법

대우 조건

연도별 논문 인용수

저널 & 학회 논문 출판 수

연구실 논문 목록

CCN3/IMP3/HIF1α positive feedback loop enhances malignant progression of triple-negative breast cancer

Nidogen-1 suppresses cell proliferation, migration, and glycolysis via integrin β1-mediated HIF-1α downregulation in triple-negative breast cancer

Discrepancies between home blood pressure and ambulatory blood pressure monitoring in apparent treatment-resistant hypertension: analysis from the Korean resistant hypertension cohort

연구실 보유 특허

대학별 입시끈

우리 연구실 모집

교수님 소개

신인철

연구실 키워드

알럼나이가 간 회사

연구실 지원 방법

대우 조건

연도별 논문 인용수

저널 & 학회 논문 출판 수

연구실 논문 목록

CCN3/IMP3/HIF1α positive feedback loop enhances malignant progression of triple-negative breast cancer

Nidogen-1 suppresses cell proliferation, migration, and glycolysis via integrin β1-mediated HIF-1α downregulation in triple-negative breast cancer

Discrepancies between home blood pressure and ambulatory blood pressure monitoring in apparent treatment-resistant hypertension: analysis from the Korean resistant hypertension cohort

연구실 보유 특허

최근 3년간 연구실적 높은 연구실

대학별 입시끈

우리 연구실 모집