MCF10A Human Breast Epithelial Cells Research Articles

Tobacco smoke condensate down‐regulates estrogen sulfotransferase (SULT1E1) expression in MCF10A human breast epithelial cells through an aryl hydrocarbon receptor‐mediated mechanism

Tobacco smoke has been implicated as a risk factor for breast cancer. We evaluated the effect of tobacco smoke condensate (TSC) on expression of the estrogen-inactivating enzyme SULT1E1 in the MCF10A human breast epithelial cell line. Because TSC contains components that are known aryl hydrocarbon receptor (AhR) agonists, effects of TSC treatment were compared to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and effects on SULT1E1 expression were compared to those on CYP1A1. Treatment for 24–72 h with 0.05–10 μg/ml TSC produced concentration-dependent increases in CYP1A1 mRNA content, decreases in SULT1E1 mRNA content, and increases in expression from a transfected AhR-responsive reporter plasmid. Treatment with 10 μg/ml TSC and 30 nM TCDD produced comparable increases in CYP1A1 mRNA levels (~300-fold) and decreases in SULT1E1 mRNA levels (~90%). Treatment with the AhR antagonist 3′-methoxy-4′-nitroflavone (PD168641, at 1 μM) completely inhibited TCDD-inducible CYP1A1 expression and partially reversed TCDD-mediated SULT1E1 suppression. PD168641 also inhibited TSC-mediated CYP1A1 induction and SULT1E1 suppression as indicated by rightward shifts of the TSC concentration-response curves. These findings support a role for the AhR in TSC-mediated regulation of CYP1A1 and SULT1E1 expression in human breast epithelial cells. Supported by NIH Grant (ARRA) R21ES016373 (to M.R.-M.).

Roles of G proteins in Human Breast Cell Invasion

Although mounting evidence suggests a role for G12 proteins, Gα12 and Gα13, in tumor progression, a direct role of G12 proteins has not been determined. This study aims to elucidate the molecular mechanism for tumorigenic/invasive potential of Gα12/13 in MCF10A human breast epithelial cells. Here we report, for the first time, that Gα12/13 induce upregulation of matrix metalloproteinase(MMP)‐2 leading to the invasive/migratory phenotypes in MCF10A cells. We further show that p53 is an important transcription factor for induction of MMP‐2 transcriptional activation by Gα12/13. Using human breast tissue samples, we demonstrate that the expression levels of Gα12 and MMP‐2 are strongly correlated with the pathogenically diagnosed cancer. To elucidate a causal relationship between inflammation and the control of invasiveness of breast cells, the effect of the inflammatory lipid sphingosine‐1‐phosphate(S1P) on the regulation of invasive/migratory phenotypes of MCF10A cells was investigated. S1P causes induction of MMP‐9 in vitro/in vivo, and enhances invasion/migration which is triggered by S1P3‐Gαq coupling. Activation of PLC‐β4 and intracellular Ca2+ release are required for S1P‐induced MMP‐9 upregulation. Taken together, this study elucidated the role of G12 proteins and S1P in regulating processes for MMP expression and malignant phenotypic conversion of breast cells.

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

Identification of H-Ras-Specific Motif for the Activation of Invasive Signaling Program in Human Breast Epithelial Cells

Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR), consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.

Rapamycin sensitizes Akt inhibition in malignant human breast epithelial cells

Akt and mTOR are therapeutic targets for the treatment of cancer. The effects of inhibiting mTOR, with rapamycin, and Akt, with A-443654, concurrently, on cell morphology, cell proliferation, the cell cycle, and apoptosis were examined using the benign MCF10A and malignant MCF10CA1a human breast epithelial cells. Rapamycin and A-443654 in combination produced the greatest morphological changes and inhibited cell proliferation by G2/M arrest. Rapamycin and A-443654 in combination induced apoptosis at earlier times and at lower A-443654 concentrations in MCF10CA1a tumor cells than in the benign MCF10A cells. Rapamycin and A-443654 increased p53 and p15 INK4B protein levels, decreased anti-apoptotic Bcl-2 levels, and increased Bad levels in the MCF10CA1a tumor cells by ∼5-fold. These results suggest that the combined inhibition of Akt and mTOR may have beneficial therapeutic and safety margin effects.

Abstract 5196: Granulocyte-colony stimulating factor (G-CSF) plays a causative role for H-Ras-mediated invasion and migration of human breast epithelial cells

Abstract The whole human genome microarray on normal MCF10A, N-Ras-transformed, non-invasive MCF10A and H-Ras-transformed invasive MCF10A human breast epithelial cells revealed that the granulocyte-colony stimulating factor (G-CSF) was most highly upregulated in an H-Ras-specific manner. The present study investigated whether G-CSF plays a causative role for H-Ras-mediated MCF10A cell invasion and migration. Importantly, siRNA-mediated knockdown of G-CSF expression significantly reduced H-Ras-induced matrix metalloproteinase(MMP)-2 expression as well as invasion/migration. Activations of Rac1, MKK3/6 and p38 MAPK were inhibited by siRNA-knockdown of G-CSF. The induction of G-CSF expression conferred the invasive/migratory phenotype to parental MCF10A cells. Expression of MMP-2 and activation of signaling molecules Rac1, MKK3/6 and p38 MAPK were increased by G-CSF induction. Taken together, this study revealed G-CSF gene as a candidate marker for metastatic potential of breast epithelial cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5196.

Abstract 5197: Comparative proteome analysis reveals flotillin-1 as an H-Ras-interacting lipid raft protein critical for breast epithelial cell invasion

Abstract Metastatic spread is the major cause of death from breast cancer. The objective of our study is to unveil signal transduction pathways specific to breast epithelial cell invasion and migration apart from cell proliferation/transformation. We previously showed that the active H-Ras, but not N-Ras, induces invasive/migratory phenotypes of MCF10A human breast epithelial cells via activation of Rac1/p38 signaling leading to MMP-2 up-regulation, while both H-Ras and N-Ras induce cell proliferation and transformation. In the present study, we hypothesized that the Ras isoform-specific signaling pathway is initiated by differential involvement of lipid raft proteins. To address this, we characterized proteome profiles of lipid raft proteins from three cell lines: normal MCF10A; N-Ras-transformed, non-invasive MCF10A; and H-Ras-transformed, invasive MCF10A cells. Among twenty-four lipid raft proteins that were up-regulated in an H-Ras-specific manner, we tested whether flotillin-1 plays a causative role for H-Ras-mediated MCF10A cell invasion/migration. Here, we show that flotillin-1 interacts with H-Ras and H-Ras signaling increases flotillin-1 expression, suggesting a signaling amplification loop between flotillin-1 and H-Ras. Importantly, siRNA-mediated flotillin-1 knockdown significantly reduced H-Ras-mediated motility/invasion accompanied with down-regulation of Rac1/p38 and MMP-2. We also show that flotillin-1 co-localizes with H-Ras in the plasma membrane, while the interaction between flotillin-1 and N-Ras was negligible. Taken together, this study identified flotillin-1 as an H-Ras-interacting lipid raft protein critical for activation of H-Ras-specific signaling resulting invasive phenotype in breast epithelial cells. Consistently, flotillin-1 mRNA was significantly higher in malignant breast cancer tissues, supporting our hypothesis that flotillin-1 may contribute to breast carcinoma invasion. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5197.

Abstract 4376: Biological, biochemical, and molecular targets for dietary catechin-induced suppression of breast cell chronic carcinogenesis

Abstract This study is to identify biological, biochemical, and molecular targets for dietary catechins in early prevention of breast cell carcinogenesis induced chronically by environmental carcinogens. Breast cell carcinogenesis is a multiyear, multistep, and multipath disease process and is the most common type of cancer among women in northern America and northern Europe. Epidemiologic and experimental evidence suggests that dietary constituents in fruits and vegetables prevent human cancers; dietary prevention is a cost-efficient strategy to reduce the risk of this disease. However, the mechanisms for dietary prevention of cellular carcinogenesis induced by chronic exposure to environmental carcinogens remain to be elucidated. To identify dietary components for prevention of chronic human breast cell carcinogenesis, we studied biological, biochemical, and molecular target endpoints that were induced by low doses of carcinogens and suppressed by dietary catechins. Immortalized, non-cancerous human breast epithelial MCF10A cells were repeatedly exposed to carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or benzo[α]pyrene (B[α]P), each at picomolar concentrations, to induce cancer-associated biological, biochemical, and molecular properties in cells. We then used these changes as target endpoints to reveal the activity of various dietary catechins in suppression of NNK- and B[α]P-induced cellular carcinogenesis. We identified that cells acquiring the biological properties of reduced dependence on growth factors, anchorage-independent growth, and acinar-conformational disruption were reduced by treatment with dietary catechins. We identified that hydroxysteroid (11-beta) dehydrogenase 2 (HSD11B2) played a novel role in NNK- and B[α]P-induced cellular carcinogenesis, and its expression acted as a molecular target endpoint in catechin-induced suppression of cellular carcinogenesis. The ability of catechins to reduce gene expression of cytochrome P450 enzymes CYP1A1 and CYP1B1, which can bioactivate NNK and B[α]P, may be involved in the preventive mechanism for catechins in suppression of cellular carcinogenesis. Our model system with biological, biochemical, and molecular target endpoints verified the value of dietary catechins for prevention of human breast cell carcinogenesis induced by repeated exposures to low doses of multiple environmental carcinogens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4376.

Abstract 1956: A novel mouse model of breast cancer metastasis to bone

Abstract Patients with advanced stage breast cancer develop bone metastasis, which leads to hypercalcemia, fractures, nerve compression, paralysis and pain. Breast cancer bone lesions can be osteolytic or osteoblastic, however osteolytic lesions are predominant. Currently, there is no experimental model available to examine osteotropism or trafficking of breast cancer cells to the bone from the primary tumor. TGFβ signaling has been suggested to be responsible for osteotropism. TGFβ stimulates Gli2 and PTHrP expression, which have been shown to be responsible for osteolysis. We have shown that MCF10A, AT-1 and CA1d human breast epithelial cells are responsive to TGFβ and express very high levels of Gli2 and PTHrP. Orthotopic grafting to the cleared fatpad of SCID mice shows that MCF10A and AT-1 cells do not form tumors, even when grafted with normal fibroblasts. CA1d carcinoma cells that are co-grafted with normal fibroblasts show rapid tumor formation with 100% incidence, whereas CA1d cells grafted alone rarely form tumors in the orthotopic site. The tumors can be localized real-time and non-invasively using optical imaging of GFP. X-ray imaging revealed loss of bone density in sacral/caudal vertebrae and hindlimbs. Ca1d grafts generate undifferentiated primary tumors that metastasize to lung and bone. Future studies will determine if CA1d cells stimulate osteolysis. This model may provide a valuable model to examine osteotropism of breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1956.

Regulation of estrogen sulfotransferase (SULT1E1) expression by proliferation state in MCF10A human breast epithelial cells: Potential role of microRNAs

The MCF10A cell line represents a model of the normal human breast epithelial cell. Estrogen sulfotransferase (SULT1E1), a major estrogen‐inactivating enzyme, is up‐regulated in confluent relative to preconfluent MCF10A cells. The mechanism responsible for SULT1E1 up‐regulation in confluent MCF10A cells was investigated. Transient or stable tranfection of reporter constructs containing up to 7 kb of the SULT1E1 5′‐flanking region failed to recapitulate confluency‐induced SULT1E1 up‐regulation. The half‐life of SULT1E1 mRNA in preconfluent MCF10A cells was estimated to be ~4h based on analysis of SULT1E1 mRNA levels in MCF10A cells that were replated after reaching confluency. Microarray analysis revealed that 85 microRNAs were significantly up‐regulated and 24 microRNAs were down‐regulated in confluent relative to preconfluent MCF10A cells. Computational analysis of the SULT1E1 3′‐untranslated region identified 4 putative candidate binding sites for confluency‐regulated microRNAs. The confluency‐related decrease in two of these microRNAs (miR‐221 and miR‐100*) corresponded with the confluency‐induced increase in SULT1E1 mRNA observed in MCF10A cells. These results support a role for the post‐transcriptional up‐regulation of SULT1E1 mRNA expression in confluent relative to preconfluent MCF10A cells. Supported by NIH grant (ARRA) ES016373 (MRM) and EHS Center grant ES06636.

Polychlorinated biphenyl induced ROS signaling delays the entry of quiescent human breast epithelial cells into the proliferative cycle

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants that can produce reactive oxygen species (ROS) by autoxidation of dihydroxy-PCBs and redox-cycling. We investigate the hypothesis that PCB induced perturbations in ROS signaling regulate the entry of quiescent cells into the proliferative cycle. Quiescent MCF-10A human breast epithelial cells were incubated with 0-3 micromolar of 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), 2, 2′, 4, 4′, 5, 5′-hexachlorobiphenyl (PCB 153), and Aroclor 1254 for 4 days. Cells were replated at a lower density and analyzed for cell cycle phase distributions, ROS levels, MnSOD expression, and cyclin D1 protein levels. Quiescent cells incubated with 4-Cl-BQ showed the maximal delay in entering S phase. This delay was associated with a decrease in MnSOD activity, protein and mRNA levels, and an increase in cellular ROS levels. Results from the mRNA turnover assay showed that the 4-Cl-BQ treatment selectively enhanced the degradation of the 4.2 kb MnSOD transcript, while the half-life of the 1.5 kb transcript did not change. Accumulation of cyclin D1 protein levels in replated cells was suppressed in cells treated with 4-Cl-BQ. Pretreatment of quiescent cells with polyethylene glycol-conjugated superoxide dismutase and catalase suppressed 4-Cl-BQ induced increase in ROS levels, which was consistent with an increase in cyclin D1 accumulation, and entry into S phase. These results showed 4-Cl-BQ induced perturbations in ROS signaling inhibit the entry of quiescent cells into S phase.

Grape seed proanthocyanidin suppression of breast cell carcinogenesis induced by chronic exposure to combined 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone and benzo[a]pyrene

Breast cancer is the most common type of cancer among women in northern America and northern Europe; dietary prevention is a cost-efficient strategy to reduce the risk of this disease. To identify dietary components for the prevention of human breast cancer associated with long-term exposure to environmental carcinogens, we studied the activity of grape seed proanthocyanidin extract (GSPE) in suppression of cellular carcinogenesis induced by repeated exposures to low doses of environmental carcinogens. We used combined carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), at picomolar concentrations, to repeatedly treat noncancerous, human breast epithelial MCF10A cells to induce cellular acquisition of cancer-related properties of reduced dependence on growth factors, anchorage-independent growth, and acinar-conformational disruption. Using these properties as biological target endpoints, we verified the ability of GSPE to suppress combined NNK- and B[a]P-induced precancerous cellular carcinogenesis and identified the minimal, noncytotoxic concentration of GSPE required for suppressing precancerous cellular carcinogenesis. We also identified that hydroxysteroid-11-beta-dehydrogenase 2 (HSD11B2) may play a role in NNK- and B[a]P-induced precancerous cellular carcinogenesis, and its expression may act as a molecular target endpoint in GSPE's suppression of precancerous cellular carcinogenesis. And, the ability of GSPE to reduce gene expression of cytochrome-P450 enzymes CYP1A1 and CYP1B1, which can bioactivate NNK and B[a]P, possibly contributes to the preventive mechanism for GSPE in suppression of precancerous cellular carcinogenesis. Our model system with biological and molecular target endpoints verified the value of GSPE for the prevention of human breast cell carcinogenesis induced by repeated exposures to low doses of multiple environmental carcinogens.

The role of collagen reorganization on mammary epithelial morphogenesis in a 3D culture model

Collagen-based three-dimensional (3D) in vitro models that recapitulate the structural and functional context of normal and malignant tissues provide a relevant surrogate to animal models in the study of developmental and carcinogenic processes. Human breast epithelial MCF10A cells embedded in a collagen gel formed both acinar and tubular structures only when the gel was detached (floating) from the cell culture plate's well, and allowed to be contracted by the cells. Epithelial phenotype depended upon the time and the location within the gel, as ducts formed exclusively on the upper layer of the gel while ductal branching occurred earlier in the central area of the gel, and gradually progressed toward the periphery. The addition of fibroblasts accelerated tubulogenesis. MCF10A cells facilitated the organization of thick collagen fibers packed into large bundles at the tip of the ducts and parallel to the direction of ductal elongation. In gels that were not detached from the well's wall, MCF10A cells organized in monolayer and collagen fibers were aligned along the axis of outstretched sprouts stemming from those cellular aggregates. Partial gel release induced uniaxial tubulogenesis associated with orderly aligned collagen fibers. These results suggest that proper collagen organization is necessary for epithelial morphogenesis to occur, and that biomechanical interactions between fibers and cells mediated duct formation, elongation and branching.

Lycopene Inhibits Proliferation, Invasion and Migration of Human Breast Cancer Cells

Breast cancer has been estimated as one of the most common causes of cancer death among women. The major cause of death from breast cancer is the metastatic spread of the disease from the primary tumor to distant sites in the body. Lycopene is one of the major carotenoids in fruits and vegetables including tomatoes. Epidemiological studies have shown that the dietary intake of lycopene is associated with decreased risk of cancer. Although mounting evidence shows the chemopreventive effect of lycopene, the role of lycopene in the prevention of metastatic potential of breast cancer has not been determined yet. In the present study, we investigated the inhibitory effect of lycopene on invasive and migratory phenotypes of two highly aggressive breast cancer cell lines, H-Ras-transformed MCF10A human breast epithelial cells (H-Ras MCF10A) and MDA-MB-231 human breast cancer cells. Here, we report that lycopene significantly inhibits invasion and migration as well as proliferation of H-Ras MCF10A and MDA-MB-231 cells. This study suggested an in vitro anti-cancer and anti-metastatic potential of lycopene. We also showed that activations of ERKs and Akt were inhibited by lycopene in H-Ras MCF10A cells, suggesting that the ERKs and Akt signaling pathways may be involved in lycopene-induced anti-proliferative and/or anti-invasive/migratory effects in these cells. Taken in conjunction with the fact that breast cancer metastasis is one of the most lethal malignancies in women, our findings may provide useful information for the application of lycopene in establishing strategy to prevent the metastatic breast cancer.

The G12 family proteins upregulate matrix metalloproteinase-2 via p53 leading to human breast cell invasion

Although mounting evidence suggests a role for G(12) proteins, G(α12) and G(α13), in tumor progression, a direct role of G(12) proteins has not been determined. This study aims to elucidate the molecular mechanism for a tumorigenic and invasive potential of G(α12) and G(α13) in MCF10A human breast epithelial cells. Here, we report, for the first time, that G(α12) and G(α13) induce upregulation of matrix metalloproteinase (MMP)-2 leading to the invasive and migratory phenotypes in MCF10A cells. We further show that p53 is an important transcription factor for induction of MMP-2 transcriptional activation by G(α12/13). G(α12/13)-induced MMP-2 upregulation, invasion, and migration are dependent on the activation of Ras, Rac1, MKK3/6, p38, and Akt. Using human breast tissue samples, we demonstrate that the expression levels of G(α12) and MMP-2 are strongly correlated with the pathogenically diagnosed cancer (P < 0.0001). Moreover, the expression of G(α12) shows a strong correlation with that of MMP-2 in human breast cancer tissues, implicating the in vivo tumorigenic potential of G(α12). Taken together, this study elucidated the role of G(12) proteins in regulating processes for MMP-2 expression and malignant phenotypic conversion of MCF10A human breast epithelial cells, providing a molecular basis for the promoting role of G(α12) and G(α13) in breast cell invasion.

Morphologic transformation of human breast epithelial cells MCF-10A: dependence on an oxidative microenvironment and estrogen/epidermal growth factor receptors

BackgroundMCF-10A, immortalized but non-transformed human breast epithelial cells, are widely used in research examining carcinogenesis. The studies presented here were initiated with the observation that MCF-10A cells left in continuous culture for prolonged periods without re-feeding were prone to the development of transformed foci. We hypothesized that the depletion of labile culture components led to the onset of processes culminating in the observed cell transformation. The purpose of this study was to define the factors which promoted transformation of this cell line.ResultsChanges in levels of phenol red (PHR), hydrocortisone (HC), and epidermal growth factor (EGF) with or without estrogen treatment indicated that both oxidative stress- and estrogen receptor alpha (ERα)-mediated pathways contribute to cell transformation. Gene array and Western blotting analyses of cells maintained in our laboratory and of those from other sources documented detectable ERα and ERbeta (ERβ) in this ERα-negative cataloged cell line. Results also indicate the possibility of a direct association of EGF receptor (EGFR) and ERα in these cells as well as the formation and high induction of a novel ternary complex that includes ERβ (ERα/ERβ/EGFR) in cells grown under conditions facilitating transformation.ConclusionsOur studies resulted in the development of a growth protocol where the effects of chronic, physiologically relevant alterations in the microenvironment on cellular transformation were examined. From our results, we were able to propose a model of transformation within the MCF-10A cell line in which oxidative stress, ER and EGFR play essential roles. Overall, our work indicates that the immediate microenvironment of cells exerts powerful growth cues which ultimately determine their transformation potential.

ErbB2-enhanced invasiveness of H-Ras MCF10A breast cells requires MMP-13 and uPA upregulation via p38 MAPK signaling

Overexpression of ErbB2 has been frequently found in mammary carcinoma. We have previously shown that the aberrant activation of H-Ras induces human breast cell invasion and migration. The present study was aimed at investigating the effect of ErbB2 overexpression on H-Ras-induced breast cell invasion and to elucidate the underlying mechanisms. Herein, we show that overexpression of ErbB2 promotes invasive and migratory abilities of H-Ras-activated MCF10A human breast epithelial cells through upregulation of matrix metalloproteinase (MMP)-13 and urokinase-type plasminogen activator (uPA). We also demonstrate that the p38 MAPK is an important signaling molecule in the ErbB2-induced upregulation of MMP-13 and uPA and invasion/migration of H-Ras MCF10A cells overexpressing ErbB2. The present study elucidating the molecular mechanism underlying ErbB2-induced promotion of H-Ras MCF10A cell invasion may provide invaluable information for understanding breast cancer progression and establishing therapeutic interventions for breast cancer.

Polychlorinated-biphenyl-induced oxidative stress and cytotoxicity can be mitigated by antioxidants after exposure

PCBs and PCB metabolites have been suggested to cause cytotoxicity by inducing oxidative stress, but the effectiveness of antioxidant intervention after exposure has not been established. Exponentially growing MCF-10A human breast and RWPE-1 human prostate epithelial cells continuously exposed for 5 days to 3 μM PCBs [Aroclor 1254 (Aroclor), PCB153, and the 2-(4-chlorophenyl)-1,4-benzoquinone metabolite of PCB3 (4ClBQ)] were found to exhibit growth inhibition and clonogenic cell killing, with 4ClBQ having the most pronounced effects. These PCBs were also found to increase steady-state levels of intracellular O 2 ·− and H 2O 2 (as determined by dihydroethidium, MitoSOX red, and 5-(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate oxidation). These PCBs also caused 1.5- to 5.0-fold increases in MnSOD activity in MCF-10A cells and 2.5- to 5-fold increases in CuZnSOD activity in RWPE-1 cells. Measurement of MitoSOX red oxidation with confocal microscopy coupled with colocalization of MitoTracker green in MCF-10A and RWPE-1 cells supported the hypothesis that PCBs caused increased steady-state levels of O 2 ·− in mitochondria. Finally, treatment with either N-acetylcysteine (NAC) or the combination of polyethylene glycol (PEG)-conjugated CuZnSOD and PEG–catalase added 1 h after PCBs significantly protected these cells from PCB toxicity. These results support the hypothesis that exposure of exponentially growing human breast and prostate epithelial cells to PCBs causes increased steady-state levels of intracellular O 2 ·− and H 2O 2, induction of MnSOD or CuZnSOD activity, and clonogenic cell killing that could be inhibited by a clinically relevant thiol antioxidant, NAC, as well as by catalase and superoxide dismutase after PCB exposure.

Phloretin Induces Apoptosis in H‐Ras MCF10A Human Breast Tumor Cells through the Activation of p53 via JNK and p38 Mitogen‐Activated Protein Kinase Signaling

Mutations in Ras play a critical role in the development of human cancers, including breast cancer. We investigated the possible antiproliferative effects of the naturally occurring dihydrochalcone phloretin [2',4',6'-trihydroxy-3-(4-hydroxyphenyl)-propiophenone] on H-Ras-transformed MCF10A human breast epithelial (H-Ras MCF10A) cells. Phloretin suppressed H-Ras MCF10A cell proliferation in a dose-dependent manner and induced nuclear condensation in the cells, indicating that phloretin-induced cell death occurs mainly via the induction of apoptosis. Prominent upregulation of p53 and Bax and cleavage of poly (ADP)-ribose polymerase were also detected in the phloretin-treated cells. Finally, phloretin markedly increased caspase-3 activity as well as JNK and p38 mitogen-activated protein kinase signaling. Our findings suggest that the phloretin-induced apoptosis of breast tumor cells contributes to the chemopreventive potential of phloretin against breast cancer.