Zeying Fan

Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro

The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G1 checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27kip1 or p21waf1. It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells.

Metformin induces unique biological and molecular responses in triple negative breast cancer cells

Triple negative (TN) breast cancer is more frequent in women who are obese or have type II diabetes, as well as young Women of Color. These cancers do not express receptors for the steroid hormones estrogen or progesterone, or the type II receptor tyrosine kinase (RTK) Her-2 but do have upregulation of basal cytokeratins and the epidermal growth factor (EGFR). These data suggest that aberrations of glucose and fatty acid metabolism, signaling through EGFR and genetic factors may promote the development of TN cancers. The anti-type II diabetes drug metformin has been associated with a decreased incidence of breast cancer, although the specific molecular subtypes that may be reduced by metformin have not been reported. Our data indicates that metformin has unique anti-TN breast cancer effects both in vitro and in vivo. It inhibits cell proliferation (with partial S phase arrest), colony formation and induces apoptosis via activation of the intrinsic and extrinsic signaling pathways only in TN breast cancer cell lines. At the molecular level, metformin increases P-AMPK, reduces P-EGFR, EGFR, P-MAPK, P-Src, cyclin D1 and cyclin E (but not cyclin A or B, p27 or p21), and induces PARP cleavage in a dose- and time-dependent manner. These data are in stark contrast to our previously published biological and molecular effects of metformin on luminal A and B, or Her-2 type breast cancer cells. Nude mice bearing tumor xenografts of the TN line MDA-MB-231, treated with metformin, show significant reductions in tumor growth (p=0.0066) and cell proliferation (p=0.0021) as compared to untreated controls. Metformin pre-treatment, before injection of MDA-MB-231 cells, results in a significant decrease in tumor outgrowth and latency. Given the unique anti-cancer activity of metformin against TN disease, both in vitro and in vivo, it should be explored as a therapeutic agent against this aggressive form of breast cancer.

Downregulation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells.

Receptor tyrosine kinase activity is essential for erbB2 (HER2/neu) promotion of breast carcinogenesis, metastasis and therapeutic resistance. erbB2 kinase can be activated by dimerization with another erbB receptor, most of which bind ligands. Of these, the erbB2/erbB3 heterodimer is the most potent oncogenic complex. erbB2 reportedly requires erbB3 to promote cellular proliferation, although this may occur without changes in erbB2 tyrosine kinase activity in some model systems. Our investigations focus on the role(s) of erbB3 in erbB2‐associated kinase activity and tamoxifen resistance. Using tumor‐derived cell lines from wild type rat c‐neu transgenic mice and human breast cancers, we demonstrate that erbB3 plays a critical role in the activation of erbB2 tyrosine kinase activity and erbB2‐associated tumorigenesis. Mechanistically, downregulation of erbB3 by specific siRNA reduces erbB2 tyrosine phosphorylation, decreases the PI‐3K/Akt signaling, and inhibits mammary/breast cancer cell proliferation and colony formation. Specific erbB3 siRNA sensitizes erbB2 transfected MCF‐7 cells (MCF‐7/erbB2) to tamoxifen‐associated inhibition of both cell growth and colony formation and enhances tamoxifen‐induced apoptosis, in contrast to control siRNA transfected MCF‐7/erbB2 cells which are tamoxifen‐resistant. Our data indicates that erbB2/erbB3 heterodimerization is a prerequisite for erbB2 tyrosine kinase activation in mammary/breast cancer cells and that downregulation of erbB3 inhibits erbB2‐associated procarcinogenic activity via inactivation of the PI‐3K/Akt pathway. Furthermore, erbB3 also contributes to erbB2‐mediated tamoxifen resistance and therefore may be a clinically relevant therapeutic target in addition to erbB2.

Potent anti-proliferative effects of metformin on trastuzumab-resistant breast cancer cells via inhibition of erbB2/IGF-1 receptor interactions.

We have shown that erbB2 altered breast cancer cells are less sensitive to the anti-proliferative effects of metformin than triple negative cells, and have described the differences of molecular mechanisms of metformin action by tumor subtypes. We hypothesized that metformin may be more effective against trastuzumab-resistant erbB2-overexpressing breast cancer cells because it targets the critical signaling pathways that are altered with resistance. BT474, SKBR3 and derived trastuzumab-resistant sublines BT474-HR20 (HR20) and SKBR3-pool2 (pool2) were used to test this hypothesis. Metformin treatment resulted in significantly more inhibition of proliferation and clonogenicity in resistant sublines. It decreased erbB2/insulin-like growth factor-1 receptor (IGF-1R) complexes (present only in the resistant sublines) without altering erbB2 expression, and reduced the expression and activity of erbB3 and IGF-1R in the trastuzumab-resistant but not parental cells. Trastuzumab-resistant sublines were resistant to rapamycin induced changes in mTOR activity and cell growth. In contrast, both BT474 and HR20 cells were highly sensitive to inhibitors of Src (Dasatinib) and PI-3K (LY294002). The pool2 cells showed higher sensitivity than SKBR3 cells to LY294002, but not Dasatinib. On the basis of these data, metformin appears to be significantly more effective against trastuzumab-resistant as compared to sensitive breast cancer cells. Metformin disrupts erbB2/IGF-1R complexes, erbB3 and IGF-1R expression and activity, as well as Src kinase and/or PI-3K/Akt signaling. This action appears to be independent of mTOR signaling. Our findings provide a rationale to study the effects of metformin on patients with erbB2 positive tumors treated with trastuzumab, with or without resistance.

Glucose promotes breast cancer aggression and reduces metformin efficacy

Metformin treatment has been associated with a decrease in breast cancer risk and improved survival. Metformin induces complex cellular changes, resulting in decreased tumor cell proliferation, reduction of stem cells, and apoptosis. Using a carcinogen-induced rodent model of mammary tumorigenesis, we recently demonstrated that overfeeding in obese animals is associated with a 50% increase in tumor glucose uptake, increased proliferation, and tumor cell reprogramming to an “aggressive” metabolic state. Metformin significantly inhibited these pro-tumorigenic effects. We hypothesized that a dynamic relationship exists between chronic energy excess (glucose by dose) and metformin efficacy/action. Media glucose concentrations above 5 mmol/L was associated with significant increase in breast cancer cell proliferation, clonogenicity, motility, upregulation/activation of pro-oncogenic signaling, and reduction in apoptosis. These effects were most significant in triple-negative breast cancer (TNBC) cell lines. High-glucose conditions (10 mmol/L or above) significantly abrogated the effects of metformin. Mechanisms of metformin action at normal vs. high glucose overlapped but were not identical; for example, metformin reduced IGF-1R expression in both the HER2+ SK-BR-3 and TNBC MDA-MB-468 cell lines more significantly at 5, as compared with 10 mmol/L glucose. Significant changes in gene profiles related to apoptosis, cellular processes, metabolic processes, and cell proliferation occurred with metformin treatment in cells grown at 5 mmol/L glucose, whereas under high-glucose conditions, metformin did not significantly increase apoptotic/cellular death genes. These data indicate that failure to maintain glucose homeostasis may promote a more aggressive breast cancer phenotype and alter metformin efficacy and mechanisms of action.

Estrogenic Promotion of ErbB2 Tyrosine Kinase Activity in Mammary Tumor Cells Requires Activation of ErbB3 Signaling

Increasing evidence suggests molecular interactions between erbB2 and other receptor tyrosine kinases, and estrogenic compounds and their cognate receptors. We have recently reported that downregulation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells. On the basis of these data, we hypothesized that erbB3 may play a major role connecting these two sentinel pathways. Interactions were studied using mammary/breast cancer cell lines from wild-type rat c-neu gene transgenic mice and humans. Estradiol promoted cell proliferation and activated erbB2/neu tyrosine kinase, Akt, and mitogen-activated protein kinase signaling exclusively in mammary and breast epithelial cell lines with coexpression of both erbB2 and erbB3. Estradiol action was independent of the transgene promoter (MMTV-LTR) activity, both in vitro and in vivo, as well as c-neu transgene or endogenous erbB2 gene expression. Estrogen induction of cell growth promotion, erbB2/neu activation, and downstream signaling was abrogated by blockade of estrogen receptor (ER) with the pure ER antagonist ICI 182,780 or knockdown of erbB3 expression via specific siRNA. These data suggest that activation of both ER and erbB2/erbB3 signaling is requisite for estrogen-induced mitogenesis and erbB2/neu tyrosine kinase activation.(Mol Cancer Res 2009;7(11):1882–92)

Metformin attenuates transforming growth factor beta (TGF-β) mediated oncogenesis in mesenchymal stem-like/claudin-low triple negative breast cancer

ABSTRACT Mesenchymal stem-like/claudin-low (MSL/CL) breast cancers are highly aggressive, express low cell-cell adhesion cluster containing claudins (CLDN3/CLDN4/CLDN7) with enrichment of epithelial-to-mesenchymal transition (EMT), immunomodulatory, and transforming growth factor-β (TGF-β) genes. We examined the biological, molecular and prognostic impact of TGF-β upregulation and/or inhibition using in vivo and in vitro methods. Using publically available breast cancer gene expression databases, we show that upregulation and enrichment of a TGF-β gene signature is most frequent in MSL/CL breast cancers and is associated with a worse outcome. Using several MSL/CL breast cancer cell lines, we show that TGF-β elicits significant increases in cellular proliferation, migration, invasion, and motility, whereas these effects can be abrogated by a specific inhibitor against TGF-β receptor I and the anti-diabetic agent metformin, alone or in combination. Prior reports from our lab show that TNBC is exquisitely sensitive to metformin treatment. Mechanistically, metformin blocks endogenous activation of Smad2 and Smad3 and dampens TGF-β-mediated activation of Smad2, Smad3, and ID1 both at the transcriptional and translational level. We report the use of ID1 and ID3 as clinical surrogate markers, where high expression of these TGF-β target genes was correlated to poor prognosis in claudin-low patients. Given TGF-βs role in tumorigenesis and immunomodulation, blockade of this pathway using direct kinase inhibitors or more broadly acting inhibitors may dampen or abolish pro-carcinogenic and metastatic signaling in patients with MCL/CL TNBC. Metformin therapy (with or without other agents) may be a heretofore unrecognized approach to reduce the oncogenic activities associated with TGF-β mediated oncogenesis.

Genome-wide functional genetic screen with the anticancer agent AMPI-109 identifies PRL-3 as an oncogenic driver in triple-negative breast cancers

Triple-negative breast cancers (TNBC) are among the most aggressive and heterogeneous cancers with a high propensity to invade, metastasize and relapse. Here, we demonstrate that the anticancer compound, AMPI-109, is selectively efficacious in inhibiting proliferation and inducing apoptosis of multiple TNBC subtype cell lines as assessed by activation of pro-apoptotic caspases-3 and 7, PARP cleavage and nucleosomal DNA fragmentation. AMPI-109 had little to no effect on growth in the majority of non-TNBC cell lines examined. We therefore utilized AMPI-109 in a genome-wide shRNA screen in the TNBC cell line, BT-20, to investigate the utility of AMPI-109 as a tool in helping to identify molecular alterations unique to TNBC. Our screen identified the oncogenic phosphatase, PRL-3, as a potentially important driver of TNBC growth, migration and invasion. Through stable lentiviral knock downs and transfection with catalytically impaired PRL-3 in TNBC cells, loss of PRL-3 expression, or functionality, led to substantial growth inhibition. Moreover, AMPI-109 treatment, downregulation of PRL-3 expression or impairment of PRL-3 activity reduced TNBC cell migration and invasion. Histological evaluation of human breast cancers revealed PRL-3 was significantly, though not exclusively, associated with the TNBC subtype and correlated positively with regional and distant metastases, as well as 1 and 3 year relapse free survival. Collectively, our study is proof-of-concept that AMPI-109, a selectively active agent against TNBC cell lines, can be used as a molecular tool to uncover unique drivers of disease progression, such as PRL-3, which we show promotes oncogenic phenotypes in TNBC cells.

Abstract 4604: PTP4A3 is oncogenic and modulates triple negative breast cancer growth

Triple negative breast cancers (TNBCs) are among the most aggressive and heterogenous breast cancers characterized by a high propensity to invade, metastasize, and relapse; the exact mechanisms by which TNBCs mediate progression is unclear and thus, new insights into the biology of TNBC are desperately needed. We have developed a novel anti-cancer compound (AMPI-109) that is selectively efficacious against TNBC cell-lines. We conducted a genome-wide functional genomic shRNA screen to identify modifiers of AMPI-109 sensitivity and identified PTP4A3 (P3), a protein tyrosine phosphatase implicated in invasive, node positive TNBC tumors and associates with poor survival outcome, as a mediator of AMPI-109 action. In silico modeling predicted that AMPI-109 binds the catalytic site of P3 with favorable energetics. To investigate whether AMPI-109 could impact the activity of P3, we performed an in vitro phosphatase assay and demonstrated that AMPI-109 impaired the catalytic activity of P3. To investigate an oncogenic role of P3 in TNBC, we conducted stable lentiviral knockdown of P3 (ShP3) in BT-20 TNBC cells and demonstrated that loss of P3 leads to substantial growth inhibition (48%, p = 0.0055), similar in magnitude to AMPI-109 at 100 nM dose (66%, p = 0.00005). AMPI-109 also decreased P3 mRNA and protein levels. Accordingly, we blocked de novo protein synthesis and observed enhanced P3 protein degradation in the presences of AMPI-109. This degradation was reversible in the presence of the proteasome inhibitor MG-132, suggesting that AMPI-109 may induce P3 degradation through a proteasome-dependent mechanism. Functionally, high expression of P3 has been implicated in driving metastatic phenotypes in colorectal cancer but its role in TNBC cell migration has not been investigated. Wound-healing assays demonstrated that both AMPI-109 and ShP3 significantly impeded the migratory capacity of BT-20 cells (AMPI-109: 85% reduction, shP3: 80% reduction). AMPI-109 and ShP3 also conferred a higher rate of apoptosis induction as measured by activation of pro-apoptotic caspases-3 and 7 suggesting that the growth inhibitory phenotypes observed could potentially be attributed to increases in apoptotic cell death. To determine how P3 mechanistically exerts its oncogenic effect in the context of modulating cell signaling pathways, we carried out co-expression analysis of mRNAs that tracked with P3 expression across a suite of TNBC cell lines. We identified TRAPPC9, an activator of the NF-kappa B pathway, as correlating positively with P3 (r =0.652). Collectively, our results identify P3 as a target of AMPI-109 and that P3 has an oncogenic role in TNBC. Future studies will further characterize the relationship between AMPI-109, P3 and the NF-kappa B pathway. Furthermore, because of the specificity of AMPI-109 for TNBC cells, it may represent a new tool for understanding the molecular basis underlying the aggressive nature of this disease. Citation Format: Hamid H. Gari, Rahul Ray, Scott Lucia, Christopher C. Porter, Christy M. Gearheart, Susan Fosmire, Gregory D. DeGala, Zeying Fan, Yuanbin Ru, Ann D. Thor, James R. Lambert. PTP4A3 is oncogenic and modulates triple negative breast cancer growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4604. doi:10.1158/1538-7445.AM2014-4604

Abstract P5-10-05: Novel Mechanisms of Metformin Action in TN Breast Cancer: Upregulation of miRNA 141 and 192, are Associated with a Decrease in Targets GRB2 and MSN Involved in Signaling and Motility Respectively

BACKGROUND: We have previously shown that the anti-diabetic agent, metformin, inhibits cellular proliferation, colony formation and cell signaling, with an induction of S-phase arrest and apoptosis in triple negative (TN) breast cancer cell lines in vitro, and cell outgrowth and proliferation in vivo. These changes are associated with profound shifts in phosphorylated cell signaling intermediates, including EGFR, IGF-1R, Akt, MAPK and others (Liu, et al Cell Cycle 2009). We have also shown that Stat3 activity (via phosphorylation at serine and tyrosine sites) is critical to the effects of metformin against TN cells (Deng, et al Cell Cycle 2012). In unpublished studies, we have noted that metformin significantly inhibits cellular motility in vitro. In this study we focus on mechanisms underlying the effects of metformin in TN cancer cell signaling and motility. METHODS: TN breast cancer cell lines MDA-MB-468 and MDA-MB-231were used to study mechanisms of metformin action in vitro. Invasion chamber and motility assays were used to quantitate the effects of metformin as compared to untreated controls, with and without a variety of inhibitors at normal or supraphysiological glucose concentrations. Affymetrix chips Human Gene 1.1 and miRNA 2.0 were used to identify genes and miRNA up or down regulated by metformin treatment at 6 and 24 hr at glucose concentrations of 5, 10 or 17 mM. Appropriate primers and antibodies were used to validate these data at the transcript and protein levels respectively by qRT-PCR and western blot analyses. Lentiviral expression vectors and mimics were used to manipulate miRNA expression levels. Direct targeting of the GRB2 and MSN 3′ UTR9s were performed using luciferase reporters. RESULTS: TargetScan and Diana miR-Path analysis of miRNA9s up-regulated at 6 hr following metformin treatmetns identified genes that were down regulated with metformin treatment such as GRB2 (a predicted target of miR-141) and MSN (a predicted target of miR-192), as possible mechanisms of drug action. GRB2 regulated the phosphorylation of numerous receptor tyrosine kinase (RTK) gene pathway intermediates, including those downstream of EGFR, IGF-1R and MAPK. Knock-down of miR-141 by an antagomir showed that miR-141 regulated GRB2. Invasion chamber and motility assays confirm that the metformin associated reduction in TN cell motility and invasion capacity involved miR-193 and MSN. CONCLUSIONS: Metformin acts by diverse molecular mechanisms; some of these may be cell type specific. In TN breast cancer cells, metformin causes a rapid increase in miR-141, which targets GRB2, a gene that encodes a critical regulator upstream of many growth factors. Metformin treatment also upregulates miR-192, which represses MSN to reduce motility and invasion. Supported by Komen Breast Cancer Foundation Grant KG100575 (SME, JKR, ZF, NSS, ADT) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-05.