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Natural killer (NK) cells are lymphocytes that are involved in controlling tumors or microbial infections through the production of interferon gamma (IFN-γ). Granulocyte colony-stimulating factor (G-CSF) inhibits IFN-γ secretion by NK cells, but the mechanism underlying this effect remains unclear. Here, by comparing the multi-omics profiles of human NK cells before and after in vivo G-CSF treatment, we identify a pathway that is activated in response to G-CSF treatment, which suppresses IFN-γ secretion in NK cells. Specifically, glucocorticoid receptors (GRs) activated by G-CSF inhibit secretion of IFN-γ by promoting interactions between SOCS1 promoters and enhancers, as well as increasing the expression of SOCS1. Experiments in mice confirm that G-CSF treatment significantly downregulates IFN-γ secretion and upregulates GR and SOCS1 expression in NK cells. In addition, GR blockade by the antagonist RU486 significantly reverses the effects of G-CSF, demonstrating that GRs upregulate SOCS1 and inhibit the production of IFN-γ by NK cells.

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-beta (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-β’s 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.

// Hamid H. Gari 1 , Christy M. Gearheart 2 , Susan Fosmire 2 , Gregory D. DeGala 1 , Zeying Fan 1 , Kathleen C. Torkko 1 , Susan M. Edgerton 1 , M. Scott Lucia 1 , Rahul Ray 3 , Ann D. Thor 1 , Christopher C. Porter 2 and James R. Lambert 1 1 Department of Pathology, University of Colorado School of Medicine, Aurora, CO, USA 2 Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA 3 Department of Medicine, Boston University School of Medicine, Boston, MA, USA Correspondence to: James R. Lambert, email: // Keywords : AMPI-109, PRL-3, triple-negative breast cancer, phosphatase, functional genomics Received : December 22, 2015 Accepted : February 09, 2016 Published : February 17, 2016 Abstract 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.

Background: Metabolic shifts in cancer and stem cells are well recognized (the so called Warburg Effect). Tumor cells frequently demonstrate alterations in lipid metabolism. The anti-diabetic drug metformin has been shown to have broad anti-cancer activity, reducing both the incidence and mortality of several types of cancer. Metformin inhibits pro-oncogenic cell signaling and metabolic pathways, and has demonstrated unique anti-stem cell activity. We have reported that metformin is particularly active against triple negative (TN) breast cancer cells, inducing S phase arrest and apoptosis. Metformin down regulates de novo fatty acid synthesis, significantly reducing fatty acid synthase (FASN) and FASN protein expression. Mechanistically, these shifts appear secondary to a metformin associated up regulation of miR-193a, which directly targets the FASN 3"UTR (In press, Wahdan-Alaswad et al, Hormones and Cancer). Cancer cells require high cholesterol for membrane synthesis, rigidity, the formation of lipid rafts (membrane regions which organize critical receptor/signaling molecules including the epidermal growth factor receptor (EGFR)), and to provide precursor molecules for hormones and sterols. While de novo cholesterol biosynthesis and high systemic cholesterol levels have been associated with an increase in breast cancer risk and a worse prognosis for women with the disease, clinical studies of statins (drugs to inhibit the mevalonate synthesis pathway and thus block cholesterol synthesis) have shown mixed results. Silvente-Poirot S et al have suggested that the contradictory effects of anti-cholesterol strategies may be due to the complexity of the genes and enzymes controlling the "sterolome" (Science, 2014). Results: The effects of metformin on de novo cholesterol biosynthesis in the TN breast cancer cells, MDA-MB-231 and MDA-MB-468, were studied using expression profiling and miRNA microarrays. Metformin treatment for 24 h down regulated the expression of 21 genes encoding enzymes in the cholesterol biosynthesis pathway at high levels (up to 9 fold), which has been confirmed by qRT-PCR. Metformin treatment for 6 h also resulted in the upregulation of 17 miRNA, which are predicted to control many of the down regulated genes. These data suggest a post-translational inhibition of cholesterol biosynthesis, similar to what we have reported for fatty acid biosynthesis (see above). The down regulated enzymes include critical steps in complex cholesterol biosynthesis pathway occurring in the cytosol, endoplasmic reticulum and the nuclear envelope. Western blots have confirmed that miRNA 141 and miR192 reversed the metformin induced reduction in p-MEK1/2, p-Akt and pMAPK. Conclusions: Metformin regulates cancer cells by multiple pathways including reduction in cell growth, increases in apoptosis and alterations in cellular metabolism. The effects on the cholesterol pathway are both extensive and affect multiple downstream pathways including EGFR, Akt, and MAPK. Partial funding was provided by the Susan G. Komen Foundation. Citation Format: Ann D Thor, Zeying Fan, Reema Wahdan-Alaswad, Steve M Anderson, Jennifer K Richer, Susan M Edgerton. Metformin blocks de novo synthesis of cholesterol in triple negative breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-08-02.

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.

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′ UTR's were performed using luciferase reporters. RESULTS: TargetScan and Diana miR-Path analysis of miRNA's 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.

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)

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 G(1) 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 p27(kip1) or p21(waf1). 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.

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.

Background: Co-morbidity of thyroid disease and breast cancer has been recognized for over a century. Up to two-thirds of women with breast cancer have clinical or occult thyroid disease, as compared to one fifth of unaffected females. Thyroid disease typically proceeds breast cancer by at least a decade, and many (nearly half) are on long term thyroid hormone supplementation at the time of diagnosis. The exogenous administration of thyroid hormone, for hypothyroidism or status post thyroidectomy for neoplasia, has been associated with an increased risk of breast cancer and in some studies, a worse prognosis. Causality, however, has been controversial. Results: We studied over 800 consecutive node negative invasive breast cancer patients diagnosed between 1976 and 1993 from a single institution. Of these, 92% were chemo-naive and only 14% received tamoxifen, reducing interactions with treatment for the study design. We showed untoward significant interactions with both univariate and multivariate analyses, between thyroid hormone supplementation, disease free and disease specific survival only in patients with steroid receptor positive tumors. These interactions were strongest in pre-menopausal, as compared to post-menopausal patients. We thus hypothesized that thyroid hormone (TH), either indirectly or directly, promoted carcinogenesis via the steroid receptors (SR) for estrogen and/or progesterone and have interrogated mechanisms of these interactions in vitro. Estrogen (E2) induce significant cellular proliferation, whereas TH alone (at concentrations of T4 1 x 10-6:T3 at 2.5 x 10-7 M) induced only mild cell proliferation in SR+ (MCF-7 andT47D), but not SR- breast cancer cell lines (MDA-MB-468 and SKBR3). In contrast, TH induced significant proliferation in SR + cells at significantly lower doses (down to T4 1 x 10-12: T3 2.5 x 10-13 M) if administered with low dose estrogen (E2: 1 x 10 -10). These mitogenic effects were demonstrated by MTS and DNA quantification assays, as well as flow cytometry, which revealed the induction of G2/M arrest and an increase in the percentage of cells in S phase with TH alone (at higher doses) or TH + E2 (at lower doses). Tamoxifen partially mitigated the pro-growth effects of TH + E2, whereas ICI 182,780 completely abrogated these effects. Using Western blots we have shown that that TH (with or without E2) upregulated ERa expression and activation, Cyclin A, B,D1, and E, E2F1, MAPK/ERK1/2 but not AKT signaling. TH +/- E2 also promoted clonogenicity, motility, a more basal phenotype (CD24+/CD44+) and mammosphere formation in cells grown under non-adherent conditions. Conclusions: We have demonstrated in Stage I breast cancer patients with minimal systemic treatment, that the administration of TH was associated with a significantly shortened disease free and disease specific survival, only in patients whose tumors expressed SR. TH administered to SR+ cell lines, particularly when co-administered with E2 at therapeutic/physiologic doses, upregulates and activates ER, cell proliferation, a more aggressive and motile basaloid phenotype. Citation Format: Ann D Thor, Zeying Fan, Reema Wahdan-Alaswad, Susan M Edgerton. Thyroid hormone up-regulates estrogenic and pro-carcinogenic signaling, inducing a basaloid and more motile phenotype in only steroid receptor positive breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-11.

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.

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 receptor (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 incidence. 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.

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

Background: HER2 alterations occur in one-fourth of breast cancers and are associated with an aggressive tumor phenotype. The anti-HER2 agent trastuzumab reduces cell proliferation, angiogenesis, DNA repair and induces antibody-dependent cellular cytotoxicity. Objective response rates and median duration of response for eligible patients given trastuzumab alone is low (12-34% and 9 months respectively), hence, it is administered in combination with other agents. Mechanisms of trastuzumab resistance include: altered receptor antibody interactions, increased signaling through other EGFR type I growth factor receptors, modulation of p27 kip1 and increased insulin-like growth factor 1 receptor (IGF-1) signaling. We have reported that metformin inhibits HER2 expression, tyrosine kinase activity (phosphorylated HER2 at both auto- and Src- phosphorylation sites), AKT/mTOR signaling, Cyclin D1 and E2F1 with G1 arrest in HER2 overexpressing breast cancer cells. We sought to determine if metformin would enhance trastuzumab associated cytotoxicity and modulate acquired resistance in vitro.Methods: We used trastuzumab sensitive (SKBR3, BT474) and resistant cell lines (BT-474/HR20, SKBR3/P2, MCF-7/713 and MCF-7/HER2Δ16) to study the effects of metformin, metformin + trastuzumab and trastuzumab alone. Assays included MTS for proliferation, clonogenicity studies, Western blots, and pull down experiments with Western blots.Results: Trastuzumab sensitive breast cancer cells were less sensitive to metformin alone, compared to trastuzumab resistant HER2 subtype breast cancer cell lines. Trastuzumab sensitive cells showed enhanced growth and clonogenicity inhibition when treated by both metformin and trastuzumab. At the molecular level, these bi-treated cells showed decreases in HER2/pHER2, erbB3/perbB3, and inactivation of AKT and MAPk signaling. Metformin as low as 20mM increased the efficacy of trastuzumab. All HER2 resistant cell lines showed higher levels of IGF1R and HER2/IGF1R complexes, as compared to sensitive parental lines. In these resistant lines Metformin decreased cell proliferation and clonogenicity, HER2/IGF1R complexes (identified through pull down experiments) and protein expression of HER2/pHER2, erbB3/perbB3, IGF1R/pIGF1R as well as downstream signaling via Akt and IGF1 pathways. Metformin overcame trastuzumab resistance as demonstrated by growth and clonogenicity assays.Conclusions: HER2 amplified trastuzumab resistant breast cancer cells showed greater sensitivity to metformin than sensitive parental lines. Metformin reversed trastuzumab resistance and decreased HER2:IGF1R complexes, HER2/pHER2 and IGF1R expression, whereas sensitive parental lines showed no complex formation. These preclinical data suggest a combination of metformin and trastuzumab may have clinical benefit, improving the efficacy and reducing the emergence of or reversing trastuzumab resistance in HER2 positive breast cancer patients.Herceptin graciously provided by Genentech Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1133.

Background: Type II diabetes and obesity are important risk factors for post-menopausal luminal A (LA) and triple negative (TN) pre-menopausal breast cancers, particularly in African American (AA) and Hispanic women. Breast cancer patients with these chronic metabolic diseases also have a worse prognosis, independent of other factors. We have published that metformin inhibits cell growth (S phase arrest) and induces apoptosis, only in TN cell lines in vivo and in vitro. It is less active, growth inhibitory (G1 arrest) and does not induce apoptosis in other breast cancer cell subtypes (Cell Cycle, 2009).Methods: We investigated the effects of glucose as a mitogen at physiologic (5mM), metabolic syndrome (7mM) or diabetic levels (10mM), with or without insulin (100 ng/ml) or leptin (100 ng/ml) using cell lines representing all molecular subtypes of breast cancer. Metformin was then used in combination with the above, to determine whether it would block the mitogenic or signaling effects of supraphysiological glucose, insulin or leptin.Results: The LA (MCF-7) and 2 of 5 TN cell lines (derived from AA patients; HCC 1806 and MDA 468) showed the most cell growth in response to glucose >5mM (75% for MCF-7, 30-50% for AA TN lines). Three TN cell lines from Caucasians (MDA231, HCC1937, BT20), the HER2 (SKBR3) and luminal B (BT-474) cells showed less growth induction with glucose >5mM. In TN lines only, glucose associated mitogenesis was associated with increased EGFR, pEGFR, IGF1R, pIGF1R, AKT and pAKT and decreases in AMPK, pAMPK, p38, IRS2, and the cyclins D1, E and A in a dose dependent manner. Metformin abrogated glucose induced cell growth and the aforementioned protein expression/phosphorylation changes involving EGFR, IGF1R, and AKT, increased AMPK and pAMPK and induced a profound reduction in Cyclin D1 across all glucose concentrations in TN cell lines from AA women. It reduced but did not eliminate glucose associated mitogenesis in the TN cell lines from Caucasian patients. Metformin had a more variable effect on cell lines of other molecular subtypes grown under high glucose conditions. The 5 TN breast cancer cell lines were uniformly resistant to both leptin and insulin associated mitogenesis, across a wide range of glucose concentrations. In contrast, both leptin and insulin significantly promoted LA breast cancer cell growth. These effects were resistant to metformin treatment. Leptin and insulin had the least growth promoting effects on HER2 breast cancer cell lines, whereas they induced modest growth induction in LB cell lines.Conclusions: All TN cell lines showed significant mitogenesis in response to glucose >5mM, whereas they were uniformly resistant to both leptin and insulin. The glucose associated mitogenesis was more pronounced in lines derived from AA patients, as were the anti-mitgenic effects of metformin. LA cells showed marked growth induction by glucose, leptin and insulin, whereas HER2 cell lines showed general resistance to all of these factors. These data suggest that metabolic and hormonal shifts with obesity and diabetes, as well as metformin response vary by the molecular subtype of breast cancer cells and ethnicity. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5154.

Abstract #6026 Breast cancers of the basal subtype (ER, PR and HER2 negative; EGFR and/or CK5/6 positive) are typically high grade, rapidly proliferative, associated with a poor outcome and are resistant to many chemotherapeutic regimens. They occur more frequently in women with BRCA-1 mutations, African Americans and obese individuals. At the molecular level, basal cancers have impairment of BRCA-1 and p53 signaling and are driven, in part, through EGFR which activates the PI3K/AKT/mTOR pathways. Treatment of basal carcinoma cell lines with the FDA approved anti-diabetic drug metformin significantly reduces cellular proliferation, induces cell cycle arrest (late G1/S phase) and apoptosis in vivo and in vitro. Similar effects were not observed in breast cancer cells of the luminal A, B or HER2 subtypes. Metformin induces a wide spectrum of metabolic and molecular changes in basal carcinoma cell lines in vitro, including: a reduction in glucose uptake, enhanced lactate release, a decrease in cellular proliferation and colony formation, a decrease in EGFR, phospho-EGFR and downstream signaling including pAKT, pMAPK and psrc, inhibition of cyclins D1 and E, reduced E2F1, PARP cleavage and activation of caspases 3, 8 and 9 in a dose and time dependent manner. Nude mice with basal cell line MDA-MB-231 xenografts treated with metformin showed significantly reduced tumor growth and prolonged survival as compared to control untreated mice. Pre-treatment with metformin prior to xenograft inoculation reduced both tumor formation and growth as compared to mice treated with metformin after implantation or untreated control mice, suggesting that metformin may have some use in basal cancer prevention. Epidemiologic data has shown that women treated with metformin have a reduced rate of breast cancer and, for those patients with breast cancer, an improved disease free and overall survival. In aggregate, these data suggest that further study of metformin to prevent or treat breast cancer in some patient subgroups may be indicated. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6026.