Malathi Banda

Metabotropic glutamate receptor-1: a potential therapeutic target for the treatment of breast cancer

Metabotropic glutamate receptors are G-protein-coupled receptors normally expressed in the central nervous system where they mediate neuronal excitability, synaptic plasticity, and feedback inhibition of neurotransmitter release. However, recent data suggest that these receptors are also expressed and functional in some cancers, most notably melanoma. We detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in triple negative breast cancer cells and evaluated its role in regulating the pro-proliferative phenotype of these cells. mGluR1 inhibitors (Riluzole or BAY36-7620) inhibited the proliferation of triple negative breast cancer cells in a time- and dose-dependent manner and this inhibition correlated with increased apoptosis as demonstrated by increase in PARP cleavage products and Annexin V staining. mGluR1 knockdown using Lentiviral constructs expressing shRNA targeting GRM1 also inhibited proliferation compared to non-silencing controls. In addition, treatment of mice bearing MDA-MB-231 xenografts with Riluzole or BAY36-7620, by intraperitoneal injection, resulted in a significant reduction in tumor volume of up to 80%. Moreover, Riluzole was effective against triple negative breast cancer xenografts in mice at doses equivalent to those currently being used in humans for the treatment of amyotrophic lateral sclerosis. Our observations implicate mGluR1 and glutamate signaling as a promising new molecular target for the treatment of breast cancer. Even more promising, Riluzole, because it is an oral drug that can be administered with low toxicity, represents a promising approach in the treatment of triple negative breast cancer.

Regulation of the Expression and Activity of the Antiangiogenic Homeobox Gene GAX/MEOX2 by ZEB2 and MicroRNA-221

ABSTRACT Tumors secrete proangiogenic factors to induce the ingrowth of blood vessels from the stroma. These peptides bind to cell surface receptors on vascular endothelial cells (ECs), triggering signaling cascades that activate and repress batteries of downstream genes responsible for the angiogenic phenotype. To determine if microRNAs (miRNAs) affect regulation of the EC phenotype by GAX, a homeobox gene and negative transcriptional regulator of the angiogenic phenotype, we tested the effect of miR-221 on GAX expression. miR-221 strongly upregulated GAX, suggesting that miR-221 downregulates a repressor of GAX. We next expressed miR-221 in ECs and identified ZEB2, a modulator of the epithelial-mesenchymal transition, as being strongly downregulated by miR-221. Using miR-221 expression constructs and an inhibitor, we determined that ZEB2 is upregulated by serum and downregulates GAX, while the expression of miR-221 upregulates GAX and downregulates ZEB2. A mutant miR-221 fails to downregulate ZEB2 or upregulate GAX. Finally, using chromatin immunoprecipitation, we identified two ZEB2 binding sites that modulate the ability of ZEB2 to downregulate GAX promoter activity. We conclude that miR-221 upregulates GAX primarily through its ability to downregulate the expression of ZEB2. These observations suggest a strategy for inhibiting angiogenesis by either recapitulating miR-221 expression or inhibiting ZEB2 activation.

Metabotropic glutamate receptor-1 contributes to progression in triple negative breast cancer.

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy.

ACB-PCR measurement of spontaneous and furan-induced H-ras Codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver

Furan is a rodent liver carcinogen, but the mode of action for furan hepatocarcinogenicity is unclear. H‐ras codon 61 mutations have been detected in spontaneous liver tumors of B6C3F1 mice, and the fraction of liver tumors carrying H‐ras codon 61 CAA to AAA mutation increased in furan‐treated mice. Allele‐specific competitive blocker PCR (ACB‐PCR) has been used previously to quantify early, carcinogen‐induced increases in tumor‐associated mutations. The present pilot study investigated whether furan drives clonal expansion of pre‐existing H‐ras mutant cells in B6C3F1 mouse liver. H‐ras codon 61 CAA to CTA and CAA to AAA mutations were measured in DNA isolated from liver tissue of female mice treated with 0, 1, 2, 4, or 8 mg furan/kg body weight, five days per week for three weeks, using five mice per treatment group. Spontaneous levels of mutation were low, with two of five control mice having an H‐ras codon 61 CTA or AAA mutant fraction (MF) greater than 10−5. Several furan‐treated mice had H‐ras codon 61 AAA or CTA MFs greater than those measured in control mice and lower bound estimates of induced MF were calculated. However, no statistically‐significant differences were observed between treatment groups. Therefore, while sustained exposure to furan is carcinogenic, at the early stage of carcinogenesis examined in this study (three weeks), there was not a significant expansion of H‐ras mutant cells. Environ. Mol. Mutagen. 54:659–667, 2013.

Abstract 5147: Characterization of the tissue-specific properties of cancer driver mutations suggests spontaneous mutation in normal tissues drives tumor susceptibility

Cancer driver mutations (CDMs) are being developed as theranostic biomarkers. This study used high-sensitivity quantification by ACB-PCR, with a sensitivity of 10−5, to elucidate the tissue-specific properties of CDMs. We employed the paradigm of comparing levels of specific hotspot CDMs in normal human tissues and tumors. Specifically, we measured KRAS G12D, KRAS G12V, PIK3CA E545K, and PIK3CA H1047R mutant fractions (MFs) in normal breast, colon, lung, and thyroid and quantified the same mutations in mammary ductal carcinomas (DCs), colonic adenocarcinomas, lung adenocarcinomas, and papillary thyroid carcinomas. Three major findings were: 1) these mutations occur in normal human tissues at relatively high frequencies, 2) there is considerable variability in CDM MFs in normal tissues across individuals, and 3) tumors frequently carry CDMs at levels greater than that present in normal tissue, but below that detected by standard DNA sequencing. Understanding the prevalence of mutant subpopulations is important because they can drive resistance to molecularly-targeted therapies. The PIK3CA H1047R profile for normal breast and DC were similar, with large mutant subpopulations present in both tissue types. Because the PIK3CA H1047R mutation is the most prevalent point mutation reported in breast cancer, we conclude that it can drive breast carcinogenesis as a subpopulation, potentially through a paracrine mechanism, and may be a useful biomarker of breast cancer susceptibility. Other properties of CDMs elucidated were gender differences, the accumulation of mutation with donor age, and the correlation between MF and maximum tumor dimension (MTD). Male lung had significantly greater PIK3CA H1047R MFs than female lung. The PIK3CA H1047R MF in normal breast was positively correlated with donor age, and the mutation in DCs was positively correlated with MTD. By contrast, KRAS MFs showed non-significant decreases in normal lung with increasing donor age. For colorectal adenocarcinomas, KRAS G12V MF was negatively correlated with MTD, consistent with previous findings that KRAS G12V MF decreases during adenoma to adenocarcinoma progression. This suggests that the selective advantage provided by KRAS G12V mutation is context-dependent and can be either positive or negative. Across the four mutational targets and four tissue types, a significant positive correlation was observed between the variability (Log10 standard deviation) of MF measured in normal tissues and the prevalence with which the mutation reportedly occurs in tumors, as per The Cancer Genome Atlas database. This suggests that level and inter-individual variation in CDMs can be used to identify promising tissue-specific, mutational biomarkers of cancer susceptibility. This is not a formal dissemination of information by FDA and does not represent agency position or policy. Citation Format: Barbara L. Parsons, Meagan B. Myers, Karen L. McKim, Malathi Banda. Characterization of the tissue-specific properties of cancer driver mutations suggests spontaneous mutation in normal tissues drives tumor susceptibility. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5147.

Abstract 4267: Quantification of somatic hotspot mutations in KRAS, HRAS, BRAF, and PIK3CA: Comparing normal human breast and ductal carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Data within the Catalogue of Somatic Mutations in Cancer indicates that a number of different hotspot oncogene mutations occur infrequently in breast tumors as compared to other tumor types (e.g., colon and lung). We hypothesized that greater sensitivity is required to detect somatic mutations in breast tumors, due to their highly-heterogeneous clonal structure. Therefore, the Allele-specific Competitive Blocker PCR method (with a sensitivity of 10-5), was used to quantify mutations in DNA isolated from normal human breast and ductal carcinoma (DC) samples (n = 10). The specific mutations quantified were: KRAS codon 12 GGT to GAT (G12D) and GGT to GTT (G12V), HRAS codon 12 GGC to GAC (G12D), BRAF codon 600 GTG to GAG (V600E), and PIK3CA codon 1047 CGT to CAT (H1047R). In addition, comparison of normal and malignant tissue was employed in order to define the lower bound on abnormal levels of mutation, as tumors that possess a mutant fraction (MF) greater than the upper 95% confidence interval of that present in normal breast. Interestingly, different patterns of mutation were observed for the different hotspot point mutations. We previously demonstrated that significantly higher levels of KRAS mutation are present in colon and lung tumors relative to the cognate normal tissue; however, the frequencies of KRAS codon 12 GGT to GAT and GGT to GTT mutation in normal breast and in DCs were quite similar (∼10-5 for the KRAS codon 12 GGT to GAT mutation in both tissues). Only one normal sample and two DCs had BRAF codon 600 GTG to GAG MFs >10-5. With respect to HRAS codon 12 GGC to GAC mutation, however, ∼80% of DCs had a MF greater than the upper 95% confidence interval of that present in normal breast tissue. Importantly, no breast tumor had a HRAS codon 12 GGC to GAC MF ≥10-1 (i.e., that detectable by DNA sequencing). Unexpectedly high levels of PIK3CA codon 1047 CGT to CAT mutation (>10-2) were observed in 4/10 normal breast samples and 4/10 DCs, suggesting that preexisting PIK3CA mutations in normal breast contribute to breast cancer susceptibility. In summary, all DC samples analyzed contained somatic mutations, none of which would be detectable by DNA sequencing. Consequently, these results indicate somatic mutations are more prevalent in breast cancer than is currently recognized. Ongoing analyses of additional tumors, corresponding to different breast cancer subtypes, will provide a better understanding of the role of somatic mutations in breast cancer. Citation Format: Malathi Banda, Meagan B. Myers, Karen L. McKim, Yiying Wang, Barbara L. Parsons. Quantification of somatic hotspot mutations in KRAS , HRAS , BRAF , and PIK3CA : Comparing normal human breast and ductal carcinoma. [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 4267. doi:10.1158/1538-7445.AM2014-4267

Abstract 1206: Metabotropic glutamate receptor 1 functions as an oncogene in the progression of triple negative breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: L-glutamate is the major excitatory neurotransmitter in the central nervous system and activates both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) are a family of G protein-coupled receptors. mGluR1 (gene: GRM1) is included in Group I of these receptors and have been shown to activate phosphatidylinositol-calcium second messenger system. Aberrant extracellular glutamate signaling has been implicated in carcinogenesis; specifically, the aberrant expression of mGluR1 in melanocytes plays a critical role in the development of melanoma. Previously in our laboratory, we detected mGRM1 expression in triple negative breast cancer cells. We therefore evaluated its role in regulating the phenotype of these cells and found that mGluR1 expression is oncogenic in triple negative breast cancer (TNBC) progression. Methods: We determined the role of mGluR1 in TNBC progression using the MCF-10 triple negative series of cell lines, which represent the progression from normal mammary epithelium (MCF10A) to atypical hyperplasia (MCF10AT1) to ductal carcinoma in situ (MCF10.DCIS.com), and finally to malignant (MCF10.CA1D). GRM1 was overexpressed in MCF10A and MCF10AT1 and silenced MCF10.DCIS.com and MCF10.CA1D. We then determined whether mGRM1 has a transforming role through in vitro studies of proliferation, invasion, migration and anchorage-independent growth. We also inhibited mGluR1 signaling using two pharmacologic inhibitors: Riluzole, which is FDA-approved for amyotropic lateral sclerosis, and BAY36-7620, which is a specific noncompetitive inhibitor of mGluR1. Effects were evaluated on proliferation and anchorage independent growth. Finally, MCF10AT1 cells were transduced with a lentiviral construct driving mGluR1 expression and injected into athymic nude mice. The growth and histology of the resultant xenografts were compared with control LacZ transduced cells. Results: mGluR1 overexpression increased proliferation, anchorage-independent growth, and invasiveness in MCF10AT1 and not in MCF10A cells, while knockdown of mGluR1 expression resulted in a decrease in proliferation, anchorage independent growth and invasiveness in MCF10.CA1D cells. Pharmacologic inhibition of mGluR1 signaling in MCF10.CA1D cells resulted in a decrease in proliferation and anchorage independent growth. Transduction of MCF10AT1 cells with GRM1 resulted in transformation to carcinoma in 10/11 of the resultant xenografts compared to 2/9 for wild type and 3/11 for LacZ controls. Conclusions: mGluR1 expression and activity increases cell proliferation, anchorage independent growth, and invasion in vitro. In vivo, mGluR1 drives progression of MCF10AT1 cells from hyperplastic lesions to frank carcinoma. We therefore conclude that mGRM1 plays a role of an oncogene in the progression of TNBC and represents a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1206. doi:1538-7445.AM2012-1206

Abstract 1673: mGluR1 as a potential therapeutic target in the treatment of triple negative breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Metabotropic glutamate receptors are expressed throughout the central nervous system where they initiate a host of signaling events that regulate neuron function. We have identified the presence of metabotropic glutamate receptor 1 (mGluR1) in breast cancer cells and demonstrated a role for these receptors in regulating the pro-proliferative phenotype of these cells, both in vitro and in vivo. In this study, we investigate the signaling mechanism(s) by which mGluR1 mediates this pro-proliferative effect, as well as investigating a potential role for mGluR1 in mediating anti-apoptotic signaling events in triple negative breast cancer. Methods: We studied the effect of mGluR1 expression on cell growth using MDA-MB-231 cells stably transduced with plasmids expressing shRNA against GRM1, the gene coding for mGluR1 protein. Cell growth was measured by MTT assay and a role for mGluR1 in mediating apoptosis was assessed by measuring PARP cleavage products and Annexin V staining, using Riluzole or BAY36-7620 (BAY), non-competitive inhibitors of mGluR1 activity. To examine potential signaling mechanisms mediating cell growth, MDA-MB-231 cells were grown in glutamate-free media and stimulated with the mGluR1 agonist, L-Quisqualic acid, in the presence or absence of BAY. PKC and phosphorylated Akt levels were examined by Western analysis. Since mGluR1 is known to protect nerve cells from oxidative stress and increased oxidative stress can result in p53-induced apoptosis, we measured the effect of Riluzole on p53 expression and oxidative stress levels in MDA-MB-231 cells. Oxidative stress was measured using immunofluorescent staining with carboxy-H2DCFDA and p53 by Western blot analysis. Results: MDA-MB-231 cells were transduced with Lentiviral constructs expressing shRNA against GRM1 and cell growth assessed by MTT assay. Growth of these cells was inhibited 50% by shRNA against GRM1 compared to non-silencing scrambled control. In addition, inhibition of mGluR1 activity by BAY in MDA-MB-231 cells prevented signaling through Akt which ultimately resulted in increased p53 expression and superoxide production and apoptosis. Incubation of MDA-MB-231 or BT549 cells with varying doses of Riluzole resulted in increased PARP cleavage by 24 hours. FACS analysis also showed a three- and ten-fold increase in Annexin V staining of MDA-MB-231 cells after 24 and 48 hr incubation with Riluzole, respectively. BAY also induced Annexin V staining of these cells by almost 3-fold at the 48 hr timepoint. Conclusion: Our results showing that inhibition of mGluR1 inhibits proliferation and increases apoptosis and oxidative stress implicate mGluR1, or one of its downstream signaling molecules, as potential new molecular targets for the treatment of breast cancer. Because it is an FDA-approved oral drug with low toxicity, Riluzole represents a promising approach in the treatment of triple negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1673. doi:10.1158/1538-7445.AM2011-1673

Abstract 3950: The role of microRNA-130a in regulating HOXA5 expression in breast cancer progression

Introduction: Micro RNAs (miRNAs) are key post -transcriptional and translational regulators of gene expression and regulate diverse physiological activities involving development, cell growth, differentiation, apoptosis, and pathological processes such as heart disease and cancer. In tumorigenesis miRNAs function as oncogenes or tumor suppressors. HOXA5 belongs to the family of homeobox (HOX) genes, which encode a highly conserved family of transcription factors that are known to regulate body patterning during embryogenesis. HOXA5 plays a major role in mesenchymal-epithelial interactions during organogenesis. Sukumar et al have demonstrated that HOXA5 loss of function leads to loss of p53 expression in mammary epithelial cells, which in turn contributes to malignant transformation, which is why understanding the mechanisms by which HOXA5 expression is dysregulated in breast cancer is critical. We identified a consensus binding sequence for miR-130a in the 3′-UTR of the HOXA5 coding region. Previous studies in our laboratory have shown that miR-130a downregulates HOXA5 expression in vascular endothelial cells. In this study we characterize the role of miR130a in downregulating HOXA5 expression that in turn can decrease p53 expression and function in breast cancer cells resulting in tumor proliferation, progression and metastasis. Methods: We determined whether mitogenic factors in serum affect miR130a and HOXA5 mRNA expression in the breast epithelial cell line MCF10A. Correlation between the expression of miR130a and HOXA5 mRNA expression in the MCF-10 breast progression series cell lines that model the progression from normal mammary epithelium (MCF-10A), ductal carcinoma in situ (MCF10.DCIS.com), to fully malignant (MCF10.CA1) was examined. In addition, we silenced miR-130a using a specific 2’-O-methyl-modified inhibitor and determined whether knocking it out increases HOXA5 mRNA expression in MCF10A cells. The expression levels of miR130a and HOXA5 were measured using quantitative reverse transcriptase real time PCR (QRT-PCR). Results: First, we observed that miR130a expression increases by 2.2 fold and HOXA5 mRNA levels decreases by 0.2 fold following treatment with 5% horse serum in MCF10A cells. Next, we noted that miR-130a expression increases and HOXA5 mRNA expression decreases as tumorigenicity increases in MCF10A progression series cell lines. Finally, knocking out miR-130a activity increased HOXA5 mRNA expression in MCF10A cells, demonstrating that HOXA5 is regulated by miR130a in these cells. Conclusion: Overall our results indicate that miR130a regulates HOXA5 in breast cancer cells and thus may have a role in breast tumor progression. This study will enable us to decipher the role of mir130a in breast tumor progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3950. doi:10.1158/1538-7445.AM2011-3950

Abstract 1578: The role of metabotropic glutamate receptor-1 in breast cancer tumor progression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Metabotropic glutamate receptors (mGluRs) are normally expressed in the central nervous system where they mediate neuronal excitability, synaptic plasticity and feedback inhibition of neurotransmitter release. However, recent studies suggest that these receptors are also present and functional in various cancer types, including melanoma and prostate cancer cells. In addition, brain endothelial cells were recently shown to express mGluR1 receptors and to mediate VEGF-induced endothelial cell proliferation both in vivo and in vitro. These results suggest a role for mGluR1 in mediating angiogenesis, a process that is required for cancer tumors to grow and metastasize. Both VEGF levels and angiogenesis are independent prognostic indicators in primary breast cancer patients with their presence being associated with early relapse. Therefore, we wished to examine a potential role for mGluR1 in mediating VEGF-induced breast cancer tumor progression. Methods: To do this, we analyzed several breast cancer cell lines as well as primary endothelial cells for mGluR1 protein and mRNA expression and looked at their potential role in mediating cell growth, angiogenesis and tumor formation in a mouse xenograph model. The role of mGluR1 on cell growth was determined using an MTT assay and by counting isolated nuclei with a Coulter counter in the presence and absence of BAY36-7620 and Riluzole, specific non-competitive inhibitors of mGluR1. The angiogenic potential of endothelial cells and the role of mGluR1 in mediating this process were measured using a matrigel tube formation assay in the presence and absence of BAY36-7620 and Riluzole. Lastly, the effect of mGluR1 on tumor formation and angiogenesis in vivo was determined by measuring tumor growth and blood vessel formation in a xenograft model derived from cell lines expressing high levels of mGluR1 and treated with either the mGluR1 inhibitors or vehicle. Results: The results of this study show strong mRNA and protein expression of mGluR1 in several breast cancer cell lines as well as in HUVECs and human dermal micro-endothelial cells (HDMEC). Cell growth, in the presence of either BAY36-7620 or Riluzole, inhibited cell growth in all these cells in a dose-response manner and completely inhibited VEGF-induced matrigel tube formation of HUVECs and HDMEC. In addition, tumor growth in a xenograft model using MBA231 cells, which expressed high levels of mGluR1, was inhibited by 30% and 50% in the presence of 9 mg/kg and 18 mg/kg Riluzole, respectively. Conclusion: By inhibiting both breast cancer cell growth and angiogenesis, these results implicate mGluR1 as a major player in the development and progression of breast cancer and thus suggests that mGluR1 may serve as a useful therapeutic target in the treatment of breast cancer. 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 1578.