Metabotropic glutamate receptor 1 disrupts mammary acinar architecture and initiates malignant transformation of mammary epithelial cells.

Metabotropic glutamate receptor 1 (mGluR1/Grm1) is a member of the G-protein-coupled receptor superfamily, which was once thought to only participate in synaptic transmission and neuronal excitability, but has more recently been implicated in non-neuronal tissue functions. We previously described the oncogenic properties of Grm1 in cultured melanocytes in vitro and in spontaneous melanoma development with 100 % penetrance in vivo. Aberrant mGluR1 expression was detected in 60-80 % of human melanoma cell lines and biopsy samples. As most human cancers are of epithelial origin, we utilized immortalized mouse mammary epithelial cells (iMMECs) as a model system to study the transformative properties of Grm1. We introduced Grm1 into iMMECs and isolated several stable mGluR1-expressing clones. Phenotypic alterations in mammary acinar architecture were assessed using three-dimensional morphogenesis assays. We found that mGluR1-expressing iMMECs exhibited delayed lumen formation in association with decreased central acinar cell death, disrupted cell polarity, and a dramatic increase in the activation of the mitogen-activated protein kinase pathway. Orthotopic implantation of mGluR1-expressing iMMEC clones into mammary fat pads of immunodeficient nude mice resulted in mammary tumor formation in vivo. Persistent mGluR1 expression was required for the maintenance of the tumorigenic phenotypes in vitro and in vivo, as demonstrated by an inducible Grm1-silencing RNA system. Furthermore, mGluR1 was found be expressed in human breast cancer cell lines and breast tumor biopsies. Elevated levels of extracellular glutamate were observed in mGluR1-expressing breast cancer cell lines and concurrent treatment of MCF7 xenografts with glutamate release inhibitor, riluzole, and an AKT inhibitor led to suppression of tumor progression. Our results are likely relevant to human breast cancer, highlighting a putative role of mGluR1 in the pathophysiology of breast cancer and the potential of mGluR1 as a novel therapeutic target.

RET rearrangements are actionable alterations in breast cancer.

Fusions involving the oncogenic gene RET have been observed in thyroid and lung cancers. Here we report RET gene alterations, including amplification, missense mutations, known fusions, novel fusions, and rearrangements in breast cancer. Their frequency, oncogenic potential, and actionability in breast cancer are described. Two out of eight RET fusions (NCOA4-RET and a novel RASGEF1A-RET fusion) and RET amplification were functionally characterized and shown to activate RET kinase and drive signaling through MAPK and PI3K pathways. These fusions and RET amplification can induce transformation of non-tumorigenic cells, support xenograft tumor formation, and render sensitivity to RET inhibition. An index case of metastatic breast cancer progressing on HER2-targeted therapy was found to have the NCOA4-RET fusion. Subsequent treatment with the RET inhibitor cabozantinib led to a rapid clinical and radiographic response. RET alterations, identified by genomic profiling, are promising therapeutic targets and are present in a subset of breast cancers.

Riluzole exerts distinct antitumor effects from a metabotropic glutamate receptor 1-specific inhibitor on breast cancer cells.

Recent evidence suggests that glutamate signaling plays an important role in cancer. Riluzole is a glutamate release inhibitor and FDA-approved drug for the treatment of amyotrophic lateral sclerosis. It has been investigated as an inhibitor of cancer cell growth and tumorigenesis with the intention of repurposing it for the treatment of cancer. Riluzole is thought to act by indirectly inhibiting glutamate signaling. However, the specific effects of riluzole in breast cancer cells are not well understood. In this study, the anti-cancer effects of riluzole were explored in a panel of breast cancer cell lines in comparison to the metabotropic glutamate receptor 1-specific inhibitor BAY 36-7620. While both drugs inhibited breast cancer cell proliferation, there were distinct functional effects suggesting that riluzole action may be metabotropic glutamate receptor 1-independent. Riluzole induced mitotic arrest independent of oxidative stress while BAY 36-7620 had no measurable effect on mitosis. BAY 36-7620 had a more pronounced and significant effect on DNA damage than riluzole. Riluzole altered cellular metabolism as demonstrated by changes in oxidative phosphorylation and cellular metabolite levels. These results provide a better understanding of the functional action of riluzole in the treatment of breast cancer.

A functionally significant SNP in TP53 and breast cancer risk in African-American women.

A coding region polymorphism exists in the TP53 gene (Pro47Ser; rs1800371) in individuals of African descent, which reduces p53 tumor suppressor function in a mouse model. It has been unclear whether this functionally significant polymorphism alters cancer risk in humans. This analysis included 6907 women with breast cancer and 7644 controls from the AMBER, ROOT, and AABC consortia. We used multivariable logistic regression to estimate associations between the TP53 Pro47Ser allele and overall breast cancer risk. Because polymorphisms in TP53 tend to be associated with cancer risk in pre-menopausal women, we also limited our analyses to this population in the AMBER and ROOT consortia, where menopausal status was known, and conducted a fixed effects meta-analysis. In an analysis of all women in the AMBER, ROOT, and AABC consortia, we found no evidence for association of the Pro47Ser variant with breast cancer risk. However, when we restricted our analysis to only pre-menopausal women from the AMBER and ROOT consortia, there was a per allele odds ratio of 1.72 (95% confidence interval 1.08-2.76; p-value = 0.023). Although the Pro47Ser variant was not associated with overall breast cancer risk, it may increase risk among pre-menopausal women of African ancestry. Following up on more studies in human populations may better elucidate the role of this variant in breast cancer etiology. However, because of the low frequency of the polymorphism in women of African ancestry, its impact at a population level may be minimal.

Role of Biomarkers in the Development of PARP Inhibitors.

Defects in DNA repair lead to genomic instability and play a critical role in cancer development. Understanding the process by which DNA damage repair is altered or bypassed in cancer may identify novel therapeutic targets and lead to improved patient outcomes. Poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) has an important role in DNA repair, and novel therapeutics targeting PARP1 have been developed to treat cancers with defective DNA repair pathways. Despite treatment successes with PARP inhibitors (PARPi), intrinsic and acquired resistances have been observed. Preclinical studies and clinical trials in cancer suggest that combination therapy using PARPi and platinating agents is more effective than monotherapy in circumventing drug resistance mechanisms. Additionally, identification of biomarkers in response to PARPi will lead to improved patient selection for targeted cancer treatment. Recent technological advances have provided the necessary tools to examine many potential avenues to develop such biomarkers. This review examines the mechanistic rationale of PARP inhibition and potential biomarkers in their development for personalized therapy.

Emerging Role of Genomic Rearrangements in Breast Cancer: Applying Knowledge from Other Cancers.

Significant advances in our knowledge of cancer genomes are rapidly changing the way we think about tumor biology and the heterogeneity of cancer. Recent successes in genomically-guided treatment approaches accompanied by more sophisticated sequencing techniques have paved the way for deeper investigation into the landscape of genomic rearrangements in cancer. While considerable research on solid tumors has focused on point mutations that directly alter the coding sequence of key genes, far less is known about the role of somatic rearrangements. With many recurring alterations observed across tumor types, there is an obvious need for functional characterization of these genomic biomarkers in order to understand their relevance to tumor biology, therapy, and prognosis. As personalized therapy approaches are turning toward genomic alterations for answers, these biomarkers will become increasingly relevant to the practice of precision medicine. This review discusses the emerging role of genomic rearrangements in breast cancer, with a particular focus on fusion genes. In addition, it raises several key questions on the therapeutic value of such rearrangements and provides a framework to evaluate their significance as predictive and prognostic biomarkers.

Fulvestrant treatment alters MDM2 protein turnover and sensitivity of human breast carcinoma cells to chemotherapeutic drugs.

The human homologue of mouse double minute 2 (MDM2) is overexpressed in tumors and contributes to tumorigenesis through inhibition of p53 activity. We investigated the effect of the anti-estrogen fulvestrant on MDM2 expression and sensitivity of estrogen receptor positive human breast cancer cell lines to chemotherapeutics. Fulvestrant down-regulated MDM2 through increased protein turnover. Fulvestrant blocked estrogen-dependent up-regulation of MDM2 and decreased basal expression of MDM2 in the absence of estradiol. As combinations of fulvestrant with doxorubicin, etoposide or paclitaxel were synergistic, altering cell cycle distribution and increasing cell death, this provides rationale for testing combinatorial chemotherapy with fulvestrant as a novel therapeutic strategy for patients with advanced breast cancer.

Inhibitory effects of different forms of tocopherols, tocopherol phosphates, and tocopherol quinones on growth of colon cancer cells.

Tocopherols are the major source of dietary vitamin E. In this study, the growth inhibitory effects of different forms of tocopherols (T), tocopheryl phosphates (TP), and tocopherol quinones (TQ) on human colon cancer HCT116 and HT29 cells were investigated. δ-T was more active than γ-T in inhibiting colon cancer cell growth, decreasing cancer cell colony formation, and inducing apoptosis; however, α-T was rather ineffective. Similarly, the rate of cellular uptake also followed the ranking order δ-T > γ-T ≫ α-T. TP and TQ generally had higher inhibitory activities than their parent compounds. Interestingly, the γ forms of TP and TQ were more active than the δ forms in inhibiting cancer cell growth, whereas the α forms were the least effective. The potencies of γ-TQ and δ-TQ (showing IC50 values of ∼0.8 and ∼2 µM on HCT116 cells after a 72 h incubation, respectively) were greater than 100-fold and greater than 20-fold higher, respectively, than those of their parent tocopherols. Induction of cancer cell apoptosis by δ-T, γ-TP, and γ-TQ was characterized by the cleavage of caspase 3 and PARP1 and DNA fragmentation. These studies demonstrated the higher growth inhibitory activity of δ-T than γ-T, the even higher activities of the γ forms of TP and TQ, and the ineffectiveness of the α forms of tocopherol and their metabolites against colon cancer cells.

P53 regulates the migration of mesenchymal stromal cells in response to the tumor microenvironment through both CXCL12-dependent and -independent mechanisms.

Mesenchymal stromal cells (MSCs) are multipotent fibroblast-like cells located in the bone marrow that localize to areas of tissue damage including wounds and solid tumors. Within the tumor microenvironment, MSCs adopt the phenotype of carcinoma-associated fibroblasts (CAFs) and stimulate tumor growth. Production of the chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF-1), by MSCs is required for their in vitro migration in response to tumor cells and has also been implicated in stimulation of tumor growth. The tumor suppressor p53 regulates cellular migration, CXCL12 production and the promotion of tumor growth by carcinoma-associated fibroblasts (CAFs). We investigated the role of p53 in MSC migration to tumors. P53 inhibits the migration of MSCs in response to tumor cells in conjunction with a decrease in CXCL12 transcription. Conversely, decreased p53 activity leads to enhanced MSC migration. Interestingly, increased p53 activity inhibits MSC migration even in the context of high concentrations of exogenous CXCL12. These data show that stromal p53 status impacts the recruitment of MSCs to solid tumors through both regulation of CXCL12 production as well as other mechanisms. Stromal p53 may influence other important aspects of tumor biology such as tumor growth and metastasis through mechanisms distinct from CXCL12.

The metabolic demands of cancer cells are coupled to their size and protein synthesis rates.

BACKGROUND: Although cells require nutrients to proliferate, most nutrient exchange rates of the NCI60 panel of cancer cell lines correlate poorly with their proliferation rate. Here, we provide evidence indicating that this inconsistency is rooted in the variability of cell size. RESULTS: We integrate previously reported data characterizing genome copy number variations, gene expression, protein expression and exchange fluxes with our own measurements of cell size and protein content in the NCI60 panel of cell lines. We show that protein content, DNA content, and protein synthesis per cell are proportional to the cell volume, and that larger cells proliferate slower than smaller cells. We estimate the metabolic fluxes of these cell lines and show that their magnitudes are proportional to their protein synthesis rate and, after correcting for cell volume, to their proliferation rate. At the level of gene expression, we observe that genes expressed at higher levels in smaller cells are enriched for genes involved in cell cycle, while genes expressed at higher levels in large cells are enriched for genes expressed in mesenchymal cells. The latter finding is further corroborated by the induction of those same genes following treatment with TGFß, and the high vimentin but low E-cadherin protein levels in the larger cells. We also find that aromatase inhibitors, statins and mTOR inhibitors preferentially inhibit the in vitro growth of cancer cells with high protein synthesis rates per cell. CONCLUSIONS: The NCI60 cell lines display various metabolic activities, and the type of metabolic activity that they possess correlates with their cell volume and protein content. In addition to cell proliferation, cell volume and/or biomarkers of protein synthesis may predict response to drugs targeting cancer metabolism.

Metabotropic glutamate receptor 1 expression and its polymorphic variants associate with breast cancer phenotypes.

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.