Sonia C. Dolfi

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.

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.

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.

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 diphosphateribose) 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.

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.

Abstract 81: Role of GRM1 in breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Despite epidemiologic studies suggesting a link between melanoma and breast cancer, molecular mechanisms leading to these associations have not been elucidated. Overexpression of metabotropic glutamate receptor 1 (GRM1) has been implicated in melanomagenesis. Its primary action occurs through activation of the MAPK signaling pathway and other pathways through intracellular calcium release. A recent study implicated GRM1 as playing a role in angiogenesis in hormone receptor negative breast cancer. However, the role of GRM1 in hormone receptor positive disease is yet to be described. Given the associations between melanoma and breast cancer, we evaluated three SNPs in GRM1 for genotype associations with breast cancer clinicopathologic variables using DNA isolated from peripheral blood of 1,096 breast cancer patients. Our study found that a SNP in GRM1 resulting in a proline to serine substitution correlates with age at diagnosis of breast cancer and reflects the receptor status of the breast cancer, e.g. for progesterone receptor positive (PR+) disease, proline carriers were diagnosed 8 years earlier than serine carriers. Genotype-specific associations with risk of developing specific molecular subtypes of breast cancer were observed, i.e. serine carriers were twice as likely to develop estrogen receptor positive (ER+) and PR+ disease. Furthermore, carrying the proline isoform also correlated with higher risk of recurrence of breast cancer and shorter time to recurrence. Immunohistochemical evaluation of breast microarrays showed that GRM1 expression is higher in breast cancers as compared to normal tissue and is higher in ER+ as compared to ER- breast cancers. This parallels higher expression observed in ER+ breast cancer cells lines in comparison to ER- lines. In vitro studies with breast cancer cell lines were also performed to evaluate the expression of GRM1 as a function of hormone exposure and cell line receptor status. Treatment of ER+ cell lines with estradiol resulted in increased GRM1, while tamoxifen decreased expression. Gene expression data on ER+ breast cancers treated with tamoxifen were reanalyzed for association between GRM1 expression and recurrence. Analysis of breast cancers treated with tamoxifen from an independent cohort demonstrated that high expressors of GRM1 have a worse distant recurrence free survival. The results of these studies implicate GRM1 in hormone receptor positive breast cancer biology. Additional knowledge regarding the interaction between GRM1 and hormone signaling could have significant implications in breast cancer risk, prevention, and treatment. 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 81. doi:1538-7445.AM2012-81

Abstract 3018: RET rearrangements as promising therapeutic targets in breast cancer

Background: Receptor tyrosine kinase alterations have played a significant role in therapeutic decisions for cancer due to their oncogenic nature and response to targeted small molecule kinase inhibitors. Increased genomic profiling of tumors using hybrid-capture based next-generation sequencing approaches now reveal the presence of previously unknown fusions and alterations involving kinases in a diverse set of cancers. Here we report the presence and therapeutic significance of recurrent and novel fusions involving RET, a known oncogenic tyrosine kinase receptor, in breast cancer. Methods: Comprehensive genomic profiling on formalin-fixed, paraffin embedded patient tumor tissues was performed using FoundationOne platform that covers the entire coding region for 315 cancer-related genes and introns of 28 genes involved in rearrangements at a depth of 500-1000X (Foundation Medicine, MA). Out of 23 rearrangements, two representative RET fusion expression vectors were synthesized and expressed in non-tumorigenic cell lines (breast MCF10A and mouse 3T3 fibroblasts) and were evaluated for RET kinase signaling, drug response, and tumorigenicity. Results: RET gene fusions, the canonical NCOA4-RET and a novel, noncanonical RASGEF1A-RET fusion, were identified in two separate breast cancers and both include exons required to retain the intact kinase domain of Ret. The novel RASGEF1A-RET fusion includes the non-coding region of RASGEF1A potentially resulting in a truncated RET protein using an alternate internal start site in exon 11 of RET. In vitro characterization of both fusions expressed in mouse 3T3 and human MCF10a cell lines revealed constitutive kinase activation and subsequent downstream signaling as evidenced by phosphorylation of Ret, Erk and Akt. This is the first reported noncanonical RET rearrangement resulting in a 5’ truncated but functional RET kinase. Non-tumorigenic cell lines with stable expression of either rearrangement showed transformed phenotypes assessed by changes in morphology, enhanced growth rate, colony forming ability, and tumor formation in mice. RET fusion-transformed cells were exquisitely sensitive to treatment with RET inhibitors when evaluated in both short-term and long-term functional assays. NCOA4-RET was found by CGP in an index case of metastatic ER+/HER2+ breast cancer that had radiographic evidence of disease progression while on trastuzumab, pertuzumab, and anastrazole. Subsequent treatment with cabozantinib plus anastrazole led to a rapid clinical and radiographic response. Conclusion: CGP techniques involving hybrid-capture based approaches can identify previously unreported but recurrent RET gene fusions in breast cancer. Here, we show that RET fusions including both canonical and non-canonical complex rearrangements are functional and may represent promising therapeutic targets in selected breast cancer patients. Citation Format: Bhavna S. Paratala, Jeffrey S. Ross, Casey B. Williams, Whitney Petrosky, Kirstin A. Williams, Jon Chung, Sonia C. Dolfi, Shridar Ganesan, Siraj Ali, Brian Leyland-Jones, Kim M. Hirshfield. RET rearrangements as promising therapeutic targets in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3018. doi:10.1158/1538-7445.AM2017-3018

Abstract 4129: Novel oncogenic BRAF fusions and impact on targeted therapies

BRAF mutations are driver events in a number of cancers including thyroid cancer and melanoma. The most common, BRAF V600E, alters normal BRAF protein activity in the mitogen-activated protein kinase (MAPK) pathway by constitutively activating BRAF and inducing proliferative signaling and tumor growth. Small molecule tyrosine kinase inhibitors targeting tumors with the V600E mutation have been evaluated in clinical trials and are now approved for melanoma. While BRAF missense mutations have been extensively characterized for oncogenic potential and actionability in genomically-guided therapy, BRAF gene fusions have been underappreciated for not only their functional role in cancer but also in differential drug response. More recently, data suggest that alternative approaches may be needed for treatment of patients with BRAF fusion-containing tumors. We have identified two novel BRAF fusions in tumors from patients with papillary thyroid cancer and melanoma. Both fusions result in an in-frame fusion of a novel gene partner at the 5’ end of the fusion, an intact BRAF kinase domain at the 3’ end, and loss of the BRAF auto-inhibitory domain. We hypothesized that these novel BRAF fusions act as oncogenic drivers, and the mechanism of BRAF activation differs from that caused by V600E mutations and may be fusion partner-specific. These fusions have been engineered in the laboratory and tested for tumorigenic potential and functional activity. BRAF fusion expression in non-transformed cells induces colony formation similar to the V600E mutation indicating tumorigenic potential. These BRAF fusions also constitutively activate the MAPK pathway in the absence of stimulation as demonstrated by phosphorylated ERK and MEK proteins. Additionally, BRAF fusion-expressing cells form tumors in vivo similarly to the BRAF V600E-expressing cells. These tumors are highly proliferative as demonstrated by strong Ki67 immunohistochemical staining and display MAPK pathway activation as evidenced by phosphorylated ERK. BRAF fusion-expressing cells have differential sensitivity to MAPK pathway inhibitors compared to cells with the V600E mutation as measured by reduced MAPK signaling. Inhibition of the MAPK pathway is relevant in targeting BRAF fusion-containing cells but may not follow the same paradigm as point mutations. Collectively, our data suggest that BRAF fusions are functional and represent novel therapeutic targets, but may need an alternative approach as compared to tumors with BRAF missense mutations. Citation Format: Sonia C. Dolfi, Ann Silk, Bhavna Paratala, Whitney Petrosky, Srilatha Simhadri, Atul Kulkarni, Shridar Ganesan, Kim M. Hirshfield. Novel oncogenic BRAF fusions and impact on targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4129. doi:10.1158/1538-7445.AM2017-4129

Abstract C151: Contribution of serine, folate, and glycine metabolism to the ATP, NADPH, and purine requirements of cancer cells.

Background: Recent observations on cancer cell metabolism indicate increased serine synthesis from glucose as a marker of poor prognosis. Expression of genes in this pathway also correlate with sensitivity to the antifolate methotrexate (Vazquez A, et al, Cancer Res. 2013 Jan 15;73(2):478-82). Using a large-scale model of human cell metabolism we have predicted that serine synthesis can be routed to a pathway for ATP production. The pathway is composed by reactions from the serine synthesis, one carbon (folate) metabolism and the glycine cleavage system (SOG pathway) and its flux is predicted to increase at high proliferation rates. Results: Here we show that the SOG pathway is upregulated at the level of gene expression in a subset of human tumors and its level of expression correlates with gene signatures of cell proliferation and Myc targets activation. To investigate the activity of the SOG pathway at the level of metabolic fluxes we estimated the metabolic fluxes of the NCI60 panel of tumor derived cell lines, using previously reported exchange fluxes and a flux balance model of cell metabolism. We show that the estimated rates of reactions in the SOG pathway are highly correlated with the proliferation rates of these cell lines. We also find that the SOG pathway contributes significantly to the energy requirements of biosynthesis, the NADPH requirements of fatty acid synthesis and to the synthesis of purines. Finally, when the PC-3 prostate cancer cell line is subject to treatment with the antifolate methotrexate, we observe a decrease in the ATP levels, an inhibition of the proliferation rate and a decrease in the ribonucleotides and fatty acids synthesized from glucose. Conclusions: Taken together our results indicate that the SOG pathway activity increases with the rate of cell proliferation and it contributes to the biosynthetic requirements of purines, ATP and NADPH. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C151. Citation Format: Philip M. Tedeschi, Elke K. Markert, Murugesan Gounder, HongXia Lin, Sonia C. Dolfi, Leo Li-Ying Chan, Jean Qiu, Kim M. Hirshfield, Laszlo G. Boros, Joseph R. Bertino, Zoltan N. Oltvai, Alexei Vazquez. Contribution of serine, folate, and glycine metabolism to the ATP, NADPH, and purine requirements of cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C151.

Abstract 681: Prognostic role of Perp in breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Perp, a tetraspan plasma membrane protein, is activated by p53 during apoptosis and is also regulated by p63 where it functions as an essential component of the desmosome and contributes to epithelial integrity. A single nucleotide polymorphism (SNP) in Perp was identified by its effect on apoptotic efficiency in irradiated lymphoblastoid cell lines. Therefore, this SNP may have functional significance in clinical outcomes of cancer. In this study, this SNP in PERP was evaluated as a prognostic marker and in its relationship to other known prognostic factors. Genotyping was performed on DNA isolated from peripheral blood of 1020 patients with breast cancer and genotypes were linked with annotated clinical information. Analysis of recurrence free survival was performed on a subset limited to 790 Caucasians. Among Caucasian cases, homozygous SNP genotypes were nearly equally distributed (AA: 25% and GG: 26%). Using a COX proportional hazards survival analysis, the AA genotype was found to be an independent predictor of worse recurrence-free survival (RFS, hazard ratio [HR]: 1.95, 95% CI: 1.38-2.76, p=0.0001). RFS was similar between AG and GG carriers and was more favorable as compared to AA carriers. For patients receiving breast-conserving surgery followed by radiation, patients diagnosed at or above 51 years of age or patients with stage III disease, the deleterious effect of AA genotype on RFS was more significant. In addition, the AA genotype associated with decreased RFS in subgroups of Caucasian cases regardless of stratification by hormone receptor status, HER2 status, and whether they received chemotherapy or hormonal therapy. Adjusting for effects due to stage and chemotherapy, this association remained significant in estrogen receptor positive and progesterone receptor negative cases. We also analyzed the NCI-60 cell line panel for an association between this polymorphism and response to 132 standard chemotherapeutic drugs. A significant association was observed with respect to genotype and response to anti-mitotic agents. Collectively, cell lines with the AA genotype were more sensitive to anti-mitotic agents than the GG genotype. Clinical data suggest that the AA genotype of Perp has a functional phenotype leading to increased risk of recurrence. Further studies are needed to determine the mechanism behind this association, but ultimately this SNP may be useful as a prognostic marker in breast cancer and could be used as a tool to optimize treatment strategies. 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 681. doi:1538-7445.AM2012-681