Roemeling, Christina A. Von

Abstract Introduction: A significant number of histologically triple negative breast cancer (TNBC) cells express substantial estrogen receptor (ER) mRNA but do not express ER protein as a result of rapid proteolytic degradation. Here we assessed whether proteasome inhibitors could sensitize such cells to tamoxifen-induced growth suppression. Methods: Data from the Katzenellenbogen laboratory elegantly demonstrate that in high ER mRNA MDA-MB-468 cells, ER protein is rapidly degraded via proteosomal degradation (Bhatt et al. MCB, 2012).Utilizing established human TNBC cell lines that express high ER mRNA (BT-20, MDA-MB-468, and newly established human TNBC cell lines from our laboratory MCJB2 and MCJB11), we defined a therapeutic strategy using the proteasome inhibitor carfilzomib in combination with the antiestrogen tamoxifen that demonstrates antitumor synergy. Results: We found that administration of carfilzomib in TNBC cell lines with high ER mRNA restored ER protein expression, and that combining this treatment with tamoxifen treatment led to enhanced growth suppression. We found that the TNBC MDA-MB-231 cells, which express no ER mRNA, show some growth suppression, but that tamoxifen provided no further growth suppression with these cells. We have previously shown that combined epigenetic therapy [methyltransferase inhibitor (decitabine) plus a histone deacetylase inhibitor (HDACi; romidepsin)] provided antitumor synergy in MDA-MB-231 cells via upregulation of the soluble Wnt suppressor, secreted frizzled protein 1 (sFRP1; Cooper et al. CCR, 2012). In addition, we show that this epigenetic therapy restores silenced ER mRNA and protein levels in MDA-MB-231 cells, and that combination of this epigenetic therapy with tamoxifen in MDA-MB-231 cells led to enhanced growth suppression by tamoxifen. We are currently testing these therapeutic strategies in vivo using established TNBC cell lines and our newly developed patient derived xenograft (PDX) mouse models that express either ERhigh or ERlow mRNA. Conclusions: If validated in animal models, we propose that ER mRNA expression level in TNBC patients could guide therapeutic choice, whereby, ERlow mRNA TNBC tumors could respond to epigenetic therapy followed by antiestrogen while ERhigh mRNA TNBC tumors could respond to a proteosome inhibitor followed by antiestrogen therapy. These results identify a novel epigenetic biomarker as predictive of therapeutic response. Citation Format: Christina A. von Roemeling, Laura A. Marlow, James Miller, Adam Mathias, Louis Dawson, Steadman Harrison, Beth A. Hollister, Derek C. Radisky, John Alton Copland. Estrogen receptor mRNA-directed therapy for triple-negative breast cancer. [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 LB-95. doi:10.1158/1538-7445.AM2014-LB-95

Abstract Introduction: Renal cell carcinoma (RCC) is the most common malignancy of the kidney, responsible for 13,000 deaths in the United States annually. Patients who present with the clear cell (ccRCC) variant of this disease demonstrate a worse prognosis as compared to other subtypes. Due to its asymptomatic nature, patients often do not demonstrate signs of illness until the disease has progressed to an advanced stage. It is estimated that 30% of patients present with metastatic disease at initial diagnosis, and 20-30% of patients with localized cases treated surgically relapse with metastatic disease likely due to the presence of undetectable micrometastases. The treatment for metastatic disease is limited due to its profound resistance against chemotherapy, radiation, and current FDA approved targeted therapies. Despite advances in treatment strategies for patients with metastatic RCC, there remains a need for identification of tumor-specific therapeutic targets. Additionally, elucidation of key signatures which contribute to the development of metastatic disease is paramount. Our group has identified secreted neuronal pentraxin 2 (NPTX2) as a novel oncogenic factor that is highly overexpressed specifically in ccRCC at all stages of disease, and facilitates tumor cell invasion through its interaction with the ionotropic glutamate receptor 4 subunit (GluR4). Methods: NPTX2 expression was evaluated by Affymetrix gene array and immunohistochemistry analysis of patient tumor tissue compared to matched normal samples. Nextbio meta-analysis was used to evaluate NPTX2 expression patterns among RCC subtypes and disease staging. Lentiviral mediated knock-down of NPTX2 and GluR4 was performed in representative ccRCC cell lines and resulting changes in proliferation, viability, morphology, and invasion were evaluated. NPTX2-GluR4 interactions were evaluated via immunoprecipitation, immunofluorescence, and intracellular calcium assays. Results: NPTX2 mRNA and protein is consistently elevated in the clear cell variant of RCC at all stages of disease, and is important for tumor cell viability. We correlate NPTX2 expression with an invasive phenotype, and demonstrate that over-expression of NPTX2 in tumor cells leads to actin cytoskeletal remodeling and increased invasion. We identify GluR4 as a mediator for NPTX2 activity which leads to increased intracellular calcium influx in tumor cells. Conclusions: NPTX2 is a tumor-specific factor that is consistently over-expressed in ccRCC. We further identify GluR4 as a downstream mediator of NPTX2 activity, whereby it modulates intracellular calcium levels, actin cytoskeletal remodeling, and tumor cell invasion. Inhibition of NPTX2 or GluR4 expression via shRNA leads to decreased tumor cell proliferation and invasion, and induces programed cell death. We propose NPTX2 as a new candidate for targeted therapy not previously described in cancer, whose inhibition may demonstrate a clinical benefit in patients suffering from metastatic ccRCC. Citation Format: Christina A. von Roemeling, Derek C. Radisky, Laura A. Marlow, Simon J. Cooper, Stefan K. Grebe, Panagiotis Z. Anastasiadis, Han W. Tun, John A. Copland. Neuronal Pentraxin 2: a novel tumor-specific molecular target that mediates clear cell renal cell carcinoma malignancy. [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 LB-111. doi:10.1158/1538-7445.AM2014-LB-111

Abstract Introduction: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC, which is the most prevalent kidney cancer among adults. Of the patients who develop this disease, over 1/3 will already present with advanced or metastatic disease during initial diagnosis due to the lack of symptoms in early stages. RCC demonstrates considerable resistance to radiation and traditional chemotherapeutics. Despite advances in the development of targeted anti-cancer treatments, no current drug therapy leads to significant improvement in long term survival in patients with advanced disease with the rare exception of those who respond to immunotherapy. Additionally, patients inevitably develop drug resistance. Therefore new molecular targets for therapy are sorely needed. Our group has identified stearoyl coA desaturase (SCD1) as a novel molecular target in ccRCC. We examined its role in tumor cell growth and viability in vitro and in vivo independently as well as in combination with current FDA approved regimens. Methods: Patient normal and ccRCC tissue samples and cell lines were examined for SCD1 expression. Genetic knockdown models and targeted inhibition of SCD1 through use of a small molecule inhibitor were analyzed for growth, apoptosis, and alterations in gene expression using gene array analysis. Therapeutic models of synergy were evaluated utilizing pharmacologic inhibition of SCD1 with the tyrosine kinase inhibitors (TKI) sunitinib and pazopanib, and the mTOR inhibitor temsirolimus. Results: Our studies identify increased SCD1 expression in all stages of ccRCC. Both genetic knockdown and pharmacologic inhibition of SCD1 decrease tumor cell proliferation and induce apoptosis in vitro and in vivo. Upon gene array, quantitative real-time PCR, and protein analysis of SCDi treated or SCD1 lentiviral knockdown samples, induction of endoplasmic reticulum (ER) stress response signaling was observed, providing mechanistic insight for SCD1 activity in ccRCC. Furthermore, combinatorial application of SCDi with temsirolimus synergistically inhibits tumor growth in vitro and in vivo. Conclusions: Increased SCD1 expression supports ccRCC viability and therefore we propose it as a novel molecular target for therapy either independently or in combination with an mTOR inhibitor for patients whose disease cannot be remedied with surgical intervention, such as in cases of advanced or metastatic disease. These findings support the further evaluation of targeting SCD1 as an anti-cancer strategy in a phase I clinical trial. Citation Format: Christina A. von Roemeling, Laura Marlow, Thomas Caulfield, Kevin Wu, Winston Tan, Han Tun, John A. Copland, Adam Mathias, Chuck Harrison, Louis Dawson, Beth Hollister. Stearoyl-CoA desaturase 1 is a novel molecular therapeutic target for clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-210. doi:10.1158/1538-7445.AM2013-LB-210

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and has the highest propensity to manifest as metastatic disease. Recent characterizations of the genetic signature of ccRCC have revealed several factors correlated with tumor cell migration and invasion; however, the specific events driving malignancy are not well defined. Furthermore, there remains a lack of targeted therapies that result in long-term, sustainable response in patients with metastatic disease. We show here that neuronal pentraxin 2 (NPTX2) is overexpressed specifically in ccRCC primary tumors and metastases, and that it contributes to tumor cell viability and promotes cell migration through its interaction with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR4. We propose NPTX2 as a novel molecular target for therapy for patients with ccRCC diagnosed with or at risk of developing metastatic disease. Cancer Res; 74(17); 4796–810. ©2014 AACR.

Abstract Purpose: We set out to identify Stearoyl-CoA desaturase 1 (SCD1) as a novel molecular target in clear cell renal cell carcinoma (ccRCC) and examine its role in tumor cell growth and viability in vitro and in vivo independently as well as in combination with current U.S. Food and Drug Administration (FDA)-approved regimens. Experimental Design: Patient normal and ccRCC tissue samples and cell lines were examined for SCD1 expression. Genetic knockdown models and targeted inhibition of SCD1 through use of a small molecule inhibitor, A939572, were analyzed for growth, apoptosis, and alterations in gene expression using gene array analysis. Therapeutic models of synergy were evaluated utilizing pharmacologic inhibition of SCD1 with the tyrosine kinase inhibitors (TKI) sunitinib and pazopanib, and the mTOR inhibitor temsirolimus. Results: Our studies identify increased SCD1 expression in all stages of ccRCC. Both genetic knockdown and pharmacologic inhibition of SCD1 decreased tumor cell proliferation and induced apoptosis in vitro and in vivo. Upon gene array, quantitative real-time PCR, and protein analysis of A939572-treated or SCD1 lentiviral knockdown samples, induction of endoplasmic reticulum stress response signaling was observed, providing mechanistic insight for SCD1 activity in ccRCC. Furthermore, combinatorial application of A939572 with temsirolimus synergistically inhibited tumor growth in vitro and in vivo. Conclusions: Increased SCD1 expression supports ccRCC viability and therefore we propose it as a novel molecular target for therapy either independently or in combination with an mTOR inhibitor for patients whose disease cannot be remedied with surgical intervention, such as in cases of advanced or metastatic disease. Clin Cancer Res; 19(9); 2368–80. ©2013 AACR.

Abstract Metastatic solid tumors are aggressive and mostly drug resistant, leading to few treatment options and poor prognosis as seen with clear cell renal cell carcinoma (ccRCC) and triple-negative breast cancer (TNBC). Therefore, the identification of new therapeutic regimes for the treatment of metastatic disease is desirable. ccRCC and TNBC cell lines were treated with the HDAC inhibitor romidepsin and the methyltransferase inhibitor decitabine, two epigenetic modifying drugs approved by the U.S. Food and Drug Administration for the treatment of various hematologic malignancies. Cell proliferation analysis, flow cytometry, quantitative PCR, and immunoblotting techniques were used to evaluate the antitumor synergy of this drug combination and identify the reexpression of epigenetically silenced tumor suppressor genes. Combinatorial treatment of metastatic TNBC and stage IV ccRCC cell lines with romidepsin/decitabine leads to synergistic inhibition of cell growth and induction of apoptosis above levels of individual drug treatments alone. Synergistic reexpression of the tumor suppressor gene secreted frizzled-related protein one (sFRP1) was observed in combinatorial drug-treated groups. Silencing sFRP1 (short hairpin RNA) before combinatorial drug treatment showed that sFRP1 mediates the growth inhibitory and apoptotic activity of combined romidepsin/decitabine. Furthermore, addition of recombinant sFRP1 to ccRCC or TNBC cells inhibits cell growth in a dose-dependent manner through the induction of apoptosis, identifying that epigenetic silencing of sFRP1 contributes to renal and breast cancer cell survival. Combinatorial treatment with romidepsin and decitabine in drug resistant tumors is a promising treatment strategy. Moreover, recombinant sFRP1 may be a novel therapeutic strategy for cancers with suppressed sFRP1 expression. Mol Cancer Ther; 11(10); 2105–15. ©2012 AACR.

Abstract The Forkhead transcription factor, FoxO3a, is a known suppressor of primary tumor growth and is negatively regulated by growth factors via phosphorylation and mislocalization from the nucleus to the cytoplasm. Here we show for the first time that FoxO3a is nuclear in anaplastic thyroid carcinoma (ATC) and it transcriptionally upregulates cyclin A thereby promoting tumor growth in human ATC cells. Silencing FoxO3a in ATC cell lines leads to down-regulation of cyclin A mRNA and protein while over-expression of FoxO3a leads to upregulation of cyclin A protein. Transient co-transfection of cyclin A promoter linked to luciferase with FoxO3a leads to enhanced luciferase activity, which can be ablated when mutating the FoxO3a site on the cyclin A promoter. This combined data implicates an entirely novel function for FoxO3a in modulating cancer progression by promoting cell cycle progression and tumor growth. ATC is a poorly differentiated carcinoma with average survival upon diagnosis of 3–4 months with 99% lethality, notably the most aggressive and deadliest cancers known. This data also suggest that any therapies under development to inactivate FOXO3a may be effective at blocking tumor expansion. 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 1038. doi:10.1158/1538-7445.AM2011-1038

Abstract Renal cell carcinoma (RCC) accounts for 3% of all malignancies reported each year making it the sixth leading cause of cancer death in the United States. Within this year more than 50,000 individuals will be diagnosed with RCC and around 12,000 will die from this disease. Out of these RCCs the most frequently diagnosed form is clear cell renal cell carcinoma (ccRCC) which accounts for approximately 80% of all RCCs. We have previously identified through genomic profiling that loss of the type II and III TGFβ receptors in the progression of this disease lead to loss of TGFβ responsiveness and suggests a role for dysregulated TGFβ signaling in RCC progression and metastasis. Restoration of the TGFβ receptor type III (TβRIII) leads to apoptosis in RCC cell lines (Margulis et al. 2007) suggesting that re-expression of TβRIII is an attractive method for intervention of ccRCC. Recently we identified that loss of TβRIII is an early event in ccRCC and that this loss is not through epigenetic silencing but due to the loss of transcriptional regulation. The transcription factor GATA3 was identified to be a positive regulator of TβRIII expression through its actions on the proximal promoter of TβRIII. Through deletion analysis we have now identified a region within the proximal promoter that contains a number of putative transcription factor binding sites that are capable of negatively regulating the expression of TβRIII. We have identified that one of these transcription factors is over-expressed in ccRCC patient matched renal tissue samples and that silencing of this transcription factor through lentiviral shRNA, leads to a three fold induction of endogenous TβRIII in ccRCC cell lines. Furthermore, loss of this transcription factor in ccRCC cell lines leads to decreased cell proliferation when compared to non-target infected controls. Further evaluation of these putative negatively regulating transcription factors and their actions upon the proximal promoter of TβRIII are being analyzed utilizing EMSA and ChIP assays. These investigations will identify novel molecular targets that through their dysregulation will have the potential to up-regulate TβRIII in ccRCC inducing apoptosis within these cells. Together these findings will allow for a deeper molecular understanding of the events taking place in ccRCC. 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 2173. doi:10.1158/1538-7445.AM2011-2173

Abstract Introduction: Despite significant progress over the last decade in the development of therapeutics for metastatic cancers including breast cancer (MBC), non small cell lung cancer (NSCLC), and renal cell carcinoma (RCC), most patients will ultimately succumb to their malignancy; more effective therapeutics are sorely needed. The microtubule (MT) is a well-validated target for anti-cancer therapeutics. Ixabepilone (Ixa), a MT stabilizer is approved for MBC and has demonstrated efficacy against other solid tumors. mTOR is another validated target for anti-cancer therapeutics. Temsirolimus (Tem) inhibits mTOR and is approved for advanced RCC. We tested the hypothesis that the addition of Ixa to Tem might augment its anti-tumor activity in vitro. Methods: The cell lines, A498 (RCC), MDA-MB-231 (MBC), and A549 (NSCLC) were treated with Tem alone, Ixa alone, or the combination. All cell lines were also treated with the combination of Tem and Ixa in a fixed ratio of 1:20; Tem doses ranged from 0.125 [nM] to 5 [nM] and Ixa doses ranged from 2.5 [nM] to 100 [nM]. The effects of the treatments were evaluated with clonogenic assays. Synergism was evaluated using CalucSyn 2.0 in accord with the isobologram method of Chou and Talalay. Results: Both Tem and Ixa inhibited cell growth in all tested cell lines as single agents in dose-dependent fashions. Moreover, the combination of Tem and Ixa synergistically enhanced cytotoxicity in all tested lines (see table below).Cell LineED50 [nM]CI (lowest value)A4980.449280.404A5490.285440.263MDA-MB-2310.483550.373 Conclusion: The combination of Ixa and Tem displayed promising activity in all tested cell lines. These findings support the further evaluation of this combination in a phase I clinical trial. 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 4480.