Anastasiadis, Panagiotis Z.

Abstract Despite a good response rate to front-line treatment, most of the patients with epithelial ovarian cancer (EOC) will eventually develop an incurable state of platinum (Pt)-resistant disease. Pt resistance is a multifactorial event driven by different molecular mechanisms such as increased ability to tolerate or repair DNA lesions, tendency to plasticity with acquisition of mesenchymal phenotype, and ability to modify/reprogram cellular metabolism. We recently identified in EOC relevant alterations in all of these factors, largely contributing to EOC aggressiveness. Focusing our attention on microRNAs (miRNAs), the master regulator of gene expression, we identified a cluster of miRNAs located on ChrXq27.3 (miR-506 family) whose downregulation is associated with advanced-stage EOC early relapse, and we found it to be the main contributor of a 35-miRNA based predictor (MiROvaR) of EOC risk of progression/relapse. We also contributed in defining altered choline metabolism in EOC that, through overexpression of choline kinase alpha (ChoK/CHKA) enzyme whose activity sustains tumor addiction to antioxidants, fosters EOC aggressiveness also by increasing chemoresistance. By in vitro and ex vivo studies performed in human EOC cell lines, non-tumoral immortalized ovarian cells and ex vivo EOC patient-derived ascitic tumor cells (PDAT), here we analyzed the role of miR-506 in regulating molecular network associated with drug resistance and evaluated the druggability of the cholinic phenotype, exploring its possible crosstalk with the immune system. We confirmed miR-506 to directly regulate the predicted target RAD17, a gene with a key role in guiding BRCA1 to DNA damage sites and stabilizing active signaling. Since an inactivation of DNA repair genes (BRCA1/2) is associated with better prognosis due to sensitization to DNA-damaging drugs, we investigated whether miR-506 can regulate DNA repair machinery by phenocopying/supporting the effects of BRCA1/2 mutation on Pt and PARP inhibitor sensitivity. We observed that miR-506 forced expression or RAD17 knockdown sensitized EOC cells to Pt and PARP inhibitors and, interestingly, showed a synthetic lethal effect with inhibitors of cell-cycle checkpoint kinases CHK1 and WEE1. Association of miR-506 cluster expression (RT-qPCR) with the BRCAness-phenotype is currently ongoing on a retrospective FFPE case material. We also recently link EOC cells plasticity, miRNAs expression, and Pt-sensitivity. Indeed, E-cadherin can exert a new and unexpected functional role in EOC cells by contributing to Pt susceptibility. We determined that overexpression of PLEKHA7 (Pleckstrin homology domain-containing family A member 7), a component of the zonula adherens in polarized epithelia and expressed at low levels in EOC biopsies, inhibits cell tumorigenicity and sensitizes EOC cells to Pt treatment. We will evaluate PLEKHA7 ability to regulate miRNAs driving tumor progression, including some of the miR-506 family. Concerning chemoresistance associated with metabolic rewiring, we showed that CHKA silencing altered the cellular oxidative stress redox status, decreasing glutathione content and raising reactive oxygen species (ROS) levels. Interestingly, the increase in intracellular ROS species improved EOC cells’ sensitivity to Pt/taxane treatments, contributing to cell death triggering. Moreover, upon CHKA silencing both in vitro and in PDAT cells, we observed an enhanced sensitization of cancer cells to TRAIL-mediated citotoxicity due to an augmented expression of pro-apoptotic/reduced expression of anti-apoptotic TRAIL receptor members, thus further increasing tumor cells’ weakness. Exploiting tumor’s reliance on these altered molecular and metabolic pathways is expected to be an appropriate strategy to improve efficacy of current treatment, possibly circumventing drug resistance. Funded by CARIPLO Foundation, AIRC and 5x1000 Institutional Research Funding. Citation Format: Marina Bagnoli, Roberta Nicoletti, Andrea Rizzo, Francesca Roggiani, Alessandro Satta, Mariangela Figini, Wei Zhang, Panagiotis Z. Anastasiadis, Egidio Iorio, Silvana Canevari, Antonella Tomassetti, Francesco Raspagliesi, Delia Mezzanzanica. An integrated molecular and metabolic approach for harnessing early relapse and chemoresistance in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A39.

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 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.