Pancreatic cancer has the highest mortality rates of all cancer types. One potential explanation for the aggressiveness of this disease is that cancer cells have been found to communicate with one another using membrane-bound vesicles known as exosomes. These exosomes carry pro-survival molecules and increase the proliferation, survival, and metastatic potential of recipient cells, suggesting that tumor-derived exosomes are powerful drivers of tumor progression. Thus, to successfully address and eradicate pancreatic cancer, it is imperative to develop therapeutic strategies that neutralize cancer cells and exosomes simultaneously. Curcumin, a turmeric root derivative, has been shown to have potent anti-cancer and anti-inflammatory effects in vitro and in vivo. Recent studies have suggested that exosomal curcumin exerts anti-inflammatory properties on recipient cells. However, curcumin’s effects on exosomal pro-tumor function have yet to be determined. We hypothesize that curcumin will alter the pro-survival role of exosomes from pancreatic cancer cells toward a pro-death role, resulting in reduced cell viability of recipient pancreatic cancer cells. The main objective of this study was to determine the functional alterations of exosomes released by pancreatic cancer cells exposed to curcumin compared to exosomes from untreated pancreatic cancer cells. We demonstrate, using an in vitro cell culture model involving pancreatic adenocarcinoma cell lines PANC-1 and MIA PaCa-2, that curcumin is incorporated into exosomes isolated from curcumin-treated pancreatic cancer cells as observed by spectral studies and fluorescence microscopy. Furthermore, curcumin is delivered to recipient pancreatic cancer cells via exosomes, promoting cytotoxicity as demonstrated by Hoffman modulation contrast microscopy as well as AlamarBlue and Trypan blue exclusion assays. Collectively, these data suggest that the efficacy of curcumin may be enhanced in pancreatic cancer cells through exosomal facilitation.

Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-β-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and β-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance.

Prostate cancer (PCa) remains the most frequently diagnosed male malignancy in Western countries and the second most common cause of male cancer death in the United States. The relatively elevated PCa incidence and mortality among African American men makes this cancer type a challenging health disparity disease. To increase the chance for successful trea tment, earlier detection and prediction of tumor aggress iveness will be important and need to be resolved. This study demonstrates that small membrane-bound vesicles shed from the tumor called exosomes contain ethnically and tumor-specific biomarkers, and could be exploited for their diagnostic and therapeutic potential.

BackgroundCurrent therapeutic options for advanced pancreatic cancer have been largely disappointing with modest results at best, and though adjuvant therapy remains controversial, most remain in agreement that Gemcitabine should stand as part of any combination study. The inhibitor of apoptosis (IAP) protein Survivin is a key factor in maintaining apoptosis resistance, and its dominant-negative mutant (Survivin-T34A) has been shown to block Survivin, inducing caspase activation and apoptosis.MethodsIn this study, exosomes, collected from a melanoma cell line built to harbor a tetracycline-regulated Survivin-T34A, were plated on the pancreatic adenocarcinoma (MIA PaCa-2) cell line. Evaluation of the presence of Survivin-T34A in these exosomes followed by their ability to induce Gemcitabine-potentiative cell killing was the objective of this work.ResultsHere we show that exosomes collected in the absence of tetracycline (tet-off) from the engineered melanoma cell do contain Survivin-T34A and when used alone or in combination with Gemcitabine, induced a significant increase in apoptotic cell death when compared to Gemcitabine alone on a variety of pancreatic cancer cell lines.ConclusionThis exosomes/Survivin-T34A study shows that a new delivery method for anticancer proteins within the cancer microenvironment may prove useful in targeting cancers of the pancreas.

Exosomes are endosomal-derived nanovesicles released by normal and tumor cells which have been shown to transfer functionally active protein, lipids, mRNAs and miRNAs between cells. Varying in molecular profiles, biological roles, functional roles and protein contents, exosomes have been described as Bmulti-purpose carriers^playing a role in supporting the survival and growth of tumor cells. The IAP Survivin has been found to be present in tumor exosomes. However, the existence of other IAPs in tumor exosomes is still unknown. Survivin, cIAP1, cIAP2 and XIAP mRNA and protein are differently expressed in a panel of tumor cell lines: DLCL2, HeLa, MCF-7, Panc-1, and PC3. Exosomes were isolated from conditioned media collected from the cells from which RNA and protein were extracted. Our results provide evidence that like Survivin, XIAP, cIAP1 and cIAP2 proteins are found in tumor exosomes. The mRNA expression, however, is differentially expressed across the tumor cell lines. The presence of these bioactive molecules in exosomes may not only serve as warning signals, but also play a role in providing protection to the cancer cells against changes that are constantly occurring in the tumor microenvironment.

Ovarian cancer is the fifth-leading cause of cancer death among women. The dissemination of ovarian tumors and growth as spheroids accompanies late stage disease. In cell culture, ovarian tumor cell spheroids can exhibit elevated resistance to environmental stressors such as reactive oxygen species. Homeostatic balance of the antioxidant response is a protective mechanism that prevents anoikis, a form of programmed cell death. Signaling pathways activated by integrin receptors suppress anoikis. Rgnef (ARHGEF28/p190RhoGEF) is a guanine nucleotide exchange factor that is activated downstream of integrins. We find that Rgnef protein levels are elevated in late-stage serous ovarian cancer, high Rgnef mRNA levels are associated with decreased progression-free and overall survival, and genomic ARHGEF28 loss is associated with increased patient survival. Using transgenic and transplantable Rgnef knockout mouse models, we find that Rgnef is essential for supporting three-dimensional ovarian spheroid formation in vitro and tumor growth in mice. Using RNA-sequencing and bioinformatic analyses, we identify a conserved Rgnef-supported anti-oxidant gene signature including GPX4, Nqo1, and Gsta4; common targets of the NF-kB transcription factor. Antioxidant treatment enhanced growth of Rgnef-knockout spheroids and Rgnef re-expression facilitated NF-κB-dependent tumorsphere survival. These studies reveal a new role for Rgnef in ovarian cancer to facilitate NF-κB-mediated gene expression protecting cells from oxidative stress.

Objectives The inhibitor of apoptosis (IAP) proteins are critical modulators of chemotherapeutic resistance in various cancers. To address the alarming emergence of chemotherapeutic resistance in pancreatic cancer, we investigated the efficacy of the turmeric derivative curcumin in reducing IAP protein and mRNA expression resulting in pancreatic cancer cell death. Methods The pancreatic adenocarcinoma cell line PANC-1 was used to assess curcumin’s effects in pancreatic cancer. Curcumin uptake was measured by spectral analysis and fluorescence microscopy. AlamarBlue and Trypan blue exclusion assays were used to determine PANC-1 cell viability following curcumin treatment. Visualization of PANC-1 cell death was performed using Hoffman Modulation Contrast microscopy. Western blot and PCR analyses were used to evaluate curcumin’s effects on IAP protein and mRNA expression. Results Curcumin enters PANC-1 cells and is ubiquitously present within the cell following treatment. Furthermore, curcumin reduces cell viability and induces morphological changes characteristic of cell death. Additionally, curcumin decreases IAP protein and mRNA expression in PANC-1 cells. Conclusions These data demonstrate that PANC-1 cells are sensitive to curcumin treatment. Furthermore, curcumin as a potential therapeutic tool for overcoming chemotherapeutic resistance mediated by IAPs, supports a role for curcumin as part of the therapeutic approach for pancreatic cancer.

= 175 words 7 Figures and 2 Tables AbstractGene copy number changes, cancer stem cell (CSC) increases, and platinum chemotherapy resistance contribute to poor prognosis in patients with recurrent high grade serous ovarian cancer (HGSOC). CSC phenotypes involving Wnt-b-catenin and aldehyde dehydrogenase activities, platinum resistance, and tumor initiating frequency are here associated with spontaneous genetic gains, including genes encoding KRAS, MYC and FAK, in a new murine model of ovarian cancer (KMF). Noncanonical FAK signaling was sufficient to sustain human and KMF tumorsphere proliferation, CSC survival, and platinum resistance. Increased FAK tyrosine phosphorylation occurred in HGSOC patient tumors surviving neoadjuvant platinum and paclitaxel chemotherapy and platinum resistant tumorspheres acquired FAK dependence for growth. Importantly, combining a pharmacologic FAK inhibitor with platinum overcame chemoresistance and triggered apoptosis in vitro and in vivo. Knockout, rescue, genomic and transcriptomic analyses collectively identified more than 400 genes regulated along a FAK/b-catenin/Myc axis impacting stemness and DNA repair in HGSOC, with 66 genes gained in a majority of Cancer Genome Atlas samples. Together, these results support combinatorial testing of FAK inhibitors for the treatment of recurrent ovarian cancer. Running title (40 characters including spaces): FAK dependent signaling in ovarian cancerKeywords (5): b-catenin, FAK, Myc, ovarian cancer, platinum chemotherapy 3 Graphical Summary Key Points• High grade serous ovarian carcinoma tumors contain PTK2 (FAK) 8q24.3 gains associated with prognostic differences.• KMF, a new murine ovarian cancer model with K-Ras, Myc, and FAK gene gains and intrinsic platinum resistance.• FAK activation in tumors surviving platinum chemotherapy promotes cancer stem cell survival.• FAK facilitates a b-catenin-Myc signaling axis controlling gene expression supporting platinum resistance.• FAK activity is essential for KMF tumor growth and is a targetable cellular adaptation of platinum resistance. We thank our colleagues for helpful discussion and comments. We thank R. Winters (FCCC BRF manager) and D. Flieder (FCCC Pathology) for identifying, reviewing the cases, and for procuring patient tumor samples used in this study.