Malyn May Asuncion Valenzuela

Plasma-Derived Exosomal Survivin, a Plausible Biomarker for Early Detection of Prostate Cancer

Background Survivin is expressed in prostate cancer (PCa), and its downregulation sensitizes PCa cells to chemotherapeutic agents in vitro and in vivo. Small membrane-bound vesicles called exosomes, secreted from the endosomal membrane compartment, contain RNA and protein that they readily transport via exosome internalization into recipient cells. Recent progress has shown that tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions via immune escape, tumor invasion and angiogenesis. Furthermore, exosome analysis may provide novel biomarkers to diagnose or monitor PCa treatment. Methods Exosomes were purified from the plasma and serum from 39 PCa patients, 20 BPH patients, 8 prostate cancer recurrent and 16 healthy controls using ultracentrifugation and their quantities and qualities were quantified and visualized from both the plasma and the purified exosomes using ELISA and Western blotting, respectively. Results Survivin was significantly increased in the tumor-derived samples, compared to those from BPH and controls with virtually no difference in the quantity of Survivin detected in exosomes collected from newly diagnosed patients exhibiting low (six) or high (nine) Gleason scores. Exosome Survivin levels were also higher in patients that had relapsed on chemotherapy compared to controls. Conclusions These studies demonstrate that Survivin exists in plasma exosomes from both normal, BPH and PCa subjects. The relative amounts of exosomal Survivin in PCa plasma was significantly higher than in those with pre-inflammatory BPH and control plasma. This differential expression of exosomal Survivin was seen with both newly diagnosed and advanced PCa subjects with high or low-grade cancers. Analysis of plasma exosomal Survivin levels may offer a convenient tool for diagnosing or monitoring PCa and may, as it is elevated in low as well as high Gleason scored samples, be used for early detection.

Exosomes Secreted from Human Cancer Cell Lines Contain Inhibitors of Apoptosis (IAP)

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 “multi-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.

Curcumin Induces Pancreatic Adenocarcinoma Cell Death Via Reduction of the Inhibitors of Apoptosis.

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 curcumins 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 after curcumin treatment. Visualization of PANC-1 cell death was performed using Hoffman Modulation Contrast microscopy. Western blot, and polymerase chain reaction analyses were used to evaluate curcumins effects on IAP protein and mRNA expression. Results Curcumin enters PANC-1 cells and is ubiquitously present within the cell after 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. Futthermore, curcumin is a potential therapeutic tool for overcoming chemotherapeutic resistance mediated by IAPs. Together, this data supports a role for curcumin as part of the therapeutic approach for the treatment of pancreatic cancer.

Antimetabolite Treatment for Pancreatic Cancer

Pancreatic cancer is a deadly and aggressive disease. Less than 1% of diagnosed patients survive 5 years with an average survival time of only 4–8 months. The only option for metastatic pancreatic cancer is chemotherapy where only the antimetabolites gemcitabine and 5-fluorouracil are used clinically. Unfortunately, efforts to improve chemotherapy regimens by combining, 5-fluorouracil or gemcitabine with other drugs, such as cisplatin or oxaliplatin, have not increased cell killing or improved patient survival. The novel antimetabolite zebularine shows promise, inducing apoptosis and arresting cellular growth in various pancreatic cancer cell lines. However, resistance to these antimetabolites remains a problem highlighting the need to discover and develop new antimetabolites that will improve a patient’s overall survival.

Early diagnostic value of survivin and its alternative splice variants in breast cancers.

35 Background: The inhibitor of apoptosis (IAP) protein Survivin and its splice variants are differentially expressed in breast cancer tissues and have recently been shown to be released from tumor cells via small membrane-bound vesicles called exosomes. Tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions by impacting immune escape, tumor invasion and angiogenesis. We, therefore, hypothesize that analysis of exosomal Survivin and its splice variants may provide a novel biomarker for early diagnosis of breast cancer in addition to current recommended methods. METHODS Twenty paired breast cancer patients sera and tumor tissue, and ten normal control sera were used for analysis. ELISA was performed to quantitate serum levels of Survivin. Exosomes were isolated from the sera using Exoquick. RT-PCR, western blots with densitometry, and immunohistochemistry followed by confocal microscopy were then performed. RESULTS For each breast cancer patient serum, Survivin levels were significantly higher compared to control (p<0.05). While Survivin and the DEx3 splice variant expression and localization were similar, differential expression of Survivin and the 2B splice variant protein and mRNA existed in the exosomes and tissue samples. Survivin and -DEx3 proteins were the predominant forms detected in 100% (20/20) of the breast cancer tissues evaluated, whereas a more variable expression of Survivin-2B, with enhanced levels found in areas of necrosis. We also for the first time here show the exosomal localization of Survivin and its splice variants DEx3 and 2B in sera from breast cancer patients. CONCLUSIONS The result of the proposed project supports our hypothesis that differential expression of exosomal-Survivin and its alternative splice variants may serve as a diagnostic marker in breast cancer patients. For future direction, we plan to study the prognostic value of exosomal-Survivin and its splice variants on a large panel of primary breast cancers within a setting of well-followed clinical outcomes.

Cell death in response to antimetabolites directed at ribonucleotide reductase and thymidylate synthase

New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor) and 5-fluorouracil (thymidylate synthase inhibitor) were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment. While all antimetabolite drugs killed PANC-1 cells in a time- and dose-dependent manner, neither family significantly altered the cytosolic protein level of the four inhibitors of apoptosis (IAPs) investigated. Survivin, XIAP, cIAP1, and cIAP2 were found localized to exosomes where no significant difference in expression was recorded. This inability for significant and long-lasting expression may be a reason why pancreatic cancer lacks responsiveness to these and other cancer-killing agents. Continued investigation is required to determine the responsibilities of these IAPs in their role in chemoresistance in pancreatic adenocarcinoma.

Abstract 1108: B-cell lymphoma-derived exosomes are reservoirs of inhibitors of apoptosis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA B-cells, along with other normal and cancerous cells, secrete 50-150 nm vesicles called exosomes. Exosomes carry proteins, mRNA, and miRNA that can be functional upon cellular uptake and are often specific to cell of origin, offering potential as disease biomarkers. Our lab has identified within exosomes from solid tumor cells a cancer-specific protein, Survivin, which is a member of the Inhibitors of Apoptosis (IAP) family. Whether Survivin and other IAP family members such as XIAP, cIAP1 or cIAP2 are also expressed in exosomes from hematopoietic malignancies is yet to be determined. In this study, we aim to investigate the exosomes released from B-cell lymphoma cells and determine the IAP content under basal and sublethal stress conditions. Three human non EBV-transformed B-cell lymphoma lines developed at Wayne State University were used. WSU-FSCCL (follicular small cleaved cell), WSU-DLCL-2 (diffuse large cell), and WSU-WM (Waldenstrom Macroglobulinemia) represent different levels of aggressiveness. Cells were cultured for 24 or 48 hours before exosome isolation from the conditioned media using either ultracentrifugation on a sucrose cushion or ExoQuick (System Biosciences). Sublethal levels of UV irradiation were determined for each cell line by analyzing DNA content by propidium iodide staining with a FACSCalibur. Protein quantity of exosomes was measured using a BCA assay, and protein content was studied using Western blotting. Corresponding gene expression was identified using RT-PCR. Here we report IAP protein and mRNA (except XIAP) within exosomes from basal cells. We are currently probing exosomes from stressed cells for comparison. We hypothesize that the IAP exosomal profile may change under stressed conditions. The specificity and variability in exosomal content offers capabilities not only as disease biomarkers, but also in the immunomodulating activity of exosomes which have shown roles in both stimulating and inhibiting the immune system, We have found evidence that exosomal Survivin plays an immune role in skewing Th1/Th2 populations and decreasing cytotoxic function. Changes in exosome content between basal and stressed cells may have roles in the immunomodulation of the tumor microenvironment which facilitates the evasion of lymphoma cells to immune destruction, as well as possible prognostic benefit as to whether treatments are being effective or detrimental. Citation Format: Heather R. Ferguson Bennit, Malyn M. Asuncion Valenzuela, Jessica S. Jutzy, Nathan R. Wall. B-cell lymphoma-derived exosomes are reservoirs of inhibitors of apoptosis. [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 1108. doi:10.1158/1538-7445.AM2014-1108

Glaucarubulone glucoside from Castela macrophylla suppresses MCF-7 breast cancer cell growth and attenuates benzo[a]pyrene-mediated CYP1A gene induction.

Quassinoids often exhibit antioxidant and antiproliferative activity. Emerging evidence suggests that these natural metabolites also display chemopreventive actions. In this study, we investigated the potential for the quassinoid glaucarubulone glucoside (Gg), isolated from the endemic Jamaican plant Castela macrophylla (Simaroubaceae), to display potent cytotoxicity and inhibit human cytochrome P450s (CYPs), particularly CYP1A enzymes, known to convert polyaromatic hydrocarbons into carcinogenic metabolites. Gg reduced the viability of MCF‐7 breast adenocarcinoma cells (IC50 = 121 nm) to a greater extent than standard of care anticancer agents 5‐fluorouracil, tamoxifen (IC50 >10 μm) and the tamoxifen metabolite 4‐hydroxytamoxifen (IC50 = 2.6 μm), yet was not cytotoxic to non‐tumorigenic MCF‐10A breast epithelial cells. Additionally, Gg induced MCF‐7 breast cancer cell death. Gg blocked increases in reactive oxygen species in MCF‐10A cells mediated by the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) metabolite B[a]P 1,6‐quinone, yet downregulated the expression of genes that promote antioxidant activity in MCF‐7 cells. This implies that Gg exhibits antioxidant and cytoprotective actions in non‐tumorigenic breast epithelial cells and pro‐oxidant, cytotoxic actions in breast cancer cells. Furthermore, Gg inhibited the activities of human CYP1A according to non‐competitive kinetics and attenuated the ability of B[a]P to induce CYP1A gene expression in MCF‐7 cells. These data indicate that Gg selectively suppresses MCF‐7 breast cancer cell growth without impacting non‐tumorigenic breast epithelial cells and blocks B[a]P‐mediated CYP1A induction. Taken together, our data provide a rationale for further investigations of Gg and similar plant isolates as potential agents to treat and prevent breast cancer. Copyright

Abstract 4856: Survivin packaging into exosomes is lipid raft-dependent

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have shown that Survivin is exported out of the cancer cell via exosomes despite its lack of a secretory signaling peptide. However, the mechanism of Survivin exosomal loading is not yet known. Exosomal protein sorting can be endosomal sorting complex required for transport (ESCRT)-dependent or ESCRT-independent. ESCRT dependent protein sorting requires monoubiquitination of the target protein, which serves as an exosome sorting signal. The ESCRT independent mechanism is through preferential aggregation of the proteins which is based on intrinsic physical properties to lipid-rafts present in exosomes. Our lab has recently shown that exosomal Survivin is associated with both Hsp70 and Hsp90, as well as PRDX1 which have also been shown associated with detergent-resistant lipid rafts. Here, we determine whether Survivin is lipid-raft dependent and whether Hsp70 and Hsp90 and/or PRDX1 are required for exosomal Survivin loading. Exosomes were collected from conditioned media taken from HeLaS cells and isolated by centrifugation. Colocalizations of Survivin with Hsp70, Hsp90 and PRDX1 in the cell and exosomes were detected by immnocytochemistry visualized by confocal microscopy and immunoprecipitation. HeLaS cells were treated with increasing concentrations of methyl-β-cyclodextrin to disrupt lipid rafts as a way to determine protein lipid raft association. Hsp70, Hsp90 and PRDX1 knock-down using siRNAs will be accomplished to verify the requirement of these proteins for Survivin exosome loading and lipid-raft dependents. Here we show that Survivin colocalizes with Hsp70, Hsp90 and PRDX1 in HeLaS cells as well as their exosomes. Increasing concentrations of methyl-β-cyclodextrin disrupts lipid rafts in the HeLaS cells and releases exosomes which also subsequently contain decreasing PRDX1 and Survivin protein amounts. The release of Survivin into the tumor microenvironment is an important finding as this IAP may contribute to the aggressiveness of the cancer as well as the development of chemoresistance. Despite lacking a secretory signaling peptide, Survivin is being released into the extracellular space via exosomes using chaperones. Knowing how Survivin is exosomally packaged may prove useful in selecting patient specific treatment regimens. Citation Format: Malyn May Asuncion Valenzuela, Heather R. Ferguson Bennit, Carlos Joel Diaz Osterman, Salma Khan, Carlos Casiano, Nathan R. Wall. Survivin packaging into exosomes is lipid raft-dependent. [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 4856. doi:10.1158/1538-7445.AM2014-4856

Abstract 3207: Curcumin induces pancreatic cancer cell death by targeting IAPs

Background Pancreatic cancer has among the highest mortality rates compared to other cancer types, with 94% of patients dying within five years of diagnosis. Contributing to the high mortality rate, patients with pancreatic cancer do not present significant symptoms until the disease is advanced. Moreover, treatment strategies have not improved substantially over the last decade. For this reason, a recent trend has led toward focusing on the use of herb-derived compounds, such as curcumin (turmeric derivative), to explore alternative treatments that will improve current pancreatic cancer therapy. Objective The objectives of this study were (1) to determine the effects of curcumin on pancreatic cancer cell metabolism, viability, cell cycle and morphology, and (2) to determine the mechanism by which curcumin induces cell death in pancreatic cancer cells by analyzing intracellular expression levels of inhibitor of apoptosis proteins (IAPs). Design/method Pancreatic adenocarcinoma cell lines (Panc-1 and MiaPaCa-2) were used to assess the effects of curcumin in pancreatic cancer. Effects on metabolism, viability and cell cycle were assayed by Alamar Blue, Trypan Blue and Propidium Iodide (PI) staining/flow cytometry (FACSCalibur). Morphological imaging was performed using Hoffman Modulation microscopy. Apoptosis was detected by Annexin V/PI flow cytometry (MACSQuant) and the expression of IAPs was assessed by Western blot. Results These data show that curcumin decreases pancreatic cancer cell metabolism and viability. Cell cycle stages were arrested in pancreatic cancer cells treated with curcumin compared to the non-treated controls. Apoptotic cells were detected by microscopy and confirmed by Annexin V/PI staining with subsequent flow cytometry analysis. IAPs levels are reduced in pancreatic cancer cell lines treated with curcumin compared to non-treated controls. Conclusion These results demonstrate that pancreatic adenocarcinoma cell lines are sensitive to curcumin treatment. Moreover, curcumin9s possible mechanism of action is inducing apoptosis by targeting IAPs. Collectively, these data suggest a potential role for curcumin in pancreatic cancer therapy. Citation Format: Carlos J. Diaz Osterman, Malyn May Asuncion Valenzuela, Heather R. Ferguson Bennit, Salma Khan, Nathan R. Wall. Curcumin induces pancreatic cancer cell death by targeting IAPs. [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 3207. doi:10.1158/1538-7445.AM2014-3207