Jonathan R. Aspe

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.

Survivin is released from cancer cells via exosomes

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provides evidence that Survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of Survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to Survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and Survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the Survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3 Gy) is significantly higher than control. These data identify a novel secretory pathway by which Survivin can be actively released from cells in both the basal and stress-induced state.

Extracellular, cell-permeable survivin inhibits apoptosis while promoting proliferative and metastatic potential

The tumour microenvironment is believed to be involved in development, growth, metastasis, and therapy resistance of many cancers. Here we show survivin, a member of the inhibitor of apoptosis protein (IAP) family, implicated in apoptosis inhibition and the regulation of mitosis in cancer cells, exists in a novel extracellular pool in tumour cells. Furthermore, we have constructed stable cell lines that provide the extracellular pool with either wild-type survivin (Surv-WT) or the previously described dominant-negative mutant survivin (Surv-T34A), which has proven pro-apoptotic effects in cancer cells but not in normal proliferating cells. Cancer cells grown in conditioned medium (CM) taken from Surv-WT cells absorbed survivin and experienced enhanced protection against genotoxic stresses. These cells also exhibited an increased replicative and metastatic potential, suggesting that survivin in the tumour microenvironment may be directly associated with malignant progression, further supporting survivins function in tumourigenesis. Alternatively, cancer cells grown in CM taken from the Surv-T34A cells began to apoptose through a caspase-2- and caspase-9-dependent pathway that was further enhanced by the addition of other chemo- and radiotherapeutic modalities. Together our findings suggest a novel microenvironmental function for survivin in the control of cancer aggressiveness and spread, and should result in the genesis of additional cancer treatment modalities.

Enhancement of Gemcitabine sensitivity in pancreatic adenocarcinoma by novel exosome-mediated delivery of the Survivin-T34A mutant

Background: Current 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. Methods: In 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. Results: Here 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. Conclusion: This 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.

Survivin-T34A: molecular mechanism and therapeutic potential.

The inhibitor of apoptosis protein survivin’s threonine 34 to alanine (T34A) mutation abolishes a phosphorylation site for p34(cdc2)–cyclin B1, resulting in initiation of the mitochondrial apoptotic pathway in cancer cells; however, it has little known direct effects on normal cells. The possibility that targeting survivin in this way may provide a novel approach for selective cancer gene therapy has yet to be fully evaluated. Although a flurry of work was undertaken in the late 1990s and early 2000s, only minor advances on this mutant have recently taken place. We recently described that cells generated to express a stable form of the mutant protein released this survivin-T34A to the conditioned medium. When this conditioned medium was collected and deposited on naive tumor cells, conditioned medium T34A was as effective as some chemotherapeutics in the induction of tumor cell apoptosis, and when combined with other forms of genotoxic stressors potentiated their killing effects. We hope with this review to revitalize the T34A field, as there is still much that needs to be investigated. In addition to determining the therapeutic dose and the duration of drug therapy required at the disease site, a better understanding of other key factors is also important. These include knowledge of target cell populations, cell-surface receptors, changes that occur in the target tissue at the molecular and cellular level with progression of the disease, and the mechanism and site of therapeutic action.

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 1118: Stress-induced differential Survivin release in prostate cancer health disparities

African-American men have the highest rate of prostate cancer (PCa) incidence in the U.S. In addition, their prostate cancer mortality rate is more than twice as high as the rate for white Americans. The causes of higher rates of prostate cancer among African-American males are largely unknown. Many studies are underway to unravel the impact of a wide variety of potential risk factors, including dietary and other lifestyle differences, occupational exposures, hormonal and genetic differences. Several lines of evidence suggest that one of the main events associated with the conversion to an aggressive phenotype is increased resistance to apoptosis (3) mainly due to upregulation of antiapoptotic genes, including Bcl-2, Bcl-XL, Mcl-1, and Survivin. Recently, we reported that Survivin is released in PCa sera and found to be in the exosomes. Therefore, we speculate that Survivin could be a potential target protein released differentially in prostate cancers in African American (AA) and Caucasian (CC) populations in response to stress. In this study, we have collected serum samples from prostate cancer patients of AA and CC, measured Survivin levels by ELISA. Exosome islation was perfomed by Exoquick approach and quantified by acetylcholinesterase activity assays. We have applied an in-vitro approach to compare the differences between cancer cell lines derived from AA (MDA-MB-PCa) and CC (PC3) patients in terms of stress and stress activated Survivin release by exosomes. Under oxidative stress and drug treatments, we have identified Survivin highly expressed in exosome derived from MDA-MB-PCa compared to PC3 cell lines. In addition, we found a relative overexpression of Survivin protein in AA-PCa compared to CC-PCa both in sera and exosomes. Based on our data, Survivin may be a key stress-induced protein in AA-PCa patients. It is possible that attempts to block Survivin release will not only interrupt the cancer proliferation and interfere with anti-apoptotic pathways, thus preventing cancer progression. Citation Format: Salma Khan, David Turay, Jessica Ms Jutzy, Jonathan R. Aspe, Tino W. Sanchez, Saeid Mirshahidi, Carlos A. Casiano, Nathan R. Wall. Stress-induced differential Survivin release in prostate cancer health disparities. [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 1118. doi:10.1158/1538-7445.AM2014-1118

Abstract A21: Survivin-T34A enhances gemcitabine treatment in pancreatic cancer.

Pancreatic cancer is the fourth most common cause of cancer fatality in American men and women, with a less than 5% survival rate. Currently, if diagnosed early, surgical resection remains the only viable cure. However, only 20% of pancreatic cancer patients meet these criteria. It is therefore necessary to discover new therapies or therapeutic combinations in order to impact significantly this deadly disease. The antimetabolite agent gemcitabine is currently being employed to treat pancreatic cancer. While gemcitabine has shown significant benefit in clinical applications, its ability to more than modestly impact pancreatic cancer is limited. It has been speculated that combinatory treatments using gemcitabine could improve survival rates. Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is expressed in virtually all cancer cells, but not detectable in most normal cells outside of development. Mutation of survivin’s threonine 34 to alanine (survivinT34A) abolishes a phosphorylation site for p34cdc2-cyclin B1 resulting in the initiation of the mitochondrial apoptotic pathway in cancer cells with little to no direct effects on normal cells. The possibility that targeting survivin in this manner may provide a novel approach for selective cancer gene therapy has yet to be fully evaluated. We have recently described that cells generated to express a stable form of the mutant protein, released this survivinT34A to the conditioned medium. When this conditioned medium was collected and deposited on naive tumor cells, conditioned medium containing survivin-T34A was as effective as chemotherapy in induction of tumor cell apoptosis. When combined with other forms of genotoxic stress, survivin-T34A potentiated their killing effects. We further determined that survivin-T34A is trafficked by microvesicles called exosomes which were released into the conditioned media. We showed strong evidence that exosomes containing survivin-T34A elicited cellular death and synergistically enhanced cellular death during combination with low doses of gemcitabine and we propose that these findings may lead to novel modalities for cancer therapies. Citation Format: Jonathan R. Aspe, Simone DeShields, Nathan R. Wall. Survivin-T34A enhances gemcitabine treatment in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A21.

Abstract B97: Exosomal survivin, a potential tool for early detection of pancreatic cancer health disparity?

Introduction: Pancreatic cancer is a fatal disease that needs immediate attention to develop better screening or early diagnostic tools. African American populations have 42% higher pancreatic cancer death rates than Caucasians. These differences may modify various phases of the multistage process of carcinogenesis by molecular, genetic, and biologic aspects of cancer health disparities. Survivin is expressed in many cancers and its downregulation sensitizes cancer 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 early or monitor pancreatic cancer prognosis in African American populations. Methods: Caucasian-derived Panc1 (control) and African American-derived (KCI-Moh) cell lines were treated with sublethal dosages of cobalt chloride (CoCl2) and gemcitabine. Conditioned media and cell lysates were collected. Exosomes were purified from both cell lines using ultracentrifugation and their quantities and qualities were quantified and visualized from the purified exosomes using ELISA and Western blotting, respectively. Results: Survivin was significantly higher in the exosomes purified from the KCI-Moh cell lines conditioned media compared to those from Panc1 (with and without treatment) although higher quantities of exosomes were noted with the different treatments in Panc1. Exosomal survivin levels were higher in KCI-Moh with CoCl2 as well as gemcitabine compared to controls. Conclusions: These studies demonstrate that survivin exists in exosomes from both Panc1 and KCI-Moh cell lines, and higher concentrations of survivin were observed in KCI-Moh-derived conditioned media. However, increased exosomal survivin levels were observed after Gemcitabine treatment. The relative exosome quantity was significantly higher when treated with CoCl2 and Gemcitabine in Panc1. This differential expression of survivin in the media and exosomes from KCI-Moh may be responsible for chemoresistance or aggressiveness in African American-derived cell lines. Further analysis of plasma exosomal survivin levels may offer a convenient tool for diagnosing or monitoring pancreatic cancers and may be used for early detection in African American populations. Citation Format: Salma Khan, Jessica M.S. Jutzy, Jonathan R. Aspe, Nathan R. Wall. Exosomal survivin, a potential tool for early detection of pancreatic cancer health disparity? [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B97.