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Ryan N Fuller, Janviere Kabagwira, Paul A Vallejos, Andrew D Folkerts, Nathan R Wall Division of Biochemistry, Department of Basic Science, Loma Linda University School of Medicine, Loma Linda, CA, 92350, USACorrespondence: Nathan R Wall, Center for Health Disparities & Molecular Medicine, 11085 Campus Street, Mortensen Hall 160, Loma Linda University, Loma Linda, CA, 92350, USA, Tel +909-558-4000 x81397, Email nwall@llu.eduBackground: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with poor prognosis, as it is difficult to predict or circumvent, and it develops chemoresistance quickly. One cellular mechanism associated with chemoresistance is alternative splicing dysfunction, a process through which nascent mRNA is spliced into different isoforms. Survivin (Baculoviral IAP Repeat-Containing Protein 5 (BIRC5)), a member of the inhibitor of apoptosis (IAP) protein family and a cell cycle-associated oncoprotein, is overexpressed in most cancers and undergoes alternative splicing (AS) to generate six different splicing isoforms.Methods: To determine if survivin splice variants (SSV) could be involved in PDAC chemoresistance, a Gemcitabine (Gem) resistant (GR) cell line, MIA PaCa-2 GR, was created and assessed for its SSV levels and their potential association with GR. Cross-resistance was assessed in MIA-PaCa-2 GR cells to FIRINOX (5-fluorouracil (5-FU), irinotecan, and oxaliplatin). Once chemoresistance was confirmed, RT-qPCR was used to assess the expression of survivin splice variants (SSVs) in PDAC cell lines. To confirm the effect of SSVs on chemoresistance, we used siRNA to knockdown all SSVs or SSV 2β.Results: The MIA PaCa-2 GR cell line was 40 times more resistant to Gem and revealed increased resistance to FIRINOX (5-fluorouracil (5-FU), irinotecan, and oxaliplatin); when compared to the parental MIA-PaCa-2 cells. RT-qPCR studies revealed an 8-fold relative expression increase in SSV 2β and a 2- to 8-fold increase in the other five SSVs in the GR cells. Knockdown of all SSV or SSV 2β only, using small inhibitory RNA (siRNA), sensitized the GR cells to Gem, indicating that these SSVs play a role in PDAC chemoresistance.Conclusion: These findings provide evidence for the potential role of SSV 2β and other SSVs in innate and acquired PDAC chemoresistance. We also show that the expression of SSVs is not affected by the type of chemoresistance, therefore targeting survivin splice variants in combination with chemotherapy could benefit a wide range of patients.Keywords: chemoresistance, survivin splice variants, siRNA, FOLFIRINOX, Gemcitabine

New stem cell and extracellular-vesicle-based therapies have the potential to improve outcomes for the increasing number of patients with heart failure. Since neonates have a significantly enhanced regenerative ability, we hypothesized that extracellular vesicles isolated from Islet-1+ expressing neonatal human cardiovascular progenitors (CPCs) will induce transcriptomic changes associated with improved regenerative capability when co-cultured with CPCs derived from adult humans. In order to test this hypothesis, we isolated extracellular vesicles from human neonatal Islet-1+ CPCs, analyzed the extracellular vesicle content using RNAseq, and treated adult CPCs with extracellular vesicles derived from neonatal CPCs to assess their functional effect. AKT, ERBB, and YAP1 transcripts were elevated in adult CPCs treated with neonatal CPC-derived extracellular vesicles. YAP1 is lost after the neonatal period but can stimulate cardiac regeneration. Our results demonstrate that YAP1 and additional transcripts associated with improved cardiovascular regeneration, as well as the activation of the cell cycle, can be achieved by the treatment of adult CPCs with neonatal CPC-derived extracellular vesicles. Progenitor cells derived from neonates secrete extracellular vesicles with the potential to stimulate and potentially improve functional effects in adult CPCs used for cardiovascular repair.

Gastric cancer (GC) peritoneal carcinomatosis (PC) is associated with a poor prognosis. Although grade, histology, and stage are associated with PC, the cumulative risk of PC when multiple risk factors are present is unknown. This study aimed to develop a cumulative GCPC risk score based on individual demographic/tumor characteristics. Patient-level data (2004–2014) from the California Cancer Registry were reviewed by creating a keyword search algorithm to identify patients with gastric PC. Multivariable logistic regression was used to assess demographic/tumor characteristics associated with PC in a randomly selected testing cohort. Scores were assigned to risk factors based on beta coefficients from the logistic regression result, and these scores were applied to the remainder of the subjects (validation cohort). The summed scores of each risk factor formed the total risk score. These were grouped, showing the percentages of patients with PC. The study identified 4285 patients with gastric adenocarcinoma (2757 males, 64.3%). The median age of the patients was 67 years (interquartile range [IQR], 20 years). Most of the patients were non-Hispanic white (n = 1748, 40.8%), with proximal (n = 1675, 39.1%) and poorly differentiated (n = 2908, 67.9%) tumors. The characteristics most highly associated with PC were T4 (odds ratio [OR], 3.12; 95% confidence interval [CI], 2.19–4.44), overlapping location (OR 2.27; 95% CI 1.52–3.39), age of 20–40 years (OR 3.42; 95% CI 2.24–5.21), and Hispanic ethnicity (OR 1.86; 95% CI 1.36–2.54). The demographic/tumor characteristics used in the risk score included age, race/ethnicity, T stage, histology, tumor grade, and location. Increasing GCPC score was associated with increasing percentage of patients with PC. Based on demographic/tumor characteristics in GC, it is possible to distinguish groups with varying odds for PC. Understanding the risk for PC based on the cumulative effect of high-risk features can help clinicians to customize surveillance strategies and can aid in early identification of PC.

Peritoneal carcinomatosis (PC), metastasized from colorectal cancer (CRC), remains a highly lethal disease. Outcomes of PC is significantly influenced by the amount of intra-abdominal tumor burden and therefore diagnostic tests that facilitate earlier diagnosis could improve PC treatment and patient outcomes.Using mass-spectrometry-based proteomics, we characterized the protein features of circulating exosomes in the context of CRC PC, CRC with liver metastasis, and primary CRC limited to the colon. We profiled exosomes isolated from patient plasma to identify exosome-associated protein cargoes released by these cancer types.Analysis of the resulting data identified metastasis-specific exosome protein signatures. Bioinformatic analyses confirmed enrichment of proteins annotated to vesicle-associated processes and intracellular compartments, as well as representation of cancer hallmark functions and processes.This research yielded distinct protein profiles for the CRC patient groups and suggests the utility of plasma exosome proteomic analysis for a better understanding of PC development and metastasis.

Extracellular vesicles (EVs) are emerging targets for biomarker discovery. However, suitable clinical collection and storage methods for blood plasma-derived EVs have not been fully described. We investigated the long- and short-term effects of temperature and time on the plasma-derived EV concentration, size, protein, and RNA content. EVs isolated from freshly collected human blood stored for various periods of time at either room temperature (RT) or 4°C were analyzed for concentration, size, and protein content. In addition, EVs isolated from plasma stored at −80°C were compared to frozen EV pellets. We found that the number, size, protein, and RNA content of EVs were similar between blood samples stored at RT for 24h compared to samples processed shortly after collection or stored at 4°C vs. −80°C. It is therefore feasible to isolate and analyze comparable, stable EVs from blood plasma stored for varying periods of time and at various temperatures.

Aberrant lipid metabolism in prostate tumors is linked to aggressive disease and poor outcomes. The purpose of this study was to identify mechanisms by which lipid metabolism in prostate cancer cells influences the tumor immune microenvironment, with emphasis on the activation of macrophages. Our hypothesis that fatty acid synthase-driven de novo lipogenesis promotes immune evasion via alternative M2-like macrophage activation was tested using human monocyte (U937)-derived macrophages co-cultured with prostate cancer cells in the presence of fatty acid synthase inhibitors. Monocytes were differentiated to macrophages in vitro using phorbol-12-myristate-13-acetate for 48 hours prior to co-culture with prostate cancer cells. Macrophage polarization following co-culture with prostate cancer cells was evaluated using multi-parameter flow cytometry to detect established M1 (CD80, CD86) and M2 (CD163, CD206) markers. Furthermore, the expression of fatty acid synthase and pan-macrophage marker CD68 in human prostate tumor tissue microarrays was evaluated using multiplex immunohistochemistry. The lipogenic enzyme and macrophage staining correlated with disease progression, reaching peak levels in metastatic tissues. These findings indicate that tumoral fatty acid synthase is an important mediator of tumor-immune crosstalk, particularly in the context of aggressive and metastatic prostate cancer. These data further suggest that fatty acid synthesis represents a targetable complement to immune-enhancing therapies by modulating tumor cell metabolism, underscoring the need for further evaluation of agents targeting lipid metabolism in prostate cancer. Citation Format: Shannalee R. Martinez, Carla Barrientos Risso, Ralphdy Vergne, Nathan R. Wall, Julie Dutil, Carlos A. Casiano, Gilberto Ruiz-Deya, Carlos Joel Diaz Osterman. Tumor lipogenesis influences macrophage polarization in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2559.

The inherent abilities of natural killer (NK) cells to recognize and kill target cells place them among the first cells with the ability to recognize and destroy infected or transformed cells. Cancer cells, however, have mechanisms by which they can inhibit the surveillance and cytotoxic abilities of NK cells with one believed mechanism for this: their ability to release exosomes. Exosomes are vesicles that are found in abundance in the tumor microenvironment that can modulate intercellular communication and thus enhance tumor malignancy. Recently, our lab has found cancer cell exosomes to contain the inhibitor of apoptosis (IAP) protein survivin to be associated with decreased immune response in lymphocytes and cellular death. The purpose of this study was to explore the effect of survivin and lymphoma-derived survivin-containing exosomes on the immune functions of NK cells. NK cells were obtained from the peripheral blood of healthy donors and treated with pure survivin protein or exosomes from two lymphoma cell lines, DLCL2 and FSCCL. RNA was isolated from NK cell samples for measurement by PCR, and intracellular flow cytometry was used to determine protein expression. Degranulation capacity, cytotoxicity, and natural killer group 2D receptor (NKG2D) levels were also assessed. Lymphoma exosomes were examined for size and protein content. This study established that these lymphoma exosomes contained survivin and FasL but were negative for MHC class I-related chains (MIC)/B (MICA/B) and TGF-β. Treatment with exosomes did not significantly alter NK cell functionality, but extracellular survivin was seen to decrease natural killer group 2D receptor (NKG2D) levels and the intracellular protein levels of perforin, granzyme B, TNF-α, and IFN-γ.

Recent military conflicts in Iraq and Afghanistan have resulted in the significant increase in blast-related traumatic brain injury (TBI), leading to increased Department of Defense interest in its potential long-term effects ranging from the mildest head injuries termed subconcussive trauma to the most debilitating termed chronic traumatic encephalopathy (CTE). Most patients with mild TBI will recover quickly while others report persistent symptoms called postconcussive syndrome. Repeated concussive and subconcussive head injuries result in neurodegenerative conditions that may hinder the injured for years. Fundamental questions about the nature of these injuries and recovery remain unanswered. Clinically, patients with CTE present with either affective changes or cognitive impairment. Genetically, there have been no clear risk factors identified. The discovery that microglia of the cerebral cortex discharged small extracellular vesicles in the injured and adjacent regions to a TBI may soon shed light on the immediate impact injury mechanisms. The combination of neuroimaging and advanced research techniques may, one day, fill critical knowledge gaps and lead to significant TBI research and treatment advancements.

Survivin, a member of the inhibitor of apoptosis (IAP) protein family plays a significant role in cell fate and function. It is significantly overexpressed in tumor cells and has been identified in most cancer cell types. A novel extracellular population has recently been identified and its function is still unknown. Emerging evidence continues to shed light on the important role the tumor microenvironment (TME) has on tumor survival and progression. This new population of survivin has been seen to enhance the tumor phenotype when internalized by recipient cells. In this paper, we sought to better understand the mechanism by which survivin is taken up by cancer cells and the possible role it plays in this phenomenon. We isolated the exosomal carriers of extracellular survivin and using a lipophilic stain, PKH67, we tracked their uptake with immunofluorescence and flow cytometry. We found that by blocking exosomal survivin, exosome internalization is reduced, signifying a novel function for this protein. We also discovered that the common membrane receptors, transferrin receptor, endothelin B receptor, insulin receptor alpha, and membrane glucocorticoid receptor all facilitate exosomal internalization. This understanding further clarifies the protein-protein interactions in the TME that may influence tumor progression and identifies additional potential chemotherapeutic targets.

Cancer-derived exosomes are constitutively produced and secreted into the blood and biofluids of their host patients providing a liquid biopsy for early detection and diagnosis. Given their ubiquitous nature, cancer exosomes influence biological mechanisms that are beneficial to the tumor cells where they are produced and the microenvironment in which these tumors exist. Accumulating evidence suggests that exosomes transport proteins, lipids, DNA, mRNA, miRNA and long non coding RNA (lncRNA) for the purpose of cell-cell and cell-extracellular communication. These exosomes consistently reflect the status as well as identity of their cell of origin and as such may conceivably be affecting the ability of a functional immune system to recognize and eliminate cancer cells. Recognizing and mapping the pathways in which immune suppression is garnered through these tumor derived exosome (TEX) may lead to treatment strategies in which specific cell membrane proteins or receptors may be targeted, allowing for immune surveillance to once again help with the treatment of cancer. This Review focuses on how cancer exosomes interact with immune cells in the blood.

// James R.W. McMullen 1 , Matthew Selleck 2 , Nathan R. Wall 1 and Maheswari Senthil 2 1 Department of Basic Sciences, Center for Health Disparities & Molecular Medicine, Division of Biochemistry, Loma Linda University Medical Center, Loma Linda, CA, USA 2 Department of Surgery, Division of Surgical Oncology, Loma Linda University Medical Center, Loma Linda, CA, USA Correspondence to: Nathan R. Wall, email: // Maheswari Senthil, email: // Keywords : peritoneal carcinomatosis, liquid biopsy, biomarker, exosomes Received : January 21, 2017 Accepted : March 15, 2017 Published : March 22, 2017 Abstract Peritoneal Carcinomatosis (PC) is a late stage manifestation of several gastrointestinal malignancies including appendiceal, colorectal, and gastric cancer. In PC, tumors metastasize to and deposit on the peritoneal surface and often leave patients with only palliative treatment options. For colorectal PC, median survival is approximately five months, and palliative systemic therapy is able to extend this to approximately 12 months. However, cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) with a curative intent is possible in some patients with limited tumor burden. In well-selected patients undergoing complete cytoreduction, median survival has been reported as high as 63 month. Identifying patients earlier who are either at risk for, or who have recently developed PC may provide them with additional treatment options such as CRS/HIPEC. PC is diagnosed late by imaging findings or often times during an invasive procedures such as laparoscopy or laparotomy. In order to improve the outcomes of PC patients, a minimally invasive, accurate, and specific PC screening method needs to be developed. By utilizing circulating PC biomarkers in the serum of patients, a “liquid biopsy,” may be able to be generated to allow a tailored treatment plan and early intervention. Exosomes, stable patient-derived nanovesicles present in blood, urine, and many other bodily fluids, show promise as a tool for the evaluation of labile biomarkers. If liquid biopsies can be perfected in PC, manifestations of this cancer may be more effectively treated, thus offering improved survival.

Heather R Ferguson Bennit,1,2 Amber Gonda,1,3 Laura J Oppegard,2 David P Chi,2 Salma Khan,1,2 Nathan R Wall1,2 1Center for Health Disparities & Molecular Medicine, 2Division of Biochemistry, Department of Basic Sciences, 3Department of Anatomy, Loma Linda University School of Medicine, Loma Linda, CA, USA Abstract: Exosomes are nanosized lipid vesicles secreted into blood and other body fluids and serve as vehicles for intercellular communication. Despite being an important component of the tumor microenvironment (TME), exosomal targeting and uptake into recipient cells are still not fully understood. Few studies have looked at lymphoma exosomes and their interactions with circulating blood cells. In this study, we examine the exosomal uptake distribution among peripheral blood leukocytes (PBLs) using vesicles derived from a diffuse large B cell lymphoma cell line, WSU-DLCL2. Lymphoma cells survive, proliferate, and are protected from the cytotoxic effects of chemotherapeutic agents by soluble factors or by direct contact with inflammatory and stromal cells within the TME. In an attempt to close the gap in knowledge concerning lymphoma TME immunosuppression, we have treated normal human PBLs with PKH67-labeled lymphoma exosomes and monitored the uptake by measuring fluorescence at different time points using flow cytometry and fluorescent microscopy. Our results show that of the four populations examined, B cells and monocytes demonstrated uptake of PKH67-labeled exosomes, while T cells and NK cells displayed significantly less uptake. Keywords: exosome, non-Hodgkin’s lymphoma, B cell

Despite significant clinical and basic science advancements, cancer remains a devastating disease that affects people of all ages, races, and backgrounds. The pathogenesis of cancer has recently been described to result from eight biological capabilities or hallmarks and two enabling characteristics. These eight hallmarks are: deregulation of cellular energetics, avoiding immune destruction, enabling replicative immortality, inducing angiogenesis, sustaining proliferative signaling, evading growth suppressors, resisting cell death, and activating invasion and metastasis. The enabling characteristics are: genome instability and mutation and tumor-promoting inflammation. Survivin, the fourth most common transcript found in cancer cells, is a protein that is thought to be involved in the enhanced proliferation, survival, and metastasis and possibly other key hallmarks of cancer cells. Understanding how this gene is turned on and off is vitally important for attempt improving cancer management and therapy. Our work has identified a novel transcriptional regulator of survivin called Yin Yang 1 (YY1), which has been observed to activate some gene promoters and repress others and is gaining increasing interest as a target of cancer therapy. Our work shows for the first time that YY1 represses survivin transcription by physically interacting with the survivin promoter. Furthermore, YY1 appears to contribute to basal survivin transcriptional activity, indicating that disruption of its binding may in part contribute to survivin overexpression after cellular stress events including chemotherapy and radiotherapy.

Traditional clinicopathologic variables do not predict recurrence and disease progression in colorectal cancer (CRC) accurately. Development of a liquid biopsy as a means to obtain real-time, clinically meaningful biomarkers to assess progression and response to treatment is critical. Current liquid biopsy techniques utilizing circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have significant limitations. Exosomes are emerging as a promising tool for liquid biopsy due to their stability and role in cancer cell to cell communication. To that end, the purpose of this study is to describe patterns of highly abundant microRNA (miRNA) found in serum exosomes of patients with metastatic colon cancer as a step toward developing an exosomal miRNA signature for liquid biopsy. Next generation sequencing was performed on exosomes isolated from serum of stage IV colon cancer patients (n=9) to determine the miRNA read counts. miRNA data from primary tumors (n=53) of stage IV colon cancer patients and available paired peritumoral tissue (n=8) were obtained from The Cancer Genome Atlas (TCGA) database for qualitative comparison. There were 515 total miRNA shared in common between the serum exosomes and primary tumors and 277 miRNA uniquely present in serum exosomes. In our initial analysis, we identified five miRNA, two in the common pool (mir-486-1 and mir-486-2) and three in the unique group (miR-486-5p, miR-92a-3p, and miR-146a-5p) that were most abundant in serum exosomes and may serve as biomarkers. mir-486-1 and mir-486-2 were the most highly abundant miRNAs in exosomes, but were only expressed at low levels in the tissue. This inverse relationship has been demonstrated by other investigators, with decreased levels of tissue mir-486 associated with more advanced stages of disease and poorer survival. Exosomal miR-92a-3p has been shown to correlate with tumor grade and stage in CRC patients. Delivery of miR-146a-5p by exosomes has been shown to promote tumorigenesis and generate immunosuppressive microenvironment in CRC. In general, the top 10 most abundant exosomal miRNA were not among the most abundant tissue level miRNAs. Our group is currently analyzing the differential expression of serum exosomal miRNA from 52 additional patients with non-metastatic colon cancer (n=14), liver metastases (n=16) and PC (n=12) in comparison to healthy volunteer serum (n=10) to better characterize miRNA patterns at different stages of disease. In summary, highly abundant miRNA found in serum exosomes of patients with metastatic colon cancer demonstrate a different pattern of expression than miRNA found in the primary colon cancer itself. Further analysis of these miRNA patterns from serum exosomes of colon cancer patients with non-metastatic disease, visceral metastases and PC compared to normal volunteers will aid in the development of a serum exosomal miRNA signature. Citation Format: Audrey H. Choi, Paul A. Vallejos, Mei Li M. Kwong, Janviere Kabagwira, Matthew J. Selleck, Nathan R. Wall, Maheswari Senthil. Liquid biopsy in colon cancer utilizing exosomal miRNA biomarkers: An initial analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6446.

Patients with peritoneal metastases from colorectal cancer (CRC) have poor prognosis. Systemic chemotherapy by itself is often ineffective in the treatment of peritoneal carcinomatosis. Cytoreductive surgery (CRS) with heated intraperitoneal chemotherapy (HIPEC) is offered to appropriately selected patients. HIPEC consists of direct application of high-dose chemotherapy to the peritoneal cavity under hyperthermic conditions for 90-120 minutes. Although there is strong evidence to demonstrate improved survival with cytoreductive surgery, the role of HIPEC needs further evaluation. Furthermore, despite optimal cytoreduction, a subset of patients will suffer early peritoneal recurrence which indicates that the intraperitoneal chemotherapy was ineffective in controlling the microscopic disease. In vitro studies focused on optimizing the drug, duration of exposure, and temperature based on the biology of the cancer are critically needed to inform the design of future clinical trials. The combination of the HIPEC treatment modality with curcumin as the therapeutic agent is attractive since the naturally occurring curcumin has fewer side effects than drugs currently used for HIPEC. Additionally, although curcumin possesses antitumor effects on various cancers including colon, its efficacy is hindered by a low systemic bioavailability. For HIPEC, curcumin could be delivered directly into the peritoneal cavity, thereby increasing bioavailability. We hypothesize that curcumin used alone or in combination with standard regimen mitomycin C treatment in HIPEC will exert antitumor effects superior to the standard regimen HIPEC. To assess this hypothesis, we developed an in vitro model in which colon cancer cell lines (SK-CO-1, COLO205 and SNU-C1) were treated with curcumin (25 µM) or mitomycin C (1 µM) alone or in combination at different exposure times (1, 2 or 3 hours), and then incubated at either 37°C (normothermia) or 42 °C (hyperthermia), simulating conditions used in clinical practice. Cell lines were chosen in order to determine the effects of p53 (SNU-C1 & COLO205), KRAS (SK-CO-1) and BRAF (COLO205) mutation status on this proposed intraperitoneal chemotherapy strategy. After treatment, cells were maintained at 37°C for 24, 48, and 72 hours after which cell viability was assessed using Annexin V/PI, trypan blue and Hoffman microscopy. Our results indicate that hyperthermia enhances the efficacy of curcumin treatment as evidenced by a significant two-fold decrease in cell viability compared to normothermia in vitro in all three cell lines. No mutation-associated effect was quantified. Though additional comparisons are still forthcoming, the addition of curcumin to HIPEC may be a promising novel therapeutic option. Citation Format: Janviere Kabagwira, Paul A. Vallejos, Audrey H. Choi, Chase Sugiono, Vola-Masoandro Andrianarijaona, Jazmine Chism, William HR Langridge, Maheswari Senthil, Nathan R. Wall. Hyperthermic conditions improve curcumin efficacy for the treatment of peritoneal carcinomatosis of colon cancer origin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1451.

The tumor microenvironment is replete with factors secreted and internalized by surrounding cells. Exosomes are nano-sized, protein-embedded, membrane-bound vesicles that are released in greater quantities from cancer than normal cells and taken up by a variety of cell types. These vesicles contain proteins and genetic material from the cell of origin and in the case of tumor-derived exosomes, oncoproteins and oncogenes. With increasing understanding of the role exosomes play in basic biology, a more clear view of the potential exosomes are seen to have in cancer therapeutics emerges. However, certain essential aspects of exosome function, such as the uptake mechanisms, are still unknown. Various methods of cell–exosome interaction have been proposed, but this review focuses on the protein–protein interactions that facilitate receptor-mediated endocytosis, a broadly used mechanism by a variety of cells.

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 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 curcumin's 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.

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.

Background Natural medicine monomers (NMMs) isolated from plants have been recognized for their roles in treating different human diseases including cancers. Many NMMs exhibit effective anti-cancer activities and can be used as drugs or adjuvant agents to enhance the efficacy of chemotherapy and radiotherapy. Some NMMs, such as paclitaxel and camptothecin, have been extensively studied for decades and are now used as anti-cancer medicines due to their remarkable curative effects, such as inhibiting cancer cell proliferation and metastasis, and inducing cell death and differentiation. Methods After extensively reviewing papers related to NMM studies in cancers, we grouped NMMs into six categories based on their chemical structures. We summarized the anti-cancer activities of these NMMs and current knowledge of molecular mechanisms for them to exert their functions. Results and conclusion Many NMMs from plants can effectively inhibit cancer cells with low or tolerable toxicity to patients. Some NMMs have been well-characterized for their anti-cancer activities and have already been used as clinical drugs or adjuvant agents; however, the mechanisms underlying the cancer suppressive activities of most NMMs remain poorly understood. Many NMMs can be used as initial structural scaffolds to design and develop novel therapeutics against cancers. This review summarizes reports related to signaling pathways mediated by different NMMs and can provide a theoretical basis for clinical application and new drug development of NMMs.

The tumor microenvironment is an area of intense interaction between normal and malignant cells. Factors and cell types within this environment can play a crucial role in the progression or regression of the tumor. Of primary interest are tumor-infiltrating T lymphocytes, which have been shown to have a key role in modifying the dynamics of the tumor microenvironment to promote or prevent tumor growth. While there is much in vitro and in vivo evidence for a modification of the tumor infiltrating T cell population toward a pro-tumor environment, what induces these changes within the tumor microenvironment has remained elusive. Our lab previously identified a role for the Inhibitor of Apoptosis protein Survivin as a secreted protein in the extracellular milieu, where it is capable of entering malignant cells and inducing a more aggressive phenotype. We hypothesized that tumorsecreted Survivin could be responsible for modulation of T lymphocytes in the tumor microenvironment. We first isolated tumor released Survivin and confirmed its ability to be taken up by T cells as it is by malignant cells by confocal microscopy, flow cytometry and Western blotting. Subsequently, we evaluated Survivin’s affect on T cell proliferation and found that tumor-released Survivin impairs T cell expansion, but does not alter its activation after exposure to appropriate stimuli. Assessment of phenotypic changes within the cytotoxic and helper T lymphocyte populations showed an increase in anti-inflammatory type 2 T cells and a reduction in type 1 T cells, whichcorrelates to what has been observed in cancer patients. Although often modified in the tumor microenvironment in patients, we did not observe any changes in regulatory T cells or Th17 cells in the presence of Survivin. The results of this study provide evidence of Survivin’s role as an extracellular mediator of the tumor microenvironment, specifically its role in inducing a pro-tumor type 2 T cell population.

The mechanisms for regulation of the Inhibitor of Apoptosis (IAP) Survivin in cells undergoing stress associated with tumor development and the tumor microenvironment are not well understood. The stress response transcription factors HIF-1α and Yin Yang 1 (YY1) were hypothesized to contribute to the upregulation of Survivin in tumor cells. As expected, U2OS cells overexpressing HIF-1α showed a 2- to 3-fold transactivation when transfected. Surprisingly, when YY1 was overexpressed in this survivin promoter reporter system, luciferase expression was repressed 30- to 40-fold. YY1 involvement in survivin promoter repression was confirmed using siRNA directed against YY1. These studies showed that knockdown of YY1 releases the survivin promoter from the observed repression and leads to a 3- to 5-fold increase in promoter activity above basal levels. A U2OS cell line containing a stable YY1 Tet-off system was used to determine whether a temporal increase in YY1 expression affects Survivin protein levels. A low to moderate decrease in Survivin protein was observed 24 h and 48 h after Tet removal. Studies also confirmed that YY1 is capable of directly binding to the survivin promoter. Collectively, these findings identify novel basal transcriptional requirements of survivin gene expression which are likely to play important roles in the development of cancer and resistance to its treatment.

Tumor angiogenesis is a hallmark of advanced cancers and promotes invasion and metastasis. Over 90% of head and neck squamous cell carcinomas (HNSCC) express angiogenic factors such as vascular endothelial growth factor (VEGF). Several preclinical studies support the prognostic implications of angiogenic markers for HNSCC and currently this is an attractive treatment target in solid tumors. Since radiotherapy is one of the most commonly used treatments for HNSCC, it is imperative to identify the interactions between antiangiogenic therapy and radiotherapy, and to develop combination therapy to improve clinical outcome. The mechanisms between antiangiogenic agents and ionizing radiation are complicated and involve many interactions between the vasculature, tumor stroma and tumor cells. The proliferation and metastasis of tumor cells rely on angiogenesis/blood vessel formation. Rapid growing tumors will cause hypoxia, which up-regulates tumor cell survival factors, such as hypoxia-inducing factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), giving rise to more tumor proliferation, angiogenesis and increased radioresistance. Thus, agents that target tumor vasculature and new tumor vessel formation can modulate the tumor microenvironment to improve tumor blood flow and oxygenation, leading to enhanced radiosensitivity. In this review, we discuss the mechanisms of how antiangiogenic therapies improve tumor response to radiation and data that support this combination strategy as a promising method for the treatment of HNSCC in the future.

African-American men with prostate cancer typically develop more aggressive tumors than men from other racial/ethnic groups, resulting in a disproportionately high mortality from this malignancy. This study evaluated differences in the expression of inhibitors of apoptosis proteins (IAPs), a known family of oncoproteins, in blood-derived exosomal vesicles (EV) between African-American and European-American men with prostate cancer. The ExoQuick™ method was used to isolate EV from both plasma and sera of African-American (n = 41) and European-American (n = 31) men with prostate cancer, as well as from controls with no cancer diagnosis (n = 10). EV preparations were quantified by acetylcholinesterase activity assays, and assessed for their IAP content by Western blotting and densitometric analysis. Circulating levels of the IAP Survivin were evaluated by ELISA. We detected a significant increase in the levels of circulating Survivin in prostate cancer patients compared to controls (P

African American (AA) men suffer from a disproportionately high incidence and mortality of prostate cancer (PCa) compared with other racial/ethnic groups. Despite these disparities, African American men are underrepresented in clinical trials and in studies on PCa biology and biomarker discovery. We used immunoseroproteomics to profile antitumor autoantibody responses in AA and European American (EA) men with PCa, and explored differences in these responses. This minimally invasive approach detects autoantibodies to tumor-associated antigens that could serve as clinical biomarkers and immunotherapeutic agents. Sera from AA and EA men with PCa were probed by immunoblotting against PC3 cell proteins, with AA sera showing stronger immunoreactivity. Mass spectrometry analysis of immunoreactive protein spots revealed that several AA sera contained autoantibodies to a number of proteins associated with both the glycolysis and plasminogen pathways, particularly to alpha-enolase (ENO1). The proteomic data is deposited in ProteomeXchange with identifier PXD003968. Analysis of sera from 340 racially diverse men by enzyme-linked immunosorbent assays (ELISA) showed higher frequency of anti-ENO1 autoantibodies in PCa sera compared with control sera. We observed differences between AA-PCa and EA-PCa patients in their immunoreactivity against ENO1. Although EA-PCa sera reacted with higher frequency against purified ENO1 in ELISA and recognized by immunoblotting the endogenous cellular ENO1 across a panel of prostate cell lines, AA-PCa sera reacted weakly against this protein by ELISA but recognized it by immunoblotting preferentially in metastatic cell lines. These race-related differences in immunoreactivity to ENO1 could not be accounted by differential autoantibody recognition of phosphoepitopes within this antigen. Proteomic analysis revealed differences in the posttranslational modification profiles of ENO1 variants differentially recognized by AA-PCa and EA-PCa sera. These intriguing results suggest the possibility of race-related differences in the antitumor autoantibody response in PCa, and have implications for defining novel biological determinants of PCa health disparities.

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.

Thymine hydroxylase (TH) is a member of the alpha-ketoglutarate-dependent nonheme iron dioxygenase family that includes a series of DNA repair proteins including alkB. Substantial interest in this family of enzymes derives from their capacity to modify DNA bases and precursors by oxidation. Previously, a sequence has been published for cloned Rhodotorula glutinis TH. However, the minimal reported activity of this enzyme, coupled with inconsistencies with previously published mass spectrometry data, compelled us to reexamine TH. The sequence reported here differs from the previously reported sequence at two amino acid positions and is consistent with previously reported mass spectrometry data. The cloned enzyme characterized in this report displayed substantial activity, indicating that the sequence differences are critical for activity. The substrate selectivity of TH against a series of pyrimidine analogues is consistent with that reported for the wild-type enzyme and, in part, explains the mode of selection of uracil analogues. A preliminary model of the active site has been constructed for the purposes of comparing TH with other members of this family. TH and alkB share in common the capacity to oxidize N-methyl groups. However, TH has the added capacity to oxidize the 5-methyl group of thymine, a property that is potentially important for enzymes that could act on DNA and modify DNA-protein interactions.

IL-7 is critical for B cell production in adult mice; however, its role in human B lymphopoiesis is controversial. One challenge was the inability to differentiate human cord blood (CB) or adult bone marrow (BM) hematopoietic stem cells (HSCs) without murine stroma. Here, we examine the role of IL-7 in human B cell development using a novel, human-only model based on coculturing human HSCs on primary human BM stroma. In this model, IL-7 increases human B cell production by >60-fold from both CB and adult BM HSCs. IL-7-induced increases are dose-dependent and specific to CD19+ cells. STAT5 phosphorylation and expression of the Ki-67 proliferation Ag indicate that IL-7 acts directly on CD19+ cells to increase proliferation at the CD34+ and CD34− pro-B cell stages. Without IL-7, HSCs in CB, but not BM, give rise to a small but consistent population of CD19lo B lineage cells that express EBF (early B cell factor) and PAX-5 and respond to subsequent IL-7 stimulation. Flt3 ligand, but not thymic stromal-derived lymhopoietin (TSLP), was required for the IL-7-independent production of human B lineage cells. As compared with CB, adult BM shows a reduction of in vitro generative capacity that is progressively more profound in developmentally sequential populations, resulting in an ∼50-fold reduction in IL-7-dependent B lineage generative capacity. These data provide evidence that IL-7 is essential for human B cell production from adult BM and that IL-7-induced expansion of the pro-B compartment is increasingly critical for human B cell production during the progression of ontogeny.

Insulin-like growth factor II (IGF-II) plays a pivotal role in fetal and cancer development by signaling through the IGF-I and insulin receptors and activating the estrogen signaling cascade. We previously showed that precursor IGF-II (proIGF-II, the predominant form expressed in cancer) and not mature IGF-II (mIGF-II) blocks resveratrol (RSV) (a phytoalexin/anticancer agent)-induced cell death in MCF-7 cells. We hypothesize that proIGF-II regulates antiapoptotic proteins and/or the mitochondria to inhibit RSV actions and promote cell survival. This study examines the effect of mIGF-II and proIGF-II on survivin expression and mitochondrial polarization in response to RSV. RSV inhibits survivin expression and stimulates mitochondrial depolarization, caspase 7 activation and cell death. These effects were completely blocked by the addition of proIGF-II. RSV treatment had no effect on transfected MCF-7 cells constitutively expressing proIGF-II, while IGF-II siRNA transfection decreased survivin levels. Our results provide new insights for the potential use of proIGF-II as target for new anticancer therapies.

HL60 and U937 (acute myeloid leukemia (AML) cell lines) were assessed for sensitivity to YM155, and found to have distinct sensitive and resistant phenotypes, respectively. In HL60 cells, YM155 inhibition of growth proliferation was due to apoptosis which was measured by annexin V/PI staining. YM155 induced apoptosis through activation of intrinsic and extrinsic pathways that also culminated in caspase-3 activity and PARP cleavage. YM155 sensitivity was partially associated with this compound's ability to down-regulate survivin transcription since this was more pronounced in the HL60 cell line. However, marked differences were also observed in XIAP, Bcl-2, and Mcl-1L, and Mcl-1s. Furthermore, YM155 treatment completely inhibited production of total Akt protein in HL60, but not U937 cells. Importantly, Akt activity (pAkt-Ser473) levels were maintained in YM155 treated U937 cells which may help stabilize other anti-apoptotic proteins. Combination treatments with an Akt inhibitor, MK-2206, reduced levels of pAkt-Ser473 in U937 cells and synergistically sensitized them to YM155 cytotoxicity. Collectively our results indicate that Akt signaling may be an important factor mediating YM155 response in AML, and combinatorial therapies with Akt inhibitors could improve treatment efficacy in YM155-resistant cells.

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.

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.

A checkpoint surveying the entry into mitosis responds to defects in spindle microtubule assembly/stability. This has been used to trigger apoptosis in cancer cells, but how the spindle checkpoint couples to the cell survival machinery has remained elusive. Here, we report that microtubule stabilization engenders a survival pathway that depends on elevated activity of p34 cdc2 kinase and increased expression of the apoptosis inhibitor and mitotic regulator, survivin. Pharmacologic, genetic, or molecular ablation of p34 cdc2 kinase after microtubule stabilization resulted in massive apoptosis independent of p53, suppression of tumor growth, and indefinite survival without toxicity in mice. By ablating this survival checkpoint, inhibitors of p34 cdc2 kinase could safely improve the efficacy of microtubule-stabilizing agents used to treat common cancers.

Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala(3)-Ile(19) exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence, nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys(57)-Trp(67) identified a cytosolic pool of survivin, which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala(3)-Ile(19), Met(38)-Thr(48), Pro(47)-Phe(58) and Cys(57)-Trp(67) identified both survivin pools within the same mitotic cell. A ratio of approximately 1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34(cdc2), and was phosphorylated by p34(cdc2) on Thr(34), in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34(cdc2) and was not phosphorylated on Thr(34). Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.

Traumatic brain injuries (TBI) are among the most misdiagnosed and underreported types of head trauma. The potential long-term impact of undiagnosed or incorrectly identified concussions and other head injuries are potentially devastating, as evidenced by the increasing societal burden exhibited by soldiers returning from combat and athletes in contact sports. Concussions and TBI are notoriously difficult to correctly diagnose and prognosis for these injuries is poorly understood. In order to increase the likelihood of successful diagnosis, treatment, and prediction of outcomes, a definitive differential diagnosis will need to be established. The establishment of a "trauma-specific profile" or a panel of known trauma markers will significantly aid in this goal. Small membrane vesicles called exosomes have been shown to contain proteins and injury-specific biomarkers. In the future it is possible that they could become an important tool, utilized for their diagnostic and therapeutic potential.

Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of cancer-related deaths in American men. African American (AA) men are more likely to be diagnosed with aggressive PCa at a younger age and twice as likely to die from the disease as European American (EA) men. In order to reduce PCa mortality and its associated racial disparities, there is a critical need to identify and target pathways responsible for PCa aggressiveness. An unexplored target is the plasminogen system, which is key to cancer cell migration and tissue invasion during metastasis. During inflammation, activation of the plasminogen pathway degrades extracellular fibrin networks; however, in the context of cancer, this activation promotes cancer tissue invasion and metastasis. Mounting evidence shows that the glycolytic enzyme alpha-enolase (ENO1) plays a vital role in both increased energy metabolism and plasminogen activation during cancer progression and metastasis. Using immunoseroproteomic profiling of anti-tumor autoantibody responses in AA and EA men with PCa, we identified several tumor-associated autoantigens with functions in glycolysis and plasminogen signaling, including ENO1, annexin A2, fructose bisphosphate aldolase, glucose-regulated protein 78, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In a cohort of PCa (N=157) and non-PCa sera (N=183), we found a significantly higher frequency of autoantibodies to ENO1 in patients with PCa (p Citation Format: Tino Wilson Sanchez, Guangyu Zhang, Jitian Li, Liping Dai, Saied Mirshahidi, Nathan R. Wall, Clayton Yates, Colwick Wilson, Susanne Montgomery, Jian-Ying Zhang, Carlos A. Casiano. Targeting the tumor-associated autoantigen alpha-enolase in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1130. doi:10.1158/1538-7445.AM2017-1130

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related male deaths in the U.S. This cancer is more aggressive in African American (AA) men, who are twice as likely to die from the disease compared to other racial groups. Despite racial disparities in PCa mortality, immunoproteomic studies profiling sera from thousands of men for the presence of autoantibodies to tumor-associated antigens (TAA) have mostly used sera from European or Asian ancestry. These autoantibodies can serve as both cancer biomarkers and potential tools in anti-tumor immunotherapy. In this study we profiled sera of AA and European American (EA) men with (N=157) and without (N=183) PCa against protein lysates from aggressive PCa cell lines using one and two-dimensional Western blotting. Sera from AA-PCa patients showed stronger immunoreactivity to proteins in PC3 cell lysates compared to EA-PCa sera. Common bands of immunoreactivity were observed around 37, 40, 50, 55, 60, and 70 kD from the sera of multiple patients. Several AA-PCa sera showed common immunoreactivity against a 50 kD protein band (16 out of 24, 67%) which was identified by serological proteomic analysis (SERPA) as alpha-enolase (ENO1), an enzyme that participates in both the glycolysis and plasminogen pathways. ELISA using purified ENO1 was conducted on 360 sera and showed significantly elevated frequency in men with PCa compared to non-PCa controls. Interestingly, SERPA analysis of other strongly immunoreactive spots outside the 50 kD region recognized of the other highly reactive PCa sera revealed additional candidate TAAs involved in the glycolysis and plasminogen pathways. These included key glycolytic enzymes GAPDH, fructose bisphosphate aldolase, phosphoglycerate kinase and lactate dehydrogenase, and other members of the plasminogen system such as GRP78 and annexin A2. Autoantibodies to stress survival proteins HSP60 and STIP1 were also identified in multiple PCa sera. We then probed by immunoblotting representative sera recognizing some of these candidate TAAs against a panel of 12 PCa and non-PCa cell lines. Anti-STIP1 sera showed elevated immunoreactivity predominantly in docetaxel-resistant cell lines. In the anti-ENO1 positive sera, we observed a race-related differential immune response, with anti-ENO1 sera from AA-PCa men showing elevated immunoreactivity mostly in metastatic cell lines, and anti-ENO1 sera from EA-PCa men showing uniform immunoreactivity across the entire panel of prostate cell lines. Interestingly, while several anti-ENO1 positive sera from AA-PCa men had increased immunoreactivity to this protein in the metastatic PCa cell lines LNCaP, MDA-PC2b, PC3 and DU145, they lacked reactivity against ENO1 in the docetaxel-resistant PC3-DR and DU145-DR cells, or purified ENO1 used in the ELISA. Anti-ENO1 EA-PCa sera did not display this differential pattern of immunoreactivity. To better understand these differences in anti-ENO1 reactivity, we interrogated the pattern of ENO1 post-translational modifications (PTMs) from PC3, PC3-DR and purified ENO1. Several different acetylated, methylated, phosphorylated, citrullinated, and glycosylated ENO1 PTMs were identified in the PC3 cells that were not present in the PC3-DR cells nor purified ENO1 which might explain the differential reactivity of AA-PCa sera against these three sources of ENO1. Taken together, these data suggest the anti-ENO1 autoantibodies from AA-PCa men may target a modified form of ENO1 that is different from that recognized by EA-PCa men. These discrepancies point to racial differences in immune response to TAAs in prostate tumors, and may have implications for identifying immunobiological factors contributing to PCa health disparities. Citation Format: Tino W. Sanchez, Guangyu Zhang, Jitian Li, Liping Dai, Rowaid Kellow, Saied Mirshahidi, Nathan R. Wall, Clayton Yates, Colwick Wilson, Susanne Montgomery, Jian-Ying Zhang, Carlos Casiano. Differential autoimmune response to glycolysis and plasminogen antigens from aggressive prostate cancer cells in African American and European American men with prostate cancer. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B05.

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.

Previous studies have shown that bryostatin 1 induces a decrease in the expression of the antiapoptotic protooncogene Bcl-2 in the human acute lymphoblastic leukemia (ALL) cell line Reh. This down-regulation has been shown to reduce drug resistance of the Reh cells to anti-tubulin polymerization agents. In the present study we investigated the effect of bryostatin 1 alone and in combination with novel anti-tubulin agents (dolastatin 10 and auristatin PE) and the chemotherapeutic vincristine on the inhibitor of apoptosis protein cIAP-1. Cells were cultured with bryostatin 1 (1 nM), dolastatin 10 (0.1 ng/ml), auristatin PE (0.1 ng/ml), or vincristine (0.5 ng/ml) alone or the combination of these anti-tubulins with bryostatin 1. Western blots were conducted to assess the effects of the above agents on cIAP-1 protein level. Flow-cytometric analysis [7-amino-actinomycin D (7AAD)] was conducted to assess apoptosis as well as staining for morphology using tetrachrome stain. Our results show that cIAP-1 is induced in a time-dependent fashion after bryostatin 1 exposure up to 72 h. However, upon treatment of cells with a combination of bryostatin 1 and dolastatin 10 or auristatin PE, the induction of cIAP-1 was abolished, leading to a significant increase in apoptosis. The initial 24- and 48-h reduction in cIAP-1 protein level recorded in the bryostatin 1 and vincristine combination recovered to control levels by 72 h. We believe that this phenomenon is responsible for the reduced apoptosis recorded in this combination. Results of this study should prove useful in guiding the clinical application of these novel agents in the treatment of ALL.

The ratio of Bax to Bcl-2 protein can determine whether cells will die via apoptosis or be protected from it. Reh was found to express a high basal level of Bcl-2 but was lacking of Bax protein expression. Treatment with bryostatin 1 induced a down-regulation in Bcl-2 protein that was not accompanied by an obvious Bax protein induction or apoptosis. These results suggest that a decreased level of Bcl-2 alone in this cell line is not sufficient for apoptosis induction. In an effort to identify the mechanism whereby apoptosis could be induced in this ALL model, we treated Reh cells with three microtubule inhibitors: dolastatin 10, auristatin PE and vincristine, in the presence and absence of bryostatin 1. When used alone, only dolastatin 10 induced apoptosis that was detected morphologically, and by flow cytometry. Western blots revealed that dolastatin 10-induced apoptosis was accompanied by the induction of Bax protein and the reduction in Bcl-2 protein. Auristatin PE and vincristine induced both Bax and Bcl-2 protein, leaving the Bax:Bcl-2 ratio constant. Reh cells pretreated for 24 h with bryostatin 1 followed by dolastatin 10, auristatin PE or vincristine showed significant apoptosis which was accompanied by Bcl-2 protein down regulation and Bax protein up regulation. We conclude that: (1) expression of bax is necessary for apoptosis-induction in this model; (2) a decrease in Bcl-2 level alone is not sufficient and might not be necessary for apoptosis-induction; and (3) the ratio of Bax:Bcl-2 plays a critical role in susceptibility to apoptosis in Reh cells. The results from this study should prove useful in guiding the clinical application of these novel agents in the treatment of acute lymphoblastic leukemia.

We studied the antitumor effects of dolastatin 10, its structural modification, auristatin PE (TZT-1027), and vincristine alone and in combination with bryostatin 1 on a human diffuse large cell lymphoma line (WSU-DLCL2) in vitro and in vivo. WSU-DLCL2 cells were cultured in RPMI 1640 at a concentration of 2 x 10(5)/ml using a 24-well plate. Agents were added to triplicate wells, and cell count, viability, mitosis and apoptosis were assessed. Dolastatin 10 showed no apparent inhibition of cell growth at concentrations less than 500 pg/ ml. Auristatin PE showed significant growth inhibition at concentrations as low as 10 pg/ml, while vincristine had a minimal effect at 50 pg/ml. Dolastatin 10, auristatin PE and vincristine-treated cultures, at 50 pg/ml, exhibited 11, 1.7; 45, 11.8%; and 39, 25% mitosis and apoptosis, respectively. In the WSU-DLCL2 SCID mouse xenograft model, the efficacy of these agents alone or in combination with bryostatin 1 was evaluated. Tumor growth inhibition (T/C), tumor growth delay (T-C) and log10 kill for dolastatin 10, auristatin PE, vincristine and bryostatin 1 were 30%, 14 days and 1.4; 0.0%, 55 days and 5.5; 29.6%, 16 days and 1.6; and 39%, 7 days and 0.7, respectively. When given in combination, two out of five mice treated with auristatin PE + bryostatin 1 were free of tumors for 150 days and were considered cured. Dolastatin 10 + bryostatin 1 and vincristine + bryostatin 1 combinations were highly active but no cure was observed. We conclude that: (i) auristatin PE is more effective in this model than dolastatin 10, vincristine or bryostatin 1, (ii) auristatin PE can be administered at a concentration 10 times greater than dolastatin 10, and (iii) there is a synergistic effect between these agents and bryostatin 1, which is more apparent in the bryostatin 1 + auristatin PE combination. The use of these agents should be further explored clinically in the treatment of lymphoma.