Lysyl Oxidase 3 Is a Dual-Specificity Enzyme Involved in STAT3 Deacetylation and Deacetylimination Modulation.

In mammalian cells, histone deacetylase (HDAC) and Sirtuin (SIRT) are two families responsible for removing acetyl groups from acetylated proteins. Here, we describe protein deacetylation coupled with deacetylimination as a function of lysyl oxidase (LOX) family members. LOX-like 3 (Loxl3) associates with Stat3 in the nucleus to deacetylate and deacetyliminate Stat3 on multiple acetyl-lysine sites. Surprisingly, Loxl3 N-terminal scavenger receptor cysteine-rich (SRCR) repeats, rather than the C-terminal oxidase catalytic domain, represent the major deacetylase/deacetyliminase activity. Loxl3-mediated deacetylation/deacetylimination disrupts Stat3 dimerization, abolishes Stat3 transcription activity, and restricts cell proliferation. In Loxl3-/- mice, Stat3 is constitutively acetylated and naive CD4+ T cells are potentiated in Th17/Treg cell differentiation. When overexpressed, the SRCR repeats from other LOX family members can catalyze protein deacetylation/deacetylimination. Thus, our findings delineate a hitherto-unknown mechanism of protein deacetylation and deacetylimination catalyzed by lysyl oxidases.

Potential functional applications of extracellular vesicles: a report by the NIH Common Fund Extracellular RNA Communication Consortium.

The NIH Extracellular RNA Communication Program's initiative on clinical utility of extracellular RNAs and therapeutic agents and developing scalable technologies is reviewed here. Background information and details of the projects are presented. The work has focused on modulation of target cell fate by extracellular vesicles (EVs) and RNA. Work on plant-derived vesicles is of intense interest, and non-mammalian sources of vesicles may represent a very promising source for different therapeutic approaches. Retro-viral-like particles are intriguing. Clearly, EVs share pathways with the assembly machinery of several other viruses, including human endogenous retrovirals (HERVs), and this convergence may explain the observation of viral-like particles containing viral proteins and nucleic acid in EVs. Dramatic effect on regeneration of damaged bone marrow, renal, pulmonary and cardiovascular tissue is demonstrated and discussed. These studies show restoration of injured cell function and the importance of heterogeneity of different vesicle populations. The potential for neural regeneration is explored, and the capacity to promote and reverse neoplasia by EV exposure is described. The tremendous clinical potential of EVs underlies many of these projects, and the importance of regulatory issues and the necessity of general manufacturing production (GMP) studies for eventual clinical trials are emphasized. Clinical trials are already being pursued and should expand dramatically in the near future.

Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells.

BACKGROUND: Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype. METHODS: EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB. RESULTS: This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs. CONCLUSIONS: Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

The emergence of extracellular vesicles in urology: fertility, cancer, biomarkers and targeted pharmacotherapy.

Extracellular vesicles (EV) are small membrane-bound vesicles enriched in a selective repertoire of mRNA, miRNA, proteins and cell surface receptors from parental cells and are actively involved in the transmission of inter and intracellular signals. Cancer cells produce EV that contain cargo including DNA, mRNA, miRNA and proteins that allow EV to create epigenetic changes in target cells both locally and systemically. Cancer-derived EV play critical roles in tumorigenesis, cancer cell migration, metastasis, evasion of host immune defense, chemoresistance, and they promote a premetastatic niche favourable to micrometastatic seeding. Their unique molecular profiles acquired from originator cells and their presence in numerous body fluids, including blood and urine, make them promising candidates as biomarkers for prostate, renal and bladder cancers. EV may ultimately serve as targets for therapy and as platforms for personalized medicine in urology. As urologic malignancy comprises 28% of new solid tumour diagnoses and 15% of cancer-related deaths, EV-related research is rapidly emerging and providing unique insights into disease progression. In this report, we review the current literature on EV in the setting of genitourinary fertility and malignancy.

Extracellular Vesicle-Mediated Reversal of Paclitaxel Resistance in Prostate Cancer.

Prostate cancer (PCa) is the most common solid tumor in males and the second leading cause of cancer-related deaths in males in the United States. The current first line therapy for metastatic PCa is androgen deprivation therapy and is initially effective against the disease. However, castrate resistant prostate cancer (CRPC) develops in many men within 18-36 months, rendering this treatment ineffective. Chemotherapy, with a class of drugs known as taxanes is the standard-of-care cytotoxic option in metastatic castrate resistant PCa (mCRPC). However, the overall survival advantage for chemotherapy in mCRPC is only 2.2 months and the cancer cells often become resistant to these drugs as well. Once patients fail chemotherapy the progression to death is inevitable. Extracellular vesicles (EVs) are involved in cell signaling and play a role in cancer progression. Previous work has demonstrated that EVs are involved in the development of drug resistance in cancer cells. We report the reversal of taxane resistance and tumorigenic phenotype in PCa cells after EVs treatment. This study suggests that EVs represent a potentially novel therapeutic treatment option for CRPC.

Epigenetic/Genetic Mismatch: Using Transdifferentiation as a Potential Cancer Therapy to Exploit the Cell Type Specificity of Cancer.

Every cell type capable of proliferation can be malignantly transformed. However, there appears to be no naturally occurring universal set of genetic mutations capable of converting every cell type to a malignant state. Any specific cell type is generally resistant to transformation by the cancer mutations accumulated by cells of different lineages, presumably due to epigenetic differences. Evidence for this idea derives from experiments in which the developmental fates of cancer cells are altered to reduce malignancy. Reprogramming cancer cells to more primitive developmental states using pluripotency factors (IPS) or somatic nuclear transfer suppresses the malignant phenotype, as does subsequent directed differentiation to mature cells of lineages distinct from the originating cell. Direct transdifferentiation to an alternative cell fate also reduces tumorigenicity. In contrast, after reprogramming, cells induced to redifferentiate toward the original tumor cell type are tumorigenic. In these types of experiments an epigenetic/genetic mismatch often results in suppression of malignancy or cell death. Elucidating the specific transcription and cell signaling network incompatibilities will identify new targets for cancer therapy. Moreover, novel strategies to induce an incompatible transdifferentiated state, in which expression of thousands of genes are altered, will prove useful in controlling malignancies that otherwise easily evolve resistance to single target-based therapeutics. Engineering small molecules, genetic vectors, cytokines, growth factors, targeted extracellular vesicles, and cell fusion will help realize transdifferentiation-based therapeutics for cancer.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Raf kinase inhibitor protein (RKIP) blocks signal transducer and activator of transcription 3 (STAT3) activation in breast and prostate cancer.

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding-protein (PEBP) family that modulates the action of many kinases involved in cellular growth, apoptosis, epithelial to mesenchymal transition, motility, invasion and metastasis. Previously, we described an inverse association between RKIP and signal transducers and activators of transcription 3 (STAT3) expression in gastric adenocarcinoma patients. In this study, we elucidated the mechanism by which RKIP regulates STAT3 activity in breast and prostate cancer cell lines. RKIP over expression inhibited c-Src auto-phosphorylation and activation, as well as IL-6-, JAK1 and 2-, and activated Raf-mediated STAT3 tyrosine and serine phosphorylation and subsequent activation. In MDA-231 breast cancer cells that stably over express RKIP, IL-6 treatment blocked STAT3 phosphorylation and transcriptional activation. Conversely, in RKIP knockdown MDA-231 cells: STAT3 phosphorylation and activation increased in comparison to parental MDA-231 cells. RKIP over expression resulted in constitutive physical interaction with STAT3 and blocked c-Src and STAT3 association. The treatment of DU145 prostate, but not PC3 prostate or MDA-231 breast, cancer cell lines with ENMD-1198 or MKC-1 dramatically increased expression of RKIP. Overexpression of RKIP sensitized PC3 and MDA-231 cells to MTI-induced apoptosis. Moreover, MTI treatment resulted in a decrease in Src-mediated STAT3 tyrosine phosphorylation and activation, an effect that was significantly enhanced by RKIP over expression. In stable RKIP over expressing MDA-231 cells, tumor xenograft growth induced by activated STAT3 is inhibited. RKIP synergizes with MTIs to induce apoptosis and inhibit STAT3 activation of breast and prostate cancer cells. RKIP plays a critical role in opposing the effects of pro-oncogenic STAT3 activation.

Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging.

Mesenchymal stem cells (MSCs) contribute to the recovery of tissue injury, providing a paracrine support. Cell-derived extracellular vesicles (EVs), carrying membrane and cytoplasmatic constituents of the cell of origin, have been described as a fundamental mechanism of intercellular communication. We previously demonstrated that EVs derived from human MSCs accelerated recovery following acute kidney injury (AKI) in vivo. The aim of the present study was to investigate the biodistribution and the renal localization of EVs in AKI. For this purpose, two methods for EV labeling suitable for in vivo tracking with optical imaging (OI), were employed using near infrared (NIR) dye (DiD): i) labeled EVs were generated by MSCs pre-incubated with NIR dye and collected from cell supernatants; ii) purified EVs were directly labeled with NIR dye. EVs obtained with these two procedures were injected intravenously (i.v.) into mice with glycerol-induced AKI and into healthy mice to compare the efficacy of the two labeling methods for in vivo detection of EVs at the site of damage. We found that the labeled EVs accumulated specifically in the kidneys of the mice with AKI compared with the healthy controls. After 5 h, the EVs were detectable in whole body images and in dissected kidneys by OI with both types of labeling procedures. The directly labeled EVs showed a higher and brighter fluorescence compared with the labeled EVs produced by cells. The signal generated by the directly labeled EVs was maintained in time, but provided a higher background than that of the labeled EVs produced by cells. The comparison of the two methods indicated that the latter displayed a greater specificity for the injured kidney.

Role of Raf kinase inhibitor protein in Helicobacter pylori-mediated signaling in gastric cancer.

Helicobacter pylori is a helical bacterium that colonizes the stomach in over half of the world's population. Infection with this bacterium has been linked to peptic ulcer disease and gastric cancer. The bacterium has been shown to affect regulatory pathways in its host cells through specific virulence factors that control gene expression. Infection with H. pylori increases levels of phosphorylation of Raf kinase inhibitor protein (pRKIP) in gastric adenocarcinoma (AGS) cells in vitro and in vivo. We investigated the role of H. pylori in the phosphorylation of RKIP as a possible mechanism to downregulate pro-survival signals in gastric adenocarcinoma. pRKIP induces RKIP transcriptional activity, which serves to induce apoptosis of damaged cells to prevent further tumorigenesis. Infection of wild type and RKIP knockout mice with H. pylori for 2 months further confirmed roles of RKIP and pRKIP in the prevention of gastric cancer progression. Loss of RKIP in AGS cells results in increased expression of the Cag A virulence factor after H. pylori infection and RKIP overexpression inhibits H. pylori-mediated STAT3 phosphorylation and STAT3 and NF-κB transcriptional activity. We examined the role of mTOR (mammalian target of rapamycin) after H. pylori infection on the phosphorylation of RKIP. Cells treated with rapamycin, an inhibitor of mTOR, displayed less expression of pRKIP after H. pylori infection. Microarray antibody analysis was conducted on wild-type and RKIP-knockdown AGS cells and showed that in the absence of RKIP, there was increased expression of pro-tumorigenic proteins such as EGFR, Raf-1, and MAPKs. Although further work is needed to confirm the interaction of RKIP and mTOR in AGS cells as a result of H. pylori infection, we hypothesize that H. pylori-mediated induction of pro-survival signaling in gastric epithelial cells induces a feedback response through the activation of RKIP. The phosphorylated, or active, form of RKIP is important in protecting gastric epithelial cells from tumorigenesis after H. pylori infection.

Clinical implications for loss or diminution of expression of Raf-1 kinase inhibitory protein and its phosphorylated form in ductal breast cancer.

Raf Kinase inhibitory protein (RKIP) is a well-established metastasis suppressor that is frequently downregulated in aggressive cancers. The impact of RKIP and its phosphorylated form on disease-free survival (DFS) and other clinicopathological parameters in breast cancer is yet to be discovered. To this end, we examined RKIP expression in 3 independent breast cancer cohorts. At the Protein level, loss or reduced total RKIP expression was associated with large-sized tumors characterized by high proliferative index, high-grade and diminished estrogen (ER) and progesterone receptor expression. Loss or diminution of RKIP expression was significantly associated with shorter DFS in all cohorts. Moreover, the complete loss of p-RKIP was an independent prognostic factor using multivariate analysis in operable invasive ductal breast cancer. We show for the first time that ER, partly, drives RKIP expression through MTA3-Snail axis. Consistent with this finding, we found that, at the mRNA level, RKIP expression varied significantly across the different molecular subtypes of breast cancer with the Luminal (ER+) subtype expressing high levels of RKIP and the more aggressive Claudin-low (ER-) subtype, which depicted the highest epithelial to mesenchymal transition (EMT) registered the lowest RKIP expression levels. In conclusion, loss of expression/diminution of RKIP or its phosphorylated form is associated with poor diseases-free survival in breast cancer. Determining the expression of RKIP and p-RKIP adds significant prognostic value to the management and subtyping of this disease.

RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients.

BACKGROUND: A major obstacle in treating colorectal cancer (CRC) is the acquired resistance to chemotherapeutic agents. An important protein in the regulation of cancer cell death and clinical outcome is Raf kinase inhibitor protein (RKIP). In contrast, activated signal transducer and activator of transcription 3 (STAT3) is a protein that promotes tumor cell survival by inhibiting apoptosis and has an important role in cancer progression in many of cancer types. The aim of this study was to evaluate the regulation of RKIP and STAT3 after treatment with clinically relevant chemotherapeutic agents (camptothecin (CPT) and oxaliplatin (OXP)) and the cytokine interleukin-6 (IL-6) in HCT116 colon cancer cells as well as evaluate the association between RKIP and STAT3 with clinical outcome of Stage II colon cancer patients. METHODS: HCT-116 colon cancer cells were treated with CPT, OXP, and IL-6 separately or in combination in a time and dose-dependent manner and examined for phosphorylated and non-phosphorylated RKIP and STAT3 via Western blot analysis. STAT3 transcriptional activity was measured via a luciferase reporter assay in HCT116 cells treated with CPT, IL-6 or transfected with JAK 1, 2 separately or in combination. We extended these observations and determined STAT3 and RKIP/ pRKIP in tumor microarrays (TMA) in stage II colon cancer patients. RESULTS: We demonstrate IL-6-mediated activation of STAT3 occurs in conjunction with the phosphorylation of RKIP in vitro in human colon cancer cells. OXP and CPT block IL-6 mediated STAT3 activation and RKIP phosphorylation via the inhibition of the interaction of STAT3 with gp130. We determined that STAT3 and nuclear pRKIP are significantly associated with poor patient prognosis in stage II colon cancer patients. CONCLUSIONS: In the analysis of tumor samples from stage II colon cancer patients and the human colon carcinoma cell line HCT116, pRKIP and STAT3, 2 proteins potentially involved in the resistance to conventional treatments were detected. The phosphorylation of pRKIP and STAT3 are induced by the cytokine IL-6 and suppressed by the chemotherapeutic drugs CPT and OXP. Therefore, these results suggest that STAT3 and pRKIP may serve as prognostic biomarkers in stage II colon cancer patients and may improve chemotherapy.

Reversal of chemosensitivity and induction of cell malignancy of a non-malignant prostate cancer cell line upon extracellular vesicle exposure.

BACKGROUND: Extracellular vesicle (EV) trafficking is a fundamental cellular process that occurs in cells and is required for different aspects of pathophysiology. EV trafficking leads to changes in cellular function including apoptosis, angiogenesis and proliferation required for increased tumor formation. RESULTS: We report several phenotypic changes mediated by EVs isolated from non-malignant and malignant prostate cells as well as patient biopsied prostate tumor samples. EVs can reverse the resistance of prostate cancer cells to camptothecin EVs isolated from non-malignant PrECs (Prostate Epithelial Cells) can reverse soft agar colony formation of malignant DU145 cells, with the reciprocal effect observed. Isolation of EVs from 2 Gleason grade 8 prostate cancer patients significantly induced soft agar colony formation of non-malignant PrECs. We have identified proteins via antibody and Mass spectrometry analysis that may be responsible for the phenotypic changes. Mass spectrometry analysis of protein lysates using ProteoIQ revealed protein candidates associated with gene ontology annotations that may be responsible for this phenotypic change. Ingenuity Pathway Analysis was used to identify statistically relevant canonical pathways and functions associated the protein IDs and expression values obtained using ProteoIQ. Western blot analysis confirmed the increase of 14-3-3 zeta, pRKIP and prohibitin protein levels in PrEC cells co-cultured with patient EVs. 14-3-3 proteins were also found as common proteins of 3 other Gleason grade 8 patients. CONCLUSION: Our study provides a rational basis to further investigate putative proteins, such as 14-3-3 and prohibitin and genetic factors that may be responsible for phenotypic changes that are associated with prostate cancer progression.

Raf kinase inhibitor RKIP inhibits MDA-9/syntenin-mediated metastasis in melanoma.

Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, functions as a positive regulator of melanoma progression and metastasis. In contrast, the Raf kinase inhibitor, RKIP, a negative modulator of RAF-stimulated MEKK activation, is strongly downregulated in metastatic melanoma cells. In this study, we explored a hypothesized inverse relationship between MDA-9 and RKIP in melanoma. Tumor array and cell line analyses confirmed an inverse relationship between expression of MDA-9 and RKIP during melanoma progression. We found that MDA-9 transcriptionally downregulated RKIP in support of a suggested cross-talk between these two proteins. Furthermore, MDA-9 and RKIP physically interacted in a manner that correlated with a suppression of FAK and c-Src phosphorylation, crucial steps necessary for MDA-9 to promote FAK/c-Src complex formation and initiate signaling cascades that drive the MDA-9-mediated metastatic phenotype. Finally, ectopic RKIP expression in melanoma cells overrode MDA-9-mediated signaling, inhibiting cell invasion, anchorage-independent growth, and in vivo dissemination of tumor cells. Taken together, these findings establish RKIP as an inhibitor of MDA-9-dependent melanoma metastasis, with potential implications for targeting this process therapeutically.

Regulation of RKIP function by Helicobacter pylori in gastric cancer.

Helicobacter pylori (H. pylori) is a gram-negative, spiral-shaped bacterium that infects more than half of the world's population and is a major cause of gastric adenocarcinoma. The mechanisms that link H. pylori infection to gastric carcinogenesis are not well understood. In the present study, we report that the Raf-kinase inhibitor protein (RKIP) has a role in the induction of apoptosis by H. pylori in gastric epithelial cells. Western blot and luciferase transcription reporter assays demonstrate that the pathogenicity island of H. pylori rapidly phosphorylates RKIP, which then localizes to the nucleus where it activates its own transcription and induces apoptosis. Forced overexpression of RKIP enhances apoptosis in H. pylori-infected cells, whereas RKIP RNA inhibition suppresses the induction of apoptosis by H. pylori infection. While inducing the phosphorylation of RKIP, H. pylori simultaneously targets non-phosphorylated RKIP for proteasome-mediated degradation. The increase in RKIP transcription and phosphorylation is abrogated by mutating RKIP serine 153 to valine, demonstrating that regulation of RKIP activity by H. pylori is dependent upon RKIP's S153 residue. In addition, H. pylori infection increases the expression of Snail, a transcriptional repressor of RKIP. Our results suggest that H. pylori utilizes a tumor suppressor protein, RKIP, to promote apoptosis in gastric cancer cells.

Expression of phosphorylated raf kinase inhibitor protein (pRKIP) is a predictor of lung cancer survival.

BACKGROUND: Raf-1 kinase inhibitor protein (RKIP) has been reported to negatively regulate signal kinases of major survival pathways. RKIP activity is modulated in part by phosphorylation on Serine 153 by protein kinase C, which leads to dissociation of RKIP from Raf-1. RKIP expression is low in many human cancers and represents an indicator of poor prognosis and/or induction of metastasis. The prognostic power has typically been based on total RKIP expression and has not considered the significance of phospho-RKIP. METHODS: The present study examined the expression levels of both RKIP and phospho-RKIP in human lung cancer tissue microarray proteomics technology. RESULTS: Total RKIP and phospho-RKIP expression levels were similar in normal and cancerous tissues. phospho-RKIP levels slightly decreased in metastatic lesions. However, the expression levels of phospho-RKIP, in contrast to total RKIP, displayed significant predictive power for outcome with normal expression of phospho-RKIP predicting a more favorable survival compared to lower levels (P = 0.0118); this was even more pronounced in more senior individuals and in those with early stage lung cancer. CONCLUSIONS: This study examines for the first time, the expression profile of RKIP and phospho-RKIP in lung cancer. Significantly, we found that phospho-RKIP was a predictive indicator of survival.

Microvesicle induction of prostate specific gene expression in normal human bone marrow cells.

PURPOSE: Transfer of genetic material from cancer cells to normal cells occurs via microvesicles. Cell specific phenotypes can be induced in normal cells by the transfer of material in microvesicles, leading to genetic changes. We report the identification and expression of prostate specific genes in normal human marrow cells co-cultured with human prostate cancer cells. MATERIALS AND METHODS: We harvested prostate tissue from 11 patients with prostate cancer. In 4 cases prostate tissue was co-cultured across from human marrow for 2 or 7 days but separated from it by a 0.4 µM polystyrene membrane. In 5 cases conditioned medium from patient cancer tissue was collected and ultracentrifuged, and microvesicles were collected for co-culture (3) and vesicle characterization (3). Explanted human marrow was harvested from cultures and RNA extracted. Real-time reverse transcriptase-polymerase chain reaction was done for select prostate specific genes. RESULTS: Marrow exposed to human prostate tumor or isolated microvesicles in culture in 4 and 3 cases, respectively, showed at least 2-fold or greater prostate gene expression than control marrow. In 1 case in which normal prostate was co-cultured there were no prostate gene increases in normal marrow. CONCLUSIONS: Prostate cancer tumor cells co-cultured with human bone marrow cells induce prostate specific gene expression. The proposed mechanism of transfer of genetic material is via microvesicles. This represents an opportunity for novel therapeutic agents, such as antibodies, to block microvesicle release from cancer cells or for agents that may block cells from accepting microvesicles.

Novel therapeutic applications of nitric oxide donors in cancer: roles in chemo- and immunosensitization to apoptosis and inhibition of metastases.

The treatment of primary tumors results in an initial response to approved conventional therapeutics. However, recurrences and malignancies develop as a result of tumors' acquisition of anti-apoptotic mechanisms of resistance. Hence, there is an urgent need of novel therapeutics that can reverse resistance. One approach of interest is the inhibition of cell survival and anti-apoptotic pathways by sensitizing agents that can render resistant tumor cells sensitive to respond to various cytotoxic therapies. We have found that nitric oxide donors, similar to DETANONOate, inhibit cell survival anti-apoptotic pathways, such as the constitutively activated NF-kappaB and sensitize drug-resistant tumor cells to apoptosis by both chemotherapy and immunotherapy. Sensitization by DETANONOate was shown to inhibit the transcription repressor Yin Yang1 (YY1) shown to regulate resistance to both Fas ligand and TRAIL. In addition, DETANONOate-induced inhibition of NF-kappaB results downstream in the inhibition of several anti-apoptotic gene products, thus facilitating the activation of the apoptotic pathways with both chemotherapy and immunotherapy. In addition, DETANONOate induces the expression of the metastatic tumor suppressor gene product, Raf-1 Kinase Inhibitor Protein (RKIP), which inhibits the survival pathways induced by NF-kappaB and Raf-1/MEK which also contributes to the sensitizing activity. This indicates a novel finding that RKIP may also play an important role in the prevention of metastasis. Inhibition of NF-kappaB activation by DETANONOate results downstream in the inhibition of the RKIP transcription repressor Snail, resulting in upregulation of RKIP. Inhibition of Snail results in downstream inhibition of the metastatic cascade initiated by the epithelial-mesenchymal transition (EMT). Thus, nitric oxide donors have the dual functions of both sensitizing tumor cells to chemotherapy and immunotherapy and are also involved in the regulation and inhibition of metastasis.

Inverse association between Raf Kinase Inhibitory Protein and signal transducers and activators of transcription 3 expression in gastric adenocarcinoma patients: implications for clinical outcome.

PURPOSE: Raf Kinase Inhibitory Protein (RKIP) plays a pivotal role in cancer by regulating apoptosis induced by chemotherapeutic agents, or immune-mediated stimuli and is a metastasis suppressor protein. The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is frequently activated in gastric adenocarcinomas, thereby promoting tumor growth. We examined the expression patterns of RKIP and STAT3 with regard to human gastric cancer, predicting that elevated RKIP status may favor clinical outcome. EXPERIMENTAL DESIGN: Tissue microarrays were created from samples from 143 patients with gastric adenocarcinomas. The microarrays were immunohistochemically stained for RKIP and STAT3, and the intensity and extent of the staining was semiquantitatively scored. RESULTS: In intestinal-type gastric adenocarcinomas, RKIP and STAT3, expression were inversely associated. Cytoplasmic RKIP expression directly correlated with patient survival. Nuclear STAT3 expression inversely correlated with survival. In the diffuse tumor type, no significant correlation was found between RKIP and patient outcome. In the intestinal-type gastric adenocarcinoma, multivariate analysis adjusted for treatment types revealed RKIP and tumor stage to be significant independent predictors of survival. In the diffuse tumor type, stage was the only significant predictor of survival. CONCLUSION: These results indicate the predictive and protective role of cytoplasmic RKIP expression in gastric adenocarcinoma of the intestinal subtype. In contrast, nuclear STAT3 expression is associated with poor patient prognosis in the intestinal subtype. Significantly, we show an inverse association between RKIP and STAT3 and a positive correlation between RKIP and patient survival.

Regulation of tumor cell sensitivity to TRAIL-induced apoptosis by the metastatic suppressor Raf kinase inhibitor protein via Yin Yang 1 inhibition and death receptor 5 up-regulation.

Raf-1 kinase inhibitor protein (RKIP) has been implicated in the regulation of cell survival pathways and metastases, and is poorly expressed in tumors. We have reported that the NF-kappaB pathway regulates tumor resistance to apoptosis by the TNF-alpha family via inactivation of the transcription repressor Yin Yang 1 (YY1). We hypothesized that RKIP overexpression may regulate tumor sensitivity to death ligands via inhibition of YY1 and up-regulation of death receptors (DRs). The TRAIL-resistant prostate carcinoma PC-3 and melanoma M202 cell lines were examined. Transfection with CMV-RKIP, but not with control CMV-EV, sensitized the cells to TRAIL-mediated apoptosis. Treatment with RKIP small interfering RNA (siRNA) inhibited TRAIL-induced apoptosis. RKIP overexpression was paralleled with up-regulation of DR5 transcription and expression; no change in DR4, decoy receptor 1, and decoy receptor 2 expression; and inhibition of YY1 transcription and expression. Inhibition of YY1 by YY1 siRNA sensitized the cells to TRAIL apoptosis concomitantly with DR5 up-regulation. RKIP overexpression inhibited several antiapoptotic gene products such as X-linked inhibitor of apoptosis (XIAP), c-FLIP long, and Bcl-x(L) that were accompanied with mitochondrial membrane depolarization. RKIP overexpression in combination with TRAIL resulted in the potentiation of these above effects and activation of caspases 8, 9, and 3, resulting in apoptosis. These findings demonstrate that RKIP overexpression regulates tumor cell sensitivity to TRAIL via inhibition of YY1, up-regulation of DR5, and modulation of apoptotic pathways. We suggest that RKIP may serve as an immune surveillance cancer gene, and its low expression or absence in tumors allows the tumor to escape host immune cytotoxic effector cells.