PDF file - 171K, Effect of ectopic expression of RKIP on cytoskeletal organization in melanoma cells

PDF file - 604K, MDA-9 activates ERK1/2

PDF file - 652K, Characterization of MDA-9 overexpressed FM-516 clones

PDF file - 622K, Effects of RKIP expression on cell growth

PDF file - 205K, Expression of RKIP mRNA in different melanoma cells

PDF file - 1.8MB, Expression of MDA-9 and RKIP in clinical samples

PDF file - 180K, Effect of RKIP expression on B-RAF mutant cells

PDF file - 413K, ERK activation negatively regulate RKIP expression

In mammalian cells, histone deacetylase (HDAC) and Sirtuin(SIRT) are twofamilies 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.

Background: Multiple myeloma (MM) remains an incurable disease. We have reported that MM cell lines and patient-derived MM cells overexpress the gene product Raf-Kinase Inhibitor Protein (RKIP) in its inactivated phosphorylated (p-RKIP) form.1 Active RKIP inhibits both the Raf/MEK/ERK and NF-κB pathways and resulting in the inhibition of proliferation, metastasis, and sensitization to chemo and immune drugs.2

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.

Abstract Background: The purpose of this study was to delineate differences in the mechanism of action (MOA) and to identify unique molecular targets responsible for the antineoplastic effects of PT-112 when compared to oxaliplatin. PT-112 is a novel platinum-based chemotherapeutic agent currently under clinical development that has demonstrated superior efficacy in resistant cell lines and xenograft models. In this study we focus on the differential mechanistic effects of PT-112 and oxaliplatin treatment of HCT-116 colon cancer cells and report data derived from multiple cancer signaling pathways. Results: Treatment of HCT-116 with the IC50 dose of PT-112 and oxaliplatin for 24 and 48h resulted in growth inhibition and apoptosis induction as measured by MTT assay, pro-caspase 8 and PARP cleavage. At equipotent doses, oxaliplatin treatment resulted in greater DNA damage as measured by H2A.X serine phosphorylation when compared to PT-112, whereas PT-112 induced markedly greater degrees of expression of several proteins in multiple pathways, including p53, p16, and FasL. PT-112's activity may derive more directly from p53 expression, when compared to oxaliplatin, as revealed by growth inhibition assays using and a p53-null HCT-116 cell line. Additionally, PT-112 triggered the cleavage of executioner pro-caspases 3, 6 and 7 to a greater extent vs oxaliplatin. When compared to oxaliplatin, PT-112 treatment resulted in the significant inhibition of gp130 and JAK/STAT signaling and transcriptional activation mediated by IL-6. A decrease, relative to oxaliplatin, in TNF-mediated NF-κB signaling and expression of cell-cycle factors E2F3, CDK4 and CDK1, as well as cell survival protein c-FLIPshort were also observed. PT-112 was more potent than oxaliplatin in inducing the release of High-mobility group protein B1, a marker for immunogenic cell death (ICD) processes. Additionally, the inhibition of the malignant phenotype, as assessed by anchorage independent growth, was significantly greater in cells incubated with PT-112 when compared to oxaliplatin. When treating non-malignant 1459 colon cells or biopsied benign normal colon tissue grown in culture, PT-112 did not result in apoptosis at concentrations much higher than those that were used in the HCT-116 experiments. In contrast, oxaliplatin remained equivalently potent in the non-malignant cell lines. Conclusions: PT-112's ability to affect numerous intracellular proteins, and the evidence of extracellular initiation of anticancer signaling, along with the apparent reduced dependence on DNA-damage, makes it an attractive and versatile compound, particularly as it relates to potential drug resistance mechanisms. PT-112's inhibition of STAT3 activation and induction of ICD also offers the intriguing proposition of downstream immune-potentiating therapeutic effects. These results underline a unique rationale for further clinical evaluation of PT-112. In conclusion, our study has demonstrated that PT-112 treatment simultaneously regulates multiple cellular targets including apoptosis, cell survival, tumor suppressor and cell cycle proteins and pathways in a manner that is clearly differentiated from oxaliplatin. Citation Format: Justin Q. Wang, Tyler Ames, Emily Arciero, Marie-Therese Hehenberger, Devasis Chatterjee. Characterization of molecular targets of the novel platinum agent PT-112 in human colon cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C32.

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.

e16111 Background: Castrate resistant prostate cancer (CRPC) is the second leading cause of cancer-related death and nearly all men develop castrate resistance. Several new therapies, including enzalutamide- and taxane-based chemotherapy have improved outcomes for CRPC. However, resistance to both therapies develops in over 40% of patients. Tumor cells release extracellular vesicles (EV), which can alter the tumor microenvironment and promote disease progression. We examined the ability of EV isolated from non-malignant cells and human mesenchymal stem cells (hMSC EV) to inhibit the malignant phenotype and reverse drug resistance. Methods: EV were isolated from hMSC EV, non-malignant, malignant prostate cells and from biopsied tissue from patients with high grade prostate cancer (PCa). EV were co-cultured with recipient non- or malignant prostate cells and/or paclitaxel and enzalutamide sensitive or resistant cells after which: the induction or transfer of proteins was determined via mass spectrometry and Western blot analysis; and anchorage independent and tumor xenograft growth were assessed and sensitivity to enzalutamide and paclitaxel was monitored via MTT assay. Results: High Gleason grade PCa patient EV significantly induced anchorage independent growth and cell migration and the malignant phenotype of non-malignant prostate cells. Antibody analysis and mass spectrometry determined proteins that may be responsible for EV-mediated phenotypic changes. We identified increased protein levels of putative mediators of the EV-induced changes including 14-3-3 zeta in (log p value < 0.05 as determined by the Comparison Analysis Tool in the IPA software) in non-malignant cells co-cultured with PCa patient EV. Knockdown of 14-3-3 zeta lead to partial reversal of soft agar growth, indicating that it is a potential candidate for targeted therapy. Co-culture of malignant PCa cells with non-malignant prostate EV or EV isolated from hMSC significantly inhibited tumor xenograft growth and reversed enzalutamide and taxane resistance. Conclusions: Our study provides a rational basis to evaluate the therapeutic use of non malignant-derived EV or hMSC EV to inhibit CRPC progression and reverse enzalutamide or taxane resistance.

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.

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.

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.