Microfluidic Generated EGF-Gradients Induce Chemokinesis of Transplantable Retinal Progenitor Cells via the JAK/STAT and PI3Kinase Signaling Pathways

A growing number of studies are evaluating retinal progenitor cell (RPC) transplantation as an approach to repair retinal degeneration and restore visual function. To advance cell-replacement strategies for a practical retinal therapy, it is important to define the molecular and biochemical mechanisms guiding RPC motility. We have analyzed RPC expression of the epidermal growth factor receptor (EGFR) and evaluated whether exposure to epidermal growth factor (EGF) can coordinate motogenic activity in vitro. Using Boyden chamber analysis as an initial highthroughput screen, we determined that RPC motility was optimally stimulated by EGF concentrations in the range of 20-400ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGFinduced motility. Using bioinformatics analysis of the EGF ligand in a retina-specific gene network pathway, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3kinase signaling pathways are necessary to drive RPC motility. Using engineered microfluidic devices to generate quantifiable steady-state gradients of EGF coupled with live-cell tracking, we analyzed the dynamics of individual RPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic with optimal activity observed in response to low concentration gradients. Our combined results show that EGFR expressing RPCs exhibit enhanced chemokinetic motility in the presence of low nanomole levels of EGF. These findings may serve to inform further studies evaluating the extent to which EGFR activity, in response to endogenous ligand, drives motility and migration of RPCs in retinal transplantation paradigms

MicroRNA-4719 and microRNA-6756-5p Correlate with Castration-Resistant Prostate Cancer Progression through Interleukin-24 Regulation

Prostate cancer (PCa) is the second leading cause of cancer death in the United States. The five-year survival rate for men diagnosed with localized PCa is nearly 100%, yet for those diagnosed with aggressive PCa, it is less than 30%. The pleiotropic cytokine Interleukin-24 (IL-24) has been shown to specifically kill PCa cells compared to normal cells when overexpressed in both in vitro and in vivo studies. Despite this, the mechanisms regulating IL-24 in PCa are not well understood. Since specific microRNAs (miRNAs) are dysregulated in PCa, we used miRNA target prediction algorithm tools to identify miR-4719 and miR-6556-5p as putative regulators of IL-24. This study elucidates the expression profile and role of miR-4719 and miR-6756-5p as regulators of IL-24 in PCa. qRT-PCR analysis shows miR-4719 and miR-6756-5p overexpression significantly decreases the expression of IL-24 in PCa cells compared to the negative control. Compared to the indolent PCa and normal prostate epithelial cells, miR-4719 and miR-6756-5p are significantly overexpressed in castration-resistant prostate cancer (CRPC) cell lines, indicating that their gain may be an early event in PCa progression. Moreover, miR-4719 and miR-6756-5p are significantly overexpressed in the CRPC cell line of African-American males (E006AA-hT) compared to CRPC cell lines of Caucasian males (PC-3 and DU-145), indicating that miR-4719 and miR-6756-5p may also play a role in racial disparity. Lastly, the inhibition of expression of miR-4719 and miR-6756-5p significantly increases IL-24 expression and inhibits proliferation and migration of CRPC cell lines. Our findings indicate that miR-4719 and miR-6756-5p may regulate CRPC progression through the targeting of IL-24 expression and may be biomarkers that differentiate between indolent and CRPC. Strategies to inhibit miR-4719 and miR-6756-5p expression to increase IL-24 in PCa may have therapeutic efficacy in aggressive PCa

Modulation of the Pol II CTD Phosphorylation Code by Rac1 and Cdc42 Small GTPases in Cultured Human Cancer Cells and Its Implication for Developing a Synthetic-Lethal Cancer Therapy

Rho GTPases, including Rho, Cdc42, Rac and ROP subfamilies, are key signaling molecules in RNA polymerase II (Pol II) transcriptional control. Our prior work has shown that plant ROP and yeast Cdc42 GTPases similarly modulate Ser2 and Ser5 phosphorylation status of the C-terminal domain (CTD) of the Pol II largest subunit by regulating CTD phosphatase degradation. Here, we present genetic and pharmacological evidence showing that Cdc42 and Rac1 GTPase signaling modulates a similar CTD Ser2 and Ser5 phosphorylation code in cultured human cancer cells. While siRNA knockdown of Cdc42 and Rac1, respectively, in HeLa cells increased the level of CTD Ser phosphatases RPAP2 and FCP1, they both decreased the level of CTD kinases CDK7 and CDK13. In addition, the protein degradation inhibitor MG132 reversed the effect of THZ1, a CDK7 inhibitor which could decrease the cell number and amount of CDK7 and CDK13, accompanied by a reduction in the level of CTD Ser2 and Ser5 phosphorylation and DOCK4 and DOCK9 (the activators for Rac1 and Cdc42, respectively). Conversely, treatments of Torin1 or serum deprivation, both of which promote protein degradation, could enhance the effect of THZ1, indicating the involvement of protein degradation in controlling CDK7 and CDK13. Our results support an evolutionarily conserved signaling shortcut model linking Rho GTPases to Pol II transcription across three kingdoms, Fungi, Plantae and Animalia, and could lead to the development of a potential synthetic-lethal strategy in controlling cancer cell proliferation or death

Predicted molecular signaling guiding photoreceptor cell migration following transplantation into damaged retina

To replace photoreceptors lost to disease or trauma and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal transplantation of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). Significant obstacles to advancement of photoreceptor cell-replacement include low migration rates of transplanted cells into host retina and an absence of data describing chemotactic signaling guiding migration of transplanted cells in the damaged retinal microenvironment. To elucidate chemotactic signaling guiding transplanted cell migration, bioinformatics modeling of PPC transplantation into light-damaged retina was performed. The bioinformatics modeling analyzed whole-genome expression data and matched PPC chemotactic cell-surface receptors to cognate ligands expressed in the light-damaged retinal microenvironment. A library of significantly predicted chemotactic ligand-receptor pairs, as well as downstream signaling networks was generated. PPC and RPC migration in microfluidic ligand gradients were analyzed using a highly predicted ligand-receptor pair, SDF-1α - CXCR4, and both PPCs and RPCs exhibited significant chemotaxis. This work present a systems level model and begins to elucidate molecular mechanisms involved in PPC and RPC migration within the damaged retinal microenvironment

Translation Control by p53

The translation of mRNAs plays a critical role in the regulation of gene expression and therefore, in the regulation of cell proliferation, differentiation and apoptosis. Unrestricted initiation of translation causes malignant transformation and plays a key role in the maintenance and progression of cancers. Translation initiation is regulated by the ternary complex and the eukaryotic initiation factor 4F (eIF4F) complex. The p53 tumor suppressor protein is the most well studied mammalian transcription factor that mediates a variety of anti-proliferative processes. Post-transcriptional mechanisms of gene expression in general and those of translation in particular play a major role in shaping the protein composition of the cell. The p53 protein regulates transcription and controls eIF4F, the ternary complex and the synthesis of ribosomal components, including the down-regulation of rRNA genes. In summary, the induction of p53 regulates protein synthesis and translational control to inhibit cell growth

IL-24 Promotes Apoptosis through cAMP-Dependent PKA Pathways in Human Breast Cancer Cells

Interleukin 24 (IL-24) is a tumor-suppressing protein, which inhibits angiogenesis and induces cancer cell-specific apoptosis. We have shown that IL-24 regulates apoptosis through phosphorylated eukaryotic initiation factor 2 alpha (eIF2α) during endoplasmic reticulum (ER) stress in cancer. Although multiple stresses converge on eIF2α phosphorylation, the cellular outcome is not always the same. In particular, ER stress-induced apoptosis is primarily regulated through the extent of eIF2α phosphorylation and activating transcription factor 4 (ATF4) action. Our studies show for the first time that cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation is required for IL-24-induced cell death in a variety of breast cancer cell lines and this event increases ATF4 activity. We demonstrate an undocumented role for PKA in regulating IL-24-induced cell death, whereby PKA stimulates phosphorylation of p38 mitogen-activated protein kinase and upregulates extrinsic apoptotic factors of the Fas/FasL signaling pathway and death receptor 4 expression. We also demonstrate that phosphorylation and nuclear import of tumor suppressor TP53 occurs downstream of IL-24-mediated PKA activation. These discoveries provide the first mechanistic insights into the function of PKA as a key regulator of the extrinsic pathway, ER stress, and TP53 activation triggered by IL-24

Mechanism of Action and Applications of Interleukin 24 in Immunotherapy

Interleukin 24 (IL-24) is an important pleiotropic immunoregulatory cytokine, whose gene is located in human chromosome 1q32-33. IL-24’s signaling pathways have diverse biological functions related to cell differentiation, proliferation, development, apoptosis, and inflammation, placing it at the center of an active area of research. IL-24 is well known for its apoptotic effect in cancer cells while having no such effect on normal cells. IL-24 can also be secreted by both immune and non-immune cells. Downstream effects of IL-24, after binding to the IL-20 receptor, can occur dependently or independently of the JAK/STAT signal transduction pathway, which is classically involved in cytokine-mediated activities. After exogenous addition of IL-24, apoptosis is induced in tumor cells independently of the JAK/STAT pathway. We have shown that IL-24 binds to Sigma 1 Receptor and this event induces endoplasmic reticulum stress, calcium mobilization, reactive oxygen species generation, p38MAPK activity, and ceramide production. Here we review IL-24’s role in autoimmunity, infectious disease response, wound repair, and vascular disease. Detailed understanding of the pleiotropic roles of IL-24 signaling can assist in the selection of more accurate therapeutic approaches, as well as targeting of appropriate cell types in treatment strategy development, and ultimately achieve desired therapeutic effects

mda-7/IL-24, novel anticancer cytokine: Focus on bystander antitumor, radiosensitization and antiangiogenic properties and overview of the phase I clinical experience (Review)

Subtraction hybridization applied to a ‘differentiation therapy’ model of cancer employing human melanoma cells resulted in the cloning of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). Initial studies confirm an inverse correlation between mda-7 expression and melanoma development and progression. Forced expression of mda-7 by means of a plasmid or via a replication incompetent adenovirus (Ad.mda-7) promotes growth suppression and induces apoptosis in a broad array of human cancers. In contrast, mda-7 does not induce growth suppressive or toxic effects in normal cells. Based on structure (containing an IL-10 signature motif), secretion by cells (including subsets of T-cells) and location on chromosome 1q (in an area containing IL-10- family genes), mda-7 has now been renamed mda-7/IL-24. Studies by several laboratories have uncovered many of mda-7/ IL-24\u27s unique properties, including cancer-specific apoptosisinduction, cell cycle regulation, an ability to inhibit angiogenesis, potent ‘bystander antitumor activity’ and a capacity to enhance the sensitivity of tumor cells to radiation, chemo- therapy and monoclonal antibody therapy. Moreover, based on its profound cancer tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant clinical activity. In these contexts, mda-7/IL-24 represents a unique cytokine gene with potential for therapy of human cancers. The present review focuses on three unique properties of mda-7/IL-24, namely its potent ‘bystander antitumor activity’, ability to sensitize tumor cells to radiation, and its antiangiogenesis properties. Additionally, an overview of the phase I clinical trial is provided. These studies affirm that mda-7/IL-24 has promise for the management of diverse cancers

Signal transduction pathways in the mitogenic response to PGF 2 alfa LIF and related cytokines in Swiss 3T3 cells, including cyclins Ds and CDKs expression by diverse signalling pathways

Mammalian cell division, is a highly complex process, regulated and coordinated by mechanisms that are conserved through most species. The physiological control of eucariotic cell proliferation initiation is external, and it is excerted by humoral factors, made by the same or other cells, under certain requirements of the organism. Progression through the different phases of the cell cycle, is governed by a regulatory machinery conserved through most species, that not only coordinates the various events that made up the cell cycle, but also connects the cell cycle with extracellular signals, that regulates cell proliferation. Beginning with a given mitogenic stimulus acting through a specific receptor in a target cell, signalling mechanisms cascades are generated in the membrane and in the citosol of that cell. These early events, act on the cell cycle machinery, finally leading to cell division. The expression of proteins that regulate the cell cycle is in part induced by mitogen-stimulated signalling mechanisms. The passage from G0 to S phase, depends on the activity of cyclin-dependent kinases (CDKs). These kinases are CDK4 and CDK6, and they are activated when they form complexes with cyclins D (D1, D2 and D3), induced in the G1 phase. Cyclins D are considered as "sensors" of the extracellular medium, since their induction is triggered by mitogenic stimuli. The activated complexes cyclin D-CDK4 and cyclin D-CDKG catalyse the phosphorilation of the Rb protein. In Swiss 3T3 cells, PGF2α is capable of inducing DNA synthesis, by means of multiple signalling mechanisms, in the absence of other factors. However its mitogenic effect is potentiated by TGFβ1 addition. We have shown that PGF2α triggers cyclin D1 mRNA/protein expression prior to cellular entry into the S phase, but fails to raise CDK4 or cyclin D3 levels, while 1-oleoyl-2acetyllglycerol (OAG), a protein kinase C (PKC) and tyrosine kinase (TK) activator, induces only cyclin D1 expression with no mitogenic response. In contrast, in PKC-depleted or -inhibited cells, PGF2α, but not OAG, increases cyclin D1 expression with no mitogenic response. Finally, OAG, in the presence of orthovanadate (Na3VO4)or TGFβ1, induces DNA synthesis. Thus, it appears that PGF2α triggers cyclin D1 expression via two independent signalling events that complement with TGFβ1-triggered events to induce DNA synthesis. TGFβ1 cannot trigger cyclin D1 expression, but, stabilise cyclin D1 mRNA, after PGF2α-triggered its expression. Leukaemia inhibitory factor (LIF) was originally described on the basis of its ability to stimulate the differentiation of murine M1 leukemic cells into granulocytes and macrophages. In Swiss 3T3 cells, both LIF and prostaglandin F2α (PGF2α) trigger initiation of DNA synthesis and cell proliferation. LIF appears to exert its action through signals and processes markedly different from those elicited by PGF2α. While pre-treatment the cell culture with either GF 109203 (bysoindolmalemide), a specific PKC inhibitor, or 12-tetradecanoyl-13-phorbolacetate, which causes PKC down modulation, or lovastatin, known to block mevalonic acid synthesis and protein isoprenylation, totally impairs PGF2α mitogenic action. None of these treatments inhibited LIF-induced DNA replication. Agents capable of rising intracellular cAMP, enhanced both LIF and PGF2α ability to cause cellular entry into the S phase. However, H89 and PKI, both PKA inhibitors, prevented cAMP-mediated potentiation, but did not affect LIF induction of cellular entry into S phase. PD98059, a MEK (MAPKK)inhibitor, prevents PGF2α-mitogenic response but does not block LIF-induced initiation of DNA synthesis. Immunofluorescence studies revealed that LIF and PGF2α responses exhibit marked differences in STAT cytoplasmic-nuclear translocation. After 15 to 30 min, LIF causes STAT1 but not STAT3 or STAT5 translocation. In contrast, PGF2α failed to induce translocation of any of those transcriptional factors. Thus, it appears that LIF triggers mitogenic action through independent signalling events such as those involving PKC, PKA, MEK, p38MAPK and protein isoprenilation. In addition, its mitogenic effect is markedly potentiated by PKC, PKA, and probably PTK mediated signalling mechanisms. Western blot analyses of cyclin D1, D2 and D3 expression (implicated in most mitogen actions), revealed that PGF2α, after 7-9 h, caused an increase in cyclin D1 protein levels, and a later increase in cyclin D2 levels. In contrast, LIF failed to increase either cyclin D1, D2, D3, CDK4 or CDK6 protein levels. Finally, oncostatin M(OSM), a cytokine closely related to LIF, exerts its action through signals and processes markedly similar to those elicited by LIF. This conclusion is based in the following facts: both cytokines causes STAT1 tranlocation; the effect of Prostaglandin E1 and insulin, when added separately or in combination, enhances the effect of either LIF or OSM; PGF2α enhances the effect of LIF or OSM on DNA synthesis, both at subsaturant or saturant concentration. Moreover, LIF and OSM added together at subsaturating concentrations had an additive effect on DNA synthesis. LIF and OSM added together at saturating concentration had an similar effect to that of these same cytokines when added separately. Interleukin -6 and CNTF, fail to cause either cyclin D expression or mitogenic response. The results obtained suggest that the PGF2α-stimulated mitogenesis would occur through cyclin D1 expression, mediated by DAG/PKC and TK dependent mechanisms, while calcium dependent mechanisms would be involved in other processes. Finally, the LlF stimulated mitogenesis is not depend on signalling mechanisms such as those that act through PKC, PKA, MEK, p38MAPK and isoprenilated proteins, and also independently of the expression of cyclins D, CDK4 and CDK6.Fil:Sauane, Moira. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

MicroRNA-4719 and microRNA-6756-5p Correlate with Castration-Resistant Prostate Cancer Progression through Interleukin-24 Regulation

Prostate cancer (PCa) is the second leading cause of cancer death in the United States. The five-year survival rate for men diagnosed with localized PCa is nearly 100%, yet for those diagnosed with aggressive PCa, it is less than 30%. The pleiotropic cytokine Interleukin-24 (IL-24) has been shown to specifically kill PCa cells compared to normal cells when overexpressed in both in vitro and in vivo studies. Despite this, the mechanisms regulating IL-24 in PCa are not well understood. Since specific microRNAs (miRNAs) are dysregulated in PCa, we used miRNA target prediction algorithm tools to identify miR-4719 and miR-6556-5p as putative regulators of IL-24. This study elucidates the expression profile and role of miR-4719 and miR-6756-5p as regulators of IL-24 in PCa. qRT-PCR analysis shows miR-4719 and miR-6756-5p overexpression significantly decreases the expression of IL-24 in PCa cells compared to the negative control. Compared to the indolent PCa and normal prostate epithelial cells, miR-4719 and miR-6756-5p are significantly overexpressed in castration-resistant prostate cancer (CRPC) cell lines, indicating that their gain may be an early event in PCa progression. Moreover, miR-4719 and miR-6756-5p are significantly overexpressed in the CRPC cell line of African-American males (E006AA-hT) compared to CRPC cell lines of Caucasian males (PC-3 and DU-145), indicating that miR-4719 and miR-6756-5p may also play a role in racial disparity. Lastly, the inhibition of expression of miR-4719 and miR-6756-5p significantly increases IL-24 expression and inhibits proliferation and migration of CRPC cell lines. Our findings indicate that miR-4719 and miR-6756-5p may regulate CRPC progression through the targeting of IL-24 expression and may be biomarkers that differentiate between indolent and CRPC. Strategies to inhibit miR-4719 and miR-6756-5p expression to increase IL-24 in PCa may have therapeutic efficacy in aggressive PCa