Murugabaskar Balan

Inhibition of type I and type III interferons by a secreted glycoprotein from Yaba-like disease virus

Type I (IFN-α/β) and type III (IFN-λs) IFNs are important components of the host antiviral response. Although type III IFNs possess intrinsic antiviral activity similar to that of type I IFNs, they signal through a specific unique receptor complex, and their functional importance for antiviral resistance is largely uncharacterized. Here, we report the first virus defense mechanism that directly targets type III IFNs. Y136 from Yaba-like disease virus, a yatapoxvirus, is a secreted glycoprotein related to protein B18 from Vaccinia virus, a known type I IFN-binding protein and a member of the Ig superfamily. Surprisingly, whereas B18 inhibits only type I IFNs, Y136 inhibits both type I and type III IFNs. Y136 inhibits IFN-induced signaling and suppresses IFN-mediated biological activities including up-regulation of MHC class I antigen expression and induction of the antiviral state. These data demonstrate that poxviruses have developed unique strategies to counteract IFN-mediated antiviral protection and highlight the importance of type III IFNs in antiviral defense. These results suggest that type III IFNs may be an effective treatment for some poxviral infections.

Brain Injury Expands the Numbers of Neural Stem Cells and Progenitors in the SVZ by Enhancing Their Responsiveness to EGF

There is an increase in the numbers of neural precursors in the SVZ (subventricular zone) after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor) receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor)-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries.

Novel Roles of c-Met in the Survival of Renal Cancer Cells through the Regulation of HO-1 and PD-L1 Expression

Background: Tyrosine kinase receptor c-Met plays critical roles in the growth of RCC. Results: c-Met-mediated Ras activation and HO-1 overexpression promote anti-apoptotic signals, and increase PD-L1 expression, which inhibits immune cell-mediated killing of RCC cells. Conclusion: c-Met-induced HO-1 and PD-L1 expression promotes RCC cell survival. Significance: HO-1 and PD-L1 can serve as novel therapeutic targets for c-Met-induced RCC. The receptor tyrosine kinase c-Met is overexpressed in renal cancer cells and can play major role in the growth and survival of tumor. We investigated how the c-Met-mediated signaling through binding to its ligand hepatocyte growth factor (HGF) can modulate the apoptosis and immune escape mechanism(s) of renal cancer cells by the regulations of novel molecules heme oxygenase-1 (HO-1) and programmed death-1 ligand 1 (PD-L1). We found that HGF/c-Met-mediated signaling activated the Ras/Raf pathway and down-regulated cancer cell apoptosis; and it was associated with the overexpression of cytoprotective HO-1 and anti-apoptotic Bcl-2/Bcl-xL. c-Met-induced HO-1 overexpression was regulated at the transcriptional level. Next, we observed that c-Met induction markedly up-regulated the expression of the negative co-stimulatory molecule PD-L1, and this can be prevented following treatment of the cells with pharmacological inhibitors of c-Met. Interestingly, HGF/c-Met-mediated signaling could not induce PD-L1 at the optimum level when either Ras or HO-1 was knocked down. To study the functional significance of c-Met-induced PD-L1 expression, we performed a co-culture assay using mouse splenocytes (expressing PD-L1 receptor PD-1) and murine renal cancer cells (RENCA, expressing high PD-L1). We observed that the splenocyte-mediated apoptosis of cancer cells during co-culture was markedly increased in the presence of either c-Met inhibitor or PD-L1 neutralizing antibody. Finally, we found that both c-Met and PD-L1 are significantly up-regulated and co-localized in human renal cancer tissues. Together, our study suggests a novel mechanism(s) by which c-Met can promote increased survival of renal cancer cells through the regulation of HO-1 and PD-L1.

A novel CXCR3-B chemokine receptor-induced growth-inhibitory signal in cancer cells is mediated through the regulation of Bach-1 protein and Nrf2 protein nuclear translocation.

Background: The chemokine receptor CXCR3-B initiates inhibitory signals. Results: CXCR3-B-mediated signal induced apoptosis and inhibited autophagy of breast cancer cells. It is associated with nuclear translocation of Bach-1, nuclear export of Nrf2, and down-regulation of HO-1. Conclusion: A CXCR3-B-mediated signal promotes apoptosis of breast cancer cells. Significance: Induction of CXCR3-B-mediated signaling can serve as a novel therapeutic approach for the treatment of breast cancer. Chemokines and their receptors play diverse roles in regulating cancer growth and progression. The receptor CXCR3 can have two splice variants with opposite functions. CXCR3-A promotes cell growth, whereas CXCR3-B mediates growth-inhibitory signals. However, the negative signals through CXCR3-B in cancer cells are not well characterized. In this study, we found that CXCR3-B-mediated signaling in MCF-7 and T47D breast cancer cells induced apoptotic cell death. Signals through CXCR3-B decreased the levels of the antiapoptotic proteins Bcl-2 and Bcl-xL and increased the expression of apoptotic cleaved poly(ADP-ribose) polymerase. Along with up-regulation in apoptosis, CXCR3-B signals were associated with a decrease in cellular autophagy with reduced levels of the autophagic markers Beclin-1 and LC3B. Notably, CXCR3-B down-regulated the expression of the cytoprotective and antiapoptotic molecule heme oxygenase-1 (HO-1) at the transcriptional level. There was an increased nuclear localization of Bach-1 and nuclear export of Nrf2, which are important negative and positive transcription factors, respectively, for HO-1 expression. We also observed that CXCR3-B promoted the activation of p38 MAPK and the inhibition of ERK-1/2. CXCR3-B could not induce cancer cell apoptosis at the optimal level when we either inhibited p38 activity or knocked down Bach-1. Further, CXCR3-B-induced apoptosis was down-regulated when we overexpressed HO-1. Together, our data suggest that CXCR3-B mediates a growth-inhibitory signal in breast cancer cells through the modulations of nuclear translocation of Bach-1 and Nrf2 and down-regulation of HO-1. We suggest that the induction of CXCR3-B-mediated signaling can serve as a novel therapeutic approach where the goal is to promote tumor cell apoptosis.

High-throughput drug screen identifies chelerythrine as a selective inducer of death in a TSC2-null setting.

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrines effects, indicating that chelerythrine-induced cell death is ROS dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress–dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors. Implications: This study demonstrates that TSC2-deficient tumor cells are hypersensitive to oxidative stress–dependent cell death, and provide critical proof of concept that TSC2-deficient cells can be therapeutically targeted without the use of a rapalog to induce a cell death response. Mol Cancer Res; 13(1); 50–62. ©2014 AACR.

Egr-1 is a Critical Regulator of EGF-Receptor-Mediated Expansion of Subventricular Zone Neural Stem Cells and Progenitors During Recovery from Hypoxia–Hypoglycemia

We recently established that the EGF-R (epidermal growth factor receptor) (EGF-R) is an essential regulator of the reactive expansion of SVZ (subventricular zone) NPs (neural precursors) that occurs during recovery from hypoxic-ischemic brain injury. The purpose of the current studies was to identify the conditions and the transcription factor (s) responsible for inducing the EGF-R. Here, we show that the increase in EGF-R expression and the more rapid division of the NPs can be recapitulated in in vitro by exposing SVZ NPs to hypoxia and hypoglycemia simultaneously, but not separately. The EGF-R promoter has binding sites for multiple transcription factors that includes the zinc finger transcription factor, Egr-1. We show that Egr-1 expression increases in NPs, but not astrocytes, following hypoxia and hypoglycemia where it accumulates in the nucleus. To determine whether Egr-1 is necessary for EGF-R expression, we used SiRNAs (small interfering RNA) specific for Egr-1 to decrease Egr-1 expression. Knocking-down Egr-1 decreased basal levels of EGF-R and it abolished the stress-induced increase in EGF-R expression. By contrast, HIF-1 accumulation did not contribute to EGF-R expression and FGF-2 only modestly induced EGF-R. These studies establish a new role for Egr-1 in regulating the expression of the mitogenic EGF-R. They also provide new information into mechanisms that promote NP expansion and provide insights into strategies for amplifying the numbers of stem cells for CNS (central nervous system) regeneration.

Honokiol inhibits c-Met-HO-1 tumor-promoting pathway and its cross-talk with calcineurin inhibitor-mediated renal cancer growth

Honokiol (HNK) is a small molecule with potent anti-inflammatory and anti-tumorigenic properties; yet the molecular targets of HNK are not well studied. Hyperactivation of the receptor tyrosine kinase c-Met and overexpression of the cytoprotective enzyme heme oxygenase-1 (HO-1) play a critical role in the growth and progression of renal cell carcinoma (RCC). Interestingly, the calcineurin inhibitor (CNI) cyclosporine A (CsA), an immunosuppressant used to prevent allograft rejection, can also increase the risk of RCC in transplant patients. We studied the potential role of c-Met signaling axis on CNI-induced renal tumor growth and tested the anti-tumor efficacy of HNK. Importantly, CNI treatment promoted c-Met induction and enhanced c-Met-induced Ras activation. We found that HNK treatment effectively down-regulated both c-Met phosphorylation and Ras activation in renal cancer cells. It inhibited the expression of both c-Met- and CNI-induced HO-1, and promoted cancer cell apoptosis. In vivo, HNK markedly inhibited CNI-induced renal tumor growth; and it decreased the expression of phospho-c-Met and HO-1 and reduced blood vessel density in tumor tissues. Our results suggest a novel mechanism(s) by which HNK exerts its anti-tumor activity through the inhibition of c-Met-Ras-HO-1 axis; and it can have significant therapeutic potential to prevent post-transplantation cancer in immunosuppressed patients.

Immunoevasion rather than intrinsic oncogenicity may confer MSCs from non-obese diabetic mice the ability to generate neural tumors

Mesenchymal stem cells (MSCs) have generated a great amount of interest in the field of regenerative medicine and for management of inflammation-related disorders [1–5]. Multiple clinical trials utilizing MSCs in the treatment of human diseases (e.g., type 1 diabetes, myocardial infarction, graft versus host disease) have demonstrated their efficacy [6–8], and transplanted MSCs have been shown overall to be well tolerated without adverse effects. Nevertheless, concerns have been raised over the potential risk of MSC-induced tumor development [9–11], while longterm screenings of patients in MSC-based trials are still lacking. We previously observed the development of tumors in murine recipients of syngeneic MSCs obtained from NOD mice, but not with BALB/c-MSCs [12]. The present report aims to elucidate the degree to which malignant transformation is attributable to MSC evasion of recipient immunosurveillance. This question is of paramount importance in order to ensure safe administration of MSCs for therapeutic applications. Furthermore, these data may shed light on the increased incidence of tumors including neural tumors in diabetic patients [13].

Abstract 4583: Potential role of neuropilin-2 in the regulation of c-MET-induced PD-L1 expression in renal cancer cells

Neuropilin-1 and -2 (NRP1 and NRP2) are the transmembrane glycoproteins that act as co-receptors for class III semaphorins and several members of the vascular endothelial growth factor (VEGF) family. In conjunction with VEGF receptors and plexins, NRPs and semaphorins modulate various biological responses. Recent studies suggest that NRPs are up-regulated in many types of cancer cells, and may play a major role in tumor growth. In addition to semaphorins and VEGF, NRP1 and NRP2 can also bind to hepatocyte growth factor (HGF), which is the ligand for the tyrosine kinase receptor c-MET. c-MET is highly over-expressed in renal cancer cells; however, the impact of NRPs on c-MET-mediated tumorigenic pathways have not been studied. Recently, we have reported that HGF-/c-MET-induced signaling increases the expression of immunosuppressive and negative-co-stimulatory molecule programmed death-1 ligand (PD-L1) on renal cell carcinoma (RCC) cells, and it protects tumor cells from immune-mediated killing. In this study, we investigated whether NRPs and its ligands can modulate the expression of HGF-/c-MET-induced PD-L1. We have observed that NRP2, and to a lesser extent NRP1, is over-expressed in human RCC cell lines (786-O, ACHN and Caki-1) compared with normal renal epithelial cells at both mRNA and protein level. siRNA-mediated silencing of NRP1 and NRP2 promoted apoptosis of renal cancer cells, suggesting possible role(s) of NRPs in tumor growth. The knock-down of NRP2 down-regulated the cell surface expression of PD-L1 possibly through the reduced interaction of growth-promoting ligands (like, HGF) with NRP2. Interestingly, SEMA3F, a class III semaphorin ligand for both NRP1 and NRP2, has been reported to have anti-tumorigenic effects. We observed that SEMA3F was significantly down-regulated in RCC cells compared with normal renal epithelial cells; and when we added SEMA3F to the cultures of renal cancer cells, it down-regulated both basal and HGF-/c-MET-induced PD-L1 expression. Together, our findings suggest that NRPs (primarily NRP2) can play a critical role in regulating HGF-/c-MET-induced PD-L1 expression in renal cancer cells to mediate immune escape of tumor cells. Therefore targeting NRP2 and/or its activation through SEMA3F/synthetic ligands may restrict c-MET-PD-L1-induced tumor promoting pathways in renal cancer cells. Citation Format: Murugabaskar Balan, Soumitro Pal. Potential role of neuropilin-2 in the regulation of c-MET-induced PD-L1 expression in renal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4583.

Abstract A4: CXCR3-B-mediated signaling suppresses c-MET-mediated angiogenic signals through the down-regulation of HO-1 and VEGF expression

Chemokine receptor CXCR3 gene encodes two splice variants with opposite functions; CXCR3-A promotes cell growth whereas CXCR3-B is growth inhibitory. Down-regulation of CXCR3-B promotes survival of many cancer cell types including Renal Cell Carcinoma (RCC) cells. However, the molecular signaling events that mediate down-regulation of CXCR3-B are not fully understood. Growth factors mediated signaling events govern Alternative Splicing (AS) of genes. The receptor tyrosine kinase c-MET is over-expressed in RCC cells and signaling through its ligand Hepatocyte Growth Factor (HGF) can play a major role in the growth and survival of tumor. In this study, we investigated the role of c-MET-mediated signaling in the modulation of AS of CXCR3 gene and analyzed the impact of CXCR3-B-mediated growth inhibitory signals on c-MET-mediated angiogenic pathway in RCC cells. We found that HGF treatment down-regulated CXCR3-B expression in both primary renal epithelial (RPTEC) and RCC (786-O and ACHN) cells. We found that c-MET-mediated signaling increased the expression of gene AS regulating protein SAM68 (also a prognostic marker for RCC) and modulated the association of SAM68 with CXCR3 gene. Utilizing gene over expression approach, we found that CXCR3-B-mediated signaling down-regulated the expression of cytoprotective molecule Hemeoxygenease-1 (HO-1) and angiogenic factor VEGF at both transcription and protein levels. We also found that CXCR3-B-mediated signaling down-regulated c-MET-induced secretion of angiogenic factors from RCC cells, as observed by the decrease in the tube formation ability of endothelial (HUVEC) cells grown with supernatant obtained from CXCR3-B over-expressing RCC cells culture. Together, we suggest that CXCR3-B-mediated signaling acts as a checkpoint for c-MET-mediated angiogenic signals in RCC. We propose a novel function for c-MET-mediated signaling in promoting angiogenesis in renal cancer cells through the down-regulation of CXCR3-B expression. Citation Format: Murugabaskar Balan, Soumitro Pal. CXCR3-B-mediated signaling suppresses c-MET-mediated angiogenic signals through the down-regulation of HO-1 and VEGF expression. [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 A4.