Linking Autophagy and the Dysregulated NFκB/ SNAIL/YY1/RKIP/PTEN Loop in Cancer: Therapeutic Implications.

The role of autophagy in the pathogenesis of various cancers has been well documented in many reports. Autophagy in cancer cells regulates cell proliferation, viability, invasion, epithelial-to-mesenchymal transition (EMT), metastasis, and responses to chemotherapeutic and immunotherapeutic treatment strategies. These manifestations are the result of various regulatory gene products that govern autophagic, biochemical, and molecular mechanisms. In several human cancer cell models, the presence of a dysregulated circuit-namely, NFκB/SNAIL/YY1/RKIP/PTEN-that plays a major role in the regulation of tumor cell unique characteristics just listed for autophagy-regulated activities. Accordingly, the autophagic mechanism and the dysregulated circuit in cancer cells share many of the same properties and activities. Thus, it has been hypothesized that there must exist a biochemical/molecular link between the two. The present review describes the link and the association of each gene product of the dysregulated circuit with the autophagic mechanism and delineates the presence of crosstalk. Crosstalk between autophagy and the dysregulated circuit is significant and has important implications in the development of targeted therapies aimed at either autophagy or the dysregulated gene products in cancer cells.

Potential Therapeutic Applications of MDA-9/Syntenin-NF-κB-RKIP Loop in Human Liver Carcinoma.

BACKGROUND: Overexpression of MDA-9/Syntenin occurs in multiple human cancer cell lines and is associated with higher grade of tumor classification, invasiveness and metastasis. In some cases, its role in cancer biology depends on relationships between MDA-9/Syntenin and NF-κB. OBJECTIVE: This study aims to analyze the presence of a regulation loop like that between MDA-9/Syntenin - NF-κB - RKIP in human liver carcinoma. METHODS: Transient transfection was performed with siRNA anti-MDA-9/Syntenin. Expression of different factors was evaluated by Real time-PCR and Western blotting, while NF-κB activation by TransAM assay. Invasion capacity was analyzed by Matrigel Invasion Assay and the effects of agents on cell viability were examined by MTS assay. RESULTS: We have examined basal expression of MDA-9/Syntenin in three cell lines of human liver carcinoma (HA22T/VGH, Hep3B and HepG2). In all cell lines there was an inverse relationship between MDA-9/Syntenin and RKIP expression levels, and a positive correlation between MDA-9/Syntenin expression and NF-κB activation levels. By silencing with a siRNA anti-MDA-9/Syntenin we observed in all cell lines a very strong increase of RKIP at mRNA level. Interestingly, in all cell lines, inhibition of MDA- 9/Syntenin expression induced NF-κB downregulation and contemporary a reduction in invasion ability MMP-2 dependent. Finally, we showed a good additive effect of MDA- 9/Syntenin siRNA when associated with Curcumin or Doxorubicin on cell growth inhibition. CONCLUSION: Our data confirm the key role of MDA-9/Syntenin in HCC biology. The presence of a regulation loop among MDA-9/Syntenin, NF-κB and RKIP provide new pharmacological approaches.

Dual roles of nitric oxide in the regulation of tumor cell response and resistance to photodynamic therapy.

Photodynamic therapy (PDT) against cancer has gained attention due to the successful outcome in some cancers, particularly those on the skin. However, there have been limitations to PDT applications in deep cancers and, occasionally, PDT treatment resulted in tumor recurrence. A better understanding of the underlying molecular mechanisms of PDT-induced cytotoxicity and cytoprotection should facilitate the development of better approaches to inhibit the cytoprotective effects and also augment PDT-mediated cytotoxicity. PDT treatment results in the induction of iNOS/NO in both the tumor and the microenvironment. The role of NO in cytotoxicity and cytoprotection was examined. The findings revealed that NO mediates its effects by interfering with a dysregulated pro-survival/anti-apoptotic NF-κB/Snail/YY1/RKIP loop which is often expressed in cancer cells. The cytoprotective effect of PDT-induced NO was the result of low levels of NO that activates the pro-survival/anti-apoptotic NF-κB, Snail, and YY1 and inhibits the anti-survival/pro-apoptotic and metastasis suppressor RKIP. In contrast, PDT-induced high levels of NO result in the inhibition of NF-kB, Snail, and YY1 and the induction of RKIP, all of which result in significant anti-tumor cytotoxicity. The direct role of PDT-induced NO effects was corroborated by the use of the NO inhibitor, l-NAME, which reversed the PDT-mediated cytotoxic and cytoprotective effects. In addition, the combination of the NO donor, DETANONOate, and PDT potentiated the PDT-mediated cytotoxic effects. These findings revealed a new mechanism of PDT-induced NO effects and suggested the potential therapeutic application of the combination of NO donors/iNOS inducers and PDT in the treatment of various cancers. In addition, the study suggested that the combination of PDT with subtoxic cytotoxic drugs will result in significant synergy since NO has been shown to be a significant chemo-immunosensitizing agent to apoptosis.

Gene set enrichment analysis of the NF-κB/Snail/YY1/RKIP circuitry in multiple myeloma.

The presence of a dysregulated NF-κB/Snail/YY1/RKIP loop was recently established in metastatic prostate cancer cells and non-Hodgkin's lymphoma; however, its involvement in multiple myeloma (MM) has yet to be investigated. Aim of the study was to investigate the role of the NF-κB/Snail/YY1/RKIP circuitry in MM and how each gene is correlated with the remaining genes of the loop. Using gene set enrichment analysis and gene neighbours analysis in data received from four datasets included in the Multiple Myeloma Genomics Portal of the Multiple Myeloma Research Consortium, we identified various enriched gene sets associated with each member of the NF-κB/Snail/YY1/RKIP circuitry. In each dataset, the 20 most co-expressed genes with the circuitry genes were isolated subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment. Among many, we highlighted on FNDC3B, TPD52, BBX, MBNL1 and MFAP2. Many co-expressed genes participated in the regulation of metabolic processes and nucleic acid binding, or were transcription factor binding genes and genes with metallopeptidase activity. The transcription factors FOXO4, GATA binding factor, Sp1 and AP4 most likely affect the expression of the NF-κB/Snail/YY1/RKIP circuitry genes. Computational analysis of various GEO datasets revealed elevated YY1 and RKIP levels in MM vs. the normal plasma cells, as well as elevated RKIP levels in MM vs. normal B lymphocytes. The present study highlights the relationships of the NF-κB/Snail/YY1/RKIP circuitry genes with specific cancer-related gene sets in multiple myeloma.

Silencing of human phosphatidylethanolamine-binding protein 4 enhances rituximab-induced death and chemosensitization in B-cell lymphoma.

Rituximab is the first line drug to treat non Hodgkin's lymphoma (B-NHL) alone or in combination with chemotherapy. However, 30-40% of B-NHL patients are unresponsive to rituximab or resistant after therapy. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a novel member of PEBP family and functions as an anti-apoptotic molecule. In this study, we found hPEBP4 to be expressed in up to 90% of B-cell lymphoma patients, but in only 16.7% of normal lymph nodes. Interestingly, hPEBP4 overexpression inhibited rituximab-mediated complement dependent cytotoxicity (R-CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in B-NHL cells while downregulation of hPEBP4 augmented the therapeutic efficacy of rituximab both in vitro and in vivo. Furthermore, hPEBP4 silencing sensitized the primary B-acute lymphocytic leukemia (B-ALL) cells to R-CDC. During rituximab-mediated complement dependent cytotoxicity, hPEBP4 was recruited to the cell membrane in a PE-binding domain dependent manner and inhibited R-CDC induced calcium flux and reactive oxygen species (ROS) generation. These events contributed to the decrease of cell death induced by R-CDC in B-cell lymphomas. Meanwhile, hPEBP4 knockdown potentiated the chemosensitization of the rituximab in B-cell lymphoma cells by regulating the expression of Bcl-xl, Cycline E, p21(waf/cip1) and p53 and the activation of caspase-3 and caspase-9. Considering that hPEBP4 conferred cellular resistance to rituximab treatment and was preferentially expressed in lymphoma tissue, it could be a potential valuable target for adjuvant therapy for B-cell lymphoma.

Synergistic cytotoxicity, inhibition of signal transduction pathways and pharmacogenetics of sorafenib and gemcitabine in human NSCLC cell lines.

INTRODUCTION: Lung cancer is one of the most lethal tumors and, although standard chemotherapy produces clinical response, there has been little improvement in prognosis. Therefore, research effort has focused on target-specific agents, such as sorafenib, which blocks both the RAF/MEK/ERK signalling pathways and receptors involved in neovascularization and tumor progression, including VEGFR-2 and c-Kit. We investigated whether sorafenib would be synergistic with gemcitabine against NSCLC cell lines. MATERIALS AND METHODS: Human lung cancer cells A549, CALU-1, CALU-6, H23 and HCC 827 were treated with sorafenib and gemcitabine, alone or in combination, and the cytotoxicity was assessed with CellTiter 96 Non-radioactive cell proliferation kit. Cell cycle and apoptosis were investigated with flow cytometry and fluorescence microscopy, respectively. Moreover, the effects of drugs on Akt (S473), c-Kit (Y823) and ERK (pTpY185/187) phosphorylation were studied with ELISA. Finally, quantitative PCR analysis was performed to assess whether sorafenib and gemcitabine modulated the expression of genes related to drug activity. RESULTS: Gemcitabine and sorafenib synergistically interacted on the inhibition of cell proliferation, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index. Sorafenib reduced c-Kit and ERK activation and gemcitabine inhibited Akt phosphorylation. Moreover quantitative PCR showed that sorafenib modulated the expression of targets related to gemcitabine activity, while gemcitabine induced the expression of RKIP. CONCLUSIONS: These data demonstrate that sorafenib and gemcitabine synergistically interact against NSCLC cells, through suppression of Akt, c-Kit and ERK phosphorylation, induction of apoptosis and modulation of dCK, RRM1, RRM2 and RKIP gene expression. The association between traditional cytotoxic agents with new target-specific agents, such as sorafenib, is a challenge for both clinical and preclinical future investigations in lung cancer treatments.

The expression of RKIP, RhoGDI, galectin, c-Myc and p53 in gastrointestinal system of Cr(VI)-exposed rats.

Hexavalent chromium (CrVI) is considered to be a risk factor in the formation of human cancer. Raf kinase inhibitor protein (RKIP), Rho-GDIα, galectin, c-Myc and p53 play important roles in cancer formation. The purpose of this study was to determine if Cr(VI) induces the formation of gastrointestinal cancer. We explored the expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 in the colon and stomach in rats exposed to chromium (CrVI). Thirty Wistar rats were divided into six groups which were chronically fed with 250, 500, 750, 1000 and 1250 ppm Na(2) Cr(2) O(7) and water for 60 days. The level of Cr(VI) was determined by electrothermal atomic absorption spectrometry. The expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 of stomach and colon was measured by western blot. The gene expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 of the stomach and colon was determined by RT-PCR. The results showed that the expression of p53 and Rho-GDIα was decreased in the stomach and colon of rats with Cr(VI) treatment. The expression of RKIP was decreased in the stomach and colon of rats treated with high-dose Cr(VI). The expression of c-Myc and gelectin-1 was increased in the stomach and colon of rats with Cr(VI) treatment. We concluded that the anomalous expression of RKIP, Rho-GDIα, galectin, c-Myc and p53 might be a dangerous index of cancer formation in the stomach and colon of rats with Cr(VI) exposure.

Frequent alteration of the Yin Yang 1/Raf-1 kinase inhibitory protein ratio in hepatocellular carcinoma.

The transcription factor Yin Yang 1 (YY1) can favor several aspects of tumorigenesis. In turn, Raf-1 Kinase Inhibitor Protein (RKIP) inhibits the oncogenic activities of MAPK and NF-κB pathways and promotes drug-induced apoptosis. Mutual influences between YY1 and RKIP may exist, and there are already separate evidences that relevant increases in YY1 and reductions in RKIP occur in hepatocellular carcinoma (HCC). However, the levels of the two factors have never been concomitantly examined in HCC. We evaluated by RT-PCR the mRNA levels of YY1, YY1AP, RKIP, and survivin in 35 clinical HCCs (91% HCV-related), in their adjacent cirrhotic tissues and in 6 healthy livers. Immunohistochemical analyses were also performed. The ratio of YY1 to RKIP mRNA was constantly profoundly inverted in the tumors compared with the adjacent nontumoral tissues. A similar result occurred frequently at protein level. Hyperactivation of YY1 in tumors was corroborated by its nuclear localization and the finding that in the tumors there were also increases in YY1AP, a YY1 coactivator not expressed in normal liver, and in survivin, as a possible target of YY1. The frequent alteration in the YY1-RKIP balance might represent a marker of malignant progression and be exploited for therapeutic interventions in HCC.

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.

American ginseng (Panax quinquefolius L.) extract alters mitogen-activated protein kinase cell signaling and inhibits proliferation of MCF-7 cells.

Ginseng has been shown to inhibit cancer cell proliferation and tumor growth, however the mechanisms underlying this inhibition have yet to be elucidated. An inhibitory effect of hot water-extracted American ginseng (Panax quinquefolius L.) root on cell proliferation was demonstrated using MCF-7 human breast cancer cells treated with a wide concentration range of the ginseng extract (GE) for 6 days. The effects of GE were concentration-dependent with an IC50 of 0.49 microg/microl and the minimum exposure time to elicit an inhibitory response was 24 hours. Using an antibody microarray, it was determined that several key cell survival proteins were altered in GE-treated cells, including several members of the mitogen-activated protein kinase (MAPK) family. A GE-induced decrease in phospho-MEK1/2 and -ERK1/2 and an increase in phospho-Raf-1 were observed and verified using Western blot analysis. Furthermore, mRNA and protein expression of the Raf-1 kinase inhibitor protein (RKIP) was shown to be transiently, yet significantly, upregulated following GE treatment. These results suggest that American ginseng may act to inhibit breast cancer cell proliferation by increasing the expression of RKIP, resulting in inhibition of the MAPK pathway. This novel mechanism has implications in the potential prevention and treatment of breast cancer.