Upneet K. Sokhi

Human polynucleotide phosphorylase selectively and preferentially degrades microRNA-221 in human melanoma cells

MicroRNAs (miRNA), small noncoding RNAs, affect a broad range of biological processes, including tumorigenesis, by targeting gene products that directly regulate cell growth. Human polynucleotide phosphorylase (hPNPaseold-35), a type I IFN-inducible 3′-5′ exoribonuclease, degrades specific mRNAs and small noncoding RNAs. The present study examined the effect of this enzyme on miRNA expression in human melanoma cells. miRNA microarray analysis of human melanoma cells infected with empty adenovirus or with an adenovirus expressing hPNPaseold-35 identified miRNAs differentially and specifically regulated by hPNPaseold-35. One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27kip1, displayed robust down-regulation with ensuing up-regulation of p27kip1 by expression of hPNPaseold-35, which also occurred in multiple human melanoma cells upon IFN-β treatment. Using both in vivo immunoprecipitation followed by Northern blotting and RNA degradation assays, we confirm that mature miR-221 is the target of hPNPaseold-35. Inhibition of hPNPaseold-35 by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-β–mediated growth inhibition, accentuating the importance of hPNPaseold-35 induction and miR-221 down-regulation in mediating IFN-β action. Moreover, we now uncover a mechanism of miRNA regulation involving selective enzymatic degradation. Targeted overexpression of hPNPaseold-35 might provide an effective therapeutic strategy for miR-221–overexpressing and IFN-resistant tumors, such as melanoma.

MDA-9/syntenin: a positive gatekeeper of melanoma metastasis.

Melanoma differentiation associated gene-9 (MDA-9), synonymous with syntenin, is an adapter protein that provides a central role in regulating cell-cell and cell-matrix adhesion. MDA-9/syntenin transduces signals from the cell-surface to the interior through its interaction with a plethora of additional proteins and actively participates in intracellular trafficking and cell-surface targeting, synaptic transmission, and axonal outgrowth. Recent studies demarcate a seminal role of MDA-9/syntenin in cancer metastasis. In the context of melanoma, MDA-9/syntenin functions as a positive regulator of melanoma progression and metastasis through interactions with c-Src and promotes the formation of an active FAK/c-Src signaling complex leading to NF-k B and matrix metalloproteinase (MMP) activation. The present review provides a current perspective of our understanding of the important features of MDA-9/syntenin and its significant role in tumor cell metastasis with special focus on molecular mechanism of action.

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.

Melanoma Differentiation Associated Gene-7/Interleukin-24 Potently Induces Apoptosis in Human Myeloid Leukemia Cells through a Process Regulated by Endoplasmic Reticulum Stress

Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 (IL-24), a member of the IL-10 cytokine gene family, preferentially induces cell death in neoplastic epithelial cells types while sparing their normal counterparts. The effects of mda-7/IL-24 in acute myeloid leukemia (AML) cells have not been extensively characterized. Treatment with recombinant GST-MDA-7/IL-24 potently induced apoptosis in diverse myeloid leukemia cell types including U937, HL60, MV4-11, EOL-1, and MLL/ENL cells. MDA-7/IL-24 also markedly induced apoptosis in and suppressed the colony-forming capacity of primary AML blasts but exerted minimal toxicity toward normal CD34+ hematopoietic progenitor cells. MDA-7/IL-24 lethality was associated with pronounced endoplasmic reticulum (ER) stress induction in leukemia cell lines and primary AML blasts, manifested by the accumulation of growth arrest and DNA damage-inducible protein 34 (GADD34), 78-kDa glucose-regulated protein (GRP78)/BiP, inositol-requiring enzyme 1α (IRE1α), and eukaryotic initiation factor 2α phosphorylation. It is noteworthy that short hairpin RNA (shRNA) knockdown of IRE1α, GADD34, or GRP78/BiP significantly enhanced MDA-7/IL-24-mediated apoptosis, indicating a protective role for these molecules against MDA-7/IL-24 lethality. MDA-7/IL-24 also down-regulated the antiapoptotic protein Mcl-1 and sharply increased expression of the proapoptotic proteins Bim and Noxa. Ectopic Mcl-1 expression or shRNA knockdown of Bim or Noxa significantly attenuated MDA-7/IL-24-mediated leukemia cell death. Finally, knockdown of Bax or Bak significantly reduced MDA-7/IL-24 lethality. Together, these findings indicate that MDA-7/IL-24 potently induces apoptosis in human myeloid leukemia cells through a process regulated by ER stress induction, Mcl-1 down-regulation, and Bim and Noxa up-regulation. They also suggest that MDA-7/IL-24 warrants further investigation in myeloid leukemia.

Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells

In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca2+. Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda-7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.

Human Polynucleotide Phosphorylase (hPNPaseold-35): An evolutionary conserved gene with an expanding repertoire of RNA degradation functions

Human polynucleotide phosphorylase (hPNPaseold-35) is an evolutionary conserved RNA-processing enzyme with expanding roles in regulating cellular physiology. hPNPaseold-35 was cloned using an innovative ‘overlapping pathway screening’ strategy designed to identify genes coordinately regulated during the processes of cellular differentiation and senescence. Although hPNPaseold-35 structurally and biochemically resembles PNPase of other species, overexpression and inhibition studies reveal that hPNPaseold-35 has evolved to serve more specialized and diversified functions in humans. Targeting specific mRNA or non-coding small microRNA, hPNPaseold-35 modulates gene expression that in turn has a pivotal role in regulating normal physiological and pathological processes. In these contexts, targeted overexpression of hPNPaseold-35 represents a novel strategy to selectively downregulate RNA expression and consequently intervene in a variety of pathophysiological conditions.

Selected Approaches for Rational Drug Design and High Throughput Screening to Identify Anti-Cancer Molecules

Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specificpromoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.

Identification of Genes Potentially Regulated by Human Polynucleotide Phosphorylase (hPNPaseold-35) Using Melanoma as a Model

Human Polynucleotide Phosphorylase (hPNPaseold-35 or PNPT1) is an evolutionarily conserved 3′→5′ exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPaseold-35 in cellular physiology, we knocked-down and overexpressed hPNPaseold-35 in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPaseold-35 resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPaseold-35 caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPaseold-35 knockdown and overexpression datasets allowed us to identify 77 potential “direct” and 61 potential “indirect” targets of hPNPaseold-35 which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPaseold-35 expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes.

Examination of Epigenetic and other Molecular Factors Associated with mda-9/Syntenin Dysregulation in Cancer Through Integrated Analyses of Public Genomic Datasets

mda-9/Syntenin (melanoma differentiation-associated gene 9) is a PDZ domain containing, cancer invasion-related protein. In this study, we employed multiple integrated bioinformatic approaches to identify the probable epigenetic factors, molecular pathways, and functionalities associated with mda-9 dysregulation during cancer progression. Analyses of publicly available genomic data (e.g., expression, copy number, methylation) from TCGA, GEO, ENCODE, and Human Protein Atlas projects led to the following observations: (a) mda-9 expression correlates with both copy number and methylation level of an intronic CpG site (cg1719774) located downstream of the CpG island, (b) cg1719774 methylation is a likely prognostic marker in glioma, (c) among 22 cancer types, melanoma exhibits the highest mda-9 level, and lowest level of methylation at cg1719774, (d) cg1719774 hypomethylation is also associated with histone modifications (at the mda-9 locus) indicative of more active transcription, (e) using Gene Set Enrichment Analysis (GSEA), and the Virtual Gene Overexpression or Repression (VIGOR) analytical scheme, we were able to predict mda-9s association with extracellular matrix organization (e.g., MMPs, collagen, integrins), IGFBP2 and NF-κB signaling pathways, phospholipid metabolism, cytokines (e.g., interleukins), CTLA-4, and components of complement cascade pathways. Indeed, previous publications have shown that many of the aforementioned genes and pathways are associated with mda-9s functionality.

Tumor Necrosis Factor dynamically regulates the mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes

During rheumatoid arthritis (RA), Tumor Necrosis Factor (TNF) activates fibroblast-like synoviocytes (FLS) inducing in a temporal order a constellation of genes, which perpetuate synovial inflammation. Although the molecular mechanisms regulating TNF-induced transcription are well characterized, little is known about the impact of mRNA stability on gene expression and the impact of TNF on decay rates of mRNA transcripts in FLS. To address these issues we performed RNA sequencing and genome-wide analysis of the mRNA stabilome in RA FLS. We found that TNF induces a biphasic gene expression program: initially, the inducible transcriptome consists primarily of unstable transcripts but progressively switches and becomes dominated by very stable transcripts. This temporal switch is due to: a) TNF-induced prolonged stabilization of previously unstable transcripts that enables progressive transcript accumulation over days and b) sustained expression and late induction of very stable transcripts. TNF-induced mRNA stabilization in RA FLS occurs during the late phase of TNF response, is MAPK-dependent, and involves several genes with pathogenic potential such as IL6, CXCL1, CXCL3, CXCL8/IL8, CCL2, and PTGS2. These results provide the first insights into genome-wide regulation of mRNA stability in RA FLS and highlight the potential contribution of dynamic regulation of the mRNA stabilome by TNF to chronic synovitis.