Search

Staphyloccocus aureus is one of the major pathogens in orthopedic periprosthetic joint infection (PJI), a devastating complication of total joint arthroplasty that often results in chronic and persistent infections that are refractory to antibiotics and require surgical interventions. Biofilm formation has been extensively investigated as a reason for persistent infection. The cellular composition, activation status, cytokine profile, and role of the immune response during persistent S. aureus PJI are incompletely understood. In this study, we used histology, multiparametric flow cytometry, and gene expression analysis to characterize the immune response in a clinically relevant orthopedic PJI model. We tested the hypothesis that persistent S. aureus infection induces feedback mechanisms that suppress immune cell activation, thereby affecting the course of infection. Surprisingly, persistent infection was characterized by strikingly high cytokine gene expression indicative of robust activation of multiple components of innate and adaptive immunity, along with ongoing severe neutrophil-dominated inflammation, in infected joint and bone tissues. Activation and expansion of draining lymph nodes and a bone marrow stress granulopoiesis reaction were also maintained during late phase infection. In parallel, feedback mechanisms involving T-cell inhibitory receptors and exhaustion markers, suppressive cytokines, and regulatory T cells were activated and associated with decreased T-cell proliferation and tissue infiltration during the persistent phase of infection. These results identify the cellular and molecular components of the mouse immune response to persistent S. aureus PJI and indicate that neutrophil infiltration, inflammatory cytokine responses, and ongoing lymph node and bone marrow reactions are insufficient to clear infection and that immune effector mechanisms are suppressed by feedback inhibitory pathways. These immune-suppressive mechanisms are associated with diminished T-cell proliferation and tissue infiltration and can be targeted as part of adjuvant immunotherapeutic strategies in combination with debridement of biofilm, antibiotics, and other therapeutic modalities to promote eradication of infection. © 2021 American Society for Bone and Mineral Research (ASBMR).

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

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

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

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

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

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

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

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

The importance of a local tissue immune response in healing injured tissues such as skin and lung is well established. Little is known about whether sterile wounds elicit lymph node (LN) responses and inflammatory responses after injury of musculoskeletal tissues that are mechanically loaded during the repair response. We investigated LN and tissue immune responses in a tibial implant model of joint replacement surgery where wounded tissue is subjected to movement and mechanical loading postoperatively. Draining inguinal and iliac LNs expanded postoperatively, including increases in regulatory T cells and activation of a subset of T cells. Thus, tissue injury was actively sensed in secondary lymphoid organs, with the potential to activate adaptive immunity. Joint tissues exhibited three temporally distinct immune response components, including a novel interferon (IFN) response with activation of signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) pathways. Fibrovascular tissue formation was not associated with a macrophage type 2 (M2) reparative immune response, but instead with delayed induction of interleukin-1 family (IL-1β, IL-33, IL-36), IL-17, and prostaglandin pathway genes concomitant with transforming growth factor (TGF)-β and growth factor signaling, fibroblast activation, and tissue formation. Tissue remodeling was associated with activity of the HOX antisense intergenic RNA (HOTAIR) pathway. These results provide insights into immune responses and regulation of tissue healing after knee arthroplasty that potentially can be used to develop therapeutic strategies to improve healing, prevent arthrofibrosis, and improve surgical outcomes. © 2021 American Society for Bone and Mineral Research (ASBMR).

Macrophages tailor their function to the signals found in tissue microenvironments, taking on a wide spectrum of phenotypes. In human tissues, a detailed understanding of macrophage phenotypes is limited. Using single-cell RNA-sequencing, we define distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF+inflammatory macrophages is enriched in RA tissues and shaped by resident fibroblasts and the cytokine TNF. These macrophages promote fibroblast invasiveness in an EGF receptor dependent manner, indicating that inflammatory intercellular crosstalk reshapes both cell types and contributes to fibroblast-mediated joint destruction. In anex vivotissue assay, the HBEGF+inflammatory macrophage is targeted by several anti-inflammatory RA medications, however, COX inhibition redirects it towards a different inflammatory phenotype that is also expected to perpetuate pathology. These data highlight advances in understanding the pathophysiology and drug mechanisms in chronic inflammatory disorders can be achieved by focusing on macrophage phenotypes in the context of complex interactions in human tissues.One Sentence SummaryA newly identified human macrophage phenotype from patients with the autoimmune condition RA is found to promote joint tissue invasiveness and demonstrates variable sensitivities to anti-inflammatory medications used to treat the disease.

Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human tissues is limited. Using single-cell RNA sequencing, we defined distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF

Enhancers regulate gene expression and have been linked with disease pathogenesis. Little is known about enhancers that regulate human disease-associated genes in primary cells relevant for pathogenesis. Here we use BAC transgenics and genome editing to dissect, in vivo and in primary immune cells, enhancers that regulate human TNFAIP3, which encodes A20 and is linked with autoimmune diseases. A20 expression is dependent on a topologically associating subdomain (sub-TAD) that harbors four enhancers, while another >20 enhancers in the A20 locus are redundant. This sub-TAD contains cell- and activation-specific enhancers, including an enhancer (termed TT>A) harboring a proposed causal SLE-associated SNV. Deletion of the sub-TAD or the TT>A enhancer results in enhanced inflammatory responses, autoantibody production, and inflammatory arthritis, thus establishing functional importance in vivo and linking enhancers with a specific disease phenotype. These findings provide insights into enhancers that regulate human A20 expression to prevent inflammatory pathology and autoimmunity., The human TNFAIP3 gene, which encodes for A20, is associated with autoimmune diseases. Here, the authors use BAC transgenics combined with CRISPR- and recombineering-mediated genome editing to dissect in vivo and in primary immune cells, the role of enhancers regulating TNFAIP3.

As a strategy to identify gene expression changes affected by human polynucleotide phosphorylase (hPNPaseold-35), we performed gene expression analysis of HeLa cells in which hPNPaseold-35 was overexpressed. The observed changes were then compared to those of HO-1 melanoma cells in which hPNPaseold-35 was stably knocked down. Through this analysis, 90 transcripts, which positively or negatively correlated with hPNPaseold-35 expression, were identified. The majority of these genes were associated with cell communication, cell cycle, and chromosomal organization gene ontology categories. For a number of these genes, the positive or negative correlations with hPNPaseold-35 expression were consistent with transcriptional data extracted from the TCGA (The Cancer Genome Atlas) expression datasets for colon adenocarcinoma (COAD), skin cutaneous melanoma (SKCM), ovarian serous cyst adenocarcinoma (OV), and prostate adenocarcinoma (PRAD). Further analysis comparing the gene expression changes between Ad.hPNPaseold-35 infected HO-1 melanoma cells and HeLa cells overexpressing hPNPaseold-35 under the control of a doxycycline-inducible promoter, revealed global changes in genes involved in cell cycle and mitosis. Overall, this study provides further evidence that hPNPaseold-35 is associated with global changes in cell cycle-associated genes and identifies potential gene targets for future investigation. J. Cell. Physiol. 229: 1952–1962, 2014. © 2014 Wiley Periodicals, Inc.

Polynucleotide phosphorylase (PNPase) is an evolutionarily conserved 3′ → 5′ phosphate-dependent exoribonucease belonging to the PDX family of proteins. It consists of two catalytic RNase PH domains (PNP1 and PNP2), an α-helical domain and two RNA-binding domains. The PNP1 and PNP2 domains share substantial sequence and structural homology with RNase PH (RPH), which is another PDX family member found in all the three major kingdoms of life, suggesting that these three domains originated from a common ancestor. Phylogenetic analysis (based on the PNPase/RNase PH sequence information for 43 vertebrate taxa) shows that PNP2 and RPH are sister taxa which arose through duplication of the ancestral PNP1 domain. Also, all three domains (PNP1, PNP2 and RPH), along with the KH and S1 domains have undergone significant and directional sequence change, as determined by branch and site-specific d N /d S analyses. In general, codons that show d N /d S ratios that are significantly greater than 1.0 are outside the ordered regions (α-helices and β-sheets) of these protein domains. In addition, sites that have been selected for mutagenesis in these proteins lie embedded in regions where there is a preponderance of codons with d N /d S values that are not significantly different from 0.0. Overall, this report is an attempt to further our understanding of the evolutionary history of these three protein domains, and define the evolutionary events that led to their refinement in the vertebrate lineage leading to mammals.

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. Cancer Res; 72(23); 6217–26. ©2012 AACR.

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-specific-promoter 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.

Human polynucleotide phosphorylase (hPNPase(old-35)) is an evolutionary conserved RNA-processing enzyme with expanding roles in regulating cellular physiology. hPNPase(old-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 hPNPase(old-35) structurally and biochemically resembles PNPase of other species, overexpression and inhibition studies reveal that hPNPase(old-35) has evolved to serve more specialized and diversified functions in humans. Targeting specific mRNA or non-coding small microRNA, hPNPase(old-35) modulates gene expression that in turn has a pivotal role in regulating normal physiological and pathological processes. In these contexts, targeted overexpression of hPNPase(old-35) represents a novel strategy to selectively downregulate RNA expression and consequently intervene in a variety of pathophysiological conditions.

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.

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 (cg17197774) located downstream of the CpG island, b) cg17197774 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 cg17197774, d) cg17197774 hypomehtylation 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 VIGOR (Virtual Gene Over-expression or Repression ) analytical scheme, we were able to predict mda-9’s 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-9’s functionality.

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., PLoS ONE, 12 (7), ISSN:1932-6203

As a strategy to identify gene expression changes affected by human polynucleotide phosphorylase (hPNPase(old-35)), we performed gene expression analysis of HeLa cells in which hPNPase(old-35) was overexpressed. The observed changes were then compared to those of HO-1 melanoma cells in which hPNPase(old-35) was stably knocked down. Through this analysis, 90 transcripts, which positively or negatively correlated with hPNPase(old-35) expression, were identified. The majority of these genes were associated with cell communication, cell cycle, and chromosomal organization gene ontology categories. For a number of these genes, the positive or negative correlations with hPNPase(old-35) expression were consistent with transcriptional data extracted from the TCGA (The Cancer Genome Atlas) expression datasets for colon adenocarcinoma (COAD), skin cutaneous melanoma (SKCM), ovarian serous cyst adenocarcinoma (OV), and prostate adenocarcinoma (PRAD). Further analysis comparing the gene expression changes between Ad.hPNPase(old-35) infected HO-1 melanoma cells and HeLa cells overexpressing hPNPase(old-35) under the control of a doxycycline-inducible promoter, revealed global changes in genes involved in cell cycle and mitosis. Overall, this study provides further evidence that hPNPase(old-35) is associated with global changes in cell cycle-associated genes and identifies potential gene targets for future investigation.

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 Ca (2+). 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.

RNA degradation plays a fundamental role in maintaining cellular homeostasis whether it occurs as a surveillance mechanism eliminating aberrant mRNAs or during RNA processing to generate mature transcripts. 3'-5' exoribonucleases are essential mediators of RNA decay pathways, and one such evolutionarily conserved enzyme is polynucleotide phosphorylase (PNPase). The human homologue of this fascinating enzymatic protein (hPNPaseold-35) was cloned a decade ago in the context of terminal differentiation and senescence through a novel "overlapping pathway screening" approach. Since then, significant insights have been garnered about this exoribonuclease and its repertoire of expanding functions. The objective of this review is to provide an up-to-date perspective of the recent discoveries made relating to hPNPaseold-35 and the impact they continue to have on our comprehension of its expanding and diverse array of functions.

Human Polynucleotide Phosphorylase (hPNPase(old-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 hPNPase(old-35) in cellular physiology, we knocked-down and overexpressed hPNPase(old-35) in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPase(old-35) resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPase(old-35) caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPase(old-35) knockdown and overexpression datasets allowed us to identify 77 potential "direct" and 61 potential "indirect" targets of hPNPase(old-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 hPNPase(old-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.

RNA degradation plays a fundamental role in maintaining cellular homeostasis whether it occurs as a surveillance mechanism eliminating aberrant mRNAs or during RNA processing to generate mature transcripts. 3'-5' exoribonucleases are essential mediators of RNA decay pathways, and one such evolutionarily conserved enzyme is polynucleotide phosphorylase (PNPase). The human homologue of this fascinating enzymatic protein (hPNPaseold-35) was cloned a decade ago in the context of terminal differentiation and senescence through a novel "overlapping pathway screening" approach. Since then, significant insights have been garnered about this exoribonuclease and its repertoire of expanding functions. The objective of this review is to provide an up-to-date perspective of the recent discoveries made relating to hPNPaseold-35 and the impact they continue to have on our comprehension of its expanding and diverse array of functions.

The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p = 0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced overexpression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p = 0.0015-0.030) and in vivo (p = 0.016) cellular growth, invasion (p = 0.029-0.049), and colony formation (p = 0.023-0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser(473)), STAT3 (Tyr(705)), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated ß-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.

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 ( hPNPase old-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 hPNPase old-35 identified miRNAs differentially and specifically regulated by hPNPase old-35 . One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27 kip1 , displayed robust down-regulation with ensuing up-regulation of p27 kip1 by expression of hPNPase old-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 hPNPase old-35 . Inhibition of hPNPase old-35 by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-β–mediated growth inhibition, accentuating the importance of hPNPase old-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 hPNPase old-35 might provide an effective therapeutic strategy for miR-221–overexpressing and IFN-resistant tumors, such as melanoma.

Human polynucleotide phosphorylase (hPNPaseold-35) is an evolutionarily conserved 3′, 5′ exoribonuclease predominantly involved in mRNA degradation. It is a type I IFN-inducible early response gene that was identified as a gene upregulated in terminally differentiated and senescent fibroblasts. Overexpression of hPNPaseold-35 via a replication incompetent adenovirus (Ad.hPNPaseold-35) arrests multiple cancer cell types in the G1 phase of the cell cycle, with cells eventually undergoing apoptosis. A potential mechanism of this growth inhibition and eventual apoptosis induction is the ability of hPNPaseold-35 to selectively degrade c-myc mRNA. This was further proven by the overexpression of c-myc that partially but significantly protects human melanoma cells from Ad.hPNPaseold-35-mediated growth inhibition, indicating that c-myc down-regulation might directly mediate the growth-inhibitory properties of Ad.hPNPaseold-35. Since the protection offered by c-myc overexpression was incomplete, additional targets of hPNPaseold-35 are likely to exist that may play a role in mediating its growth-inhibitory effects. The goal of the present study was to identify these additional candidate genes that may be directly or indirectly regulated by hPNPaseold-35, which would help us better understand the physiological implications of this interesting enzymatic protein. In order to identify the target genes a whole genome cDNA microarray was performed to assess gene expression changes that occur when hPNPaseold-35 was silenced in HO-1 melanoma cells. The genes that are upregulated in the hPNPaseold-35 knockdown cell line might be potential direct degradation targets of hPNPaseold-35 according to our current hypothesis. Finally, validation of these targets may provide us a unique opportunity for defining specific molecules and pathways that may represent new targets of hPNPaseold-35 contributing to its growth-inhibitory effects, which may be further used for developing approaches to enhance its therapeutic efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2184. doi:1538-7445.AM2012-2184

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.