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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.

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.

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.

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.

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

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.

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.

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.