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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.

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.

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.

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.