Gene amplification and protein overexpression of MDM2, which is often found in certain types of cancers, indicate that MDM2 plays an important role in tumorigenesis. Interestingly, several clinical reports have demonstrated that amplification of the MDM2 gene correlates with the metastatic stage. Using an antibody array assay, we identified E-cadherin as an MDM2-binding protein and confirmed that E-cadherin is a substrate for the MDM2 E3 ubiquitin ligase. We demonstrate that MDM2 interacts in vivo with E-cadherin, resulting in its ubiquitination and degradation. This regulation appears to be clinically relevant, as we found a significant correlation between high MDM2 and low E-cadherin protein levels in resected tumor specimens recovered from breast cancer patients with lymph node metastases. Ectopic expression of MDM2 in breast cancer cells was found to disrupt cell-cell contacts and enhance cell motility and invasive potential. We found that E-cadherin and MDM2 colocalized on the plasma membrane and in the early endosome, where ubiquitin moieties were attached to E-cadherin. Blocking endocytosis with dominant-negative mutants of dynamin abolished the association of MDM2 with E-cadherin, prevented E-cadherin degradation, and attenuated cell motility as observed by fluorescence microscopy. Thus, we provide evidence to support a novel role for MDM2 in regulating cell adhesions by a mechanism that involves degrading and down-regulating the expression of E-cadherin via an endosome pathway. This novel MDM2-regulated pathway is likely to play a biologically relevant role in cancer metastasis.MDM2 has been shown to play an important role in a variety of physiological and pathological processes. Overexpression of the human homologue of MDM2, referred to as HDM2, occurs in diverse human malignancies including soft tissue sarcomas and cancers of the brain, breast, ovary, cervix, lung, colon, and prostate (40, 54). Overexpression in tumors correlates with a poor prognosis for those patients (4,5,24,25,39,43,44,50,53,56,58,61). Furthermore, several studies have shown that amplification of the MDM2 gene occurs more frequently in metastatic and recurrent cancers than in primary tumors (23,46). One study examined 100 tumor samples from patients with esophageal squamous cell carcinoma and found that MDM2 expression was the most significant risk factor for distant metastases (52), implicating its potential role in metastasis. Hypoxia, a common condition within solid tumors, has been shown to lead to the up-regulation of MDM2 expression and to increase the metastatic efficiency of tumor cells (77). Additionally, MDM2 expression was shown to correlate with increased levels of vascular endothelial growth factor, which may facilitate the intravasation and metastatic seeding of tumor cells (42,68,80). Thus, MDM2 expression appears to correlate with an increased risk of distant metastases, which may contribute to an overall poorer prognosis for patients with tumors that overexpress MDM2. However, the precise role and underlying mecha...

Importance Cytotoxic CD8+ T lymphocytes (TILs) participate in immune control of ovarian cancer; however, little is known about prognostic patterns of CD8+ TILs by histotype and in relation to other clinical factors. Objective To define the prognostic role of CD8+ TILs in epithelial ovarian cancer. Design Prospective survival cohort. Setting Multi-center observational. Participants Over 5,500 patients, including 3,196 high-grade serous ovarian carcinomas (HGSOCs), followed prospectively for over 24,650 person-years. Exposure(s) Following immunohistochemistry, CD8+ TILs were identified within the epithelial components of tumor islets. Patients were grouped based on the estimated number of CD8+ TILs per high-powered field: negative (none), low (1–2), moderate (3–19), and high (≥20). CD8+ TILs in a subset of patients were also assessed in a quantitative, uncategorized manner, and the functional form of associations with survival was assessed using penalized B-splines. Main Outcome Measure(s) Overall survival time. Results Among the five major invasive histotypes, HGSOCs showed the most infiltration. CD8+ TILs in HGSOCs were significantly associated with longer overall survival; median survival was 2.8 years for patients with no CD8+ TILs and 3.0 years, 3.8 years, and 5.1 years for patients with low, moderate, or high levels of CD8+ TILs, respectively (p-trend=4.2 × 10−16). A survival benefit was also observed among women with endometrioid and mucinous carcinomas, but not the other histotypes. Among HGSOCs, CD8+ TILs were favorable regardless of extent of residual disease following cytoreduction, known standard treatment, and germline BRCA1 pathogenic mutation, but were not prognostic for BRCA2 mutation carriers. Evaluation of uncategorized CD8+ TIL counts showed a near linear functional form. Conclusions and Relevance This study demonstrates the histotype-specific nature of immune infiltration and provides definitive evidence for a dose-response relationship between CD8+ TILs and HGSOC survival. That the extent of infiltration is prognostic, not merely its presence or absence, suggests that understanding factors which drive infiltration will be key to unravelling outcome heterogeneity in this cancer.

Emerging evidence indicates that microRNAs (miRNAs) play essential roles in regulating osteoblastogenesis and bone formation. However, the role of miRNA in osteoblast mechanotransduction remains to be defined. In this study, we aimed to investigate whether miRNAs regulate mechanical stimulation-triggered osteoblast differentiation and bone formation through modulation of Runx2, the master transcription factor for osteogenesis. We first investigated the role of mechanical loading both in a mouse model and in an osteoblast culture system and the outcomes clearly demonstrated that mechanical stimuli can regulate osteogenesis and bone formation both in vivo and in vitro. Using bioinformatic analyses and subsequent confirmation by quantitative real-time PCR (qRT-PCR), we found that multiple miRNAs that potentially target Runx2 were responding to in vitro mechanical stimulation, among which miR-103a was fully characterized. miR-103a and its host gene PANK3 were both downregulated during cyclic mechanical stretch (CMS)-induced osteoblast differentiation, whereas Runx2 protein expression was upregulated. Overexpression of miR-103a significantly decreased and inhibition of miR-103a increased Runx2 protein level, suggesting that miR-103a acts as an endogenous attenuator of Runx2 in osteoblasts. Mutation of putative miR-103a binding sites in Runx2 mRNA abolishes miR-103a-mediated repression of the Runx2 3 0 -untranslated region (3 0 UTR) luciferase reporter activity, suggesting that miR-103a binds to Runx2 3 0 UTR. Osteoblast marker gene profiling and osteogenic phenotype assays demonstrated that miR-103a negatively correlates with CMSinduced osteogenesis. Further, the perturbation of miR-103a also has a significant effect on osteoblast activity and matrix mineralization. More importantly, we found an inhibitory role of miR-103a in regulating bone formation in hindlimb unloading mice, and pretreatment with antagomir-103a partly rescued the osteoporosis caused by mechanical unloading. Taken together, our data suggest that miR-103a is the first identified mechanosensitive miRNA that regulates osteoblast differentiation by directly targeting Runx2, and therapeutic inhibition of miR-103a may be an efficient anabolic strategy for skeletal disorders caused by pathological mechanical loading.

BET proteins are a group of epigenetic regulators controlling transcription through reading acetylated histone tails and recruiting transcription complexes. They are considered as potential therapeutic targets in many distinct diseases. A novel synthetic bromodomain and extraterminal domain (BET) inhibitor, JQ1, was proved to suppress oncogene transcription and inflammatory responses. The present study was aimed to investigate the effects of JQ1 on inflammatory response and bone destruction in experimental periodontitis. We found that JQ1 significantly suppressed lipopolysaccharide (LPS)-stimulated inflammatory cytokine transcription, including interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), as well as receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast markers, such as c-Fos, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP) and cathepsin K in vitro. JQ1 also inhibited toll-like receptors 2/4 (TLR2/4) expression and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation and nuclear translocation. Chromatin immunoprecipitation and quantitative polymerase chain reaction (ChIP-qPCR) revealed that JQ1 neutralized BRD4 enrichment at several gene promoter regions, including NF-κB, TNF-α, c-Fos, and NFATc1. In a murine periodontitis model, systemic administration of JQ1 significantly inhibited inflammatory cytokine expression in diseased gingival tissues. Alveolar bone loss was alleviated in JQ1-treated mice because of reduced osteoclasts in periodontal tissues. These unprecedented results suggest the BET inhibitor JQ1 as a prospective new approach for treating periodontitis.

AimsTo evaluate the suitability of malignant pleural effusion (MPE) and plasma as surrogate samples for epidermal growth factor receptor (EGFR) mutation detection, and compare three different detection methods.MethodsMatched tissue and plasma samples were collected from patients with advanced non-small cell lung cancer (NSCLC) (stage IIIB/IV adenocarcinoma/adenosquamous carcinoma), with matched MPE samples collected from a subgroup. DNA was extracted from tissue, MPE cell block, MPE supernatant and plasma before mutation detection by amplification refractory mutation system (ARMS) (all samples), Sanger sequencing and mutant-specific immunohistochemistry (IHC) (tissue and MPE cell blocks only).ResultsSensitivity of MPE cell block, MPE supernatant and plasma versus tissue: 81.8% (9/11), 63.6% (7/11) and 67.5% (27/40); specificity was 80.0% (8/10), 100% (10/10) and 100% (46/46), respectively. Sensitivity of Sanger sequencing versus ARMS: 81.8% (27/33) for tissue, 40% (4/10) for MPE cell blocks; specificity was 100% (36/36 and 12/12) for both. Sensitivity of mutant-specific IHC versus ARMS: 54.8% (17/31) for tissue, 50.0% (6/12) for MPE cell blocks; specificity was 97.1% (34/35) and 100% (14/14), respectively.ConclusionsMPE and plasma are valid surrogates for NSCLC tumour EGFR mutation detection when tissue is not available. ARMS is most suitable for mutation detection in tissue and MPE cell blocks; however, mutant-specific IHC could be a complementary method when DNA-based molecular testing is unavailable.

Racemic phenanthroindolizidine alkaloids tylophorine, antofine, and deoxytylophorinine, and optically pure alkaloids S-(+)-tylophorine and R-(-)-tylophorine were synthesized and evaluated for their antiviral activities against tobacco mosaic virus (TMV). Further salinization modifications based on tylophorine increased stability and water solubility and improved the antiviral activity in application. The bioassay results showed that most of these synthesized compounds showed higher antiviral activity against TMV in vitro and in vivo than commercial Ningnanmycin. Especially, tylophorine salt derivatives 10, 11, 13, 17, and 22 emerged as potential inhibitors of plant virus. These findings demonstrate that these phenanthroindolizidine alkaloids and their salt derivatives represent a new template for antiviral studies and could be considered for novel therapy against plant virus infection.

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