Zhong et al. describe two novel roles for soluble TREM2 (sTREM2) in regulation of proinflammatory responses and prevention of cellular apoptosis in microglia.

The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death, reflecting the need for better understanding the oncogenesis, and developing new diagnostic and therapeutic targets for the malignancy. Emerging evidence suggests that small nucleolar RNAs (snoRNAs) have malfunctioning roles in tumorigenesis. Our recent study demonstrated that small nucleolar RNA 42 (SNORA42) was overexpressed in lung tumors. Here, we investigate the role of SNORA42 in tumorigenesis of NSCLC. We simultaneously assess genomic dosages and expression levels of SNORA42 and its host gene, KIAA0907, in 10 NSCLC cell lines and a human bronchial epithelial cell line. We then determine in vitro functional significance of SNORA42 in lung cancer cell lines through gain- and loss-of-function analyses. We also inoculate cancer cells with SNORA42-siRNA into mice through either tail vein or subcutaneous injection. We finally evaluate expression level of SNORA42 on frozen surgically resected lung tumor tissues of 64 patients with stage I NSCLC by using quantitative reverse transcriptase PCR assay. Genomic amplification and associated high expression of SNORA42 rather than KIAA0907 are frequently observed in lung cancer cells, suggesting that SNORA42 overexpression is activated by its genomic amplification. SNORA42 knockdown in NSCLC cells inhibits in vitro and in vivo tumorigenicity, whereas enforced SNORA42 expression in bronchial epitheliums increases cell growth and colony formation. Such pleiotropy of SNORA42 suppression could be achieved at least partially through increased apoptosis of NSCLC cells in a p53-dependent manner. SNORA42 expression in lung tumor tissue specimens is inversely correlated with survival of NSCLC patients. Therefore, SNORA42 activation could have an oncogenic role in lung tumorigenesis and provide potential diagnostic and therapeutic targets for the malignancy.

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Triggering Receptor Expressed on Myeloid cells 2 (TREM2), which is expressed on myeloid cells including microglia in the CNS, hasrecently been identified as a risk factor for Alzheimer's disease (AD). TREM2 transmits intracellular signals through its transmembrane binding partner DNAX-activating protein 12 (DAP12). Homozygous mutations inactivating TREM2 or DAP12 lead to Nasu-Hakola disease; however, how AD risk-conferring variants increase AD risk is not clear. To elucidate the signaling pathways underlying reduced TREM2 expression or loss of function in microglia, we respectively knocked down and knocked out the expression of TREM2 in in vitro and in vivo models. We found that TREM2 deficiency reduced the viability and proliferation of primary microglia, reduced microgliosis in Trem2 Ϫ / Ϫ mouse brains, induced cell cycle arrest at the G 1 /S checkpoint, and decreased the stability of ␤-catenin, a key component of the canonical Wnt signaling pathway responsible for maintaining many biological processes, including cell survival. TREM2 stabilized ␤-catenin by inhibiting its degradation via the Akt/GSK3␤ signaling pathway. More importantly, treatment with Wnt3a, LiCl, or TDZD-8, which activates the ␤-catenin-mediated Wnt signaling pathway, rescued microglia survival and microgliosis in Trem2 Ϫ / Ϫ microglia and/or in Trem2 Ϫ / Ϫ mouse brain. Together, our studies demonstrate a critical role of TREM2-mediated Wnt/␤-catenin pathway in microglial viability and suggest that modulating this pathway therapeutically may help to combat the impaired microglial survival and microgliosis associated with AD.

FUS1 is a tumor suppressor gene located on human chromosome 3p21, and expression of Fus1 protein is highly regulated at various levels, leading to lost or greatly diminished tumor suppressor function in many lung cancers. Here we show that selected microRNAs (miRNA) interact with the 3′-untranslated region (3′UTR) of FUS1, leading to down-regulation of protein expression. Using computational methods, we first predicted that FUS1 is a target of three miRNAs, miR-93, miR-98, and miR-197, and then showed that exogenous overexpression of these miRNAs inhibited Fus1 protein expression. We then confirmed that the three miRNAs target the 3′UTR region of the FUS1 transcript and that individual deletion of the three miRNA target sites in the FUS1 3′UTR restores the expression level of Fus1 protein. We further found that miR-93 and miR-98 are expressed at higher levels in small-cell lung cancer cell lines (SCLC) than in non-small-cell lung cancer cell lines (NSCLC) and immortalized human bronchial epithelial cells (HBEC), and that miR-197 is expressed at higher levels in both SCLCs and NSCLCs than in HBECs. Finally, we found that elevated miR-93 and miR-197 expression is correlated with reduced Fus1 expression in NSCLC tumor specimens. These results suggest that the three miRNAs are negative regulators of

Delivery of therapeutic genes to disseminated tumor sites has been a major challenge in the field of cancer gene therapy due to lack of an efficient vector delivery system. Among the various vectors currently available, liposomes have shown promise for the systemic delivery of genes to distant sites with minimal toxicity. In this report, we describe an improved extruded DOTAP:cholesterol (DOTAP:Chol) cationic liposome that efficiently delivers therapeutic tumor suppressor genes p53 and FHIT, which are frequently altered in lung cancer, to localized human primary lung cancers and to experimental disseminated metastases. Transgene expression was observed in 25% of tumor cells per tumor in primary tumors and 10% in disseminated tumors. When treated with DOTAP:Chol-p53 and -FHIT complex, significant suppression was observed in both primary (P < 0.02) and metastatic lung tumor growth (P < 0.007). Furthermore, repeated multiple treatments revealed a 2.5-fold increase in gene expression and increased therapeutic efficacy compared to single treatment. Finally, animal survival experiments revealed prolonged survival (median survival time: 76 days, P < 0.001 for H1299; and 96 days, P = 0.04 for A549) when treated with liposome-p53 DNA complex. Our findings may be of importance in the development of treatments for primary and disseminated human lung cancers.

The lack of a specific targeting strategy against cancer stem cells in current cancer treatment regimens is at least partly responsible for life-threatening cytotoxicity for patients undergoing traditional chemotherapy. An effective cancer stem cell targeting system is urgently required for the next generation of cancer medicine. Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and it has recently been identified as a cancer stem cell marker. In this study, we isolated a 40-base RNA aptamer that binds to EpCAM from a random oligonucleotide library using systematic evolution of ligands by exponential enrichment. The aptamer was further truncated to 19 bases. This 19-nt RNA aptamer interacts specifically with a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM, but not with those not expressing EpCAM, as analyzed using flow cytometry and confocal microscopy. The binding affinity of the EpCAM RNA aptamer to human cancer cells is approximately 55 nM. Importantly, this EpCAM RNA aptamer is efficiently internalized after binding to cell surface EpCAM. To our knowledge, this is the first RNA aptamer against a cancer stem cell surface marker being developed. Such cancer stem cell aptamers will greatly facilitate the development of novel targeted nanomedicine and molecular imaging agents for cancer theranostics. (Cancer Sci 2011; 102: 991-998) T he epithelial cell adhesion molecule EpCAM (also known as CD326 or ESA) is a pleiotropic molecule, capable of both promoting and preventing epithelial cell-cell adhesion.(1) It is a 30-40 kDa type I glycosylated membrane protein expressed at a low level in a variety of human epithelial tissues. EpCAM is overexpressed in most solid cancers.(2-4) For example, intense expression of EpCAM is found in more than 98% patients with colorectal cancer.(5) Two decades of studies have shed light on the roles that EpCAM plays in tumorigenesis. Rather than antagonising apoptosis, EpCAM acts by inducing proliferation with a direct impact on cell cycle control, upregulating the proto-oncogene c-myc and cyclins A and E, and signal transduction into the cell nucleus by way of the wnt pathway. (2,3,(6)(7)(8) Recently, it has been recognized that a small proportion of cancer cells possess unlimited proliferation potential and are able to self-renew and to generate differentiated cancer cell progeny. These so-called cancer stem cells (also known as cancer initiating cells) are resistant to chemotherapy and radiotherapy.(9) It is thought that cytotoxic drugs and radiation kill mainly the bulk tumor cells but spare the cancer stem cells and thus a cure or even long-term control of macroscopic solid cancers by chemotherapy is still an exception rather than the rule.(10) Therefore, in order to eradicate cancer, one must target and eliminate cancer stem cells.Epithelial cell adhesion molecule has been identified to be a cancer stem cell marker in a number of solid cancers, including breast cancer, (11) colorectal c...