Tumor suppressor microRNAs (miRNA) provide a new opportunity to treat cancer. This approach, "miRNA replacement therapy," is based on the concept that the reintroduction of miRNAs depleted in cancer cells reactivates cellular pathways that drive a therapeutic response. Here, we describe the development of a therapeutic formulation using chemically synthesized miR-34a and a lipid-based delivery vehicle that blocks tumor growth in mouse models of non-small-cell lung cancer. This formulation is effective when administered locally or systemically. The antioncogenic effects are accompanied by an accumulation of miR-34a in the tumor tissue and downregulation of direct miR-34a targets. Intravenous delivery of formulated miR-34a does not induce an elevation of cytokines or liver and kidney enzymes in serum, suggesting that the formulation is well tolerated and does not induce an immune response. The data provide proof of concept for the systemic delivery of a synthetic tumor suppressor mimic, obviating obstacles associated with viral-based miRNA delivery and facilitating a rapid route for miRNA replacement therapy into the clinic. Cancer Res; 70(14); 5923-30. ©2010 AACR.

MicroRNAs (miRNAs) have recently emerged as an important new class of cellular regulators that control various cellular processes and are implicated in human diseases, including cancer. Here, we show that loss of let-7 function enhances lung tumor formation in vivo, strongly supporting the hypothesis that let-7 is a tumor suppressor. Moreover, we report that exogenous delivery of let-7 to established tumors in mouse models of non-small cell lung cancer (NSCLC) significantly reduces tumor burden. These results demonstrate the therapeutic potential of let-7 in NSCLC and point to miRNA replacement therapy as a promising approach in cancer treatment.

The macrolide fungicide rapamycin has shown significant antiproliferative action toward a variety of tumor types. In this study, we used BK5.erbB2 transgenic mice as an animal model to examine the therapeutic effect of rapamycin as a potential treatment for gallbladder cancer. Homozygous BK5.erbB2 mice overexpressing the wild-type rat erbB2 gene in basal epithelial cells of the gallbladder have an f70% incidence of gallbladder adenocarcinoma by 2 to 3 months of age. Groups of mice (f2-3 months of age) were treated with rapamycin by i.p. injection (once daily for 14 days) and then sacrificed 24 h after the last treatment. Rapamycin significantly reduced the incidence and severity of gallbladder carcinoma in BK5.erbB2 mice in a dose-dependent manner. Tumors responsive to treatment exhibited a higher number of apoptotic cells. Furthermore, rapamycin treatment led to decreased levels of phosphorylated p70 S6 kinase (Thr 389

Biliary tract cancer (BTC) is the second most common primary hepatobiliary cancer after hepatocellular cancer. At the time of diagnosis, most BTC are at an advanced stage and are unresectable. There is presently no effective curative treatment of the advanced disease nor is there any effective clinical therapy that will prevent the development of BTC. All of these factors render gallbladder cancer nearly incurable with a poor survival rate. The aim of our study was to provide a better understanding of the mechanisms involved in the development of gallbladder carcinoma as the advancement of more effective treatment options would significantly improve prognosis. In the present study, we examined the effect of gefitinib, a selective epidermal growth factor receptor/tyrosine kinase inhibitor (EGFR/TKI), on the development of gallbladder carcinoma in BK5.erbB2 mice. In addition, we examined the effect of another quinazoline derivative, GW2974, which is able to block the activation of both the EGFR and erbB2, in this model. Animals were treated with either 400 ppm gefitinib or 200 ppm GW2974 as a supplement in the diet using either a chemopreventive or therapeutic protocol. The results show that both compounds were potent chemopreventive and therapeutic agents in this mouse model of human BTC. The results also suggest that activation of the EGFR plays an important role in development of BTC in this model and that targeting both the EGFR and erbB2 may be an effective strategy for treatment of this disease.

Recent work from our laboratory has shown that elevated src kinase activity enhances tumor promotion, malignant progression, and metastasis during multistage skin carcinogenesis. In this study, we have generated "gene-switch" src(530) transgenic mice to further analyze the role of this nonreceptor tyrosine kinase in multistage carcinogenesis. Target transgenic mice that have an activated form of the human c-src (src(530)) gene fused with GAL4 binding sites upstream of the thymidine kinase (TK) promoter were generated. Two lines of epidermis-specific transactivator mice were used that targeted the expression of GLVPc or GLp65 transactivators, fusion molecules containing a truncated progesterone receptor with a GAl4-DNA binding domain, with either a mouse loricrin (ML) or human keratin 14 (HK14) promoter, respectively. The transactivator mice (ML.GLVPc or HK14.GLp65) and the target mice (TK.src(530)) were mated to generate bitransgenic mice, and src(530) transgene expression was induced by topical application of RU486 (mifepristone, a progesterone receptor antagonist). In both ML.GLVPc/TK.src(530) and HK14.GLp65/TK.src(530) bitransgenic mice, histological analysis revealed that only the bitransgenic mice had marked epidermal hyperplasia and hyperkeratosis after treatment with RU486. Neither the nontransgenic mice nor the mice hemizygous for either the transactivator transgene or the target transgene alone showed any response to treatment with RU486. In addition, no differences were observed in the skin of the bitransgenic mice versus nontransgenic littermates without treatment of RU486. Interestingly, in HK14.GLp65/TK.src(530) bitransgenic mice, squamous cell carcinomas (SCCs) arose along the periphery of the area of the punch biopsies in 25% of the bitransgenic mice several weeks after taking the biopsy and subsequent to RU486 treatment. Collectively, the data support a role of c-src activation in epidermal hyperproliferation. Furthermore, the data support the conclusion that src activation can substitute for an initiating event in the presence of a tumor promoting stimulus (i.e., wounding). Finally, inducible src(530) transgenic mice provide a new tool for dissecting the role of src activation in multistage carcinogenesis by allowing temporal control of the expression of this oncogene.

Background & Aims Gallbladder carcinoma (GBCa), a type of biliary tract cancer (BTC), has proven challenging to treat demonstrating the need for more effective therapeutic strategies. In our current study, we examined the therapeutic effects of the histone deacetylase (HDAC) inhibitor PCI-24781 against GBCa that developed in BK5.erbB2 mice. Methods PCI-24781 [50 mg/kg/day] and control solution were delivered to BK5.erbB2 mice for four weeks. The therapeutic effect of PCI-24781 was evaluated via ultrasound biomicroscopy (USBM) throughout the experiment and histological analyses at the end of the experiment. To investigate potential mechanisms for the therapeutic effects of PCI-24781 on GBCa in BK5.erbB2 mice, PCI-24781-treated gallbladders were subjected to Western blot and RT-PCR analysis. The inhibitory effect of PCI-24781 on the growth of BTC cells was compared to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and gemcitabine. To study the role of miRNAs in GBCa tumorigenesis, the expression profile of 368 miRNAs in GBCas from BK5.erbB2 (both treated and untreated) and wild-type mice was analyzed. Results Treatment of BK5.erbB2 mice with PCI-24781 for one month prevented 79% of GBCa cases from progression and showed a clinical effect in 47% of cases. We also confirmed a potent inhibitory effect on tumor cell growth in human BTC cell lines treated with PCI-24781. This effect was associated with down-regulation of erbB2 mRNA and erbB2 protein/activity and up-regulation of acetylated histone and acetylated tubulin. Treatment with PCI-24781 resulted in decreased expression of Muc4, an intramembrane ligand for erbB2, in BTC cells. PCI-24781 had more effect on inhibiting growth of BTC cells than SAHA. In addition, PCI-24781 effectively inhibited the growth of gemcitabine-resistant cells. MiRNA profiling revealed that the expression of several miRNAs were significantly altered in GBCa in the BK5.erbB2 mouse compared to normal gallbladder, including up-regulated miR21, which was down-regulated by PCI-24781. Conclusion These results indicate that PCI-24781 potently inhibits the growth of BTC cells by decreasing erbB2 expression and activity as well as regulating altered miRNA expression. PCI-24781 may have potential value as a novel chemotherapeutic agent against human BTC in which erbB2 is overexpressed.

Biliary tract cancer is still challenging to treat and manage due to its poor sensitivity to conventional therapies and the inability to prevent or detect the early tumor formation. The most well known risk factor for gallbladder cancer is the presence of chronic inflammation, usually related to gallstones. It has been suggested that cyclooxygenase-2 (COX-2) plays a variety of roles in the gastrointestinal tract, including pathogenic processes such as neoplasia. Recently, we have generated transgenic mice that overexpress rat ErbB-2 under the control of bovine keratin 5 promoter (BK5.ErbB-2 mice). Homozygous BK5.ErbB-2 mice develop adenocarcinoma of gallbladder with an f90% incidence. In addition to the activation of ErbB-2 and epidermal growth factor receptor, mRNA and protein levels of COX-2 were up-regulated in the gallbladder carcinomas that developed in these transgenic mice. The aim of this study was to examine the effects of a COX-2 inhibitor, CS-706, on the development of gallbladder carcinomas using the BK5.ErbB-2 mouse model. Ultrasound image analysis as well as histologic evaluation revealed a significant therapeutic effect of CS-706 on the gallbladder tumors, either as reversion to a milder phenotype or inhibition of tumor progression. The antitumor effect was associated with inhibition of prostaglandin E 2 synthesis. CS-706 treatment also down-regulated the activation of ErbB-2 and epidermal growth factor receptor, resulting in decreased levels of phosphorylated Akt and COX-2 in gallbladder cancers of BK5.ErbB-2 mice. Based on our results, targeting COX-2 could provide a potentially new and effective therapy alone or in combination with other therapeutic agents for patients with biliary tract cancer.

The erbB family of receptor tyrosine kinases are known to play important roles in normal epithelial development and epithelial neoplasia. Considerable evidence also suggests that signaling through the epidermal growth factor receptor (EGFR) plays an important role in multistage skin carcinogenesis in mice; however, less is known about the role of erbB2. In this study, to further examine the role of both erbB2 and EGFR in epithelial carcinogenesis, we examined the effect of a dual erbB2/EGFR tyrosine kinase inhibitor, GW2974, given in the diet on skin tumor promotion during two-stage carcinogenesis in wildtype and BK5.erbB2 mice. In BK5.erbB2 mice, erbB2 is overexpressed in the basal layer of epidermis and leads to heightened sensitivity to skin tumor development. GW2974 effectively inhibited skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in wild-type and BK5.erbB2 mice, although a more marked effect was seen in BK5.erbB2 mice. In addition, this inhibitory effect was reversible when GW2974 treatment was withdrawn. GW2974 inhibited 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperproliferation, which correlated with reduced activation of both the EGFR and erbB2. These results support the hypothesis that both the EGFR and erbB2 play an important role in the development of skin tumors during two-stage skin carcinogenesis, especially during the tumor promotion stage. Furthermore, the marked sensitivity of BK5.erbB2 mice to the inhibitory effects of GW2974 during tumor promotion suggest greater efficacy for this compound when erbB2 is overexpressed or amplified as an early event in the carcinogenic process. Cancer Prev Res; 3(8); 940-52. ©2010 AACR.