Cholangiocarcinomas (CCAs) are aggressive cancers, with high mortality and poor survival rates. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of late diagnosis secondary to relatively poor accuracy of diagnostic means. MicroRNAs (miRs) are involved in every cancer examined, but they have not been evaluated in primary CCA. In this study, miR arrays were performed on five primary CCAs and five normal bile duct specimens (NBDs). Several miRs were dysregulated and miR-21 was overexpressed in CCAs. miR-21 differential expression in these 10 specimens was verified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). To validate these findings, qRT-PCR for miR-21 was then performed on 18 additional primary CCAs and 12 normal liver specimens. MiR-21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the receiver operating characteristic curve of 0.995. Inhibitors of miR-21 increased protein levels of programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinases 3 (TIMP3). Notably, messenger RNA levels of TIMP3 were significantly lower in CCAs than in normals. Conclusions: MiR-21 is overexpressed in human CCAs. Furthermore, miR-21 may be oncogenic, at least in part, by inhibiting PDCD4 and TIMP3. Finally, these data suggest that TIMP3 is a candidate tumor suppressor gene in the biliary tree. (Hepatology 2009.)

Liver cancer is the fourth leading cause of cancer-related mortality and is distinguished by a relative paucity of chemotherapy options. It has been hypothesized that intratumor genetic heterogeneity may contribute to the high failure rate of chemotherapy. Here, we evaluated functional heterogeneity in a cohort of primary human liver cancer organoid lines. Each primary human liver cancer surgical specimen was used to generate multiple cancer organoid lines, obtained from distinct regions of the tumor. A total of 27 liver cancer lines were established and tested with 129 cancer drugs, generating 3,483 cell survival data points. We found a rich intratumor, functional (drug response) heterogeneity in our liver cancer patients. Furthermore, we established that the majority of drugs were either ineffective, or effective only in select organoid lines. In contrast, we found that a subset of drugs appeared pan-effective, displaying at least moderate activity in the majority of these cancer organoid lines. These drugs, which are FDA approved for indications other than liver cancers, deserve further consideration as either systemic or local therapeutics. Of note, molecular profiles, obtained for a reduced sample set, did not correlate with the drug response heterogeneity of liver cancer organoid lines. Taken together, these findings lay the foundation for in-depth studies of pan-effective drugs, as well as for functional personalized oncology approaches. Lastly, these functional studies demonstrate the utility of cancer organoid drug testing as part of a drug discovery pipeline.

Hookworm inspired, autonomous latching on the gastrointestinal mucosa extends the in vivo retention of a drug delivery device.

MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study we aimed at elucidating regulatory pathways and mechanisms through which miR‐494, one of the miR species found to be down‐regulated in cholangiocarcinoma (CCA), participates in cancer homeostasis. miR‐494 was identified as down‐regulated in CCA based on miR arrays. Its expression was verified with quantitative real‐time reverse‐transcription polymerase chain reaction (qRT‐PCR). To enforce miR expression, we employed both transfection methods, as well as a retroviral construct to stably overexpress miR‐494. Up‐regulation of miR‐494 in cancer cells decreased growth, consistent with a functional role. mRNA arrays of cells treated with miR‐494, followed by pathway analysis, suggested that miR‐494 impacts cell cycle regulation. Cell cycle analyses demonstrated that miR‐494 induces a significant G1/S checkpoint reinforcement. Further analyses demonstrated that miR‐494 down‐regulates multiple molecules involved in this transition checkpoint. Luciferase reporter assays demonstrated a direct interaction between miR‐494 and the 3′‐untranslated region of cyclin‐dependent kinase 6 (CDK6). Last, xenograft experiments demonstrated that miR‐494 induces a significant cancer growth retardation in vivo . Conclusion: Our findings demonstrate that miR‐494 is down‐regulated in CCA and that its up‐regulation induces cancer cell growth retardation through multiple targets involved in the G1‐S transition. These findings support the paradigm that miRs are salient cellular signaling pathway modulators, and thus represent attractive therapeutic targets. miR‐494 emerges as an important regulator of CCA growth and its further study may lead to the development of novel therapeutics. (HEPATOLOGY 2011)

MicroRNA (miRs) have emerged as salient regulators in cancer homeostasis and, recently, as putative therapeutics. Cholangiocarcinomas (CCA) are aggressive cancers with survival usually measured in months. mRNA arrays followed by pathway analysis revealed that miR-494 is a major modulator of the cell cycle progression from gap 2 (G₂) to mitosis (M). We performed fluorescence activated cell sorting (FACS) as well as differential interference contrast (DIC) microscopy, and confirmed that miR-494 induces a significant arrest in G₂/M in CCA cells. Furthermore, we verified that miR-494 modulates the protein level of six genes involved in the G₂/M transition: Polo-like Kinase 1 (PLK1), pituitary tumor-transforming gene 1 (PTTG1), Cyclin B1 (CCNB1), cell-division cycle 2 (CDC2), cell-division cycle 20 (CDC20) and topoisomerase II α (TOP2A). Next, we identified direct binding of miR-494 to the open reading frame (ORF) and downregulation of PTTG1 and TOP2A. In summary, our findings suggest that miR-494 has a global regulatory role in cell cycle progression, exerted by concerted effects on multiple proteins involved in gap 1 (G₁) to synthesis (S), as described previously, as well as G₂ to M progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. In addition, our data suggest that miRs act within a narrow range. miR expression above the upper threshold does not appear to induce further effects, which is reassuring in terms of off-target effects of miR surrounding noncancerous tissue.

Pseudogene INTS6P1 regulates its cognate gene INTS6 through competitive binding of miR-17-5p in hepatocellular carcinoma