Roberts, Lewis R.

Abstract [Background and Aim] TRAIL is a promising therapeutic agent for human malignancies that can activate apoptosis via mitochondria-independent (Type I) or mitochodria-dependent (Type II) death receptor signaling. However, many malignant cells are resistant to TRAIL cytotoxicity and fail to trigger Type I or Type II killing. Using cholangiocarcinoma cells, we identified a role for Hedgehog signaling via GLI transcription factors in the regulation of TRAIL cytotoxicity, and provide new mechanistic insights whereby Hedghog inhibition increases apoptosis. [Materials and Methods] The human cholangiocarcinoma cell lines KMCH, HuCCT-1, and Mz-ChA-1 were employed. Hedgehog signaling was inhibited with cyclopamine or an shRNA construct targeting smootherned (SMO). Type II TRAIL signaling was inhibited with shRNA constructs for Bid or Bim and siRNA for Bax and Bak. Antiapoptotic mediators including Inhibitor of Apoptosis Proteins (IAPs) and Bcl-2 family proteins were examined by immunoblot analysis and/or real time RT-PCR. Apoptosis was quantified morphologically by DAPI and fluorescence microscopy, and biochemically by caspase-3/7 activity. GLI transcription factor binding to the XIAP promoter was examined by EMSA and chromatin immuneprecipitation (ChIP) assay. [Results] Pharmacologic or genetic inhibition of Hedgehog signaling down-regulates both XIAP and cIAP-1 mRNA and protein expression. Knockdown of XIAP, but not cIAP-1, by small-interfering RNA markedly sensitized cells to TRAIL-induced apoptosis, suggesting XIAP is the functional target. Putative GLI-binding sites were identified in the XIAP promoter, and EMSA and ChIP assays demonstrated direct binding by GLI proteins. Sensitization to TRAIL-induced apoptosis was not altered by knockdown of the BH3-only proteins Bim and Bid, or by knockdown of Bax plus Bak, implicating a mitochondria-independent Type I pathway of apoptosis. [Conclusion] These data provide evidence for up-regulation of XIAP by an oncogenic Hedgehog pathway and demonstrate that Hedgehog inhibition sensitizes cells to TRAIL via decreasing XIAP levels. Hedgehog inhibition thereby promotes Type I death receptor signaling and sensitizes cells to apoptosis, which suggests Hedgehog and XIAP inhibition may be a therapeutic approach for TRAIL-resistant neoplasms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2011-4105

Abstract The mammalian target of rapamycin (mTOR) consists of two distinct complexes, mTORC1 and mTORC2. mTOR plays a pivotal role in Hepatocellular Carcinoma (HCC). Previous studies indicated that inhibition of mTORC1 only moderately enhanced histone deacetylase inhibitors (HDACis)-mediated anti-tumor activity. This limited effect may be attributable to the increase of AKT activation through S6K1-IRS1-PI3K negative feedback loop triggered by selective mTORC1 inhibition. While as AKT is the downstream effector of mTORC2, we hypothesize that dual targeting of mTORC1/C2 would be more efficient in suppressing AKT and in enhancing anti-tumor activity of HDACi. In this study, the interactions between mTOR kinase inhibitors (mTORKis) and HDACis were examined in vitro using established HCC cell lines and in vivo using patient-derived HCC tumor xenografts on SCID mice. Our results demonstrate that inhibition of both mTORC1/2 not only effectively blocked mTORC1 signaling, but also abrogated negative-feedback activation of AKT caused by mTORC1 inhibition. Furthermore, inactivation of mTORC1/C2 by mTORKis substantially enhanced HDACi-induced anti-tumor activity. Either dysfunction of mTORC2 by knockdown of Rictor (a component of mTORC2), or diminution of AKT1 significantly lowered the threshold of HDACi-induced apoptosis. Lastly, in vivo study indicated that the combination of AZD8055 and Vorinostat yielded much greater tumor-growth inhibition compared to either agent alone in primary HCC xenografts without obvious adverse effects. Conclusion: Dual mTORC1/2 inhibition is more efficient than selective mTORC1 inhibition in enhancing anti-tumor activity of HDACi due to abrogation of negative-feedback activation of AKT induced by selective mTORC1 inhibition. Our findings suggest that combining regimen of mTORKi and HDACi may be an effective therapeutic strategy for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5363. doi:10.1158/1538-7445.AM2011-5363

Abstract Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines (Huh7, HepG2, SNU182, PLC/PRF/5, Hep3B, SNU423 and SNU449) compared to primary hepatocytes. To determine if miR-199a-3p has a tumor suppressive role, miR-199a-3p mimetic was transfected into the 7 HCC cell lines. miR-199a-3p mimetic reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A miR-199a-3p mimetic formulated with chemical modifications to enhance stability while preserving processing reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available miR-199a-3p mimetic. Furthermore, only the duplex miR-199a-3p mimetic, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling (Orian-Rousseau, et al., Genes Dev. 2002) and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Indeed, miRNA target algorithms predict miR-199a-3p to be a putative target of CD44. Immunoblotting confirmed that only the HCC lines that were sensitive to the effects of miR-199a-3p mimetic (SNU449 and SNU423) were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC tissue specimens. The anti-proliferative and anti-invasive properties of miR-199a-3p mimetic on CD44+ HCC may be a useful targeted therapy for CD44+ HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1183. doi:10.1158/1538-7445.AM2011-1183

Abstract The tumor microenvironment, including stromal myofibroblasts and associated matrix proteins, regulates cancer cell invasion and proliferation. Here, we report that neuropilin-1 (NRP-1) orchestrates communications between myofibroblasts and soluble fibronectin that promote α5β1 integrin–dependent fibronectin fibril assembly, matrix stiffness, and tumor growth. Tumor growth and fibronectin fibril assembly were reduced by genetic depletion or antibody neutralization of NRP-1 from stromal myofibroblasts in vivo. Mechanistically, the increase in fibronectin fibril assembly required glycosylation of serine 612 of the extracellular domain of NRP-1, an intact intracellular NRP-1 SEA domain, and intracellular associations between NRP-1, the scaffold protein GIPC, and the nonreceptor tyrosine kinase c-Abl that augmented α5β1 fibronectin fibril assembly activity. Analysis of human cancer specimens established an association between tumoral NRP-1 levels and clinical outcome. Our findings indicate that NRP-1 activates the tumor microenvironment, thereby promoting tumor growth. These results not only identify new molecular mechanisms of fibronectin fibril assembly but also have important implications for therapeutic targeting of the myofibroblast in the tumor microenvironment. Cancer Res; 72(16); 4047–59. ©2012 AACR.

Abstract Background/Aim: Glioma-associated oncogene 1 (GLI1) is one of the most important transcription factors in the hedgehog (Hh) signaling pathway and has been shown to be involved in embryonic development and tumor progression. The epithelial-to-mesenchymal transition (EMT) is an important cellular program which contributes to invasion and metastasis of cancer. We have previously shown that GLI1 may promote HCC metastasis via EMT. Here, we demonstrate that GLI1 upregulates SNAI1 expression, induces EMT, and promotes HCC recurrence in vivo and in vitro. Methods: Gli1 mRNA was assessed in 139 HCCs by microarray and its correlation with HCC recurrence and SNAI1 mRNA expression was analyzed. In vitro, the mRNA and protein expression of GLI1 was measured in Gli1-negative Huh7 cells transfected with GU1 and GU1-expressing SNU398 cells transfected with antisense oligonucleotides targeting GU1 (GLI1 ASO). GLI1-dependent transcriptional activity was assessed with GU reporter luciferase assay and electrophoretic mobility shift assay (EMSA). The interaction between Gli1, SNAI1 and EMT was assessed by real-time PCR, Western immunoblotting and immunofluorescence. Effects of GLI1 on proliferation, viability, migration, and invasion of tumor cells were also assessed. Results: GLI1 mRNA was detected in 74 HCC tumors and found to be upregulated in 43 (58.1 %) HCCs. Increased Gli1 mRNA in HCCs was associated with tumor recurrence after resection. There was a positive correlation between GLI1 and SNAI1 mRNA expression. In HCC cells, GLI1 overexpression increased Gli1-dependent transcriptional activity, proliferation, viability, migration, and invasion of tumor cells. GU1 also increased SNAI1 expression and induced the EMT. Opposite effects were observed in GU1-knockdown models. Conclusion: Together, these findings identify a novel mechanism mediating HCC recurrence that includes GLI1-mediated activation of EMT via upregulation of SNAI1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B20.

SummaryBackgroundThe extent to which potentially curative therapies are used in patients with hepatocellular cancer (HCC) and their related outcomes are unknown in the US.AimTo determine the rate and outcomes of potentially curative treatment in patients with HCC.MethodsEleven US centers followed patients with HCC between 2001 and 2007. We determined rates of liver transplantation, surgical resection, or tumour ablation during follow‐up, examined differences in adjusted survival of patients receiving these treatments, and determined the factors associated with receipt of potentially curative treatment.ResultsOf the 267 patients, 76 (28%) patients had early HCC, defined as Child A or B cirrhosis, with a solitary HCC or ≤3 nodules, each ≤3 cm. Of these, 53 (69.7%) received curative treatment. Thirty six percent of patients with non‐early HCC received curative treatment. Compared to patients with non‐early HCC who did not receive curative treatment, patients with early HCC and curative treatment had the best survival [hazard ratio, HR = 0.19 (95% CI, 0.08–0.42)] followed by patients with advanced HCC who received curative treatment [HR = 0.37 (95% CI, 0.22–0.64)]. Baseline performance status was significantly associated with receipt of curative treatment as well as survival after adjusting for demographics, clinical characteristics, and HCC stage.ConclusionsIn this multicenter database, most of the patients with early HCC received potentially curative treatment. However, only 28% of patients had early HCC. One‐third of patients with non‐early HCC also underwent curative therapy. Potentially curative treatment improved survival and this effect was seen in patients with early as well as non‐early HCC.

AbstractLearning Objectives:After completing this course, the reader will be able to:Identify the components of the “postembolization syndrome”: elevated liver function tests, right upper quadrant pain, nausea and vomiting, and fever.Distinguish the postembolization syndrome from rare complications of embolization that would merit an extended hospitalization.CME This article is available for continuing medical education credit at CME.TheOncologist.comBackground.There is scant evidence to guide the management of patients after hepatic artery embolization (HAE). We examined length of stay (LOS), laboratory patterns, medication usage, morbidity, and mortality of patients hospitalized after HAE for metastatic neuroendocrine tumors.Methods.Data were abstracted retrospectively from electronic medical records on LOS, liver function tests (LFTs), i.v. antibiotics, analgesia, peak temperature, bacteremia, hepatic abscess formation, carcinoid crisis, and metastatic burden on cross-sectional imaging.Results.In 2005–2009, 72 patients underwent 174 HAEs for carcinoid and islet cell tumors. The median LOS was 4 days (range, 1–8 days). There was no correlation between peak LFTs and tumor burden. Declines in LFTs were not uniform before hospital discharge; 25%, 37%, 30%, 53%, and 67% of patients were discharged before their respective aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total and direct bilirubin levels began to decline, with no readmissions for acute hepatic failure. The median i.v. analgesia dose was 60 mg oral morphine equivalents (range, 3–1,961 mg). Pre-HAE i.v. antibiotics were administered in 99% of cases; post-HAE fever occurred in 37% of patients, with no documented bacteremia. One patient developed a hepatic abscess after HAE. There were two carcinoid crises. The single in-hospital death was associated with air in the portal veins.Conclusions.The duration and intensity of in-hospital care following HAE should be managed on an individual basis. A downward trend in LFTs is not required before discharge. Modest use of i.v. analgesia suggests that many patients could exclusively receive oral analgesics. Given the rarity of serious complications, hospital stays could be shortened, thereby reducing costs and nosocomial risks.