Michael Mullendore

An orally bioavailable small-molecule inhibitor of Hedgehog signaling inhibits tumor initiation and metastasis in pancreatic cancer

Recent evidence suggests that blockade of aberrant Hedgehog signaling can be exploited as a therapeutic strategy for pancreatic cancer. Our previous studies using the prototype Hedgehog small-molecule antagonist cyclopamine had shown the striking inhibition of systemic metastases on Hedgehog blockade in spontaneously metastatic orthotopic xenograft models. Cyclopamine is a natural compound with suboptimal pharmacokinetics, which impedes clinical translation. In the present study, a novel, orally bioavailable small-molecule Hedgehog inhibitor, IPI-269609, was tested using in vitro and in vivo model systems. In vitro treatment of pancreatic cancer cell lines with IPI-269609 resembled effects observed using cyclopamine (i.e., Gli-responsive reporter knockdown, down-regulation of the Hedgehog target genes Gli1 and Ptch, as well as abrogation of cell migration and colony formation in soft agar). Single-agent IPI-269609 profoundly inhibited systemic metastases in orthotopic xenografts established from human pancreatic cancer cell lines, although Hedgehog blockade had minimal effect on primary tumor volume. The only discernible phenotype observed within the treated primary tumor was a significant reduction in the population of aldehyde dehydrogenase–bright cells, which we have previously identified as a clonogenic tumor-initiating population in pancreatic cancer. Selective ex vivo depletion of aldehyde dehydrogenase–bright cells with IPI-269609 was accompanied by significant reduction in tumor engraftment rates in athymic mice. Pharmacologic blockade of aberrant Hedgehog signaling might prove to be an effective therapeutic strategy for inhibition of systemic metastases in pancreatic cancer, likely through targeting subsets of cancer cells with tumor-initiating (“cancer stem cell”) properties. [Mol Cancer Ther 2008;7(9):2725–35]

Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway

Although activating mutations in RAS oncogenes are known to result in aberrant signaling through multiple pathways, the role of microRNAs (miRNAs) in the Ras oncogenic program remains poorly characterized. Here we demonstrate that Ras activation leads to repression of the miR-143/145 cluster in cells of human, murine, and zebrafish origin. Loss of miR-143/145 expression is observed frequently in KRAS mutant pancreatic cancers, and restoration of these miRNAs abrogates tumorigenesis. miR-143/145 down-regulation requires the Ras-responsive element-binding protein (RREB1), which represses the miR-143/145 promoter. Additionally, KRAS and RREB1 are targets of miR-143/miR-145, revealing a feed-forward mechanism that potentiates Ras signaling.

Hedgehog inhibition prolongs survival in a genetically engineered mouse model of pancreatic cancer

Background and aims: Pancreatic cancer is among the most dismal of human malignancies. Current therapeutic strategies are virtually ineffective in controlling advanced, metastatic disease. Recent evidence suggests that the Hedgehog signalling pathway is aberrantly reactivated in the majority of pancreatic cancers, and that Hedgehog blockade has the potential to prevent disease progression and metastatic spread. Methods: Here it is shown that the Hedgehog pathway is activated in the Pdx1-Cre;LsL-KrasG12D;Ink4a/Arflox/lox transgenic mouse model of pancreatic cancer. The effect of Hedgehog pathway inhibition on survival was determined by continuous application of the small molecule cyclopamine, a smoothened antagonist. Microarray analysis was performed on non-malignant human pancreatic ductal cells overexpressing Gli1 in order to screen for downstream Hedgehog target genes likely to be involved in pancreatic cancer progression. Results: Hedgehog inhibition with cyclopamine significantly prolonged median survival in the transgenic mouse model used here (67 vs 61 days; p = 0.026). In vitro data indicated that Hedgehog activation might at least in part be ascribed to oncogenic Kras signalling. Microarray analysis identified 26 potential Hedgehog target genes that had previously been found to be overexpressed in pancreatic cancer. Five of them, BIRC3, COL11A1, NNMT, PLAU and TGM2, had been described as upregulated in more than one global gene expression analysis before. Conclusion: This study provides another line of evidence that Hedgehog signalling is a valid target for the development of novel therapeutics for pancreatic cancer that might be worth evaluating soon in a clinical setting.

Ligand-dependent Notch signaling is involved in tumor initiation and tumor maintenance in pancreatic cancer

Purpose: Aberrant activation of the Notch signaling pathway is commonly observed in human pancreatic cancer, although the mechanism(s) for this activation has not been elucidated. Experimental Design: A panel of 20 human pancreatic cancer cell lines was profiled for the expression of Notch pathway-related ligands, receptors, and target genes. Disruption of intracellular Notch signaling, either genetically by RNA interference targeting NOTCH1 or pharmacologically by means of the γ-secretase inhibitor GSI-18, was used for assessing requirement of Notch signaling in pancreatic cancer initiation and maintenance. Results: Striking overexpression of Notch ligand transcripts was detectable in the vast majority of pancreatic cancer cell lines, most prominently JAGGED2 (18 of 20 cases, 90%) and DLL4 (10 of 20 cases, 50%). In two cell lines, genomic amplification of the DLL3 locus was observed, mirrored by overexpression of DLL3 transcripts. In contrast, coding region mutations of NOTCH1 or NOTCH2 were not observed. Genetic and pharmacologic inhibition of Notch signaling mitigated anchorage-independent growth in pancreatic cancer cells, confirming that sustained Notch activation is a requirement for pancreatic cancer maintenance. Further, transient pretreatment of pancreatic cancer cells with GSI-18 resulted in depletion in the proportion of tumor-initiating aldehyde dehydrogenase–expressing subpopulation and was associated with inhibition of colony formation in vitro and xenograft engraftment in vivo, underscoring a requirement for the Notch-dependent aldehyde dehydrogenase–expressing cells in pancreatic cancer initiation. Conclusions: Our studies confirm that Notch activation is almost always ligand dependent in pancreatic cancer, and inhibition of Notch signaling is a promising therapeutic strategy in this malignancy.

A resource for analysis of microRNA expression and function in pancreatic ductal adenocarcinoma cells.

MicroRNAs (miRNAs) are 21-24 nucleotide RNA molecules that regulate the translation and stability of target messenger RNAs. Abnormal miRNA expression is a common feature of diverse cancers. Several previous studies have classified miRNA expression in pancreatic ductal adenocarcinoma (PDAC), although no uniform pattern of miRNA dysregulation has emerged. To clarify these previous findings as well as to set the stage for detailed functional analyses, we performed global miRNA expression profiling of 21 human PDAC cell lines, the most extensive panel studied to date. Overall, 39 miRNAs were found to be dysregulated and have at least two- fold or greater differential expression in PDAC cell lines compared to control non-transformed pancreatic ductal cell lines. Several of these miRNAs show comparable dysregulation in first- passage patient-derived xenografts. Initial functional analyses demonstrate that enforced expression of miRNAs derived from the miR-200 family and the miR-17-92 cluster, both of which are overexpressed in PDAC cell lines, enhances proliferation. In contrast, inhibition of the miR-200 family diminishes anchorage independent growth. Consistent with a known role for the miR-200 family in negatively regulating an epithelial-to-mesenchymal transition (EMT), the abundance of these miRNAs correlated positively with E-cadherin expression and negatively with the EMT-associated transcription factors and established miR-200 targets ZEB1/ZEB2. Finally, restituted expression of miR-34a, a miRNA whose expression is frequently lost in PDAC cell lines, abrogates growth, demonstrating that the anti-proliferative activity of this miRNA is operative in PDAC. These results, and the widespread availability of PDAC cell lines wherein the aforementioned data were generated, provide a valuable resource for the pancreatic cancer research community and will greatly facilitate functional studies essential for elucidating the consequences of miRNA dysregulation in pancreatic cancer.

In vivo characterization of a polymeric nanoparticle platform with potential oral drug delivery capabilities

Nanotechnology has enabled significant advances in the areas of cancer diagnosis and therapy. The field of drug delivery is a sterling example, with nanoparticles being increasingly used for generating therapeutic formulations of poorly water-soluble, yet potent anticancer drugs. Whereas a number of nanoparticle-drug combinations are at various stages of preclinical or clinical assessment, the overwhelming majorities of such systems are injectable formulations and are incapable of being partaken orally. The development of an oral nano-delivery system would have distinct advantages for cancer chemotherapy. We report the synthesis and physicochemical characterization of orally bioavailable polymeric nanoparticles composed of N-isopropylacrylamide, methylmethacrylate, and acrylic acid in the molar ratios of 60:20:20 (designated NMA622). Amphiphilic NMA622 nanoparticles show a size distribution of <100 nm (mean diameter of 80 ± 34 nm) with low polydispersity and can readily encapsulate a number of poorly water-soluble drugs such as rapamycin within the hydrophobic core. No apparent systemic toxicities are observed in mice receiving as much as 500 mg/kg of the orally administered void NMA622 for 4 weeks. Using NMA622-encapsulated rapamycin (“nanorapamycin”) as a prototype for oral nano-drug delivery, we show favorable in vivo pharmacokinetics and therapeutic efficacy in a xenograft model of human pancreatic cancer. Oral nanorapamycin leads to robust inhibition of the mammalian target of rapamycin pathway in pancreatic cancer xenografts, which is accompanied by significant growth inhibition (P < 0.01) compared with control tumors. These data indicate that NMA622 nanoparticles provide a suitable platform for oral delivery of water-insoluble drugs like rapamycin for cancer therapy. [Mol Cancer Ther 2008;7(12):3878–88]

Snail and Sonic Hedgehog activation in neuroendocrine tumors of the ileum

The transcription factor Snail represses E-cadherin and induces epithelial-mesenchymal transition, a process also exploited by invasive cancer cells. Aberrant Hedgehog (Hh) signaling was recently observed in a variety of epithelial cancers and it has been shown that the Hh target gene Gli1 induces expression of Snail. In this study, we examined whether Snail and Sonic Hedgehog (SHH) are expressed in neuroendocrine tumors (NETs) of the ileum. Using immunohistochemistry, we found expression of Snail in 22 out of 37 (59%) of evaluated NET samples, but not in adjacent normal tissues. Snail expression was mostly restricted to the invasive front of the tumors. Six of seven liver metastases analyzed were positive for Snail. Intratumoral expression of SHH was detected in 27 out of 37 (73%) tumors. As opposed to Snail, cells expressing SHH were found to be distributed more randomly throughout the tumors. Out of 30 primary NETs, 16 (53%) showed both Snail and SHH expression. Furthermore, we found downregulation of E-cadherin in Snail-expressing cells by immunofluorescence. Real-time RT-PCR revealed conservation of the Hh target genes Gli1, Gli2, and Ptch in the pancreatic carcinoid cell line BON-1, which were downregulated upon Hh inhibition with cyclopamine. Moreover, Hh inhibition attenuated in vitro cell growth in a dose-dependent manner. In conclusion, we describe for the first time that Snail and SHH are overexpressed in a large subset of NETs of the ileum. Aberrant activation of these pathways might be involved in invasion and metastatic spread in NETs.

Homozygous deletions of methylthioadenosine phosphorylase in human biliary tract cancers

The p16INK4A/CDKN2A gene on chromosome 9p21 is a site of frequent allelic loss in human cancers, and in a subset of cases, homozygous deletions at this locus encompass the telomeric methylthioadenosine phosphorylase (MTAP) gene. The MTAP gene product is the principal enzyme involved in purine synthesis via the salvage pathway, such that MTAP-negative cancers are solely dependent on de novo purine synthesis mechanisms. Inhibitors of the de novo pathway can then be used to selectively blockade purine synthesis in cancer cells while causing minimal collateral damage to normal cells. In this study, we determine that 10 of 28 (35%) biliary tract cancers show complete lack of Mtap protein expression. In vitro analysis using a selective inhibitor of the de novo purine synthesis pathway, l-alanosine, shows robust growth inhibition in MTAP-negative biliary cancer cell lines CAK-1 and GBD-1 accompanied by striking depletion of intracellular ATP and failure to rescue this depletion via addition of exogenous methylthioadenosine, the principal substrate of the MTAP gene product; in contrast, no significant effects were observed in MTAP-expressing HuCCT1 and SNU308 cell lines. Colony formation studies confirmed that l-alanosine reduced both number and size of CAK-1 colonies in soft agar assays. Knockdown of Mtap protein by RNA interference in l-alanosine-resistant HuCCT1 cells conferred sensitivity to this agent, confirming that intracellular Mtap protein levels determine response to l-alanosine. Inhibitors of de novo purine synthesis can be a potential mechanism-based strategy for treatment of biliary tract cancers, one third of which show complete loss of MTAP function. [Mol Cancer Ther 2005;4(12):1860–6]

Serial Analysis of Gene Expression Identifies Connective Tissue Growth Factor Expression as a Prognostic Biomarker in Gallbladder Cancer

Background: Gallbladder cancer (GBC) is an uncommon neoplasm in the United States, but one with high mortality rates. This malignancy remains largely understudied at the molecular level such that few targeted therapies or predictive biomarkers exist. Experimental Design: We built the first series of serial analysis of gene expression (SAGE) libraries from GBC and nonneoplastic gallbladder mucosa, composed of 21-bp long-SAGE tags. SAGE libraries were generated from three stage-matched GBC patients (representing Hispanic/Latino, Native American, and Caucasian ethnicities, respectively) and one histologically alithiasic gallbladder. Real-time quantitative PCR was done on microdissected epithelium from five matched GBC and corresponding nonneoplastic gallbladder mucosa. Immunohistochemical analysis was done on a panel of 182 archival GBC in high-throughput tissue microarray format. Results: SAGE tags corresponding to connective tissue growth factor (CTGF) transcripts were identified as differentially overexpressed in all pairwise comparisons of GBC (P < 0.001). Real-time quantitative PCR confirmed significant overexpression of CTGF transcripts in microdissected primary GBC (P < 0.05), but not in metastatic GBC, compared with nonneoplastic gallbladder epithelium. By immunohistochemistry, 66 of 182 (36%) GBC had high CTGF antigen labeling, which was significantly associated with better survival on univariate analysis (P = 0.0069, log-rank test). Conclusions: An unbiased analysis of the GBC transcriptome by SAGE has identified CTGF expression as a predictive biomarker of favorable prognosis in this malignancy. The SAGE libraries from GBC and nonneoplastic gallbladder mucosa are publicly available at the Cancer Genome Anatomy Project web site and should facilitate much needed research into this lethal neoplasm.

Personalized Chemotherapy Profiling Using Cancer Cell Lines from Selectable Mice

Purpose: High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells. Experimental Design: We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Results: Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)–approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Conclusion: Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice. Clin Cancer Res; 19(5); 1139–46. ©2012 AACR.