Collins Karikari

An in vivo platform for translational drug development in pancreatic cancer

Effective development of targeted anticancer agents includes the definition of the optimal biological dose and biomarkers of drug activity. Currently available preclinical models are not optimal to this end. We aimed at generating a model for translational drug development using pancreatic cancer as a prototype. Resected pancreatic cancers from 14 patients were xenografted and expanded in successive groups of nude mice to develop cohorts of tumor-bearing mice suitable for drug therapy in simulated early clinical trials. The xenografted tumors maintain their fundamental genotypic features despite serial passages and recapitulate the genetic heterogeneity of pancreatic cancer. The in vivo platform is useful for integrating drug screening with biomarker discovery. Passages of tumors in successive cohorts of mice do not change their susceptibility to anticancer agents and represent a perpetual live bank, facilitating the application of new technologies that will result in the creation of an integrated stable database of tumor-drug response data and biomarkers.

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]

Restitution of tumor suppressor microRNAs using a systemic nanovector inhibits pancreatic cancer growth in mice

Mis-expression of microRNAs (miRNA) is widespread in human cancers, including in pancreatic cancer. Aberrations of miRNA include overexpression of oncogenic miRs (Onco-miRs) or downregulation of so-called tumor suppressor TSG-miRs. Restitution of TSG-miRs in cancer cells through systemic delivery is a promising avenue for pancreatic cancer therapy. We have synthesized a lipid-based nanoparticle for systemic delivery of miRNA expression vectors to cancer cells (nanovector). The plasmid DNA–complexed nanovector is approximately 100 nm in diameter and shows no apparent histopathologic or biochemical evidence of toxicity upon intravenous injection. Two miRNA candidates known to be downregulated in the majority of pancreatic cancers were selected for nanovector delivery: miR-34a, which is a component of the p53 transcriptional network and regulates cancer stem cell survival, and the miR-143/145 cluster, which together repress the expression of KRAS2 and its downstream effector Ras-responsive element binding protein-1 (RREB1). Systemic intravenous delivery with either miR-34a or miR-143/145 nanovectors inhibited the growth of MiaPaCa-2 subcutaneous xenografts (P < 0.01 for miR-34a; P < 0.05 for miR-143/145); the effects were even more pronounced in the orthotopic (intrapancreatic) setting (P < 0.0005 for either nanovector) when compared with vehicle or mock nanovector delivering an empty plasmid. Tumor growth inhibition was accompanied by increased apoptosis and decreased proliferation. The miRNA restitution was confirmed in treated xenografts by significant upregulation of the corresponding miRNA and significant decreases in specific miRNA targets (SIRT1, CD44 and aldehyde dehydrogenase for miR34a, and KRAS2 and RREB1 for miR-143/145). The nanovector is a platform with potential broad applicability in systemic miRNA delivery to cancer cells. Mol Cancer Ther; 10(8); 1470–80. ©2011 AACR.

Epigenetic Silencing of MicroRNA miR-107 Regulates Cyclin-Dependent Kinase 6 Expression in Pancreatic Cancer

Aberrant expression of microRNAs (miRNAs) has emerged as an important hallmark of cancer. However, the putative mechanisms regulating miRNAs per se are only partially known. It is well established that many tumor suppressor genes in human cancers are silenced by chromatin alterations, including promoter methylation and histone deacetylation. We postulated that miRNAs undergo similar epigenetic inactivation in pancreatic cancer. Two human pancreatic cancer cell lines – MiaPACA-2 and PANC-1 – were treated with the demethylating agent, 5-aza-2′-deoxycytidine (5-Aza-dC) or the histone deacetylase inhibitor, trichostatin A, as well as the combination of the two. Expression of miRNAs in control and treated cell lines was assessed using a custom microarray platform. Fourteen miRNAs were upregulated two-fold or greater in each of the cell lines following exposure to both chromatin-modifying agents, including 5 that were in common (miR-107, miR-103, miR-29a, miR-29b, and miR-320) to both MiaPACA-2 and PANC-1. The differential overexpression of miR-107 in the treated cancer cell lines was confirmed by Northern blot assays. Methylation-specific PCR assays for assessment of CpG island methylation status in the 5′ promoter region of the miR-107 primary transcript demonstrated complete loss of methylation upon exposure to 5-Aza-dC. Enforced expression of miR-107 in MiaPACA-2 and PANC-1 cells downregulated in vitro growth, and this was associated with repression of the putative miR-107 target, cyclin-dependent kinase 6, thereby providing a functional basis for the epigenetic inactivation of this miRNA in pancreatic cancer.

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.

Systemic Administration of Polymeric Nanoparticle-Encapsulated Curcumin (NanoCurc) Blocks Tumor Growth and Metastases in Preclinical Models of Pancreatic Cancer

Curcumin or diferuloylmethane is a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa). A large volume (several hundreds) of published reports has established the anticancer and chemopreventative properties of curcumin in preclinical models of every known major cancer type. Nevertheless, the clinical translation of curcumin has been significantly hampered due to its poor systemic bioavailability, which mandates that patients consume up to 8 to 10 g of the free drug orally each day to achieve detectable levels in circulation. We have engineered a polymeric nanoparticle encapsulated curcumin formulation (NanoCurc) that shows remarkably higher systemic bioavailability in plasma and tissues compared with free curcumin upon parenteral administration. In xenograft models of human pancreatic cancer established in athymic mice, administration of parenteral NanoCurc significantly inhibits primary tumor growth in both subcutaneous and orthotopic settings. The combination of parenteral NanoCurc with gemcitabine results in enhanced tumor growth inhibition versus either single agent, suggesting an additive therapeutic influence in vivo. Furthermore, this combination completely abrogates systemic metastases in orthotopic pancreatic cancer xenograft models. Tumor growth inhibition is accompanied by significant reduction in activation of nuclear factor-κB, as well as significant reduction in expression of matrix metalloproteinase-9 and cyclin D1, in xenografts treated with NanoCurc and gemcitabine. NanoCurc is a promising new formulation that is able to overcome a major impediment for the clinical translation of curcumin to cancer patients by improving systemic bioavailability, and by extension, therapeutic efficacy. Mol Cancer Ther; 9(8); 2255–64. ©2010 AACR.

Convergent structural alterations define SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeler as a central tumor suppressive complex in pancreatic cancer

Defining the molecular genetic alterations underlying pancreatic cancer may provide unique therapeutic insight for this deadly disease. Toward this goal, we report here an integrative DNA microarray and sequencing-based analysis of pancreatic cancer genomes. Notable among the alterations newly identified, genomic deletions, mutations, and rearrangements recurrently targeted genes encoding components of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, including all three putative DNA binding subunits (ARID1A, ARID1B, and PBRM1) and both enzymatic subunits (SMARCA2 and SMARCA4). Whereas alterations of each individual SWI/SNF subunit occurred at modest-frequency, as mutational “hills” in the genomic landscape, together they affected at least one-third of all pancreatic cancers, defining SWI/SNF as a major mutational “mountain.” Consistent with a tumor-suppressive role, re-expression of SMARCA4 in SMARCA4-deficient pancreatic cancer cell lines reduced cell growth and promoted senescence, whereas its overexpression in a SWI/SNF-intact line had no such effect. In addition, expression profiling analyses revealed that SWI/SNF likely antagonizes Polycomb repressive complex 2, implicating this as one possible mechanism of tumor suppression. Our findings reveal SWI/SNF to be a central tumor suppressive complex in pancreatic cancer.

The Axl receptor tyrosine kinase confers an adverse prognostic influence in pancreatic cancer and represents a new therapeutic target.

Pancreatic cancer is a near uniformly lethal disease and a better understanding of the molecular basis of this malignancy may lead to improved therapeutics. The Axl receptor tyrosine kinase is implicated in cellular transformation and tumor progression, although its role in pancreatic cancer has not been previously documented. The immunohistochemical expression of Axl protein was assessed in a panel of 99 archival pancreatic cancers. Axl labeling was present in 54 of 99 (55%), and was absent in 45 of 99 (45%) cases, respectively. Axl expression in pancreatic cancer was significantly associated with lymph node metastases (P

Tumor Engraftment in Nude Mice and Enrichment in Stroma- Related Gene Pathways Predict Poor Survival and Resistance to Gemcitabine in Patients with Pancreatic Cancer

Purpose: The goal of this study was to evaluate prospectively the engraftment rate, factors influencing engraftment, and predictability of clinical outcome of low-passage xenografts from patients with resectable pancreatic ductal adenocarcinoma (PDA) and to establish a bank of PDA xenografts. Experimental Design: Patients with resectable PDA scheduled for resection at the Johns Hopkins Hospital were eligible. Representative pieces of tumor were implanted in nude mice. The status of the SMAD4 gene and content of tumor-generating cells were determined by immunohistochemistry. Gene expression was carried out by using a U133 Plus 2.0 array. Patients were followed for progression and survival. Results: A total of 94 patients with PDA were resected, 69 tumors implanted in nude mice, and 42 (61%) engrafted. Engrafted carcinomas were more often SMAD4 mutant, and had a metastatic gene expression signature and worse prognosis. Tumors from patients resistant to gemcitabine were enriched in stroma-related gene pathways. Tumors sensitive to gemcitabine were enriched in cell cycle and pyrimidine gene pathways. The time to progression for patients who received treatment with gemcitabine for metastatic disease (n = 7) was double in patients with xenografts sensitive to gemcitabine. Conclusion: A successful xenograft was generated in 61% of patients attempted, generating a pool of 42 PDA xenografts with significant biological information and annotated clinical data. Patients with PDA and SMAD4 inactivation have a better engraftment rate. Engraftment is a poor prognosis factor, and engrafted tumors have a metastatic gene expression signature. Tumors from gemcitabine-resistant patients were enriched in stromal pathways. Clin Cancer Res; 17(17); 5793–800. ©2011 AACR.

Aberrant MicroRNA-155 Expression Is an Early Event in the Multistep Progression of Pancreatic Adenocarcinoma

Background/Aims: Pancreatic intraepithelial neoplasia (PanIN) is the most common noninvasive precursor to invasive pancreatic adenocarcinoma. Misexpression of microRNAs (miRNAs) is commonly encountered in invasive neoplasia; however, miRNA abnormalities in PanIN lesions have not been documented. Methods: Three candidate miRNAs (miR-21, miR-155, and miR-221) previously reported as overexpressed in pancreatic cancers were assessed in 31 microdissected PanINs (14 PanIN-1, 9 PanIN-2, 8 PanIN-3) using quantitative reverse transcription PCR (qRT-PCR). Subsequently, miR-155 was evaluated by locked nucleic acid in situ hybridization (LNA-ISH) in PanIN tissue microarrays. Results: Relative to microdissected non-neoplastic ductal epithelium, significant overexpression of miR-155 was observed in both PanIN-2 (2.6-fold, p = 0.02) and in PanIN-3 (7.4-fold, p = 0.014), while borderline significant overexpression of miR-21 (2.5-fold, p = 0.049) was observed in PanIN-3 only. In contrast, no significant differences in miR-221 levels were observed between ductal epithelium and PanIN lesions by qRT-PCR. LNA-ISH confirmed the aberrant expression of miR-155 in PanIN-2 (9 of 20, 45%) and in PanIN-3 (8 of 13, 62%), respectively, when compared with normal ductal epithelium (0 of 10) (p < 0.01). Conclusions: Abnormalities of miRNA expression are observed in the multistep progression of pancreatic cancer, with miR-155 aberrations demonstrable at the stage of PanIN-2, and miR-21 abnormalities at the stage of PanIN-3 lesions.