Surajit Dhara

Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells

Invasive pancreatic cancer is thought to develop through a series of noninvasive duct lesions known as pancreatic intraepithelial neoplasia (PanIN). We used cDNA microarrays interrogating 15,000 transcripts to identify 49 genes that were differentially expressed in microdissected early PanIN lesions (PanIN-1B/2) compared with microdissected normal duct epithelium. In this analysis, a cluster of extrapancreatic foregut markers, including pepsinogen C, MUC6, KLF4, and TFF1, was found to be up-regulated in PanIN. Up-regulation of these genes was further validated using combinations of real-time reverse transcription-PCR, in situ hybridization, and immunohistochemistry in a total of 150 early PanIN lesions from 81 patients. Identification of these gastrointestinal transcripts in human PanIN prompted assessment of other foregut markers by both semiquantitative and real-time reverse transcription-PCR, revealing similar up-regulation of Sox-2, Gastrin, HoxA5, GATA4/5/6, Villin and Forkhead 6 (Foxl1). In contrast to frequent expression of multiple gastric epithelial markers, the intestinal markers intestinal fatty acid binding protein, CDX1 and CDX2 were rarely expressed either in PanIN lesions or in invasive pancreatic cancer. Hedgehog pathway activation induced by transfection of immortalized human pancreatic ductal epithelial cells with Gli1 resulted in up-regulation of the majority of foregut markers seen in early PanIN lesions. These data show frequent up-regulation of foregut markers in early PanIN lesions and suggest that PanIN development may involve Hedgehog-mediated conversion to a gastric epithelial differentiation program.

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.

A low molecular weight PSMA-based fluorescent imaging agent for cancer

We synthesized YC-27 3 to provide a fluorescent imaging agent for the prostate-specific membrane antigen (PSMA), a marker for hormone-independent prostate cancer and tumor neovasculature, with suitable pharmacokinetics for use in vivo. Immediate precursor trifluoroacetate salt of 2-(3-{5-[7-(5-amino-1-carboxy-pentylcarbamoyl)-heptanoylamino]-1-carboxy-pentyl}-ureido)-pentanedioic acid 2 was conjugated with a commercially available near-infrared light-emitting dye (IRDye 800CW) to provide 3 in 72% yield. YC-27 3 demonstrated a PSMA inhibitory activity of 0.37nM and was capable of generating target-to-nontarget ratios of at least 10 in PSMA-expressing PC3-PIP vs. PSMA-negative PC3-flu tumors in vivo. YC-27 3 may be useful for study of PSMA-expressing tissue in preclinical models or for intraoperative guidance.

The High-Mobility Group A1a/Signal Transducer and Activator of Transcription-3 Axis: An Achilles Heel for Hematopoietic Malignancies?

Although HMGA1 (high-mobility group A1; formerly HMG-I/Y) is an oncogene that is widely overexpressed in aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only beginning to emerge. HMGA1 encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. To determine how HMGA1 leads to neoplastic transformation, we looked for genes regulated by HMGA1 using gene expression profile analysis. Here, we show that the STAT3 gene, which encodes the signaling molecule signal transducer and activator of transcription 3 (STAT3), is a critical downstream target of HMGA1a. STAT3 mRNA and protein are up-regulated in fibroblasts overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a in fibroblasts. HMGA1a also binds directly to a conserved region of the STAT3 promoter in vivo in human leukemia cells by chromatin immunoprecipitation and activates transcription of the STAT3 promoter in transfection experiments. To determine if this pathway contributes to HMGA1-mediated transformation, we investigated STAT3 expression in our HMGA1a transgenic mice, all of which developed aggressive lymphoid malignancy. STAT3 expression was increased in the leukemia cells from our transgenics but not in control cells. Blocking STAT3 function induced apoptosis in the transgenic leukemia cells but not in controls. In primary human leukemia samples, there was a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function in human leukemia or lymphoma cells led to decreased cellular motility and foci formation. Our results show that the HMGA1a-STAT3 axis is a potential Achilles heel that could be exploited therapeutically in hematopoietic and other malignancies overexpressing HMGA1a.

The High-Mobility Group A1 Gene Up-Regulates Cyclooxygenase 2 Expression in Uterine Tumorigenesis

Uterine cancer is the most common cancer of the female genital tract and is the fourth most frequent cause of cancer death in women in the U.S. Despite the high prevalence of uterine cancers, the molecular events that lead to neoplastic transformation in the uterus are poorly understood. Moreover, there are limited mouse models to study these malignancies. We generated transgenic mice with high-mobility group A1 gene (HMGA1a) expression targeted to uterine tissue and all female mice developed tumors by 9 months of age. Histopathologically, the tumors resemble human uterine adenosarcoma and are transplantable. To determine whether these findings are relevant to human disease, we evaluated primary human uterine neoplasms and found that HMGA1a mRNA and protein levels are increased in most high-grade neoplasms but not in normal uterine tissue, benign tumors, or most low-grade neoplasms. We also found that HMGA1a up-regulates cyclooxygenase 2 (COX-2) expression in transgenic tumors. Moreover, both HMGA1a and COX-2 expression are up-regulated in high-grade human leiomyosarcomas. Using chromatin immunoprecipitation, HMGA1a binds directly to the COX-2 promoter in human uterine cancer cells in vivo and activates its expression in transfection experiments. We also show that blocking either HMGA1a or COX-2 in high-grade human uterine cancer cells blocks anchorage-independent cell growth in methylcellulose. These findings show that HMGA1a functions as an oncogene when overexpressed in the uterus and contributes to the pathogenesis of human uterine cancer by activating COX-2 expression. Although a larger study is needed to confirm these results, HMGA1a may be a useful marker for aggressive human uterine cancers.

Effect of Nitroxoline on Angiogenesis and Growth of Human Bladder Cancer

BACKGROUND Angiogenesis plays an important role in tumor growth and metastasis; therefore, inhibition of angiogenesis is a promising strategy for developing new anticancer drugs. Type 2 methionine aminopeptidase (MetAP2) protein is likely a molecular target of angiogenesis inhibitors. METHODS Nitroxoline, an antibiotic used to treat urinary tract infections, was identified from a high-throughput screen of a library of 175,000 compounds for MetAP2 inhibitors and from a parallel screen using the Johns Hopkins Drug Library to identify currently used clinical drugs that can also inhibit human umbilical vein endothelial cells (HUVEC) proliferation. To investigate the mechanism of action of nitroxoline, inhibition of MetAP2 activity and induction of senescence were assessed in HUVEC. To test the antiangiogenic activity of nitroxoline, endothelial tube formation in Matrigel and microvessel formation in Matrigel plugs in vivo were assessed. Antitumor efficacy of nitroxoline was evaluated in mouse models of human breast cancer xenograft (n = 10) and bladder cancer orthotopic xenograft (n = 11). Furthermore, the mechanism of action of nitroxoline was investigated in vivo. RESULTS Nitroxoline inhibited MetAP2 activity in vitro (half maximal inhibitory concentration [IC(50)] = 54.8 nM, 95% confidence interval [CI] = 22.6 to 132.8 nM) and HUVEC proliferation (IC(50) = 1.9 μM, 95% CI = 1.54 to 2.39 μM). Nitroxoline inhibited MetAP2 activity in HUVEC in a dose-dependent manner and induced premature senescence in a biphasic manner. Nitroxoline inhibited endothelial tube formation in Matrigel and reduced microvessel density in vivo. Mice (five per group) treated with nitroxoline showed a 60% reduction in tumor volume in breast cancer xenografts (tumor volume on day 30, vehicle vs nitroxoline, mean = 215.4 vs 86.5 mm(3), difference = 128.9 mm(3), 95% CI = 32.9 to 225.0 mm(3), P = .012) and statistically significantly inhibited growth of bladder cancer in an orthotopic mouse model (tumor bioluminescence intensities of vehicle [n = 5] vs nitroxoline [n = 6], P = .045). CONCLUSION Nitroxoline shows promise as a potential therapeutic antiangiogenic agent.

Upregulation of MMP-2 by HMGA1 Promotes Transformation in Undifferentiated, Large-Cell Lung Cancer

Although lung cancer is the leading cause of cancer death worldwide, the precise molecular mechanisms that give rise to lung cancer are incompletely understood. Here, we show that HMGA1 is an important oncogene that drives transformation in undifferentiated, large-cell carcinoma. First, we show that the HMGA1 gene is overexpressed in lung cancer cell lines and primary human lung tumors. Forced overexpression of HMGA1 induces a transformed phenotype with anchorage-independent cell growth in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA1 expression blocks anchorage-independent cell growth in the H1299 metastatic, undifferentiated, large-cell human lung carcinoma cells. We also show that the matrix metalloproteinase-2 (MMP-2) gene is a downstream target upregulated by HMGA1 in large-cell carcinoma cells. In chromatin immunoprecipitation experiments, HMGA1 binds directly to the MMP-2 promoter in vivo in large-cell lung cancer cells, but not in squamous cell carcinoma cells. In large-cell carcinoma cell lines, there is a significant, positive correlation between HMGA1 and MMP-2 mRNA. Moreover, interfering with MMP-2 expression blocks anchorage-independent cell growth in H1299 large-cell carcinoma cells, indicating that the HMGA1–MMP-2 pathway is required for this transformation phenotype in these cells. Blocking MMP-2 expression also inhibits migration and invasion in the H1299 large-cell carcinoma cells. Our findings suggest an important role for MMP-2 in transformation mediated by HMGA1 in large-cell, undifferentiated lung carcinoma and support the development of strategies to target this pathway in selected tumors. (Mol Cancer Res 2009;7(11):1803–12)

Molecular Imaging of Metastatic Potential

If molecular imaging is to prove clinically useful it will have to surpass current, primarily anatomic techniques in terms of sensitivity and the ability to detect minimal changes in tissue. One of the most important tests for molecular imaging is to determine whether it can image the metastatic potential of tumors. Like all predictive endeavors, the imaging of such “potential” is a daunting task, but one that only molecular imaging—rather than standard, anatomic techniques—is likely to solve. Although difficult, imaging of metastatic potential is also arguably the most important task for molecular imaging of cancer because it is generally the dissemination of malignant tissue, not its prolonged residence in an inopportune site, which kills the patient. Below are examples of uses of molecular imaging of metastases as well as of metastatic potential, the former being a far more developed area of clinical inquiry.

CDC2/CDK1 expression in esophageal adenocarcinoma and precursor lesions serves as a diagnostic and cancer progression marker and potential novel drug target

Esophageal adenocarcinoma arises through well-defined precursor lesions (Barrett esophagus), although only a subset of these lesions advances to invasive adenocarcinoma. The lack of markers predicting progression in Barrett esophagus, typical presentation at advanced stage, and limitations of conventional chemotherapy result in >90% mortality for Barrett-associated adenocarcinomas. To identify potential prognostic markers and therapeutic targets, we compared gene expression profiles from Barrett-associated esophageal adenocarcinoma cell lines (BIC1, SEG1, KYAE, OE33) and normal esophageal epithelial scrapings utilizing the Affymetrix U133_A gene expression platform. We identified 560 transcripts with >3-fold up-regulation in the adenocarcinoma cell lines compared with normal epithelium. Utilizing tissue microarrays composed of normal esophageal squamous mucosa (n = 20), Barrett esophagus (n = 10), low-grade dysplasia (n = 14), high-grade dysplasia (n = 27), adenocarcinoma (n = 59), and node metastases (n = 27), we confirmed differential up-regulation of three proteins (Cdc2/Cdk1, Cdc5, and Igfbp3) in adenocarcinomas and Barrett lesions. Protein expression mirrored histologic progression; thus, 87% of low-grade dysplasias had at least focal surface Cdc2/Cdk1 and 20% had >5% surface staining; 96% of high-grade dysplasias expressed abundant surface Cdc2/Cdk1, while invasive adenocarcinoma and metastases demonstrated ubiquitous expression. Esophageal adenocarcinoma cell lines treated with the novel CDC2/CDK1 transcriptional inhibitor, tetra-O-methyl nordihydroguaiaretic acid (EM-1421, formerly named M4N) demonstrated a dose-dependent reduction in cell proliferation, paralleling down-regulation of CDC2/CDK1 transcript and protein levels. These findings suggest a role for CDC2/CDK1 in esophageal adenocarcinogenesis, both as a potential histopathologic marker of dysplasia and a putative treatment target.

Inactivation of the Cdkn2a locus cooperates with HMGA1 to drive T-cell leukemogenesis

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia with high relapse rates compared to B-lineage ALL. We previously showed that HMGA1a transgenic mice develop aggressive T-ALL, indicating that HMGA1 causes leukemic transformation in vivo. HMGA1 is also highly expressed in embryonic stem cells, hematopoietic stem cells and diverse, refractory human cancers. Disruption of the CDKN2A tumor suppressor locus occurs in most cases of T-ALL and is thought to contribute to leukemic transformation. To determine whether loss of function of CDKN2A cooperates with HMGA1 in T-ALL, we crossed HMGA1a transgenics onto a Cdkn2a null background. We discovered that T-ALL is markedly accelerated in HMGA1a transgenic Cdkn2a null mice. In addition, these mice recapitulate salient clinical and pathologic features of human T-ALL. HMGA1 is also highly overexpressed in human T-ALL. These findings suggest that HMGA1 plays a causative role in T-ALL and could represent a rational therapeutic target.