SMURF1 Amplification Promotes Invasiveness in Pancreatic Cancer

Pancreatic cancer is a deadly disease, and new therapeutic targets are urgently needed. We previously identified DNA amplification at 7q21-q22 in pancreatic cancer cell lines. Now, by high-resolution genomic profiling of human pancreatic cancer cell lines and human tumors (engrafted in immunodeficient mice to enrich the cancer epithelial fraction), we define a 325 Kb minimal amplicon spanning SMURF1, an E3 ubiquitin ligase and known negative regulator of transforming growth factor β (TGFβ) growth inhibitory signaling. SMURF1 amplification was confirmed in primary human pancreatic cancers by fluorescence in situ hybridization (FISH), where 4 of 95 cases (4.2%) exhibited amplification. By RNA interference (RNAi), knockdown of SMURF1 in a human pancreatic cancer line with focal amplification (AsPC-1) did not alter cell growth, but led to reduced cell invasion and anchorage-independent growth. Interestingly, this effect was not mediated through altered TGFβ signaling, assayed by transcriptional reporter. Finally, overexpression of SMURF1 (but not a catalytic mutant) led to loss of contact inhibition in NIH-3T3 mouse embryo fibroblast cells. Together, these findings identify SMURF1 as an amplified oncogene driving multiple tumorigenic phenotypes in pancreatic cancer, and provide a new druggable target for molecularly directed therapy

Genomic Profiling Identifies GATA6 as a Candidate Oncogene Amplified in Pancreatobiliary Cancer

Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies

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

Background: 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. Results: 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 <0.01), and a shorter median survival (12 versus 18 months, p <0.01), than in tumors with negative labeling. Stable knockdown of Axl resulted in significant reduction in cell viability (p <0.001), anchorage independent growth (p = 0.0031), as well as attenuation of migratory (p <0.001) and invasive properties (p <0.005), compared to vector-transfected cells. Profound inhibition of p42/p44 MAP kinase and PI-3kinase/Akt effector pathways was observed in MIAPaCa-2 cells with loss of Axl function. The reduction in invasion and migration upon Axl knockdown was mirrored by a decrease in the amounts of activated (GTP-bound) GTPase proteins Rho and Rac, significant downregulation in transcript levels of the epithelial mesenchymal transition (EMT)-associated transcription factors slug, snail and twist, and significant decrease in matrix metalloproteinase MMP-9 mRNA levels. Materials: The immunohistochemical expression of Axl protein was assessed in a panel of 99 archival pancreatic cancers. Endogenous Axl expression was stably downregulated by lentiviral short hairpin shRNA directed against AXL mRNA in MIAPaCa-2 cells, and the effects on cell viability, anchorage independent growth, invasion, migration and intracellular effector pathways was assessed, by comparing to lentiviral vector-transfected cells. Conclusion: Expression of Axl tyrosine kinase in pancreatic cancers confers an adverse prognostic influence, and represents a new therapeutic target in this malignancy

Array-Based Comparative Genomic Hybridization Identifies Localized DNA Amplifications and Homozygous Deletions in Pancreatic Cancer

Pancreatic cancer, the fourth leading cause of cancer death in the United States, is frequently associated with the amplification and deletion of specific oncogenes and tumor-suppressor genes (TSGs), respectively. To identify such novel alterations and to discover the underlying genes, we performed comparative genomic hybridization on a set of 22 human pancreatic cancer cell lines, using cDNA microarrays measuring ∼26,000 human genes (thereby providing an average mapping resolution of <60 kb). To define the subset of amplified and deleted genes with correspondingly altered expression, we also profiled mRNA levels in parallel using the same cDNA microarray platform. In total, we identified 14 high-level amplifications (38–4934 kb in size) and 15 homozygous deletions (46–725 kb). We discovered novel localized amplicons, suggesting previously unrecognized candidate oncogenes at 6p21, 7q21 (SMURF1, TRRAP), 11q22 (BIRC2, BIRC3), 12p12, 14q24 (TGFB3), 17q12, and 19q13. Likewise, we identified novel polymerase chain reaction-validated homozygous deletions indicating new candidate TSGs at 6q25, 8p23, 8p22 (TUSC3), 9q33 (TNC, TNFSF15), 10q22, 10q24 (CHUK), 11p15 (DKK3), 16q23, 18q23, 21q22 (PRDM15, ANKRD3), and Xp11. Our findings suggest candidate genes and pathways, which may contribute to the development or progression of pancreatic cancer