Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer

Abstract

SMAD4 is inactivated in the majority of pancreatic ductal adenocarcinomas (PDAC) with concurrent mutational inactivation of the INK4A/ARF tumor suppressor locus and activation of the KRAS oncogene. Here, using genetically engineered mice, we determined the impact of SMAD4 deficiency on the development of the pancreas and on the initiation and/or progression of PDAC-alone or in combination with PDAC-relevant mutations. Selective SMAD4 deletion in the pancreatic epithelium had no discernable impact on pancreatic development or physiology. However, when combined with the activated KRAS G12D allele, SMAD4 deficiency enabled rapid progression of KRAS G12D -initiated neoplasms. While KRAS G12D alone elicited premalignant pancreatic intraepithelial neoplasia (PanIN) that progressed slowly to carcinoma, the combination of KRAS G12D and SMAD4 deficiency resulted in the rapid development of tumors resembling intraductal papillary mucinous neoplasia (IPMN), a precursor to PDAC in humans. SMAD4 deficiency also accelerated PDAC development of KRAS G12D INK4A/ARF heterozygous mice and altered the tumor phenotype; while tumors with intact SMAD4 frequently exhibited epithelial-to-mesenchymal transition (EMT), PDAC null for SMAD4 retained a differentiated histopathology with increased expression of epithelial markers. SMAD4 status in PDAC cell lines was associated with differential responses to transforming growth factor-␤ (TGF-␤) in vitro with a subset of SMAD4 wild-type lines showing prominent TGF-␤-induced proliferation and migration. These results provide genetic confirmation that SMAD4 is a PDAC tumor suppressor, functioning to block the progression of KRAS G12D -initiated neoplasms, whereas in a subset of advanced tumors, intact SMAD4 facilitates EMT and TGF-␤-dependent growth.[Keywords: Smad4; pancreatic cancer; epithelial-to-mesenchymal transition mouse models; TGF-␤] Supplemental material is available at http://www.genesdev.org. PDAC (pancreatic ductal adenocarcinoma) ranks as the fourth leading cause of cancer mortality in the United States and carries a median survival of <6 mo (Li et al. 2004). Hallmarks of this disease include the infiltration of the tumor with a proliferative stroma (desmoplasia), early invasion and metastasis, and pronounced genomic instability (Solcia et al. 1995). PDAC is characterized by a highly recurrent pattern of genetic lesions consisting of activating mutations of KRAS and inactivation of INK4A (via mutation, deletion, or promoter methylation) in virtually all cases, inactivation of the p53-ARF pathway in ∼87% of cases (including tumors with deletions of the INK4A/ARF locus), and SMAD4 inactivation in ∼53% (Hansel et al. 2003). Hence, SMAD4 status can be considered as a distinguishing molecular feature of two major classes of PDAC. Significant ongoing efforts are being directed toward the elucidation of how specific signature mutations contribute to the genesis and progression of PDAC and influence its tumor biological features.

TAp73 loss favors Smad-independent TGF-β signaling that drives EMT in pancreatic ductal adenocarcinoma

“…We confirmed that TGF-β induced Bgn expression in 4A cells and not in W1 cells (Supplementary Figure S4a), suggesting that Bgn expression, after TGF-β treatment, is dependent on TAp73. The tumor-suppressor role of TGF-β in pancreatic cancer is mainly mediated by SMAD4, a transcription factor, inactivated in half of invasive PDA 41 and known to induce Bgn expression after TGF-β treatment. 42 Interestingly in TAp73-deficient cells, we observed a decrease in SMAD4 (Figure 6a) consistent with the presence of p53-responsive elements in all promoters of Smad genes.…”

MicroRNA 483-3p suppresses the expression of DPC4/Smad4 in pancreatic cancer

“…2D), suggesting that miR-483-3p can regulate DPC4/Smad4-regulated signaling pathways. It is known that DPC4/Smad4 is involved in TGF-b-induced epithelial-mesenchymal transition (EMT) [32], and that pancreatic cancers with wild type DPC4/Smad4 show frequent EMT [33]. To test the possibility that miR-483-3p could regulate the DPC4/ Smad4-mediated EMT process, we transfected PANC1 cells with miR-483-3p mimics or scramble oligos and simultaneously treated them with TGF-b.…”

Utilization of Liquid Chromatography Mass Spectrometry Analyses to Identify LKB1–APC Interaction in Modulating Wnt/β-Catenin Pathway of Lung Cancer Cells

“…Xenograft tumor tissue processing and immunohistochemical (IHC) staining were performed as previously described (24). The IHC analysis was performed as previously described using anti-LKB1 (Cell Signaling Technology), anti-c-Myc (Santa Cruz Biotechnology), anti-survivin (Santa Cruz Biotechnology), and anti-COX2 (Santa Cruz Biotechnology) antibodies.…”

“…MTT (Amresco) was added to 25 mL of cell suspension in 500 mL of medium and incubated for 2 hours as previously described (24).…”

“…Western blotting was performed as previously described (23,24). Primary antibodies used were anti-smooth muscle actin (SMA, ab5698), anti-N-cadherin (ab12221; Abcam), anti-E-cadherin (sc-8426), anti-vimentin (sc-32322), anti-APC (sc-54), anti-CD44 (sc-18849), anti-LKB1 (sc-5638 and sc-32245), anti-COX2 (sc-0736), anti-survivin (sc-752), anti-VEGF (sc-152), anti-cyclin D1 (sc-718), antic-Myc (9E10; sc-40), and total b-catenin (sc-1496; Santa Cruz Biotechnology), anti-GSK3b (#9315), anti-p-GSK3b (#9336, Cell Signal), anti-active-b-catenin (Millipore), and mouse anti-b-actin (Sigma Aldrich).…”