Smad4 Inactivation Promotes Malignancy and Drug Resistance of Colon Cancer

Abstract

SMAD4 is localized to chromosome 18q21, a frequent site for loss of heterozygosity in advanced stage colon cancers. Although Smad4 is regarded as a signaling mediator of the TGFb signaling pathway, its role as a major suppressor of colorectal cancer progression and the molecular events underlying this phenomenon remain elusive. Here, we describe the establishment and use of colon cancer cell line model systems to dissect the functional roles of TGFb and Smad4 inactivation in the manifestation of a malignant phenotype. We found that loss of function of Smad4 and retention of intact TGFb receptors could synergistically increase the levels of VEGF, a major proangiogenic factor. Pharmacologic inhibition studies suggest that overactivation of the TGFbinduced MEK-Erk and p38-MAPK (mitogen-activated protein kinase) auxiliary pathways are involved in the induction of VEGF expression in SMAD4 null cells. Overall, SMAD4 deficiency was responsible for the enhanced migration of colon cancer cells with a corresponding increase in matrix metalloprotease 9 enhanced hypoxiainduced GLUT1 expression, increased aerobic glycolysis, and resistance to 5 0 -fluoruracil-mediated apoptosis.Interestingly, Smad4 specifically interacts with hypoxia-inducible factor (HIF) 1a under hypoxic conditions providing a molecular basis for the differential regulation of target genes to suppress a malignant phenotype. In summary, our results define a molecular mechanism that explains how loss of the tumor suppressor Smad4 promotes colorectal cancer progression. These findings are also consistent with targeting TGFb-induced auxiliary pathways, such as MEK-ERK, and p38-MAPK and the glycolytic cascade, in SMAD4-deficient tumors as attractive strategies for therapeutic intervention. Cancer Res; 71(3); 998-1008. Ó2011 AACR.

Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids

“…Our results are further confirmed by the observation that in addition to loss of P53, SMAD4 inactivation plays a key role in late stages of CRC, such as migration and metastatic outgrowth potential. SMAD4 inactivation appeared to block differentiation, which is in line with previous findings where SMAD4 loss was associated with cell spreading, liver metastasis, and a poor disease prognosis (2,(20)(21)(22)(23). Together, our study depicts metastasis as an extremely inefficient process where at least four genetic alterations are required for tumor cells to seed and grow out at distant sites independently of stem cell niche factors.…”

Genetic disruption ofUSP9Xsensitizes colorectal cancer cells to 5-fluorouracil

“…3A). As has been previously observed, 19 apoptosis was suppressed in isogenic cells deficient in SMAD4; the effect of FBW7 genotype on apoptosis was variable.…”

“…These results are consistent with previous reports that SMAD4 is required for 5-FU-induced apoptosis. 19 In subcellular fractions of USP9X-deficient cells, we could observe the translocation of the pro-death BCL2 family member BCLX L from the cytosol into the mitochondria in tandem with the movement of cytochrome c from the mitochondria into the cytosol, and the degradation of mitochondrial MCL1, from 24 to 48 h after the start of 5-FU treatment (Fig. 3C).…”

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

“…The cells were cultured in low FBS media and incubated for predetermined times to monitor wound closing. Wound closure was recorded by phase-contrast microscopy according to previously published protocols (22,25).…”

“…Transient transfections and luciferase reporter assays were conducted using the Dual-Glo Luciferase Assay System from Promega (Cat. #E2920), as previously described (22,23,25).…”