Loss of SMAD4 is sufficient to promote tumorigenesis in a model of dysplastic Barrett's esophagus.

ABSTRACT Background and Aims Esophageal adenocarcinoma (EAC) develops from its precursor Barrett’s esophagus, through intermediate stages of low and high grade dysplasia. However, knowledge of genetic drivers and molecular mechanisms implicated in disease progression is limited. Herein, we investigated the effect of SMAD4 loss on TGFβ signaling functionality and in vivo tumorigenicity in high grade dysplastic Barrett’s cells. Methods In vivo xenograft model was utilized to test tumorigenicity of SMAD4 knockdown or knockout in CP-B high grade dysplastic Barrett’s cells. RT2 PCR arrays were used to analyse TGFβ signaling functionality and Low Coverage Whole Genome Sequencing was performed to detect copy number alterations, upon SMAD4 loss. Results We found that SMAD4 knockout significantly alters the TGF-β pathway target gene expression profile. SMAD4 knockout positively regulates potential oncogenes such as CRYAB, ACTA2 and CDC6, whereas the CDKN2A/B tumor suppressor locus was negatively regulated. We verified that SMAD4 in combination with CDC6-CDKN2A/B or CRYAB genetic alterations in patient tumors have significant predictive value for poor prognosis. Importantly, we investigated the effect of SMAD4 inactivation in Barrett’s tumorigenesis. We found that genetic knockdown or knockout of SMAD4 was sufficient to promote tumorigenesis in dysplastic Barrett’s esophagus cells, in vivo. Progression to invasive EAC was accompanied by distinctive and consistent copy number alterations in SMAD4 knockdown or knockout xenografts. Conclusions Altogether, up-regulation of oncogenes, down-regulation of tumor suppressor genes and chromosomal instability within the tumors following SMAD4 loss, implicates SMAD4 as a protector of genome integrity in EAC development and progression. Foremost, SMAD4 loss promotes tumorigenesis from dysplastic Barrett’s towards EAC.