Loss of SMAD4 Is Sufficient to Promote Tumorigenesis in a Model of Dysplastic Barrett’s Esophagus

Abstract

Loss of the tumor-suppressor gene SMAD4 promotes progression of high-grade Barrett's esophagus toward esophageal adenocarcinoma. This study provides an Q7 in vivo model of dysplastic Barrett's esophagus progression toward invasive esophageal adenocarcinoma upon SMAD4 inactivation.BACKGROUND & 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 transforming growth factor b (TGF-b) signaling functionality and in vivo tumorigenicity in high-grade dysplastic Barrett's cells. METHODS:An in vivo xenograft model was used to test tumorigenicity of SMAD4 knockdown or knockout in CP-B highgrade dysplastic Barrett's cells. RT 2 Q8polymerase chain reaction arrays were used to analyze TGF-b 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-b pathway target gene expression profile. SMAD4 knockout positively regulates potential oncogenes such as CRYAB, ACTA2, and CDC6, whereas the CDKN2A/B tumorsuppressor locus was regulated negatively. 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, downregulation of tumor-suppressor genes, and chromosomal instability within the tumors after SMAD4 loss implicates SMAD4 as a protector of genome integrity in EAC development and progression. Foremost, SMAD4 loss promotes tumorigenesis from dysplastic Barrett's toward EAC.

Drivers of Esophageal Adenocarcinoma and Opportunities for Cancer Interception

“…To assess the role of SMAD4 in malignant transformation, wild-type CP-B cells were then compared with SMAD4 knockout and knockdown lines in a tumor-formation assay using a xenograft model. 2 Although only 1 of 17 mice injected with control cells formed a tumor, all of the SMAD4 knockdown and many of the SMAD4 knockout cell lines established tumors in the xenograft model, illustrating that SMAD4 loss, in the context of a dysplastic cell line, was sufficient to form tumors. 2 Furthermore, xenografts were used to rederive cell lines that were reinjected into mice resulting in rapid development of metastatic disease.…”

“…10 The current study by Gotovac et al 2 provides a deeper understanding of the underlying mechanism for the development of genomic instability and aggressive cancer. 2 Using an established BE cell line (CP-B) from a patient with BE dysplasia, CRISPR/Cas9 technology was used to knockout the SMAD4 gene. Lentiviral vectors were also used for RNAi-mediated SMAD4 knockdown.…”

“…Molecular approaches to stratifying BE risk offer intriguing opportunities for cancer interception, defined as active intervention to reduce cancer risk. 1 In the paper by Gotovac et al, 2 the investigators model progression from dysplastic BE to EAC to better understand the functional significance of SMAD4 loss. Their experiments, focused on alterations in SMAD4, provide insights into how molecular alterations resulting in genomic instability can lead to rapid progression to cancer and metastasis.…”

“…Lentiviral vectors were also used for RNAi-mediated SMAD4 knockdown. Comparison of the wild-type and SMAD4 knockdown revealed SMAD4 loss was associated with increased expression of CDC6, 2 a gene with proto-oncogenic activity. Gotovac et al 2 also demonstrated that the increased CDC6 was associated with loss of tumor suppressor CDKN2A/B, a factor already associated with BE.…”

“…Comparison of the wild-type and SMAD4 knockdown revealed SMAD4 loss was associated with increased expression of CDC6, 2 a gene with proto-oncogenic activity. Gotovac et al 2 also demonstrated that the increased CDC6 was associated with loss of tumor suppressor CDKN2A/B, a factor already associated with BE. To assess the role of SMAD4 in malignant transformation, wild-type CP-B cells were then compared with SMAD4 knockout and knockdown lines in a tumor-formation assay using a xenograft model.…”