Surgery (St Vincent's) - Research Publications

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Start date: 2021 × End date: 2021 × Author: Clemons, Nicholas × Author: Duong, Cuong ×

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    SMAD4 as a potential gatekeeper for genomic instability and mTOR-mediated tumorigenesis in esophageal adenocarcinoma.
    Milne, JV ; Gotovac, JR ; Fujihara, KM ; Duong, CP ; Phillips, WA ; Clemons, NJ (AMER ASSOC CANCER RESEARCH, 2021-07)
    Abstract Esophageal cancer is the 8th most common cancer worldwide and has the 6th highest mortality rate of all cancers. The 5-year survival rate following esophageal adenocarcinoma (EAC) diagnosis is dismal at less than 15 percent, indicating a dire need for improved therapeutic strategies and early detection. EAC develops stepwise following exposure to chronic gastric reflux: From pre-malignant Barrett's metaplasia, through stages of low- and high-grade dysplasia until developing into invasive cancer. Mutation or loss of common tumor suppressor genes TP53 and SMAD4 act as markers for cancer progression, occurring in high-grade dysplastic tissue and invasive EAC, respectively. Our novel in vivo tumorigenesis model demonstrates progression of Barrett's metaplasia to EAC, in which SMAD4-deficient Barrett's metaplasia cells form tumors in immunodeficient mice after a period of latency and in a dose-dependent manner. This delayed tumor growth onset suggests further drivers are required for oncogenesis, and these SMAD4-deficient cells and tumors display a greater degree of genomic instability than wildtype-SMAD4 controls. A genome-wide CRISPR-Cas9 knockout screen unveiled a synthetic lethal relationship between SMAD4-deficiency and cell cycle checkpoint inhibition, suggesting a role for SMAD4 in maintaining genomic stability and a potential novel therapeutic avenue for SMAD4-deficient EAC. Additionally, a concurrent in vivo CRISPR-Cas9 tumorigenesis screen produced tumors 4-fold faster than the previous model and identified regulators of mTOR signaling as co-operative drivers of tumorigenesis in EAC. Wildtype-SMAD4 cells failed to generate tumors despite undergoing the same genetic perturbations, indicating a potential gatekeeping effect of SMAD4 in mTOR-mediated EAC tumorigenesis. In sum, loss of SMAD4 acts as a double-edged sword, increasing genomic instability and thereby rendering EAC cells sensitive to cell cycle checkpoint inhibition, whilst simultaneously co-operating with modulated mTOR signaling to promote tumorigenesis in EAC xenograft models. Citation Format: Julia V. Milne, Jovana R. Gotovac, Kenji M. Fujihara, Cuong P. Duong, Wayne A. Phillips, Nicholas J. Clemons. SMAD4 as a potential gatekeeper for genomic instability and mTOR-mediated tumorigenesis in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2671.
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    Loss of SMAD4 Is Sufficient to Promote Tumorigenesis in a Model of Dysplastic Barrett's Esophagus
    Gotovac, JR ; Kader, T ; Milne, J ; Fujihara, KM ; Lara-Gonzalez, LE ; Gorringe, KL ; Kalimuthu, SN ; Jayawardana, MW ; Duong, CP ; Phillips, WA ; Clemons, NJ (ELSEVIER INC, 2021)
    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 Mothers against decapentaplegic homolog 4 (SMAD4) loss on transforming growth factor β (TGF-β) 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 high-grade dysplastic Barrett's cells. RT2 polymerase chain reaction arrays were used to analyze TGF-β 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 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, down-regulation 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.