Supplementary Material for: Downregulation of SIRT4 Expression Is Associated with Poor Prognosis in Esophageal Squamous Cell Carcinoma

<b><i>Objective:</i></b> SIRT4, a mitochondria-localized sirtuin, represses glutamine metabolism by inhibiting glutamate dehydrogenase (GDH). The current study aimed to evaluate the clinical and biological significance of SIRT4 in esophageal squamous cell carcinoma (ESCC). <b><i>Methods:</i></b> The study comprised 172 patients with surgically resected ESCC in two independent cohorts. <i>SIRT4</i> mRNA expression was analyzed in Cohort 1 (n = 79) and SIRT4 protein expression in Cohort 2 (n = 93). The association of SIRT4 expression with clinicopathological parameters and prognosis was assessed. Furthermore, the biological role of SIRT4 in ESCC cell lines was examined. <b><i>Results:</i></b> SIRT4 expression was not correlated with any clinicopathological parameters in both cohorts. In Cohort 1, low-<i>SIRT4</i>-expression cases had poorer overall survival than high-<i>SIRT4</i>-expression cases (p = 0.016). In Cohort 2, SIRT4-negative cases had poorer overall survival and disease-free survival than SIRT4-positive cases (p = 0.011 and 0.0026). Multivariate analysis revealed that SIRT4 expression was an independent prognostic factor for overall survival (HR = 2.06, p = 0.038). The rate of distant recurrence was significantly higher in SIRT4-negative cases than in SIRT4-positive cases (39.4 vs. 7.4%; p = 0.0023). In vitro, <i>SIRT4</i> knockdown significantly increased GDH activity and promoted cell proliferation and migration. <b><i>Conclusion:</i></b> SIRT4 is a potential prognostic biomarker in ESCC.