AUTHORS' REPLY

Abstract

<h3>Background</h3> Glutamate-cysteine ligase modifier (GCLM) subunits, combined with catalytic (GCLC) subunits, has been known for the first rate-limiting enzyme for glutathione (GSH) synthesis. Increasing studies have shown that the non-metabolic functions of many metabolic enzymes also play a crucial role in tumor progress. However, the non-metabolic functions of GCLM remain unexplored. Hence, we wonder if there are unorthodox functions of GCLM under redox stress participating colorectal Carcinoma(CRC) tumorigenesis. <h3>Methods</h3> Western blot, real-time quantitative PCR and immunohistochemical analysis were used to detect the relative expression of GCLM in cell lines and patients‘ specimens. The MTS assay, migration and invasion assay, sphere formation assay were performed to detect the functions of GCLM <i>in vitro</i>, and the xenograft models and PDX models in nude mice <i>in vivo</i>. Cytosolic and nuclear extraction, immunofluorescent analysis were used to show the location of GCLM. Chromatin immunoprecipitation (ChIP) assay was used to screen and verify the GCLM target genes. <h3>Results</h3> We found that glucose deprivation condition upregulated the expression of GCLM rather than hypoxia or H2O2. Compared to adjacent tumor tissues, the expression of GCLM was upregulated in CRC tumor tissues. The high expression of GCLM predicted poor prognosis in CRC patients. Inhibition of GCLM results in decreased proliferation rate, migration and invasion ability and sphere formation in HCT116 and DLD1 <i>in vitro</i>. Similarly, GCLM inhibition suppressed CRC tumorigenesis and metastasis in xenograft models and PDX models<i> in vivo</i>. The cancer cell stem cell markers were also downregulated after GCLM inhibition. Interestingly, we found that glucose starvation could rescue the anti-cancer phenotypes of GCLM inhibition and led to nucleus-translocation and accumulation of GCLM. ChIP assay showed GCLM could interact with octamer-binding transcription factor 4 (<i>OCT4)</i> promoter<i>,</i> a cancer stem cell marker, to increase its expression, and the target gene of <i>OCT4</i> showed a positive correlation with GCLM expression in CRC patients. <h3>Conclusions</h3> Our data showed that GCLM displayed nucleus accumulation in the condition of glucose deprivation which increased the transcription of <i>OCT4.</i> This study revealed an unorthodox oncogenic function of GCLM in colorectal carcinoma and suggested GCLM as a potential therapeutic target in CRC.