AKT And GSK3β Drive SOX2 Overexpression In Esophagus Squamous Cell Carcinoma Through Phosphorylation-Dependent Protein Stabilizaiton

Esophagus Squamous Cell Carcinoma(ESCC)is a malignant tumor that primarily originates in epithelial cell of esophagus.China is the highest morbidity and mortality of esophageal cancer in the world for a long time.Thus,it is urgent to fully and deeply investigare the underlying molecular mechanisms that drive pathogenesis and progression of ESCC.SOX2 is broadly recognized as a crucial driver for various tumors including ESCC.SOX2 has been shown to be broadly over-expressed in ESCC tumor samples.SOX2 gene amplification has been identified in 15-20% of ESCC samples,providing a mechanism for SOX2 overexpression in ESCCs.However,because SOX2 overexpression has been observed in ～60-70% ESCCs,additional mechanisms besides gene amplification must exist and remain to be explored.In this thesis,we mainly focus on exploring the molecular mechanisms that promote SOX2 protein stability in ESCCs,aiming to provide novel strategies for precision treatment of SOX2-positive cancers.Previous study from our laboratory revealed a critical role for AKT in promoting SOX2 protein stability in embryonic stem cells.We therefore investigated if AKT also plays a critical role in driving SOX2 overexpression in ESCC.We demonstrated that AKT is a major driver for SOX2 overexpression in ESCCs and stabilizes SOX2 via its ability to phosphorylate SOX2 proteins at the residue T116.We further identified UBR5 as a crucial ubiquitin E3 ligase that induces SOX2 degradation by ubiquitinating SOX2 at residue K115.AKT can protect SOX2 from degradation by phosohorylating SOX2 at threonine 116 and blocking the interaction of UBR5 with SOX2.We also provided evidence that AKT inhibitor can effectively downregulate SOX2 and suppress espphageal cancer cell proliferation and stemness.Taken together,our study provides new insight into SOX2 overexpression in cancer and evidence for targeting AKT as a potential therapeutic strategy for SOX2-positive cancer.Having established that AKT stabilizes SOX2 in ESCCs,we explored if there are additional signaling pathways that drive SOX2 overexpression in ESCCs.To this end,we employed a kinase inhibitor library that target the major signaling pathways in mammalian cells.We demonstrated that GSK3β-specific inhibitors can markedly down-regulate the level of SOX2 protein in various ESCC cell lines.Subsequent mechanistic study revealed that GSK3β promotes SOX2 stability by phosphorylating SOX2 at residue S251 and does so by inhibiting SOX2 degradation induced by CRL4 A ubiquitin E3 ligase.S251 phosphorylation by GSK3β inhibits the interaction of SOX2 with CRL4 A complex.Importantly,we found that both SOX2 and GSK3β are highly co-expressed in the basal layer of esophageal epithelium,and inhibition of GSK3β leads to substantial reduction of SOX2 protein levels.Moreover,tisse microarray analysis revealed that SOX2 and GSK3β are highly expressed in ESCCs.Notably,GSK3β overexpression is positively correlated with SOX2 protein levels in tumor samples.We also provided evidences that GSK3β inhibitor can effectively suppress esophageal cancer cell proliferation in subcutaneous xenogragt mouse model.Taken together,we uncover a novel role for GSK3β in driving SOX2 overexpression in ESCCs and provide evidence that targeting GSK3β likely a feasible treatment strategy for SOX2-positive cancers.