LZTS2 Inhibits PI3K/AKT Activation And Radioresistance In Nasopharyngeal Carcinoma By Interacting With P85

Objective: Nasopharyngeal carcinoma(NPC)is the most common head and neck cancer derived from the epithelial cells at the nasopharynx.Radiotherapy remains the main treatment because of its specific anatomic location.Despite the clinical outcome has dramatically improved because of the advancement in modern radiotherapy technology and diagnostic imaging,loco-regional recurrence and distant metastasis still occurs at 20% ~ 30% patients of NPC due to radioresistance.Therefore,it is necessary to explore novel biomarkers and molecular mechanisms of radioresistance to further improve the prognosis for NPC patients.Leucine zipper tumor suppressor 2(LZTS2),also known as LAPSER1,is located on human chromosome 10q24.3.Accumulating evidences have suggested LZTS2 may be a potential tumor suppressor protein involved in tumorigenesis.The phosphatidylinositol-3-kinase(PI3K)signalling pathway has been extensively studied in the past decades since it is crucial to a wide spectrum of cellular processes,such as cell growth and proliferation,protein synthesis,glucose homeostasis and cell survival.The dysregulation and activation of PI3K/AKT signaling has been found in a variety of cancers,especially in nasopharyngeal carcinoma.Koeffler and colleagues reported that PI3 K signaling is prominently activation pathway in NPC using whole-exome sequencing,suggesting the activation of this pathway may contribute to NPC oncogenesis.In this study,we identified PI3 K subunit p85 as a novel LZTS2-interacting protein using an unbiased proteomics technology.However,it remains largely unknown how LZTS2 regulates PI3K/AKT signaling pathway and biological function of LZTS2 in radiosensitivity and tumorigenesis.Therefore,this project intends to uncover the mechanism of how LZTS2 negatively controls the PI3K/AKT pathway,and explore the role of LZTS2 in radioresistance of nasopharyngeal cancer.This study will provide a novel molecular target for radiosensitization of nasopharyngeal cancer.Methods: To evaluate the clinical significance of LZTS2 in NPC,we first detected the expression of LZTS2 protein level in nasopharyngeal carcinoma cell lines through Western blot.Next we performed the immunohistochemistry staining for LZTS2 in NPC tissue microarray.Kaplan-Meier analysis showed the relationship between expression of LZTS2 and overall survival of nasopharyngeal carcinoma patients.Tandem affinity purification and mass spectrometry method was used to identify candidate LZTS2-binding proteins in cells.Coimmunoprecipitation experimentswas used to validate the interaction between LZTS2 and p85.Competitive binding experiment explored whether LZTS2 affects the expression or translocation of p85.To further determine whether LZTS2 negatively controls the PI3 K signaling pathways,we examined the protein levels of downstream activated AKT and GSK3? of PI3 K pathway.To investigate the biological functions of LZTS2,cell growth,proliferation assay and cell cycle analysis of nasopharyngeal carcinoma were carried out through inhibiting LZTS2(si RNA).Clonogenic survival assay and ?-H2 AX foci formation experiment were conducted to assess the radiosensitivity.And in vivo xenografts nude mouse model experiments were performed to further validate the function of LZTS2.Moreover,a series of functional experiments was further conducted to determine whether LZTS2 participates in these biological processes is dependent on p85.Results: We found that the protein level of LZTS2 was obviously lower in six NPC cell lines compared to normal nasopharyngeal epithelium cell line NP69.NPC patients with low LZTS2 expression had shorter overall survival than those of patients with high LZTS2 levels.Then,we identified p85 as a LZTS2-interacting protein through an unbiased tandem affinity purification and coimmunoprecipitation.More importantly,we also uncovered a new role of LZTS2 in radioresistance in NPC.In our work,we showed that LZTS2 significantly suppresses tumorigenesis in NPC in vitro and in vivo by the inhibition of PI3K/AKT signaling pathway.LZTS2 enhances the radiosensitivity of nasopharyngeal carcinoma cells by the regulation of G2/M checkpoint,DNA damage repair and cell survival.These effects induced by LZTS2 knockdown is mainly dependent on p85.Conclusions: we found that LZTS2 is downregulated and predicts poor prognosis in nasopharyngeal carcinoma patients.Furthermore,we identified PI3 K subunit p85 as a novel LZTS2-interacting protein using an unbiased proteomics approach.Moreover,we demonstrated that LZTS2 competes with p110 for p85 binding and negatively regulates PI3K/AKT signaling pathway.Functionally,we showed that LZTS2 suppresses tumorigenesis and radioresistance in nasopharyngeal carcinoma in p85 dependent manner.Taken together,our results not only shed new light on the understanding of the molecular mechanisms by which PI3K/AKT signaling is activated,but also provide a potential therapeutic target for radiosensitization of NPC.