SHON Promotes The Oncogenicity Of Breast Cancer Cells Through Activation Of PI3K/AKT/mTOR Signaling Pathway By Destabilizing Tumor Suppressor PTEN

Backgrounds:Breast cancer is the most prevalent malignant tumor in women worldwide.According to the statistics of the 2018 Global Cancer Report,there were about 2.1 million(11.6%)new cases of breast cancer and 0.63 million(6.6%)dead cases of breast cancer worldwide in 2018.Among the breast cancer patients,approximately 70%-75% are expressing estrogen receptor(ER)and/or progesterone receptor(PR)which are considered as hormone receptor-positive breast cancer.According to the National Comprehensive Cancer Network(NCCN)guideline and the 2018 Chinese Breast Cancer Expert Consensus guideline,patients diagnosed with hormone receptor-positive breast cancer should be given appropriate endocrine therapy in accordance with the guidelines.Although endocrine therapy can significantly reduce the risk of recurrence and metastasis of hormone receptor-positive breast cancer,approximately 30% of patients with metastatic hormone receptor-positive breast cancer achieve a complete or partial non-response to endocrine therapy because of intrinsic or acquired resistance.The mechanism of endocrine therapy resistance mainly includes ER structure or function abnormality,growth factor signaling pathway interaction,abnormal RNA regulation and metabolic changes of drugs.Therefore,it is important to explore a biomarker that can predict the sensitivity of hormone receptor-positive breast cancer patients to endocrine therapy.We have identified a novel secreted hominoid-specific oncogene(SHON)which locates on chromosome 7 with a predicted open reading frame of 282 bp encoding a 93-amino acid residue protein.SHON has been shown to express both on human normal tissues and carcinoma cell lines.Besides,SHON is an estrogen regulated oncogene and plays an important role in breast cancer progression.SHON has also been shown to activate many important cellular signaling pathways including NF-?B,TGF?,MAPK and PI3K/AKT/mTOR to promote cell proliferation,cell invasion and migration,cell survival and reduce cell apoptosis.At the same time,SHON can also induce the transformation of normal breast epithelial cells MCF10 A into cancer cells,and enhance their capacity of proliferation and anchorage-independent growth characteristics.Moreover,expression of SHON is correlated with tumor grades and can be used as a prognostic biomarker to predict hormone receptor-positive breast cancer patients' response to endocrine therapy.The expression of SHON in pathological sections of 1650 breast cancer patients was studied by Nottingham Medical Research Center in the United Kingdom using immunohistochemical method,and it was concluded that the overexpression of SHON in the cell nucleus of ER positive breast cancer could enhance the sensitivity of tamoxifen treatment and prolong breast cancer specific survival(BCSS).Objectives:In this study,the biological function of SHON and the molecular mechanism of SHON-induced activation of PI3K/AKT/mTOR signaling pathway were investigated by constructing SHON overexpression and SHON gene knockdown hormone receptor positive breast cancer cell lines.Methods:(1)Overexpression of SHON in MCF7 and T47 D cell lines were established by cell transfection technology,and the effect of SHON overexpression on breast cancer cell lines was verified by total cell number assays,MTT cell proliferation assays,colony formation assays,transwell invasion assays and wound healing assays.(2)RNA interference technology was utilized to knock down the SHON gene in MCF7 and T47 D cell lines,and the effect of SHON gene knockdown on breast cancer cell lines was verified through the above functional assays.(3)The influence of SHON overexpression or knockdown on phosphorylation of PI3K/AKT/mTOR pathway was investigated by Western blot analysis.(4)Explore the influence of Wortmannin,a PI3K inhibitor,on the effect of oncogenicity induced by SHON.(5)Explore the direct target of SHON by Western blot analysis.(6)The effect of upregulation of PTEN on the oncogenicity induced by SHON was verified by Western blot analysis and various functional assays.Results:(1)Overexpression of SHON in MCF7 and T47 D cell lines was established by cell transfection technology,and the correct establishment of cell lines was verified by RT-PCR assays.It was proved by total cell number assays,MTT cell proliferation assays,colony formation assays,transwell invasion assays and wound healing assays that SHON overexpression could enhance the oncogenicity of MCF7 and T47 D cell lines.(2)SHON gene knockdown cell line was established by RNA interference technology,and the correct cell line was verified by RT-PCR assays.Through the above functional assays,knockdown of endogenous SHON can reduce the oncogenicity of MCF7 and T47 D cell lines.(3)Western blot analysis showed that the expression levels of key protein AKT and phosphorylated-AKT involved in the PI3K/AKT/mTOR signaling pathway were increased after the overexpression of SHON.Consistent with this result,the levels of total AKT and phosphorylated-AKT were lower in the cells with si RNAmediated depletion of SHON compared to that of negative si RNA control cells.SHON,therefore,stimulated the activation of PI3K/AKT/mTOR signaling pathway in human mammary carcinoma cells.(4)Various functional assays have proved that Wortmannin,a PI3K inhibitor,can reduce the oncogenicity induced by SHON.(5)Western blot analysis proved that PTEN was the direct target of SHON.SHON restrained the expression of PTEN.The expression level of PTEN was decreased in overexpression of SHON groups compared to the vector control groups.Consistent with this result,the expression level of PTEN was increased in the si RNA-mediated depletion of SHON groups compared with that of negative si RNA control groups.(6)Western blot analysis and various functional assays proved SHON-induced cancer-promoting function can be reversed by upregulation of PTEN in breast cancer cells.Conclusion:(1)SHON-stimulated oncogenicity in breast cancer cells was mediated by activation of PI3K/AKT/mTOR signaling pathway.(2)Wortmannin,a PI3K inhibitor,can significantly reduce the oncogenicity induced by SHON.(3)PTEN was the direct target of SHON,SHON repressed the stability of tumor suppressor PTEN to activate the PI3K/AKT/mTOR signaling pathway.(4)SHON-induced cancer-promoting function can be reversed by upregulation of PTEN in breast cancer cells.