The Functions And Underlying Mechanisms Of Sestrin2 In Melanoma Anoikis

BackgroundMelanoma is a skin neoplasm arising from by melanocytes,characterized by highly invasive and metastasis.The morality of melanoma ranks 1st in all skin tumors.It was reported that about 232000 new cases of melanoma were diagnosed per year worldwide.Although the incidence of melanoma in China was lower than the Western World,about 20000 cases of new melanoma patients were found in China every year.In the early stage,melanoma can be removed by surgery.However,melanoma progresses rapidly and during metastasis,melanoma cells manage to escape from the primary tumor,survive the treacherous transit through the lymphovascular system,and eventually form distant disseminations.The majority of melanoma deaths is caused by metastasis.After leaving the primary niche,the vast majority of cancer cells suffer from metabolic disorders immediately,including deficiency in glucose uptake,ATP production,intracellular ROS accumulation,which eventually leads to cell apoptosis,termed as “anoikis”.Only very few cells are able to adapt to metabolic stress through a series of molecular events and resist to anoikis and successfully disseminate and colonize in the distant organs.When suspended,tumor cells can express higher levels of the tyrosine kinase receptors such as IGF1 R and EGFR in the cell membrane surface,which activates PI3K-Akt pathway to promote the cell survival and resist anoikis.Suspension tumor cells also resist anoikis through metabolic reprogramming,such as enhancing glutamine decomposition,promoting fatty acid oxidation.Besides,they also improve their ability to get used to the suspension environment by activating cell stress related pathways such as PERK-ATF4-HO1.Sestrin proteins belong to stress induced proteins and exert cell protective roles when cells experience various environmental stimulus such as lack of energy,shortage of oxygen,ROS accumulation and DNA damage.Recently,various studies revealed that Sestrin family proteins protect cells from stress injury and apoptosis resulting from multiple environmental stimulus including DNA damage,oxidative stress,energy stress and oxygen deficiency.Sestrin2 is one important member of Sestrin proteins attracking increasing attention these years due to its involvement in multiple kinds of tumors like lung cancer,colon cancer,liver cancer and prostate cancer.Some studies on melanoma also identified the protective role of Sestrin2 in UV-induced apoptosis.Over activation of MAPK pathway is tightly associated with melanoma proliferation.BRAF inhibitor,vemurafenib,PLX4032,exerts anti-melanoma effects through inhibiting MAPK pathway.It was reported vemurafenib led to a nice response rate,but inevitably most cases developed resistance,leading to the failure of treatment.Vemurafenib is able to trigger intrinsic apoptosis in melanoma cells,however,few of these cells rely on metabolic reprogramming and oncogene activation to improve abilities to resist stress to acquire therapy resistance.Therefore,it is an urgent need to explore the possible mechanisms underlying melanoma cells of acquiring stress resistance.Sestrin2 is a stress-inducible metabolic regulator that is conserved through metazoan species.Cell-based studies illustrated Sestrin2 as twofold pseudo-symmetric with two globular subdomains.The N-terminal domain of Sestrin2(SesnA)exerts antioxidant function that suppresses reactive oxygen species(ROS).In addition to its antioxidant activity,the C-terminal domain(SesnC)modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1.Moreover,Sestrin2 plays a role in regulating cell metabolism and sustain energy homeostasis via AMPK,Keap1-Nrf2 and Ucp1 pathways.Given the important functions of Sestrin2 in metabolism regulating and ROS detoxifying,we aim to explore whether Sestrin2 is involved in mediating melanoma anoikis resistance and the possible underlying mechanisms.Methods1.Real-time PCR was adopted to examine the mRNA expression levels of Sestrin2 in melanocyte cell lines(NHEM?Hermes),primary melanoma cell lines(W793B?WM35)and metastatic melanoma cell lines(A375?A2058?UACC62?UACC257?451Lu?HTB67).2.Western Blot was performed to determine the protein expression levels of Sestrin2 in melanocyte cell lines(NHEM?Hermes),primary melanoma cell lines(W793B?WM35)and metastatic melanoma cell lines(A375?A2058?UACC62?UACC257?451Lu?HTB67).3.Immunohistochemistry was performed to examine Sestrin2's expression in malignant melanoma tissue microarray containing 16,32 and 45 cases of melanocytic nevus,primary melanoma,and metastatic melanoma,respectively.Next,we use immunofluorescence to further confirm Sestrin2 's expression melanocytic nevus,primary melanoma,and metastatic melanoma.4.TCGA database was analyzed to examine the relation between Sestrin2 expression and melanoma prognosis.5.Metastatic melanoma cell lines including A375,A2058,HTB67,UACC257,451Lu were placed in suspension culture on the plates coated overnight with poly-HEMA(10mg/ml)at 37? for drying.6.Real-time PCR,Western Blot and immune fluorescence were performed to examine mRNA and protein expression level of Sestrin2 in A375,A2058,HTB67,UACC257,451 Lu cells cultured in attached and suspended conditions for 48 h.7.Lentivirus carrying shRNA targeting Sestrin2 was used to infect A2058 and 451Lu cells to knockdown endogenous Sestrin2 expression.Cells were infected with lentivirus for 48 hours for transient infection or were selected by 2ug/ml puromycin for stable selection.8.CCK8 cell counting kit was used to determine the effects of Sestrin2 on melanoma cell growth cultured in attached and suspended conditions at 24 hours and 48 hours.9.Flow cytometry was peroformed to determine the number of apoptosis cells after 7AAD and PI staining in Sestrin2 knockdown and control groups after cultured in suspended and attached conditions for 48 hours.10.Flow cytometry was peroformed to examine intracellular ROS levels in alive cells after DCFHA and PI staining in Sestrin2 knockdown and control groups after cultured in suspended and attached conditions for 48 hours.11.Western blot was performed to examine the protein expression levels of intrinsic apoptosis pathway Bax,Bcl-2 and cleaved parp in Sestrin2 knockdown and control groups after cultured in suspended and attached conditions for 48 hours.12.Western blot was performed to examine the protein expression levels of intrinsic apoptosis pathway Bax,Bcl-2,cleaved parp and Sestrin2 in vemurafenib sensitive melanoma cells after vemurafenib treatment.13.Western blot was performed to examine the protein expression levels of Sestrin2 in vemuafenib sensitive cells A2058 P and 451 Lu P and vemurafenib resistant cells A2058 Rs and 451 Lu Rs cells.14.CCK8 was performed to examine IC50 value of vemurafenib after overexpression Sestrin2 in vemuafenib sensitive cells A2058 P and 451 Lu P and knockdown Sestrin2 n in vemurafenib resistant cells A2058 Rs and 451 Lu Rs cells.Results 1.Higher expression levels of stress-induced protein Sestrin2 in metastatic melanoma cell lines and tissues.The cell lines we examined including melanocyte cell lines(NHEM?Hermes),primary melanoma cell lines(W793B?WM35)and metastatic melanoma cell lines(A375?A2058?UACC62?UACC257?451Lu?HTB67)expressed Sestrin2,and the mRNA expression levels consistent with the protein expression levels.As indicated by the results in cell lines,normal human melanocyte cell lines(NHEM?Hermes)and primary melanoma cell lines(W793B?WM35)showed low expression of Sestrin2,however,in metastatic melanoma cell lines(A375?A2058?UACC62?UACC257?451Lu?HTB67)expressed higher level of Sestrin2.Next,we found that tissues including melanocytic nevus,primary melanoma,and metastatic melanoma expressed Sestrin2,the expression levels of Sestrin2 was higher in metastatic melanoma tissues as compared with melanocytic nevus,primary melanoma.2.Sestrin2 mediats melanoma anoikis resistance.Six metastatic melanoma cell lines including A375,A2058,UACC62,451 Lu,HTB67 were cultured in attached and suspended conditions and these cells were harvested for Quantitative Real-time PCR and western bolt to determine the Sestrin2 mRNA and protein expression levels.According to the results,all suspended melanoma cells showed higher expression of Sestrin2 compared to attached controls,Sestrin2's expression in A2058,UACC257 and 451 Lu melanoma cells was significantly increased after suspension culture at both mRNA and protein levels.We picked A2058 and 451 Lu for further research,Lentivirus carrying shRNA targeting Sestrin2 was used to infect A2058 and 451 Lu cells to knockdown endogenous Sestrin2 expression,we found Sestrin2's expression level was significantly decreased.The viability of Sestrin2 decreased cells was significantly lower the control groups when cultured in suspended conditions.After 48 hours suspended culturing,we observed the morphology of suspended melanoma cells,Sestrin2's expression decreased cells show fewer and smaller tumor sphere compared with control groups.The detection of apoptosis cell found that Sestrin2 knockdown cells show significantly higher apoptosis levels than cells in control groups when culturing in suspended conditions.3.Sestrin2 governing melanoma anoikis resistance through detoxifying ROS and regulating Bax and Bcl-2.Flow cytometry was peroformed to examine intracellular ROS levels in alive cells after DCFHA and PI staining in Sestrin2 knockdown and control groups after cultured in suspended and attached conditions for 48 hours,we found Sestrin2 knockdown cells show higher intracellular ROS levels as compared to control groups.We postulated that Sestrin2 protects suspended melanoma cells through detoxifying intracellular ROS.We pre-treated Sestrin2 knockdown cells with N-Acetyl-L-cysteine(NAC),Sestrin2 knockdown cell anoikis was reversed.Western bolt results indicated that Sestrin2 also influenced Bcl-2 family proteins and Akt phosphorylation to promote melanoma cells anoikis resistance.4.Sestrin2 mediates melanoma vemurafenib therapy resistanceVemurafenib sensitive and resistant melanoma cells show Sestrin2's expression,vemurafeib resistant show significantly higher Sestrin2's expression levels.We infected Vemurafenib sensitive and resistant melanoma cells with lentivirus carrying Sestrin2 plasmid and shRNA targeting to overexpress or knockdown Sestrin2's expression.In vemurafeib resistant melanoma cells,knockdown Sestrin2 increased therapy sensitivity with lower IC50 value.And in vemurafeib sensitive melanoma cells,Sestrin2's overexpression decreased therapy sensitivity with higher IC50 value.ConclusionsIn this study,we proved Sestrin2 expression was higher in metastatic melanoma cell lines and tissues than primary melanoma cell lines and tissues.For the first time,we demonstrated that suspension conditions can trigger Sestrin2's expression in metastatic melanoma cells.Furthermore,increased Sestrin2 exerted cell protective roles against anoikis.Moreover,our study revealed the possible mechanism that Sestrin2 mediated melanoma anoikis resistance through detoxifying intracellular ROS and regulating Bcl-2 family protein members including Bax,Bcl-2 as well as Akt phosphorylation.Besides,we found Sestrin2 promoted vemurafenib reistance,and knockdown Sestrin2's expression significantly improved the curative effect of vemurafeib.