SOX4 Promotes Proliferative Signals By Regulating Glycolysis Through AKT Activation In Melanoma Cells

BackgroundCutaneous melanoma(CM),originated from melanocyte,is the most lethal form of skin cancer because of its high metastatic potential.It is estimated that there will be 20,000 new cases of melanoma in China,and the CM incidence rate continues to rise.There have been many advances in our understanding of melanoma,from a greater appreciation of epidemiologic trends and risk factors to an increased understanding of the molecular genomics and biology of melanoma.With the presence of mutations,aberrations,translocations and deletions,malignant transformation can take place,giving rise to melanoma.Although CM patients at early stages can be cured with surgical excision,a proportion of patients still die of this disease once metastases.The prognosis of melanoma patients at early stage is relatively good if the tumor is excised timely,and the 5-year survival is around 90%.However,once the occurrence of metastasis,the prognosis will be poor with the 5-year survival less than 10%.Recently,some studies have suggested that mutant genes and abnormal activated pathways may be the crucial events in the carcinogenesis of melanocytes and finally contribute to tumor progression.The importance of metabolic alterations in melanoma has been increasingly recognized.Tumorigenesis is dependent on the cellular metabolism reprogram directly or indirectly as consequences of oncogenic mutations and dysregulated signalings.Cancer cell metabolism has the ability to acquire necessary nutrients from a frequently nutrient-poor environment and use these nutrients to maintain viability and build new biomass.The alterations in intracellular and extracellular metabolites that can accompany cancer-associated metabolic reprogram have effects on gene expression;cellular differentiation and the tumor microenvironment.Specific metabolic activities can participate in the process of transformation or support the biological progression that enables tumor growth.Defining the pathways that are activating or limiting for cancer progression and understanding the context-specific of metabolic preferences and liabilities in tumor cells are important for clinical benefit,which may provide new insight into cancer biology and can guide more effective management of cancer patients by targeting cellular metabolism.The sex-determining region Y-related high-mobility group box transcription factor 4(SOX4)plays a key role in embryonic development and cell differentiation,which was at first recognized as the “classical” transcription factor among the SOX family.SOX4 has been recently reported to function as either a tumor suppressor gene or oncogene,depending on the tumor specific type,the cellular context,and the stage of disease,and thus influences key processes involved in apoptosis,cellular proliferation and metastasis.However,its downstream transcriptional target genes involved in melanoma development,particularly from the perspective of energy metabolism,remain unclear.The present study is aimed to investigate SOX4 expression in melanoma tissues,and in step wise to explore the association between SOX4 expression and survival of melanoma patients.Next,we investigated the effects of shRNA mediated SOX4 gene silencing on glycolysis of human malignant melanoma cell lines(A2058 and WM35).Mechanistically,we identified that SOX4 can regulate important enzymes in glycolytic metabolism through acutely transforming retrovirus AKT8 in rodent T-cell lymphoma(AKT)activation.We also found p70 ribosomal S6 kinase(p70S6K)and eukaryotic initiation factor 4E-binding protein 1(4E-BP1)as substrates involved in SOX4/AKT-mediated mTOR complex 1(mTORC1)activity.Knockdown of SOX4 expression leads to decreased micropthalmia-associated transcription factor(MITF)and peroxisome proliferator-activated receptor gamma co-activator 1(PGC1?)expression,as well as reduced melanoma cell and tumor growth,highlighting its potential as a therapeutic target in melanoma management.Methods1.Detection of SOX4 expression in human melanoma tissue microarray by immunohistochemistry,including 82 cases of human melanoma samples and 18 cases of human melanocytic nevus samples,respectively.Further analysis of Affymetrix arrays from the two independent publically available databases was performed to test the expression of SOX4 in melanoma and melanocytic nevus.2.We preformed Kaplan-Meier survival curves for outcome of patients with melanoma stratified by SOX4 expression in The Cancer Genome Atlas data,which included a set of 459 patients with melanoma.3.We then transfected WM35 and A2058 cell lines with SOX4 shRNA,and efficacy was confirmed by real-time PCR and western blotting.Furthermore,SOX4 overexpression of WM35 and A2058 cell lines were generated by plasmid transfection and efficiency was also confirmed.4.To determine whether SOX4 affects key processes related to melanoma biology,such as apoptosis and cell cycle,we analyzed the expression of key genes in apoptosis(Bcl2,BAX and cleaved caspase-3)and cell cycle(p15,cyclinD1 and cyclinE2)of A2058 and WM35 cell lines after SOX4 knockdown and overexpression by western blotting.5.To determine the function of transcription factor SOX4 in melanoma energetic metabolism reprogram,we monitored glucose consumption and lactate production as readouts of glycolysis.To investigate the underlying target molecules,we detected the expression of glucose transporters and glycolytic pathway-related enzymes,such as glucose transporter type 1(GLUT1),hexokinase 2(HK2),pyruvate kinase M2 isoform(PKM2),and lactate dehydrogenase A(LHDA)on both mRNA and protein levels by real-time PCR and western blotting.6.Considering the important role of AKT signals in the regulation of energetic metabolism,we then investigated whether it affected as the downstream molecules in SOX4-stimulated glycolysis.We also identified the downstream effector molecules of AKT signals,such as mTORC1 and glycogen synthase kinase 3?(GSK3?)pathway.7.To address the impact of SOX4 on melanoma cell proliferation,we analyzed the ability of prolifertaion in vitro by cell counting kit(CCK8)and in vivo by nude mice injection of melanoma cells after SOX4 knockdown.Results1.Human melanoma tissue microarray(82 cases of human melanoma samples and 18 cases of human melanocytic nevus samples)by immunohistochemistry showed that SOX4 expression in metastasis melanoma and primary melanoma was significantly higher than those in melanocytic nevus(P < 0.05).Nevertheless,we did not observe any difference in SOX4 expression between primary melanoma and metastatic melanoma nor any significant correlation between nuclear SOX4 expression and sex,age,and tumor stage.Consistently,the results of Affymetrix arrays from the two independent NCBI GEO databases showed significant higher expression of SOX4 in melanoma compared with melanocytic nevus.2.Kaplan-Meier survival curves showed that melanoma patients with a high SOX4 expression level(n = 229)had a significantly worse survival outcome compared with those with a lower SOX4 expression level(n = 230)(Log-rank test: P < 0.05).3.Apoptosis analysis by flow cytometry revealed that SOX4 knockdown leads to a significantly higher proportion of apoptotic cells in both WM35 and A2058 cell lines.Meanwhile,SOX4 knockdown group showed a significant lower cell numbers in the S phase compared with the control group.Considering the fact that the basic apoptosis level is relatively low,cisplatin was used to induce apoptosis in both WM35 and A2058 cell lines.Cell death was significantly increased 48 hours after cisplatin treatment,with a significant increase in the apoptotic subpopulation in the SOX4 overexpression group,compared with the vector control group,suggesting that SOX4 overexpression can inhibit apoptosis.As expected,SOX4 overexpression resulted in an increase in the population of cells in the S phase(P < 0.05).Consistently,western blotting analysis showed higher expression of BAX and cleaved caspase-3 and lower expression of Bcl2 in SOX4 knockdown group,when compared with the control group.Decreased expression of both CyclinD1 and CyclinE2 and increased expression of cyclin-dependent kinase inhibitor p15 were observed after SOX4 knockdown.Concurrently,SOX4 overexpression resulted in downregulation of BAX,cleaved caspase-3 and p15 expression and upregulation of CyclinD1,CyclinE2 and Bcl2 expression.4.We found significantly decreased levels of glucose consumption and lactate production in both SOX4 shRNA-1 and SOX4 shRNA-2 groups,which suggested that SOX4 knockdown leads to reduced glycolysis in melanoma cells.Western blotting analysis revealed that key enzymes in glycolysis,i.e.,PKM2,HK2,GLUT1 and LDHA,which showed a uniformly reduced expression on protein levels in response to SOX4 knockdown.5.We also found significantly increased levels of glucose consumption and lactate production after SOX4 overexpression in A2058 and WM35 cell lines.Western blottings revealed that increased expression of GLUT1,HK2,PKM2 and LHDA in response to SOX4 overexpression.6.Knockdown of SOX4 revealed significant decreased AKT phosphorylation at Ser473 and Thr308 in A2058 and WM35 cell lines.SOX4 overexpression resulted in increased activation of phosphorylation on AKT at Ser473 and Thr308.The concomitant treatment of LY294002 and SOX4 overexpression by plasmid revealed a higher level of phosphorylation on AKT at Ser473 and Thr308 sites when compared with LY294002 treatment alone.7.Western blotting analysis revealed that SOX4 knockdown resulted in decreased levels of both total and phosphorylated mTOR,as well as the phosphorylation levels of downstream members p70S6 K at Thr389 and Ser371 and 4E-BP1 at Thr37/46 in both A2058 and WM35 melanoma cells.Meanwhile,SOX4 overexpression lead to a significant increase in the expression of p-mTOR and p-p70S6 K at Thr389 and Ser371 site and p-4E-BP1 at Thr37/46 site.Nevertheless,we did not observe any significant change in GSK3? phosphorylation after SOX4 knockdown.8.Western blotting analysis also showed the decreased MITF and PGC1? expression after SOX4 knockdown and the increased MITF and PGC1? expression after SOX4 overexpression.9.We found that SOX4 knockdown in both A2058 and WM35 cells exhibited reduced proliferative capacity by CCK8.In vivo experiments showed that nude mice injected with SOX4 knockdown group in A2058 cell lines exhibited significantly slower tumor growth compared with those injected with the control group.ConclusionIn the present study,we identified that SOX4 was overexpressed in melanoma tissues compared with melanocytic nevus.SOX4 expression was found elevated in most melanoma cell lines compared with human primary melanocytes and human melanocytes cell lines.We also found higher level of SOX4 expression in melanoma tissues than melanocytic nevus tissue in National Center for Biotechnonlogy Information Databases.Consistenly,Kaplan-Meier curves indicated that melanoma patients with higher SOX4 expression level had a significantly shorter survival time compared with those with lower SOX4 expression level(Log-rank test: P < 0.05).Through the knockdown or overexpression of SOX4 in melanoma cell lines,we confirmed SOX4 as a key transcription factor in tumor development,affecting key processes such as apoptosis,cell cycle,proliferation,and energetic metabolism.We found that SOX4 knockdown attenuate tumor growth by increasing apoptosis and inducing cell cycle arrest and also regulate the expression of key genes as Bcl2,BAX,cleaved caspase-3,cyclinE2,cyclinD1,and p15.Here we also found that SOX4 regulates glucose consumption and lactate production,as well as GLUT1,PKM2,HK2,and LDHA expression in WM35 and A2058 melanoma cells.Furthermore,application of LY294002 alleviated the upregulation of glucose consumption,lactate production,and AKT activation by SOX4 overexpression,suggesting that SOX4 enables glycolysis via AKT activation in melanoma cells.We also found that p70S6 K and 4E-BP1 as downstream substrates involved in SOX4-mediated mTORC1 activity in melanoma cells.In addition,knockdown of SOX4 expression lead to decreased expression of MITF and PGC1? as well as reduced melanoma cell and tumor growth.These findings revealed that the metabolic mechanisms promoting melanoma growth,which could have clinical implications for metabolism-targeted therapies against melanoma.