| Renal cell carcinoma(RCC),one of the most common types of urological malignancies,and its persistent prevalence continues to present a formidable challenge to urological research and treatment efforts.This issue becomes increasingly complicated when considering the diverse histological subtypes of RCC,among which ccRCC emerges as the most frequently observed.As ccRCC evolves into its advanced stages,the disease frequently metastasizes,an ominous progression that unfortunately correlates with substantially poor clinical prognoses.This poses a daunting conundrum for clinicians and researchers alike,who strive to impede the progression of this aggressive disease.Furthermore,RCC presents a unique therapeutic challenge,as it exhibits a notorious resistance to traditional treatment options such as radiation and chemotherapy.Adding to the complexity,an alarmingly high proportion of patients,even those who have undergone seemingly successful surgical intervention,confront a recurrence of the disease during their subsequent follow-up periods.This issue underscores the notorious resilience and unpredictability of RCC,amplifying the challenges clinicians face in both diagnosis and treatment.Given the intricate dynamics and elusive nature of RCC,it stands as a formidable adversary in the medical field.Therefore,the urgency for the identification of new biological markers cannot be overstated.Such markers could potentially revolutionize our ability to detect RCC early and could also offer invaluable insights that could be leveraged to design patient-specific therapeutic strategies,ultimately improving the overall clinical outcomes.Belonging to the class of serine-threonine kinases,Ribosomal S6 protein kinases(RSKs)are critical components of the Ras-mitogen-activated protein kinase(MAPK)signaling cascade.Their function spans across a wide range of cellular activities such as promoting cell growth,facilitating cell multiplication,ensuring cell survival,and regulating cellular motility.Ribosomal S6 protein kinase 4(RSK4),a member of the RSK family,has drawn significant attention in this context.A wealth of studies have been devoted to investigating the patterns of RSK4 mRNA expression across human tissues,which have offered key insights into its role under various physiological and pathological conditions.From this body of work,RSK4 has emerged as a potential candidate for a tumor suppressor gene,as evidenced by some scientific reports.Certain investigations have highlighted a decrease in RSK4 expression in a subset of cancers,while others have found that an upregulated RSK4 expression appears to restrict the invasive and metastatic tendencies of tumor cells.Curiously,the scenario in RCC appears to deviate from the norm.RSK4 expression in RCC appears to be significantly higher than in regular kidney tissue.Moreover,a higher incidence of invasive and metastatic behaviors has been noted in RCC cases where RSK4 overexpression is present.These observations intimate that RSK4 might have an instrumental role to play in driving the progression of RCC tumors.Despite these intriguing leads,our understanding of the exact functions and influences of RSK4 in the context of RCC remains a topic of active research and is yet to be fully elucidated.At the same time,given that the term "renal clear cell carcinoma(ccRCC)" is due to the accumulation of lipid and glycogen in the tumor cells,renal cell carcinoma is closely related to metabolism.In addition,HIF pathway activation leads to the upregulation of lipid metabolism genes such as fatty acid synthase(FASN),which leads to lipid accumulation in tumor cells also contributes to the formation of cytoplasmic transparent features in ccRCC.When modeling RSK4 whole body knockout mice,it was found that RSK 4-KO mice on conventional diet were significantly obese and increased visceral fat;blood glucose and serum TG increased,and HDL-C decreased;liver volume increased significantly,and HE slices showed fat droplets and fatty liver changes.This suggests an important role of RSK4 in lipid metabolism,but the regulatory mechanism remains unclear.Therefore,we also considered the factors of substance metabolism in the study of ccRCC.The focal point of our study was to explore blood vessel density within RCC and decipher the potential underpinning mechanisms,relying on RCC cell lines as the primary research tools.Intriguingly,our data highlight a probable relationship between heightened RSK4 levels and the aggressive phenotype often observed in RCC.This study illuminates that an overexpression of RSK4 can stimulate an increase in the secretion of vascular endothelial growth factor(VEGF),as well as contribute to enhanced tube formation assays’efficiency.Moreover,the overexpression of RSK4 also facilitates the phosphorylation of RUNX1,which subsequently leads to the upregulation of EPHA2,a gene playing a pivotal role in angiogenesis downstream.Collectively,our results contribute to a broader comprehension of the mechanisms driving RCC tumorigenesis,casting light on potential molecular pathways involving RSK4,RUNX1,and EPHA2 in the specific context of RCC cell lines.This enhanced understanding could pave the way for further in-depth investigations and possible therapeutic interventions in the future.Purpose1.To clarify the role of RSK4 in angiogenesis and the invasion of ccRCC tumors.2.To elucidate the molecular mechanisms of RSK4-RUNX1-EPHA2 thereby affects angiogenesis and malignant progression of ccRCC.3.Through the screening of small molecule inhibitors specific for RSK4 and verify the role of inhibiting ccRCC.4.Initially explore the role of RSK4 in metabolic reprogramming.Methods1.The expression of RSK4 in ccRCC patients was determined by IHC staining and rt-PCR.2.Patient information was collected to analyze the correlation between RSK4 and prognosis of ccRCC patients.3.RSK4 overexpression(hRSK4)and shRSK4 underexpression(shRSK4)renal cancer cell lines were established,and the effects of RSK4 on invasion,migration and proliferation of renal cancer cells were detected by Transwell and CCK-8.4.Immunohistochemistry were used to detect the correlation between MVD and the protein expression of RSK4 in RCC.5.Tube formation assay were used to detect HUVEC cells with hRSK4 RCC cells,ELISA was used to further substantiate the role of RSK4 in steering VEGF expression.6.The interaction between RSK4 and RUNX1 was determined by co-immunoprecipitation(Co-IP),immunofluorescence(IF)and Western Blot(WB),and the regulation of EPHA2 transcription by RUNX1 was determined by luciferase reporter gene(Luc)activity analysis.7.The promoting effect of RSK4 on ccRCC metastasis and its relationship with angiogenesis was detected by in vivo lung metastasis assay.8.The role of RSK4 in metabolic reprogramming was examined by RSK4 knockout mice.Results1.RSK4 is highly expressed in RCCs and is associated with the poor survival of RCC patients.2.Overexpression of RSK4 can promote the proliferation,invasion and migration ability of renal cancer cells.3.Continuing our assessment with the tube formation assay,we noted a substantial increase in the mean number of complete tubular structures that Human Umbilical Vein Endothelial Cells(HUVECs)formed in the medium enriched with overexpressed RSK4,relative to the control conditions.RSK4-overexpression enhanced angiogenesis in vitro,and regulation of angiogenesis by RSK4 depends on VEGF.4.We initiated RSK4 overexpression in luciferase-tagged 786-O RCCs.The survival rates recorded for the RSK4-overexpression group were found to be lower when compared to the control group.Further analysis using IHC on CD34 in metastatic tumors demonstrated increased MVD in hRSK4 tumors.5.Proteomics analysis singled out Ser-249 of RUNX1 as the most notable phosphorylation site,prompting us to concentrate on this site for subsequent investigations.6.In a bid to further substantiate this association,we employed an endogenous coimmunoprecipitation assay,which corroborated RSK4’s direct binding to RUNX1 in the 786-O RCC cell line.7.Transcription factor prediction analysis led us to a potential RUNX1 binding site between-501 and-491,which is situated upstream of the transcriptional initiation site in the EPHA2 promoter.The transcriptional regulatory impact of RUNX1 on EPHA2 was evaluated through a luciferase reporter gene,which displayed the reversal of RUNX1’s transcriptional activation effect on EPHA2 post-binding site mutation.8.After screening the specific inhibitor YY-57 in the small molecule compound library,the reversal experiment found that YY57 could inhibit the proliferation,invasion and migration of ACHN cells,and the formation of HUVEC cells co-cultured with renal cancer cells.9.RSK4-KO mice became obese under regular diet,mainly visceral fat,blood glucose and serum TG and HDL-C;the volume of liver significantly increased,and the HE section showed lipid droplet storage and accumulation with fatty liver.Suggesting an important role of RSK 4 in metabolic reprogramming.Conclusions1.High expression of RSK4 is an independent prognostic factor for RCC patients.2.RSK4-overexpression enhanced angiogenesis in vitro,and regulation of angiogenesis by RSK4 depends on VEGF.3.RSK4 may promote angiogenesis in RCC by regulating downstream of RUNX1 through phosphorylation of EPHA2.4.YY-57 a small molecule inhibitor,can effectively inhibit the metastatic ability and tubular capacity of RCC,suggesting that RSK4 can be combined to improve the tumor treatment effect.5.Spontaneous obesity of RSK4-KO mice suggests that RSK4 is related to metabolic reprogramming. |
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