CUL3^SPOP Promotes The Ubiquitination And Degradation Of Tumor Suppressor Protein LATS1 To Enhance Kidney Cancer Progression

Kidney cancer is one of the most malignancy tumors,accounting for 4%of all cancers.Despite the considerable improvement in cancer diagnosis and treatment,patients with advanced kidney cancer still have poor prognosis mainly due to frequent tumor recurrence and metastasis.Therefore,a better understanding of the mechanisms involved in pathogenesis of kidney cancer and discover of potential therapeautic targets were especially necessary for the wide applied precision targeted therapy.The LATS1 protein has been known as a repressor on cell proliferation and overexpression of LATS1 can lead to G2/M phase arrest to inhibit tumor gpowth.In an immunohistochemical study,lower LATS1 protein expression was found in renal cancer tissue.Ubiquitin signalings regulate many cellular processes depending on ubiquitination and degradation of diverse cellular proteins.As a member of E3 ligase family,speckle-type POZ protein(SPOP)is overexpressed and misallocated in the cytoplasm of virtually all clear-cell renal cell carcinoma(ccRCC),which may promote proliferation and induce kidney tumorigenesis.Our previous cell experiment demonstrated that down-regulation of SPOP suppressed cell proliferation,invasion,and induced G2/M phase arrest in kidney cancer cells.Therefore,we presume that SPOP promotes ubiquitination and degradation of LATS1 to promote kidney cancer progressionIn the first part of this study,we detected the biological function of SPOP on kidney cancer cell proliferation,cell invasion and cell cycle progression.Here we found that overexpression of SPOP promoted cell proliferation and invasion in kidney cancer cells.Conversely,down-regulation of SPOP suppressed cell proliferation,invasion,and induced G2/M phase arrest in kidney cancer cells.In the second part of this study,we demonstrated that the tumor suppressor LATS1 as a novel ubiquitin substrate of SPOP could interacte with Cullin3,and depletion of Cullin 3 could upregulate the abudance of LATS1 largely via prolonging LATS1 half-life.Notably,SPOP specifically interacted with LATS1,and promoted the poly-ubiquitination and subsequent degradation of LATS1 in a degron-dependent manner.More importantly,when expressed at comparable levels,SPOP could suppress LATSI-WT,but not ADegl+2,-mediated descent of cell invasion and G2/M phase arrest.In the third part of this study,we observed that over-expression of SPOP in 786-0 cells dramatically elevated the growth of tumor xenografts in mouse models.We performed western blot and immunohistochemistry(IHC)on the xenografted tumors and found that LATS1 was down-regulation in the SPOP over-expression xenografted tumors.On the other hand,depletion of SPOP by shRNA-mediated knockdown in 786-0 cells suppressed the growth of tumor xenografts in mouse models.To further investigate the clinical relevance between SPOP and LATS1 in kidney cancer,IHC was used to measure the expression level of SPOP and LATS1 in kidney cancer tissues.We found that SPOP was over-expressed in 79.8%of kidney cancer clinical tissues and LATS1 was down-expressed in 78.7%of kidney cancer clinical tissues.Moreover,we identified that cytoplasmic expression of SPOP and LATS1 in the paraffin-embedded kidney tumor samples was inversely correlated.Therefore,our studies identified a novel role of SPOP in kidney cancer progression partly through promoting degradation of LATS1 and targetting SPOP would be a promising strategy to combat kidney cancer in future therapies.