KLHL29-induced DDX3X Degradation Mediates Tumor Progression And Chemosensitivity In Triple-negative Breast Cancer

Objective: Breast cancer is the most common malignancy and the leading cause of cancer-related mortality among females worldwide.Triple-negative breast cancer(TNBC)accounts for approximately 15–20% of breast cancer cases and is defined by the negative expression of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2).TNBC represents the most challenging subtype of breast cancer,because of its high heterogeneity and metastatic potential and the absence of effective targeted therapy and predictive biomarkers.Chemotherapy remains the standard of care for TNBC,however,a considerable number of patients exhibit limited responsiveness or eventually develop drug resistance.Thus,elucidating the mechanisms underlying TNBC progression and chemoresistance remains an active area of investigation.The Kelch-like gene family(KLHLs)encodes a group of evolutionarily conserved proteins.Members of the KLHL family are found to participate in a variety of cellular processes,such as mitotic progression,DNA damage repair,immune response,and oxidative stress.Several KLHL proteins have been shown to play important roles in tumorigenesis and cancer progression.The role of Kelch-like family member 29(KLHL29)in the occurrence and development of breast cancer has not been reported,and it is still unclear whether KLHL29 can be a potential target for breast cancer diagnosis and treatment.Methods:I.Verify the expression level and prognosis value of KLHL29 in TNBC:1.By analyzing the multi-omics data from the TNBC cohort at FUSCC and transcriptomic data of TNBC samples in TCGA dataset,we identified an understudied KLHL family gene,KLHL29,whose m RNA levels were significantly downregulated in TNBC tissues compared with adjacent normal tissues.2.We evaluated the prognostic significance of KLHL29 expression in patients with TNBC by Kaplan–Meier plotter database.We collected surgical samples from TNBC patients and performed IHC staining analysis of KLHL29 expression and carried out Kaplan–Meier analysis.KLHL29 downregulation was associated with unfavorable prognosis.II.Determine the biological functions of KLHL29 in TNBC:1.We used the cell viability assay and colony-formation assay to test the role of KLHL29 in TNBC cell proliferation and colony-forming ability.2.We used the flow cytometry analysis to assess the role of KLHL29 in TNBC apoptosis.3.We used the transwell assay to evaluate the role of KLHL29 in TNBC cell migration and invasion.4.We used mouse xenograft experiment to determine the role of KLHL29 in the growth of MDA-MB-231 cell-derived xenograft tumors.III.Confirm the interaction between KLHL29 and DDX3X:1.We conducted IP assays coupled with mass spectrometry(MS)analysis of potential KLHL29-interacting proteins.2.We analyzed the proteomic data from the TNBC cohort at FUSCC and CPTAC proteomic database to verify the expression level of DDX3 X in TNBC.3.We evaluated the prognostic significance of DDX3 X expression in patients with TNBC.4.We determined the biological functions of DDX3 X in TNBC.5.We performed reciprocal co-IP assays to show the interaction between KLHL29 and DDX3 X.6.We generated a series of KLHL29 deletion mutants to test which domain DDX3 X can bind.7.We conducted the immunofluorescence confocal microscopic analysis to test co-localization condition of KLHL29 and DDX3 X.IV.Investigate whether KLHL29 regulates DDX3 X levels:1.We explored whether KLHL29 regulates DDX3 X protein levels and m RNA levels by Western blotting and q PCR assays.2.We determined the clinical correlation between KLHL29 and DDX3 X by performing IHC staining analysis in TNBC specimens.3.We treated TNBC cells with the proteasome inhibitor MG132 or the autophagy inhibitor Baf-A1,to test whether DDX3 X is subjected to the proteasomal degradation in TNBC cells.4.We examined the half-life and ubiquitination level of DDX3 X protein by conducting cycloheximide chase assay and ubiquitination assay.V.Explore whether CUL3 is involved in KLHL29-mediated degradation of DDX3X:1.We performed reciprocal co-IP assays to show the interaction between CUL3 and DDX3 X.2.We explored whether CUL3 regulated DDX3 X protein levels by Western blotting assay.3.We conducted ubiquitination assays to examine whether the ubiquitination level of DDX3 X protein was regulated by CUL3.4.We investigated the possible role of KLHL29’s BTB domain in the regulation of DDX3 X by constructing a BTB domain-deleted variant(KLHL29-ΔBTB).VI.Investigate whether the KLHL29-DDX3 X axis regulates cell cycle progression in TNBC:1.We conducted transcriptomic analysis to explore the potential molecular mechanism behind the role of DDX3 X in TNBC.2.We explored the relationship between KLHL29 and cell cycle regulation pathway through the single-sample gene set enrichment analysis based on the FUSCC-TNBC dataset.3.We conducted a series of flow cytometry analysis to determine the role of KLHL29 and DDX3X on cell cycle in TNBC.4.We tested whether DDX3 X regulated genes encoding cyclins.5.We explored the relationship between DDX3 X and CCND1 by RIP assay and RNA stability experiment.VII.Explore the clinical translational value of KLHL29 and DDX3 X.1.We evaluated the anti-cancer efficacy of RK33 in TNBC cells.2.We calculated half-maximal inhibitory concentration of RK33 to assess the predictive value of KLHL29 and DDX3 X on RK33 sensitivity.3.We evaluated the combination effects of RK33 and cisplatin on TNBC cell growth.4.We investigated whether RK33 and cisplatin synergistically inhibit TNBC cell growth.5.We tested the combination effects of RK33 with cisplatin in xenograft mouse models.Results:1.KLHL29 m RNA levels were significantly downregulated in TNBC tissues compared with adjacent normal tissues based on FUSCC-TNBC cohort and TCGA database.Low expression of KLHL29 was associated with poor relapse-free survival and overall survival in TNBC.Multivariate cox regression analysis confirmed that low expression of KLHL29 was an independent risk factor for poor prognosis in patients with TNBC.2.To determine the biological functions of KLHL29 in TNBC,we first generated KLHL29-stably overexpressing BT549 and CAL51 cells,and we used two independent si RNAs in MDA-MB-231 and SUM159 cells to knock down KLHL29.Ectopic KLHL29 suppressed TNBC cell proliferation,inhibited the colony-forming ability,augmented apoptosis and impaired the migration and invasion of TNBC cells.KLHL29 knockdown enhanced cell proliferation,colony formation,inhibited apoptosis and increased the migration and invasion of TNBC cells.The tumor suppressive role of KLHL29 was further validated using a xenograft tumor model.3.We conducted IP assays coupled with MS analysis to identify potential KLHL29-interacting proteins.The DEAD-box helicase family member DDX3 X was one of the top candidates.DDX3 X was upregulated in TNBC tissues compared with corresponding normal tissues.High expression of DDX3 X was associated with poor relapse-free survival and overall survival in TNBC.DDX3 X could act as an oncoprotein in TNBC.Reciprocal co-IP assays showed that KLHL29 could interact with DDX3 X.Domain-mapping experiment demonstrated that DDX3 X bound to the Kelch domain of KLHL29.The immunofluorescence confocal microscopic analysis confirmed that KLHL29 was partially co-localized with DDX3 X.4.KLHL29 could regulate DDX3 X protein levels,but KLHL29 didn’t influence m RNA levels of DDX3 X.KLHL29 expression was negatively correlated with DDX3 X expression by performing IHC staining analysis in TNBC specimens.KLHL29-mediated DDX3 X reduction was through proteasomal degradation,as MG132 treatment could abrogate this reduction.Ectopic expression of KLHL29 enhances the ubiquitination level and shorten half-life of DDX3 X protein.5.Reciprocal co-IP assays indicated that KLHL29 interacted with CUL3 via the BTB domain.CUL3 is involved in KLHL29-mediated ubiquitin-proteasome degradation of DDX3 X.We constructed a BTB domain-deleted variant(KLHL29-ΔBTB)to investigate the possible role of BTB domain in the regulation of DDX3 X.KLHL29-ΔBTB could interact with DDX3 X,it fails to bind to CUL3.KLHL29-ΔBTB could not increase the interaction between CUL3 and DDX3 X,reduce DDX3 X levels,enhance ubiquitination level or regulate biological functions of TNBC,suggesting the critical role of BTB domain.6.Bioinformation analyses indicated that KLHL29 and DDX3 X were correlated with cell cycle-associated pathways based on transcriptomic analysis and FUSCC-TNBC dataset.The flow cytometry analysis of cell cycle determined that ectopic KLHL29 and depletion of DDX3 X induced cell cycle arrest at G0/G1 phase.Overexpression of KLHL29 and knockdown of DDX3 X repressed the expression of CCND1,genes encoding cyclins that promote the G1/S transition.We found that DDX3 X could bind to the transcripts of CCND1 by RIP assays.RIP assay and RNA stability experiment confirmed that DDX3 X can bind and stabilize CCND1.7.The DDX3 X inhibitor,RK33,has promising anti-cancer activity in different types of cancer.Our results confirmed that RK33 treatment dramatically reduced both m RNA and protein levels of DDX3 X and induced cell cycle arrest at G0/G1 phase.DDX3 X depletion or KLHL29 overexpression markedly increased IC50 values of RK33.Furthermore,our results demonstrated that the combination of RK33 and cisplatin can effectively suppress TNBC in vitro and in vivo.Conclusions:In conclusion,our study uncovers KLHL29 as a tumor suppressor in TNBC.KLHL29 recruits the CUL3 E3-ligase to promote ubiquitination and proteasomal degradation of DDX3 X,resulting in the repression of cyclin genes and cell cycle arrest.TNBC expresses low levels of KLHL29 and high levels of DDX3 X.Thus,targeting DDX3 X by the small molecule RK33 sensitizes TNBC to platinum-based treatment by arresting cell cycle at G0/G1 phase,which can be exploited to develop a novel combinational therapy for TNBC.