Cell softness reveals tumorigenic potential via ITGB8/AKT/glycolysis signaling in a mice model of orthotopic bladder cancer

Background:Bladder cancer,characterized by a high potential of tumor recurrence,has high lifelong monitoring and treatment costs.To date,tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types.Nonetheless,the existence of soft tumor cells in bladder tumors remains elusive.Thus,our study aimed to develop a microbarrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells.Methods:The stiffness of bladder cancer cells was determined by atomic force microscopy(AFM).The modified microfluidic chip was utilized to separate soft cells,and the 3D Matrigel culture system was to maintain the softness of tumor cells.Expression patterns of integrinβ8(ITGB8),protein kinase B(AKT),and mammalian target of rapamycin(mTOR)were determined by Western blotting.Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59(TRIM59).The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models.Results:Using our newly designed microfluidic approach,we identified a small fraction of soft tumor cells in bladder cancer cells.More importantly,the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens,in which the number of soft tumor cells was associated with tumor relapse.Furthermore,we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells.Simultaneously,we detected a remarkable up-regulation in ITGB8,TRIM59,and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts.Conclusions:The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness.Meanwhile,the soft tumor cells become more sensitive to chemotherapy after stiffening,that offers new insights for hampering tumor progression and recurrence.

DOK3 promotes proliferation and inhibits apoptosis of prostate cancer via the NF-κB signaling pathway

Background: DOK3 (Downstream of kinase 3) is involved primarily with immune cell infiltration. Recent research reported the role of DOK3 in tumor progression, with opposite effects in lung cancer and gliomas;however, its role in prostate cancer (PCa) remains elusive. This study aimed to explore the role of DOK3 in PCa and to determine the mechanisms involved. Methods: To investigate the functions and mechanisms of DOK3 in PCa, we performed bioinformatic and biofunctional analyses. Samples from patients with PCa were collected from West China Hospital, and 46 were selected for the final correlation analysis. A lentivirus-based short hairpin ribonucleic acid (shRNA) carrier was established for silencing DOK3. A series of experiments involving the cell counting kit-8, bromodeoxyuridine, and flow cytometry assays were performed to identify cell proliferation and apoptosis. Changes in biomarkers from the nuclear factor kappa B (NF-κB) signaling pathway were detected to verify the relationship between DOK3 and the NF-κB pathway. A subcutaneous xenograft mouse model was performed to examine phenotypes after knocking down DOK3 in vivo . Rescue experiments with DOK3 knockdown and NF-κB pathway activation were designed to verify regulating effects. Results: DOK3 was up-regulated in PCa cell lines and tissues. In addition, a high level of DOK3 was predictive of higher pathological stages and worse prognoses. Similar results were observed with PCa patient samples. After silencing DOK3 in PCa cell lines 22RV1 and PC3, cell proliferation was significantly inhibited while apoptosis was promoted. Gene set enrichment analysis revealed that DOK3 function was enriched in the NF-κB pathway. Mechanism experiments determined that knockdown of DOK3 suppressed activation of the NF-κB pathway, increased the expressions of B-cell lymphoma-2 like 11 (BIM) and B-cell lymphoma-2 associated X (BAX), and decreased the expression of phosphorylated-P65 and X-linked inhibitor of apoptosis (XIAP). In the rescue experiments, pharmacological activation of NF-κB by tumor necrosis factor-α (TNF-α) partially recovered cell proliferation after the knockdown of DOK3. Conclusion: Our findings suggest that overexpression of DOK3 promotes PCa progression by activating the NF-κB signaling pathway.

Sleep duration and testosterone levels in community older men: results from the West China Health and Aging Trend study

To the Editor:Testosterone is a fundamental male sex hormone produced by the testicular Leydig cells.Testosterone levels are affected by age,peaking in the 20s and 30s and gradually declining thereafter.[1]Low testosterone level may predispose men,especially older men,to a poor prognosis or death in coronavirus disease 2019(COVID-19).[2]Therefore,testosterone levels have a significant impact on the health status of older men.The identification of modifiable non-drug factors affecting testosterone levels in older men is important for improving their health.

Complete androgen blockade vs.medical castration alone as adjuvant androgen deprivation therapy for prostate cancer patients following radical prostatectomy:a retrospective cohort study

Background:Till date,the optimal treatment strategy for delivering adjuvant androgen deprivation therapy(ADT)in localized and locally advanced prostate cancer(PCa),as a lower stage in PCa progression compared with metastatic PCa,is still unclear.This study compares the efficacy of castration alone with complete androgen blockade(CAB)as adjuvant ADT in patients with localized and locally advanced PCa undergoing radical prostatectomy(RP).Methods:Patients diagnosed with PCa,without lymph node or distant metastasis,who received RP in West China Hospital between January 2009 and April 2019,were enrolled in this study.We performed survival,multivariable Cox proportional hazard regression,and subgroup analyses.Results:A total of 262 patients were enrolled,including 107 patients who received castration alone and 155 patients who received CAB.The survival analysis revealed that there was no significant difference between the two groups(hazard ratios[HR]=1.07,95%confidence intervals[95%CI]=0.60-1.90,P=0.8195).Moreover,the multivariable Cox model provided similarly negative results before and after adjustment for potential covariant.Similarly,there was no significant difference in the clinical recurrence between the two groups in both non-adjusted and adjusted models.Furthermore,our subgroup analysis showed that CAB achieved better biochemical recurrence(BCR)outcomes than medical castration alone as adjuvant ADT for locally advanced PCa(P for interaction=0.0247,HR=0.37,95%CI=0.14-1.00,P=0.0497).Conclusion:Combined androgen blockade achieved better BCR outcomes compared with medical castration alone as adjuvant ADT for locally advanced PCa without lymph node metastasis.

Blockade of integrin signaling reduces chemotherapy-induced premature senescence in collagen cultured bladder cancer cells

Background:Diminished sensitivity towards chemotherapy remains the major impediment to the clinical treatment of bladder cancer.However,the critical elements in control of chemotherapy resistance remain obscure.Methods:We adopted improved collagen gels and performed cytotoxicity analysis of doxorubicin(DOX)and mitomycin C(MMC)of bladder cancer cells in a 3D culture system.We then detected the expression of multidrug resistant gene ABCB1,dormancy-associated functional protein chicken ovalbumin upstream-transcription factor 1(COUPTF1),cell proliferation marker Ki-67,and cellular senescence marker senescence-associatedβ-galactosidase(SA-β-Gal)in these cells.We further tested the effects of integrin blockade or protein kinase B(AKT)inhibitor on the senescent state of bladder cancer.Also,we examined the tumor growth and survival time of bladder cancer mouse models given the combination treatment of chemotherapeutic agents and integrinα2β1 ligand peptide TFA(TFA).Results:Collagen gels played a repressive role in bladder cancer cell apoptosis induced by DOX and MMC.In mechanism,collagen activated the integrinβ1/AKT cascade to drive bladder cancer cells into a premature senescence state via the p21/p53 pathway,thus attenuating chemotherapy-induced apoptosis.In addition,TFA had the ability to mediate the switch from senescence to apoptosis of bladder cancer cells in xenograft mice.Meanwhile,TFA combined with chemotherapeutic drugs produced a substantial suppression of tumor growth as well as an extension of survival time in vivo.Conclusions:Based on our finding that integrinβ1/AKT acted primarily to impart premature senescence to bladder cancer cells cultured in collagen gel,we suggest that integrinβ1 might be a feasible target for bladder cancer eradication.