Evaluation of the Small Heat Shock Protein Family Members HSPB2 and HSPB3 in Bladder Cancer Prognosis and Progression

Abstract

Bladder cancer (BlCa) represents the sixth most commonly diagnosed type of male malignancy. Due to the clinical heterogeneity of BlCa, novel markers would optimize treatment efficacy and improve prognosis. The small heat shock proteins (sHSP) family is one of the major groups of molecular chaperones responsible for the maintenance of proteome functionality and stability. However, the role of sHSPs in BlCa remains largely unknown. The present study aimed to examine the association between HSPB2 and HSPB3 expression and BlCa progression in patients, and to investigate their role in BlCa cells. For this purpose, a series of experiments including reverse transcription-quantitative PCR, Western blotting, MTT assay and flow cytometry were performed. Initial analyses revealed increased vs. human transitional carcinoma cells, expression levels of the HSPB2 and HSPB3 genes and proteins in high grade BlCa cell lines. Therefore, we then evaluated the clinical significance of the HSPB2 and HSPB3 genes expression levels in bladder tumor samples and matched adjusted normal bladder specimens. Total RNA from 100 bladder tumor samples and 49 paired non-cancerous bladder specimens were isolated, and an accurate SYBR-Green based real-time quantitative polymerase chain reaction (qPCR) protocol was developed to quantify HSPB2 and HSPB3 mRNA levels in the two cohorts of specimens. A significant downregulation of the HSPB2 and HSPB3 genes expression was observed in bladder tumors as compared to matched normal urothelium; yet, increased HSPB2 and HSPB3 levels were noted in muscle-invasive (T2–T4) vs. superficial tumors (TaT1), as well as in high-grade vs. low-grade tumors. Survival analyses highlighted the significantly higher risk for post-treatment disease relapse in TaT1 patients poorly expressing HSPB2 and HSPB3 genes; this effect tended to be inverted in advanced disease stages (muscle-invasive tumors) indicating the biphasic impact of HSPB2, HSPB3 genes in BlCa progression. The pro-survival role of HSPB2 and HSPB3 in advanced tumor cells was also evident by our finding that HSPB2, HSPB3 genes expression silencing in high grade BlCa cells enhanced doxorubicin toxicity. These findings indicate that the HSPB2, HSPB3 chaperone genes have a likely pro-survival role in advanced BlCa; thus, they can be targeted as novel molecular markers to optimize treatment efficacy in BlCa and to limit unnecessary interventions.