Various Anti-HSPA2 Antibodies Yield Different Results in Studies on Cancer-Related Functions of Heat Shock Protein A2

Abstract

Heat shock proteins (HSPs) constitute a major part of the molecular chaperone system and play a fundamental role in cell proteostasis. The HSPA (HSP70) family groups twelve highly homologous HSPA proteins. Certain HSPAs are regarded as important cancer-related proteins, prospective therapeutic targets for cancer treatment, and also as potential cancer biomarkers. Heat Shock Protein A2 (HSPA2), a testis-enriched chaperone and one of the least characterized members of the HSPA family, has recently emerged as an important cancer-relevant protein with potential biomarker significance. Nevertheless, conflicting conclusions have been recently drawn both according to HSPA2 role in cancer cells, as well as to its prognostic value. In this work we have shown that one of the serious limitations in HSPA2 protein research is cross-reactivity of antibodies marketed as specific for HSPA2 with one or more other HSPA(s). Among non-specific antibodies were also those recently used for HSPA2 detection in functional and biomarker studies. We showed how using non-specific antibodies can generate misleading conclusions on HSPA2 expression in non-stressed cancer cells and tumors, as well as in cancer cells exposed to proteotoxic stress. Our findings addressed concerns on some published studies dealing with HSPA2 as a cancer-related protein.

HSPA2 Chaperone Contributes to the Maintenance of Epithelial Phenotype of Human Bronchial Epithelial Cells but Has Non-Essential Role in Supporting Malignant Features of Non-Small Cell Lung Carcinoma, MCF7, and HeLa Cancer Cells

“…It is clear that HSPA2 is critical for the development and function of male germ cells [ 4 , 19 ]. Several papers showed that HSPA2 has a universal contribution to cancer cell phenotype (review in [ 20 ]), but it is not expressed in corresponding non-tumorigenic cells [ 27 , 28 ]. In this study, for the first time, we directly compared the phenotypic effects of HSPA2 deficiency in cancer and corresponding normal cells.…”

“…Taking together our previous observations that HSPA2 deficit neither affects the growth nor chemoresistance of two NSCLC cell lines [ 18 ], our studies put the current belief on the universal cancer-related role of HSPA2 under question. This paper is the only one that exploited different methods for the inhibition of the HSPA2 gene expression (transient siRNA- or stable shRNA-mediated knockdown (using retroviral or lentiviral vectors; or CRISPR/Cas9 knockout) and used a validated anti-HSPA2 antibody with proven specificity [ 20 ]. Furthermore, we found that independently of the initial level of the endogenous HSPA2, the downregulation of its expression did not lead to growth inhibition.…”

“…Bearing in mind that HSPA2 is a member of one of the most conservative protein families in nature, we recently tested the specificity of commercial anti-HSPA2 antibodies. Only one out of six analyzed antibodies (including those previously used in biomarker studies) ensured the specific detection of HSPA2 and did not cross-react with other HSPA paralog(s) [ 20 ]. Unfortunately, no information on potential cross-reactivity with other HSPA paralog(s) was provided for anti-HSPA2 antibodies used in a vast majority of functional studies on HSPA2 role in cancer cells (for details, we refer to [ 20 ]).…”

“…Cells were seeded in 6-cm dishes with a maximum density of 50–70%. Total protein extracts were prepared as described previously [ 20 ]. Briefly, cells were lysed by scraping in RIPA buffer (1× PBS, 1% NP-40, 0.1% SDS, 0.5% SDC, 50 mM NaF, 1 mM PMSF) supplemented with protease inhibitor mixture.…”

“…HSPA2 was initially considered as testis-specific chaperone crucial for male germ cell development and function [ 4 , 19 ]. Recently, it has emerged as a novel cancer-related protein due to its elevated expression in various tumors and reported prognostic significance (for summary, we refer to [ 20 ]). However, HSPA2 is also expressed in a cell-type specific manner in a number of human somatic organs [ 21 , 22 ].…”