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.

Keywords: HSPA2; antibody specificity; cancer biomarker; heat shock protein family A; immunohistochemistry; proteotoxic stress.

Figure 1

Reactivity of six commercial anti-HSPA2 antibodies to different recombinant HSPAs. Immunoblots show binding of commercial anti-HSPA2 antibodies to recombinant HSPA1 (69.92 kDa), HSPA2-GST (96.03 kDa), HSPA6 (71.028 kDa) and HSPA8 (70.898 kDa) proteins (predicted molecular weight in kDa is indicated). In total, 10 ng of recombinant protein was blotted and detected using anti-HSPA2 antibody (n = 2).

Figure 2

Western blot detection of endogenous HSPA2 in NCI-H1299 cells using commercial anti-HSPA2 antibodies. (a) Reactivity of commercial anti-HSPA2 antibodies to native HSPA2 protein in wild-type (wt) and lentivirally transduced cell sublines: sh-luc, control cells expressing non-targeting shRNA sequence; sh-A1.S, cells deficient in HSPA1 (expressing HSPA1-targeting shRNA sequence); sh-A2.4, cells expressing HSPA2-targeting sequence (shRNAs sequences were described in [18]. SPA810, antibody distributed by Enzo, Life Sciences. (b) HSPA2 overexpression in lentivirally modified cell variants; pLVX, control cells transduced with empty vector pLVX-Puro; pLVX-A2, cells transduced with pLVX-Puro vector bearing HSPA2 protein coding sequence. (c) Detection of HSPA2 in cells overproducing HSPA1 protein (pCMV1-A1) and in control cells (pCMV). Altogether, 25–35 µg of total protein extracts were blotted and immunodetected using the anti-HSPA2 antibody. Representative immunoblots are shown (n ≥ 3), actin—a protein loading control.

Figure 3

Response of HSPAs system to proteotoxic stress in MCF7 cells exposed to MG132 proteasome inhibitor. (a) Reactivity of antibodies used for detection of stress-inducible HSPA1 and HSPA6 and constitutive HSPA8 proteins against human recombinant HSPA1, HSPA2-GST, HSPA6 and HSPA8 proteins. SPA810 and SPA754, antibodies distributed by Enzo, Life Sciences. (b) Detection of endogenous HSPA1, HSPA6, HSPA8 and (c) HSPA2 in non-treated and MG132-treated cells. Cells were incubated with the inhibitor for 24 h, total protein extracts (30–35 µg) were used for Western blot analysis. Representative immunoblots are shown (n ≥ 3), actin was used as a protein loading control.

Figure 4

Results of immunohistochemical staining with the use of specific and validated custom anti-HSPA2 antibody [12] and commercial anti-HSPA2 antibodies in NSCLC tumor samples were incubated with primary antibody overnight at 4 °C. Two pairs of HSPA2-positive and -negative samples were analyzed; representative images are shown. The summary on antibody cross-reactivity and on technical details are collected in Table 2 and Table 3, respectively. The scale bar represents 100 µm.