The human testis-enriched HSPA2 interacts with HIF-1α in epidermal keratinocytes, yet HIF-1α stability and HIF-1-dependent gene expression rely on the HSPA (HSP70) activity.

The Hypoxia-Inducible Factor 1 (HIF-1) is essential for cellular adaptation to reduced oxygen levels. It also facilitates the maintenance and re-establishment of skin homeostasis. Among others, it is involved in regulating keratinocyte differentiation. The stability of the oxygen-liable HIF-1α subunit is regulated by various non-canonical oxygen-independent mechanisms, which among others involve Heat Shock Proteins of the A family (HSPA/HSP70). This group of highly homologous chaperones and proteostasis-controlling factors includes HSPA2, a unique member crucial for spermatogenesis and implicated in the regulation of keratinocyte differentiation. HIF-1 can control the HSPA2 gene expression. In this study, we revealed that HIF-1α is the first confirmed client of HSPA2 in human somatic cells. It colocalises and interacts directly with HSPA2 in the epidermis in situ and immortalised keratinocytes in vitro. Using an in vitro model based on HSPA2-overexpressing and HSPA2-deficient variants of immortalised keratinocytes we showed that changes in HSPA2 levels do not affect the levels and intracellular localisation of HIF-1α or influence the ability of HIF-1 to modulate target gene expression. However, HIF-1α stability in keratinocytes appears critically reliant on HSPAs as a group of functionally overlapping chaperones. In addition to HSPA2, HIF-1α colocalises and forms complexes with HSPA8 and HSPA1, representing housekeeping and stress-inducible HSPA family paralogs, respectively. Chemical inhibition of HSPA activity, but not paralog-specific knockdown of HSPA8 or HSPA1 expression reduced HIF-1α levels and HIF-1-dependent gene expression. These observations suggest that pharmacological targeting of HSPAs could prevent excessive HIF-1 signalling in pathological skin conditions.

An Increase in HSF1 Expression Directs Human Mammary Epithelial Cells toward a Mesenchymal Phenotype.

HSF1 is a well-known heat shock protein expression regulator in response to stress. It also regulates processes important for growth, development or tumorigenesis. We studied the HSF1 influence on the phenotype of non-tumorigenic human mammary epithelial (MCF10A and MCF12A) and several triple-negative breast cancer cell lines. MCF10A and MCF12A differ in terms of HSF1 levels, morphology, growth in Matrigel, expression of epithelial (CDH1) and mesenchymal (VIM) markers (MCF10A are epithelial cells; MCF12A resemble mesenchymal cells). HSF1 down-regulation led to a reduced proliferation rate and spheroid formation in Matrigel by MCF10A cells. However, it did not affect MCF12A proliferation but led to CDH1 up-regulation and the formation of better organized spheroids. HSF1 overexpression in MCF10A resulted in reduced CDH1 and increased VIM expression and the acquisition of elongated fibroblast-like morphology. The above-mentioned results suggest that elevated levels of HSF1 may direct mammary epithelial cells toward a mesenchymal phenotype, while a lowering of HSF1 could reverse the mesenchymal phenotype to an epithelial one. Therefore, HSF1 may be involved in the remodeling of mammary gland architecture over the female lifetime. Moreover, HSF1 levels positively correlated with the invasive phenotype of triple-negative breast cancer cells, and their growth was inhibited by the HSF1 inhibitor DTHIB.

Heat shock protein A2 is a novel extracellular vesicle-associated protein.

70-kDa Heat Shock Proteins (HSPA/HSP70) are chaperones playing a central role in the proteostasis control mechanisms. Their basal expression can be highly elevated as an adaptive response to environmental and pathophysiological stress conditions. HSPA2, one of poorly characterised chaperones of the HSPA/HSP70 family, has recently emerged as epithelial cells differentiation-related factor. It is also commonly expressed in cancer cells, where its functional significance remains unclear. Previously, we have found that proteotoxic stress provokes a decrease in HSPA2 levels in cancer cells. In the present study we found that proteasome inhibition-related loss of HSPA2 from cancer cells neither is related to a block in the gene transcription nor does it relate to increased autophagy-mediated disposals of the protein. Proteotoxic stress stimulated extracellular release of HSPA2 in extracellular vesicles (EVs). Interestingly, EVs containing HSPA2 are also released by non-stressed cancer and normal cells. In human urinary EVs levels of HSPA2 were correlated with the levels of TSG101, one of the main EVs markers. We conclude that HSPA2 may constitute basic components of EVs. Nevertheless, its specific role in EVs and cell-to-cell communication requires further investigation.

Inhibition of the Heat Shock Protein A (HSPA) Family Potentiates the Anticancer Effects of Manumycin A.

Manumycin A (MA) is a well-tolerated natural antibiotic showing pleiotropic anticancer effects in various preclinical in vitro and in vivo models. Anticancer drugs may themselves act as stressors to induce the cellular adaptive mechanism that can minimize their cytotoxicity. Heat shock proteins (HSPs) as cytoprotective factors can counteract the deleterious effects of various stressful stimuli. In this study, we examined whether the anticancer effects of MA can be counteracted by the mechanism related to HSPs belonging to the HSPA (HSP70) family. We found that MA caused cell type-specific alterations in the levels of HSPAs. These changes included concomitant upregulation of the stress-inducible (HSPA1 and HSPA6) and downregulation of the non-stress-inducible (HSPA2) paralogs. However, neither HSPA1 nor HSPA2 were necessary to provide protection against MA in lung cancer cells. Conversely, the simultaneous repression of several HSPA paralogs using pan-HSPA inhibitors (VER-155008 or JG-98) sensitized cancer cells to MA. We also observed that genetic ablation of the heat shock factor 1 (HSF1) transcription factor, a main transactivator of HSPAs expression, sensitized MCF7 cells to MA treatment. Our study reveals that inhibition of HSF1-mediated heat shock response (HSR) can improve the anticancer effect of MA. These observations suggest that targeting the HSR- or HSPA-mediated adaptive mechanisms may be a promising strategy for further preclinical developments.

Evaluation of the Role of ITGBL1 in Ovarian Cancer.

In our previous microarray study we identified two subgroups of high-grade serous ovarian cancers with distinct gene expression and survival. Among differentially expressed genes was an Integrin beta-like 1 (ITGBL1), coding for a poorly characterized protein comprised of ten EGF-like repeats. Here, we have analyzed the influence of ITGBL1 on the phenotype of ovarian cancer (OC) cells. We analyzed expression of four putative ITGBL1 mRNA isoforms in five OC cell lines. OAW42 and SKOV3, having the lowest level of any ITGBL1 mRNA, were chosen to produce ITGBL1-overexpressing variants. In these cells, abundant ITGBL1 mRNA expression could be detected by RT-PCR. Immunodetection was successful only in the culture media, suggesting that ITGBL1 is efficiently secreted. We found that ITGBL1 overexpression affected cellular adhesion, migration and invasiveness, while it had no effect on proliferation rate and the cell cycle. ITGBL1-overexpressing cells were significantly more resistant to cisplatin and paclitaxel, major drugs used in OC treatment. Global gene expression analysis revealed that signaling pathways affected by ITGBL1 overexpression were mostly those related to extracellular matrix organization and function, integrin signaling, focal adhesion, cellular communication and motility; these results were consistent with the findings of our functional studies. Overall, our results indicate that higher expression of ITGBL1 in OC is associated with features that may worsen clinical course of the disease.

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.

Heat Shock Protein A2 (HSPA2) is a member of the HSPA (HSP70) chaperone family and has a critical role for male fertility. HSPA2 is present in a number of somatic organs. Limited evidence suggests that HSPA2 may be involved in regulating epithelial cell differentiation. HSPA2 also emerged as a cancer-related chaperone; however, no consensus on its functional significance has been reached so far. In this study, we compared the phenotypic effects of HSPA2 deficit in non-transformed human bronchial epithelial cells (HBEC), and in lung, breast, and cervical cancer cells. We used various techniques to inhibit the HSPA2 gene expression in order to examine the impact of HSPA2 deficiency on cell growth, migration, adhesion, and invasion. Our results show that HBEC but not cancer cells are sensitive to HSPA2 deficit. HSPA2 knockdown in HBEC cells impaired their clone-forming ability and adhesiveness. Thus, our results indicate that epithelial cells can rely on a specific activity of HSPA2, but such dependence can be lost in epithelial cells that have undergone malignant transformation.

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

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.

Heat shock proteins in the physiology and pathophysiology of epidermal keratinocytes.

Heat shock proteins (HSPs), a large group of highly evolutionary conserved proteins, are considered to be main elements of the cellular proteoprotection system. HSPs are encoded by genes activated during the exposure of cells to proteotoxic factors, as well as by genes that are expressed constitutively under physiological conditions. HSPs, having properties of molecular chaperones, are involved in controlling/modulation of multiple cellular and physiological processes. In the presented review, we summarize the current knowledge on HSPs in the biology of epidermis, the outer skin layer composed of stratified squamous epithelium. This tissue has a vital barrier function preventing from dehydratation due to passive diffusion of water out of the skin, and protecting from infection and other environmental insults. We focused on HSPB1 (HSP27), HSPA1 (HSP70), HSPA2, and HSPC (HSP90), because only these HSPs have been studied in the context of physiology and pathophysiology of the epidermis. The analysis of literature data shows that HSPB1 plays a role in the regulation of final steps of keratinization; HSPA1 is involved in the cytoprotection, HSPA2 contributes to the early steps of keratinocyte differentiation, while HSPC is essential in the re-epithelialization process. Since HSPs have diverse functions in various types of somatic tissues, in spite of multiple investigations, open questions still remain about detailed roles of a particular HSP isoform in the biology of epidermal keratinocytes.

Functional redundancy of HSPA1, HSPA2 and other HSPA proteins in non-small cell lung carcinoma (NSCLC); an implication for NSCLC treatment.

Heat shock proteins (HSPs) are a large group of chaperones considered critical for maintaining cellular proteostasis. Their aberrant expression in tumors can modulate the course of processes defined as hallmarks of cancer. Previously, we showed that both stress-inducible HSPA1 and testis-enriched HSPA2, highly homologous members of the HSPA (HSP70) family, are often overexpressed in non-small cell lung carcinoma (NSCLC). HSPA1 is among the best characterized cancer-related chaperones, while the significance of HSPA2 for cancer remains poorly understood. Previously we found that in primary NSCLC, HSPA1 was associated with good prognosis while HSPA2 correlated with bad prognosis, suggesting possible different roles of these proteins in cancer. Therefore, in this work we investigated the impact of HSPA1 and HSPA2 on NSCLC cell phenotype. We found that neither paralog-selective nor simultaneous knockdown of HSPA1 and HSPA2 gene expression reduced growth and chemoresistance of NSCLC cells. Only blocking of HSPA proteins using pan-HSPA inhibitors, VER-155008 or JG-98, exerted potent anticancer effect on NSCLC cells, albeit the final outcome was cell type-dependent. Pan-HSPA inhibition sensitized NSCLC cells to bortezomib, but not to platinum derivates. Our result suggests the inhibitors of proteasome and HSPAs seem an effective drug combination for pre-clinical development in highly aggressive NSCLC.