Overexpression of HSPA2 is correlated with poor prognosis in esophageal squamous cell carcinoma

Esophageal cancer is the seventh leading cause of cancer deaths worldwide, while its highest incidence and mortality rates are found in Asia. For example, in China, the majority of esophageal cancer diagnoses are esophageal squamous cell carcinoma (ESCC), which is ranked as the eighth leading cause of death nationwide, mostly in northern China where the incidence rate can be as high as 800 cases per 100,000 people[1]. The incidence and mortality rates of ESCC have decreased over the last few decades in China. On the contrary, it is continuing its march as the fastest growing malignancy in the Western world[2]. ESCC patients have a high mortality rate and poor prognosis because of the high prevalence of invasion and metastasis. Therefore, it is important to identify and characterize the tumor-specific molecular markers involved in early stages of ESCC that may contribute to ESCC carcinogenesis for improving the survival of this disease.

Heat shock-related 70 kDa protein 2 (HSPA2, also known as HSP70-2) is a member of the HSP70 family of heat-shock proteins, mapped to 14q24.1 in one study[3] and to 14q22 in another[4]. HSPA2 was originally found specifically in primary spermatocytes and spermatids, described as a testis-specific protein that played an important role in spermatogenesis[5, 6]. Aberrant expression of HSPA2 in testes induced primary spermatocytes to arrest in meiosis I and undergo apoptosis, which was leading to male infertility[6]. Now HSPA2 has attracted increased interest due to its possible involvement in carcinogenesis of non-testicular tissues. HSPA2 has been identified as a potential cancer-promoting protein expressed at abnormal levels in a subset of human cancers, such as breast cancer[7], cervical cancer[8], bladder urothelial cancer[9], nasopharyngeal carcinoma[10] and malignant tumors[11]. Some level of the HSPA2 gene activity was also observed in cell lines derived from several human cancers[12‐14], while silencing of the HSPA2 gene in cancer cells led to growth arrest and decrease in tumorigenic potential[8, 9, 12, 15]. A HSPA2 mutation was recognized by cytotoxic T lymphocyte (CTL) on a human renal cell carcinoma[16]. Furthermore, polymorphism in the HSPA2 gene is associated with an increase in the risk of developing type 1 diabetes[17], non-Hodgkin's lymphoma[18], lung cancer[19], systemic lupus erythematosus (SLE)[20], rheumatoid arthritis[21] and inflammatory bowel diseases[22]. Overexpression of HSPA2 is correlated with increased cell proliferation, poor differentiation and lymph node metastases in human breast cancer, cervical cancer and bladder urothelial cancer[7‐9]. The highest level of HSPA2 was also detected in cells of the basal layers of the skin, esophagus and bronchus epithelia. However, whether there is HSPA2 expression in ESCC or not, has not been reported in China and abroad so far.

Thus, in this study we used quantitative RT-PCR (qRT-PCR), western blotting and immunohistochemistry to evaluate HSPA2 expression and its correlation with the survival of ESCC patients. Consistent with these studies, we suggested that elevated HSPA2 expression was associated with poor survival of ESCC patients. Our findings provide insights that could lead to better diagnosis, prognosis and therapeutic opportunities for ESCC patients.

The HSPA2 gene was originally characterized as a counterpart of the testis-specific rodent HST70/HSP70-2 gene, which was specially and highly expressed in the testis[5, 6]. Recently, research has shown that human tumor cells and some somatic tissues can express this gene at significant levels[7‐10, 12‐14]. The polymorphism of HSPA2 at position 1267 has been suggested to be associated with carcinogenesis in many malignant cancer tissues[7‐10]. The highest level of HSPA2 is also detected in cells of the basal layers of the esophagus[23], but the function of HSPA2 in basal cells, which may be involved in the proliferation and/or differentiation of epidermal keratinocytes, is currently unknown.

In this study, the expression levels of HSPA2 mRNA and protein in the ESCC tissues were significantly higher than in adjacent non-cancerous tissues and normal esophageal tissues, consistent with other cancer reports[7‐9]. As a chaperone protein, HSPA2 is essential for the growth of spermatocytes and cancer cells, and it is known to be involved in apoptosis and regulation of cell proliferation[18]. Silencing the expression of the HSPA2 gene by RNA interference suggests that HSPA2 increases the growth rate and tumorigenic potential not only in the cell culture but also in tumor xenograft in mice[7‐9, 15]. However, the underlying mechanisms for the high expression of HSPA2 in tumors remain incompletely understood, but likely involve regulatory processes, such as cell cycling. Indeed, HSPA2 appears to be a molecular chaperone for CDC2 and is required for CDC2/cyclinB1 complex formation, whose destruction can prevent development of the CDC2 kinase activity, required to trigger G2/M phase transition[24]. During meiosis, spermatogenic cells synthesize HSPA2, knockout of HSPA2 in HSPA2 (−/−) male mice testes induce primary spermatocytes to arrest in meiosis I and undergo a dramatic increase in spermatocyte apoptosis, which was leading to male infertility[6]. HSPA2 overexpression protects V79 fibroblasts against bortezomib-induced apoptosis[25]. The mechanism of a mutated HSPA2 chaperone has a dominant effect in tumor cells by triggering the G2/M phase transition during the mitotic cell cycle, which could explain the HSPA2 abnormal expression in somatic tumors. Moreover, downregulation of lens epithelium-derived growth factor (LEDGF) is responsible for the death of HSPA2-depleted cancer cells and LEDGF possesses great oncogenic potential[26]. HSPA2 may also have a potential role in cancer pathogenesis by participating in the regulation of antitumor immunity, such as acting as a chaperone molecule for immunogenic tumor-associated peptides[27], while HSPA2 has been identified as a putative susceptibility locus in organ-specific autoimmune diseases. Lastly, a new regulatory mechanism of HSPA2 expression in tumor cells has been disclosed, which suggests that the upregulation of HSPA2 enhanced the resistance of tumor cells to hypoxia-induced apoptosis, which provides a new insight into how tumor cells overcome hypoxic stress and survive[28].

Immunohistochemical analysis of a large set of specimens revealed that HSPA2 protein was positively expressed in 75% (90/120) ESCC tissues, compared to 45.83% (55/120) in adjacent non-cancerous tissues (P <0.05). We correlated HSPA2 protein expression with various clinicopathological factors in 120 ESCC patients. Our results showed that overexpression of HSPA2 was significantly associated with primary tumor, TNM stage, lymph node metastases and recurrence respectively (all, P <0.05). We also found HSPA2 mRNA expression in advanced-stage ESCC tissues at stage III was significantly higher than in early-stage ESCC tissues at stages I and II. These data revealed a potential association between HSPA2 overexpression and malignant phenotypes, such as metastasis, consistent with other reports in breast cancer[7], cervical cancer[8] and bladder urothelial carcinoma[9]. It is well known that the HSPA2 is localized primarily in cytoplasm at physiological temperature, whereas HSPA2 migrates to the nucleus and nucleoli under heat-shock conditions[14, 23], and our results confirm this theory. The phenomenon of HSPA2 translocation into the nucleus and nucleoli has been presently found in many other heat-shock cancer cells, which is part of a cellular protective response under stress conditions, but the mechanism is currently unknown. Generally, HSPA2 is involved in intracellular trafficking and nuclear receptor binding, which also prevents inappropriate protein aggregation and mediates transport of immature (or damaged) proteins to target subcellular compartments for final packaging, degradation or repair[29]. Since HSPA2 shuttles continuously between the cytoplasm and nucleus during heat-shock, this cell migration is critical for tumor formation and metastasis.

Finally, patients with HSPA2-positive expression were significantly associated with shorter disease-free and overall survival by using the Kaplan-Meier analysis. Therefore, HSPA2 protein expression pattern might be a valuable prognostic marker for ESCC patients. More importantly, from univariate and multivariate analysis, we obtained sufficient evidence to deduce that HSPA2 expression level was an independent prognostic indicator for patients with ESCC. Overexpression of HSPA2 is correlated with poor therapeutic outcomes in human breast cancer, cervical cancer and bladder urothelial cancer; furthermore, it is now a valuable prognostic marker for breast cancer patients[7]. The polymorphism of HSPA2 acts as an attractive susceptibility marker and independent prognostic indicator in Kangri cancer patients[30]. Considering the frequency of positivity of HSPA2 in adjacent non-cancerous tissues is much higher, whether or not HSPA2 molecule can be qualified to be a prognostic biomarker will need to be validated in future clinical work.

Our present study demonstrated that elevated HSPA2 expression levels was positively associated with the progression and poor prognosis in patients with ESCC, which indicated that HSPA2 might serve as a valuable prognosis marker for ESCC patients. However, the possible underlying mechanisms for its participation in ESCC progression are still unclear. Therefore, the next step we will take will be further research of the cell signaling pathway in order to gain a better molecular mechanism understanding in this field.