Unveiling the potential of HSPA4: a comprehensive pan-cancer analysis of HSPA4 in diagnosis, prognosis, and immunotherapy

Introduction

The global incidence and mortality rates of cancer have emerged as a grave concern, with the worldwide burden of this affliction exhibiting an escalating trajectory [1]. Elucidating the mechanisms of cancer initiation and developing targeted therapeutic strategies hold significant importance. Historically, chaperone proteins have been posited to assist in the folding and refolding of proteins. When proteins exhibit defects or undergo irreversible misfolding, chaperones perform the dual function of error correction and degradation [2]. As a prominent subclass within the chaperone proteins, heat shock proteins (HSPs) have garnered significant attention in cancer research. Over the past few decades, the field of HSP research has witnessed substantial development and notable progress. Extensive researches on HSP inhibitors and vaccines have been conducted both domestically and internationally, demonstrating significant therapeutic effects [3–5]. Several studies indicate that HSPs play pivotal roles in several cancer-associated features, such as promoting tumor cell growth, supporting angiogenesis, and modulating tumor immune responses [6, 7]. Particularly noteworthy is the ongoing exploration of HSPs’ therapeutic potential in cancer treatment and immune modulation. Their emerging significance as targets for new drug development is increasingly recognized [8]. These research findings not only offer novel therapeutic strategies to researchers but also open new horizons for future medical studies.

HSPs can be classified into several families based on their apparent molecular weight and homology, including HSP90, HSP40, HSP60, and HSP70, among others [9]. HSP90 plays a role in signal transduction pathways alongside certain kinases and steroid receptors, and it may also serve as a “typical” molecular chaperone [10]. As a crucial co-chaperone protein, HSP40 is primarily involved in regulating the activity of HSP70 [11]. HSP60, with the assistance of ATP, facilitates the correct folding of 15-30% of cellular proteins [12]. HSP70 is considered to play a central role in maintaining protein homeostasis [13]. Its functions include preventing the adhesion of unfolded polypeptide chains, dissociating protein oligomers, participating in protein transport, and regulating the heat shock response. These members of the heat shock protein families play vital roles in organisms, primarily involving protein quality control and degradation. However, despite the overall functions of HSPs being well-established, there are still some areas where the specific functions of individual family members remain unclear. This article focuses on HSPA4, a key member of the HSP70 subfamily. HSPA4 is considered a type of “evil chaperone” within the HSPs family [14]. Recently, HSPA4 has come under the spotlight due to its potential oncogenic role across various cancers. Evidence suggests that HSPA4 is implicated in the pathogenesis and progression of hepatocellular carcinoma [15], colorectal cancer [16], gastric cancer [17], and head and neck cancer [14], positioning it as a potential therapeutic target and prognostic biomarker. While current research primarily focuses on the role of HSPA4 in specific cancers, its universal function across cancer types remains a relatively uncharted domain. Thus, a pan-cancer, in-depth study of HSPA4 is of paramount importance. Research into pan-cancer has unveiled the commonalities and specificities among different tumor lineages, thereby deepening our understanding of the mechanisms underlying tumor formation and progression [18]. In recent years, pan-cancer research integrating multiple omics analyses has been extensively utilized in the investigation of therapeutic targets and prognostic biomarkers [19, 20]. To delve deeper into the molecular mechanisms of tumorigenesis, we employed a myriad of bioinformatics approaches to investigate the role of HSPA4 in various cancers, aiming to assess its potential value in clinical diagnosis and prognosis. We also conducted a comprehensive analysis of HSPA4 from genetic variations, methylation, and immune-related perspectives. Our research seeks to elucidate the pivotal role of HSPA4, offering insights into its position within the HSPs family. We posit that HSPA4 stands as a robust biomarker for cancer, with applications in diagnosis and prognostic prediction, and may also emerge as a potential molecular target for future therapies.

Materials and Methods

Results

Discussion

Recent advancements in clinical cancer screening and treatment face three main challenges: the lack of symptoms in early-stage cancers, the need for personalized treatment due to cancer’s heterogeneity, and the absence of effective biomarkers for certain cancers, complicating diagnosis and treatment monitoring. HSPs have garnered significant attention in tumor biology, especially with their pivotal role in tumorigenesis and cancer progression. The mechanisms of action of HSPs in cancer therapy primarily involve their roles in modulating the immune system and influencing the TME. HSPs can bind to antigenic peptides on the surface of cancer cells, forming HSP-peptide complexes, which facilitate the uptake, processing, and presentation of tumor-specific antigens, subsequently presented by antigen-presenting cells (APCs) to CD8+ cytotoxic T lymphocytes [32]. However, HSPs can also promote the growth, proliferation, and metastasis of tumor cells and mediate tumor cell resistance to stressors, contradicting their role in host protection [33]. Additionally, HSPs may induce T-cell tolerance, thereby creating an immunosuppressive environment conducive to cancer progression. Studies have shown that a variety of HSP inhibitors demonstrate potential therapeutic efficacy in cancer treatment, potentially by disrupting the ATPase activity of HSPs within cancer cells, thereby blocking oncogenic pathways and leading to apoptosis or loss of proliferative capacity in cancer cells [3, 4, 34, 35]. Moreover, cancer vaccines based on HSPs have been proven effective against a range of tumors expressing specific antigens, making them ideal targets for cancer vaccine therapy [5]. Current clinical trials targeting HSPs through inhibitors (NCT04437420) and vaccines (NCT03650257) are actively underway, primarily focusing on exploring the potential of novel anti-cancer therapies. However, the clinical application of HSP inhibitors faces several challenges, such as the induction of heat shock responses, retinal pathologies, and gastrointestinal toxicity [36, 37]. These challenges not only impose stringent safety requirements for patients but also present significant difficulties for researchers in further developing these inhibitors. On the other hand, research on vaccines, particularly their potential in cell-mediated immune activation, still encounters many unresolved issues [38]. The regulation of cell-mediated immune responses is crucial for vaccine efficacy. Delving into the role of HSPs family members within the TME and the immune system, and designing more precise targeted treatment strategies based on these insights, can provide pivotal understanding for the development of more effective immunotherapeutic methods.

Within the HSPs family, the member encoded by the HSPA4 gene stands out. This gene demonstrates a profound association with various cancer types. In non-small cell lung cancer, HSPA4 has shown its potential as an oncogene [39]. Inhibition of the HSPA4 gene substantially diminishes the invasive and metastatic capabilities of tumor cells [39]. Similarly, in head and neck squamous cell carcinoma, HSPA4 not only emerges as a promising therapeutic target but also might serve as a prognostic biomarker [14]. In breast cancer patients, pathogenic IgG targeting glycosylated HSPA4 is produced in sentinel lymph nodes by B cells [40], potentially accelerating lymph node metastasis. The relationship between HSPA4 and hepatocellular carcinoma has also been scrutinized. Bioinformatic studies highlight that high HSPA4 expression is significantly correlated with poor hepatocellular carcinoma prognosis, underscoring its central role in immune modulation [41]. Intriguingly, the progression of colorectal cancer can be effectively curbed by decreasing HSPA4 expression [16]. In gastric cancer, specific mutations related to HSPA4 are significantly associated with tumorigenesis [17]. Despite a multitude of studies examining HSPA4’s role in specific cancer types, a holistic perspective remains lacking. Addressing this gap, we embarked on the first comprehensive investigation of HSPA4 across cancers, aiming to elucidate its broader biological significance.

Delving deep into the TIMER2.0 tool, TCGA, GTEx, and CPTAC databases, we found marked variability in HSPA4 expression across multiple cancers. Notably, bioinformatics analyses reveal that HSPA4 is significantly overexpressed in gastric, colorectal, and liver cancers. Previous studies also indicate that HSPA4 plays a pivotal role in these gastrointestinal tumors [16, 17, 41]. Furthermore, we hypothesize that the link between HSPA4 and gastrointestinal tumors might be closely related to changes in the TME’s stress response, including reduced glucose and oxygen availability and environmental acidification [42, 43]. However, the precise molecular mechanisms underlying the association between gastrointestinal tumors and HSPA4 require further investigation to elucidate. This suggests HSPA4’s crucial role in cancer progression. Conversely, its expression in KIRC and PRAD was significantly diminished. Such variation unveils the intimate relationship between HSPA4, cellular specificity, and underlying BP. Further investigations into HSPA4’s localization revealed significant enrichment in microtubules and cytoplasm. Given that microtubules play an essential role in maintaining cell morphology, material transport, and cell division, their intimate association with oncogenesis becomes evident [44]. Furthermore, our ROC analysis demonstrated a noteworthy diagnostic value of HSPA4 across various cancers, with its AUC frequently surpassing the 0.7 mark, strongly indicating its potential as a bio-marker for oncology. Delving deeper into the CPTAC dataset, a pronounced correlation was discerned between HSPA4 expression and the pathological staging of cancer. To understand this nexus with greater precision, we stratified based on HSPA4 expression levels and meticulously examined its association with patients’ OS, DSS, and PFI. OS is a widely-utilized metric primarily employed to assess the impact of treatment on the overall extension of a patient’s life. It encompasses all causes of death, thereby providing a comprehensive measure of survival. DSS focuses on deaths directly attributable to the disease itself, more accurately reflecting the mortality risk associated with the specific illness. On the other hand, PFI centers on evaluating the efficacy of treatments in delaying disease progression. Intriguingly, in our analysis, HSPA4 demonstrated correlations with these survival indices across multiple tumor types, typically exhibiting consistent trends in most cases. In the majority of cancers, an elevated expression of HSPA4 was concomitant with a diminished survival rate. However, distinctively in KIRC, heightened HSPA4 expression seemed to serve as a favorable prognostic indicator for extended survival. Collectively, augmented expression of HSPA4 is generally linked with an adverse cancer prognosis, especially pronounced in cancers like ESAD, LIHC, and LUAD. Given the paramount diagnostic and predictive prowess HSPA4 showcases as a cancer biomarker, it’s evident that while its high expression commonly suggests an unfavorable outcome, exceptions like KIRC do exist. This underscores the intricacies inherent in cancer’s pathogenesis and progression, emphasizing the need for an in-depth exploration of specific tumor types even within the broader scope of oncological research.

Upon rigorous exploration of genetic variations in the HSPA4 gene across diverse cancers, we identified significant mutations of HSPA4 in several tumor types. Specifically, in UCEC, the mutation frequency of HSPA4 notably surpassed 6%, with over 5% of these categorically classified as “mutations”. Missense mutations can induce significant alterations in protein structure and function, conferring a selective growth advantage to cancer cells [45]. Importantly, missense mutations dominated the mutation spectrum of HSPA4. From an analysis spanning 30 distinct cancer types, we detected a prevalent shallow deletion in HSPA4 mRNA across the majority of tumors. Strikingly, in KIRC, an extraordinary amplification of HSPA4 mRNA was observed, potentially playing a critical regulatory role in cancer progression. Moreover, the methylation status of the HSPA4 promoter region plays a pivotal role in tumorigenesis, progression, and its prognostic implications for patients [41]. Our data revealed that, relative to their normal tissue counterparts, there was a pronounced increase in the methylation levels of the HSPA4 promoter region in various tumor types. Conversely, in COAD and READ, a significant reduction in its methylation was identified. These findings suggest that the transcriptional activity of HSPA4 may be modulated by the methylation status of its promoter region. Collectively, our insights into the genetic variations and methylation patterns of HSPA4 across different cancers provide a novel perspective on its functional role in oncology, laying the groundwork for potential targeted therapeutic strategies in the future.

In recent years, the TME and the infiltration of immune cells therein have emerged as focal points in cancer research, a trend substantiated by numerous studies [28, 29, 46]. Notably, CAFs are believed to regulate the function of infiltrating immune cells [30, 31]. Our research indicates a pronounced positive correlation between HSPA4 gene expression and CAFs infiltration in CESC, HNSC, and PAAD, while a negative correlation was observed with TGCT. B cells and T cells are central functional players in the immune response. B cells possess the capability to produce a diverse set of antibodies, thereby playing an instrumental role in mediating the immune response. When the function and quantity of immune cells are compromised, tumor cells may exploit this vulnerability to evade immunosurveillance, posing a detrimental impact on the OS of cancer patients [47]. In our heatmap analysis focused on HSPA4 expression with respect to immune cells, a significant correlation between B cells, T cells, and various cancers emerged, suggesting their integral role in the oncogenic progression modulated by HSPA4. Further investigations revealed distinct expression patterns of HSPA4 across immune subtypes in KIRC, ESCA, COAD, STAD, READ, LUAD, and LIHC. These findings underscore the pivotal role of HSPA4 in modulating immune responses, cancer progression, and immune subtypes. Over recent years, immunotherapy has increasingly emerged as the focal point of research in the realm of cancer treatment, demonstrating remarkable efficacy in various tumor therapies [48]. A deeper understanding of HSPA4 not only unveils its central function in oncology but also offers potential avenues for cancer treatment, especially in refining immunotherapeutic strategies [49].

In our exploration of the functional role of HSPA4 in tumorigenesis, we employed single-cell transcriptomics and the CancerSEA tool. CancerSEA serves as a unique platform for delving into the functional states of cancer cells at the single-cell level, providing invaluable insights for cancer research and gene-function association analyses [24]. Our findings indicate a significant association between HSPA4 and various functional states across different tumors, with its role in cell cycle regulation being particularly pronounced. It is well-understood that tumorigenesis is closely linked to aberrant cell proliferation, a process tightly governed by the cell cycle control mechanism [50]. However, in cancer cells, the failure of damage checkpoints allows these cells to continue dividing despite accumulating genetic defects [51]. Intriguingly, previous studies have shown that knocking down HSPA4 results in a G2 phase arrest, suggesting a novel therapeutic avenue for colorectal cancer [16]. Through GSEA enrichment analysis, we further discerned that HSPA4 plays a pivotal role in various tumors, especially in pathways related to cell cycle regulation, immune checkpoint pathways, and activation responses of B/T cells. These novel findings furnish invaluable insights into the role of HSPA4 in the landscape of cancer single-cell studies.

In the present study, we harnessed the STRING database to explore genes associated with HSPA4, culminating in the successful construction of a PPI network. Our meticulous analysis identified ten core genes intricately linked with HSPA4. Crucially, upon evaluating genes that may significantly influence cancer prognosis, HSP90AA1, HSPA8, DNAJB1, and HSP90AB1 manifested pronounced correlations with HSPA4 expression. A cross-comparative analysis of genes related to HSPA4 and their interacting counterparts spotlighted five overlapping genes: GRPEL2, TCP1, DNAJA1, HSPD1, and HSPA8. Delving deeper using data from the STRING database, we embarked on a GO and KEGG enrichment analysis, aiming for a profound understanding of these genes’ functions and interplay within the cellular milieu. Our GO categorization spanned BP, MF, and CC, granting us insights into the gene’s functionality and prospective cellular localization [52]. It is noteworthy that these genes were intimately tied to the quintessential functions of the HSPs family, particularly protein folding. Protein folding, an essential BP within the cell, ensures proteins achieve their correct 3D conformations, thereby facilitating their designated roles. Despite the considerable advances in heat shock protein research, the specific role and mechanism of HSPA4 within this family warrant further elucidation. Furthermore, our KEGG pathway analysis underscored a tight association of these genes with cell cycle regulation, resonating with our earlier single-cell analysis outcomes. This paves the way for future investigations into the potential roles of these genes in cancer and other diseases.

In summary, we have conducted a comprehensive pan-cancer analysis of HSPA4 for the first time. Across various cancer types, the HSPA4 gene emerges as a pivotal player, underscoring its central role in tumorigenesis and cancer progression. Notably, HSPA4 not only shows potential as a key biomarker for malignancies, but its elevated expression is also frequently associated with poorer patient outcomes. Upon deeper investigation, genetic variations of HSPA4, specific methylation patterns, and its interaction with immune cells all appear to be critical determinants of its function in tumors. Moreover, HSPA4 plays a crucial role in maintaining protein homeostasis, which is vital for cell function and survival. This gene is closely intertwined with essential biological processes such as the cell cycle, immune responses, and protein folding. Elucidating these associated genes will empower us to devise more precise and targeted cancer therapeutic strategies. Based on these findings, there is reason to propose that HSPA4 could serve as a promising biomarker for the diagnosis and prognosis of various types of cancer, and as a potential target for immunotherapy. Although significant progress has been made in understanding HSPA4, there remain some limitations. Previous studies have largely focused on individual cancer types, lacking a comprehensive understanding of HSPA4’s overall role. Moreover, our research has primarily been based on database analysis, with a shortage of validation using a broader range of clinical samples. Given the complexity of cancer, HSPA4 may play varying roles in different cancer types, necessitating further research for deeper exploration. Future studies should emphasize conducting more clinical research to verify the role of HSPA4 in human cancers and its potential as a therapeutic target. Understanding how HSPA4 interacts with other proteins and signaling pathways, and how these interactions impact cancer development and treatment, are also crucial. Exploring the application of HSPA4 inhibitors or vaccines in cancer therapy is another important research direction. In summary, despite certain progress, many unknown areas in HSPA4 research remain to be explored further, to better harness this protein’s potential in cancer therapy.

Supplementary Materials

Supplementary Figures

Supplementary Table 1