High Expression of HSPA8 is A Favorable Prognostic Factor in Colon Cancer


 Background: Colon adenocarcinoma (COAD), a type of colon cancer, is among the most common malignant diseases afflicting people globally. Heat shock protein family, including HSPA8, are involved in tumor progression in numerous human cancers, however the association between HSPA8 and COAD has not been explored.Methods: RNA sequencing data and clinical characteristics from TCGA and GTEx, as well as protein data from CPTAC were obtained for analysis of HSPA8 expression in COAD. Furthermore, functional enrichment analysis of HSPA8-related differentially expressed genes (DEGs) was performed via GO/KEGG, immune infiltration analysis, and protein-protein interaction (PPI) network. In addition, the clinical significance of HSPA8 was analyzed by Kaplan-Meier Cox regression and prognosis assessment. R package was employed to analyze incorporated studies.Results: Compared with normal samples, the expression of HSPA8 increased remarkably in various tumors, including COAD, Moreover, high HSPA8 expression was associated with good prognosis in COAD, as well as subgroups with male, T2-T4 stage, no lymphatic and perineural invasion. (P < 0.05). High HSPA8 was a prognostic factor in Cox regression analysis. The diagnostic efficacy of HSPA8 in COAD analyzed by ROC (AUC: 0.889). Furthermore, a total of 3321 DEGs were identified between the high- and the low-expression group, of which 9 genes were up-regulated and 3312 genes were down-regulated.Conclusions: High HSPA8 expression is a potential biomarker for favorable outcomes in COAD. the results might provide clues and guidance for developing novel diagnosis, targeted therapy and prognosis of HSPA8 in COAD.

Heat shock 70 kDa protein 8 (HSPA8), a member of the heat shock protein 70 (HSP70) family, plays an integral role in the protein quality control system, including the folding of newly synthesized proteins, the refolding of misfolded proteins and targeting of proteins for subsequent degradation. HSPA8 has also been found associated with tumor progression in mounting human cancers, such as hepatocellular carcinoma [9], acute myeloid leukemia [10], endometrial carcinoma [11], and usually leads to poor outcomes. However, the role of HSPA8 expression in colon cancer remains poorly understood.
In this way, we aimed to explore the relationship between the expression level of HSPA8 and the prognosis of COAD in our study by using bioinformatics resources. Expression of HSPA8 is elevated signi cantly in many types of tumors, including COAD, however, unlike other types of malignant tumors, high HSPA8 expression is associated with a good prognosis in COAD. The result indicates that HSPA8 may could be a potential biomarker in the diagnosis, therapy and follow-up of colon cancer.

Results
High HSPA8 expression in many pan-cancer, including COAD.
The data of RNA-seq was download from UCSC XENA in TCGA and GTEx formats uniformly processed.
The expression level of HSPA8 increased signi cantly in 21 types of cancer ( Figure 1A), colon cancer (COAD) included ( Figure 1B), as for paired tumor and adjacent samples, HSPA8 was found clearly high expressed as well ( Figure 1C), the differences above all was statistically signi cant (P<0.001).
To further validate the expression of protein, we explored the patterns of HSPA8 in COAD by Human Protein Atlas and UALCAN. As was shown in Figure 1D, HSPA8 protein was not detected in normal colon tissues, while medium expressions were observed in COAD samples. We next analyzed the relationship of HSPA8 protein expression in colon cancer between normal and primary tumor by UALCAN (Figure 2A), and found that protein expression of tumor group surpassed normal tissues obviously (P<0.001).

Clinical Characteristics
The clinical data of 478 patients of COAD were obtained from TCGA, including age, race, gender, primary therapy outcome, CEA level, TNM stage, pathological stage, perineural invasion, lymphatic invasion (Table 1). Among them, HSPA8 expression was 50% high and 50% low in 478 cases, according the cut-off valve, which was the median HSPA8 expression. Correlation analysis elucidated that HSPA8 expression was signi cantly correlated with age (P=0.050), race (P=0.035) N stage (P<0.001), M stage (P=0.027), pathological stage (P=0.002), OS event (P=0.045).  Table 2). ROC curve analysis was adapted to evaluated the potential valve of HSPA8 in differentiating patients from population, the AUC was 0.889, the result showed that HSPA8 was a potential biomarker for the diagnosis of COAD ( Figure 2B). Moreover, the Wilcoxon rank sum test was employed to contrast the HSPA8 expression in patients with distinct clinicopathological characters. As a result, the expression of HSPA8 was obviously negative associated with N stage (

Performance of DEGs in COAD samples with high-and low expressed HSPA8
The gene expression pro les were divided into high-and low-expression groups according to the criteria of median HSPA8 mRNA expression (Additional le 1: S1). analyzed for differences in the median mRNA expression. A total of 3321 DEGs from gene expression RNA-seq-HTSeq-Counts, including 9 up-regulated and 3312 down-regulated, were identi ed statistically signi cant between the two groups (|log fold change (log FC) | > 1.5, P < 0.05) ( Figure 3A). The top ve up-regulated DEGs and top ve down-regulated DEGs were shown by the heat map ( Figure 3B).

GO/KEGG functional enrichment analysis and PPI network construction
In association with functional enrichment analysis via GO/KEGG, the correlation with the biological process (BP) consisted of sensory perception of bitter taste and corresponding detection of chemical stimulus involved; cellular component (CC) included nuclear nucleosome and muscle myosin complex; molecular function (MF) included bitter taste receptor activity, taste receptor activity; KEGG included taste transduction, olfactory transduction ( Figure 4A, 4B).
We also employed STRING to explore the PPI network of HSPA8 protein to analysis the interaction in COAD, the top 10 gene names and corresponding protein, as well as the annotations and score are shown in Figure 5A, 5B. Moreover, with a threshold of 0.4, the network of HSPA8 and its potential co-expressed genes in HSPA8-related DEGs was constructed by SRING, a total of 114 protein coding were screened out with the threshold of |log fold change (log FC) | > 1.5, P < 0.05 ( Figure 5C) (Additional le 1: S2).

Immune in ltration analysis in COAD
Spearman correlation analysis demonstrated that the HSPA8 expression level in the COAD tumor microenvironment was relevant to the immune in ltration quanti ed by ssGSEA, the expression level of HSPA8 had clearly positive correlation with in ltrating level of Th2 cells, but negative correlation with Plasmacytoid DC (Figure 6).

Discussion
In addition to cancer genetic, aberrant epigenetic regulation has also been reported to participate the development and progression of many types of cancer, including COAD [25]. Heat shock protein (HSP) family members, as components of epigenetic regulation complexes, are important in the development of colorectal cancer [26]. The distinct roles of HSP family members in COAD, however, remained unclear. In our study, the expression of mRNA and protein, and prognostic values of HSPA8 in COAD were explored.
HSPA8, also known as heat shock cognate 70 (HSC70), belongs to heat shock 70kDa protein family, which were notably produced under stress conditions. HSPA8, the major family member is critical for cells to be healthy and function normally [27][28][29][30], as well as plays an essential role in many biological tumor processes [31,32], consisting of cell proliferation, autophagy, immune in ltration. Several studies have elucidated that High HSPA8 expression predicted adverse outcomes of hepatocellular carcinoma [9], acute myeloid leukemia [10], endometrial carcinoma [11]. However, little has been reported about the prognostic value of HSPA8 in COAD, and the clinicopathologic implications of HSPA8 expression in COAD have not been thoroughly studied.
In this study, we found that, comparing to normal cohort, the mRNA and protein expression levels of HSPA8 increased signi cantly in COAD samples, and the high HSPA8 expression levels were associated with low N and M stage, low pathologic stage, and good overall survive. Via GO/KEGG functional enrichment analysis and PPI network construction, we found that HSPA8 regulates tumor related genes, such as STUB1, BAG family, HSP family, MYH family. The results might indicate that HSPA8 was a tumor suppressor gene.
Exosomes, which belong to extracellular vesicles (EVs) and enrich in nucleic acids, lipids, and proteins [33], play important roles in cell-to-cell communication during the tumor progression, immune response, neuron survival and many other contexts [34]. As a traditional exosome marker, HSPA8 has been analyzed in pancreatic cancer, lung cancer [8] and diabetic kidney disease [35], breast cancer [36]. Little is known about the relationship between HSPA8 and exosomes in COAD. In present study, HSPA8 had a strong interaction with HSP90AA1, an exosomal protein that implicated in maintenance and cellular adhesion for colorectal cancer cells via integrin-mediated cell adhesion pathway [37]. However further studies were need to con rm the result and explore the potential regulatory mechanism of HSPA8.
In immune cell in ltration analysis, high HSPA8 expression was positively associated with the majority of immune cells in COAD samples, including Th2 cells, Dendritic cells, Macrophages, Neutrophils, Dysbiosis is known and implicated in the growth and progression of COAD, and immune responses play indispensable roles in the procedure [38,39]. Therefore, HSPA8 could regulate immune cells and immune responses, and further affecting the progression of colon cancer, but the details of the immunoregulatory mechanisms in COAD still deserve further exploration.
There were some limitations in our study. First, although high HSPA8 expression was an independent prognostic factor for longer OS of colon cancer patients, all the data was retrieved from online databases, further studies that consist of extensive functional in vitro and in vivo work, and larger sample sizes were needed to validate our nding. Second, the retrospective nature of our study was a signi cant limitation, and further prospective investigation designed trials were warranted. Lastly, we did not explore the potential mechanisms in depth of HSPA8 in COAD, future studies to investigate the potential diagnostic and therapeutic values of HSPA8 in COAD waited to be fully elucidated.

Conclusion
In summary, the present study demonstrated that the mRNA and protein expression levels of HSPA8 markedly elevated in colon cancer samples compared with normal tissue, and over expression of HSPA8 was found signi cant associated with lower clinical cancer stages and pathologic grades of COAD. Moreover, high HSPA8 expression predicts favorable outcomes in COAD, which indicated that HSPA8 might be a promising biomarker with diagnostic or therapeutic value, and provide novel target for therapeutics. Therefore, the results above might provide clues and guidance for developing novel diagnosis, targeted therapy and prognosis of HSPA8 in colon cancer.

Data source and Bioinformatics Analysis
The Cancer Genome Atlas (TCGA) is an open and free data portal of largescale genomics project, provides a comprehensive clinic and pathological information of 33 types of cancer for researchers and scholars. In our study, the pan-cancer RNA-seq data of TCGA and GTEx with toil processed were downloaded from UCSC XENA [12,13]. And data of COAD patients were collected from TCGA, including 308 normal samples from GTEx, 41 para-carcinoma tissues and 290 COAD tissues from TCGA. Clinical data, including gender, age, weight, primary therapy outcome, CEA level, perineural invasion, lymphatic invasion, histologic grade and pathologic stage were summarized.

UALCAN and Human Protein Atlas
UALCAN is a comprehensive, user-friendly, and interactive web resource for analyzing cancer OMICS data from TCGA, MET500 and CPTAC, and has been now visited over 700,000 times by researchers from over 100 countries and cited more than 1900 times [14]. The Clinical Proteomic Tumor Analysis Consortium (CPTAC), now provides protein expression analysis, which for colon cancer is accessible [15].

Immune in ltration analysis
Immune in ltration analysis of HSPA8 was conducted using GSVA package by ssGSEA in R (3.6.3) [18]. Spearman correction was used to analyze the correlation between HSPA8 and the enrichment scores of 24 types of immune cells [19]. Independent-samples T test was used to analyze the enrichment scores of high-and low-expression groups of HSPA8.

Protein-protein interaction (PPI) network
The PPI network of DEGs was predicted using the Search Tool for the Retrieval of Interacting Genes (STRING) database, and the cut-off criterion was 0.4 [20]. The PPI network was mapped using Cytoscape (version 3.9.0) [21,22].

Prognosis analysis with multiple modes
Receiver operating characteristic (ROC) and Kaplan-Meier survival curves were employed to analyze the diagnostic and prognostic values of HSPA8 in COAD patients [12]. Secondly, univariate regression analyses were used to elucidate the association between HSPA8 expression and overall survival (OS) rates [23].

Statistical analysis
All statistical analyses and graphs were analyzed and displayed by R (3.6.3) [24]. The expression of HSPA8 in unpaired samples was analyzed by Wilcoxon rank sum test, with Wilcoxon signed-rank test used in paired samples. Logistic regression analysis was used to evaluate the relationship between clinical characteristics and expression of HSPA8. Kaplan-Meier method were used to evaluate the prognostic factors. Univariate Cox analysis was adopted to compare the impact of HSPA8 expression on survival comparing with other clinical features. The cut-off value was the median HSPA8 expression. In all tests, P value < 0.05 was considered statistically signi cant. Moreover, ROC analysis was performed on the pROC package to assess the effectiveness of the expression of HSPA8.    PPI network of HSPA8. (A, C) The network of HSPA8 and its potential co-expression genes was analysis by STRING tool. (B) the detailed information of top ten HSPA8-related genes, as well as their annotations and score were listed.