AHSA1 is a Prognostic Biomarker that Correlates with Macrophage Inltration in Pan-Cancer

Background: Activator of heat shock 90 kDa protein ATPase homolog 1 (AHSA1) is differentially expressed in several tumor types. However, its association with immune cell inltration remains elusive. Methods: AHSA1 expression was analyzed using The Cancer Genome Atlas (TCGA) pan-cancer data and normal tissue expression data from Genotype-Tissue Expression (GTEx). The clinical prognostic role of AHSA1 in pan-cancer was investigated, and an enrichment analysis of AHSA1 was performed using the R package “clusterProler.” We downloaded data regarding the immune cell inltration level of TCGA pan-cancer tissues and analyzed the association between immune cell inltration and AHSA1 expression. Results: The results of TCGA pan-cancer data analysis revealed that AHSA1 was overexpressed and associated with poor survival in patients with cancer. Furthermore, the inltration levels of tumor-associated macrophages (TAMs) were higher, while those of CD8+ T cells were lower, in the high AHSA1 expression group. Conclusions: Our study suggests that AHSA1 is an oncogene and a risk factor for patient survival in cancer. AHSA1 may contribute to high inltration levels of TAMs and low inltration levels of CD8+ T cells, thus indicating that high AHSA1 expression may be associated with the tumor immunosuppressive microenvironment.


Background
The activator of heat shock 90 kDa protein ATPase homolog 1 (AHSA1) is a chaperone of heat shock 90 kDa (HSP90) and stimulates the ATPase activity of HSP90 [1]. Notably, AHSA1 reportedly plays additional roles apart from regulating the HSP90 ATP hydrolysis rate [2]. Some researchers have revealed the critical role of AHSA1 in tumor progression. For example, knockdown of AHSA1 could inhibit the proliferation and invasion of osteosarcoma cells. However, research on AHSA1 in tumors, especially in the tumor immune microenvironment, remains scarce.
Several studies have reported that the tumor immune microenvironment has clinicopathological signi cance in predicting the therapeutic effect and prognosis of patients presenting tumors [3]. Tumorassociated macrophages (TAMs) and CD8 + T cells play a key role in tumor progression and immunotherapy [4]. Moreover, it has been con rmed that solid tumors are composed of malignant, nonmalignant, hematopoietic, and mesenchymal cells. Among the non-malignant cells, TAMs play an essential role in promoting tumor angiogenesis. The extensive heterogeneity of TAMs enables these cells to adapt or alter their phenotypes to conform to the tumor microenvironment, playing a role in cancer progression and metastasis [5].
In this study, we evaluated the expression of AHSA1 and its association with the prognosis in patients with cancer. We observed that AHSA1 was overexpressed in most tumor types. AHSA1 is predicted to be involved cell cycle-related pathways. We further examined the correlation between AHSA1 expression and the immune cell in ltration score and found that TAM in ltration increased and CD8 + T cell in ltration decreased in tissues with high AHSA1 expression. Our results present novel insights into the functional role of AHSA1 in pan-cancer, highlighting a potential mechanistic basis, whereby AHSA1 in uences TAM and CD8 + T-cell in ltration, as well as the tumor immunosuppressive microenvironment.

Data Collection and Analysis
The expression and clinical data of The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) were downloaded from the UCSC Xena database (https://xenabrowser.net/datapages/). For AHSA1, the DNA copy number and methylation data were downloaded from the cBioPortal database (https://www.cbioportal.org/). The protein level of AHSA1 was downloaded from the Ualcan database (http://ualcan.path.uab.edu/index.html).

Correlation and Enrichment Analyses
Correlation analysis between AHSA1 and other mRNAs was performed using TCGA pan-cancer data, and Pearson's correlation coe cient was calculated. The top 300 genes most positively correlated with AHSA1 expression were selected for enrichment analysis to re ect the AHSA1 function. Gene set enrichment analysis (GSEA) was conducted using the R package "clusterPro ler."

Immune Cell In ltration
We downloaded the immune cell in ltration score of TCGA from a previously published study, "The Immune Landscape of Cancer" [6]. For each TCGA tumor type, patients were divided into two groups (high and low AHSA1 expression groups), based on the median AHSA1 expression to compare levels of immune cell in ltration.

Pan-Cancer Analysis of AHSA1 Expression
We rst evaluated AHSA1 expression using RNA sequence data combined with TCGA and GTEx expression pro lers. The results revealed that AHSA1 was overexpressed in 27 cancer types, including  (Fig. 1a). In tumor tissues derived from TCGA, AHSA1 expression was highest in TGCT and lowest in KIRC (Fig. 1b). In normal tissues from GTEx, the highest AHSA1 expression was detected in the testis, with the lowest observed in the pancreas (Fig. 1c).

Genetic Alteration of AHSA1
Genetic and epigenetic alterations induce changes in gene expression. We explored genetic alterations in AHSA1 using cBioPortal and observed that patients with high AHSA1 expression presented gene alterations in uterine and cervical cancers, as well as DLBC (Fig. 4a). Copy number values were positively correlated with AHSA1 expression (Fig. 4b). In addition, the methylation level of the AHSA1 promoter was negatively correlated with AHSA1 expression (Fig. 4c). These results suggest that high copy number values and low methylation levels contribute to the high expression of AHSA1 observed in pan-cancer analysis.

Prognostic Signi cance of AHSA1
To evaluate the signi cance of AHSA1 in predicting the prognosis of patients with tumors, we performed univariate Cox regression analysis and Kaplan-Meier survival analysis using TCGA pan-cancer data. Results of the univariate Cox regression analysis indicate that AHSA1 is a risk factor for patients with ACC, HNSC, KIRP, LIHC, LUAD, MESO, PRAD, and UVM and a protective factor in LGG and OV (Fig. 5a). Kaplan-Meier survival analysis revealed that high AHSA1 expression predicted worse overall survival of patients with ACC, HNSC, KIRP, LIHC, LUAD, and UVM (Fig. 5b). We further assessed the signi cance of AHSA1 in predicting the disease-free interval (DFI), progression-free interval (PFI), and disease-speci c survival (DSS) in patients with tumors, using univariate Cox regression analysis. DFI analysis revealed that AHSA1 acts as a risk factor for ACC, KIRP, LIHC, LUAD, and MESO and a protective factor for OV (Fig. 6a). PFI analysis revealed that AHSA1 acts as a risk factor in ACC, BLCA, ESCA, HNSC, KIRP, LIHC, LUAD, PRAD, and UVM, while serving as a protective factor in LGG, OV, and STAD (Fig. 6b). Finally, the DSS analysis showed that AHSA1 acts as a risk factor in ACC, ESCA, HNSC, KIRP, LIHC, LUAD, LUSC, MESO, and UVM and a protective factor in LGG and OV (Fig. 6c).

Enrichment Analysis of AHSA1
To predict the functions of AHSA1, we performed GSEA using TCGA pan-cancer data. The results suggested that AHSA1 was signi cantly associated with cell cycle-related and immune regulation-related pathways in ACC, HNSC, KIRP, LIHC, LUAD, and UVM ( Fig. 7a-f). For example, AHSA1 was predicted to be involved in pathways such as "Cell Cycle," "Cell Cycle, Mitotic," "Adaptive Immune System," and "Innate Immune System" (Fig. 7d). These results suggest that AHSA1 is strongly associated with tumor cell cycle arrest and tumor immune microenvironment regulation.

Tumor Immune Microenvironment Analysis
We further downloaded the immune cell in ltration score of TCGA pan-cancer from a previously published article [6]. We divided each tumor into two groups according to the median expression of AHSA1 to compare possible differences in immune cell in ltration. We observed that macrophage in ltration levels were signi cantly higher in the high AHSA1 expression groups of BRCA, CESC, HNSC, KIRC, LIHC, SKCM, and STAD. Simultaneously, the number of CD8 + T cells were lower in the high AHSA1 expression groups of these tumor types ( Fig. 8a-g). These results suggest that high AHSA1 expression is associated with the tumor suppressor microenvironment.

Discussion
Immune cells in the tumor microenvironment play a vital role in restricting tumor progression. However, tumor cells can eventually escape immune surveillance and inhibit the cytotoxicity of antitumor immune cells through various mechanisms. The remodeling of immune cells by tumor cells can lead to immune escape, a known cancer marker [7].
Macrophages are multifunctional components of myeloid cells. These cells can phagocytize invading microorganisms or cell debris in injured parts, release immune-regulatory cytokines, and activate the adaptive immune system [8]. Previously, activated TAMs were suspected of demonstrating cytotoxic activity in tumor cells; however, several studies have con rmed the pre-tumor function of TAMs [9]. In recent decades, human samples with CD68 as a macrophage marker and CD163/CD204 as an M2 macrophage marker have been extensively investigated. Several immunohistochemical studies using tumor samples of different histological types and various locations have revealed that the greater the total number of TAMs, the worse the clinical prognosis of patients with tumors [10,11] .
In human cancer, cell in ltration is a regulator of natural disease progression. As a primary effector cell of anticancer immunity, CD8 + T cells are usually in a state of dysfunction or are reduced in number when in ltrating cancer tissues [12]. Depleted CD8 + T cells are characterized by impaired activity and proliferation, increased apoptotic rates, and decreased production of effector cytokines. The increase in TAM in ltration and decrease in CD8 + T-cell in ltration, observed in the tumor microenvironment, frequently indicates that the tumor is in a state of immunosuppression, which is unfavorable for implementing immunotherapy. Screening markers that can signal the immunosuppressive status of tumors help identify patients with tumors sensitive to immunotherapy.
In this study, we identi ed AHSA1 as an oncogene in most tumor types. We observed that AHSA1 was overexpressed in 27 cancer types, including ACC, BLCA, BRCA, CESC, CHOL, COAD, DLBC, ESCA, GBM,  HNSC, KICH, KIRP, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, READ, SKCM, STAD, TGCT, THCA, THYM, UCEC, and UCS, while low expression was noted in only 2 cancer types (KIRC and LAML). The univariate Cox regression analysis suggested that AHSA1 was a risk factor in patients with ACC, HNSC, KIRP, LIHC, LUAD, MESO, PRAD, and UVM, whereas in LGG and OV, AHSA1 was deemed a protective factor. We further divided each tumor into two groups according to the median expression of AHSA1 to compare the differences in immune cell in ltration. We observed that TAM in ltration levels were signi cantly elevated and the number of CD8 + T cells were reduced in the high AHSA1 expression groups in BRCA, CESC, HNSC, KIRC, LIHC, SKCM, and STAD. These results suggest that high AHSA1 expression indicates the tumor immunosuppressive status.

Conclusion
In summary, we conducted a comprehensive assessment on AHSA1, revealing the potential cancerpromoting effect of AHSA1 in and its role as an indicator of patient prognosis. Importantly, high AHSA1 expression often indicates that the tumor is immunosuppressed, which may render immune checkpoint inhibitors unsuitable for treatment. Targeting AHSA1 may increase the sensitivity to immune checkpoint inhibitors, providing a potential direction for tumor immune therapy.

Abbreviations
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Competing interests
No con ict or nancial interests.