key: cord-310091-x31g02xw authors: Zhang, Zhilan; Li, Lin; Li, Mengyuan; Wang, Xiaosheng title: Pan-cancer analysis reveals that ACE2 is positively associated with immunotherapy response and is a potential protective factor for cancer progression date: 2020-09-02 journal: Comput Struct Biotechnol J DOI: 10.1016/j.csbj.2020.08.024 sha: doc_id: 310091 cord_uid: x31g02xw The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 13 million people and has caused more than 570,000 deaths worldwide as of July 14, 2020. The SARS-CoV-2 human cell receptor ACE2 has recently received extensive attention for its role in SARS-CoV-2 infection. Many studies have also explored the association between ACE2 and cancer. However, a systemic investigation into associations between ACE2 and oncogenic pathways, tumor progression, and clinical outcomes in pan-cancer remains lacking. Using cancer genomics datasets from the Cancer Genome Atlas (TCGA) program, we performed computational analyses of associations between ACE2 expression and antitumor immunity, immunotherapy response, oncogenic pathways, tumor progression phenotypes, and clinical outcomes in 13 cancer cohorts. We found that ACE2 upregulation was associated with increased antitumor immune signatures and PD-L1 expression, and favorable anti-PD-1/PD-L1/CTLA-4 immunotherapy response. ACE2 expression levels inversely correlated with the activity of cell cycle, mismatch repair, TGF-β, Wnt, VEGF, and Notch signaling pathways. Moreover, ACE2 expression levels had significant inverse correlations with tumor proliferation, stemness, and epithelial-mesenchymal transition. ACE2 upregulation was associated with favorable survival in pan-cancer and in multiple individual cancer types. These results suggest that ACE2 is a potential protective factor for cancer progression. Our data may provide potential clinical implications for treating cancer patients infected with SARS-CoV-2. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 23 million people and has caused more than 800,000 deaths worldwide as of August 23, 2020 (https://coronavirus.jhu.edu/map.html). SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) as a host cell receptor to infect humans [1, 2, 3, 4] . ACE2 plays an important role in regulating cardiovascular and renal function [5] . This protein has recently received extensive attention for its role in SARS-CoV-2 infection [1, 2, 4] . Our recent study revealed that ACE2 is expressed in various human tissues [6] , suggesting that SARS-CoV-2 may invade various human organs besides the lungs. Moreover, SARS-CoV-2 infection may result in ACE2 upregulation [7] . However, ACE2 deficiency may exacerbate outcomes in patients with SARS-CoV-2 infection [8] . Indeed, a recent study showed that ACE2 was downregulated in virusinfected lung tissue [9] , indicating a potential protective role of ACE2 in patients with SARS-CoV-2 infection. ACE2 also plays a protective role in hypertension and heart disease [8] . Many studies have investigated the association between ACE2 and cancer [10] [11] [12] [13] [14] [15] [16] [17] [18] . For example, Yu-Jun et al. analyzed ACE2 expression in various cancers and revealed a positive association between ACE2 expression and survival prognosis in liver cancer [10] . Cai et al. described the genetic alteration, mRNA expression, and DNA methylation of ACE2 in over 30 cancer types and revealed genetic and epigenetic variations of ACE2 in various cancers [11] . Several studies demonstrated that ACE2 had antitumor effects by inhibiting tumor angiogenesis [13, 14, 16] . Zhang et al. revealed that ACE2 expression was more highly expressed in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) than in normal tissues [17] . Huang et al. found that ACE2 expression had a significant association with immune cell infiltration in various normal and cancer tissues [18] . A recent study [9] showed that ACE2 expression was associated with increased tumor immune infiltration and was a positive prognostic factor in uterine corpus endometrial and renal papillary cell cancers. Despite these previous studies, a systemic investigation into the association between ACE2 expression and antitumor immunity, oncogenic pathways, tumor progression phenotypes, and clinical outcomes in pan-cancer remains lacking. In this study, we investigated associations between ACE2 expression and antitumor immune signatures in 13 human cancer cohorts from the Cancer Genome Atlas (TCGA) program (https://cancergenome.nih.gov/). We also explored associations between ACE2 expression and multiple tumor phenotypes, including cell proliferation, stemness, epithelial-mesenchymal transition (EMT), oncogenic signaling, and clinical outcomes in these cancer cohorts. We also investigated the association between ACE2 expression and immunotherapy response in four cancer cohorts receiving the immune checkpoint blockade therapy. This study aimed to provide new insights into the association between ACE2 and cancer and the potential association between cancer and SARS-CoV-2 infection. From the genomic data commons data portal (https://portal.gdc.cancer.gov/), we Besides, we obtained gene expression profiling and clinical data in four cancer cohorts receiving anti-PD-1/PD-L1/CTLA-4 immunotherapy from their related publications, including Nathanson (melanoma) [19] , Topalian (melanoma) [20] ,Ascierto (renal cell carcinoma) [21] , and Snyder (bladder cancer) cohorts [22] . A summary of these datasets is presented in Supplementary Table S1. We evaluated the enrichment level of a pathway or tumor phenotype in a tumor sample by the single-sample gene-set enrichment analysis (ssGSEA) score [23] . The gene set included all marker genes of a pathway or tumor phenotype. A total of six cancer-associated pathways (cell cycle, mismatch repair, TGF-β, Wnt, VEGF, and Notch signaling) and three tumor phenotypes (cell proliferation, stemness, and EMT) were analyzed. We presented the marker genes of these pathways and tumor phenotypes in Supplementary Table S2. We defined high-ACE2-expression-level (upper third) and low-ACE2-expressionlevel (bottom third) tumors in each cancer type based on ACE2 expression profiles. We identified the KEGG [24] pathways highly enriched in both groups of tumors using GSEA [25] with a threshold of adjusted p-value < 0.05. Moreover, we used WGCNA [26] to detect the gene modules (gene ontology) differentially enriched between the high-and low-ACE2-expression-level tumors in pan-cancer. We identified the hub genes as the genes connected to at least 5 other genes with a connectedness weight greater than 0.25 in a gene module and built their co-expression network. We used Spearman's correlation test to evaluate the correlation (ρ) of ACE2 expression levels with the enrichment levels of pathways or tumor phenotypes, which were not normally distributed. We used Pearson's correlation test to evaluate the correlation (r) of ACE2 expression levels with the ratios of immune signatures, which was the log2-transformed values of the ratios between the mean expression levels of all marker genes in immune signatures and was normally distributed. We used the Benjamini and Hochberg method [27] to calculate the FDR for adjusting for multiple tests. We compared overall survival (OS), disease-specific survival (DSS), progression-free interval (PFI), and disease-free interval (DFI) between the high-and low-ACE2-expression-level tumors. We utilized Kaplan-Meier curves to display survival time differences and the log-rank test to evaluate the significance of survival time differences. The R package "survival" was used to perform the survival analyses. GSEA [25] identified many immune-related pathways highly enriched in the high-ACE2-expression-level tumors at least 5 cancer types. These pathways included viral myocarditis, T cell receptor signaling, systemic lupus erythematosus, primary immunodeficiency, NOD-like receptor signaling, natural killer cell-mediated cytotoxicity, Leishmania infection, Jak-STAT signaling, intestinal immune network for IgA production, hematopoietic cell lineage, graft-versus-host disease, Fc epsilon RI signaling, epithelial cell signaling in Helicobacter pylori infection, cytosolic DNAsensing, cytokine-cytokine receptor interaction, chemokine signaling, B cell receptor signaling, autoimmune thyroid disease, asthma, antigen processing and presentation, and allograft rejection (Fig. 1A) . Moreover, we found that ACE2 expression levels positively correlated with the immune-promoting/immune-inhibiting ratios in pancancer (Pearson's correlation test, r = 0.25, p = 2.37 × 10 -71 ) and in 12 individual cancer types (adjusted p-value (FDR) < 0.05) (Fig. 1B) . This suggests that ACE2 expression has a stronger positive association with the immune-promoting signature than the immune-inhibiting signature in these cancer types. Altogether, these results suggest a prominent positive association between ACE2 expression and antitumor immune signatures in cancer. We found that ACE2 had a positive expression correlation with PD-L1 in pan-cancer and in 6 individual cancer types (FDR < 0.05) ( Fig. 1C) . We expected that the ACE2 expression would have a positive association with the response to anti-PD-1/PD-L1/CTLA-4 immunotherapy. We confirmed the anticipation in four cancer cohorts receiving immune checkpoint blockade therapy. In these cohorts, the high-ACE2-expression-level (> median) tumors displayed a higher rate of immunotherapy response than the low-ACE2-expression-level (< median) tumors (67% versus 17%, 80% versus 40%, 40% versus 20%, and 46% versus 25% for Nathanson (melanoma), Topalian (melanoma), Ascierto (renal cell carcinoma), and Snyder (bladder cancer) cohorts, respectively) (Fig. 1D) . As a result, the former had better overall survival (OS) than the latter in the Nathanson cohort, which had related data available (log-rank test, p = 0.036) (Fig. 1E) . These results suggest that the ACE2 expression is likely to be a positive predictor for anti-PD-1/PD-L1 immunotherapy. GSEA [25] also identified several cancer-associated pathways highly enriched in the high-ACE2-expression-level tumors at least 5 cancer types. These pathways included axon guidance, renin angiotensin system, PPAR signaling, MAPK signaling, glycolysis, cell adhesion molecules, endocytosis, and calcium signaling. We found that the activities of these pathways were significantly and positively associated with PD-L1 expression levels in pan-cancer (FDR < 0.001) (Fig. 1F) . In individual cancer types, the elevated activities of these pathways were also likely to correlate with increased PD-L1 expression levels (FDR < 0.05) (Fig. 1F) . The elevated activities of these pathways could be responsible for the more active immunotherapy response in cancer. We quantified the activity of a pathway using the single-sample gene-set enrichment analysis (ssGSEA) [23] score of the set of genes included in the pathway. We found that ACE2 expression levels inversely correlated with the activity of cell cycle, mismatch repair, TGF-β, Wnt, VEGF, and Notch signaling pathways in 10, 7, 9, 7, 5, and 7 individual cancer types, respectively (Spearman's correlation test, FDR < 0.05) ( Fig. 2A) . Moreover, we found that ACE2 expression levels had a significant inverse correlation with the expression levels of MKI67, which is a tumor proliferation index marker, in pan-cancer and 8 individual cancer types (Pearson's correlation test, FDR < 0.05) (Fig. 2B) . Tumor stemness represents a stem cell-like tumor phenotype associated with tumor progression, metastasis, immune evasion, and drug resistance. We found that ACE2 expression levels showed a marked negative correlation with tumor stemness scores (ssGSEA scores) in pan-cancer and in 10 individual cancer types (FDR < 0.05) (Fig. 2C) . EMT plays an outstanding role in facilitating malignant transformation, tumor progression, and metastasis. We observed a marked negative correlation between ACE2 expression levels and EMT signature scores (ssGSEA scores) in 11 individual cancer types (FDR < 0.05) (Fig. 2D) . Overall, these data indicate that ACE2 could be a protective factor for cancer progression. Indeed, survival analyses showed that ACE2 upregulation was associated with favorable survival in pan-cancer (log-rank test, p < 0.01 for OS, DSS, PFI, and DFI) and in KIRC, KIRP, LUSC, and OV (log-rank test, p < 0.05 for OS, DSS, PFI, and/or DFI) (Fig. 2E) . Furthermore, we found that ACE2 expression levels significantly We identified 217 and 26 genes having marked positive and negative expression correlations with ACE2 in pan-cancer, respectively (|r| > 0.5) (Fig. 3A and Supplementary Table S3 ). WGCNA [26] identified five gene modules (indicated in blue, green, yellow, brown, and turquoise color, respectively) highly enriched in the high-ACE2-expression-level tumors and three gene modules (indicated in pink, black, and red color, respectively) highly enriched in the low-ACE2-expression-level tumors (Fig. 3B) . The GO terms highly enriched in the high-ACE2-expression-level tumors mainly included brush border, cellular response to zinc ion, cell adhesion, immune response, and chemical homeostasis within a tissue. In contrast, the GO terms highly enriched in the low-ACE2-expression-level tumors mainly included microtubulebased process, cell cycle, and nucleic acid binding (Fig. 3B) . Again, these results indicate that ACE2 expression has a significant positive association with antitumor immune response and a significant negative association with the cell cycle in cancer, suggesting the potential protective role of ACE2 from cancer progression. From the brown gene module, we identified 82 hub genes mainly associated with immune-related pathways. Among the 82 hub genes, three transcription factor (TF) genes, including EOMES, IRF4, and TBX21, were co-expressed with many other immune-related genes, such as PDCD1, TIGIT, GZMK, IL21R, and PRF1 (Fig. 3C) . The association between these TFs and immune regulation has been well recognized, such as EOMES (Eomesodermin) mediating the CD8 + T cell differentiation [28] , IRF4 (interferon regulatory factor 4) regulating immune cell development [29] , and TBX21 (T-bet) playing a pivotal role in regulating Th1 cell development [30] . We investigated the association of ACE2 expression with immune signatures, oncogenic pathways, and tumor phenotypes in diverse cancer cohorts. Our results indicate that ACE2 is a potential protective factor for cancer progression. In particular, the ACE2 downregulation correlates with worse survival and tumor advancement in KIRC, also known as clear cell renal cell carcinoma (ccRCC). Previous studies demonstrated that ACE2 exerts antitumor effects by inhibiting tumor angiogenesis [13] and promoting tumor immune infiltration [9] . Our results are consistent with these previous findings. Besides, we found that ACE2 upregulation was associated with reduced cell proliferation, stemness, and EMT, as well as the downregulation of oncogenic pathways, such as cell cycle, mismatch repair, TGF-β, Wnt, and Notch signaling. Moreover, we found that ACE2 had a positive expression correlation with PD-L1, a predictive marker for an active response to immune checkpoint inhibitors. As a result, ACE2 upregulation correlates with a favorable response to anti-PD-1/PD-L1/CTLA-4 immunotherapy. Our and others' studies indicate that ACE2 plays a protective role in cancer, hypertension, heart disease, and COVID-19 patients. An intriguing phenomenon is that ACE2 as a SARS-CoV-2 human host cell receptor is crucial for SARS-CoV-2 to invade human cells, while its deficiency may exacerbate outcomes in COVID-19 patients [8] . A potential explanation is that the ACE2 deficiency could worsen outcomes in the people with underlying conditions, such as hypertension, heart disease, and cancer, who are infected with COVID-19. Thus, using ACE2 inhibitors for preventing and treating COVID-19 may not be an advisable strategy for individuals with certain underlying conditions, such as hypertension, heart disease, and cancers. It should be noted that the negative correlation between ACE2 and cancer progression presented in this study is an association but not a causation. To prove their causal relationship, further experiments are necessary. This would be an important direction for further studies. ACE2 upregulation was associated with increased antitumor immunity and immunotherapy response, reduced tumor malignancy, and favorable survival in cancer, suggesting that ACE2 is a potential protective factor for cancer progression. Our data may provide potential clinical implications for treating cancer patients infected with SARS-CoV-2. Not applicable. Not applicable. The authors declare that they have no competing interests. Table S1 . A summary of the datasets analyzed. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2 Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Angiotensin-converting enzyme II in the heart and the kidney Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract The pivotal link between ACE2 deficiency and SARS-CoV-2 infection ACE2 correlated with immune infiltration serves as a prognostic biomarker in endometrial carcinoma and renal papillary cell carcinoma: implication for COVID-19 A profiling analysis on the receptor ACE2 expression reveals the potential risk of different type of cancers vulnerable to SARS-CoV-2 infection Genetic alteration, RNA expression, and DNA methylation profiling of coronavirus disease 2019 (COVID-19) receptor ACE2 in malignancies: a pancancer analysis Elevated expression of ACE2 in tumor-adjacent normal tissues of cancer patients ACE2 inhibits breast cancer angiogenesis via suppressing the VEGFa/VEGFR2/ERK pathway The angiotensinconverting enzyme 2 in tumor growth and tumor-associated angiogenesis in non-small cell lung cancer The ACE2/Angiotensin-(1-7)/Mas Receptor Axis: Pleiotropic Roles in Cancer Overexpression of ACE2 produces antitumor effects via inhibition of angiogenesis and tumor cell invasion in vivo and in vitro Expression of the SAR2-Cov-2 receptor ACE2 reveals the susceptibility of COVID-19 in non-small cell lung cancer Bioinformatic Analysis of Correlation between Immune Infiltration and COVID-19 in Cancer Patients Somatic Mutations and Neoepitope Homology in Melanomas Treated with CTLA-4 Blockade Clinical cancer research : an official journal of the American Association for Cancer Research The Intratumoral Balance between Metabolic and Immunologic Gene Expression Is Associated with Anti-PD-1 Response in Patients with Renal Cell Carcinoma Contribution of systemic and somatic factors to clinical response and resistance to PD-L1 blockade in urothelial cancer: An exploratory multi-omic analysis GSVA: gene set variation analysis for microarray and RNA-seq data KEGG: new perspectives on genomes, pathways, diseases and drugs Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles WGCNA: an R package for weighted correlation network analysis Controlling the false discovery rate: a practical and powerful approach to multiple testing Characterization of T-bet and eomes in peripheral human immune cells Essential role of interferon regulatory factor 4 (IRF4) in immune cell development A novel transcription factor, T-bet, directs Th1 lineage commitment Not applicable.