ALOX5AP Predicts Poor Prognosis by Enhancing M2 Macrophages Polarization and Immunosuppression in Serous Ovarian Cancer Microenvironment

Abstract

Background: Serous ovarian cancer (SOC) is a highly lethal gynecological malignancy with poor prognosis. Given the importance of the immune-related tumor microenvironment (TME) in ovarian cancer, investigating tumor-immune interactions and identifying novel prognostic and therapeutic targets in SOC is a promising avenue of research. ALOX5AP (Arachidonate 5-Lipoxygenase Activating Protein) is a key enzyme in converting arachidonic acid to leukotriene: a crucial immune-modulating lipid mediator. However, the role of ALOX5AP in SOC has yet to be studied.

Methods: ALOX5AP expression patterns across ovarian cancer and their normal tissue counterparts were cross-checked using public microarray and RNA-seq analyses and then validated in clinical samples by qRT-PCR. Kaplan-Meier survival analysis was performed in multiple independent SOC patient cohorts. Univariate and multivariate Cox regression analysis were then employed to identify clinical risk parameters associated with survival, and a genomic-clinicopathologic nomogram was built. Gene enrichment, immune infiltration, and immunosuppressor correlation analyses were then evaluated.

Results: ALOX5AP mRNA levels in SOC tissues were significantly upregulated compared to normal tissues. Elevated ALOX5AP was markedly associated with poor overall survival and progression-free survival in multiple SOC patient cohorts as well as with adverse clinicopathological features, including lymphatic invasion, unsatisfactory cytoreductive surgery, rapid relapse after primary treatment, and platinum non-responsiveness. A predictive nomogram, which integrated ALOX5AP expression and two independent prognosis factors (primary therapy outcome and tumor residual), was conducted to predict the 3-year and 5-year survival rate of SOC patients. Mechanistically, functional and pathway enrichment analyses revealed that ALOX5AP was primarily involved in immune response and regulation. Further exploration demonstrated that ALOX5AP was highly expressed in the immunoreactive subtype of ovarian cancer and closely related to immunocyte infiltration, especially M2 macrophage polarization. Additionally, ALOX5AP was enriched in the C4 (lymphocyte depleted) immune subtype of SOC and associated with crucial immune-repressive receptors in the tumor microenvironment at the genomic level.

Conclusions: ALOX5AP expression indicates a worse survival outcome and has the potential to be utilized as a prognostic predictor for SOC patients. Given the availability of well-studied ALOX5AP inhibitors, this study has immediate clinical implications for the exploitation of ALOX5AP as an immunotherapeutic target in SOC.

Keywords: ALOX5AP; immunosuppression; prognosis; serous ovarian cancer; targeted therapy.

Figure 1

ALOX5AP expression in normal and cancer tissues. Comparison of ALOX5AP expression in SOC and normal controls based on (A) RNA-Seq analysis using TCGA-OV samples and matched normal control samples from the GTEx project, (B) microarray analysis using meta GEO ovarian cancer datasets, and (C) qRT-PCR analysis using clinical SOC and normal control samples from Qilu Hospital of Shandong University. (D) ROC analysis of ALOX5AP expression for the discrimination between SOC and normal controls using TCGA-OV samples and matched normal control samples from the GTEx project. (E) Comparison of ALOX5AP mRNA levels across 33 TCGA cancer types and matched normal controls using GEPIA. (*p < 0.05).

Figure 2

Survival analysis comparing the high and low expression of ALOX5AP in different ovarian cancer cohorts. Survival curves of OS and PFS for (A) pooled ovarian cancer patient cohorts, (B) TCGA-OV cohort, (C) GSE9891 datasets (D) GSE14764 datasets, (E) GSE30161 datasets. and (F) stage 3-4 ovarian cancer patient cohorts. (OS, overall survival; PFS, progression free survival).

Figure 3

Association between ALOX5AP expression and clinicopathologic characteristics. Comparison of ALOX5AP expression in different clinical conditions including: (A) lymphatic invasion, (B) tumor residual, (C) primary therapy outcome and (D) platinum response. (E) Receiver operating characteristic (ROC) curve was drawn to analyze the predictive value of ALOX5AP for platinum-containing chemotherapy responsiveness. (F) The gene effect of ALOX5AP on cisplatin responsiveness of ovarian adenocarcinoma cell lines. Kaplan Meier curves of SOC patients receipt of platinum-based chemotherapy with high or low ALOX5AP expression in (G) pooled SOC patient cohorts, (H) TCGA-OV cohort, and (I) stage 3-4 SOC patient cohorts. (NRD, no residual disease; RD, residual disease; CR, complete response; PR, partial response).

Figure 4

Construction and performance validation of ALOX5AP based nomogram for SOC patients. (A) Nomogram for predicting overall survival for ovarian cancer patients. Each predictor is assigned a score on each axis. Compute the sum of points for all predictors and denote this value as the total points. (B, C) Calibration curve of the nomogram for predicting 3- and 5-year OS in the TCGA-OV cohort.

Figure 5

The gene enrichment analysis of ALOX5AP in SOC. (A) Volcano plot of the differentially expressed genes (DEGs) between ALOX5AP high and low groups in the TCGA-OV dataset. (B) Representative heatmap of top regulated DEGs after integrated analysis. The X-axis represents the samples, while the Y-axis denotes the differentially expressed genes. (C) The Bar graph demonstrating the top 20 clusters of enriched biological processes. (D) Visualization of the network of enriched terms colored by cluster using Metascape tool. (E) Enrichment plots from the gene set enrichment analysis (GSEA) in ALOX5AP high expressed samples. (F) Top 15 enriched signaling pathways by Gene set variation analysis (GSVA) comparison of DEGs between ALOX5AP high and low expression groups.

Figure 6

Relationship between ALOX5AP and immune infiltration. (A) ALOX5AP expression levels in different molecular subtypes of SOC. (B) ALOX5AP expression levels in different immune subtypes of SOC. (C) Lollipop plot shows the correlation between ALOX5AP expression and 24 immune cell subsets infiltration in SOC microenvironment. The size of dots indicates the absolute Spearman r value.

Figure 7

Correlation of ALOX5AP expression and M2 macrophages polarization. (A) Survival analysis of patients with high or low M2 macrophage infiltration in TCGA-OV cohort. (B) Survival time comparation in groups of different ALOX5AP expression and M2 macrophage infiltration. (C) Purity‐corrected Spearman’s correlation between ALOX5AP expression in ovarian cancer and two types of macrophages infiltration. The correlation between ALOX5AP and molecular biomarkers of (D) M2 macrophages and (E) M1 macrophages.

Figure 8

Association between Immunosuppressors and ALOX5AP expression in SOC. (A) Co-expression heatmap of ALOX5AP and immune inhibitory molecules across 33 TCGA tumor types. (B–G) Spearman correlation analysis of ALOX5AP and individual immune suppressive molecule in SOC. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).