BackgroundEsophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer and the seventh most prevalent cause of cancer-related death worldwide. Tumor microenvironment (TME) has been confirmed to play an crucial role in ESCC progression, prognosis, and the response to immunotherapy. There is a need for predictive biomarkers of TME-related processes to better prognosticate ESCC outcomes.AimTo identify a novel gene signature linked with the TME to predict the prognosis of ESCC.MethodsWe calculated the immune/stromal scores of 95 ESCC samples from The Cancer Genome Atlas (TCGA) using the ESTIMATE algorithm, and identified differentially expressed genes (DEGs) between high and low immune/stromal score patients. The key prognostic genes were further analyzed by the intersection of protein–protein interaction (PPI) networks and univariate Cox regression analysis. Finally, a risk score model was constructed using multivariate Cox regression analysis. We evaluated the associations between the risk score model and immune infiltration via the CIBERSORT algorithm. Moreover, we validated the signature using the Gene Expression Omnibus (GEO) database. Within the ten gene signature, five rarely reported genes were further validated with quantitative real time polymerase chain reaction (qRT-PCR) using an ESCC tissue cDNA microarray.ResultsA total of 133 up-regulated genes were identified as DEGs. Ten prognostic genes were selected based on intersection analysis of univariate COX regression analysis and PPI, and consisted of C1QA, C1QB, C1QC, CD86, C3AR1, CSF1R, ITGB2, LCP2, SPI1, and TYROBP (HR>1, p<0.05). The expression of 9 of these genes in the tumor samples were significantly higher compared to matched adjacent normal tissue based on the GEO database (p<0.05). Next, we assessed the ability of the ten-gene signature to predict the overall survival of ESCC patients, and found that the high-risk group had significantly poorer outcomes compared to the low-risk group using univariate and multivariate analyses in the TCGA and GEO cohorts (HR=2.104, 95% confidence interval:1.343-3.295, p=0.001; HR=1.6915, 95% confidence interval:1.053-2.717, p=0.0297). Additionally, receiver operating characteristic (ROC) curve analysis demonstrated a relatively sensitive and specific profile for the signature (1-, 2-, 3-year AUC=0.672, 0.854, 0.81). To identify the basis for these differences in the TME, we performed correlation analyses and found a significant positive correlation with M1 and M2 macrophages and CD8+ T cells, as well as a strong correlation to M2 macrophage surface markers. A nomogram based on the risk score and select clinicopathologic characteristics was constructed to predict overall survival of ESCC patients. For validation, qRT-PCR of an ESCC patient cDNA microarray was performed, and demonstrated that C1QA, C3AR1, LCP2, SPI1, and TYROBP were up-regulated in tumor samples and predict poor prognosis.ConclusionThis study established and validated a novel 10-gene signature linked with M2 macrophages and poor prognosis in ESCC patients. Importantly, we identified C1QA, C3AR1, LCP2, SPI1, and TYROBP as novel M2 macrophage-correlated survival biomarkers. These findings may identify potential targets for therapy in ESCC patients.

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Cancer stem cell (CSC) marker doublecortin-like kinase 1 (DCLK1) contributes greatly to the malignancy of gastrointestinal cancers, and DCLK1-targeted agents have potential therapeutic value. However, the molecular pathways regulated by DCLK1-S (DCLK1 isoform 4), a shortened splice variant of DCLK1, still remain obscure. Here we found that the expression of DCLK1-S is significantly increased in human esophageal squamous cell carcinoma (ESCC) tissues and associated with malignant progression and poor prognosis. Functional studies indicated that silencing total of DCLK1 mediated by CRISPR/Cas9 inhibited ESCC cell proliferation, migration, and invasion. Conversely, these changes were largely reversed after DCLK1-S rescue or overexpression. More importantly, DCLK1-S significantly enhanced primary tumor formation and metastatic lung colonization in vivo. The Cancer Genome Atlas database and molecular analysis showed that DCLK1-S was closely related to the epithelial–mesenchymal transition (EMT) process in patients with ESCC. Further RNA sequencing and Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that MAPK signaling pathway was significantly enriched. Our in vitro study proclaimed that DCLK1-S induced MMP2 expression in ESCC cells via MAPK/ERK signaling, leading to the activation of EMT. In addition, administration of ERK1/2 blocker SCH772984 attenuated the proliferative and migratory phenotype induced by DCLK1-S. In conclusion, these findings suggest that DCLK1-S may be a key molecule in MAPK/ERK/MMP2 pathway–mediated progression of ESCC, and that it has potential as a biomarker or therapeutic target to improve outcomes in patients with ESCC. Implications : DCLK1-S induces ESCC progression by activating the MAPK/ERK/MMP2 axis and may serve as a prognostic biomarker or therapeutic target for patients with ESCC.

<p>Table S1 shows the patients cohort in TCGA database.</p>

<p>Figure S2 shows DCLK1 protein expression in ESCC tissue and lymph node.</p>