Both hypoxia and immune status play important roles in OSCC [23–25]. However, there is a lack of reliable and validated hypoxia- and immune-related gene signatures. Based on hypoxia-related signatures, the samples in our study were grouped into 3 clusters (Cluster 1, Cluster 2, and Cluster 3) (Fig. 1A-C). The presence of cell hypoxia induces the expression of HIF-1α. Enhanced HIF-1α expression has previously been associated with poor prognosis and lymph nodes metastasis in OSCC patients . Further evaluation of overall patient survival resulted in the combination of Clusters 2 and 3 to form the Hypoxia_High group, while those in Cluster 1 were designated as the Hypoxia_Low group (Fig. 1D). The expression of genes from two hypoxia-related gene sets, “Increased oxygen delivery” and “Reduced oxygen consumption”, were compared between Cluster 1 and Cluster 2 & 3. 11 of the 14 genes belonging to the “Increased oxygen delivery” were expressed at higher levels in Clusters 2 and 3 in contrast to Cluster 1 (Fig. 1E). On the other hand, 7 of the 12 genes associated with “Reduced oxygen consumption” were highly expressed in Cluster 1 compared to Clusters 2 and 3 (Fig. 1F). Therefore, Cluster 1 and Clusters 2 & 3 were considered as Hypoxia_Low and Hypoxia_High groups, respectively. A total of 773 DEGs were identified across both these groups (Fig. 1E and F).
We then divided these OSCC patients into three groups (Immune_High, Immune_Medium and Immune_Low) according to the abundance of immune cells (Fig. 2A). The immune score of Immune_High group was higher than that of the Immune_Medium and Immune_Low groups. The Immune_Low group has the lowest immune score amongst the three groups (Fig. 2B & C). A total of 854 immune-related genes in OSCC were determined between Immune_High and Immune_Low groups (Fig. 2E & F). All genes were found to be enriched in immune-related functions including “leukocyte activation involved immune response”, “adaptive immune response”, “lymphocyte activation” and “Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell” (Fig. 2F). These immune processes also related to the malignant properties of OSCC. Previous reports found that a higher neutrophil-to-lymphocyte ratio was a negative predictor for overall survival for patients with OSCC . Dendritic cell immune response activation was able to be induced by IFN-γ inhibited OSCC growth in tumour-bearing mice .
To further integrate the hypoxia- and immune-related genes, the overlapping genes between the two gene sets were screened. Eight prominent mRNA signatures associated with OS from the 193 overlapping genes were selected, which were FAM122C, RNF157, RANBP17, SOWAHA, KIAA1211, RIPPLY2, INSL3, and DNAH1 (Fig. 3A-D). Among these mRNAs, KIAA1211 is known to be an oncogenic gene. Non-small cell lung cancer tissues were found to have raised KIAA1211 expressions in contrast to adjacent normal tissues. Knockdown of KIAA1211 inhibited the proliferative abilities of NSCLC cells while promoting apoptosis both in vitro and in vivo . Small cell lung cancer patients with a KIAA1211 mutation possesses longer survival period than those with wild-type KIAA1211 mutations . RIPPLY2 represented one of the mRNAs in a five-gene signature verified to be able to predict survival of endometrial cancer patients . The tumor-promoting effect of INSL3 in cancer has also been widely addressed. The plasma level of INSL3 was found to be raised in an individual with metastatic ovarian cancer . Other reports highlight the potential role of INSL3 as a marker of human testicular Leydig cell tumors . INSL3 could promote tumor growth and angiogenesis in nude mice model of thyroid cancer in a manner which appeared to be related to the action of RXFP2 and the secretion of S100A4 and (pro-) cathepsin-L . In pancreatic cancer patients, higher serum level of INSL3 was associated with increased anorexia . These mRNAs, which have previously been found to be of significant value in other cancers, should be further investigated for their role in OSCC.
These 8 mRNAs were used to construct a mRNA signature which may have prognostic potential in OSCC (Fig. 3E-H). This constructed risk score was then validated in a cohort from the GEO dataset (Fig. 4A-F). The risk score showed a significant association only with age, perineural and lymphovascular invasion, but not with other features including gender, T stage, N stage, and tumour stage (Fig. 5A-H). Therefore, this established gene signature may be an independent prognostic indicator.
Finally, the immune profile variability between the high- and low-risk groups were compared in the TCGA OSCC cohort. High-risk group samples showed different degree of immune cell infiltration (Fig. 6A). The low-risk group showed higher immune status than low-risk group, as evidenced by a higher amount of immune checkpoint expression including CTLA-4, PD-1, APM, CYT, and TILs (Fig. 6B-G). These checkpoints are indictors of OSCC risk and may function as therapeutic targets in OSCC. The genetic variants of CTLA-4 were associated with tobacco-related OSCC risk in the North Indian population . A high number of CTLA-4⁺ cells is associated with poor 5-year metastasis-free survival of OSCC patients . Anti-PD-1 antibody is an agent which may prevent the initiation and progression of OSCC while prolonging patient survival time [38–40]. Therefore, patients in low-risk group may be more sensitive to immunotherapy.
In conclusion, hypoxia statues and immune statues both play pivotal roles in the prognostic of OSCC. Combined with hypoxia- and immune-related genes, we established a hypoxia-immune-based gene signature for prognosis of OSCC, which may serve as a reliable reference for clinical decision making.