A dynamic co-expression approach reveals Gins2 as a potential upstream modulator of HNSCC metastasis

Abstract

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer that is notably associated with a high risk of lymph node metastasis, a major cause of cancer mortality. Current therapeutic options remain limited to surgery supplemented by radio- or chemotherapy; however, these interventions often result in high-grade toxicities. Distant metastasis significantly contributed to the poor prognosis and decreased survival rates. However, the underlying molecular mechanisms remain poorly understood. Disease-related "omics" data provide a comprehensive overview of gene relationships, helping to decode the complex molecular mechanisms involved. Interactions between biological molecules are complex and highly dynamic across various cellular conditions, making traditional co-expression methods inadequate for understanding these intricate relationships. In the present study, a novel three-way interaction approach was employed to uncover dynamic co-expression relationships underlying the metastatic nature of HNSCC. Subsequently, the biologically relevant triples from statistically significant ones were defined through gene set enrichment analysis and reconstruction of the gene regulatory network. Finally, the validity of biologically relevant triplets was assessed at the protein level. The results highlighted the "PI3K/AKT/mTOR (PAM) signaling pathway" as a disrupted pathway involved in the metastatic nature of HNSCC. Notably, Gins2, identified as a switch gene, along with the gene pair {Akt2, Anxa2}, formed a statistically significant and biologically relevant triplet. It suggests that Gins2 could serve as a potential upstream modulator in the PAM signaling pathway, playing a crucial role in the distant metastasis of HNSCC. In addition, survival analysis of significant switch genes indicated that two genes, C19orf33 and Usp13, may be especially important for prognostic purposes in HNSCC.

Keywords: Head and neck squamous cell carcinoma; Liquid association method; Metastasis.

Fig. 1

Analysis of LA score and assessment of accuracy using the fastLA method. (A) FDR versus –log(p-value) for the first 200,000 fastLA results, with (x, y) = (6.3, 0.1) indicating the FDR threshold of 0.1 for selecting significant triplet combinations. (B) The accuracy of fastLA was assessed by comparing the observed event rate of Z position genes (red line) with a randomly generated event rate (blue line) across various p-values, indicating a higher number of significant events compared to random chance (blue line).

Fig. 2

Association between biologically-relevant triplets and enriched biological processes or pathways. This figure illustrates the association between significant triplet combinations identified using the fastLA method and their corresponding enriched biological processes or pathways. Each triplet is mapped to specific biological terms representing underlying biological functions or pathways, highlighting the relevance of these triplets in biological contexts.

Fig. 3

Gene Regulatory Network (GRN) reconstruction of biologically-relevant triplets. This figure illustrates the Gene Regulatory Network (GRN) reconstructed using the ARACNE algorithm to detect biologically-relevant triplets. The regulatory connections among significant triplets identified through the LA method are mapped within this network. The biological relevance of six statistically significant triplets was confirmed during both Gene Set Enrichment Analysis (GSEA) and GRN reconstruction. These confirmed triplets and their interactions are depicted as a sub-network in this figure, highlighting key regulatory relationships among the identified triplets.

Fig. 4

Protein expression levels of biologically-relevant triplets assessed through UALCAN portal. The validity of biologically-relevant triplets was assessed at the protein level using the UALCAN portal. Protein expression levels of genes involved in these triplets were compared between metastasizing (M) and non-metastasizing (NM) groups across four major metastatic pathways: PTK, NRF2, mTOR, and Hippo. Significant dysregulation was observed in two proteins, GINS2 and AKT2, from the Gins2/{Akt2, Anxa2} triplet within the mTOR pathway. Additionally, dysregulation was detected in two proteins, HOMER2 and KEAP1, from the Homer2/{Keap1 and Edn3} triplet in the Hippo pathway. The box plots show the Z-values of protein expression levels for each group.

Fig. 5

Dynamic co-expression pattern in Gins2/{Akt2, Anxa2} triplet that is biologically relevant. According to the concept of three-way interactions, the dynamic changes in co-expression patterns of genes X and Y in response to fluctuations in the expression of gene Z are of central importance in the hypothetical Z/{X, Y} triplet. Therefore, the co-expression pattern of Akt2 and Anxa2 genes has been illustrated in three bins based on the expression levels of the Gins2 gene. These bins categorize the expression levels of Gins2 into low, medium, and high groups. When the normalized expression level of Gins2 is low, as commonly observed in non-metastasizing (NM) samples, there is an inverse correlation between Akt2 and Anxa2 expression levels (r_low = − 0.48). Conversely, in the high normalized expression range of Gins2, typical of metastasizing (M) samples, a direct correlation exists between Akt2 and Anxa2 expression levels (r_high = 0.42). Furthermore, the correlation between Akt2 and Anxa2 expression levels in transit state, when the normalized expression level of Gins2 is moderate, is near zero (r_transit = 0.11). These results illustrate a dynamic co-expression relationship between Akt2 and Anxa2, modulated by changes in Gins2 expression levels. The expression profile changes of Akt2 and Anxa2 between metastatic and non-metastatic HNSCC samples have been reported in Fig. S3.

Fig. 6

Kaplan-Meier survival curves illustrating the association between gene expression levels and metastasis-free survival (MFS). The Kaplan-Meier survival curves show the relationship between the expression levels of specific genes and metastasis-free survival (MFS). Low expression levels of Dmtn, Camk2a, C19orf33, and A4galt are associated with shorter MFS. Conversely, high expression levels of Usp13, Dffa, and Fam181b correspond to shorter MFS. These curves demonstrate the impact of gene expression levels on the survival outcomes of patients, highlighting the prognostic significance of these genes.

Fig. 7

Fold change in expression levels of genes C19orf33 and Usp13. This figure presents the absolute fold change in the expression levels of genes C19orf33 and Usp13. Both genes exhibit significant changes, with an absolute fold change exceeding 1.5. These substantial changes in expression levels highlight the potential prognostic significance of C19orf33 and Usp13, suggesting their importance in the context of disease progression and patient outcomes.