Multi-gene anoikis signature combined with tumor microenvironment stratifies prognosis in cutaneous melanoma with experimental validation of PTK6

Abstract

Background: Cutaneous melanoma (CM) exhibits aggressive behavior driven by anoikis resistance and dysregulated tumor microenvironment (TME). The combined prognostic value of anoikis susceptibility and immune landscape remains uncharacterized.

Methods: Using transcriptomic data from 471 TCGA-CM patients, we developed an 11-gene anoikis risk score (RS) through LASSO-Cox regression, with external validation in the GSE65904 cohort (n = 214). This risk score was integrated with ESTIMATE-quantified immune/stromal features to construct the RS-TME classifier. Functional validation focused on Protein Tyrosine Kinase 6 (PTK6), a key prognostic driver, employing in vitro and in vivo models.

Results: We established an 11-gene RS with superior predictive capacity compared to conventional clinical indicators. Consensus clustering identified two molecular subtypes where Cluster B showed significantly improved survival compared to Cluster A (p < 0.001). Single-cell RNA sequencing confirmed immune cells, particularly macrophages, possessed substantially higher anoikis scores than malignant cells. This microenvironment foundation guided development of the RS-TME classifier, which stratified patients into three prognostic groups. Low-risk/high-TME patients exhibited significantly superior survival outcomes (p < 0.001), with external validation in the GSE65904 cohort (p = 0.002). Crucially, PTK6 knockdown suppressed tumor progression through increased apoptosis, reduced cell viability, impaired migration, and decreased tumor volume, mediated by Hippo pathway activation.

Conclusions: The RS-TME classifier is the first model integrating anoikis resistance and immune microenvironment to predict survival and immunotherapy response. Simultaneously, PTK6 targeting suppresses CM progression via Hippo-mediated anoikis resistance, providing a foundation for personalized treatment strategies.

Keywords: Anoikis; Cutaneous melanoma; PTK6; Prognostic biomarkers; Tumor microenvironment.