Molecular and clinicopathological insights into oral squamous cell carcinoma: Exploring oxidative stress, ferroptosis, circadian regulation, and novel biomarkers.

Oral squamous cell carcinoma (OSCC) accounts for over 90% of oral cancers and remains a significant global health burden due to its high recurrence rate, late-stage diagnosis, and limited prognostic markers. Oxidative stress, ferroptosis, and circadian rhythm dysregulation have emerged as critical mechanisms in cancer biology, yet their integrated roles in OSCC remain underexplored. Besides, the role of other emerging biomarkers such as the protein KLOTHO or the Insulin Receptor Substrate 4 (IRS4) remains to be deciphered in OSCC. In this study, we analyzed the immunohistochemical expression of biomarkers related to oxidative stress (NOX1, NOX2), ferroptosis (TFRC, ALOX5, ACSL4, GPX4), circadian regulation (CLOCK, Bmal1, PER1, PER2), KLOTHO and IRS-4 in a cohort of 30 OSCC patients. Expression patterns were correlated with key clinicopathological variables including tumor grade, recurrence, vascular and perineural invasion, and lifestyle factors such as smoking and alcohol consumption. Elevated expression of oxidative stress-, ferroptosis and proliferative-associated molecules (including NOX1, NOX2, ALOX5, ACSL4, GPX4, and IRS4) were significantly associated with adverse prognostic features such as tumor recurrence, vascular invasion, and higher histological grade, supporting their role in promoting tumor aggressiveness. On the other hand, higher expressions of circadian rhythm regulators (CLOCK, BMAL1, PER1, PER2) and the anti-aging protein KLOTHO were predominantly observed in patients with favorable clinical parameters, including good oral hygiene, absence of relapses, no drinking habits and lower-grade tumors. Notably, co-expression analyses uncovered divergence between ferroptosis and circadian pathways in advanced tumors, suggesting mechanistic antagonism and disrupted regulatory balance. These findings underscore the prognostic value of integrating oxidative and circadian biomarkers in OSCC and highlight their potential as targets for future personalized therapies. This study supports the clinical relevance of molecular stratification to guide treatment decisions and improve patient outcomes in OSCC.