Abstract 203: The role of FOXM1 in senescence suppression and radiation resistance in oral cavity squamous cell carcinoma
Authors: Shrivastava Nitisha , Wallach Yisrael , Li Daniel , Thomas Carlos , Prystowsky Michael B. , Basu Indranil , Guha Chandan , Ow Thomas J. 
Published: June 2022

Abstract
INTRODUCTION: Advanced oral cavity squamous cell carcinoma (OCSCC) relies on surgery and radiation for cure; with ~30% experiencing locoregional failure. Earlier studies suggest OCSCCs resist radiation response by suppressing senescence. CDKN2A, coding for p16INK4a (p16), a CDK4/6 inhibitory protein, is among the most commonly lost tumor suppressors in OCSCC. Forkhead Box M1 (FOXM1) is an understudied key regulatory protein in OCSCC whose activation, similar to Rb, is controlled by CDK4/6. FOXM1 activity not only impacts cell proliferation, but also promotes DNA damage repair and suppresses senescence. The current study examines how FOXM1 signaling plays a critical role in resistance to radiation in OCSCC. We demonstrate that CDK4/6 inhibition improves radiosensitivity in OCSCC via senescence as a result of FOXM1 suppression.
METHODS: Microarray data were analyzed to investigate FOXM1 levels in OSCC tumors. Basal FOXM1 levels in immortalized OCSCC cell lines (HN5 and Cal27) were evaluated using western blot. Cells were treated with palbociclib, radiation (RT), or combination (1µM palbociclib and 4 Gy (P+RT)) to examine the effects on senescence and FOXM1 signaling. β galactosidase colorimetric (β-gal) staining was used to evaluate senescence. Western blot was used to measure protein levels and QPCR to measure FOXM1 expression. To demonstrate senescence as a function of FOXM1 inhibition, knockdown assays (KD) were performed with siRNA targeting FOXM1. To further eliminate off target drug effects knockdown was also performed with siRNA targeting CDK4/6.
RESULTS: FOXM1 was upregulated ~2.2-fold in OCSCC tumors vs. matched normal mucosa samples (N=43, p=2.85008E-06). FOXM1 protein was upregulated 7-fold (HN5) and 5-fold (Cal27) compared to normal oral keratinocytes (HOK16B). β-gal staining revealed a dose dependent senescence induction in OCSCC, where P+RT vs. RT showed a robust 5-fold (HN5) and 3-folds (Cal27) increase. Interestingly, FOXM1 protein and transcript expression doubled in samples treated with RT. P+RT vs. RT demonstrated a decrease in FOXM1 protein levels in HN5 (10-fold) and Cal27 (3-fold), with corresponding decreases in mRNA levels. Knockdown of FOXM1 induced senescence, which further increased when combined with radiation: siFOXM1+4 Gy resulted in 60% (HN5) and 50% (CAL27) β-gal positive cells. Compared to untreated controls, CDK4/6 KD demonstrated a 2.5 and 10-fold decrease in FOXM1 protein levels in HN5 and CAL27, respectively. Furthermore, a concurrent inhibition of CDK4/6 with RT not only resulted in a decrease in FOXM1 expression but also resulted in a near-complete senescence in treated cells (95% β-Gal staining).
CONCLUSION: Suppressing FOXM1 directly or via CDK4/6 inhibition appears to make OCSCC more susceptible to RT via potentiating senescence. We propose inhibition of FOXM1 as a rational and potentially effective strategy to sensitize OCSCC to radiation.
Citation Format: Nitisha Shrivastava, Yisrael Wallach, Daniel Li, Carlos Thomas, Michael B. Prystowsky, Indranil Basu, Chandan Guha, Thomas J. Ow. The role of FOXM1 in senescence suppression and radiation resistance in oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 203.