CDK4/6 Inhibition Induces Senescence and Enhances Radiation Response by Disabling DNA Damage Repair in Oral Cavity Squamous Cell Carcinoma

Abstract

Purpose: HPV(-) OCSCC resists radiation treatment. The CDKN2A gene, encoding p16INK4A, is commonly disrupted in OCSCC. p16 inhibits CDK4/CDK6, leading to cell cycle arrest, but the biological sequelae of CDK4/6 inhibition in OCSCC remains understudied. This study examines whether inhibition of CDK4/6 enhances radiation response in OCSCC.

Methods: MTT assays were performed in OCSCC cell lines HN5 and CAL27 following treatment with palbociclib. Clonogenic survival and synergy were analyzed after radiation (RT-2 or 4Gy), palbociclib (P) (0.5 µM or 1 µM), or concurrent combination treatment (P+RT). DNA damage/repair and senescence were examined. CDK4/6 were targeted via siRNA to corroborate P+RT effects. Three-dimensional immortalized spheroids and organoids derived from patient tumors (conditionally reprogrammed OCSCC CR-06 and CR-18) were established to further examine and validate responses to P+RT.

Results: P+RT demonstrated reduced viability and synergy, increased β-gal expression (~95%), and ~two-fold higher γH2AX. Rad51 and Ku80 were reduced after P+RT, indicating impairment of both HR and NHEJ. siCDK4/6 increased senescence with radiation. Spheroids showed reduced proliferation and size with P+RT. CR-06 and CR-18 further demonstrated three-fold reduced proliferation and organoids size with P+RT.

Conclusion: Targeting CDK4/6 can lead to improved efficacy when combined with radiation in OCSCC by inducing senescence and inhibiting DNA damage repair.

Keywords: CDK4; CDK6; DNA repair; PD-0332991 (palbociclib); head and neck squamous cell carcinoma (HNSCC); oral cavity; organoids; radiosensitizing; senescence.

Figure 1

Palbociclib potentiates cell killing in HPV(−) HNSCC. HNSCC cells grown for 72 h in drug medium exhibit decreased viability in a dose dependent fashion in HN5 (A) and CAL27 (B). (C,D) Surviving fraction on day 10 in HN5 (C) and CAL27 (D) post 72 h palbociclib treatment. Synergy plot demonstrating dose response matrix for palbociclib/radiation treatment in HN5 (E) and CAL 27 (F) showed a synergistic response compared to either modality alone. Gy—Gray. Pal—palbociclib

Figure 2

Palbociclib induces G1 arrest and senescence in HPV(−) OCSCC when combined with radiation treatment. OCSCC cells grown for 72 h in drug medium exhibited cell cycle arrest in G1 phase in HN5 (A) and CAL27 (B). The 72 h treatment resulted in senescence with SA β-gal staining using western blot and colorimetric assay in HN5 (C,E) and CAL27 (D,G). The uncropped blots are shown in Supplementary Materials. Percent β-gal-positive cell quantification for HN5 (F) and CAL27 (H). Bright field images captured at 200×. Scale: 200 µm. A 72 h palbociclib/RT (1 µM + 4 Gy) demonstrated highest levels of senescence (β-gal-positive blue-stained cells) in both HN5 (E,F) and CAL27 (G,H). Analysis by two tailed t-test. * p < 0.05; ** p< 0.01; **** p < 0.0001. Each data point represents the average percent of three individual experiments. Gy—Gray; β-gal—β-galactosidase; Pal—palbociclib.

Figure 3

Palbociclib induces senescence in HPV (-) OCSCC and results in increased DNA damage and repression of key DNA damage repair proteins. HN5 and CAL27 western blots demonstrated increased SA β-gal expression after 72 h palbociclib treatment (A,B,F,G).The uncropped blots are shown in Supplementary Materials. Quantification of western blots for HN5 (B–E). Palbociclib/RT (4 Gy + 1 µM palbociclib) for 72 h in HN5 demonstrated increased γ-H2AX expression consistent with increased DNA damage induction (A,C); reduced expression of NHEJ signaling molecule Ku80 (A,D) and HR signaling molecule Rad51 (A,E). Quantification of western blots for CAL27 (G–J). Palbociclib/RT (4 Gy + 1 µM palbociclib) for 72 h in CAL27 demonstrated increased γ-H2AX expression consistent with increased DNA damage induction (F,H); reduced expression of NHEJ signaling molecule Ku80 (F,I) and HR signaling molecule Rad51 (F,J). Analysis by two-tailed t-test. * p < 0.05; ** p < 0.01. Each data point represents the average represented from three individual experiments. Pal—palbociclib; Gy—Gray; SA β-gal—senescence-associated β-galactosidase; Pal—palbociclib; HR—homologous recombination; NHEJ—non homologous end joining.

Figure 4

Palbociclib induces senescence in HPV(−) OCSCC, with levels recapitulated by combined knockdown of CDK4/6 and concurrent radiation treatment. OCSCC cell lines when exposed for 72 h to palbociclib exhibited senescence (SA β-gal staining) with palbociclib and radiation treatment in a dose-dependent manner. This senescence phenotype was similar to combined knockdown of both CDK4 and CDK6 together in HN5 (A) and CAL27 (B). These effects were not recapitulated with knockdown of either CDK4 or CDK6 alone. Radiation treatment at 2 or 4 Gy exhibited effects similar to respective controls. Bright field images captured at 200×. Scale: 200 µm. Gy-Gray.

Figure 5

Palbociclib combined with RT results in decreased proliferation and increased cell death among HPV(−) OCSCC spheroids. HNSCC cells grown in 3D culture for 72 h in drug medium and radiation exhibits reduced spheroid formation and decreased spheroid size and viability in a dose-dependent fashion in HN5 (A) and CAL27 (B). Scale = 100 µm. Spheroid size was measured on day 12 by measuring pixel area of 50–100 random spheroids in Image J for HN5 (C) and CAL27 (D). Cell proliferation was calculated on the basis of total number of cells on the day of termination (day 12) relative to cells seeded on day 0 in HN5 (E) and CAL27 (G). Cell viability was evaluated by trypan blue assay on day 12 in HN5 (F) and CAL27 (H). The combination of palbociclib and RT demonstrated profound effects in both spheroid models. Analysis by one-way ANOVA. * p < 0.05; *** p < 0.005; **** p < 0.001. Each data point mean represents the average represented from three individual experiments. RT—radiation treatment; Pal—palbociclib; Gy—Gray.

Figure 6

Palbociclib in combination with radiation reduces proliferation and potentiates cell killing in patient-derived HPV(−) OCSCC organoids. Patient-derived OCSCC CR cells grown in 3D culture for 72 h in the presence of palbociclib combined with radiation treatment exhibit reduced organoid formation, decreased organoid size, and decreased cell viability in a dose-dependent fashion in CR-06 (A) and CR-18 (B) Scale = 100 µm. Organoid size was measured on day 12 by measuring pixel area of 50–100 random spheroids in Image J in CR-06 (C) and CR-18 (D). Cell proliferation was calculated on the basis of total number of cells on the day of termination (day 12) relative to cells seeded on day 0 in CR-06 (E) and CR-18 (G). Cell viability was evaluated by trypan blue assay on day 12 in CR-06 (F) and CR-18 (H). Analysis by one-way ANOVA. * p < 0.05; *** p < 0.005; **** p < 0.001. Each data point mean represents the average represented from three individual experiments. Pal—palbociclib; Gy—Gray.