The p16INK4a protein is a principal cyclin-dependent kinase inhibitor that decelerates the cell cycle. Abnormally high levels of p16INK4a are commonly observed in human papillomavirus (HPV)–positive head and neck squamous cell carcinomas (HNSCC). We and others found that p16INK4a overexpression is associated with improved therapy response and survival of patients with HNSCC treated with radiotherapy. However, the functional role of p16INK4a in HNSCC remains unexplored. Our results implicate p16INK4a in regulation of homologous recombination–mediated DNA damage response independently from its role in control of the cell cycle. We found that expression of p16INK4a dramatically affects radiation sensitivity of HNSCC cells. p16INK4a overexpression impairs the recruitment of RAD51 to the site of DNA damage in HPV-positive cells by downregulating of cyclin D1 protein expression. Consistent with the in vitro findings, immunostaining of HNSCC patient samples revealed that high levels p16INK4a expression significantly correlated with decreased cyclin D1 expression. In summary, these findings reveal an unexpected function of p16INK4a in homologous recombination–mediated DNA repair response and imply p16INK4a status as an independent marker to predict response of patients with HNSCC to radiotherapy. Cancer Res; 74(6); 1739–51. ©2014 AACR.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and responsible for approximately half a million cases every year (1). Tobacco and alcohol consumption are two environmental risk factors associated with the development of this group of cancers. Recent epidemiologic studies have also revealed that infection with high-risk human papillomavirus (HPV) is etiologically linked to HNSCC pathogenesis (2, 3). HPV-positive HNSCCs, which are predominantly found within the oropharyngeal regions with an occurrence of 25% to 47% (4), are now widely recognized as a distinct HNSCC entity due to demographic, histologic, clinical, and molecular differences (5).

The HPV oncogene products, E6 and E7, play a key role in HPV-associated carcinogenesis by inactivating of p53 and retinoblastoma tumor suppressor functions. The functional inhibition of retinoblastoma by HPV-E7 results in high expression levels of the cyclin-dependent kinase (CDK) inhibitor p16INK4a. The p16INK4a, encoded by the CDKN2A tumor suppressor gene, is one of the core components of the cell cycle that acts by disrupting the complex between cyclin D1 and CDK4/6. The cyclin D1/CDK4/6 complex phosphorylates the retinoblastoma protein that results in the release of the transcription factor E2F and the initiation of the cell-cycle progression. Conversely, the retinoblastoma/E2F repressor complex inhibits transcription of several genes, including CDKN2A (6, 7). By inactivating retinoblastoma, HPV-E7 releases the CDKN2A gene from its transcriptional inhibition that results in p16INK4a overexpression. p16INK4a overexpression has been recently proposed to serve as a surrogate marker of HPV infection in clinical samples because of a strong correlation between HPV and p16INK4a expression status in HNSCCs (8).

Despite the differences between HPV-positive and HPV-negative HNSCC tumors, currently all locally advanced patients with HNSCC are treated uniformly with radiotherapy and concurrent chemotherapy or surgery followed by (chemotherapy) radiotherapy (9, 10). Recent clinical trials have revealed that patients with HPV-infected tumors show markedly improved treatment response to conventional therapy and have a better prognosis compared with patients with HPV-negative HNSCC (3, 8). Although recent studies showed that HPV-positive HNSCC cells are more sensitive to ionizing radiation (11, 12), the molecular mechanism and the function of p16INK4a in the enhanced sensitivity to ionizing radiation of these tumors remains largely unknown.

Here, we assessed the impact of p16INK4a on DNA damage repair response in HNSCC. We found that p16INK4a hampers DNA damage response independently from its ability to inhibit CDK4/6 activity or control cell-cycle progression. Decreased expression of cyclin D1 in p16INK4a-overexpressing cells impaired the recruitment of RAD51 to damaged DNA and impeded the homologous recombination–mediated DNA repair. These findings may result in novel therapeutic applications for p16INK4a-overexpressing and HPV-positive tumors.

Immunohistochemistry and HPV detection

For immunostaining, routine formalin-fixed paraffin-embedded (FFPE) tumor tissues were available of 87 patients with oropharyngeal obtained before treatment with (chemotherapy) radiotherapy at the University Hospitals Leuven (Leuven, Belgium). The human tumor samples were acquired according protocols approved by the Ethic Board of the University Hospitals (Leuven, Belgium). Briefly, 5-μm FFPE tumor sections were deparaffinized with the DAKO PT link module and treated with primary antibodies against p16INK4a (G175-405; BD Pharmingen) or cyclin D1 (clone EP12; Dako). Immunodetection was performed with EnVision detection systems peroxidase/DAB (Dako), and sections were counterstained with hematoxylin.

DNA was extracted from FFPE tumor tissues using the QIAMP DNA FFPE Kit according to the manufacturer's protocol. The quality of extracted DNA to use for HPV PCR was checked by control amplification of 167 basepair fragment of the isocitrate dehydrogenase 2 (IDH2) gene using PCR. HPV positivity was determined by high-risk HPV–DNA-specific PCR reaction using GP5+/GP6+ primer set as described previously (13).

Cell lines and reagents

SCC154 cell line (received 2011) was purchased from the German collection of microorganisms and cell cultures (DSMZ), authentication was performed by DSMZ by short tandem repeat testing. Cells were used within 6 months of receipt after resuscitation per the DSMZ recommendation. SCC090 cell line (received 2010) was a generous gift of Dr. S. Collin, the University of Pittsburgh (Pittsburgh, PA). SCC154 and SCC090 cells were cultured and maintained in minimum essential media supplemented with 10% FBS, 1% l-glutamine, and 1% nonessential amino acids. SQD9, SC263, and CAL27 cell lines (received 2008) were a generous gift of Dr. A. Begg, the Netherlands Cancer Institute (Amsterdam, the Netherlands) and were cultured as previously described (14). No authentication was performed in our laboratory for these cells.

Lentiviral short hairpin RNAs (shRNA) against luciferase or p16INK4a (TRCN0000255853 and TRCN0000265833) were purchased from Sigma-Aldrich. Lentiviral infections were performed as previously described (15). Infected cells were selected by treatment with 4-μg puromycin (Sigma-Aldrich) for 4 days. siRNA-targeting p16INK4a was 5′AGAACCAGAGAGGCUCUGAtt-3′ (sip16-1) and 5′CGCACCGAAUAGUUACGGUtt-3′ (sip16-2; Applied Biosystems). Scrambled siRNA (Silencer Negative Control; Applied Biosystems) was used as control.

Cyclin D1 plasmid (pCMV–cyclin D1) was obtained from Addgene (plasmid 19927), and pBabe HPV-E7 was a generous gift from Dr. K. Munger, Harvard University (Cambridge, MA). pLA-p16INK4a was obtained by cloning INK4a in a lentiviral pLA vector. Transient transfections were performed using lipofectamine 2000 (Life technologies) according to the manufacturer's protocol.

DNA damage was induced by ionizing radiation using a linear accelerator (6 MV photons; Varian Medical Systems). PD-0332991 (Pfizer) was obtained from Selleckchem.

Colony formation and cell viability assay

Twenty-four hours upon drug (PD-0332991) treatment where after the drug is replaced by fresh full media or 72 hours after transfection with siRNAs, cells were exposed to increasing dose of ionizing radiation (2–10 Gy) and plated into 10-cm dishes. After 2 to 3 weeks cells were fixed with 2.5% glutaraldehyde in PBS and stained with 0.2% crystal violet. The colonies containing 50 cells or more were counted with ColCount colony counter (Oxford Optronix). Survival fractions were corrected for the plating efficiencies.

For cell viability assay cells were seeded with 20% confluence in 96-well plates and treated with drug or indicated doses of ionizing radiation and after 7 days a short-term survival assay (sulforhodamine B assay) was performed as previously described (14).

Nuclear extraction, immunoblotting, and coimmunoprecipitation

For nuclear extraction and immunoprecipitation experiments a radiation dose of 5 Gy was used as previously described (16). Whole-cell lysates were prepared in a lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitors (Roche). Nuclear extracts were prepared in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Roche). Protein concentrations were determined by Bradford assay (Bio-Rad) and 10 to 50 μg of proteins were subjected to SDS–PAGE. Immunoblotting was performed with antibodies recognizing β-actin (#4967; Cell Signaling Technology), RAD51 (clone 213; Abcam), cyclin D1 (clone A-12; Santa Cruz Biotechnology), p16INK4a (clone G175-405; BD Pharmingen), vinculin (clone hVIN-1; Sigma-Aldrich), and α-tubulin (clone 6-11B; Sigma-Aldrich).

The soluble nuclear fraction and insoluble chromatin fraction were extracted as previously described (17, 18). The chromatin fraction was sonicated before gel electrophoresis and immunoblotting.

For coimmunoprecipitations, cells were lysed with RIPA buffer. Two micrograms of rabbit anti-cyclin D1 antibody (clone H295; Santa Cruz Biotechnology) was incubated with 1.5 mg of cells lysate overnight at 4°C, and then protein A beads were added for 1 hour at 4°C. Immunoprecipitated proteins were detected by immunoblotting using antibodies specific to RAD51 (clone ab213; Abcam) and CDK4 (clone DCS156; Cell Signaling Technology).

Immunofluorescence and light microscopy

Immunofluorescence was performed as previously described (18). Briefly, the cells were plated on μClear 96-well plates (Greiner Bio-One) and fixed with 4% paraformaldehyde. After permeabilization with methanol, primary antibodies against RAD51 (clone ab213; Abcam or clone H-29; Santa Cruz Biotechnology) were followed by a secondary antibody conjugated to fluorescein isothiocyanate (Life Technologies). The DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). The immunofluorescence images were acquired using In Cell Analyzer 2000 (GE Healthcare).

For micronuclei analysis cells were irradiated with 6 Gy and fixed with 4% paraformaldehyde. Cells were stained with DAPI and analyzed by In Cell Analyzer 2000 (GE Healthcare). The frequencies of micronuclei were scored by counting the percentage of micronuclei in at least 1,000 cells per experiment.

For immunohistochemical analysis a light microscope (Olympus) with ×200 and ×400 magnification was used and pictures were taken by CellD software (Olympus).

Cell-cycle analysis

For cell-cycle analysis cells were irradiated with doses of 4 to 6 Gy as previously described (19), cells were fixed with 70% ethanol and stained with 10 μg/mL propidium iodide containing 100 μg/mL RNase A. Bromodeoxyuridine (BrdUrd) analysis was performed with the BD Pharmingen BrdUrd Flow Cytometry Kit and the Cell Proliferation ELISA BrdUrd Kit (Roche Applied Science) according to the manufacturer's instruction. Cell-cycle distribution was assessed by FACSCanto (Becton-Dickinson) and FlowJo (Treestar).

Statistical analysis

Survival rates were determined on the basis of Kaplan–Meier estimates using Statistica software version 11. The log-rank test was used to compare survival intervals. To examine association in categorical data the χ2 test and the nonparametric-rank test were used. For continuous variables, the unpaired Student t test or the nonparametric-rank test were used. All tests were considered statistically significant for P ≤ 0.05.

High levels of p16INK4a expression correlate with better therapy response in patients with HNSCC

Recent studies have demonstrated that HPV positivity in HNSCC is strongly associated with p16INK4a overexpression. To expand these observations, we performed a parallel analysis of HPV status and p16INK4a nuclear staining in a cohort of 87 patients with HNSCCs treated with radiotherapy or chemoradiotherapy at the University Hospitals of Leuven (Leuven, Belgium). We assessed p16INK4a expression by immunohistochemical analysis in 87 tumor biopsies obtained before treatment (Fig. 1A). All tumors were classified dichotomously as either p16INK4a-positive (strong, diffuse nuclear staining in more than 10% of HNSCC cells) or p16INK4a-negative (Fig. 1A). Consistent with previous reports (8, 20), 37% of the analyzed patients demonstrated high levels of nuclear p16INK4a immunostaining (Fig. 1B).

Figure 1.

High levels of p16INK4a expression correlates with better therapy response in patients with HNSCC. A, representative examples of p16INK4a immunostaining in HNSCCs. Pictures were taken with a magnification of ×200 and ×400. B, p16INK4a overexpression correlates with HPV positivity in HNSCC samples. p16INK4a and HPV statuses were assessed in the same tumor samples. C, immunoblot analysis of p16INK4a expression in the indicated cell lines using an anti-p16INK4a antibody. D, survival of patients with HNSCC with different status of p16INK4a as measured by Kaplan–Meier curves with locoregional tumor control as endpoint.

Figure 1.

High levels of p16INK4a expression correlates with better therapy response in patients with HNSCC. A, representative examples of p16INK4a immunostaining in HNSCCs. Pictures were taken with a magnification of ×200 and ×400. B, p16INK4a overexpression correlates with HPV positivity in HNSCC samples. p16INK4a and HPV statuses were assessed in the same tumor samples. C, immunoblot analysis of p16INK4a expression in the indicated cell lines using an anti-p16INK4a antibody. D, survival of patients with HNSCC with different status of p16INK4a as measured by Kaplan–Meier curves with locoregional tumor control as endpoint.

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In parallel, we determined HPV status by PCR detection of high-risk HPV–DNA using GP5+/GP6+ primers in all 87 patients of the Leuven cohort. However, we were not able to detect HPV status in 33 patients due to insufficient material or poor quality of the material (see Materials and Methods). About 25% of HNSCCs were found to be HPV-positive, which is the average HPV prevalence in Europe. We observed a significant association between HPV positivity and overexpression of p16INK4a (P value < 0.001; Yates χ2 test; Fig. 1B). The same tendency was observed for the available HNSCC cell lines. HPV-positive HNSCC cell lines, SCC154 and SCC090, had high levels of p16INK4a expression, whereas we were not able to detect p16INK4a expression in HPV-negative SQD9, SC263, and CAL27 cell lines (Fig. 1C).

A recent analysis of a cohort of patients treated solely with conventional radiotherapy in the Danish Head and Neck Cancer Group 5 trial revealed that high levels of p16INK4a expression are associated with better treatment response and survival rate of patients with HNSCC treated with conventional radiotherapy. The p16INK4a expression has been shown to be an even stronger independent prognostic factor than the classical clinical parameters of tumor stage and nodal status (20). Consistent with this report, analysis of the Leuven cohort confirmed that high levels of p16INK4a nuclear expression are associated with improved locoregional control of patients with HNSCC (Fig. 1D). Together, these observations indicate that p16INK4a overexpression has a major impact on treatment response and survival in patients with HNSCC treated with conventional radiotherapy.

p16INK4a sensitizes HPV-infected HNSCCs to ionizing radiation independent from its ability to regulate cell cycle

We next examined the effect of p16INK4a overexpression on cell sensitivity to ionizing radiation. A colony formation assay of irradiated cells normalized for plating efficiencies revealed that p16INK4a-positive SCC154 and SCC090 HNSCC cell lines are more sensitive to ionizing radiation compared with p16INK4a-negative SQD9 HNSCC cells (Fig. 2A and Supplementary Fig. S1A). A short-term cell survival assay further confirmed that cell lines with lower levels of p16INK4a expression were more resistant to ionizing radiation than cell lines with high levels of p16INK4a (Fig. 2B).

Figure 2.

p16INK4a sensitizes HPV-infected HNSCCs to ionizing radiation. A, colony formation assay of p16INK4a-positive SCC090 and SCC154 cells and p16INK4a-negative SQD9 cells after exposure to increasing doses of ionizing radiation. B, cell survival of the indicated cell lines 7 days after treatment with increasing doses of ionizing radiation determined by sulforhodamine B assay. C, colony formation assay of SCC154 cells expressing shRNAs targeting luciferase (shluc) or p16INK4a treated with increasing doses of ionizing radiation. Suppression of p16INK4a was confirmed by immunoblotting. Knockdown efficiency was assessed by densitometry in arbitrary units (below the immunoblots). D, cell survival of the indicated cell lines determined by sulforhodamine B assay 7 days after treatment with increasing doses of ionizing radiation. E, colony formation assay of SCC090 cells expressing siRNA for p16INK4a or scrambled siRNA (control) treated with increasing doses of ionizing radiation. Suppression of p16INK4a was confirmed by immunoblotting. Knockdown efficiency was assessed by densitometry in arbitrary units (below the immunoblots). For A, C, and E, survival is expressed as a percentage ± SEM of colonies formed relative to untreated cells over three experiments. Representative images (left) of the colony assays. For B and D, survival is expressed as ±SEM relative to untreated cells in three independent experiments.

Figure 2.

p16INK4a sensitizes HPV-infected HNSCCs to ionizing radiation. A, colony formation assay of p16INK4a-positive SCC090 and SCC154 cells and p16INK4a-negative SQD9 cells after exposure to increasing doses of ionizing radiation. B, cell survival of the indicated cell lines 7 days after treatment with increasing doses of ionizing radiation determined by sulforhodamine B assay. C, colony formation assay of SCC154 cells expressing shRNAs targeting luciferase (shluc) or p16INK4a treated with increasing doses of ionizing radiation. Suppression of p16INK4a was confirmed by immunoblotting. Knockdown efficiency was assessed by densitometry in arbitrary units (below the immunoblots). D, cell survival of the indicated cell lines determined by sulforhodamine B assay 7 days after treatment with increasing doses of ionizing radiation. E, colony formation assay of SCC090 cells expressing siRNA for p16INK4a or scrambled siRNA (control) treated with increasing doses of ionizing radiation. Suppression of p16INK4a was confirmed by immunoblotting. Knockdown efficiency was assessed by densitometry in arbitrary units (below the immunoblots). For A, C, and E, survival is expressed as a percentage ± SEM of colonies formed relative to untreated cells over three experiments. Representative images (left) of the colony assays. For B and D, survival is expressed as ±SEM relative to untreated cells in three independent experiments.

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To assess the contribution of p16INK4a to radiosensitivity, we suppressed p16INK4a expression in p16INK4a-positive SCC154 HNSCC cell line by lentiviral shRNA against p16INK4a. We found that p16INK4a knockdown did not alter the colony formation capacity of SCC154 cells (Supplementary Fig. S1B). In contrast, suppression of p16INK4a led to a significant increase in survival of the irradiated cells (Fig. 2C and D). We also observed an increased survival of HPV-positive SCC090 cells upon ionizing radiation when we suppressed p16INK4a in these cells by the introduction of siRNA-targeting p16INK4a (Fig. 2E and Supplementary Fig. S1C).

The presence of HPV, which inhibits retinoblastoma activity, in p16INK4a-overexpressing cells makes it unlikely that p16INK4a affects cell survival by affecting the cell-cycle progression upon DNA damage. Indeed, we found that suppression of p16INK4a did not alter the cell-cycle distribution of HPV-positive SCC154 cells either under untreated conditions or after ionizing radiation, confirming that p16INK4a does not affect the cell-cycle progression of HPV-positive cells upon DNA damage (Fig. 3A). In addition, we did not observe any difference in BrdUrd incorporation at different time points after ionizing radiation between cells that express shRNAs against shluc or shp16INK4a (Fig. 3B). These results suggest that p16INK4a affects radiation response independently from its ability to regulate cell cycle.

Figure 3.

p16INK4a affects radiosensitivity independent of its ability to regulate cell cycle. A, cell-cycle distribution of propidium iodide–stained SCC154 cells expressing shRNAs specific to luciferase (shluc) or p16INK4a after ionizing radiation with 6 Gy. B, percentage of BrdUrd-positive cells in SCC154 cells expressing shRNA for luciferase or p16INK4a after ionizing radiation with 6 Gy. C, cell-cycle distribution of propidium iodide–stained SQD9E7 cells expressing p16INK4a or empty vector (V) after ionizing radiation with 6 Gy. D, colony formation assay of SQD9E7 cells expressing p16INK4a or empty vector (V) with increasing doses of ionizing radiation. Overexpression of p16INK4a was confirmed by immunoblotting. Survival is expressed as a percentage ± SEM of colonies formed relative to untreated cells over three experiments. E, cell survival of the indicated cell lines determined by sulforhodamine B assay 7 days after ionizing radiation. Survival is expressed as ±SEM relative to untreated cells in three independent experiments. A–C, percentage of cells is presented as a mean ± SEM for three independent experiments.

Figure 3.

p16INK4a affects radiosensitivity independent of its ability to regulate cell cycle. A, cell-cycle distribution of propidium iodide–stained SCC154 cells expressing shRNAs specific to luciferase (shluc) or p16INK4a after ionizing radiation with 6 Gy. B, percentage of BrdUrd-positive cells in SCC154 cells expressing shRNA for luciferase or p16INK4a after ionizing radiation with 6 Gy. C, cell-cycle distribution of propidium iodide–stained SQD9E7 cells expressing p16INK4a or empty vector (V) after ionizing radiation with 6 Gy. D, colony formation assay of SQD9E7 cells expressing p16INK4a or empty vector (V) with increasing doses of ionizing radiation. Overexpression of p16INK4a was confirmed by immunoblotting. Survival is expressed as a percentage ± SEM of colonies formed relative to untreated cells over three experiments. E, cell survival of the indicated cell lines determined by sulforhodamine B assay 7 days after ionizing radiation. Survival is expressed as ±SEM relative to untreated cells in three independent experiments. A–C, percentage of cells is presented as a mean ± SEM for three independent experiments.

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To confirm these observations, we overexpressed p16INK4a in HPV-negative SQD9 cells. To exclude the effect of p16INK4a on DNA damage response due to its impact on cell-cycle progression, we inhibited the retinoblastoma pathway by introducing HPV-E7 to SQD9 cells. We confirmed that p16INK4a overexpression did not alter cell-cycle distribution of SQD9E7 cells after DNA damage (Fig. 3C). On the other hand, we found that overexpression of p16INK4a in SQD9E7 cells resulted in a decreased cell survival upon DNA damage (Fig. 3D and E and Supplementary Fig. S1D). Taken together, these results strongly indicate that p16INK4a overexpression contributes to radiosensitivity of HPV-positive HNSCCs. This also suggests a novel function of p16INK4a in control of DNA damage response that is independent from its ability to regulate cell-cycle progression.

p16INK4a affects radiosensitivity independent of its ability to inhibit CDK4/6 activity

Previous reports demonstrated that CDKs could modulate DNA damage response and affect cell survival upon DNA damage by phosphorylating DNA repair proteins (21–23). Therefore, we hypothesize that p16INK4a overexpression affects radiosensitivity due to direct inhibition of CDK4/6 activity. To test this idea, we examined whether a specific CDK4/6 inhibitor, PD-0332991 (24, 25), has the same effect on survival of HPV-positive cells upon ionizing radiation as we observed in case of p16INK4a overexpression.

Consistent with previous reports (24, 26), the presence of PD-0332991 did not affect cell-cycle distribution of HPV-positive SCC154 cells with inactivated retinoblastoma (Supplementary Fig. S2A). In contrast, we observed G1–S cell-cycle arrest and decreased proliferation of HPV-negative SQD9 cells after treatment with increasing doses of PD-0332991 (Supplementary Fig. S2A and S2B). Also, a slight increase in toxicity was seen with higher doses of PD-0332991 in HPV-negative SQD9 cells (Supplementary Fig. S2C). These findings confirm that the used doses of PD-0332991 efficiently inhibited CDK4/6 activity. Nonetheless, we did not find any differences in cell-cycle progression after ionizing radiation between cells expressing shRNAs against either luciferase (shluc) or p16INK4a (shp16) in the presence of PD-0332991 (Fig. 4A).

Figure 4.

p16INK4a affects radiosensitivity independent of its ability to inhibit CDK4/6 activity. A, cell-cycle distribution of PD-0332991-treated SCC154 cells expressing shluc or shp16INK4a upon ionizing radiation with 4 Gy. Cell-cycle distribution (right) is presented as a mean ± SEM for two independent experiments. B, colony formation of PD-0332991-treated SCC154 cells expressing shluc or shp16INK4a upon ionizing radiation with increasing doses of ionizing radiation. Cell survival is expressed as a percentage ± SEM of colonies formed by irradiated cells versus nonirradiated cells for three independent experiments. Representative images (below) of the colony assay. C, cell survival of PD-0332991-treated SCC154 cells expressing shluc or shp16INK4a 7 days after treatment with increasing doses of ionizing radiation as detected by sulforhodamine B assay. Cell survival is expressed as ±SEM relative to nonirradiated cells. A–C, cells were pretreated with 1 μmol/L of PD-0332991 for 24 hours before ionizing radiation, after which the drug was removed.

Figure 4.

p16INK4a affects radiosensitivity independent of its ability to inhibit CDK4/6 activity. A, cell-cycle distribution of PD-0332991-treated SCC154 cells expressing shluc or shp16INK4a upon ionizing radiation with 4 Gy. Cell-cycle distribution (right) is presented as a mean ± SEM for two independent experiments. B, colony formation of PD-0332991-treated SCC154 cells expressing shluc or shp16INK4a upon ionizing radiation with increasing doses of ionizing radiation. Cell survival is expressed as a percentage ± SEM of colonies formed by irradiated cells versus nonirradiated cells for three independent experiments. Representative images (below) of the colony assay. C, cell survival of PD-0332991-treated SCC154 cells expressing shluc or shp16INK4a 7 days after treatment with increasing doses of ionizing radiation as detected by sulforhodamine B assay. Cell survival is expressed as ±SEM relative to nonirradiated cells. A–C, cells were pretreated with 1 μmol/L of PD-0332991 for 24 hours before ionizing radiation, after which the drug was removed.

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Next, we performed a colony formation assay and a short-term cell survival analysis upon ionizing radiation of SCC154 cells pretreated with PD-0332991 for 24 hours. Inhibition of CDK4/6 activity with PD-0332991 did not affect radiosensitivity of p16INK4a knockdown cells (Fig. 4B and C and Supplementary Fig. S2D). Taken together, these results indicate that increased sensitivity of p16INK4a-overexpressing/HPV-positive cells to ionizing radiation is not due to the ability of p16INK4a to inhibit CDK4/6 activity.

p16INK4a inhibits recruitment of RAD51 to the sites of DNA damage via cyclin D1 downregulation

It is also well established that p16INK4a inhibits CDK4/6 activity by displacing cyclin D1 from cyclin D1–CDK4/6 complexes that, in turn, leads to cyclin D1 degradation (27). In fact, we found lower levels of cyclin D1 expression in p16INK4a-overexpressing SCC154 and SCC090 cells, whereas we observed higher levels of cyclin D1 in p16INK4a-negative SQD9, SC263, and CAL27 cells (Fig. 5A).

Figure 5.

p16INK4a negatively correlates with cyclin D1 protein expression. A, immunoblot analysis of cyclin D1 expression in the indicated cell lines using anti-cyclin D1 antibody. B, representative examples of cyclin D1 immunostaining in HNSCC patient samples. Images were taken with a magnification of ×200 and ×400. C, association between HPV status and cyclin D1 expression was assessed in the same tumor samples. D, association between p16INK4a overexpression and cyclin D1 expression was assessed in the same tumor samples by IHC using antibodies against cyclin D1 and p16INK4a. E, colony formation of SCC154 shluc cells transfected with an empty vector or cyclin D1 after exposure to increasing doses of ionizing radiation. Cell survival is expressed as a percentage ± SEM of colonies formed by irradiated versus nonirradiated cells. Representative images of the colony assay are shown below the graph. Expression of cyclin D1 was confirmed by immunoblotting. F, RAD51 foci formation 4 hours after 2 Gy of ionizing radiation in SCC154 shluc cells expressing empty vector or cyclin D1. Data, mean ± SEM percentage of cells containing more than 10 foci for three independent experiments.

Figure 5.

p16INK4a negatively correlates with cyclin D1 protein expression. A, immunoblot analysis of cyclin D1 expression in the indicated cell lines using anti-cyclin D1 antibody. B, representative examples of cyclin D1 immunostaining in HNSCC patient samples. Images were taken with a magnification of ×200 and ×400. C, association between HPV status and cyclin D1 expression was assessed in the same tumor samples. D, association between p16INK4a overexpression and cyclin D1 expression was assessed in the same tumor samples by IHC using antibodies against cyclin D1 and p16INK4a. E, colony formation of SCC154 shluc cells transfected with an empty vector or cyclin D1 after exposure to increasing doses of ionizing radiation. Cell survival is expressed as a percentage ± SEM of colonies formed by irradiated versus nonirradiated cells. Representative images of the colony assay are shown below the graph. Expression of cyclin D1 was confirmed by immunoblotting. F, RAD51 foci formation 4 hours after 2 Gy of ionizing radiation in SCC154 shluc cells expressing empty vector or cyclin D1. Data, mean ± SEM percentage of cells containing more than 10 foci for three independent experiments.

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We also tested whether there is a correlation between cyclin D1 and p16INK4a and between cyclin D1 and HPV status in HNSCC samples by performing immunohistochemistry (IHC) analysis of cyclin D1 expression in HNSCC biopsies from the Leuven cohort. All HNSCC tumors were classified dichotomously as either cyclin D1–positive (strong, diffuse staining in more than 25% of carcinoma cells) or cyclin D1–negative (Fig. 5B). We found that cyclin D1 overexpression correlates with HPV negativity and low p16INK4a expression (P values of ≤ 0.001; the Yates χ2 test; and the nonparametric spearman-rank test; Supplementary Fig. S3A), further confirming that HPV status and p16INK4a overexpression could affect the expression of cyclin D1 (Fig. 5C and D and Supplementary Fig. S3A). High levels of cyclin D1 in a number of p16INK4a-positive tumors (Fig. 5D) could be explained by cyclin D1 amplification, which is commonly observed in HNSCCs (28).

Previous studies revealed that elevated levels of cyclin D1 confer radiation resistance of cancer cells (29, 30) and high levels of cyclin D1 correlate with unfavorable response to radiotherapy (31, 32). Conversely, suppression of cyclin D1 sensitizes cancer cells to DNA damage (29, 33). Our results also demonstrated that the response to ionizing radiation of HNSCC cells strongly correlated with levels of cyclin D1 expression (Figs. 2A and B and 5A). Furthermore, when we overexpressed cyclin D1 in p16INK4a-positive SCC154 cells, it led to radiation resistance of these cells (Fig. 5E and Supplementary Fig. S3B). Given that p16INK4a overexpression leads to cyclin D1 downregulation, this suggests that p16INK4a could affect radiosensitivity by modulating cyclin D1 protein expression.

Recent reports have shown that cyclin D1 facilitates homologous recombination–mediated DNA repair through direct recruitment of RAD51 to the sites of DNA damage (34, 35). In fact, we found that overexpression of cyclin D1 in SCC154 cells enhanced radiation-induced RAD51 foci formation, a marker of homologous recombination competence (Fig. 5F).

We next examined how p16INK4a affects cyclin D1–RAD51 complex formation. We pulled down cyclin D1 from SCC154 or SCC090 cells expressing either shluc, or shp16. Consistent with our prior observations, we revealed higher expression levels of cyclin D1 in p16INK4a-depleted cells compared with shluc-expressing cells (Fig. 6A and B). Notably, suppression of p16INK4a led to accumulation of cyclin D1–RAD51 complexes (Fig. 6A and B). Inhibition of p16INK4a expression also led to the increased amount of RAD51 associated with chromatin, whereas p16INK4a depletion did not affect RAD51 protein levels in the soluble fraction (Fig. 6C and D). We also assessed the effect of p16INK4a overexpression in SQD9E7 cells on cyclin D1–Rad51 complex formation. In contrast with p16INK4a knockdown, overexpression of p16INK4a impaired the interaction between cyclin D1 and Rad51. A lower amount of RAD51 associated with chromatin was seen in cells overexpressing p16INK4a compared with cells expressing an empty vector (Fig. 6E and F). These results strongly indicate that p16INK4a suppresses RAD51 recruitment to the chromatin by downregulating cyclin D1 protein expression.

Figure 6.

p16INK4a inhibits recruitment of RAD51 to the sites of DNA damage by downregulating cyclin D1 protein expression. A and B, coimmunoprecipitation of cyclin D1 with RAD51 and CDK4 in SCC154 and SCC090 cells expressing shluc or shp16INK4a 4 hours after 5 Gy of ionizing radiation. Immunoblotting was performed using the indicated antibodies. C and D, association of RAD51 with chromatin in SCC154 and SCC090 cells expressing shluc or shp16INK4a 4 hours after ionizing radiation with 5 Gy. E, coimmunoprecipitation of cyclin D1 with RAD51 and CDK4 in SQD9E7 cells expressing p16INK4a or empty vector (V) 4 hours after 5 Gy of ionizing radiation. Immunoblotting was performed using the indicated antibodies. F, association of RAD51 with chromatin in SQD9E7 cells expressing shp16INK4a or empty vector (V) 4 hours after ionizing radiation with 5 Gy. For C, D, and F, soluble and chromatin fractions were isolated and immunoblotted with the indicated antibodies. A–F, the expression levels of indicated proteins were assessed by densitometry (below the immunoblots).

Figure 6.

p16INK4a inhibits recruitment of RAD51 to the sites of DNA damage by downregulating cyclin D1 protein expression. A and B, coimmunoprecipitation of cyclin D1 with RAD51 and CDK4 in SCC154 and SCC090 cells expressing shluc or shp16INK4a 4 hours after 5 Gy of ionizing radiation. Immunoblotting was performed using the indicated antibodies. C and D, association of RAD51 with chromatin in SCC154 and SCC090 cells expressing shluc or shp16INK4a 4 hours after ionizing radiation with 5 Gy. E, coimmunoprecipitation of cyclin D1 with RAD51 and CDK4 in SQD9E7 cells expressing p16INK4a or empty vector (V) 4 hours after 5 Gy of ionizing radiation. Immunoblotting was performed using the indicated antibodies. F, association of RAD51 with chromatin in SQD9E7 cells expressing shp16INK4a or empty vector (V) 4 hours after ionizing radiation with 5 Gy. For C, D, and F, soluble and chromatin fractions were isolated and immunoblotted with the indicated antibodies. A–F, the expression levels of indicated proteins were assessed by densitometry (below the immunoblots).

Close modal

p16INK4a impairs homologous recombination–mediated DNA repair

We next assessed whether p16INK4a affects the homologous recombination–mediated DNA repair pathway in HPV-positive cells by analyzing RAD51 foci formation. Consistent with the observation that p16INK4a suppression blocks RAD51 recruitment to the chromatin, we found decreased RAD51 foci formation in p16INK4a-positive SCC154 and SCC090 HNSCC cells compared with p16INK4a-negative SQD9 cells (Fig. 7A). On the other hand, depletion of p16INK4a resulted in a dramatic increase of RAD51 foci formation, indicating that p16INK4a contributes to control of the error-free homologous recombination–mediated DNA repair pathway (Fig. 7B).

Figure 7.

p16INK4a impairs homologous recombination–mediated DNA repair. A, RAD51 foci formation 4 hours after 2 Gy of ionizing radiation in the indicated cell lines. Data, mean ± SEM percentage of cells containing more than 10 foci in three experiments. Representative images of RAD51 immunostaining (supra). Scale bar, 100 μm. B, RAD51 foci formation 4 hours after 2 Gy of ionizing radiation in shluc and shp16INK4a-expressing cell lines. Data, mean ± SEM percentage of cells containing more than 10 foci in three experiments. Representative images of RAD51 immunostaining (supra). Scale bar, 100 μm. C, relative number of micronucleated cells in expressing shluc or shp16INK4a after 6 Gy of ionizing radiation. Graph, mean ± SEM of three independent experiments. Representative images of micronuclei detection (left). Arrows, micronucleated cells. Images were taken with a magnification of ×60.

Figure 7.

p16INK4a impairs homologous recombination–mediated DNA repair. A, RAD51 foci formation 4 hours after 2 Gy of ionizing radiation in the indicated cell lines. Data, mean ± SEM percentage of cells containing more than 10 foci in three experiments. Representative images of RAD51 immunostaining (supra). Scale bar, 100 μm. B, RAD51 foci formation 4 hours after 2 Gy of ionizing radiation in shluc and shp16INK4a-expressing cell lines. Data, mean ± SEM percentage of cells containing more than 10 foci in three experiments. Representative images of RAD51 immunostaining (supra). Scale bar, 100 μm. C, relative number of micronucleated cells in expressing shluc or shp16INK4a after 6 Gy of ionizing radiation. Graph, mean ± SEM of three independent experiments. Representative images of micronuclei detection (left). Arrows, micronucleated cells. Images were taken with a magnification of ×60.

Close modal

We hypothesized that a decrease in the homologous recombination–mediated DNA repair pathway could result in an increase in the error-prone nonhomologous end-joining pathway (NHEJ), which commonly leads to misrepair of double-strand breaks in DNA. Increased rate of NHEJ DNA repair results in formation of both dicentric chromosomes and acentric chromosome fragments, which could be detected by micronuclei analysis (36, 37). In concordance with our idea, a micronuclei analysis revealed increased frequency of micronucleated cells in p16INK4a-positive SCC154 cells upon ionizing radiation compared with p16INK4a-depleted cells (Fig. 7C). This suggests that p16INK4a overexpression in HPV-positive cells impairs the homologous recombination DNA repair but facilitates the NHEJ pathway, leading to increased ionizing radiation–mediated genomic instability and enhanced radiosensitivity of p16INK4a-positive cells.

The role of the tumor suppressor p16INK4a in the regulation of cell cycle and senescence has been studied extensively during last two decades (38, 39). On the other hand, cell-cycle–independent functions of p16INK4a are much less studied. Because the HPV oncogenic proteins, E6 and E7, disrupt cell-cycle regulation and lead to p16INK4a overexpression, HPV-infected tumors serve as an ideal platform to elucidate the involvement of p16INK4a in regulation of cellular processes other than control of cell cycle. Here, we found that p16INK4a is directly involved in control of DNA damage response independently from its ability to inhibit CDK4/6 activity and regulate cell-cycle progression. Conversely, p16INK4a expression impairs error-free homologous recombination–mediated DNA repair by downregulating cyclin D1 protein expression and thereby decreasing recruitment of RAD51 to the sites of DNA damage.

HPV status is well known to be an independent prognostic factor determining tumor control and survival after radiotherapy for head and neck cancer. Despite the prognostic value of HPV-positive HNSCCs, universal methods for stratifying patients with HPV status remain elusive (40) because direct methods of HPV detection are complex to implement in a clinical setup. Recent studies propose that p16INK4a expression could serve as a surrogate marker for HPV positivity (41, 42).

Our results strongly indicate that p16INK4a actively modulates the radiation response and has a broader role than its causal relationship with HPV infections. Consistent with our analysis of the Leuven cohort of patients with HNSCC, recent analyses of clinical data (8, 42) revealed that p16INK4a expression has a major impact on treatment response and survival in patients with HNSCC and have a stronger prognostic value than HPV status.

Importantly, the prognostic value of p16INK4a expression has been shown to depend on its cellular localization. Nuclear p16INK4a gave a better correlation with prognosis (42, 43). In our study, we classified p16INK4a tumors as positive only for samples with higher than 10% nuclear staining. This cutoff is quite strict and it could explain why some of HPV-positive tumors were considered as p16INK4a-negative. We also observed the high levels of p16INK4a expression in some of HPV-negative tumors. It could be due to other mechanisms of retinoblastoma pathway inactivation. Although retinoblastoma mutations are quite rare in HNSCC, deletions of the retinoblastoma-containing region are found in 15% to 50% of the tumors (44). Moreover, CCND1 has been shown to be amplified in about 30% of HNSCCs (31). Taken together, our data strongly imply p16INK4a is an independent marker for prediction of radiotherapy response in patients with HNSCC.

In addition, we confirmed previous reports demonstrating a direct involvement of cyclin D1 in DNA damage response (34, 35). There are conflicting studies on how cyclin D1 expression affects response to ionizing radiation in HNSCCs. Whereas some groups reported a negative correlation between high cyclin D1 expression and radiosensitivity (30), other groups observed that high cyclin D1 expression is associated with increased radiosensitivity in oral squamous cell cancer and early-stage of laryngeal cancer (45, 46). It is possible that the contradictory observations in these reports are due to the lack of p16INK4a and HPV detection. Moreover, the strong negative correlation between HPV status and cyclin D1 expression in the Leuven cohort and the interaction between p16INK4a, cyclin D1, and RAD51 in HPV-positive cells suggests that cyclin D1 has only a prognostic value in HPV-positive HNSCCs. Therefore, cyclin D1 IHC could be used in addition to p16INK4a as a biomarker for stratification of patients with HPV-positive HNSCC. Unfortunately, the relatively low number of patients with HPV-positive HNSCC did not allow us to make conclusions with certitude.

We believe that our data have important clinical implications by highlighting the importance of p16INK4a as a marker for stratification of patients with HNSCC. Current highly intensive chemo- and radiation treatment schedules lead to severe short- and long-term morbidity (47, 48). Clinical trials looking at treatment deintensification and organ preservative therapeutic procedures for HPV-positive tumors are ongoing. Our data suggest that p16INK4a status should be taken into account for further clinical trials of novel treatment therapies.

We also found that p16INK4a overexpression dramatically impairs the homologous recombination–DNA repair pathway, which suggests the potential use of PARP inhibitors, a promising class of anticancer agents particularly effective against homologous recombination–deficient tumors (49), for treatment of p16INK4a-positive HNSCCs. However, further in vitro and in vivo studies are required to confirm the utility of this approach for patients with HNSCC.

In summary, we found that p16INK4a hampers homologous recombination–mediated DNA repair machinery and sensitizes HPV-positive cells to radiation treatment, implicating p16INK4a expression as a potent marker for stratification of patients with HNSCC.

No potential conflicts of interest were disclosed.

Conception and design: R. Dok, P. Kalev, E.J. Van Limbergen, A. Sablina, S. Nuyts

Development of methodology: R. Dok, P. Kalev, S. Nuyts

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Dok, P. Kalev, E.J. Van Limbergen, L. Abbasi Asbagh, I. Vázquez, E. Hauben, S. Nuyts

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Dok, E.J. Van Limbergen, E. Hauben, S. Nuyts

Writing, review, and/or revision of the manuscript: R. Dok, I. Vázquez, E. Hauben, A. Sablina, S. Nuyts

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Dok, P. Kalev, L. Abbasi Asbagh

Study supervision: A. Sablina, S. Nuyts

This work was supported by grants from the Flemish League Against Cancer (VLK project and VLK van der Schueren grants) and FWO (grant number G-083313).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Jemal
A
,
Siegel
R
,
Ward
E
,
Hao
Y
,
Xu
J
,
Thun
MJ
. 
Cancer statistics, 2009
.
CA Cancer J Clin
2009
;
59
:
225
49
.
2.
Fakhry
C
,
Gillison
ML
. 
Clinical implications of human papillomavirus in head and neck cancers
.
J Clin Oncol
2006
;
24
:
2606
11
.
3.
Lassen
P
. 
The role of human papillomavirus in head and neck cancer and the impact on radiotherapy outcome
.
Radiother Oncol
2010
;
95
:
371
80
.
4.
Fakhry
C
,
Westra
WH
,
Li
S
,
Cmelak
A
,
Ridge
JA
,
Pinto
H
, et al
Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial
.
J Natl Cancer Inst
2008
;
100
:
261
9
.
5.
Marur
S
,
Forastiere
AA
. 
Head and neck cancer: changing epidemiology, diagnosis, and treatment
.
Mayo Clin Proc
2008
;
83
:
489
501
.
6.
Sellers
WR
,
Rodgers
JW
,
Kaelin
WG
 Jr
. 
A potent transrepression domain in the retinoblastoma protein induces a cell-cycle arrest when bound to E2F sites
.
Proc Natl Acad Sci U S A
1995
;
92
:
11544
8
.
7.
Bremner
R
,
Cohen
BL
,
Sopta
M
,
Hamel
PA
,
Ingles
CJ
,
Gallie
BL
, et al
Direct transcriptional repression by pRB and its reversal by specific cyclins
.
Mol Cell Biol
1995
;
15
:
3256
65
.
8.
Weinberger
PM
,
Yu
Z
,
Haffty
BG
,
Kowalski
D
,
Harigopal
M
,
Brandsma
J
, et al
Molecular classification identifies a subset of human papillomavirus–associated oropharyngeal cancers with favorable prognosis
.
J Clin Oncol
2006
;
24
:
736
47
.
9.
Dirix
P
,
Nuyts
S
. 
Evidence-based organ-sparing radiotherapy in head and neck cancer
.
Lancet Oncol
2010
;
11
:
85
91
.
10.
Nuyts
S
,
Dirix
P
,
Clement
PM
,
Poorten
VV
,
Delaere
P
,
Schoenaers
J
, et al
Impact of adding concomitant chemotherapy to hyperfractionated accelerated radiotherapy for advanced head-and-neck squamous cell carcinoma
.
Int J Radiat Oncol Biol Phys
2009
;
73
:
1088
95
.
11.
Rieckmann
T
,
Tribius
S
,
Grob
TJ
,
Meyer
F
,
Busch
CJ
,
Petersen
C
, et al
HNSCC cell lines positive for HPV and p16 possess higher cellular radiosensitivity due to an impaired DSB repair capacity
.
Radiother Oncol
2013
;
107
:
242
6
.
12.
Kimple
RJ
,
Smith
MA
,
Blitzer
GC
,
Torres
AD
,
Martin
JA
,
Yang
RZ
, et al
Enhanced radiation sensitivity in HPV-positive head and neck cancer
.
Cancer Res
2013
;
73
:
4791
800
.
13.
Smeets
SJ
,
Hesselink
AT
,
Speel
EJ
,
Haesevoets
A
,
Snijders
PJ
,
Pawlita
M
, et al
A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen
.
Int J Cancer
2007
;
121
:
2465
72
.
14.
De Schutter
H
,
Kimpe
M
,
Isebaert
S
,
Nuyts
S
. 
A systematic assessment of radiation dose enhancement by 5-Aza-2′-deoxycytidine and histone deacetylase inhibitors in head-and-neck squamous cell carcinoma
.
Int J Radiat Oncol Biol Phys
2009
;
73
:
904
12
.
15.
Moffat
J
,
Grueneberg
DA
,
Yang
X
,
Kim
SY
,
Kloepfer
AM
,
Hinkle
G
, et al
A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen
.
Cell
2006
;
124
:
1283
98
.
16.
Xu
B
,
Kim
ST
,
Lim
DS
,
Kastan
MB
. 
Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation
.
Mol Cell Biol
2002
;
22
:
1049
59
.
17.
Mladenov
E
,
Tsaneva
I
,
Anachkova
B
. 
Cell-cycle–dependent association of Rad51 with the nuclear matrix
.
DNA Cell Biol
2007
;
26
:
36
43
.
18.
Kalev
P
,
Simicek
M
,
Vazquez
I
,
Munck
S
,
Chen
L
,
Soin
T
, et al
Loss of PPP2R2A inhibits homologous recombination DNA repair and predicts tumor sensitivity to PARP inhibition
.
Cancer Res
2012
;
72
:
6414
24
.
19.
Seo
HR
,
Lee
DH
,
Lee
HJ
,
Baek
M
,
Bae
S
,
Soh
JW
, et al
Cyclin G1 overcomes radiation-induced G2 arrest and increases cell death through transcriptional activation of cyclin B1
.
Cell Death Differ
2006
;
13
:
1475
84
.
20.
Lassen
P
,
Eriksen
JG
,
Hamilton-Dutoit
S
,
Tramm
T
,
Alsner
J
,
Overgaard
J
. 
Effect of HPV-associated p16(INK4A) expression on response to radiotherapy and survival in squamous cell carcinoma of the head and neck
.
J Clin Oncol
2009
;
27
:
1992
8
.
21.
Cerqueira
A
,
Santamaria
D
,
Martinez-Pastor
B
,
Cuadrado
M
,
Fernandez-Capetillo
O
,
Barbacid
M
. 
Overall Cdk activity modulates the DNA damage response in mammalian cells
.
J Cell Biol
2009
;
187
:
773
80
.
22.
Johnson
N
,
Shapiro
GI
. 
Cyclin-dependent kinases (cdks) and the DNA damage response: rationale for cdk inhibitor-chemotherapy combinations as an anticancer strategy for solid tumors
.
Expert Opin Ther Targets
2010
;
14
:
1199
212
.
23.
Alvarez-Fernandez
M
,
Halim
VA
,
Krenning
L
,
Aprelia
M
,
Mohammed
S
,
Heck
AJ
, et al
Recovery from a DNA damage-induced G2 arrest requires Cdk-dependent activation of FoxM1
.
EMBO Rep
2010
;
11
:
452
8
.
24.
Fry
DW
,
Harvey
PJ
,
Keller
PR
,
Elliott
WL
,
Meade
M
,
Trachet
E
, et al
Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts
.
Mol Cancer Ther
2004
;
3
:
1427
38
.
25.
Toogood
PL
,
Harvey
PJ
,
Repine
JT
,
Sheehan
DJ
,
VanderWel
SN
,
Zhou
H
, et al
Discovery of a potent and selective inhibitor of cyclin-dependent kinase 4/6
.
J Med Chem
2005
;
48
:
2388
406
.
26.
Wiedemeyer
WR
,
Dunn
IF
,
Quayle
SN
,
Zhang
J
,
Chheda
MG
,
Dunn
GP
, et al
Pattern of retinoblastoma pathway inactivation dictates response to CDK4/6 inhibition in GBM
.
Proc Natl Acad Sci U S A
2010
;
107
:
11501
6
.
27.
Sherr
CJ
,
Roberts
JM
. 
CDK inhibitors: positive and negative regulators of G1-phase progression
.
Genes Dev
1999
;
13
:
1501
12
.
28.
Kaminagakura
E
,
Werneck da Cunha
I
,
Soares
FA
,
Nishimoto
IN
,
Kowalski
LP
. 
CCND1 amplification and protein overexpression in oral squamous cell carcinoma of young patients
.
Head Neck
2011
;
33
:
1413
9
.
29.
Shimura
T
,
Kakuda
S
,
Ochiai
Y
,
Nakagawa
H
,
Kuwahara
Y
,
Takai
Y
, et al
Acquired radioresistance of human tumor cells by DNA-PK/AKT/GSK3 beta-mediated cyclin D1 overexpression
.
Oncogene
2010
;
29
:
4826
37
.
30.
Milas
L
,
Akimoto
T
,
Hunter
NR
,
Mason
KA
,
Buchmiller
L
,
Yamakawa
M
, et al
Relationship between cyclin D1 expression and poor radioresponse of murine carcinomas
.
Int J Radiat Oncol Biol Phys
2002
;
52
:
514
21
.
31.
Chang
AR
,
Wu
HG
,
Park
CI
,
Jeon
YK
,
Kim
CW
. 
Expression of epidermal growth factor receptor and cyclin D1 in pretreatment biopsies as a predictive factor of radiotherapy efficacy in early glottic cancer
.
Head Neck
2008
;
30
:
852
7
.
32.
Lai
JP
,
Tong
CL
,
Hong
C
,
Xiao
JY
,
Tao
ZD
,
Zhang
Z
, et al
Association between high initial tissue levels of cyclin D1 and recurrence of nasopharyngeal carcinoma
.
Laryngoscope
2002
;
112
:
402
8
.
33.
Wang
MB
,
Yip
HT
,
Srivatsan
ES
. 
Antisense cyclin D1 enhances sensitivity of head and neck cancer cells to cisplatin
.
Laryngoscope
2001
;
111
:
982
8
.
34.
Jirawatnotai
S
,
Hu
Y
,
Michowski
W
,
Elias
JE
,
Becks
L
,
Bienvenu
F
, et al
A function for cyclin D1 in DNA repair uncovered by protein interactome analyses in human cancers
.
Nature
2011
;
474
:
230
4
.
35.
Li
Z
,
Jiao
X
,
Wang
C
,
Shirley
LA
,
Elsaleh
H
,
Dahl
O
, et al
Alternative cyclin D1 splice forms differentially regulate the DNA damage response
.
Cancer Res
2010
;
70
:
8802
11
.
36.
Oliveira
NG
,
Castro
M
,
Rodrigues
AS
,
Goncalves
IC
,
Gil
OM
,
Fernandes
AP
, et al
Wortmannin enhances the induction of micronuclei by low and high LET radiation
.
Mutagenesis
2003
;
18
:
37
44
.
37.
Fenech
M
. 
Micronuclei and their association with sperm abnormalities, infertility, pregnancy loss, preeclampsia and intrauterine growth restriction in humans
.
Mutagenesis
2011
;
26
:
63
7
.
38.
Burd
CE
,
Sorrentino
JA
,
Clark
KS
,
Darr
DB
,
Krishnamurthy
J
,
Deal
AM
, et al
Monitoring tumorigenesis and senescence in vivo with a p16(INK4a)-luciferase model
.
Cell
2013
;
152
:
340
51
.
39.
Anders
L
,
Ke
N
,
Hydbring
P
,
Choi
YJ
,
Widlund
HR
,
Chick
JM
, et al
A systematic screen for CDK4/6 substrates links FOXM1 phosphorylation to senescence suppression in cancer cells
.
Cancer Cell
2011
;
20
:
620
34
.
40.
Lewis
JS
 Jr
. 
p16 Immunohistochemistry as a standalone test for risk stratification in oropharyngeal squamous cell carcinoma
.
Head Neck Pathol
2012
;
6
:
S75
82
.
41.
Lewis
JS
,
Thorstad
WL
,
Chernock
RD
,
Haughey
BH
,
Yip
JH
,
Zhang
Q
, et al
p16-positive oropharyngeal squamous cell carcinoma: an entity with a favorable prognosis regardless of tumor HPV status
.
Am J Surg Pathol
2010
;
34
:
1088
96
.
42.
Zhao
N
,
Ang
MK
,
Yin
XY
,
Patel
MR
,
Fritchie
K
,
Thorne
L
, et al
Different cellular p16(INK4a) localisation may signal different survival outcomes in head and neck cancer
.
Br J Cancer
2012
;
107
:
482
90
.
43.
Hoffmann
M
,
Tribius
S
,
Quabius
ES
,
Henry
H
,
Pfannenschmidt
S
,
Burkhardt
C
, et al
HPV DNA, E6*I-mRNA expression and p16INK4A immunohistochemistry in head and neck cancer—how valid is p16INK4A as surrogate marker?
Cancer Lett
2012
;
323
:
88
96
.
44.
Burkhart
DL
,
Sage
J
. 
Cellular mechanisms of tumour suppression by the retinoblastoma gene
.
Nat Rev Cancer
2008
;
8
:
671
82
.
45.
Shintani
S
,
Mihara
M
,
Ueyama
Y
,
Matsumura
T
,
Wong
DT
. 
Cyclin D1 overexpression associates with radiosensitivity in oral squamous cell carcinoma
.
Int J Cancer
2001
;
96
:
159
65
.
46.
Yoo
SS
,
Carter
D
,
Turner
BC
,
Sasaki
CT
,
Son
YH
,
Wilson
LD
, et al
Prognostic significance of cyclin D1 protein levels in early-stage larynx cancer treated with primary radiation
.
Int J Cancer
2000
;
90
:
22
8
.
47.
Machtay
M
,
Moughan
J
,
Trotti
A
,
Garden
AS
,
Weber
RS
,
Cooper
JS
, et al
Factors associated with severe late toxicity after concurrent chemoradiation for locally advanced head and neck cancer: an RTOG analysis
.
J Clin Oncol
2008
;
26
:
3582
9
.
48.
Ang
KK
,
Harris
J
,
Wheeler
R
,
Weber
R
,
Rosenthal
DI
,
Nguyen-Tan
PF
, et al
Human papillomavirus and survival of patients with oropharyngeal cancer
.
N Engl J Med
2010
;
363
:
24
35
.
49.
Rouleau
M
,
Patel
A
,
Hendzel
MJ
,
Kaufmann
SH
,
Poirier
GG
. 
PARP inhibition: PARP1 and beyond
.
Nat Rev Cancer
2010
;
10
:
293
301
.