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Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer, with an annual incidence of ~600,000 new cases globally (1,2). Despite advances in surgery and radiotherapy, as well as the incorporation of chemotherapy into treatment modalities, the 5-year survival remains ~50% and has barely improved over the past decades (3). The majority of HNSCC cases are tobacco- and alcohol-associated, and in oropharyngeal cancer, infection with high-risk human papillomavirus (HPV) is an oncogenic factor (4). HPV-positive HNSCC is biologically and clinically distinct from HPV-negative HNSCC (5). Notably, most patients with a HPV-positive tumor have a more favorable prognosis, with a 30-40% higher 5-year survival rate than patients with a HPV-negative tumor (6). However, the HPV status of the tumor is not predictive for individual patient outcome. A subgroup of patients with HPV-positive tumors has a worse prognosis, characterized by a higher risk of disease recurrence and/or secondary primary tumors. Although the underlying reason for this less favorable prognosis is not completely understood, exposure to additional risk factors, such as tobacco smoking, may play a role (7). Treatment with radiation or surgery alone is typically indicated for early-stage disease, whereas combined approaches, including surgery, radiotherapy and chemotherapy is generally applied for locoregionally advanced disease (8,9). For inoperable, locally advanced HNSCC, a combination treatment with radiotherapy with cisplatin remains the standard of care. For patients that are unfit for cisplatin, treatment with cetuximab, a monoclonal antibody directed against epidermal growth factor receptor, could be an alternative to chemotherapy. Novel immunotherapies, including programmed cell death protein-1 checkpoint inhibitors such as nivolumab and pembrolizumab, could be considered as treatment option for patients with recurrent or metastatic HNSCC, irrespective of HPV-status (9). These immunotherapies have shown durable responses, but this benefit was only observed in a limited number of patients (9). Furthermore, the currently used treatment modalities often result in severe side effects and a reduction in quality of life, which is particularly important for patients with an unfavorable prognosis. Therefore, new agents that can improve survival rates of patients with HPV-negative and HPV-positive HNSCC, without causing severe side effects, are urgently needed.

Recent whole-exome sequencing studies have revealed a wide spectrum of genetic aberrations and molecular diversity in HNSCC (5,10). Frequently deregulated cellular pathways include the cell cycle and the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which regulate cell proliferation, survival and apoptosis (11,12). Genetic alterations associated with deregulation of the cell cycle machinery are detected in nearly all cases of HNSCC (13). Retinoblastoma (Rb1) tumor suppressor protein plays a critical role in regulating cellular proliferation. Cyclin D-cyclin-dependent kinase 4/6 (CDK4/6) may phosphorylate and inactivate Rb1, leading to the release and activation of E2F transcription factors necessary for G1-S phase cell cycle progression (14). In HPV-positive HNSCC, viral oncoprotein E7 drives unrestrained proliferation by promoting Rb1 degradation, which also leads to p16 upregulation (14). In HPV-negative HNSCC, Rb1 inactivation occurs through hyperactivation of the Rb1 inhibitory complex CDK4/6-Cyclin D. The CCND1 gene (encoding Cyclin D1, the regulatory subunit of the complex) is amplified, and/or the CDK4/6 inhibitor, p16, is inactivated in nearly all of these cancer types, preventing the phosphorylation of Rb1 (15-17).

The Cancer Genome Atlas (TCGA) data demonstrates that >50% of HPV-positive and HPV-negative HNSCC cases harbor activated PI3K (and related pathways) signaling, mainly due to mutations in or amplifications of PIK3CA, loss of PTEN or activation of receptor tyrosine kinases (13). PI3K activation leads to synthesis of phosphatidylinositol 3,4,5-trisphosphate (PIP3) at the plasma membrane, resulting in the recruitment of pleckstrin homology domain-containing proteins phosphoinositide dependent protein kinase-1 (PDK1) and Akt. Akt is phosphorylated by PDK1 at Thr308, resulting in activation of downstream proteins, including mammalian target of rapamycin (mTOR) complex 1. The activation of the PI3K pathway is associated with resistance to chemotherapy and other targeted therapies and plays a crucial role in cell energy metabolism (18). Therefore, inhibition of the PI3K pathway may be an important step and one of the most promising targets in anticancer therapy, including for HNSCC (19,20).

There are several CDK4/6 inhibitors (CDKi) and PI3K/Akt/mTOR pathway inhibitors (PI3Ki) available. Palbociclib (PD-0332991; Pfizer) is a selective CDKi that was first approved for the treatment of breast cancer (21). Ribociclib (LEE011; Novartis) is a selective orally bioavailable CDKi that received Food and Drug Administration (FDA) approval in March, 2017 (22). Both drugs bind to the ATP cleft of CDK4 and CDK6. Alpelisib (BYL719; Novartis) is an oral selective PI3K p110α isoform inhibitor (23,24) that has been approved by the FDA in combination with fulvestrant for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated metastatic breast cancer. This drug inhibits wild-type PI3K p110α and mutated PI3K p110α (as a result of PIK3CA mutations). Buparlisib (BKM120; Novartis) is a 2,6-dimorpholino pyrimidine derivative that significantly inhibits wild-type and mutant PI3K catalytic subunit p110 (α, β, δ and γ) (25). Gedatolisib (PF-05212384; Pfizer) is a highly potent dual inhibitor of PI3K (α, β, δ and γ) and mTOR (TORC1 and TORC2). In vitro, gedatolisib potently inhibits class I PI3Ks, PI3K-α mutants and mTOR (26).

The aim of the present study was to investigate the in vitro antiproliferative effects of several CDKi (palbociclib and ribociclib) and PI3Ki (gedatolisib, buparlisib and alpelisib) in HPV-positive and -negative HNSCC cell lines. We hypothesized that CDKi are effective inhibitors in HPV-negative HNSCC cell lines, associated with hyperactivation of the Rb1 inhibitory complex, CDK4/6-Cyclin D. In addition, PI3Ki are expected to be effective in both HPV-negative and -positive HNSCC cell lines related to active PI3K/Akt/mTOR signaling.

Materials and methods

Cell lines and culture conditions

In total, five HPV16-positive HNSCC cell lines: UD-SCC-2 (kindly provided by Thomas Hoffmann, University of Ulm, Germany), 93-VU-147T (kindly provided by Johan P. De Winter, VU Medical Center, Amsterdam, The Netherlands), UM-SCC-47 and UM-SCC-104 (both kindly provided by Thomas E. Carey, University of Michigan, USA) and UPCI-SCC-090 (kindly provided by Susanne M. Gollin, University of Pittsburgh, USA) were used in the present study. In addition, three HPV16-negative HNSCC cell lines: UPCI-SCC-72 and UPCI-SCC-003 (both kindly provided by Susanne M. Gollin) and UT-SCC-33 (kindly provided by R.A. Grenman, Turku University, Finland) were used. The MCF-7, HeLa, CaSki and SiHa cell lines were purchased from the American Type Culture Collection and the HaCaT cell line was purchased from CLS Cell Line Service GmbH. The normal oral keratinocyte (NOK) cell line was prepared from gingival tissues obtained from oral surgeries and immortalized by activation of h-TERT, as described previously (27-29). The NOK cell line was immortalized and kindly provided by Karl Munger, Tufts University Medical School, USA.

Cells were cultured at 37°C in a humidified atmosphere with 5% CO2. All HNSCC cell lines (except for the UT-SCC-33), HaCaT, HeLa and MCF-7 were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal calf serum (FCS; Bodinco BV). UT-SCC-33 and SiHa cells were cultured in MEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FCS. CasKi was cultured in Roswell Park Memorial Institute (RPMI) with 10% FCS. The NOK cell line was cultured in keratinocyte serum-free medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with bovine pituitary extract (2.6 μg/ml) and recombinant EGF (0.16 ng/ml). The clinicopathological cell line characteristics, including genetic alterations, are presented in Table SI. To confirm p16/HPV status and determine mutational status of pathways that may be relevant for inhibitor efficacy, immunocytochemical staining for p16 and mutation analysis were performed as previously described (6,30). All cell lines were regularly tested and found to be mycoplasma-free. All cell lines were confirmed to have unique genotypes, as determined using the ProfilerPlus assay (31). The presence of HPV DNA was detected using PCR as previously described (32,33). Stocks of palbocliclib and gedatolisib were provided by Pfizer, Inc. and stocks of alpelisib, buparlisib, and ribociclib were provided by Novartis International AG.

In vitro cell viability assay

The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT] to purple formazan crystals in metabolically active cells by mitochondrial dehydrogenases, predominantly succinate dehydrogenase (34,35). In this present study, cells were seeded in 96-well flat-bottom plates at densities that allowed for exponential growth throughout the experiment. The cells were placed in the cell culture incubator overnight at 37°C allowing the cells to attach, after which they were treated with different concentrations of the different test compounds (Table SII). The compounds were resolved in DMSO at a non-toxic (0.1%; 0.2% for palbociclib) concentration of DMSO at the cellular level. At the indicated time points (PI3Ki: day 3, CDKi: day 3 and 5), the MTT assay (Sigma-Aldrich; Merck KGaA) was performed as previously described (34). Purple formazan crystals were dissolved in ethanol/DMSO solution (1:1) and the absorbance was measured at a wavelength of 595 nm with a spectrophotometer. The experiments were performed in triplicate.

Western blot analysis

Cells treated with the compounds or control were lysed with RIPA buffer (Cell Signaling Technology, Inc.) containing Protease/Phosphatase Inhibitor Cocktail for 5 min on ice, followed by brief sonication (30 sec, 47 kHz, 4°C). After centrifugation (10 min, 14,000 × g, 4°C), the pellet was discarded, and the protein extracts were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Equal amounts of the extracts (10-30 μg) were separated on 8-12% SDS-PAGE gels and transferred to nitrocellulose membranes according to the manufacturers' instructions using a Mini-Protean Tetra System (Bio-Rad Laboratories, Inc.). Membranes were blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature and incubated with primary antibodies diluted in blocking buffer (5% BSA diluted in TBS/Tween 0.1%) overnight at 4°C. For detection, secondary antibodies labeled with horseradish peroxidase were incubated with the membranes for 1 h at room temperature. The bands were visualized with enhanced chemiluminescence (SuperSignal West Dura Extended Duration Substrate; Thermo Fisher Scientific, Inc.) using an Image reader LAS-3000 (FUJIFILM Wako Pure Chemical Corporation). The primary and secondary antibodies, including the dilutions used, are listed in Table SIII. The experiments were performed in triplicate.

Cell cycle analysis

Cells were seeded in 6-well culture plates, placed in the cell culture incubator at 37°C and allowed to attach overnight. The culture medium containing the inhibitor or DMSO was added to the cells. After 24 h, the cells were washed with PBS and trypsinized to form a cell pellet. Ice-cold 70% ethanol was added to the cell pellet while vortexing, ensuring cell fixation and minimizing cell clumping. Cells in 70% ethanol were stored at −20°C for a minimum of 30 min. The cells were then washed with PBS and resuspended in 0.5 ml propidium iodide (PI)/RNAse staining solution (100 μg/ml PI and 1 mg/ml RNAse in PBS). The cells were incubated for 30 min at room temperature and analyzed by flow cytometry using a FACSCanto II (BD Biosciences). Data analysis was performed using FACSdiva software version 6.1.2 (BD Biosciences). The different cell cycle regions were set to those defined by the untreated control cells for each individual cell line.

Annexin-V apoptosis assay

For the Annexin V assay, cells were seeded in 96-well imaging microplates and allowed to attach overnight at 37°C. Cells were treated with 500 nM staurosporine and two different concentrations of PI3Ki for 24 h. The cells were then stained with Hoechst 33342 (200 μg/ml; Sigma-Aldrich; Merck KGaA) in culture medium for 15 min at 37°C. Cells were washed with Annexin-V binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 5 mM CaCl2 made up in PBS) and stained with Annexin-V-FITC (2.5 μg/ml in Annexin-V binding buffer) for 15 min at 37°C. Imaging was performed using a BDpathway 855 High-Content Bioimager (BD Biosciences). Digitalization and segmentation of the acquired data were performed using Attovision software Version 1.6 (BD Biosciences). The processed data were evaluated using FACSDiva software version 6.1.2 (BD Biosciences).

β-galactosidase staining

Cells were seeded in 24-well plates and allowed to attach overnight. The cells were treated with palbociclib (2 μM), alpelisib (10 μM) or a combination of the two for 6 days. Expression of β-galactosidase was determined using the Senescence Detection Kit (Abcam; cat. no. ab65351), following the manufacturer's instructions. Briefly, cells were washed with PBS and fixed, followed by staining with the provided X-gal solution. The plate was covered and incubated in a ziplock bag at 37°C overnight. The stained cells were observed under a light microscope (magnification, ×200).

Measurement of glucose uptake and lactate release

Cells were seeded in 96-well plates and treated with PI3Ki. After 24 h, the cell culture medium was collected and pooled to a volume of 300 μl per condition. The glucose and lactate concentrations were determined using a D-Glucose Enzymatic Assay Kit and an L-Lactate Acid Enzymatic Assay Kit from BioSenTec, by measuring absorbance at 340 nm. The reaction volumes were optimized for use in 96-well plates, and a standard curve was used to determine the glucose and lactate concentrations. To measure the lactate content, the cell culture medium was deproteinized prior to the assay using 10 kDa filter units (Merck KGaA; MRCPRT010). The determined glucose and lactate concentrations were normalized to μg of protein, measured using the Pierce BSA protein assay kit (Thermo Fisher Scientific, Inc.).

Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

OCR and ECAR measurements were performed using an XF96 Extracellular Flux analyzer and Mito Stress Test and Glyco Stress test assays (all Seahorse Bioscience; Agilent Technologies, Inc.), according to the manufacturer's instructions. Briefly, the cells were seeded at optimized densities (7,300 for UM-SCC-47, 7,500 for UPCI-SCC-003 and 38,000 for UD-SCC-2) in 80 μl of growth medium in an XF96 culture plate well. After attachment overnight, the cells were treated with vehicle control or the IC50 of the PI3Ki for 24 h, washed in XF assay medium and kept in XF assay medium (with inhibitors) at 37°C in a non-CO2 incubator for 1 h prior to the assay. Mitochondrial respiration was determined using the Mito Stress Test after the subsequent injection of oligomycin (1 μM), FCCP (1 μM) and a mixture of rotenone and antimycin A (both 1 μM) (all from Sigma-Aldrich; Merck KGaA) to determine the basal and ATP-coupled respiration. Baseline respiration was determined by subtracting non-mitochondrial respiration (OCR values obtained after injection of antimycin A/rotenone) from the initial OCR. ATP-linked respiration is the OCR decrease after the injection of oligomycin. Injection of FCCP collapses the mitochondrial membrane potential and results in maximal OCR. Finally, the injection of rotenone/antimycin A (inhibitors of complex III and I, respectively) block the mitochondrial respiratory chain and strongly inhibit respiration. A Glycolysis Stress Test was performed to measure changes in ECAR following the addition of glucose (10 mM), oligomycin (1 μM) and 2-deoxyglucose (2-DG; 0.1 M) to determine glycolysis and glycolytic capacity. Glycolysis was measured after adding saturating glucose. Oligomycin inhibits mitochondrial ATP production, thereby pushing cells to use glycolysis maximally. Glycolytic capacity was calculated using the following equation: maximum rate measurement after oligomycin injection-last rate measurement before glucose injection, expressed as mpH/min. Finally, 2-DG, a glucose analog that inhibits glycolysis through competitive binding to hexokinase, was added. The decrease in ECAR after 2-DG injection confirmed that glycolysis was the cause of the increase in ECAR during the experiment. Both OCR and ECAR were corrected for total protein content using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.). Experiments were performed in triplicate with at least 7 technical replicates per condition.

Analysis of inhibitor synergism

Cell viability after treatment with (combinations of) inhibitors was determined using the MTT assay as described previously. The interaction between alpelisib and ribociclib was evaluated by comparing the observed response to the combination of inhibitors to the expected response using the SynergyFinder V3 calculator (https://synergyfinder.org/). The expected response was based on the highest single-agent (HSA) reference model, which states that the expected combination effect is the maximum of the single-drug responses at corresponding concentrations (36,37). The most synergistic area (MSA) was determined by the most synergistic 3-by-3 dose window in the dose response matrix. HSA synergy scores can be interpreted as the average excess response due to drug interactions. A synergy score >10 is considered a synergistic interaction.

Statistical analysis

GraphPad Prism software (version 8; Dotmatics) was used to perform all statistical analyses. All results are presented as the mean ± standard error of the mean. All experiments were performed in triplicate, and statistical analysis was performed using unpaired Student's t-tests and one-way ANOVA with a Dunnett's post hoc tests. P<0.05 was considered to indicate a statistically significant difference.

Results

CDK4/6 inhibition suppresses the viability of HPV-negative HNSCC cells

To identify differences in the response to CDK4/6 inhibition between cell lines, three HPV-negative and five HPV-positive cell lines were treated with ribociclib and palbociclib. In addition, immortalized human keratinocyte (HaCaT) and NOK cell lines were included in the analysis. The MCF-7 breast cancer cell line was used as a positive control as these cells are dependent on the CDK pathway for proliferation (38). All cell lines were cultured for 3 days with increasing CDKi concentrations. Since no reduction of 50% in cell viability was achieved, cell lines were treated for 5 days with increasing concentrations of CDKi. The IC50 values are listed in Table I. For comparison, cell lines were also treated with increasing concentrations of cisplatin, which is currently the standard treatment along with radiotherapy for patients with HNSCC. The positive control MCF-7 cell line showed an effective response to both CDKi in a dose-dependent manner. All HPV-negative HNSCC cell lines showed decreased cell viability following incubation with the CDKi, which was not observed in the HPV-positive HNSCC cell lines. The immortalized HaCaT and NOK cell lines were also sensitive to both CDKi. In comparison with cisplatin, CDKi had comparable IC50 values in HPV-negative HNSCC cells.

Table I IC50 values for CDKi (5 day-treatment), PI3Ki (3 day-treatment) and cisplatin 3 (day-treatment) in all cell lines.

Table I

IC50 values for CDKi (5 day-treatment), PI3Ki (3 day-treatment) and cisplatin 3 (day-treatment) in all cell lines.

A, HPV-negative HNSCC
Cell line	IC50 CDKi, μM		IC50 PI3Ki, μM			IC50 cisplatin, μM
	Ribociclib	Palbociclib	Alpelisib	Buparlisib	Gedatolisib
UPCI-SCC-72	6.5	2.1	4.7	0.6	0.005	22.9
UPCI-SCC-003	4.7	0.5	4.1	0.6	0.011	5.9
UT-SCC-33	>10a	2	>10a	1.8	0.032	5.1
B, HPV-positive HNSCC
Cell line	IC50 CDKi, μM		IC50 PI3Ki, μM			IC50 cisplatin, μM
	Ribociclib	Palbociclib	Alpelisib	Buparlisib	Gedatolisib
93-VU-147T	NA	NA	3.3	0.4	0.009	3.7
UM-SCC-104	NA	NA	5.8	0.3	0.029	17.3
UM-SCC-47	7.1	NA	8.2	0.3	0.0057	2.8
UD-SCC-2	NA	NA	>10a	1.1	0.021	11.8
UPCI-SCC-090	NA	NA	>10a	1.0	0.014	5.3
C, HPV-positive UCC
Cell line	IC50 CDKi, μM		IC50 PI3Ki, μM			IC50 cisplatin, μM
	Ribociclib	Palbociclib	Alpelisib	Buparlisib	Gedatolisib
CaSki	NA	NA	>10a	0.5	0.01	6.2
HeLa	NA	NA	9.1	0.2	0.014	1.1
SiHa	NA	NA	>10a	2.0	0.02	5.5
D, Controls
Cell line	IC50 CDKi, μM			IC50 PI3Ki, μM		IC50 cisplatin, μM
	Ribociclib	Palbociclib	Alpelisib	Buparlisib	Gedatolisib
HaCaT	4	0.3	4.7	0.3	0.0037	3.8
NOK	1.4	0.1	2.4	1.2	0.0061	0.8
MCF-7	0.7	0.6	-	-	-	-

a 50% cell viability not reached at maximum concentration. CDKi, cyclin-dependent kinase 4/6 inhibitor; PI3Ki, phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway inhibitor; HNSCC, head and neck squamous cell carcinoma; UCC, uterine cervical carcinoma; na, cannot be analyzed by GraphPad (no decrease in cell viability at maximum concentration); NOK, normal oral keratinocyte; HPV, human papillomavirus.

CDK4/6 inhibition downregulates the levels of cell cycle-relevant proteins

The cell lines MCF-7, UPCI-SCC-003 (HPV negative) and UM-SCC-47 (HPV positive) were used to investigate changes in protein expression involved in the CDK pathway induced by CDKi treatment. Upregulation of Cyclin D1 and downregulation of Rb1 and phosphorylated (p-)Rb1 were observed in the MCF-7 and UPCI-SCC-003 cell lines upon CDKi administration, which was in agreement with earlier studies (39) (Fig. 1). There was greater expression of CDK4 in UPCI-SCC-003 cells than in MCF-7 cells, but under CDKi treatment, no notable changes were observed. In the HPV-positive UM-SCC-47 cell line, the baseline expression of CDK4, cyclin D1 and p-Rb1 was lower than that in the other two cell lines, most likely as a result of the activity of viral oncoprotein E7 (31), and no marked changes were observed under CDKi treatment.

Figure 1 Assessment of the expression levels of proteins involved in the CDK-pathway by western blot analysis of whole protein extracts. The cells were treated for 1, 3 and 6 days with the IC50 value of palbociclib and ribociclib (see Table I) or control (DMSO, 1:1,000). β-actin was used as loading control. (A) Breast cancer cell line MCF-7, (B) HPV-negative HNSCC cell line UPCI-SCC-003 and (C) HPV-positive HNSCC cell line UM-SCC-47 were assessed. The experiments were performed in triplicate. CDK, cyclin-dependent kinase; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; p-, phosphorylation; Rb, retinoblastoma protein.

CDK4/6 inhibition results in G1-phase arrest without apoptosis

A known consequence of CDK4/6 inhibition is cell cycle phase arrest. For this purpose, the cell cycle distribution was analyzed by flow cytometry after a 24-h treatment with palbociclib and ribociclib (Figs. 2A and S1). In HPV-negative UPCI-SCC-003 cells, treatment with CDKi resulted in an increase in the proportion of cells in the G1 phase and a decrease in cells in the S- and G2/M phases. In addition, increased levels of the senescence marker β-galactosidase were observed in this cell line after treatment with palbociclib (Fig. S2). In HPV-positive UM-SCC-47 cells, there was no difference in the distribution between untreated and treated cells. These results underscore the western blotting results. To assess whether cells undergo apoptosis after treatment with CDKi, an Annexin-V assay was performed. In UPCI-SCC-003 and UM-SCC-47 cells (Figs. 2B, C and S3), there was no significant increase in the number of apoptotic cells after treatment.

Figure 2 Effect of cyclin-dependent kinase 4/6 inhibition on cell cycle distribution and the induction of apoptosis. (A) Cells were treated with DMSO (1:1,000), 1 μM palbociclib or 1 μM ribociclib for 1 day and cell cycle analysis was performed. (B) Cells were either treated for 1 day with 500 nM Staurosporine, a known inducer of apoptosis or with 1 μM palbociclib and 1 μM ribociclib for 1 day, followed by analysis of Annexin V staining. Representative images are shown for HPV-positive UM-SCC-47 cells. (C) Quantification of the apoptotic HPV-negative UPCI-SCC-003 and HPV-positive UM-SCC-47 cells. Each bar represents the mean ± SEM of the data obtained from three independent experiments. *P<0.05 vs. DMSO cells. HPV, human papillomavirus.

PI3Ki suppress the viability of HPV-positive and -negative HNSCC cell lines

To determine the efficacy of the PI3K pathway inhibitors, all cell lines were cultured for 3 days at increasing concentrations. The IC50 values of the cell lines are presented in Table I. All three inhibitors significantly decreased the viability of the HPV-negative HNSCC, HPV-positive HNSCC and the immortalized HaCaT and NOK cell lines. This effect was dose-dependent. Gedatolisib had the lowest and alpelisib the highest IC50 values. Notably, two HPV-positive cell lines (UD-SCC-2 and UPCI-SCC-090) and the HPV-negative UT-SCC-33 cell line were less responsive to all three PI3Ki. There was no statistically significant difference in the activity of PI3Ki between the HPV-positive and -negative cell lines (data not shown). In comparison with the IC50 values of cisplatin, PI3Ki appeared to be more potent in HPV-positive and -negative HNSCC cells.

Effects of PI3Ki on cell cycle distribution and apoptosis

To determine whether PI3Ki causes cell cycle arrest, flow cytometry analysis was performed on the HPV-negative UPCI-SCC-003 and HPV-positive UM-SCC-47 and UD-SCC-2 cell lines (Figs. 3A and S1). In UPCI-SCC-003 and UM-SCC-47 cells, treatment with alpelisib resulted in an increase in G1 and SubG1, whereas treatment with buparlisib resulted in an increase in cells in G2/M phase. In UD-SCC-2 cells, none of the inhibitors induced a notable change in the cell cycle (Figs. 3A and S1). In addition, all three PI3Ki induced an increase in apoptosis in all cell lines, which was only statistically significant for buparlisib (Figs. 3B, C and S3).

Figure 3 Effect of phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway inhibitors on the cell cycle distribution and the induction of apoptosis. (A) Cells were treated with DMSO (1:1,000), gedatolisib (15 nM), buparlisib (1 μM) or alpelisib (10 μM) and cell cycle analysis was performed after 1 day. (B) Cells were either treated for 1 day with 500 nM Staurosporine, a known inducer of apoptosis, or for 1 day with different concentration of gedatolisib, alpelisib and buparlisib, followed by analysis of Annexin V staining. Representative images are shown for UM-SCC-47 cells. (C) Quantification of the apoptotic UPCI-SCC-003, UM-SCC-47 and UD-SCC-2 cells. Each bar represents the mean ± SEM of the data obtained from three independent experiments. *P<0.05 vs. DMSO cells. HPV, human papillomavirus.

PI3K inhibition downregulates the levels of PI3K-Akt-mTOR pathway proteins

To investigate the expression of proteins involved in the PI3K pathway, western blotting of cell lysates after treatment with PI3Ki was performed. Treatment with all three inhibitors resulted in the expected downregulation p-4E-BP1 and a slight downregulation of p-Akt (Thr308) (Fig. 4). In addition, treatment with alpelisib and buparlisib resulted in the downregulation of p-Akt (Ser473), and treatment with alpelisib and gedatolisib also downregulated PI3K itself. Treatment with gedatolisib resulted in less downregulation of the proteins compared with alpelisib and buparlisib. Therefore, the cell lines were also treated with concentrations of gedatolisib above the IC50 (5, 15 and 30 nM), producing similar results (Fig. S4).

Figure 4 Assessment of the expression levels of proteins involved in the PI3K/Akt/mTOR pathway by western blot analysis of whole protein extracts. The cells were treated for 1 and 3 days with the IC50 value of (A) gedatolisib, (B) buparlisib and (C) alpelisib (See Table I) or the control (DMSO, 1:1,000). β-actin was used as the loading control. HPV-negative, UPCI-SCC-003, HPV positive UM-SCC-47 and HPV positive UD-SCC-2 head and neck squamous cell carcinoma cell lines were assessed. The experiments were performed in triplicate. HPV, human papillomavirus; mTOR, mammalian target of rapamycin; p-, phosphorylated; PI3K, phosphatidylinositol 3-kinase.

PI3K inhibition decreases cellular metabolism

The PI3K/Akt/mTOR pathway is crucial in the regulation of cell energy metabolism and is involved in both glucose uptake and the coordination of glucose fate within the cell. Therefore, it was evaluated whether the viability-inhibitory effects of PI3Ki were associated with alterations in both oxidative and glycolytic energy metabolism. To investigate the impact of PI3Ki on mitochondrial respiration, a Seahorse Mito Stress Test was performed. The baseline oxidative metabolism was the highest in UPCI-SCC-003 and lowest in UD-SCC-2 cells (Fig. 5A). Treatment with alpelisib significantly reduced basal respiration and the respiration associated with ATP production in all cell lines. Buparlisib only downregulated basal respiration and ATP production in UM-SCC-47 cells (Fig. 5A and B). Gedatolisib downregulated basal respiration in all cell lines and ATP production-coupled respiration in UD-SCC-2 cells. The impact of PI3Ki treatment on cell glycolytic capacity was also examined by performing the SeaHorse Glycolysis Stress Test. The glycolytic capacity, measured as the maximum ECAR rate achieved upon the inhibition of oxidative phosphorylation by oligomycin, was significantly downregulated by alpelisib in the three cell lines. Gedatolisib and buparlisib downregulated glycolytic capacity in UD-SCC-2 cells (Fig. 5C and D). These results were confirmed using glucose and lactate assays (Fig. S5). Taken together, these data show that alpelisib in particular has the capacity to inhibit both oxidative metabolism and glycolysis.

Figure 5 Effect of phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway inhibitors on mitochondrial oxidative metabolism and glucose metabolism. (A) Mitochondrial respiration profiles were analyzed by the Agilent SeaHorse Cell Mito Stress Test. UCPI-SCC-003, UM-SCC-47 and UD-SCC-2 cells were treated for 24 h with DMSO or the IC50 concentrations of the test drugs (See Table I). The OCR data are representative of three independent experiments. OCR was measured after the addition of oligomycin (an ATP-synthase inhibitor), FCCP (an uncoupling agent that disrupts the mitochondrial membrane potential and maximizes OCR) and a mixture of antimycin A/rotenone (shuts down mitochondrial respiration by inhibiting the electron transport chain). (B) The parameters of basal respiration and ATP-production were calculated as a percentage vs. the DMSO cells. (C) Glycolytic profiles were obtained using the Agilent SeaHorse Glycolysis Stress Test. UPCI-SCC-003, UM-SCC-47 and UD-SCC-2 cells were treated for 24 h with the IC50 concentrations of the test drugs (See Table I). The ECAR was measured under glucose starvation and after the addition of glucose, oligomycin (to maximize the glycolytic flux of the cell) and 2-DG (to shut shown the glycolytic process). The ECAR profiles are representative of three different experiments. (D) The parameters of glycolysis and glycolytic capacity were calculated as a percentage vs. DMSO cells. Each bar represents the mean ± SEM of the data obtained from three independent experiments. *P<0.05 vs. DMSO cells. 2-DG, 2-deoxyglucose; ECAR, extracellular acidification rate; HPV, human papillomavirus; OCR, oxygen consumption rate.

Effect of the combination of PI3Ki alpelisib and CDKi ribociclib on cell viability

Dual inhibition of PI3Ki and CDKi might synergistically decrease cell viability, especially in HPV-negative HNSCC cell lines. Therefore, the efficacy of the combination of PI3Ki alpelisib and CDKi ribociclib in UPCI-SCC-003 and UM-SCC-47 cells were investigated. The choice for this inhibitor combination was based on the subunit selectivity of alpelisib and the pronounced effect of this inhibitor in the aforementioned (functional) assays, as well as the enhanced solubility of ribociclib compared with palbociclib, requiring lower concentrations of DMSO. The cell lines were treated for 3 days with increasing concentrations of ribociclib (0 nM, 10 nM, 100 nM, 1 μM and 10 μM), with the addition of increasing concentrations of alpelisib (0 nM, 10 nM, 100 nM, 1 μM and 5 μM). In comparison with ribociclib alone, the combination treatment resulted in lower IC50 values (Figs. 6A-E and S6). Interactions between ribociclib and alpelisib were evaluated using the HSA reference model to determine possible synergism. For the UPCI-SCC-003 cell line, the overall HSA synergy score was 8.712, which corresponds to 8.712% of the response beyond the expectation for all concentrations (Fig. 6C), indicating a moderate synergistic effect. The MSA had a synergy score of 12.66 and the highest score of 27.2 for the combination of 10 μM ribociclib and 5 μM alpelisib. For the UM-SCC-47 cells, the overall HSA synergy score was 5.6, indicating an additive effect of the combination therapy (Fig. 6D-F). The MSA represented a synergy score of 9.15, with the highest score of 28.1 obtained for the combination of 10 μM ribociclib and 5 μM alpelisib. However, no increase in apoptotic cells was observed with the combinational treatment compared to alpelisib treatment alone (Fig. S7).

Figure 6 Combination therapy (3 days) of ribociclib with alpelisib in the HPV-negative UPCI-SCC-003 and HPV-positive UM-SCC-47 cell lines. (A and D) The dose-response curves for ribociclib (0 nM, 10 nM, 100 nM, 1 μM and 10 μM) without alpelisib are illustrated by the A0 curves. The increasing concentrations of alpelisib (0 nM, 10 nM, 100 nM, 1 μM and 5 μM) are indicated by the A10, A100, A1000 and A5000 curves. The calculated IC50 of ribociclib alone and in combination with different alpelisib concentrations (nM) are shown in (B) for UPCI-SCC-003 and in (E) for UM-SCC-47 cells. (C and F) The HSA synergy score graph shows the combined concentration of ribociclib and alpelisib which are synergistic (red), additive (white) and antagonistic (green). Each bar represents the mean ± SEM. *P< 0.05. HSA, highest single-agent; HPV, human papillomavirus.

Discussion

A large body of preclinical data indicates that the cell cycle and PI3K/Akt/mTOR pathways are deregulated and may provide therapeutic targets for patients with HNSCC (11,12,13). Several studies are currently evaluating CDKi and PI3Ki monotherapy or in combination with chemotherapy, targeted therapy, immunotherapy or radiotherapy [such as a phase III trial with buparlisib in combination with paclitaxel (NCT04338399) and a phase III trial with palbociclib combined with cetuximab (NCT04966481)]. The results thus far are diverse regarding the advantages of the combination of CDKi and standard cytotoxic chemotherapy (40). For PI3Ki, most of these compounds have not advanced to late-stage clinical trials, and the results of phase II studies are awaited in the next couple of years. A possible explanation for these results might involve the complexity of PI3K signaling and intrinsic adaptive responses, leading to inadequate pathway inhibition (41). The aim of the present study was to investigate the in vitro effects of several CDKi (palbociclib and ribociclib) and PI3Ki (alpelisib, buparlisib and gedatolisib) on the viability of five HPV-positive and three HPV-negative HNSCC cell lines, together with the consequences and underlying mechanisms.

In the present study, CDKi showed particular efficacy in HPV-negative HNSCC cell lines, of which two harbor a CCND1 amplification, represented by reduced cell viability and downregulation of the proteins, Rb1 and p-Rb1, leading to G1 cell cycle arrest without increased apoptosis. Cyclin-D1 was found to be upregulated after CDKi treatment, which was possibly induced by a positive feedback mechanism, as was also found in another study (39). The observations of the present study were also in accordance with an earlier study in which CDKi treatment with palbociclib showed efficacy in oral squamous cell carcinoma cell lines (42). Together, these data support CDKi as cytostatic agents involving the prevention of cell cycle progression by blocking hyperphosphorylation of Rb1 (43). Most likely, cells may enter senescence, an irreversible arrest of cell proliferation, while maintaining metabolic function (44,45). This was also supported by the increased levels of the senescence maker β-galactosidase in the HPV-negative UPCI-SCC-003 cell line after treatment with palbociclib, observed in the present study. These findings suggest that CDKi should be combined with other therapies to achieve cytotoxic effects at clinically relevant concentrations. This is underscored by a phase II study demonstrating a synergistic treatment response of palbociclib combined with cetuximab in patients with platinum- or cetuximab-resistant HNSCC (46). In accordance with our hypothesis, CDKi treatment did not inhibit the viability of HPV-positive HNSCC cell lines, and differences in cell cycle distribution were not detected between untreated and treated cells in the present study.

In the present study, PI3K inhibition resulted in a strong reduction in cell viability in both HPV-positive and -negative HNSCC cell lines and downregulation of PI3K/Akt/mTOR pathway protein expression. Only moderate cell cycle arrest and apoptosis were observed after treatment with PI3Ki. The differences in therapeutic efficacy between PI3Ki may be explained by several features. First, the PI3K subunits p110α and p110β are ubiquitously expressed in mammalian cells, whereas p110γ and p110δ are mainly expressed in leukocytes (47). Thus, this may have consequences when using PI3Ki specific to one or more PI3K subunits. In addition, the structure of the PI3K inhibitors determines (subunit) selectivity, protein binding affinity and flexibility, cellular uptake and thereby the activity of the protein inhibitor (48,49).

As aforementioned, alpelisib selectively inhibits p110α (23,24). In luminal HER2-amplified and PIK3CA mutant breast cancer, the initial efficacy of p110α inhibition appears to be mitigated by the rapid re-accumulation of the PI3K product, PIP3, produced by the p110β isoform. The combination of a p110β inhibitor with alpelisib therefore prevented PIP3 rebound and induced greater antitumor efficacy in a luminal breast cancer study (50). Treatment with buparlisib significantly inhibited wild-type and mutant PI3K catalytic subunit p110α, β, δ and γ (25), and showed lower IC50 values than treatment with alpelisib in the present study. However, there are concerns regarding the safety profile of buparlisib. In the BELLE-3 breast cancer trial, in which patients with advanced breast cancer following progression on prior mTOR inhibition and endocrine therapy were included and treated with fulvestrant with or without the addition of buparlisib, treatment with buparlisib resulted in frequent grade 3 and 4 adverse events (hyperglycemia, elevated liver enzymes and major psychiatric symptoms) (51). A possible explanation for the (severe) side effects of buparlisib observed in clinical trials could be the off-target effects of this inhibitor. Buparlisib was found to cause cell death in various cellular systems independent of PI3K pathway dependence by influencing the expression of mitotic genes. In addition, buparlisib inhibited microtubule dynamics following direct binding to tubulin (52). This is supported by the G2/M phase arrest and increased apoptosis observed in the present study and other studies. Specifically, in both glioma and acute myeloid leukemia cell lines, G2/M phase arrest and apoptosis were observed after treatment with buparlisib (53,54).

Gedatolisib is a highly potent dual inhibitor of PI3K (α, β, δ and γ) and mTOR (TORC1 and TORC2), showing low IC50 values in the present study. Akt-mediated stimulation of mTORC1 serves as a key point in the regulation of anabolic metabolism, by stimulating, for example, pyrimidine and de novo purine synthesis. mTORC1 activation also increases the protein synthesis capacity of cells through multiple mechanisms (55,56). Gedatolisib, on the one hand, blocks DNA and protein synthesis. On the other hand, inhibiting mTOR might repress a negative feedback loop that activates the PI3K and MAPK pathways, which has also been reported in other breast cancer cell lines (57,58). Together, these findings may explain the lower IC50 value of gedatolisib observed compared with the other two PI3K inhibitors.

Mutation analysis revealed several genetic alterations in the PI3K/Akt/mTOR pathway in the studied cell lines, in line with TCGA data (59). Cell lines with amplification of PIK3CA (UPCI-SCC-72) or amplification of AKT1 and AKT2 (UM-SCC-47) responded well to PI3Ki in the present study, according to their IC50 values. By contrast, the UPCI-SCC-090 cell line harboring a double PTEN mutation was observed to be more resistant to PI3Ki, which could be expected based on the literature (59,60). Another relatively resistant cell line (UT-SCC-33) harbors mutations in FGFR3 and HRAS, among others, suggesting the activation of other compensatory oncogenic signaling pathways. The UD-SCC-2 cell line was relatively PI3Ki-resistant, and no pathogenic mutations were observed via the sequencing panel. These results suggest that the presence of an activating PIK3CA mutation is not a prerequisite for the therapeutic effect of PI3Ki in these cell lines and that the complexity of this pathway and its interaction with other compensatory pathways, may play a role. Moreover, the oncogenic activity of HPV E5, E6 and E7 oncogenes may directly and/or indirectly interact with the PI3K pathway (61).

The PI3K pathway is central to most deregulated metabolic pathways supporting the anabolic needs of cancer cells. Therefore, the cellular metabolism of PI3Ki-treated cell lines were investigated. Cells use two major pathways to produce ATP: Glycolysis and mitochondrial respiration via oxidative phosphorylation. Cancer cells heavily depend on glycolysis, balancing ATP production with anabolic needs, which is also the case for HNSCC, supported by the increased lactate levels observed in HNSCC tumors (62). In the present study, it was found that PI3Ki decreased both glycolysis and mitochondrial oxidative metabolism in HNSCC cells.

The PI3K isoform, p110α, is suggested to be the key mediator of glucose metabolism in multiple tissues (63,64), which is supported by the strongest metabolic effect observed with p110α-specific inhibitor, alpelisib, in the present study. Although the pan-PI3Ki, gedatolisib and buparlisib, also inhibit this isoform, dual- and pan-PI3Ki in general have been found to exhibit more off-target effects (such as on mitotic genes or on members of the PI3K-related kinase family), thereby reducing efficacy in the inhibition of the actual target pathway (65,66). Isoform-selective inhibitors (such as alpelisib) may achieve greater efficacy and have the potential to block the relevant target more completely while limiting toxicities (65,66). The findings of the present study underscore this principle, as alpelisib treatment most prominently affected Akt phosphorylation and cellular metabolism. There are few studies investigating the effects of PI3K inhibition on metabolism in HNSCC cell lines. In colorectal cancer, PI3Ki LY294002 reduced glycolysis by significantly decreasing hexokinase-2 levels as a result of PI3K-Akt inhibition (67). Treatment of glioblastoma cell lines with NVP-BEZ235 resulted in a significant reduction in lactate secretion and lower glucose uptake, indicating a strong effect on glycolytic activity by this PI3Ki (68). Taken together, PI3K/Akt/mTOR pathway inhibitors were effective in both HPV-negative and HPV-positive cell lines, with varying efficacy between inhibitors, inducing apoptosis, attenuating cellular metabolism and only moderate cell cycle arrest. Although the induction of apoptosis is the most frequently described mechanism of cell death in response to PI3Ki, other cell death mechanisms, such as autophagy-mediated cell death, may also play a role, which could be considered in future research (69,70).

It has been reported that inhibition of the cell cycle or MAPK pathway in cancer cells, including breast and pancreatic cancer cells, leads to compensatory upregulation of other oncogenic signaling pathways, including the PI3K/mTOR pathway. Therefore, dual inhibition of CDKi and PI3Ki could represent an interesting treatment option (39,71,72). The cell viability inhibitory effects of combined treatment with CDKi ribociclib and the PI3Ki alpelisib in HPV-negative UPCI-SCC-003 and HPV-positive UM-SCC-47 cell lines were analyzed in the present study. The results showed a synergistic effect of the combination of ribociclib and alpelisib, which was higher in the HPV-negative cell line, as expected, since CDKi alone only affected HPV-negative HNSCC cells. Despite the apparent synergistic effect of both inhibitors on cell viability, no increase in apoptotic cells was observed with the combinational treatment compared with alpelisib treatment alone, indicating that other mechanisms may play a role in the observed synergistic reduction in cell viability. Furthermore, treatment duration and sequence (simultaneously or sequentially) are important considerations that may influence the therapeutic effect of combination therapy. Further studies to confirm compensatory mechanisms, including PI3K/Akt/mTOR pathway upregulation, after CDKi in HNSCC and the efficacy of combinational therapy at clinically relevant concentrations are required. In this respect, the effects of these targeted therapies may also be investigated in combination with radiotherapy, which may offer opportunities for treatment de-escalation, as a replacement for chemotherapy, or in combination with radiotherapy dose reduction.

In addition to PI3Ki, mTOR inhibitors (mTORi) have been investigated for the treatment of HNSCC, resulting in decreased tumor cell proliferation and apoptosis in cell lines and xenograft models (73,74). A phase II clinical trial showed promising response rates to the mTORi, temsirolimus, combined with cetuximab in patients with cetuximab-resistant HNSCC, but no improvement in progression-free survival was observed (75). Furthermore, the combination of CDKi ribociclib with mTORi everolimus showed acceptable safety profiles and promising tumor responses in breast cancer and multiple pediatric cancer types (76,77). Although it might be expected that downstream inhibition of mTOR results in fewer off-target effects than upstream PI3Ki, studies have shown that mTORi leads to compensatory feedback loops. For example, mTORi can upregulate tyrosine kinase receptors, leading to increased PI3K/Akt and MAPK signaling (58,78). Dual PI3K and mTOR inhibitors, including gedatolisib, may limit these compensatory feedback mechanisms.

The present study may be limited by the absence of PIK3CA mutations in the tested cell lines, which hampers the evaluation of these specific genetic changes in response to inhibitors. Nevertheless, the IC50 values of alpelisib and gedatolisib to PI3Kα-wildtype and -mutant cell lines were similar to that observed in other studies (26,79), thus extending the effect of PI3Ki. It will be important to identify biomarkers that can predict the response to PI3Ki in HNSCC and accelerate the integration of novel targeted agents into the treatment of these cancer types.

In conclusion, the present study described multiple mechanisms and consequences of CDK4/6 and PI3K/Akt/mTOR pathway inhibition and provided the basis for further research into the targeting of these oncogenic signaling pathways and possible resistance mechanisms. Future preclinical studies should focus on identifying optimal concentrations, treatment durations and treatment sequences, as well as the mechanisms underlying the synergistic effects of combination treatment approaches. Furthermore, the use of ex vivo culture models, which are directly derived from tumor tissue, will be an interesting next step to facilitate the translation of in vitro findings to patients (80). The combination of CDKi with PI3Ki and combinational treatment with radiotherapy could be a promising new treatment approach and may offer opportunities for treatment de-escalation, as a replacement for chemotherapy, or in combination with radiotherapy dose reduction.

Supplementary Data

Availability of data and materials

The data generated in this study may be obtained from the corresponding author.

Authors' contributions

Conceptualization and design were conducted by FV, ID, BK, AH and EJS. Acquisition and analysis of data were conducted by FV, ID, DL and RJ. FV, ID, EJS and BK confirm the authenticity of all the raw data. Data interpretation was conducted by FV, ID, DL, RJ and EJS. Writing of the original manuscript draft was conducted by FV and ID. Reviewing and editing of the manuscript was conducted by FV, ID, DL, RJ, AH, BK and EJS. Construction of the figures was conducted by FV and ID. Study supervision was conducted by AH, BK and EJS. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

Although Pfizer and Novartis funded this study and the drugs, palbociclib, ribociclib, gedatolisib, buparlisib and alpelisib, were provided by them, the funders were not involved in the study design, collection, analysis and interpretation of data, the writing of this article, or the decision to submit it for publication.

Acknowledgements

The authors would like to thank Dr Ludwig Dubois (Department of Precision Medicine, GROW Research Institute for Oncology and Reproduction, Maastricht University, Maastricht) and Dr Marike van Gisbergen (Department of Dermatology, Maastricht University Medical Centre, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht) for their helpful discussions regarding the cellular metabolism experiments.

The abstract was presented at the Annual Meeting of the American Association for Cancer Research April 8-Apr 13, 2022, in New Orleans, LA, and published as abstract no. 2573 in Cancer Res 82 (Suppl 12): 2022.

Funding

This study was funded by Pfizer (grant no. WI194733) and Novartis (grant no. CBYL719NCMUMC01).

References