Abstract
Background/Aim: The cyclin-D-CDK4/6-RB pathway is often deregulated in head and neck squamous cell carcinoma (HNSCC), making it an attractive therapeutic target. However, previous studies showed that treatment with specific CDK4/6 inhibitors (CDK4/6Is) induced ROS generation and the activation of ERK phosphorylation in HNSCC, enabling cancer cells escape from CDK4/6I-medicated cell cycle arrest. Here we aimed to explore the therapeutic effects of the combination of abemaciclib (CK4/6I) and trametinib (an ERK1/2 inhibitor) in preclinical models of HNSCCs.
Materials and Methods: The activities of trametinib as a monotherapy and a combination therapy with abemaciclib in HNSCC cells and tumors were evaluated using cell viability assays, colony formation assays, and xenograft models, respectively.
Results: Trametinib enhanced the inhibitory effect of abemaciclib in HNSCC cancer cells and xenografts, and the co-administration of abemaciclib and trametinib exhibited synergy in anti-tumor activities.
Conclusion: The combination of abemaciclib and trametinib potently repressed the growth of HNSCC cells and xenografts in a synergistic manner, thus being a potential therapeutic approach for HNSCC.
Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with about 700,000 new cancer diagnoses per year (1, 2). The main treatment options for patients with HNSCC are surgery, radiotherapy, chemotherapy, or combinations of these interventions. Despite advances in multimodality therapy, the 5-year overall survival (OS) rate remains approximately 40% to 50%. Even for patients who are cured, the treatments for HNSCC often bring about severe adverse effects and reduced quality of life. In recent years, targeted therapies have been introduced into the treatment protocol for a sub-population of patients with HNSCC, in which drugs targeting the epidermal growth factor receptor (EGFR) or anti-programmed cell death protein-1 (those targeting PD-1 are used as single-agent therapy or combined with standard HNSCC treatment regimens). However, the clinical outcomes are suboptimal, indicative of the desperate need for novel therapeutic agents in HNSCC treatment (3-5).
The cyclin-D-dependent kinase 4 and 6-retinoblastoma (CDK4/6-RB) pathway plays critical roles in the control of cell cycle progression in the G0/1 to S transition, and deregulation of this pathway has been frequently found in a variety of human cancers, including HNSCC (Figure 1) (6, 7). The molecular mechanisms underlying the deregulation of the CDK4/6-RB pathway include but are not limited to alteration of RB gene, the inactivation of CDKN2A (encoding the tumor suppressor p16INK4A protein, an specific upstream inhibitor of CDK4/6), the amplification of CCND1 (encoding cyclin D1 protein, the activating regulatory subunit of the CDK4/6 complex), the activation of oncoproteins such as gankyrin that promotes CDK4-mediated phosphorylation of RB protein (8-10), and the infection with human papillomavirus (HPV) that functionally inhibits the RB1 protein and promotes the latter’s ubiquitination-mediated degradation through RB/viral oncoprotein E7 binding (11, 12). Expression levels of the CDK4 and CDK6 proteins were also found to be significantly higher in oral premalignant and malignant HNSCC tissues than in healthy tissues (13). Therefore, CDK4/6 may represent a rational therapeutic target in HNSCC, especially HPV-negative HNSCC, which cover approximately 80% of diagnosed HNSCC.
The major impacts of trametinib and abemaciclib on proliferation pathways in head and neck squamous cell carcinoma cells. P: Phosphorylated; (+): positive regulation; (−): negative regulation.
While three specific CDK4/6 inhibitors (CDK4/6Is), namely, palbociclib, ribociclib, and abemaciclib have been approved by FDA for the treatment of breast cancer, several preclinical and clinical studies have shown that CDK4/6Is exhibited encouraging activities in HNSCC as a monotherapy or in combination therapy (14-17). In a phase II study in platinum-resistant, HPV-negative, recurrent/metastatic HNSCC, the combination of palbociclib with cetuximab resulted in a robust tumor response rate and a median overall survival (OS) time of 12.1 months, which is the longest reported OS thus far for patients with platinum-resistant, recurrent/metastatic HNSCC (18). It has also been reported that both palbociclib and abemaciclib induced the G1/G0 cell cycle arrest in HPV-negative HNSCC (15, 19, 20). Moreover, ribociclib exhibited cytotoxic potency in HPV-negative HNSCC cell lines and patient-derived xenografts (PDX) with intact RB1 protein expression and epithelial phenotype (21). However, knowledge about mechanisms underlying sensitivity and resistance to CDK4/6Is in HNSCCs remain limited. On one hand, while some studies showed that the efficacy of CDK4/6Is in HPV-negative tumors was associated with CCND1 amplification or CDKN2A deletion, such correlation was not found in patients with advanced ER+/HER2− breast cancer undergoing the combination therapy of palbociclib and letrozole (22, 23); on the other hand, recent studies in our laboratory showed that abemaciclib treatment induced ROS generation and phosphorylation of signal-regulated kinases (ERK1/2 proteins) in HNSCC cells regardless of their CCND1 and p16INK4A statuses, which might promote tumor progression (15). ERK1/2 proteins are components in the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) pathway (also known as the RAS-RAF-MEK-ERK pathway), whose aberrant activation has been frequently found in most of human cancers (if not all) (24, 25). Briefly, activated RAS oncogenic protein activates RAF, a serine/threonine kinase that phosphorylates mitogen-activated extracellular signal-regulated kinase 1 (MEK1) and MEK2 (Figure 1). Subsequently, MEKs activate ERKs, which further promote several target proteins and nuclear transcription factors by phosphorylation, thus contributing to the regulation of cell proliferation, differentiation, survival, and growth (26, 27). Trametinib is a reversible, highly selective, allosteric inhibitor of MEK1 and MEK2, and it blocks the catalytic activity of MEKs by binding to unphosphorylated MEK proteins with high affinity preventing phosphorylation and activation of MEKs. Trametinib has been used alone or in combination with dabrafenib (Tafinlar) to treat certain types of melanomas, non-small-cell lung cancer (NSCLC), thyroid cancer, and brain cancer (28). In this study, we evaluated the activity of trametinib as a monotherapy and a combination therapy with abemaciclib in preclinical models of HNSCCs. Our results showed that trametinib exhibited potency in inhibiting the growth of HNSCC cell lines and xenografts. Furthermore, the combination of trametinib and abemaciclib synergistically repressed cell growth and inhibited tumor growth in HNSCC. These results support further studies on the clinical development of the combination of CDK4/6 and ERK1/2 inhibition for HPV-negative HNSCC.
Materials and Methods
Cell lines and cell culture. CAL27 (CRL-2095) and SCC4 (CRL-1624) cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Prior to use, both cell lines were authenticated using short tandem repeat (STR) analysis and were tested to be mycoplasma-free following the recommendations of ATCC (15). Cells were grown in Dulbecco’s modified Eagle medium (DMEM)/Ham F12 medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, 1% penicillin/streptomycin (hereafter, complete media) at 37°C, in a humidified atmosphere with 5% CO2. All cell culture materials were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Inhibitors and antibodies. Trametinib and Abemaciclib were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies were obtained from different resources: anti-GAPDH (#10R-G109a, 1:10,000 dilution) was from Fitzgerald (Acton, MA, USA); antibodies against total RB (#9309S, 1:1,000) and phospho-RB (Ser780) (#9307, 1:1,000 dilution) were from Cell Signaling Technology (Danvers, MA, USA); antibodies against phospho-ERK1/2 (Thr202/Tyr204, 36-8800 1:1,000 dilution) and total ERK1/2 (#MA5-15605, 1:1,000 dilution) were from Thermo Fisher Scientific (Rockford, IL, USA). Alexa Fluor 488 and 594 conjugated secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA).
Cell viability assay. Cells were seeded in 96-well plates at a density of 3,000-5,000 per well in triplicate and grown at 37°C for 24 h. Subsequently, cells were treated with the indicated inhibitors for 72 h. Cell viability was evaluated using a WST-1 cell proliferation assay kit (Roche, Madison, WI, USA) following the manufacturer’s instructions. Cell viability was calculated as a percentage of the vehicle (DMSO)-treated control. Data were fitted into a non-linear regression model (four-parameter) using R package to obtain absolute EC50 values (R Foundation for Statistical Computing, Vienna, Austria, http://www.r-project.org).
Colony formation assay. A colony formation assay was performed as previously described. Briefly, CAL27 or SCC4 cells were seeded at a density of 3,000 to 5,000 cells per well in 12-well plates and grown at 37°C for 24 h. Cells were then treated with the indicated drugs for seven days. Growth media were changed with or without drugs every other day. After treatment, cells were washed with phosphate-buffered saline (PBS; pH 7.4), fixed with 4% paraformaldehyde, and stained with a 0.5% crystal violet solution for 2-4 h. Plates were washed with water and imaged using a digital imager (FluorChem M, ProteinSimple, San Jose, CA, USA). Colonies were quantified by assessing the absorbance of crystal violet at 570 nm after extraction with 10% acetic acid. The colony formation ability (%) was calculated relative to vehicle-treated control cells.
Western blotting. Cell and tumor samples were lysed in RIPA buffer (Alfa Aesar, Ward Hill, MA, USA) supplemented with protease inhibitors (Cat # P8340; Sigma-Aldrich, St. Louis, MO, USA) and Phosphatase inhibitors (Cat # A32959; Thermo Fisher Scientific). Equal amounts of protein (50 μg) were then subjected to SDS-PAGE (Bolt 4-12% Bis-Tris Gel; Invitrogen) separation and transferred to PVDF membranes. Membranes were incubated with respective primary antibodies diluted in 1X TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5), 5% dry milk overnight at 4°C with gentle agitation on a shaker. Subsequently, the membranes were incubated with the corresponding secondary antibody conjugated with horseradish peroxidase (HRP) and developed with enhanced chemiluminescence (ECL) solution (Thermo Fisher Scientific). The bands were visualized using a digital imager (FluorChem M; ProteinSimple).
In vivo xenograft studies. Xenograft tumors were generated as previously described. Briefly, 3×106 CAL27 cells in 1:3 dilution in matrigel (Corning Life Sciences, Bedford, MA, USA)/PBS were subcutaneously injected into the rear flank of 7-8-week-old male and female athymic (Foxn1nu) nude mice. When the tumor volume reached approximately 150 mm (Day 0), mice were randomized into 4 groups (n=8 per group): the vehicle control group [1% Hydroxyethylcellulose (HEC) in PBS], the abemaciclib (50 mg/kg) group, the trametinib (1 mg/kg) group, and the group with the combination of abemaciclib (50 mg/kg) and trametinib (1 mg/kg). Tumor size was measured with a digital caliper and tumor volume was calculated using the formula 4/3 × (l/2) × (w/2)2, where I is the perpendicular length and w is the perpendicular width of the tumor (15). Mice were sacrificed after 18 days of treatment. All animal experiments were carried out under protocols approved by the Ohio State University Institutional Animal Care and Use Committee.
Drug combination and synergy. While cell viability was assessed using WST-1 assays as aforementioned, analysis of combination drug dose-effect was performed, and combination indexes were calculated using Compusyn® software (ComboSyn, Inc., Paramus, NJ, USA) (15).
Statistical analysis. In vitro data were obtained from at least 3 independent experiments unless stated otherwise and presented as mean±standard deviation (SD). Groupwise comparisons were performed using unpaired Student’s t-tests. Data from in vivo experiments were analyzed using one-way analysis of variance (ANOVA) with repeated measurements followed by Tukey-post hoc multiple comparisons (if needed). All statistical tests were two-sided, and the significance level was α=0.05. R3.2 software (R Foundation for Statistical Computing, Vienna, Austria) was used in this study.
Results
Abemaciclib inhibited cell growth but activated the ERK pathway in HNSCC. In our previous studies, abemaciclib exhibited considerably high anti-proliferative potency in HNSCC cells in vitro. Specifically, the EC50 values for CAL27 and SCC4 cells were 0.8±0.2 μM and 6.4±1.3 μM, respectively. Interestingly, further studies in our laboratory showed that while it down-regulated CDK4/6-mediated phosphorylation of RB protein, abemaciclib activated the phosphorylation of ERK1/2 in HNSCC cells (15). As shown in Figure 2A, increased concentrations of abemaciclib (from 0 to 1.0 μM) continuously promoted the phosphorylation of Rb at Ser780 and Ser807 in both CAL27 and SCC4 cells. Meanwhile, the level of total RB protein was unchanged (in SCC4) or decreased to a much lesser extent (in CAL27), indicating that the impact of abemaciclib on the RB pathway is mainly at the posttranslational level. In contrast, as the concentration of abemaciclib increased to 0.5 and 1.0 μM, enhanced phosphorylation of ERK1/2 was detected in both cell lines (Figure 2B), whereas the levels of total Rb proteins remained comparable at different concentrations of abemaciclib (data not shown). Further studies showed that abemaciclib treatment induced the mRNA expression of MAPK1 and MAPK3 in HNSCC cells, which proceeded the phosphorylation of ERK1/2 as well as the activation of entire ERK pathway (data not shown). Taken together, these results suggest that abemaciclib-induced activation of ERK1/2 may underline the resistance to abemaciclib in human HNSCC. As such, it is arguably safe to assume that concurrent use of ERK1/2-inhbiting agents with abemaciclib could enhance the latter’s anticancer potency in HNSCC.
Abemaciclib inhibits RB phosphorylation but activates the phosphorylation of ERK1/2 proteins in HNSCC cells. CAL27 and SCC4 cells were treated abemaciclin at the indicated concentrations for 72 h, and the whole-cell lysate were then applied for western-blot analyses of RB and ERK proteins. (A) Total RB protein and phosphorylated RB proteins (at Ser780 and Ser807); (B) Phosphorylated ERK1/2 proteins (at Thr202/Tyr204). In both assays, GAPDH, glyderaldehyde-3-phosphate dehydrogenase, was used as internal control.
Trametinib enhanced the efficacy of abemaciclib in HNSCC cells. To examine the above hypothesis, we assessed the anti-proliferative activities of an ERK1/2 inhibitor, Trametinib, in HNSCC cells in the absence and presence of abemaciclib. Trametinib exhibited anti-proliferative potencies as a single agent in both CAL27 and SCC4 cells with EC50 values of approximately 5.0 μM after 72-h treatment (Figure 3A). Moreover, the combination of trametinib and abemaciclib synergistically inhibited the growth of these two oral HNSCC cells in cell viability assays (Figure 3B). As monotherapy agents, 1.0 μM abemaciclib and 0.2 μM trametinib significantly reduced the viability of CAL27 after 72-h treatment (control, 100.0±3.2% vs. abemaciclib, 67.4±6.2%, p<0.01; control, 100.0±3.2% vs. trametinib, 78.5±4.6%, p<0.05). Remarkably, the combination of 1.0 μM abemaciclib and 0.2 μM trametinib further reduced the viability of CAL27 cells (42.8±3.6% vs. control, p<0.001; vs. abemaciclin only, p<0.05; vs. trametinib only, p<0.01). Similar observations were obtained in SCC4 cells: 1.0 μM abemaciclib only led to a reduction of 10% in the viability of SCC4 cells (control, 100.0±6.5% vs. abemaciclib, 88.0±2.4%, p=0.08), consistent with our previous results showing that SCC4 was less sensitive to abemaciclib than CAL27. Contrarily, 0.2 μM trametinib led to a statistically significant reduction of 20% in the viability of SCC4 cells (78.7±7.1%, p<0.05 in comparison with the control), and the combination of 1.0 μM abemaciclib and 0.2 μM trametinib reduced the viability of SCC4 cells by 35% (66.3±4.0%, p<0.01 in comparison with the control). The synergistic effect of trametinib and abemaciclib on the viability of oral HNSCC cells was further evaluated in a cell viability assay and calculated the combination index (CI). Table I summarizes the CI values of various doses of trametinib and abemaciclib. In all tested combinations of trametinib (at a fixed dose of 0.2 μM) and abemaciclib (at doses of 0.5, 1.0, 2.0, 4.0, and 6.0 μM), the CI values in both CAL27 and SCC4 cells were far below 1 (ranging from 0.06 to 0.21), indicative of the existence of strong synergism between trametinib and abemaciclib in oral HNSCC cells.
Trametinib synergizes with abemaciclib in vitro. (A) Viability of head and neck squamous cell carcinoma (HNSCC) cells after 72 h of treatment with trametinib at select concentrations. Cell viability was assessed using a WST-1 cell proliferation assay kit (Roche, Madison, WI, USA) following the manufacturer’s instructions. Experiments were performed in triplicate. (B) Viability of HNSCC cells after 72 h of treatment with abemaciclib only (1.0 μM), trametinib only (0.2 μM), and the combination of abemaciclib (1.0 μM) and trametinib (0.2 μM). (C) Representative images of colony formation after 7 days treatment of HNSCC cells with 1.0 μM abemaciclib, 0.2 μM trametinib, or both. (D) Whole-cell lysate western-blot analyses of RB and ERK proteins in HNSCC cells after treatment with 1.0 μM abemaciclib with or without 0.2 μM trametinib for 72 h. Quantitative data are shown as mean±SD of at least three different experiments *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS: not significant.
Dose and Combination Index (CI) values of the combination treatment of abemaciclib and trametinib in head and neck squamous cell carcinoma cells.
Results from colony-formation assays of CAL27 and SCC4 cells provided additional evidence supporting the synergistic effect of trametinib and abemaciclib. As shown in Figure 3C, while treatment of 1.0 μM abemaciclib for seven days led to statistically significant reductions in the colony-formation abilities of both CAL37 and SCC4 cells (p-values <0.01), the reductions in the colony-formation abilities of both cell lines appeared to be even larger after treatment with 0.2 μM trametinib (p-values <0.001; in comparison with treatment with 1.0 μM abemaciclib). Moreover, the combination of 0.2 μM trametinib and 1.0 μM abemaciclib led to additional reductions in the colony-formation abilities of both cell lines in comparison with either 1.0 μM abemaciclib only or 0.2 μM trametinib only (all p-values <0.05).
To explore the molecular mechanisms underlying the synergistic effect of trametinib and abemaciclib, we probed the expression of select proteins in the Rb and MEK pathways using western blotting. Our results revealed that treatment with abemaciclib (1.0 μM) for 72 h significantly decreased the phosphorylation of Rb in both CAL27 and SCC4 cells. However, such treatment also induced the phosphorylation of ERK1/2 proteins in these cells. The treatment with trametinib (0.2 μM) did not cause any notable change in RB levels nor the phosphorylation of RB protein but significantly decreased the phosphorylation of ERK1/2 proteins (Figure 3D). In the presence of both abemaciclib (1.0 μM) and trametinib (0.2 μM), the abemaciclib-mediated induction of phosphorylated ERK1/2 proteins was abolished, and the “overall” levels of phosphorylated ERK in both cell lines were low or undetectable (Figure 3D), implying that trametinib is potent in down-regulating abemaciclib-induced phosphorylation of ERK/12, thus synergistically enhancing the inhibitory activity of abemaciclib.
Trametinib and abemaciclib had synergistic antitumor effects in vivo. Lastly, we investigated the in vivo anti-tumor effects of abemaciclib and trametinib combination treatment in mice bearing xenograft tumors. The combination treatment effectively suppressed tumor growth compared to vehicle control and individual drug treatments in CAL27 xenografts (Figure 4A). We did not observe any toxicities with the treatments; mice tolerated the treatment throughout the entire experimental period (18 days) and their weight remained stable (Figure 4B). These results suggest that inhibition of the phosphorylation of ERK1/2 proteins by trametinib enhances abemaciclib antitumor action in the HNSCC xenograft model.
Trametinib and abemaciclib synergistically inhibit head and neck squamous cell carcinoma tumor growth in vivo. CAL27 cells were subcutaneously injected into the rear flank of athymic (Foxn1nu) nude mice to generate xenografts as previously described. When the tumor volume reached approximately 150 mm (Day 0), mice were randomized into 4 treatment groups (n=8 per group): the vehicle control group [1% Hydroxyethylcellulose (HEC) in PBS], the abemaciclib (50 mg/kg) group, the trametinib (1 mg/kg) group, and the group with the combination of abemaciclib (50 mg/kg) and trametinib (1 mg/kg). The tumor volumes (A) and body weights (B) were measured at the indicated time points. Quantitative data are shown as mean±SEM and were analyzed using one-way analysis of variance (ANOVA) with Tukey post hoc multiple comparisons at the 21-day point. *p<0.05, **p<0.01, ***p<0.001, NS: not significant.
Discussion
It is well known that the anticancer activity of CDK4/6Is results from their inhibition of CDK4/6-mediated phosphorylation, which consequently halts cell cycle progression at the G0/G1 to S transition (14-17). It has been also reported that CDK4/6Is are able to trigger antitumor immunity by stimulating the production of type III interferons and enhance tumor antigen presentation while suppressing regulatory T-cell proliferation (29, 30). However, a few studies have also demonstrated that the sensitivity to CDK4/6Is varies among cancer cells (or tumors). While CDK4/6Is exhibited little anti-tumor potency in HPV-positive HNSCC due to the inactivation of RB by HPV infection (12), CDK4/6I-induced quiescent/senescent cells were found to readily recover upon CDK4/6I treatment withdrawal in some studies (31, 32). Moreover, some cancer cells escaped from CDK4/6I inhibition and continued to progress through the cell cycle via diverse molecular mechanisms, including (but not limited to) the activation of mitogenic signaling through the up-regulation of cyclin D, cyclin E, CDK2 proteins, the MAPK and AKT/mTOR pathways, and the promotion of immune-related and epithelial-mesenchymal transition (EMT) pathways (33-36). Taken together, emerging clinical and preclinical data support the development of early adaptive and acquired resistance to CDK4/6 inhibition, which leads to a fundamental concern with CDK4/6I therapy, i.e., how to increase durable response and prevent resistance.
Previous studies in our laboratory as well as by other groups demonstrated that CDK4/6I treatment induced ROS generation and pro-survival autophagy in HNSCC, breast cancer, and multiple myeloma cells, thus promoting cancer progression (15, 36, 37). Interestingly, the combination of CDK4/6 and autophagy inhibitors (such as hydroxychloroquine) exhibited synergy in preventing cell/tumor growth in different cancer cell lines and preclinical models, suggesting that down-regulating autophagy may represent a potential mechanism to sensitize cancer cells to CDK4/6I treatment (15, 38-40).
Our previous study also showed that CDK4/6I treatment activated ERK phosphorylation in HNSCC cells and xenografts (15). In the present study, we reported that the combination of abemaciclib (CDK4/6I) and trametinib (a specific ERK1/2 inhibitor) potently repressed the growth of HNSCC cells and xenografts in a synergistic manner. The combination of ERK1/2 inhibitor and CDK4/6I presents a compelling therapeutic approach for HNSCC, exhibiting synergistic effects in cell viability, clonogenic potential, and in vivo tumor growth inhibition. Further preclinical and clinical investigations are warranted to validate these findings and assess the translational potential of this combination therapy in the treatment of HNSCC and other cancers.
Acknowledgements
This work was partially supported by a startup research grant from the College of Pharmacy, The Ohio State University (MP). The Authors thank Zachary VanGundy (vangundy.11{at}osu.edu) for his assistance on animal experiments.
Footnotes
Authors’ Contributions
Ernest Duah: development of methodology, collecting, analyzing and interpreting data, writing, conceptualization. Ming Poi: analyzing and interpreting data, writing, conceptualization, supervision. Junan Li: analyzing and interpreting data, writing, conceptualization, supervision.
Conflicts of Interest
The Authors declare no known conflicts of interest in relation to this study.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received April 22, 2025.
- Revision received April 28, 2025.
- Accepted April 30, 2025.
- Copyright © 2025 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.