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. Author manuscript; available in PMC: 2022 Jul 4.
Published in final edited form as: Head Neck. 2021 Feb 3;43(6):1721–1729. doi: 10.1002/hed.26633

Mitogen-activated protein kinase inhibition-induced modulation of epidermal growth factor receptor signaling in human head and neck squamous cell carcinoma

Guiqin Xie 1,2, Ailin Zhu 1, Xinbin Gu 1,2
PMCID: PMC9252597  NIHMSID: NIHMS1807736  PMID: 33533173

Abstract

Background:

Epidermal growth factor receptor (EGFR) overexpression is one of the most notable characteristics in head and neck squamous cell carcinoma (HNSCC). The MAPK kinase (MEK) inhibitor trametinib has shown efficacy to treat HNSCC; however, the molecular mechanism remains unclear.

Methods:

HNSCC lines, mouse models, Western blot, and flow cytometry were employed to analyze the anticancer effects of trametinib.

Results:

The JHU-011, JHU-022, and JHU-029 HNSCC cells with different genetic alterations were highly susceptible to trametinib. Trametinib effectively reduced EGFR expression, which was accompanied by the reduction of pro-survival protein MYC, and the increased expression of a MYC-targeted cyclin-dependent kinase inhibitor p27kip1 and pro-apoptotic protein BIM. Trametinib resulted in G1 arrest of the cells, markedly reduced cell numbers in S phase, and significantly increased apoptosis. In mouse models, trametinib strongly inhibited tumors growth.

Conclusions:

The MAPK–ERK signaling inhibition by trametinib may target EGFR and the downstream proteins against HNSCC.

Keywords: EGFR, head and neck squamous cell carcinoma, MEK inhibitor, MYC, trametinib

1 |. INTRODUCTION

In the United States, ~37 000 new cases are diagnosed with head and neck squamous cell carcinoma (HNSCC) every year. Despite therapeutic advances, the 5-year survival rate remains 55%–65%.1,2 Epidermal growth factor receptor (EGFR) is a membrane-bound tyrosine kinase receptor that regulates cell proliferation and migration. EGFR overexpression in HNSCC is one of the most notable characteristics.3 The mitogen-activated protein kinase (MAPK) pathway plays critical roles in cellular proliferation and survival of cancer cells. The MAPK pathway consists of RAS, RAF, mitogen-activated protein kinase (MEK), and extracellular signal-regulated kinases (ERK), which transmits proliferative signals generated at cell surface receptors and through cytoplasmic signaling into the nucleus.4 Induction of MEK–ERK has been implicated in established HNSCC cell lines and an ex vivo tissue culture model.5–7 Overexpressed EGFR elevates MAPK–ERK signaling to drive tumor cell proliferation and cancer progression.8 Cetuximab, an EGFR monoclonal antibody, is the only Food and Drug Administration (FDA)-approved agent against EGFR for the treatment of HNSCC. However, cetuximab monotherapy has been quite disappointing, achieving only a 10%–30% response rate.9 Furthermore, patients who achieve a clear clinical tumor response eventually progress due to acquired cetuximab resistance.9,10 There is an unmet need to explore more effective targeted therapy for HNSCC.

In the MAPK pathway, MEK is the kinase that relays the signaling from surface receptors and upstream kinase to the downstream ERK.11 Trametinib is a potent inhibitor of MEK1 and MEK2 kinases to prevent Raf-dependent MEK phosphorylation, producing prolonged p-ERK1/2 inhibition.12 Trametinib has been approved by the FDA for the treatment of patients with advanced melanoma, thyroid cancer, and metastatic nonsmall cell lung cancer with a BRAF mutation.13,14 Although the mutations of the BRAF gene in HNSCC are rare, it was clearly shown that trametinib may have efficacy to treat oral squamous cancer in a clinical trial of an oral squamous cancer.15 However, the mechanism of the response of HNSCC to trametinib remains to be defined.

In the current study, we evaluated efficacy of trametinib to treat HNSCC and explored the mechanisms of the trametinib-induced cell proliferation and tumor growth inhibition. Our data indicated that MEK inhibition may target EGFR and its downstream effectors to inhibit cell proliferation and tumor growth. Thus, targeting MEK with trametinib may provide a novel approach to inhibit EGFR signaling against HNSCC.

2 |. MATERIALS AND METHODS

2.1 |. Cell culture

Three HNSCC cell lines, JHU-011 (larynx, p53 mutated), JHU-022 (larynx, wild type of p53), and JHU-029 (tongue, wild type of p53), were obtained from the Johns Hopkins University.16,17 The HNSCC lines were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution in 5% CO2 at 37°C in a humidified incubator.

2.2 |. Cell viability assay

The HNSCC cells were plated 3000–5000/well in 96-well plates and treated with trametinib following a threefold serial dilution. Three days later, cell viability was measured using a cell cytotoxicity and cell viability assay kit (#ab232855; Abcam, Cambridge, MA). Briefly, crystal violet staining assay was performed to determine cell viability according to the manufacturer’s instruction. After removal of the culture medium and gently washed with washing solution, crystal violet staining solution (50 μL) was added to each well, and incubated for 20 min at room temperature. After thorough washing, solubilization solution was added and incubated again for 20 min at room temperature on a benchtop rocker. Optical density (OD) of each well was measured at 595 nm. Data were represented in percentage of viable (attached) cells calculating against the values of vehicle-treated cells.18

2.3 |. Western blot analysis

For the HNSCC cells, cells cultured in 6 wells were collected and washed with phosphate-buffered saline. Collected cells were homogenized in a solution with 50 mM Tris buffer, 150 mM NaCl, 1 mM EDTA, 1% NP40, and proteinase–phosphatase inhibitors. Homogenized cells were centrifuged at 13 000 rpm for 3 min, lysates were transferred to a new Eppendorf tube. The protein concentrations of lysates were quantified and 35 μm of lysates was used for Western blot analysis. The antibody against MYC (ab32072) was purchased from Abcam. The antibodies against p-ERK1/2 (#9101), total-ERK (#9102), BIM (#2933), p27kip1 (p27) (#3686), and β-actin (#4970) were purchased from Cell Signaling (Beverly, MA). Horseradish peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody and specific protein bands were detected by means of an ECL system. Specific protein band intensities were quantified by the ImageJ software. For each protein from the cells treated with vehicle or trametinib, ratio of band intensity to β-actin was first calculated, then normalized to the mean of the ratios of band intensity to β-actin ratio from three independent experiments in vehicle-treated cells.

2.4 |. Flow cytometric analysis of cell cycle

Cells were seeded into six-well plates at a density of 1–4 × 105 cells/well 24 h earlier before treatment. Cells were treated with vehicle or trametinib (500 nM) for 24 h (JHU-011) or 48 h (JHU-022 or JHU-029). After treatment, cells were washed with ice-cold PBS and then fixed with cold 70% ethanol and kept at −20°C at least 2 h. Cells were washed in PBS buffer and resuspended in a PBS buffer at a density of 1 × 106 cells/mL with 0.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, MA). Analysis of cell cycle phase distribution was performed on a BD flowcytometer (BD Bioscience, San Jose, CA) and the data were analyzed using ModFit LT 3.0 program (Verity Software House, Topsham, ME). All experiments were performed in at least triplicate.

2.5 |. Flow cytometry apoptosis assay

After incubation with vehicle or trametinib (500 nM) for 48 h, cells were collected and incubated with annexin V-FITC and propidium iodide (PI) solutions in the dark for 15 min before flow cytometry assay using a BD flowcytometer (BD Bioscience). Annexin V-positive and PI-negative cells and annexin V-positive and PI-positive cells were regarded as apoptotic cells. The percentage of apoptotic cells was analyzed via FlowJo software (FlowJo LLC, Ashland, OR).

2.6 |. In vivo xenograft tumor assays

Six- to eight-week-old NOD scid gamma mouse (NSG) mice were used for xenograft studies. Five million of the cells in a volume of 200 μL medium containing 45% Matrigel basement membrane matrix (BD Biosciences; Cat. No. 354234) were inoculated subcutaneously into the right flank of mice 2–3 weeks before treatment. The treatment with vehicle or trametinib started when the median tumor size reached approximately 100 mm3. Trametinib was administered via oral gavage at a dose of 1 mg/kg/mouse per day. The tumor size was measured with a caliper daily. After 14 days of treatment, mice with tumors were euthanized and the tumors were dissected for analysis.

2.7 |. Statistical analysis

Student’s t test and one-way ANOVA were used for statistical analysis. All data are expressed as mean ± SD. All tests were two-sided and p < 0.05 was considered significant.

3 |. RESULTS

3.1 |. Trametinib inhibits cell viability in human HNSCC cell lines

To test whether trametinib exhibits efficacy against human HNSCC, we evaluated the effects of trametinib on the proliferation of three cell lines: JHU-022, JHU-011, and JHU-029 cells. All the three cell lines do not carry a BRAFV600E mutation. As shown in Figure 1(A), trametinib dose-dependently decreased cell viability of the JHU-022 cells. In the JHU-011, JHU-022, and JHU-029 cells, we found that trametinib decreased cell viability with an IC50 value of 49.8, 74.1, and 612.0 nM, respectively (Figure 1(B)). Our findings indicated that trametinib treatment inhibits the proliferation of HNSCC cells regardless of the difference in the mutation background in these three cell lines.

FIGURE 1.

FIGURE 1

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Sensitivity of head and neck squamous cell carcinoma (HNSCC) cells to trametinib. (A) Dose-escalation effects of trametinib on cell viability in JHU-022 cells. The cells were exposed to vehicle or trametinib with increased doses for 4 days. Data are shown of two duplicates. (B) IC50 of JHU-011, JHU-022, and JHU-029 cells to trametinib

3.2 |. Trametinib reduces ERK signaling in HNSCC cell lines

To validate whether trametinib effectively inhibits activity of the MEK kinase, the HNSCC cell lines were treated with trametinib (500 nM) or vehicle for 48 h. MEK activity reflected by the level of active phosphorylated ERK (p-ERK) was determined by Western blot analysis (Figure 2(A)). p-ERK level was drastically decreased in the JHU-011, JHU-022, and JHU-029 cells, while total ERK levels were not changed by trametinib in these cells (Figure 2(A)). The ratio of the p-ERK to total ERK was significantly reduced in all of the three HNSCC cells (Figure 2(B)). The results validated that trametinib suppresses the ERK signaling in HNSCC cell lines.

FIGURE 2.

FIGURE 2

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Trametinib decreases phosphorylated extracellular signal-regulated kinases (p-ERK) in JHU-011, JHU-022, and JHU-029 cells. (A) Total protein extracts were prepared from the cells treated with vehicle or trametinib (500 nM). Western blot analysis was performed for p-ERK (a), total ERK (b), and β-actin (c) in JHU-011, JHU-022, and JHU-029 cells treated with vehicle or trametinib for 48 h. (B) Protein band intensities were quantified for comparisons between the vehicle-treated cells and the trametinib-treated cells. Data of three independent experiments are presented as mean ± SD (n = 3)

3.3 |. Trametinib inhibits EGFR and its downstream effectors in HNSCC cell lines

To explore the mechanism of the inhibition of cell proliferation by trametinib, we investigated key proteins for cell proliferation. EGFR is critical for cell proliferation and EGFR overexpression is one of the most notable characteristics in HNSCC. We found that treatment with trametinib (500 nM) reduced EGFR protein in all the three HNSCC cells (Figure 3(Aa), (B)). MYC is an oncoprotein critical in cell proliferation and suppression of MYC is essential for MEK inhibitor to induce inhibition in cell proliferation.19 In the JHU-011, JHU-022, and JHU-029 cells, Western blot analysis showed that the level of MYC protein was significantly suppressed in trametinib-treated cells compared to vehicle-treated cells (Figure 3(Ab), (C)). One of the mechanisms for MYC to promote cell proliferation is to antagonize the activity of cell cycle inhibitors, such as p27.20 Indeed in the JHU-011, JHU-022, and JHU-029 cells, the levels of p27 protein were significantly increased in trametinib-treated cells compared to vehicle-treated cells (Figure 3(Ac), (D)). It is known that downregulated MYC may be resulted in increased apoptosis. BIM is a proapoptotic protein in the Bcl-2 protein family that it alone is sufficient to induce apoptosis.21 Upregulation of BIM signifies the therapeutic outcomes of target inhibition.22,23 In these three cells, we found that BIM was significantly increased in the trametinib-treated cells than in the vehicle-treated cells (Figure 3(Ad), (E)). These results indicated that trametinib may target EGFR and its downstream effectors to suppress cell proliferation in HNSCC cells.

FIGURE 3.

FIGURE 3

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Trametinib decreases epidermal growth factor receptor (EGFR) and MYC expression but increases p27 and BIM expression JHU-011, JHU-022, and JHU-029 cells. (A) Total protein extracts were prepared from the cells treated with vehicle or trametinib (500 nM). Western blot analysis was performed for EGFR (a), MYC (b), p27 (c), BIM (d), and β-actin (e) in the cells treated with vehicle or trametinib for 48 h. (B) Band intensities of EGFR were quantified for comparisons. (C) Band intensities of MYC were quantified for comparisons. (D) Band intensities of p27 were quantified for comparisons. (E) Band intensities of BIM were quantified for comparisons. Data of three independent experiments are presented as mean ± SD (n = 3)

3.4 |. Trametinib inhibits cell proliferation via cell cycle arrest and apoptosis induction

Because trametinib increased p27, which is critical for cell cycle progression, we examined the effects of trametinib on cell cycle in the three HNSCC lines. In the JHU-011 cells, the treatment increased the cells in the G1 phase from 57.6% with vehicle treatment to 78.3%, reduced the cells in the S phase and G2 from 37.9% and 4.5% with vehicle treatment to 19.7% and 2.0%, indicating that trametinib induced G1 arrest (Figure 4(A), (B)). In the JHU-022 cells treated with trametinib, the cells in the G1 phase were increased from 80.1% with vehicle treatment to 92.9%, while the cells in the S and G2 phases were reduced from 16.9% and 3.0% with vehicle treatment to 4.7% and 2.1% (Figure 4(C)). Similarly, in the JHU-029 cells treated with trametinib, the cells in the G1 phase were increased from 76.5% with vehicle treatment to 97.9%, while the cells in the S and G2 phases were reduced from 19.4% and 4.4% to 2.1% and 0.1%, respectively, with vehicle treatment (Figure 4(D)).

FIGURE 4.

FIGURE 4

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Trametinib treatment increased cell number in G1 phase and reduced cell numbers in S and G2 phases. (A) Trametinib induced cell cycle arrest in the JUH-011 cells. The cells were treated with vehicle or trametinib (500 nM) for 24 h. Flow cytometric analysis of cell cycle phases was carried out after 4′,6-diamidino-2-phenylindole (DAPI) staining. (B) Cell cycle phase analysis of the JUH-011 cells treated with vehicle or trametinib for 24 h. (C) Cell cycle phase analysis of the JUH-022 cells treated with vehicle or trametinib for 48 h. (D) Cell cycle phase analysis of the JUH-029 cells treated with vehicle or trametinib for 48 h. Data of three independent experiments are presented as mean ± SD (n = 3)

Because BIM alone is sufficient to induce apoptosis, we next evaluated apoptotic events in the JHU-022 cells. With trametinib treatment, we found that the apoptotic cells were increased from 8.74% (Figure 5(Aa), (B)) with vehicle treatment to 36.4% (Figure 5(Ab), (B)). We further confirmed the apoptotic events induced by trametinib in JHU-029: the apoptotic cells were increased from 3.55% (Figure 5(Ca), (D)) with vehicle treatment to 12.83% (Figure 5(Cb), (D)). Thus, trametinib may be responsible for cell cycle arrest and apoptosis.

FIGURE 5.

FIGURE 5

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Trametinib induces apoptosis in the JHU-022 and JHU-029 cells. Cells were treated with vehicle or trametinib (500 nM) for 48 h and collected for annexin V and propidium iodide (PI) staining followed by flow cytometric analysis for apoptotic cells in JHU-022 (A) and JHU-029 (C). The populations of apoptotic cells were quantified for JHU-022 (B) and JHU-029 (D). Data of three independent experiments are presented as mean ± SD (n = 3)

3.5 |. Treatment of trametinib inhibits the growth of JHU-022 tumors in mice

Because trametinib treatment inhibited proliferation and induced cell death of cultured JHU-022 cells, we assessed the effects in vivo using a mouse xenograft model. We evaluated trametinib-affected tumor growth of the JHU-022 cells in NSG mice. Trametinib suppressed the tumor growth (Figure 6(A)) and reduced the tumor size (Figure 6(B)) as well as the tumor weight (Figure 6(C)). Compared to tumor volumes at the time when the treatment was initiated, tumor volumes of the mice treated with trametinib were stabilized 6 days after trametinib treatment, while tumor volume of the mice treated with vehicle continuously increased (Figure 6(A)). No significant difference was observed in the body weight between the vehicle-treated and trametinib-treated mice during 2 weeks of treatment (Figure 6(D)). The in vivo data supported that trametinib effectively inhibits the growth of HNSCC tumors in mice.

FIGURE 6.

FIGURE 6

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Trametinib treatment inhibits tumor growth of JHU-022 cells in NOD scid gamma mouse (NSG) mice. (A) When the tumors reached an average size of 100 mm3, vehicle or trametinib (1 mg/kg/mouse, once daily) was administered for 14 days. Tumor growth curves plotted for a 14-day treatment period with vehicle or trametinib. (B) Images of the gross tumors (a) and dissected tumors (b) from mice treated with vehicle or trametinib (C) Average weight of the dissected tumors from NSG mice after 14-day treatment. (D) Body weight curves plotted for a 14-day treatment period with vehicle or trametinib. Data are presented as mean ± SD (n = 5)

4 |. DISCUSSION

EGFR overexpression in HNSCC is identified in the majority of head and neck squamous tumors.24 MAPK signaling pathway is a critical signaling pathway activated by EGFR for the development and progression of HNSCC.10,25,26 Our in vitro and in vivo findings revealed that targeting MEK using trametinib to block the MAPK signaling suppressed EGFR and its downstream effector MYC, which is critical for regulation of cell proliferation, while upregulated BIM proteins critical for apoptosis. We demonstrated that trametinib was capable to remarkably inhibit cell growth in all the three HNSCC cells and significantly suppressed JHU-022 tumor growth in a mouse xenograft model. Our data strongly support that trametinib-induced inhibition of MAPK–ERK signaling can be effective to treat HNSCC through modulation of EGFR–MAPK–ERK signaling.

Trametinib has been approved for the treatment of patients in several types of cancers with a BRAF mutation.13,14 However, HNSCC with a BRAF mutation is not common. The efficacy of trametinib to treat cancers without a BRAF mutation is not well defined yet. In our studies, all of the three HNSCC cells tested have no BRAFV600E mutation in COSMIC cell lines database. Nevertheless, our data demonstrated that trametinib has efficacy against all the three cells, suggesting that directly targeting MEK with trametinib could be effective to treat HNSCC. The efficacy of trametinib to treat tumors with no BRAFV600E mutation is not without precedent.27 It was reported that lung adenocarcinoma with mutations in ataxia-telangiectasia mutated is hypersensitive to trametinib.28 Our data indicate that overactivated EGFR signaling may also render cancer cells susceptible to MAPK–ERK inhibition.

Evidence shows that MEK/ERK inhibitor rapidly induces a reduction in MYC protein expression in rhabdomyosarcoma.29 In KRAS-mutant pancreatic cancer, long-term ERK inhibition is associated with MYC degradation.30 Reduced MYC expression may result in several changes that led to decreased EGFR signaling, cell proliferation, and apoptosis. MYC has been identified as a universal amplifier of expressed genes.31 Therefore, reduced MYC may be responsible for the reduced EGFR expression. MYC directly regulates cell cycle inhibitor p27 expression. It was reported that suppression of MYC is essential for MEK inhibitor to induce inhibition of cell proliferation.19 Therefore, reduced MYC may increase p27 to inhibit cell cycle progression. In addition, reduced MYC could also cause the increase in the BIM protein. BIM is a proapoptotic protein in the BCL-2 protein family that it alone is sufficient to induce drug-induced apoptosis.21 These observations implicate that trametinib-induced MEK inhibition seems to be necessary and sufficient to effectively inhibit EGFR signaling, resulting in apoptotic response and suppression of cell cycle progression, cell proliferation as well as tumor growth.

Our observation that trametinib was effective to treat HNSCC through modulation of EGFR–MAPK–ERK signaling has potential implications. Activation of MAPK–ERK pathways is one of the mechanisms limiting the effectiveness of kinase inhibitors.8 Trametinib may be used to enhance the efficacy of other inhibitors in several types of cancers including HNSCC.32 Dabrafenib is an inhibitor of mutant BRAF V600E that is the upstream kinase of MEK in the MAPK pathway. The adjuvant treatment with dabrafenib and trametinib increases therapeutic efficacy in melanoma and thyroid cancer.33,34 Similar strategy may be employed in a combination of cetuximab to increase the efficacy to treat HNSCC.

In summary, our findings strongly support that directly targeting MEK to block MAPK signaling with trametinib may be a highly feasible approach to treat HNSCC. The treatment of trametinib together with other inhibitors such as cetuximab may provide a more effective treatment in HNSCC.

ACKNOWLEDGMENTS

This work was supported by the National Institute of Health (R15DE025138, R25DE025778).

Footnotes

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

ETHICAL APPROVAL

All animal experiments were performed under protocols approved by the Howard University Animal Care and Use Committee.

DATA AVAILABILITY STATEMENT

All data generated or analyzed during this study are included in this published article.

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Data Availability Statement

All data generated or analyzed during this study are included in this published article.

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