Elsevier

Oral Oncology

Volume 59, August 2016, Pages 12-19
Oral Oncology

Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma

https://doi.org/10.1016/j.oraloncology.2016.05.007Get rights and content

Highlights

  • Late-stage OSCC express high levels of activated ALK protein compared to early-stage tumors.

  • Gefitinib reduces invasive HSC3 tumor growth in vivo.

  • Co-targeting ALK and EGFR additively and significantly reduces HSC3 tumor growth.

  • Co-treating OSCC cells with ALK inhibitor (TAE684) and EGFR inhibitor (Gefitinib) abolishes AKT activation.

  • TAE684 up-regulates p-STAT3 in OSCC cell lines.

Summary

Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14 days (p < 0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the major malignancy of the oropharynx and the sixth most common cancer in the world [1]. The incidence of HNSCC is increasing worldwide. Currently, HNSCC patients are treated with surgery, chemotherapy, radiotherapy or combination. However, these treatment modalities are less effective with advanced disease involving lymph node invasion, which is associated with a five-year survival as low as 34% [2]. A major factor in decreased survival is resistance to chemotherapy. New lines of therapy are therefore needed to improve the survival rate of HNSCC patients.

In the effort to discover novel molecular factors that contribute to advanced HNSCC and may serve as therapeutic targets, our recent studies demonstrated that anaplastic lymphoma kinase (ALK) is epigenetically deregulated in late-stage, oral SCC (OSCC) and may play a role in OSCC invasiveness [3]. We found that invasive OSCC tumors with lymph node metastasis exhibited significantly lower ALK promoter methylation compared to non-invasive OSCC, suggesting that differential ALK promoter methylation affecting expression may predict the development of metastatic OSCC [3]. Correspondingly, our analysis of head and neck cancer cohort in The Cancer Genome Atlas revealed a significant increase (p < 0.01) of ALK expression in tumors compared to normal controls [3]. These studies suggested that ALK may be a potential therapeutic target for advanced OSCC.

ALK was identified as a therapeutic target for non-small cell lung cancer (NSCLC) in 2007 when Soda and colleagues discovered an inversion on chromosome arm 2p resulting in fusion of the 5′ end of echinoderm microtubule-associated protein-like 4 (EML4) to 3′ ALK yielding the EML4-ALK fusion gene [4], [5]. However full-length ALK expression due to point mutations and constitutive activation were reported in numerous cancers including neuroblastoma, neuroectodermal tumors, melanoma and glioblastoma [5], [6], [7], [8], [9], [10], [11], [12]. NSCLCs expressing EML4-ALK fusion protein respond very well to the ALK small molecule inhibitor, Crizotinib. However, a subgroup eventually develops resistance to ALK inhibition via induction of epidermal growth factor receptor (EGFR) bypass signaling which, like ALK, signals through AKT, RAS, and STAT3 [12], [13], [14], [15].

Approximately 90% of HNSCC express EGFR, yet EGFR inhibitors have yielded little to no efficacy in clinical trials [16], [17]. Studies evaluating Erlotinib, a small molecule inhibitor of EGFR, found that a mere 10–15% of oral cancer patients with stage I disease responded to Erlotinib [18]. Moreover, patients with stages II, III, and IV disease failed to respond entirely [18]. The reason for these unexpected failures of EGFR targeted therapies remains unclear. We hypothesize that parallel signaling pathways may compensate for EGFR inhibition rendering these treatments ineffective. In this study we tested the hypothesis that EGFR inhibition is thwarted by ALK parallel signaling in tumors that progress clinically. We assessed the effects of ALK and EGFR inhibition in ALK expressing OSCC cell lines and in a mouse OSCC xenograft model. We further demonstrated anti-proliferative, anti-mobility, and signaling effects in vitro and significant tumor growth inhibition in vivo.

Section snippets

Human OSCC cell lines

OSCC cell lines, HSC3 and Cal27 were derived from human primary tongue OSCC. Cal27 cells were obtained from ATCC (Rockville, MD). HSC3 cells were kindly provided by Dr. Brian Schmidt (NYU) [19] and authenticated by Genetica DNA Laboratories (Cincinnati, OH). Cells were maintained in DMEM (Gibco, Carlsbad, CA) containing 10% FBS at 37 °C in 5% CO2.

Reagents

Gefitinib (EGFR inhibitor) and TAE684 (ALK inhibitor) were obtained from Selleck Chemicals (Houston, TX). TAE684 (50 mg/ml) and Gefitinib (5 mg/ml) were

Activated ALK is expressed in late-stage human OSCC and in OSCC cell lines/xenografts

Our previous studies showed differential expression in ALK was associated with late-stage OSCC [3]. We also showed that OSCC cell lines exhibit different levels of ALK mRNA expression, which corresponded with cell invasiveness [3]. To further examine the role of ALK in OSCC, we examined expression of activated ALK (the phosphorylated form, phospho-ALK) in a human OSCC tissues containing early-stage (stage I) and late-stage (stage 4) OSCCs and in OSCC cell lines, HSC3 and Cal27. IHC analysis of

Discussion

The invasion of OSCC into loco-regional structures is a deadly consequence for patients with this malignancy. As of yet, effective therapies that target invasive OSCC are lacking. Although EGFR is expressed in the majority of OSCCs, drugs targeting this tyrosine kinase receptor have surprisingly led to little effect. Although this might be due to increased copy number of EGFR, which is associated with poor prognosis in head and neck cancer, studies showed that EGFR gene amplification, polysomy

Conflict of interest statement

The authors have no conflict of interest.

Acknowledgements

This work was supported by the University of Texas Health Science Center of San Antonio (UTHSCSA), Cancer Therapy and Research Center (CTRC), Experimental and Developmental Therapeutics Pilot Award and The Owen’s Foundation Cancer Research Pilot Award. Immunohistochemistry was performed by the CTRC Core Pathology Laboratory supported by the NCI Cancer Center Support Grant (P30 CA054174). Aaron Horning was supported by a predoctoral fellowship from NCI-funded NRSA T32CA148724.

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