Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma
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.
References (32)
- et al.
Pleiotrophin signaling through anaplastic lymphoma kinase is rate-limiting for glioblastoma growth
J Biol Chem
(2002) - et al.
Enhanced antitumorigenic effects in glioblastoma on double targeting of pleiotrophin and its receptor ALK
Neoplasia
(2009) - et al.
Cannabinoids attenuate cancer pain and proliferation in a mouse model
Neurosci Lett
(2011) - et al.
Clinical significance of genetic alterations and expression of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas
Oral Oncol
(2011) - et al.
Tissue microarray analysis reveals the expression and prognostic significance of phosphorylated AktThr(3)(0)(8) in oral squamous cell carcinoma
Oral Surg Oral Med Oral Pathol Oral Radiol
(2013) - et al.
Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family
Cell Biosci
(2012) Cancer facts and figures
(2015)Cancer facts and figures
(2012)- et al.
Epigenetic deregulation of the anaplastic lymphoma kinase gene modulates mesenchymal characteristics of oral squamous cell carcinomas
Carcinogenesis
(2013) - et al.
EML4-ALK: honing in on a new target in non-small-cell lung cancer
J Clin Oncol
(2009)
Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology
J Mol Diag
Anaplastic lymphoma kinase: role in cancer pathogenesis and small-molecule inhibitor development for therapy
Expert Rev Anticancer Ther
Activation of anaplastic lymphoma kinase is responsible for hyperphosphorylation of ShcC in neuroblastoma cell lines
Oncogene
Significance of ALK gene expression in neoplasms and normal tissues
Ai Zheng
ALK receptor activation, ligands and therapeutic targeting in glioblastoma and in other cancers
Front Oncol
Identification of oncogenic point mutations and hyperphosphorylation of anaplastic lymphoma kinase in lung cancer
Neoplasia
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