Activation of the insulin-like growth factor-1 receptor alters p27 regulation by the epidermal growth factor receptor in oral squamous carcinoma cellsby Mark J. Jameson, Linnea E. Taniguchi, Kyle K. VanKoevering, Menachem M. Stuart, Christian R. Francom, Rolando E. Mendez, Andrew D. Beckler, Hans T. Carlson, Christopher Y. Thomas, Ashraf A. Khalil

J Oral Pathol Med



Epidermal growth factor receptor expression in squamous cell carcinoma of the hypopharynx

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Alcides Chaux, Enrico Munari, Sheila Faraj, Nilda Gonzalez-Roibon, Rajni Sharma, Antonio Cubilla, George Netto

P200 Association of the syalilation pattern and expression profile of two different epidermal growth factor receptors with degree malignization of oral squamous cell carcinoma

L.F. Jacinto-Alemán, J.C. Hernández-Guerrero, C. Trejo-Solís, H. Domínguez-Malagon, C. Ledesma-Montes


Activation of the insulin-like growth factor-1 receptor alters p27 regulation by the epidermal growth factor receptor in oral squamous carcinoma cells

Mark J. Jameson1, Linnea E. Taniguchi1, Kyle K. VanKoevering1, Menachem M. Stuart1, Christian R.

Francom1, Rolando E. Mendez1, Andrew D. Beckler1,3, Hans T. Carlson1, Christopher Y. Thomas2,4,

Ashraf A. Khalil1 1Division of Head and Neck Surgical Oncology, Department of Otolaryngology – Head and Neck Surgery, University of Virginia Health

System, Charlottesville, VA, USA; 2Division of Hematology and Oncology, Department of Medicine, University of Virginia Health System,

Charlottesville, VA, USA; 3Present address: Department of Otolaryngology – Head and Neck Surgery, Vanderbilt University, Nashville,

TN, USA; 4Present address: Department of Hematology and Oncology, Wake Forest School of Medicine, Winston-Salem, NC, USA

BACKGROUND: Although oral squamous cell carcinomas (OSCCs) commonly overexpress the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors (TKIs) exhibit poor efficacy clinically. Activation of the insulin-like growth factor-1 receptor (IGF1R) induces resistance of OSCC cells to EGFR-TKIs in vitro.

This study seeks to evaluate the changes in cell cycle status inOSCCcells in response togefitinib and IGF1Ractivation.

METHODS: SCC-25 OSCC cells were used for in vitro analyses.

RESULTS: Gefitinib caused a 50% reduction in S-phase population, and IGF1R activation caused a 2.8-fold increase; combined treatment yielded a baseline S-phase population. Gefitinib treatment increased the cyclindependent kinase inhibitor p27, and this was not abrogated by IGF1R activation. pT157-p27 was noted by immunoblot to be decreased on gefitinib treatment, but this was reversed with IGF1R activation. T157 phosphorylation contributes to cytoplasmic localization of p27 where it can promote cell proliferation and cell motility.

Using both subcellular fractionation and immunofluorescence microscopy techniques, IGF1R stimulation was noted to increase the relative cytoplasmic localization of p27; this persisted when combined with gefitinib.

CONCLUSIONS: IGF1R activation partially reverses the cell cycle arrest caused by gefitinib in OSCC cells. While

IGF1R stimulation does not eliminate the gefitinibinduced increase in total p27, its phosphorylation state and subcellular localization are altered. This may contribute to the ability of the IGF1R to rescue OSCC cells from EGFR-TKI treatment and may have important implications for the use of p27 as a biomarker of cell cycle arrest and response to therapy.

J Oral Pathol Med (2013) 42: 332--338

Keywords: cell cycle regulation; cyclin-dependent kinase inhibitor; growth factor; oral cavity cancer; therapeutic resistance


The epidermal growth factor receptor (EGFR) is an established therapeutic target in head and neck squamous cell carcinoma (HNSCC). Several targeted anti-EGFR agents have been developed, but their efficacy in HNSCC is limited due to frequent intrinsic or acquired resistance. To date, molecular markers that predict sensitivity of HNSCC to anti-EGFR drug have not been identified (1). However,

EGFR inhibition in HNSCC is known to cause cell cycle arrest, which is dependent on increased p27 expression (2). p27 is a cell cycle regulator that was initially identified as a CDK inhibitor of the cyclin E/CDK2 complex (3). It mediates cell cycle arrest by impairing the ability of cyclin E to promote G1-S transition. This function is regulated primarily by the amount of nuclear p27; p27 degradation via ubiquitination causes increased cyclin E activity and cell cycle progression. Thus, p27 expression has been interpreted as a surrogate for overall cell cycle status. Studies of

HNSCC have correlated greater disease burden and/or poorer survival outcomes with decreased p27 expression at multiple subsites, including oral cavity (OSCC; 4–6), oropharynx (6), larynx (7), and hypopharynx (8).

More recently, detailed study of p27 has demonstrated additional functional roles and highly complex regulation. p27 can inhibit or promote cell cycle progression based on

Correspondence: Mark J Jameson, MD, PhD, Division of Head and Neck

Surgical Oncology, Department of Otolaryngology – Head and Neck

Surgery, University of Virginia Health System, PO Box 800713,

Charlottesville, VA 22908-0713, USA. Tel: 1 434 924 2040, Fax: 1 434 982 3965, E-mail:

Accepted for publication September 17, 2012

J Oral Pathol Med (2013) 42: 332–338 © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd doi: 10.1111/jop.12014 protein level and subcellular localization [for review, see

Wander et al. (9)]. Serine, threonine, and tyrosine phosphorylation of p27 regulate its protein–protein interactions, nuclear import/export, and degradation, thus impacting its function. Phosphorylation at threonine-157 (pT157) results in accumulation in the cytoplasm where p27 interacts with cyclin D/CDK4 and RhoA to increase cell cycle progression and cell migration, respectively; in the setting of malignancy, these could portend therapeutic resistance and metastasis (10,11). Thus, the interpretation of changes in total p27 expression in response to treatmentwith a targeted therapeutic agent is unlikely to correlate directly with biologic outcome.

We have recently demonstrated that, in OSCC cell lines, the activation of the insulin-like growth factor-1 receptor (IGF1R) can overcome the growth inhibition caused by

EGFR tyrosine kinase inhibitors (TKIs; 12). Although there is no established marker of this phenomenon, IGF1Rinduced resistance to EGFR-TKIs was associated with increased Akt activity and reduced apoptosis. Noting that (i) the growth inhibitory (nuclear) function of p27 is required for EGFR-TKI efficacy, (ii) IGF1R activation causes resistance to EGFR-TKIs, (iii) the IGF1R is a potent activator of Akt, and (iv) Akt phosphorylates p27 at T157 with resultant cytoplasmic sequestration of p27 and cell cycle progression, we evaluated regulation of p27 by