Cancer Letters

Cancer Letters

Volume 306, Issue 1, 1 July 2011, Pages 85-91
Cancer Letters

Constitutively active Harvey Ras confers resistance to epidermal growth factor receptor–targeted therapy with cetuximab and gefitinib

https://doi.org/10.1016/j.canlet.2011.02.035Get rights and content

Abstract

Kirsten Ras (K-Ras) mutations have been implicated as a key predictive marker of resistance to therapies targeting the epidermal growth factor receptor (EGFR). To determine whether Harvey Ras (H-Ras) mutations also can confer resistance to EGFR-targeted therapy, we expressed a constitutively active H-Ras (Ras G12V) in A431 human vulvar squamous carcinoma cells. Compared with corresponding control cells, A431-Ras cells exhibited marked resistance to the EGFR inhibitors cetuximab and gefitinib, reducing inhibition of Akt and Erk phosphorylation, inhibition of HIF-1α expression and transcriptional activity, and antitumor effects in vitro and in vivo. Our data indicate that constitutively active H-Ras can also confer resistance to anti-EGFR therapy in cancer cells.

Introduction

The epidermal growth factor receptor (EGFR), a 170-kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity [1], is often highly expressed in a variety of human tumors of epithelial origin, including cancers of the colon, lung, head and neck, esophagus, stomach, prostate, bladder, kidney, pancreas and ovary [2]. Activation of EGFR triggers signal transduction through several well-characterized downstream pathways, including the Ras/Raf/Erk, PI3K/Akt/mTOR, PLCγ, PKC, and JAK/STAT pathways, leading to gene transcription that is responsible for a variety of cellular functions [3]. For example, activation of several of these pathways leads to increased expression of hypoxia-inducible factor-1alpha (HIF-1α) [4], [5], [6], [7], [8], which forms a heterodimer with HIF-1β; together this protein complex regulates more than 100 targeted genes that are critical for both bioenergetic and biosynthetic metabolism in cancer cells and are responsible for many metastatic properties of cancer cells, such as angiogenesis, invasion, drug resistance, increased cell proliferation, and reduced apoptosis [9], [10].

Several therapeutic strategies to inhibit EGFR signaling have been devised, and five agents that target EGFR are approved for use in the treatment of patients with several types of cancers. These agents include the monoclonal antibodies cetuximab and panitumumab and the tyrosine kinase inhibitors gefitinib, erlotinib, and lapatinib (a dual inhibitor to EGFR and HER2) [2]. The antitumor mechanisms of these agents have been well explored, resulting in a large body of experimental evidence revealing that they prevent ligand-induced receptor activation and subsequently inhibit downstream signaling, resulting in cell cycle arrest, apoptosis induction, and angiogenesis inhibition in preclinical models; however, despite objective responses in some patients, the clinical benefits of these agents in prolonging patients’ overall survival have been modest [2]. Understanding of the mechanisms of tumor resistance to the EGFR-targeting agents has been an active topic of research in the area in recent years.

Resistance to EGFR-targeted therapy has been linked to Ras mutations. The Ras gene subfamily consists of the Harvey, Kirsten, and neuroblastoma Ras genes (H-Ras, K-Ras, and N-Ras), which encode proteins with GTP/GDP binding and GTPase activity [11], [12], [13], [14]. Ras proteins alternate between an inactive form bound to GDP (Ras-GDP) and an active form bound to GTP (Ras-GTP); the proteins are activated by a guanine nucleotide-exchange factor (GEF) and inactivated by a GTPase-activating protein (GAP) [15]. Functioning as molecular switches in regulating cell survival, proliferation, and differentiation, Ras proteins are essential mediators that convey extracellular signals from surface receptors to intracellular signaling pathways [16]. Oncogenic mutant Ras proteins are locked into constitutively GTP-bound conformation and thus are independent of EGFR signaling. Recent clinical studies have identified K-Ras mutations as a key predictive marker of resistance to EGFR-targeted therapy [17], [18], [19], [20], [21], [22], [23], [24], [25]. Indeed, due to the high percentage of K-Ras mutations in patients with colon cancer (ranging from 30% to 35%) [17], [18], [19], [20], [21], [22], [23], [24], [25] and the lack of response by these tumors to cetuximab, patient biopsy specimens are now routinely assessed for K-Ras mutations [26]. Metastatic colon cancer patients with K-Ras mutations are excluded from anti-EGFR therapy because of the likelihood of de novo resistance.

Despite their great biochemical and biological similarities, the Ras proteins may not be fully functionally identical or redundant [27], [28]. H-Ras mutations are less common than K-Ras mutations in human cancer; however, rates of H-Ras mutations are not negligible, and genetic analysis of tumor specimens has revealed H-Ras mutations in approximately 10–22% of head and neck cancers [29], [30], 11% of bladder cancers, 9% of cervical cancers, and in smaller percentages of several other cancers [31]. It is unknown whether H-Ras mutations, like K-Ras mutations, can confer resistance to EGFR-targeted therapy. In the present study, we examined the impact of the expression of constitutively active H-Ras on the antitumor effects of cetuximab and gefitinib both in vitro and in vivo.

Section snippets

Reagents

The EGFR-blocking monoclonal antibody cetuximab and the small molecule EGFR tyrosine kinase inhibitor gefitinib were provided by ImClone Systems (New York, NY, USA) and AstraZeneca (Wilmington, DE, USA), respectively [32]. Antibodies directed against total Akt, ser473-phosphorylated Akt, and the phosphorylated Erk p42/p44 were obtained from Cell Signaling Technology (Beverly, MA, USA). The rabbit anti-MAPK (Erk2) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The

Constitutively active H-Ras blocks cetuximab-mediated inhibition of Akt and Erk phosphorylation

Previous studies have demonstrated that EGFR-targeting agents such as cetuximab and gefitinib inhibit the activation-specific phosphorylation of EGFR downstream targets such as Akt and Erk [38], [39]. Therefore, we examined the ability of cetuximab to inhibit the phosphorylation of these EGFR downstream targets after the introduction of constitutively active H-Ras G12V into A431 cells. Fig. 1 shows that A431-Ras cells had higher basal phosphorylation levels of Akt and Erk than did A431V control

Discussion

Our in vitro and in vivo preclinical studies confirmed that expression of a constitutively active H-Ras (G12V) can confer resistance to EGFR-targeting agents in a manner similar to that of mutant K-Ras protein, which has been reported to play a key role in mediating resistance to EGFR-blocking antibodies in the treatment of colorectal cancers [17], [18], [19], [20], [21], [22], [23], [24], [25]. It is noteworthy that expression of H-Ras mutant did not confer complete resistance in vitro, and

Conflict of interest

None declared.

Acknowledgments

This work was supported in part by a US National Institutes of Health (NIH) R01 award (CA129036 to Z.F.) and by the Cancer Center Support Grant CA016672 from US National Cancer Institute (NCI). We thank Bryan Tutt of the Department of Scientific Publications at The University of Texas MD Anderson Cancer Center for editorial assistance.

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    1

    Present address: Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Victoria 3050, Australia.

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