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.

Cetuximab, an epidermal growth factor receptor (EGFR)-blocking antibody, was approved for treatment of metastatic colorectal cancer over a decade ago; however, patients' responses to cetuximab vary substantially due to intrinsic and acquired resistance to cetuximab. Here, we report our findings using Affymetrix HG-U133A array to examine changes in global gene expression between DiFi, a human colorectal cancer cell line that is highly sensitive to cetuximab, and two other cell lines: DiFi5, a DiFi subline with acquired resistance to cetuximab, and DiFi-AG, a DiFi subline with acquired resistance to the EGFR tyrosine kinase inhibitor AG1478 but sensitivity to cetuximab. We identified prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes cyclooxygenase-2 (COX-2), as the gene with the greatest difference between the cetuximab-resistant DiFi5 cells and the cetuximab-sensitive DiFi cells and DiFi-AG cells. Reverse transcription polymerase chain reaction and Western blotting validated upregulation of COX-2 in DiFi5 but not in DiFi or DiFi-AG cells. We developed COX-2 knockdown stable clones from DiFi5 cells and demonstrated that genetic knockdown of COX-2 partially re-sensitized DiFi5 cells to cetuximab. We further confirmed that cetuximab in combination with a COX-2 inhibitor led to cell death via apoptosis or autophagy not only in DiFi5 cells but also in another colorectal cancer cell line naturally resistant to cetuximab. Our findings support further evaluation of the strategy of combining cetuximab and a COX-2 inhibitor for treatment of metastatic colorectal cancer.

Cetuximab inhibits HIF-1-regulated glycolysis in cancer cells, thereby reversing the Warburg effect and leading to inhibition of cancer cell metabolism. AMP-activated protein kinase (AMPK) is activated after cetuximab treatment, and a sustained AMPK activity is a mechanism contributing to cetuximab resistance. Here, we investigated how acetyl-CoA carboxylase (ACC), a downstream target of AMPK, rewires cancer metabolism in response to cetuximab treatment. We found that introduction of experimental ACC mutants lacking the AMPK phosphorylation sites (ACC1_S79A and ACC2_S212A) into head and neck squamous cell carcinoma (HNSCC) cells protected HNSCC cells from cetuximab-induced growth inhibition. HNSCC cells with acquired cetuximab resistance contained not only high levels of T172-phosphorylated AMPK and S79-phosphorylated ACC1 but also an increased level of total ACC. These findings were corroborated in tumor specimens of HNSCC patients treated with cetuximab. Cetuximab plus TOFA (an allosteric inhibitor of ACC) achieved remarkable growth inhibition of cetuximab-resistant HNSCC xenografts. Our data suggest a novel paradigm in which cetuximab-mediated activation of AMPK and subsequent phosphorylation and inhibition of ACC is followed by a compensatory increase in total ACC, which rewires cancer metabolism from glycolysis-dependent to lipogenesis-dependent.

Therapeutic targeting of ASCT2, a glutamine transporter that plays a major role in glutamine uptake in cancer cells, is challenging because ASCT2 also has a biological role in normal tissues. In this study, we report our novel finding that ASCT2 is physically associated in a molecular complex with epidermal growth factor receptor (EGFR), which is often overexpressed in human head and neck squamous cell carcinoma (HNSCC). Furthermore, we found that ASCT2 can be co-targeted by cetuximab, an EGFR antibody approved for treating metastatic HNSCC. We demonstrated that cetuximab downregulated ASCT2 in an EGFR expression-dependent manner via cetuximab-mediated EGFR endocytosis. Downregulation of ASCT2 by cetuximab led to decreased intracellular uptake of glutamine and subsequently a decreased glutathione level. Cetuximab thereby sensitized HNSCC cells to reactive oxygen species (ROS)-induced apoptosis and, importantly, it is independent of effective inhibition of EGFR downstream signaling by cetuximab. In contrast, knockdown of EGFR by siRNA or inhibition of EGFR kinase with gefitinib, an EGFR kinase inhibitor, failed to sensitize HNSCC cells to ROS-induced apoptosis. Our findings support a novel therapeutic strategy for EGFR-overexpressing and cetuximab-resistant cancers by combining cetuximab with an oxidative therapy.

The human epidermal growth factor receptor (EGFR) is an important target for cancer treatment. Currently, only the EGFR antibodies cetuximab and panitumumab are approved for the treatment of patients with colorectal cancer. However, a major clinical challenge is a short-term response owing to development of acquired resistance during the course of the treatment.

Searching for novel molecular markers that dependably predict or indicate responses of human cancer cells to epidermal growth factor receptor (EGFR)-targeted therapy is strongly warranted. The purpose of the current study was to evaluate hypoxia-inducible factor-1alpha (HIF-1alpha) as a novel response marker compared with previously explored markers following treatment with an EGFR-blocking monoclonal antibody (cetuximab) and a small-molecule EGFR tyrosine kinase inhibitor (gefitinib) in a group of cancer cell lines containing wild-type or tyrosine kinase domain-mutated EGFR.