Purpose: Everolimus is a selective mammalian target of rapamycin (mTOR) inhibitor with promising anticancer activity. In order to identify a rationally based dose and schedule for cancer treatment, we have conducted a tumor pharmacodynamic phase I study in patients with advanced solid tumors. Patients and Methods: Fifty-five patients were treated with everolimus in cohorts of 20, 50, and 70 mg weekly or 5 and 10 mg daily. Dose escalation depended on dose limiting toxicity (DLT) rate during the first 4-week period. Pre- and on-treatment steady-state tumor and skin biopsies were evaluated for total and phosphorylated (p) protein S6 kinase 1, eukaryotic initiation factor 4E (elF-4E) binding protein 1 (4E-BP1), eukaryotic initiation factor 4G (eIF-4G), AKT, and Ki-67 expression. Plasma trough levels of everolimus were determined on a weekly basis before dosing during the first 4 weeks. Results: We observed a dose- and schedule-dependent inhibition of the mTOR pathway with a near complete inhibition of pS6 and peIF-4G at 10 mg/d and ≥ 50 mg/wk. In addition, pAKT was upregulated in 50% of the treated tumors. In the daily schedule, there was a correlation between everolimus plasma trough concentrations and inhibition of peIF4G and p4E-BP1. There was good concordance of mTOR pathway inhibition between skin and tumor. Clinical benefit was observed in four patients including one patient with advanced colorectal cancer achieving a partial response. DLTs occurred in five patients: one patient at 10 mg/d (grade 3 stomatitis) and four patients at 70 mg/wk (two with grade 3 stomatitis, one with grade 3 neutropenia, and one with grade 3 hyperglycemia). Conclusion: Everolimus achieved mTOR signaling inhibition at doses below the DLT. A dosage of 10 mg/d or 50 mg/wk is recommended for further development.

Purpose: Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by onset of resistance. Experimental Design: We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab. Results: Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET–dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR–MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells. Conclusions: These results suggest that overexpression of TGF-α through induction of EGFR–MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer. Clin Cancer Res; 19(24); 6751–65. ©2013 AACR.

Purpose: EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) have been found to be effective against lung cancer, but clinical resistance to these agents has developed as their usage has increased. Metformin is a widely used antidiabetic drug and also displays significant growth-inhibitory and proapoptotic effects in several cancer models, alone or in combination with chemotherapeutic drugs. Experimental Design: The effects of gefitinib, a selective EGFR-TKI, and metformin on a panel of non–small cell lung cancer (NSCLC) cell lines were assessed by using MTT, bromide assay, flow cytometry, anchorage-independent growth, coimmunoprecipitation, and Western blot analysis. Results: The combination of metformin with gefitinib induced a strong antiproliferative and proapoptotic effect in NSCLC cell lines that harbored wild-type LKB1 gene. Treatment with metformin as single agent, however, induced an activation and phosphorylation of mitogen-activated protein kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. The inhibition of EGFR phosphorylation and of downstream signaling by adding gefitinib to metformin treatment abrogated this phenomenon and induced a strong apoptotic effect in vitro and in vivo. Conclusions: Metformin and gefitinib are synergistic in LKB1 wild-type NSCLC cells. However, further studies are required to investigate better the effect of metformin action on the RAS/RAF/MAPK pathway and the best context in which to use metformin in combination with molecular targeted agents. Clin Cancer Res; 19(13); 3508–19. ©2013 AACR.

Purpose: The EGFR-independent activation of the RAS/RAF/MEK/MAPK pathway is one of the resistance mechanisms to cetuximab. Experimental Design: We have evaluated, in vitro and in vivo, the effects of BAY 86-9766, a selective MEK1/2 inhibitor, in a panel of human colorectal cancer cell lines with primary or acquired resistance to cetuximab. Results: Among the colorectal cancer cell lines, five with a KRAS mutation (LOVO, HCT116, HCT15, SW620, and SW480) and one with a BRAF mutation (HT29) were resistant to the antiproliferative effects of cetuximab, whereas two cells (GEO and SW48) were highly sensitive. Treatment with BAY 86-9766 determined dose-dependent growth inhibition in all cancer cells, including two human colorectal cancer cells with acquired resistance to cetuximab (GEO-CR and SW48-CR), with the exception of HCT15 cells. Combined treatment with cetuximab and BAY 86-9766 induced a synergistic antiproliferative and apoptotic effects with blockade in the MAPK and AKT pathway in cells with either primary or acquired resistance to cetuximab. The synergistic antiproliferative effects were confirmed using other two selective MEK1/2 inhibitors, selumetinib and pimasertib, in combination with cetuximab. Moreover, inhibition of MEK expression by siRNA restored cetuximab sensitivity in resistant cells. In nude mice bearing established human HCT15, HCT116, SW48-CR, and GEO-CR xenografts, the combined treatment with cetuximab and BAY 86-9766 caused significant tumor growth inhibition and increased mice survival. Conclusion: These results suggest that activation of MEK is involved in both primary and acquired resistance to cetuximab and the inhibition of EGFR and MEK could be a strategy for overcoming anti-EGFR resistance in patients with colorectal cancer. Clin Cancer Res; 20(14); 3775–86. ©2014 AACR.

Purpose: This study assessed biomarkers for cetuximab efficacy in tissue samples collected during a phase I dose-escalation study exploring every second week administration of cetuximab as first-line therapy in patients with metastatic colorectal cancer (mCRC). Patients and Methods: Sixty-two patients received cetuximab monotherapy for 6 weeks, followed by cetuximab plus infusional fluorouracil, leucovorin, and irinotecan until disease progression. Patients in the control arm received cetuximab as a 400 mg/m² initial dose then 250 mg/m² per week; patients in the dose-escalation arms received 400 to 700 mg/m² every second week. Tumor and skin biopsies were taken for immunohistochemical and microarray expression analyses (tumor only) at baseline and week 4. Plasma was collected for proteomic analysis at baseline and week 4. KRAS tumor mutation status was assessed. Results: In subsets of paired skin samples from 35 patients, cetuximab treatment was associated with substantial downregulation of phospho(p)-EGFR, p-MAPK and proliferation and substantial upregulation of p27^Kip1 and p-STAT3 levels. No marked difference in these effects was noted for different schedules of administration and dose levels. In the cetuximab monotherapy phase, responses were seen only in patients whose tumors were wild-type for KRAS (eight of 29 v zero of 19 for KRAS mutant tumors; P = .015). Progression-free survival was longer for patients with KRAS wild-type compared with KRAS mutant tumors (log-rank P = .048). Genomics/proteomics analyses (42 and 45 patients, respectively) identified candidate biomarkers associated with response. Conclusion: Biomarker analysis supported the functional equivalence of weekly and every second week administration of cetuximab and provided further confirmation that patients with KRAS wild-type mCRC were those most likely to benefit from cetuximab treatment.