Irinotecan is a major drug for treatment of metastatic colorectal cancer and a promising agent for other applications like gastric cancer. Its clinical activity is currently limited by both intrinsic (natural) and acquired drug resistance. A better understanding of the underlying resistance mechanisms is needed to develop novel therapeutic strategies. Exposure of tumor cells to irinotecan or its active metabolite SN-38 is accompanied by EGFR activation, either by stimulation of EGFR autophosphorylation or by Src-mediated phosphorylation. Accordingly, combinations of irinotecan and EGFR inhibitors have been associated with supra-additive activity. We now show that acquired resistance to SN-38 is accompanied by increased expression of EGFR, HER2, HER3 and Src proteins in two colorectal cancer cell models as well as by Src activation. One SN-38 resistant model (HT-29) showed increased sensitivity to erlotinib, an EGFR inhibitor, and afatinib, a dual EGFR/HER2 inhibitor, while the other SN-38 resistant model (HCT-116) showed increased resistance to erlotinib but unchanged or increased sensitivity to afatinib. Unexpectedly, both models showed increased or unaltered resistance to the Src inhibitor dasatinib. Therefore, tyrosine kinase upregulation is not necessarily accompanied by increased sensitivity to targeted agents. Taken together, our findings demonstrate that prolonged exposure to topoisomerase I inhibitors is accompanied by upregulation of different signal transduction pathways which can alter tumor sensitivity to molecular targeted agents. These results suggest that chemotherapy exposure may lead to creation of novel targets which could be exploited therapeutically.

Irinotecan is a major anticancer agent specifically targeting DNA topoisomerase I. Its cytotoxicity is mediated via a two-step process involving accumulation of reversible DNA‑topoisomerase I complexes associated with transient DNA single-strand breaks which subsequently are converted into permanent DNA double-strand breaks by the replication fork during S phase. Irinotecan may be selectively active for treatment of colorectal cancers that show microsatellite instability (MSI) due to deficiencies in mismatch repair enzymes, compared to tumors that are microsatellite stable but show chromosome instability (CIN). Although the clinical activity of irinotecan is principally limited by acquired drug resistance, surprisingly little is known about the influence of prolonged irinotecan exposure on the cell cycle dynamics. We have developed two colon cancer cell lines resistant to SN-38, the active metabolite of irinotecan, one derived from HT-29 (CIN), the other from HCT-116 (MSI). We here show that besides classical resistance mechanisms, SN-38 resistance is accompanied by an increased generation doubling time, a decreased S phase fraction and an increased G2 fraction in vitro as in tumor xenografts for both CIN and MSI models. As a consequence, SN-38-resistant cells and tumors show cross-resistance to the S-phase selective agent 5-fluorouracil. The resistance is accompanied by increased basal levels of γ-H2AX and phospho-Chk2 without notable changes in the levels of phospho-Chk1. Taken together, our results show that prolonged irinotecan exposure is accompanied by stable modifications of cell cycle dynamics which could have profound impact on tumor sensitivity to a wide range of antitumor agents and may influence tumor progression in patients.

Topoisomerase I is a privileged target for widely used anticancer agents such as irinotecan. Although these drugs are classically considered to be DNA-damaging agents, increasing evidence suggests that they might also influence the tumor environment. This study evaluates in vivo cellular and molecular modifications induced by irinotecan, a topoisomerase I-directed agent, in patient-derived colon tumors subcutaneously implanted in athymic nude mice. Irinotecan was given intraperitoneally at 40 mg/kg five times every 5 d, and expression profiles were evaluated at d 25 in tumors from treated and untreated animals. Unexpectedly, the in vivo antitumor activity of irinotecan was closely linked to a downregulation of hypoxia-inducible factor-1α (HIF1A) target genes along with an inhibition of HIF1A protein accumulation. The consequence was a decrease in tumor angiogenesis leading to tumor size stabilization. These results highlight the molecular basis for the antitumor activity of a widely used anticancer agent, and the method used opens the way for mechanistic studies of the in vivo activity of other anticancer therapies.

Purpose: Epidermal growth factor receptor (EGFR) and VEGF(R) signaling show extensive cross-talk, providing a rationale for joint targeting of the two pathways. However, combinations of monoclonal antibodies (mAb) targeting EGFR and VEGF showed disappointing activity in patients with colorectal cancer (CRC). We speculated that inhibition of surface receptors and ligands might only partly prevent oncogenic signaling whereas small-molecule tyrosine kinase inhibitors (TKI) would also influence intracellular signaling. Experimental Design: Mice with CRC xenografts were treated with two TKIs, vargatef and afatinib, or with two mAbs, bevacizumab and cetuximab, and their influence on tumor growth, viability, in vivo DNA synthesis, and the presence of phosphorylated EGFR and VEGFR was determined. The activity of the TKIs was further characterized in CRC cells with different KRAS status. Results: Vargatef and afatinib together showed strong tumor growth inhibition toward HT-29 xenografts compared with either drug alone, which was associated with a 5-fold increase in apoptotic tumor cell death. In comparison, bevacizumab and cetuximab together were exclusively cytostatic with no more activity than either drug alone. Exposure to the two TKIs was accompanied by a marked decrease of tumor-associated intracellular phospho-VEGFR1 and phospho-EGFR, whereas similar exposure to the two mAbs had no detectable effect. A synergistic activity of vargatef plus afatinib was observed in all eight CRC cell lines examined, independent of KRAS status. Conclusions: Our results indicate that attenuation of intracellular EGFR and/or VEGF signaling is required for cytotoxic activity. These findings provide a rationale for trials of the TKIs, even in patients with mutant KRAS. Clin Cancer Res; 17(20); 6522–30. ©2011 AACR.

The last decade has witnessed the approval of monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors (TKIs) for targeting of oncogenic signaling pathways. Generally, the clinical activity of these agents has been less than expected, in part due to unsuspected feed-back loops and cross-talk between different signaling pathways, thereby suggesting the interest of inhibiting multiple pathways. The extensive degree of EGFR-VEGF(R) pathway cross-talk identifies these pathways as particularly promising for joint targeting. Activation of the EGFR pathway increases the production of tumor-derived VEGF that acts on endothelial cells in a paracrine manner to promote angiogenesis. Accordingly, exposure to EGFR inhibitors is accompanied by attenuation of VEGF expression while resistance to EGFR inhibitors is frequently associated with enhanced VEGF levels. Recent data have expanded the biological activities of the two pathways by documenting a role for VEGF signaling in tumor cell survival and demonstrating the expression of EGFR by some tumor-associated endothelial cells. At least part of these signaling events are intracrine (intracellular and autocrine) and thus not readily accessible for the mAbs which target extracellular ligands and membrane receptors. This may explain why two major clinical trials combining EGFR and VEGF-targeted mAbs gave disappointing results and suggest a need for compounds that are able to inhibit intracrine signaling. Clinical application of new combinations should be preceded by preclinical development guided by functional biomarker analysis to identify active drug combinations and to facilitate the identification of patient subgroups likely, or not, to respond to dual pathway inhibition.

Abstract Backgound: Combining different classes of targeted agents represents a major challenge in modern cancer therapy. Agents targeting EGFR- or VEGF-signaling are approved for treatment of metastatic colorectal cancer paving the way for novel therapeutic strategies combining the two classes of agents. Unexpectedly, large clinical studies with combinations of EGFR-targeted antibodies and bevacizumab showed no added benefit compared to bevacizumab alone. It is currently unclear whether these findings are linked to incompatibility between the antibodies or rather to incompatibility between the two classes of targeted agents. Experimental: Combinations of two small orally active molecules, the irreversible EGFR and HER2 inhibitor BIBW2992 (B2) and the triple angiokinase inhibitor BIBF1120 (B1) were characterized in human colorectal cancer cell lines in vitro and in mice using the HT-29 colorectal xenograft model that is BRAF V600E mutated. Growth inhibitory and cytotoxic effects were characterized by flow cytometry complemented by immunological analyses for p27Kip1, phospho-Erk and phospho-Akt in cellular models and by quantitative immunohistochemistry analyses of EdU incorporation and TUNEL staining in tumor xenografts. Results: Exposure of colorectal cancer cells to B1 or to B2 resulted in growth inhibition linked to prolonged p27Kip1-mediated G1 arrest, which in most cell lines was accompanied by delayed apoptosis. Combinations of B1 and B2 resulted in increased cell death in all cellular models studied. In xenografts, four weeks treatment with combinations of B1 and B2 were associated with a clear synergistic effect on tumor growth inhibition compared to either agent alone. In contrast, no additional activity was observed for combinations of cetuximab and bevacizumab in the same xenograft model. Quantitative immunohistochemistry analyses revealed that all 4 agents inhibited in vivo DNA synthesis, as shown by EdU incorporation, and that none of the combinations tested showed superiority. TUNEL staining was not increased following single agent treatment with B1, cetuximab and bevacizumab while B2 resulted in increased TUNEL staining. Combinations of B1 and B2 were characterized by a pronounced increase in TUNEL staining, while no increase was observed for combinations of cetuximab and bevacizumab. Conclusions: We here show that combinations of small molecules that simultaneously target the HER and the angiogenic pathways show synergistic cytotoxic and tumor-growth inhibitory activities in the HT-29 colorectal xenograft model which is refractory to the combinations of cetuximab and bevacizumab. Therefore, the lack of superior clinical activity for combinations of cetuximab/panitumumab and bevacizumab is not due to an inherent incompatibility between the 2 classes of targeted agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A240.