Sequencing of type I insulin-like growth factor receptor inhibition with chemotherapy in breast cancer cells.

Abstract

The type-I insulin like growth factor (IGF-IR) is either activated and/or overexpressed in a wide range of tumor types and contributes to tumorigenicity, proliferation, survival, metastasis and drug resistance. Disruption of type I insulin-like growth factor receptor (IGF-IR) signaling alone or in combination with other cytotoxic agents has emerged as an important strategy in cancer therapy. Recent findings suggest that the combination of chemotherapy and targeted therapy may be sequencedependent. Since several anti-IGF-IR antibodies and small kinase inhibitors are being evaluated in phase I, II or III clinical trials, some in combination with cytotoxic chemotherapy, it is important to determine the optimal schedule for antibodies in combination with chemotherapy. Here, we sought to determine the optimal sequence of combining anti-IGF-IR inhibition using anti-IGF-IR antibody or small-molecule inhibitor with chemotherapeutic drugs in human cancer cells in vitro and in vivo. Data presented in this study provide direct evidence that anti-IGF-IR antibody with chemotherapy is sequence-dependent. DOX followed by anti-IGF-IR antibody (scFv-Fc or EM164) was the most effective combination strategy to inhibit cell monolayer growth and anchorage-independent growth. This sequential combination triggered increased Poly (ADP-ribose) polymerase (PARP) cleavage compared to other treatment sequences. The reverse sequence, antibody followed by DOX treatment, protected cells from chemotherapy by decreasing apoptosis, arresting cells in S phase, and inhibiting the mRNA, protein and activity of topoisomerase IIα. Finally, our in vivo data show that recovery of IGF-IR prior to DOX therapy resulted in the best therapeutic

responses. Low doses of AVE1642 that allowed IGF-IR expression to recover at one week were more effective in combination with DOX than higher antibody doses. In addition, we show that a novel small-molecule IGF-IR kinase inhibitor, cis- 3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5- a]pyrazin-8-ylamine (PQIP) inhibits both the IGF-IR and insulin receptor (IR) with similar potency by blocking Akt and MAPK activation in human cancer cell lines. At doses that inhibit proliferation, the compound also inhibits the cell motility and induces cell apoptosis. PQIP enhances the cytotoxicity of doxorubicin in inhibiting cell proliferation but not in inducing cell apoptosis. Furthermore, our sequencing study shows that both combining PQIP with doxorubicin simultaneously and doxorubicin followed by PQIP significantly inhibits the anchorage-independent growth, while PQIP followed by doxorubicin does not enhance the cytotoxicity of doxorubicin. In summary, the timing of IGF-IR inhibition affects responses to chemotherapy. The optimal sequence was DOX followed by anti-IGF-IR antibody, while the opposite sequence inhibited DOX effects. Thus, short course suppression in combination with DOX might be the optimal combination and provides a rationale for the design of future clinical trials. PQIP can be used alone or in combination with chemotherapy for breast cancer treatment.