Sequencing of type I insulin-like growth factor receptor inhibition affects chemotherapy response in vitro and in vivo.

PURPOSE: The aim of this study was to determine the optimal sequence of combining anti-type I insulin-like growth factor receptor (IGF1R) antibodies with chemotherapeutic drugs in cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: MCF-7 and LCC6 cells were treated with subcytotoxic concentrations of doxorubicin with or without anti-IGF1R antibodies (scFv-Fc or EM164 and its humanized version AVE1642). Treatments were given simultaneously, doxorubicin followed by anti-IGF1R antibody, or anti-IGF1R antibody followed by doxorubicin, with measurement of in vitro proliferation, apoptosis, and anchorage-independent growth. The effects of sequencing on LCC6 xenograft growth and metastasis were studied. RESULTS: Doxorubicin followed by anti-IGF1R 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 cleavage compared with other treatment sequences. The reverse sequence, antibody followed by doxorubicin treatment, protected cells from chemotherapy by decreasing apoptosis, arresting cells in S phase, and inhibiting the level and activity of topoisomerase IIalpha. Finally, our in vivo data show that recovery of IGF1R prior to doxorubicin therapy resulted in the best therapeutic responses. Low doses of AVE1642 that allowed IGF1R expression to recover at one week were more effective in combination with doxorubicin than higher antibody doses. CONCLUSION: The timing of IGF1R inhibition affects responses to chemotherapy. The optimal sequence was doxorubicin followed by anti-IGF1R antibody, whereas the opposite sequence inhibited doxorubicin effects. Thus, the dose and sequencing of anti-IGF1R therapies should be considered in the design of future clinical trials.

Detection and downregulation of type I IGF receptor expression by antibody-conjugated quantum dots in breast cancer cells.

The type I insulin-like growth factor (IGF) receptor (IGF1R) is a transmembrane tyrosine kinase involved in breast cancer proliferation, survival, and metastasis. Several monoclonal antibodies directed against the receptor are in clinical trials. In order to develop a methodology to detect and measure IGF1R levels in breast cancer cells, we covalently conjugated an IGF1R antibody, AVE-1642, with quantum dots (Qdots), which are nanocrystals that emit fluorescence upon excitation. AVE-1642 Qdots only bound to cells that express IGF1R, and measured IGF1R levels by fluorescence emission at 655 nm. After binding to the cell surface, AVE-1642 Qdots underwent receptor mediated endocytosis, localized to endosome, and later translocated into the nucleus. Treating MCF-7 cells with AVE-1642 Qdots, but not unconjugated Qdots alone, downregulated IGF1R levels and rendered cells refractory to IGF-I stimulation. Furthermore, cell proliferation was slightly inhibited by AVE-1642 Qdots, but not the unconjugated Qdots. Our data suggest that AVE-1642 Qdots can be used to detect IGF1R expression and measure changes in cell surface receptor levels. In addition, the inhibitory effect of AVE-1642 Qdots to cell proliferation implies that it may serve as a traceable therapeutic agent.

A functional androgen receptor is not sufficient to allow estradiol to protect bone after gonadectomy in estradiol receptor-deficient mice.

Although the role of estradiol in maintaining bone mass is well established, the relative contributions of the estradiol receptors ERalpha and ERbeta and of the androgen receptor (AR) remain controversial. To determine the role of ERalpha-mediated, ERbeta-mediated, and non-ER-mediated mechanisms in maintaining bone mass, gonadectomy and estradiol treatment were studied in ER-knockout mice. Estradiol treatment of ovariectomized ERalphabeta(-/-) mice failed to prevent bone loss, precluding significant effects of estradiol on bone through non-ER-signaling pathways. In contrast, estradiol prevented ovariectomy-induced bone loss in ERbeta(-/-) mice, as in WT males and females, indicating that ERalpha is the major mediator of estradiol effects in bone. No response of bone to estradiol was detected in orchidectomized ERalpha(-/-) mice, suggesting estradiol cannot protect bone mass via the AR in vivo. In contrast to female ERalphabeta(-/-) and male ERalpha(-/-) mice, female ERalpha(-/-) mice were partially protected against ovariectomy-induced bone loss by estradiol, confirming that ERbeta mediates estradiol effects in bone, but only in females and with a lower efficacy than ERalpha. We conclude that ERalpha is the main effector of estradiol's protective function in bone in both male and female mice, and that, in its absence, AR is not sufficient to mediate this response.