th IGF-R and IR may promote tumor growth. We investigated the phosphorylation status of IGF-R and IR in HCC cell lines (Fig. B). Compared with other tumor cell lines we tested (), all 6 HCC cell lines showed higher IR phosphorylation than IGF-R, suggesting the signifi- cance of IR activity in HCC. Furthermore, all 3 HCC cell lines (HepG, Hep3B, and HuH-7) that are sensitive to OSI-906 had much higher phosphorylation levels of both IGF-R and IR than insensitive cell lines (Fig. B). This suggests that sensitivity to OSI-906 associates Docetaxel with acti- vation of both IGF-R and IR in HCC cell lines. Recently, we reported that in some tumor cell lines, inhibition of IGF-R signaling by neutralizing antibodies was associ- ated with a compensatory increase in IR signaling (), and dual inhibition of both IGF-R and IR was required for maximal inhibition of tumor growth for tumors where both IGF-R and IR were phosphorylated. Compensatory signaling between IGF-R and IR was bidirectional as knockdown of IR was associated with increased phos- phorylation of IGF-R.

To test whether the IR pathway can also compensate for the loss of IGF-R activities in HCC cell lines, we treated OSI-906 sensitive HuH-7 cells with either the anti–IGF-R Piroxicam antibody Mab39 or OSI-906 and monitored the phosphorylation status of IR and IGF-R (Fig. C). OSI-906 fully inhibited phosphorylation of both IGF-R and IR. In contrast, specific inhibition of IGF-R phosphorylation upon treatment with Mab39 was accompanied by an increase in IR phosphorylation, indi- cating the potential for compensatory signaling. We inves- tigated differential inhibition of IGF-R/IR on down- stream signaling, including IRS-, AKT, and PRAS40 phosphorylation, for OSI-906 compared with Mab39. Immunoblotting showed that phosphorylation of AKT and the AKT substrate PRAS40 was inhibited to a greater extent by OSI-906 than by Mab39 (Fig. C). Furthermore, while OSI-906 fully inhibited IR and IRS- phosphoryla- tion, the induced IR phosphorylation upon Mab39 treat- ment was associated with an inability to inhibit IRS- tyrosine phosphorylation to the same extent as OSI-906 (Fig. D).

These data indicate that signaling through the IRS-/AKT pathway may also be mediated by IR in HCC tumor cells, and inhibition of IR is needed to achieve maximal inhibition of this cellular survival pathway in HCC. studies have shown that cross-talk between the IGF and EGF signaling pathways can occur, resulting in limited sensitivity when either pathway is targeted individually. Therefore, we hypothesized that the combination of erlo- tinib and risedronate 115436-72-1 OSI-906 (Fig. A) might be especially efficacious at inhibiting select HCC cell proliferation. A panel of HCC tumor cell lines exhibiting varying sensitivity to OSI-906 was treated with the combination of erlotinib and OSI-906, and effects of compound treatment were monitored by cell proliferation assays (Fig. ). Addition of erlotinib further increased sensitivity of Hep3B, Jhh-7 cells, OSI-906–sensitive HCC cell lines (Fig. B and C). We assessed combination synergy using the BLISS method, which would allow us to capture cooperative effects both on potency and maximal activity (33). Using this approach, we find the combination of erlotinib and OSI-906 to be synergistic in these tumor cell lines. More- over, the combination of erlotinib and OSI-906 also showed good synergy for inhibiting cell proliferation for Jhh- and PLC/PRF/5 cells, HCC cell lines that are not sensitive to OSI-906 as a single agent (data not shown).

Taken together, these data indicate that the activity for OSI-906 may be augmented in HCC tumor cells through combination with an EGFR antagonist. Blocking compensatory buy risedronate pathways increases sensitivity to OSI-906 It was shown previously that a subset of HCC tumor cells was sensitive to epidermal growth factor receptor (EGFR) pathogens antagonists including erlotinib (36), which can also inhibit AKT phosphorylation. In addition, several IGF axis gene expression in HCC cell lines We sought to determi