Using a kinome-wide siRNA screen, we identified the InsR/IGF-1R pathway as a mechanism of escape from hormone dependence in ER+ breast cancer. RNAi-mediated knockdown of InsR and/or IGF-1R inhibited growth of ER+ breast cancer cells adapted to hormone deprivation, but dual knockdown additively suppressed PI3K/AKT signaling. Pharmacological blockade of InsR/IGF-1R with OSI-906 inhibited PI3K/AKT and LTED cell growth. OSI-906 also prevented the emergence of hormone-independent tumors, and suppressed growth of ER+ xenografts in ovariectomized mice. Blockade of IGF-1R alone was insufficient to prevent emergence of hormone-independent cells or suppress tumor growth, suggesting that dual inhibition of InsR and IGF-1R is necessary to prevent escape of ER+ breast cancer cells from estrogen dependence. Combined inhibition of ER and InsR/IGF-1R suppressed hormone-independent tumor growth more effectively than each intervention alone. Finally, an insulin/IGF-1-induced gene expression signature was predictive of RFS in patients with ER+ breast cancer treated with adjuvant tamoxifen.
While the IGF-1R has been implicated in tamoxifen resistance (7
), we show herein the importance of InsR in acquired resistance to endocrine therapy, as a dual inhibitor of InsR/IGF-1R was clearly superior at abrogating hormone independence compared to a neutralizing IGF-1R antibody. There is evidence of hyperactivation of the InsR/IGF-1R/PI3K/mTOR pathway in LTED cells (13
), which is likely to be causally associated with resistance to estrogen deprivation. Both InsR and IGF-1R knockdown inhibited hormone-independent growth (), suggesting both receptors are important in endocrine-resistant cells. Of note, IGF-1R was not a hit in the siRNA screen; however, false negatives are unavoidable in screens of this nature. IGF-1R knockdown using an independent siRNA suppressed hormone-independent growth (). While dual knockdown additively suppressed PI3K/AKT, InsR knockdown inhibited MCF-7/LTED growth more effectively than dual InsR/IGF-1R knockdown, but this difference did not reach statistical significance (). We speculate that the increased effect of InsR knockdown may be due to downregulation of both InsR homodimers and InsR/IGF-1R heterodimers.
The InsR/IGF-1R TKI OSI-906 is in early clinical trials, where it has been well-tolerated (40
). Consistent with observations of hyperglycemia in patients treated with other IGF-1R inhibitors (8
), hyperglycemia was reported in a fraction of patients treated with OSI-906 in phase I trials (41
). However, this side effect did not limit establishment of a maximally tolerated dose (MTD) based on dosing schedules corresponding to drug exposures predicted to inhibit IGF-1R and InsR in tissue and peripheral blood. Treatment with OSI-906 was superior to the IGF-1R antibody MAB391 at inhibiting PI3K/AKT (). Further, OSI-906 prevented the emergence of hormone-independent cells and tumors (, ), and suppressed hormone-independent tumor growth (). These results imply that blockade of IGF-1R alone is insufficient to prevent or treat endocrine-resistant breast cancer, and that both receptors should be targeted in this clinical setting. In agreement with these data, a recent report showed that OSI-906 was superior to MAB391 against human colon cancer xenografts (6
). Additionally, dual inhibition of InsR/IGF-1R was required to inhibit growth in IGF-2-driven cancers in a transgenic mouse model (43
The requirement of targeting both InsR and IGF-1R to suppress estrogen-independent tumor growth may help explain the outcome of a recent clinical trial. Patients with ER+ metastatic breast cancer who progressed on prior endocrine therapy were randomized to the AI letrozole ± the IGF-1R monoclonal antibody AMG-479. AMG-479 did not add to the clinical effect of letrozole alone (44
). Although insulin levels were not reported in the AMG-479 study, we speculate that a compensatory upregulation of insulin (as seen in and (23
)) and, in turn, InsR activation may have negated a clinical effect of the antibody.
Other studies have shown that amplified InsR signaling conveys intrinsic resistance to IGF-1R inhibitors (43
). InsR and IGF-1R crosstalk bidirectionally, suggesting that InsR can compensate for loss of IGF-1R (). Further, IGF-1R downregulation sensitizes breast cancer cells to insulin action (46
), MAB391 treatment results in a compensatory increase in InsR phosphorylation (6
), and IGF-1R knockout can sensitize cells to insulin-mediated activation of InsR, AKT, and MAPK (47
). These data further suggest a dual InsR/IGF-1R inhibitor such as OSI-906 would be a better strategy at inhibiting this receptor network. The relative contribution of InsR and IGF-1R homo- vs. heterodimers to breast cancer cell growth is unclear. IGF-1 and IGF-2 bind heterodimers and IGF-1R homodimers with high affinity, whereas insulin binds InsR homodimers but not IGF-1R homodimers or heterodimers at physiological concentrations (5
). Since OSI-906 blocked insulin- and IGF-1-induced PI3K/AKT activation and cell growth (), we speculate OSI-906 likely inhibits both InsR and IGF-1R heterodimers and homodimers. Further, insulin and IGF-1 altered both common and distinct gene expression signatures, reinforcing distinct functionality of these two pathways (). We speculate that genes commonly deregulated by short-term insulin and IGF-1 stimulation may drive resistance to endocrine therapy, since the insulin/IGF-1 gene signature was more predictive than the insulin signature of disease recurrence (). Collectively, these data suggest that homo- and hetero-dimers may promote endocrine resistance, and targeting both receptors is required for effective suppression of the InsR/IGF-1R pathway.
In summary, we have identified the InsR/IGF-1R pathway as a mechanism of escape from hormone dependence in ER+ breast cancer. Since inhibition of InsR and IGF-1R prevented the emergence of hormone-independent tumors, we propose early intervention with combined ER and InsR/IGF-1R-directed therapies in high-risk patients with ER+ breast cancer may prevent disease recurrence. Further, this study suggests that targeting InsR/IGF-1R may be more effective than targeting IGF-1R alone. As a result, dual TKIs of InsR/IGF-1R should be more effective than neutralizing IGF-1R antibodies in preventing escape of ER+ breast cancer from hormone dependence.