Selective estrogen receptor modulators (SERMs) such as tamoxifen are used to treat estrogen receptor (ER) positive breast cancers, the most common intrinsic subtype. The development of secondary resistance to SERMs is an unsolved clinical dilemma, leading to the exploration, and approval, of therapies targeting growth factor signaling pathways. The insulin-like growth factor (IGF) system is well documented to cooperate with ER and is implicated as a contributor to endocrine resistance. Unfortunately, antibodies directed against Type I IGF receptor (IGF1R) failed to demonstrate clinical efficacy in endocrine resistant breast cancer. We developed a tamoxifen resistant (TamR) model to further identify the role for this system in endocrine resistance. In these cells, IGF1R levels are low but the level of insulin receptor (InsR), a closely related receptor of IGF1R still remains functional. Thus, we hypothesize that InsR may serve as a compensatory pathway to the loss of IGF1R in TamR cells and InsR may be a target in the therapy of endocrine resistant breast cancer. Indeed, our results showed that TamR breast cancer cells are more sensitive to insulin stimulation than wild-type cells. As compared to the parental cells, insulin-stimulated TamR cells showed enhanced PI3K/AKT and MAPK/ERK activation, greater monolayer and anchorage-independent growth. Suppression of InsR expression with either lentiviral shRNA or pharmacological methods in TamR cells was able to attenuate their sensitivity towards insulin-regulated PI3K/MAPK activation and growth, suggesting InsR targeting may be necessary. To target InsR, we are developing InsR-selective small protein scaffolds - the 10th type III domain of human fibronectin (Fn3) and T7 phage gene 2 protein (Gp2) using yeast surface display. This technique has arisen as an alternative candidate to bind cells surface proteins. Compared to antibodies, protein scaffolds are smaller (<12kDa), thermally more stable, lack disulfide bonds, tolerance to mutations and have the ability to bind a large variety of proteins. Using error-prone polymerase mutagenesis, we have identified improved Fn3 and Gp2 binders with increased affinity, specificity, and stability for InsR. Yeast surface displayed (YSD) Gp2 and Fn3 libraries yield affinities for recombinant InsR in the high nanomolar range and some specificity with an ability to differentiate binding from IGF1R. Purified Gp2 binder was able to distinguish InsR expression levels between InsRlow and InsRhigh mammalian cells, verifying its specificity for cell surface InsR. Its affinity for cell surface InsR, however was much lower compared to YSD affinity. Preliminary analyses also indicated that Gp2 binders have minimal effect in InsR-regulated signaling and short-term biological functions in vitro. These InsR binding engineered protein scaffolds will further be improved and evaluated as a potential imaging, diagnostic, and therapeutic tools.
Citation Format: Jie Ying Chan, Kelly LaPara, Benjamin Hackel, Douglas Yee. Insulin receptor targeting in breast cancer through yeast surface display. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1881.