Generation of engineered small protein scaffolds and insulin receptor targeting in human breast cancer
2017-04
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Generation of engineered small protein scaffolds and insulin receptor targeting in human breast cancer
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2017-04
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The insulin-like growth factor (IGF) system is a well-studied growth regulatory pathway implicated in breast cancer tumorigenicity and drug resistance. The pivotal members of IGF system, the type I insulin-like growth factor (IGF1R) and insulin receptor (InsR) are homologous receptors necessary for signal transduction by their cognate ligands insulin, insulin-like growth factor-I and –II. A number of drugs, including monoclonal antibodies (mAbs) directed against IGF1R, small molecule tyrosine kinase inhibitors (TKIs) and IGF ligand neutralizing antibodies have been developed and tested in clinical trials. Early trials suggested benefits in delaying tumor progression, unfortunately, none of the anti-IGF1R mAbs has, thus far, shown significant benefits in phase III clinical trials. Although preclinical studies of anti-IGF1R mAbs showed promising results using endocrine-sensitive models, these antibodies were evaluated in breast cancer patients with endocrine-resistant tumors. When our group generated endocrine-resistant breast cancer models, we showed that IGF1R expression was lacking and anti-IGF1R mAb treatment was inefficacious in treating these endocrine-resistant cells. Inaccurate recapitulation of human diseases explains the disappointing outcomes of the trials. This finding also suggests that IGF1R inhibition is not an appropriate treatment in treating breast cancer patients whom are resistant to endocrine treatments. Unlike IGF1R, InsR expression is not affected in the endocrine-resistant breast cancer model. Several studies have shown a shift in gene signatures from IGF- to insulin-mediated growth and differentiation in the absence of IGF1R. Our group has shown an increase of insulin sensitivity in breast cancer cells when IGF1R is downregulated and insulin/InsR alone is sufficient to drive metastasis and tumor growth in vivo. However, InsR has been intentionally avoided as a potential target in cancer therapy due to its major function in glucose homeostasis. There are, currently no InsR-specific inhibitor or molecular diagnostics available. This reason has become the motivation for my work. The first section of the work examines the roles of InsR and efficacy of InsR inhibition in endocrine-resistant breast cancer. The model we used was a tamoxifen resistant (TamR) cells derived from estrogen receptor positive breast cancer cell lines. These TamR cells did not simply lose IGF1R expression and function, but also gained sensitivity to insulin stimulation compared to their parental cells. We used three different targeting mechanisms to disrupt the functions of InsR: 1.) InsR short hairpin RNA (shIR) to knock down endogenous InsR expression, 2.) a small InsR-blocking peptide, S961 and 3.) an InsR down-regulator mAb (clone 83-7). These methods showed consistent results that suppression of InsR function in TamR cells successfully blocked insulin-mediated signaling, monolayer proliferation, cell cycle progression and anchorage-independent growth. This strategy, however was not effective in the parental cells, which were sensitive to endocrine treatments, likely because of the presence of IGF1R/InsR hybrid receptors. Down-regulation of IGF1R with monoclonal antibody in conjunction with shIR or S961 was more effective in blocking IGF- and insulin-mediated signaling and growth in the parental cells compared with single-receptor targeting alone. Our finding showed TamR cells were stimulated by InsR and were not sensitive to IGF1R inhibition, whereas in tamoxifen-sensitive parental cells, the presence of both receptors, especially hybrid receptors, allowed cross-reactivity of ligand-mediated activation and growth. Surprisingly, the synergistic inhibitory effects were not achievable with anti-IGF1R and anti-InsR mAbs in the parental cells. When this combination treatment was tested in triple-negative breast cancer cells, an additive inhibition of ligand-mediated signaling was observed, especially in the samples treated with IGF-II, suggesting that they may potentially benefit from the combinational IGF1R/InsR therapy. Although the signaling result of IGF1R/InsR blocking antibodies seems promising in triple-negative breast cancer, no functional assay has yet been done to prove their efficacy. The second section of my dissertation focuses on the generation and characterization of InsR-evolved small protein scaffolds based on the T7 phage Gene 2 protein (Gp2). Gp2 is one of the potential small protein scaffolds for ligand engineering and has the capable for mutation to generate new binding functions. Our long-term goal is to create effective InsR inhibitors and/or diagnostic tools. Using yeast surface display and directed evolution, we identified three Gp2 variants, known as Gp2 #1, #5 and #10 with low nanomolar binding affinity to cell-surface InsR with weak cross-reactivity to IGF1R of Gp2#1. These Gp2 variants inhibited insulin-mediated monolayer proliferation in both endocrine-sensitive and -resistant breast cancer, but did not down-regulate InsR expression. Gp2 #5 and Gp2#10 disrupted InsR function by inhibiting ligand-induced receptor activation. In contrast, Gp2#1 did not block InsR phosphorylation. Notably, Gp2#1 binding was enhanced by pre-treatment of cells with insulin suggesting a unique receptor-ligand binding mode. These Gp2 variants are the first non-immunoglobulin protein scaffolds to target insulin receptor and present compelling opportunity for modulation of InsR signaling. Taken together, targeting both IGF1R and InsR is optimal in endocrine-sensitive, endocrine-resistant breast cancer and potentially triple-negative breast cancer to fully suppress the IGF system. Since double inhibition using mAbs might not be an ideal approach in endocrine-sensitive breast cancer, future studies using one of the Gp2 variants as alternative options, either as a single or in combination with other agents may provide some insights with regards to IGF targeting in breast cancer.
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University of Minnesota Ph.D. dissertation. April 2017. Major: Pharmacology. Advisors: Douglas Yee, Jill Siegfried. 1 computer file (PDF); xiv, 140 pages.
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