Preclinical evaluation of Insulin-like growth factor receptor 1 (IGF1R) and Insulin Receptor (IR) as a therapeutic targets in triple negative breast cancer

Abstract

Triple Negative Breast Cancer (TNBC), a subtype of breast cancer, has fewer successful therapeutic therapies than other types of breast cancer. Insulin-like growth factor receptor 1 (IGF1R) and the Insulin receptor (IR) are associated with poor outcomes in TNBC. Targeting IGF1R has failed clinically. We aimed to test if inhibiting both IR/IGF1R was a rationale therapeutic approach to treat TNBC. We showed that despite IGF1R and IR being expressed in TNBC, their expression is not associated with a negative survival outcome. Furthermore, targeting both IR/IGF1R with inhibitors in multiple TNBC cell lines did not inhibit cell growth. Linsitinib, a small molecule inhibitor of both IGF1R and IR, did not block tumour formation and had no effect on tumour growth in vivo. Cumulatively these data suggest that while IGF1R and IR are expressed in TNBC, they are not good therapeutic targets. A potential reason for the limited anti-cancer impact when IR/IGF1R was targeted may be because multiple signalling pathways are altered in TNBC. Therefore, targeting individual signalling pathways may not be sufficient to inhibit cancer growth.

Fig 1. Expression of IGF1R and IR mRNA across the different breast cancer subtypes in the BreastMark database using the ssp2006 classifier.

Fig 2. Kaplan Meier curves for distant disease free survival (DDFS), disease-free survival (DFS) and overall survival (OS) based on expression levels of IGF1R and IR mRNA in basal-like breast cancer, using the ssp2006 classifier on BreastMark.

Fig 3

Proliferation assays in A) MDA-MB-231, B) HDQ-P1 and C) HCC1143. Cells were serum starved (2% FCS) for 24 hours followed by addition of IGF-I (50 ng/mL), with /without linsitinib (MDA-MB-231 10 μM; HDQ-P1; 3 μM; HCC1143 5 μM) or xentuzumab (40 μg/mL) (treated with 2% FCS). Proliferation assays in D) MDA-MB-231, E) MDA-MB-468 testing the combination of xentuzumab (BI-836845) and linsitinib in 2 TNBC cell lines. F) Proliferation assays in HCC1143 cells testing the anti-proliferative effects of combining xentuzumab (40 μg/mL) and cisplatin (2 μM) or docetaxel (0.5 nM) in the CAL-51 or HCC1143 cells. G) Proliferation assays in HCC1143, CAL-51 (PIK3CA Mut) and Hs578t (PTEN null) cell lines testing anti-proliferative effect of the combining xentuzumab and NVPBEZ-235 (PI3K/mTOR inhibitor). Standard deviations are calculated from triplicate independent experiments. ‘*’ indicates a p-value of <0.05 as calculated by Students T-test.

Fig 4. Effect of linsitinib and vehicle control on tumour volume, compared to untreated control.

HCC1143 cells were implanted by subcutaneous injection into SCID mice. Animals were randomised to three arms; control, vehicle or linsitinib. Treatments, linsitinib (25 mg/kg) or vehicle were commenced 4 days post-implantation. Tumour volumes were assessed by callipers measurement. Tumour volumes were normalised to volume at day 33, as the starting point for tumour measurement, and represented as the mean and SEM. Tumour volumes arms were stopped when 5 animals remained on study.

Fig 5. Effect of linsitinib, vehicle and untreated control on animal body weight on SCID mice implanted with HCC1143 cells.

The treatment schedule is represented by the grey bars whereby white bars indicate days the animals did not receive treatment. Body weights were measured three times per week. Percentage weight change was monitored relative to the body weight at the time of implant. The mean percentage weight change and standard error of each group are shown. Statistically significant differences were calculated by two-way ANOVA using GraphPad Prism v8 and is represented by *or ** where * = <0.05, **<0.001.