Role of IL6ST in endocrine sensitivity of breast cancer

Breast cancer is the most common type and cause of cancer death in women. Oestrogen is a major growth stimulant of oestrogen receptor alpha (ERa) positive breast cancer and consequently, endocrine therapy which targets either ER or oestrogen synthesis is standard treatment. Recently, high expression of the ubiquitously expressed transmembrane receptor IL6ST (gp130) has been identified as a predictive biomarker of endocrine treatment response in breast cancer patients and was included in the ‘Endopredict’ test. However, the mechanism as to why IL6ST is associated with response to endocrine treatment is not yet understood. To investigate this further, this project evaluated the expression, function and signalling of IL6ST in ER- positive breast cancer cell lines. It then sought to investigate crosstalk between the ER and IL6ST signalling pathways. The expression of IL6ST in three ER+ breast cancer cell lines (MCF-7, ZR751 and T47D) was found to be differentially expressed across these models, while multiple soluble isoforms of IL6ST were identified. As IL6ST is the common signal transducing receptor component for the IL6ST family of cytokines, the effects of seven IL6ST cytokines (IL-6, OSM, LIF, IL- 11, CNTF, IL-27, CT-1) on these cell lines were studied. These cytokines caused differential growth and migration effects in these cell lines e.g., MCF-7 cells were growth-stimulated, while ZR751 cells were inhibited by IL-6 and OSM. IL-6 signalling itself can operate through either classic (via membrane-bound IL-6R) or transsignalling (via the soluble IL-6R) mechanisms which are activated through IL6ST. Evidence to support trans-signalling was obtained for both MCF-7 and ZR751 cell line models. Both IL-6 and OSM modulated the expression of ER and PR. The interaction between cytokines and E2 on ER+ cell lines growth was analysed. E2 eliminated the effect of IL-6 trans-signalling pathway and maintained the effect of OSM. A three-stage MCF- 7 cell model emulating the clinical development of acquired endocrine was used to understand the possible effects of IL6ST cytokines in changing their response to E2. Endocrine insensitive and resistant MCF-7/LCC1 and MCF-7/LCC9 cell lines were inhibited by OSM exposure in the absence of E2 stimulation. The intracellular pathways JAK/STAT and MAPK/ERK may have roles in endocrine resistance. These pathways are the major IL6ST-activated pathways and have the potential to crosstalk with ER signalling. Therefore, the experiments here focused on their activation and their implication in cellular processes. Both JAK/STAT and MAPK/ERK pathways were activated by IL-6 and OSM stimulation in a concentration dependent manner. Expression of STAT3 (Tyr705) and p42/p44 MAPK (ERK1/ERK2) (Thr202/Tyr204) phosphorylation were increased by trans-signalling compared to classic signalling processes in a time dependent manner and decreased by recombinant sgp130Fc (a soluble form of IL6ST). The differential activation of the two pathways in the different cell lines may be associated with modified response. Modulation of the expression of IL6ST and its consequences were investigated using a small molecule inhibitor (SC144) and a CRISPR reduction (of IL6ST) strategy. Growth modulation studies along with gene expression analysis and reverse phase protein array (RPPA) experiments were undertaken. SC144 inhibited the growth of both endocrine treatment sensitive and insensitive cell lines. The CRISPR cell lines had diminished growth responses to both IL-6 and OSM and this was associated with modified signalling pathways assayed by western analysis. RPPA analysis demonstrated STAT, MAPK and mTOR pathway activation after IL-6 treatment in CAS9 treated control cells which showed marked expression reduction in the CRISPR cell line after IL-6 exposure. RPPA analysis identified a large number of differential signalling activation components between MCF-7 and MCF7/ LCC9 cell lines as a result of SC144 treatment. Gene expression analysis of the CRISPR cells revealed modulation of several major pathways compared with the wild type. In conclusion, while IL6ST is ubiquitously expressed, its expression levels vary between cell lines. High expression of IL6ST (as in ZR751) may lead to growth inhibition by key cytokines while lower expression (as in MCF7) may be associated with proliferation. Both classic and trans-signalling pathways can operate in ERpositive breast cancer cells. The IL6ST and ER pathways interact at multiple points