Role of the IL-6/STAT3 pathway in invasive lobular carcinoma

Abstract

Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer (BC) after invasive ductal carcinoma (IDC) and accounts for 15% of BC cases. ILC is characterised by loss of E-cadherin, slow and discohesive invasive growth, with a unique pattern of metastatic sites which includes the gastrointestinal tract, peritoneum, and ovaries.

90% of ILC cases are estrogen receptor (ER) positive and are considered ideal candidates for endocrine therapies (ET), including letrozole and tamoxifen. Initially, ILC patients respond well to ET but eventually develop resistance. Retrospective analysis has shown poor outcomes in ILC compared to IDC. Therefore, it is crucial to comprehend the mechanisms of resistance to ETs to enhance the care and outcomes of ILC patients.

Interleukin-6 (IL-6) has been identified as a cancer-associated fibroblast (CAF) secreted factor in human ILC cells driving the phosphorylation and translocation of Signal Transducer and Activator of Transcription 3 (STAT3) into the nucleus where it acts as a transcription factor. The IL-6/STAT3 signalling pathway has been associated with resistance to ET in BC cell lines and patients via multiple mechanisms including signalling from the tumour microenvironment (TME). We initially characterised this pathway in a mouse model of ILC, where significant expression of Il6 was found in mouse CAFs, with no measurable expression in the mouse epithelial tumour cells. CAF-conditioned media (CM) and recombinant IL-6 stimulation of mouse epithelial cells resulted in phosphorylation of STAT3 confirming the paracrine activation of STAT3. Additionally, the effect of IL-6 on mouse ILC cell proliferation, migration, and invasion was assessed in vitro, where we observed that both CAF CM and recombinant IL-6 resulted in a decreased proliferation rate of ILC tumour cells compared to the control. Recombinant IL-6 stimulation resulted in increased migration and invasion of ILC cells.

RNA sequencing analysis of human ILC cell lines SUM44PE, MDA-MB-134VI, and two patient-derived xenografts, HCI-013 and HCI-018, following stimulation with recombinant IL-6 showed downregulation of ER expression and suppression of generic and ILC-specific ERα-dependent gene sets, as well as upregulating a subset of letrozole and tamoxifen resistance genes.

We generated a 60-gene IL-6 dependent gene signature and found a significant negative correlation between ESR1 expression and expression of IL6 and the IL-6 dependent gene signature in ER+ ILC and IDC tumours in the TCGA dataset. To further investigate and understand the clinical significance of IL-6/STAT3 pathway, we carried out immunohistochemical (IHC) analysis on 246 primary operable ILC (Glasgow cohort) BC patients for alpha-smooth muscle actin (αSMA), STAT3, pSTAT3, and the STAT3 target gene, BCL3. The analysis highlighted the significant association of αSMA and cytoplasmic STAT3 with disease-free survival as well as other clinicopathological features of the patients.

This project also aimed to investigate the effect of Letrozole treatment in ILC. IHC analysis in 32 ILC patients (Edinburgh cohort) who were treated with letrozole, and their matched primary tumour sections, for STAT3, pSTAT3, and BCL3 revealed a differential pattern of expression of nuclear STAT3 and pSTAT3 upon letrozole treatment. NanoString GeoMax Digital Spatial Proteome Profiling of the Edinburgh cohort revealed a distinct proteome in stromal and tumour compartments. Letrozole treated patients showed a reduction in proliferation, PI3K-AKT and MAPK pathway-related proteins in tumour cells and a subset of immune cell markers in the stromal compartment compared to their baseline biopsies. These findings suggest that letrozole may not only inhibit tumour growth directly by reducing estrogen levels, but also alter the tumour microenvironment.

To conclude, this work provides valuable insights into the importance of tumour-stromal IL-6 signalling in BC and suggests that inhibiting IL-6/STAT3 signalling could represent a promising therapeutic strategy for investigation in BC.