Investigating the role of the tumour microenvironment in invasive lobular carcinoma

Abstract

Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, after invasive ductal carcinoma (IDC), and is defined by loss of the adherens junction protein E-cadherin. ILC tumours are very slow growing and uniquely metastasise to the gastrointestinal tract, peritoneum and ovaries. Although prognosis for ILC and IDC patients initially is good, with roughly 90% of ILC tumours being ER+ and so ideal candidates for endocrine therapies, ILC patients have a poorer long term outcome in multivariate analyses and a poorer response to neoadjuvant chemotherapy than IDC patients. Despite the myriad molecular, histological and clinical differences that distinguish ILC tumours, ILC and IDC patients are treated in the same way in the clinic. Understanding the differences between these two subtypes and identifying therapeutic targets that are enriched in ILC tumours is necessary to improve patient care and outcome for this under-studied but common subtype of breast cancer.

ILC tumours contain a particularly high stromal content and in particular, have a very high density of cancer-associated fibroblasts (CAFs). Analysis of primary ILC and IDC patient-derived CAFs by RNA-sequencing identified 153 differentially expressed genes between ILC and IDC CAFs and a significant enrichment for the pro-tumorigenic mTORC1 pathway in ILC CAFs. A forward-phase protein array (FPPA) identified high expression of insulin-like growth factor-1 (IGF-1) and IGF-1 pathway proteins by CAFs, a pro-proliferative pathway that is enriched in ILC.

To determine the consequences of paracrine signalling from CAFs to ILC tumour cells, the ILC cell line SUM44PE was stimulated with conditioned media collected from the primary ILC CAFs. Reverse-phase protein array analysis identified a significant activation of Mitogen-Activated Protein Kinase pathway proteins and a significant and sustained phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) at Tyr705. This phosphorylation leads to STAT3 translocation to the nucleus where it acts as a transcription factor.

IL-6 was identified as the CAF-secreted factor driving STAT3 activation and RNA-sequencing was used to generate a 42-gene IL-6 dependent gene signature. In the TCGA dataset, this signature, as well as IL-6 and pSTAT3 are all significantly more highly expressed in ER+ ILC tumours compared to ER+ IDC tumours and their expression correlates with stromal content, supporting the paracrine nature of this signalling pathway in ILC.

CAF conditioned media stimulation of SUM44PE cells downregulated expression of the Estrogen receptor (ERα) at the RNA and protein level and suppressed expression of generic and ILC-specific ERα-dependent gene sets as well as upregulating endocrine therapy resistance signatures, dependent on IL-6. In ER+ ILC and IDC tumours in the TCGA dataset, there is a significant negative correlation between ESR1 expression and expression of IL6 and the IL6 dependent gene signature. This suggests that paracrine IL-6 signalling from CAFs suppresses Estrogen signalling and may contribute to endocrine-targeted therapy resistance in ILC.

IL-6 stimulation also led to increased expression of EMT gene sets and caused SUM44PE cells to become more elongated and mesenchymal-like when adhered to CAF-derived extracellular matrices, dependent on STAT3 expression. IL-6 stimulation promoted increased cell motility in 2D and in a zebrafish embryo xenograft model of metastasis, IL-6 stimulation significantly increased SUM44PE cell dissemination throughout the embryos and promoted angiogenesis, suggesting that IL-6/STAT3 signalling may contribute to the metastatic spread of ILC tumours.

Overall, this work identifies ILC-enriched CAF expressed genes and secreted proteins, as well as highlighting the IL-6/STAT3 pathway as a potential therapeutic target in ILC.