SKOR1 mediates FER kinase-dependent invasive growth of breast cancer cells

High expression of the non-receptor tyrosine kinase FER is an independent prognostic factor that correlates with poor survival in breast cancer patients. To investigate whether the kinase activity of FER is essential for its oncogenic properties, we developed an ATP analogue-sensitive knock-in allele (FERASKI). Specific FER kinase inhibition in MDA-MB-231 cells reduces migration and invasion, as well as metastasis when xenografted into a mouse model of breast cancer. Using the FERASKI system, we identified Ski family transcriptional corepressor 1 (SKOR1) as a direct FER kinase substrate. SKOR1 loss phenocopies FER inhibition, leading to impaired proliferation, migration and invasion, and inhibition of breast cancer growth and metastasis formation in mice. We show that SKOR1 Y234, a candidate FER phosphorylation site, is essential for FER-dependent tumor progression. Finally, our work suggests that the SKOR1 Y234 residue promotes Smad2/3 signaling through SKOR1 binding to Smad3. Our study thus identifies SKOR1 as a mediator of FER-dependent progression of high-risk breast cancers. Cancer Signaling networks and Molecular Therapeutic

Characterisation of the Stromal Microenvironment in Lobular Breast Cancer

SIMPLE SUMMARY: Invasive lobular breast cancer (ILC) accounts for approximately 5–15% of breast cancers, and although response rates to treatments are initially good, an ILC diagnosis is associated with adverse long-term outcomes; better treatments, specifically targeted to this sub-type of breast cancer, are required to improve patient survival. The tumor microenvironment (TME) plays an important role in determining how cancers respond to treatment, and in this study, we carried out an in-depth analysis of the TME in ILC following laser-capture microdissection of the tumor stroma, and analysis of primary cancer-associated fibroblasts (CAFs), which comprise the majority of non-malignant cells within tumors. This identified changes in genes involved in regulation of the extracellular matrix and also growth factor signaling pathways that were differentially regulated in ILC. Further analysis of breast cancer datasets showed that two of these genes which encode a secreted metalloproteinase (PAPPA) and a metalloproteinase inhibitor (TIMP2) were associated with survival outcomes in ILC. ABSTRACT: Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, and it exhibits a number of clinico-pathological characteristics distinct from the more common invasive ductal carcinoma (IDC). We set out to identify alterations in the tumor microenvironment (TME) of ILC. We used laser-capture microdissection to separate tumor epithelium from stroma in 23 ER+ ILC primary tumors. Gene expression analysis identified 45 genes involved in regulation of the extracellular matrix (ECM) that were enriched in the non-immune stroma of ILC, but not in non-immune stroma from ER+ IDC or normal breast. Of these, 10 were expressed in cancer-associated fibroblasts (CAFs) and were increased in ILC compared to IDC in bulk gene expression datasets, with PAPPA and TIMP2 being associated with better survival in ILC but not IDC. PAPPA, a gene involved in IGF-1 signaling, was the most enriched in the stroma compared to the tumor epithelial compartment in ILC. Analysis of PAPPA- and IGF1-associated genes identified a paracrine signaling pathway, and active PAPP-A was shown to be secreted from primary CAFs. This is the first study to demonstrate molecular differences in the TME between ILC and IDC identifying differences in matrix organization and growth factor signaling pathways

CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer

The origin of cancer-associated fibroblasts (CAFs) in cancer remains to be identified. Here, single-cell transcriptomics, in vivo and in vitro studies suggest that CD26+ and CD26- normal fibroblasts transform into distinct CAF subpopulations in mouse models of breast cancer

Investigating the role of the tumour microenvironment in invasive lobular carcinoma

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

Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models

The adhesion protein Kindlin-1 is over-expressed in breast cancer where it is associated with metastasis-free survival; however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in mouse models of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells led to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of spontaneous mammary tumorigenesis. There was a significant increase in IL-6 secretion from Met-1 cells when Kindlin-1 was depleted and conditioned media from Kindlin-1-depleted cells led to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6. In addition, deletion of tumor-derived IL-6 in the Kindlin-1-depleted tumors reversed the reduction of tumor-infiltrating Tregs. Overall, these data identify a novel function for Kindlin-1 in regulation of anti-tumor immunity, and that Kindlin-1 dependent cytokine secretion can impact the tumor immune environment