Title: Myoepithelial degradome in the progression of DCIS to invasive breast cancer
Funder: Cancer Research UK

Abstract: Background Half of ductal carcinoma in situ (DCIS) cases will not develop into invasive cancer. This has led to the over-treatment of women with breast cancer because we cannot identify DCIS cases at risk of progression. This is further undermined by a poor understanding of the cellular mechanisms behind progression. Pathologically, invasive breast cancer is distinguished from DCIS by the disappearance of the outer myoepithelial cell layer of the duct. However, the biology of the myoepithelial cells changes dramatically in DCIS, wherein they adopt a more invasive phenotype, characterised by enhanced motility and matrix remodelling. Aims We hypothesise that myoepithelial cells play an active role in progression, promoting invasion through degradation of the surrounding matrix. Understanding how myoepithelial cells promote invasion using unbiased degradomic and transcriptomic analysis of physiomimetic models of progression will reveal novel, functional, biomarkers to be used in the clinic to stratify patients with DCIS. Methods We have developed a 3D in vitro model system of the breast duct bilayer using patient-derived cells. Luminal expression of HER2 recapitulates DCIS, and expression of integrin ITGB6 in myoepithelial cells, a marker of high-risk DCIS, results in myoepithelial-led invasion. This invasion is dependent on protease activity and highlights the role these cells play in progression. We will adapt our model system to incorporate cells of the breast microenvironment (adipocytes, fibroblasts, and macrophages), and myoepithelial cells isolated from DCIS patients, allowing the invasive properties of myoepithelial cells to be assessed in settings that closely mimic clinical tissue. Degradomic analysis of our model system will identify the cleaved proteins that arise during myoepithelial-led invasion. Integration with RNA sequencing data will reveal protease/target pairs, whose importance for invasion will be validated using shRNA/pharmacological approaches. Alongside this, the regulation and downstream biology of these novel cleaved proteins will be investigated. Expression of proteases, and their novel cleaved protein products, will then be assessed in a clinical cohort of DCIS patients with long-term patient follow up, to evaluate their ability to predict recurrence. How this research will be used This project will demonstrate that patient-derived cells in 3D models of progression, combined with “omics” approaches, can identify functional biomarkers of progression. Using our biomarker panel, we aspire to initiate a clinical trial to assess progressive risk in biopsies from DCIS patients, stratifying patients into conventional treatment or active monitoring accordingly, thus reducing the unnecessary treatment and stress experienced by many women with indolent DCIS.

Lay abstract: Background Half of ductal carcinoma in situ (DCIS) cases will not develop into invasive cancer. This has led to the over-treatment of women with breast cancer because we cannot identify DCIS cases at risk of progression. This is further undermined by a poor understanding of the cellular mechanisms behind progression. Pathologically, invasive breast cancer is distinguished from DCIS by the disappearance of the outer myoepithelial cell layer of the duct. However, the biology of the myoepithelial cells changes dramatically in DCIS, wherein they adopt a more invasive phenotype, characterised by enhanced motility and matrix remodelling. Aims We hypothesise that myoepithelial cells play an active role in progression, promoting invasion through degradation of the surrounding matrix. Understanding how myoepithelial cells promote invasion using unbiased degradomic and transcriptomic analysis of physiomimetic models of progression will reveal novel, functional, biomarkers to be used in the clinic to stratify patients with DCIS. Methods We have developed a 3D in vitro model system of the breast duct bilayer using patient-derived cells. Luminal expression of HER2 recapitulates DCIS, and expression of integrin ITGB6 in myoepithelial cells, a marker of high-risk DCIS, results in myoepithelial-led invasion. This invasion is dependent on protease activity and highlights the role these cells play in progression. We will adapt our model system to incorporate cells of the breast microenvironment (adipocytes, fibroblasts, and macrophages), and myoepithelial cells isolated from DCIS patients, allowing the invasive properties of myoepithelial cells to be assessed in settings that closely mimic clinical tissue. Degradomic analysis of our model system will identify the cleaved proteins that arise during myoepithelial-led invasion. Integration with RNA sequencing data will reveal protease/target pairs, whose importance for invasion will be validated using shRNA/pharmacological approaches. Alongside this, the regulation and downstream biology of these novel cleaved proteins will be investigated. Expression of proteases, and their novel cleaved protein products, will then be assessed in a clinical cohort of DCIS patients with long-term patient follow up, to evaluate their ability to predict recurrence. How this research will be used This project will demonstrate that patient-derived cells in 3D models of progression, combined with “omics” approaches, can identify functional biomarkers of progression. Using our biomarker panel, we aspire to initiate a clinical trial to assess progressive risk in biopsies from DCIS patients, stratifying patients into conventional treatment or active monitoring accordingly, thus reducing the unnecessary treatment and stress experienced by many women with indolent DCIS.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS