Abstract P5-18-08: Defining molecular signatures to personalise management of patients with early breast cancer
Authors: Allen N , Allen M , Ahmed K , Gomm J , Nelan R , Nagano A , Chelala C , Gadaleta E , Thorat M , Cuzick J , Jones LJ 
Published: May 2019

Background

A review of breast screening highlighted the need to reduce overdiagnosis. Ductal Carcinoma In-Situ (DCIS) contributes significantly to this overdiagnosis. Epithelial cells in DCIS are as genetically advanced as those in invasive disease, focusing attention on the tumour microenvironment (ME). A key components of the ME in DCIS is the myoepithelial cell(MEC). These cells lie at the interface of the epithelial and stromal compartments, regulating cell function. We previously have identified changes in the MEC that contribute to tumour progression. Here we investigate the functional and clinical significance of a novel change in MEC phenotype: loss of Galectin-7 (Gal-7) expression. Gal-7 is proposed to play a role in apoptosis. We hypothesise that changes in MEC phenotype in DCIS alter the ME towards a pro-invasive phenotype, and hypothesise that loss of Gal-7 modifies the ME, destabilizes the MEC interface and ultimately may lead to loss of the MEC population through apoptosis.

Methods

Gal-7 expression and function was investigated in clinical samples and in-vitro model systems, respectively.

Gal-7 expression was assessed in a series of pure DCIS samples (low risk model) and DCIS with co-existant invasion (high risk model). Tissue sections were stained for Gal-7 and MEC expression scored on a duct-by-duct basis as positive, heterogeneous or negative.

An in-vitro model of normal primary myoepithelial cells isolated from reduction mammoplasty was used to investigate the functional impact of loss of Gal-7. These cells have high endogenous levels of Gal-7. Gal-7 was knocked down using siRNA and apoptosis assessed using cleaved caspase-3. The effect of Gal-7 on MEC layer integrity was assessed using immunofluorescence and adhesion assays.

The global impact of loss of Gal-7 was investigated using RNA sequencing.

Results

In the tissue analysis 1926 DCIS ducts were scored for MEC expression of Gal-7. Significantly more ducts showed loss of Gal-7 in DCIS with co-existant invasion, with pure DCIS showing 388 ducts positive and DCIS with invasion 144 DCIS ducts positive (p=0.0014). Pure DCIS and DCIS with invasion had 99 and 646 negative DCIS ducts respectively (p=0.0002).

In model systems of primary MEC, knockdown of Gal-7 resulted in increased expression of cleaved caspase-3, suggesting lower levels of Gal-7 increases apoptosis. In functional assays silencing Gal-7 reduces adhesion to both fibronectin and laminin extracellular matrices (p-value 0.005 and 0.001 respectively)

RNA sequencing indicates silencing Gal-7 increases LOX expression - a key regulator of the collagen matrix of the microenvironment.

Conclusion

Normal MEC strongly express Gal-7. Expression is lost in DCIS, with significantly more frequent loss in DCIS with co-existant invasion, suggesting that loss is associated with a more advanced phenotype. Functional assays indicate that loss of MEC Gal-7 enhances MEC apoptosis, which may be one mechanism by which this interface is lost during progression. Gal-7 negative MEC also show impaired adhesion to matrix proteins and lead to up-regulation of LOX, an enzyme key in promoting tumourigenesis. The incorporation of Gal-7 expression into a risk stratification algorithm has functional evidence and is currently being investigated.

Citation Format: Allen N, Allen M, Ahmed K, Gomm J, Nelan R, Nagano A, Chelala C, Gadaleta E, Thorat M, Cuzick J, Jones LJ. Defining molecular signatures to personalise management of patients with early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-18-08.