Conditional in vivo deletion of LYN kinase has little effect on a BRCA1 loss-of-function-associated mammary tumour model

ABSTRACT LYN kinase is expressed in BRCA1 loss-of-function-dependent mouse mammary tumours, in the cells of origin of such tumours, and in human breast cancer. Suppressing LYN kinase activity in BRCA1-defective cell lines as well as in in vitro cultures of Brca1-null mouse mammary tumours is deleterious to their growth. Here, we examined the interaction between LYN kinase and BRCA1 loss-of-function in an in vivo mouse mammary tumour model, using conditional knockout Brca1 and Lyn alleles. Comparison of Brca1 tumour cohorts showed little difference in mammary tumour formation between animals that were wild type, heterozygous or homozygous for the conditional Lyn allele, although this was confounded by factors including incomplete Lyn recombination in some tumours. RNA-sequencing analysis demonstrated that tumours with high levels of Lyn gene expression had a slower doubling time, but this was not correlated with levels of LYN staining in tumour cells themselves. Rather, high Lyn expression and slower tumour growth were likely a result of B-cell infiltration. The multifaceted role of LYN indicates that it is likely to present difficulties as a therapeutic target in breast cancer.

tumours, 8 Lyn low and 6 Lyn high tumours) and tumour cells (n=6 of each genotype) in primary culture.There are no significant differences between any group (Mann-Whitney), however, there does appear to be a batch effect in the cultured cells, with higher B-actin expression in Lyn wt/wt samples.Therefore only Gapdh was used for normalisation in these samples.

Disease Models & Mechanisms • Supplementary information
Table S7.Raw and normalised RNAseq counts from the 13 tumours with the highest Lyn RNAseq expression counts (Lyn high tumours) and the 13 tumours with the lowest Lyn RNAseq expression counts (Lyn low tumours) and analysis of differential expression of genes across Lyn high and Lyn low groups.
Table S8.Raw and normalised RNAseq counts and differentially expressed genes comparing tumours from PCA groups 1 and 2 with tumours from PCA groups 3 and 4.
Note positive fold changes are elevated in groups 3 and 4; negative fold changes are lowered in groups 3 and 4 / elevated in 1 and 2. Table S9.Significantly differentially expressed genes (DEGs) (adjusted p value <0.05; log2 fold change ≤0.5 or ≥2.0) for the WT vs HOM, Lyn high vs Lyn low and PCA 1 and 2 vs PCA 3 and 4 comparisons.
For each comparison, whether or not the DEG is also found to be differentially expressed in the other comparisons is indicated.

Table S5. RNAseq sample identifiers and sample features, including histological observations, diagnosis, in vivo
doubling time, LYN and Ki67 staining.Also included are the three categories used to identify sets of differentially expressed genes, namely (1) genotype of the animal from which the tumour was derived; (2) the normalised Lyn RNAseq expression score and the ranking of each tumour on the basis of that score; (3) the identifiers of each tumour used in the Principal Component Analysis (PCA ID) and its PCA group.Table S6.Raw and normalised RNAseq counts across all 39 samples and analysis of differential expression comparing tumours from the different cohort genotypes.

Fig. S3 .
Fig. S3.(relates to Fig. 2): LYN staining in white pulp of spleen.Representative white pulp areas with germinal centres from spleens of Lyn wt/wt (A -C) and Lyn fl/fl (D -F) mice.Antigens detected by immunohistochemistry are indicated on each panel.LYN expression is downregulated in proliferative germinal centres in both genotypes.Bars = 200 µm.

Fig. S4 (
Fig. S4 (relates to Fig. 2): Key distinguishing features enabling histotyping of mouse mammary epithelial tumours.Left hand column, H&E staining.Right hand column, p63 staining.A, B) Adenocarcinoma of no special type (AC) showing no metaplastic features (A) and little p63 staining (B).C, D) Adenomyoepithelioma (AME) showing characteristic abundant p63 staining with a pseudo-basal pattern and 'ectopic' pseudo-luminal expression.E, F) Metaplastic adenosquamous carcinoma (ASQC) showing squamous metaplasia, keratin pearls (asterisks) and abundant p63 staining.G, H) Metaplastic spindle cell carcinoma (MSCC) showing spindle cell metaplasia and characteristic background stain at the periphery of each spindle cell.This is seen with all antibodies, not just the p63 stain.Bars = 200 µm.Insets are magnified 2.5 times.

Fig
Fig. S7.(relates to Fig. 8): CIBERSORTx estimates of immune cell abundance in the Lyn low (n=13) vs Lyn high (n=13) (A) and PCA group 1/2 (n=29) vs PCA group 3/4 (n=10) tumours (B) based on normalised RNAseq expression data.Estimates are provided using the absolute abundance (arbitrary units) approach (Newman et al., 2019) so that the proportions of each different immune cell type are comparable.Mean±s.d.. N.S., no significant difference, multiple two-tailed t-tests with Holm-Sidak correction.If no significance indication is given, there were too few samples for statistical comparison (or none).'Zero' data points are not plotted.Raw data in Supplementary : Supplementary information