LMO2 regulates epithelial-mesenchymal plasticity of mammary epithelial cells

Abstract

Cellular plasticity in mammary epithelial cells enables dynamic cell state changes essential for normal development but can be hijacked by breast cancer cells to drive tumor progression. However, the molecular factors that maintain cellular plasticity through the regulation of a hybrid cell state (epithelial/mesenchymal) are not fully defined. As LMO2 has been previously shown to regulate metastasis, here we determined the role of LMO2 in the normal mammary epithelial cells. Using lineage tracing and knockout mouse models we find that Lmo2 lineage-traced cells persist long-term in the mammary gland, both in the luminal and basal layer but have limited proliferative potential. Lmo2 loss does not impact mammary gland development, but acute deletion decreases in vivo reconstitution. Moreover, LMO2 knockdown in mouse and human mammary epithelial cells (MECs) reduces organoid formation. We find that LMO2 maintains a hybrid cell state in MECs and LMO2 knockdown promotes mesenchymal differentiation. Transcriptional profiling of LMO2 knockdown cells reveals significant enrichment in the epithelial-mesenchymal transition (EMT) pathway and upregulation of MCAM, a negative regulator of regenerative capacity in the mammary gland. Altogether, we show that LMO2 plays a role in maintaining cellular plasticity in MECs, adding insight into the normal differentiation programs hijacked by cancer cells to drive tumor progression.

Figure 1:. LMO2 marks a long-lived population in the mammary gland

(A) Schematic diagram showing the crossing of Rosa26^mTmG reporter with Lmo2^creERT2 mice to generate Lmo2^creERT2/ Rosa26^mTmG (B) q-PCR analysis of GFP+ and GFP− cells sorted from Lmo2-cre mice (n=2) (C) Immunofluorescence analysis of adult Lmo2^creERT2/ Rosa26^mTmG mice pulsed with Tamoxifen at 8 weeks and analyzed after 36h. Representative images (left), quantification using flow cytometry (right) (n=4 mice) (see Supplementary Fig. 1B for flow cytometry plots). (D) Lmo2^creERT2/ Rosa26^mTmG pulsed at 4 weeks and analyzed at 12 weeks. Representative images (left), quantification by flow cytometry (right) (n=3 mice) (E) Flow cytometry quantification of GFP+ cells from Lmo2^creERT2/ Rosa26^mTmG mice pulsed at 8 weeks and analyzed at 16 weeks (n=4 mice). (F) Representative images of Lmo2^creERT2/ Rosa26^mTmG mice pulsed at puberty, mated 8 weeks later and analyzed at gestation day 15.5. Scale bar 10μm except F is 50μm.

Figure 2:. Lmo2 loss does not impact normal development but leads to decreased reconstitution potential

(A) Flow cytometry analysis of Live/Lineage⁻ cells from K14^Cre/Lmo2^fl/fl and K14^Cre/Lmo2^+/+ mammary glands (see Supplementary Fig. 1A for gating strategy). Representative flow plot of Live/Lineage− cells. (B) Ratio of basal to luminal cells in the mammary epithelial cell population from K14^Cre/Lmo2^fl/fl and K14^Cre/Lmo2^+/+ mammary glands (n=5 mice) (C) Limiting dilution transplantation assay with lineage depleted cells from Lmo2^+/+ and Lmo2^fl/fl mice transduced with Lenti-cre-GFP plasmid. Pie chart indicates percentage of fat-pad filled. The proportion of positive outgrowths out of the total cell number injected is shown on the right. (Note: Injections with 5000 or less lineage depleted MECs transduced with lenti-cre did not lead to any outgrowths in either Lmo2^+/+ or Lmo2^fl/fl mice.) P-value and repopulating cell frequency was calculated using ELDA. Representative images of transplantation outgrowth are displayed on the right. (D) Organoid formation in control and Lmo2 knockdown basal epithelial cells sorted from 3-month-old mice. Representative image (left), quantification (right), n=3 mice with 3 technical replicates each. (E) Organoid formation in control and Lmo2 knockdown luminal epithelial cells sorted from 3-month-old mice. Representative image (left), quantification (right), n=3 mice with 3 technical replicates each. Data are shown as mean ± SD. Statistical significance was calculated using unpaired student’s t test (B) and ordinary one-way ANOVA with multiple comparison test (D). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p<0.0001.

Figure 3:. LMO2 knockdown in MCF10A cells reduces organoid formation, hybrid cell state and induces mesenchymal differentiation

(A) Quantification (left) and representative images (right) of colony formation in control and LMO2 knockdowns MCF10A cells (n=3 biological and 3 technical replicates). (B) Quantification (left) and representative images (right) of acini formation of control and LMO2 knockdown MCF10A cells. (n=3 biological and 3 technical replicates). (C) Representative images of flow cytometry analysis of control and LMO2 knockdown MCF10A cells with previously established markers EpCAM and CD49f (top row), ECAD (middle row), CD44 and CD104 (bottom row). (D-G) Quantification of flow cytometry analysis of control and LMO2 knockdown MCF10A cells for the following populations: (D) EpCAM+/CD49f+ (E) CD49f+ (F) ECAD-high (G) CD104+/CD44+. Data are shown as mean ± SD. Statistical significance was calculated using ordinary one-way ANOVA with multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p<0.0001.

Figure 4:. Transcriptomic analysis of LMO2 knockdown in MCF10A cells

(A) Schematic of bulk-RNA sequencing of control and LMO2 knockdown MCF10A cells (n=3) at 6 and 21 days after infection. (B) Volcano plots visualizing the fold-changes and p-values between control and LMO2 knockdown after 6 days post-transduction. (C) Enrichment plots for interferon-gamma and interferon-alpha response gene sets from data displayed in B at 6 days post-transduction. (D) Representative flow cytometry analysis of MCAM expression in control and LMO2 knockdown MCF10A cells. (E) Quantification of flow cytometry analysis of MCAM expression in control and LMO2 knockdown MCF10 cells (n=9). (F) Volcano plots visualizing the p-value and fold change between control and LMO2 knockdown MCF10A cells 21 days after post-transduction (G) Enrichment plot of the epithelial-mesenchymal transition gene set from data displayed in F at 21 days post-transduction. NES = normalized enrichment score, FDR = false discovery rate. Statistical significance was calculated using ordinary one-way ANOVA with multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p<0.0001.