ΔNp63 regulates cell proliferation, differentiation, adhesion, and migration in the BL2 subtype of basal-like breast cancer

Abstract

Triple-negative breast cancers (TNBC) comprise a heterogeneous subgroup of tumors with a generally poor prognosis. Subclassification of TNBC based on genomic analyses shows that basal-like TNBCs, specifically the basal A or BL2 subtype, are characterized by the expression of ΔNp63, a transcription factor that has been attributed a variety of roles in the regulation of proliferation, differentiation, and cell survival. To investigate the role(s) of p63 in basal-like breast cancers, we used HCC1806 cells that are classified as basal A/BL2. We show that these cells endogenously express p63, mainly as the ΔNp63α isoform. TP63 gene knockout by CRISPR resulted in viable cells that proliferate more slowly and adhere less tightly, with an increased rate of migration. Analysis of adhesion-related gene expression revealed a complex set of alterations in p63-depleted cells, with both increased and decreased adhesion molecules and adhesion substrates compared to parental cells expressing p63. Examination of the phenotype of these cells indicated that endogenous p63 is required to suppress the expression of luminal markers and maintain the basal epithelial phenotype, with increased levels of both CK8 and CK18 and a reduction in N-cadherin levels in cells lacking p63. On the other hand, the level of CK5 was not decreased and ER was not increased, indicating that p63 loss is insufficient to induce full luminal-type differentiation. Taken together, these data demonstrate that p63 exerts multiple pro-oncogenic effects on cell differentiation, proliferation and adhesion in basal-like breast cancers.

ΔNp63α expression induces loss of cell adhesion in triple-negative breast cancer cells

“…The major findings from this model were related to alterations of differentiation markers, where p63 is required to suppress expression of luminal markers and maintain the basal epithelial phenotype, but p63 loss is insufficient to induce full luminal-type differentiation. We also found that p63 depletion changed expression of adhesion molecules in a similar manner to that observed here, with reciprocal changes in FAT2 , ITGA2 , CLDN1 , LAMA4 and CEACAM6 following p63 depletion [ 29 ] or p63 induction (this manuscript). However, either over-expression or gene knockout each caused a loss of cellular adhesion in the respective cell lines, indicating that the consequences of manipulating p63 levels are dependent on the cells under investigation, which likely relates to their dependence on p63 and their different genetic backgrounds.…”

“…Consistent with this notion, ΔNp63 promotes normal mammary stem cell activity by enhancement of Wnt signalling and through this mechanism governs tumour-initiating activity of basal-like breast cancer [ 28 ]. In general agreement with these findings, abrogation of endogenous ΔNp63 causes a switch towards luminal phenotype and away from the basal phenotype in basal breast cancer cells, indicating a role in lineage regulation, although p63 silencing was insufficient to cause full luminal-type differentiation [ 29 ]. Further, ΔNp63 acts as a survival factor in a subset of breast cancers by antagonizing p73-mediated apoptosis [ 23 ].…”

“…We recently reported results of CRISPR/Cas9-mediated TP63 knock-out in the HCC1806 basal-A breast cancer cell line [ 29 ]. The major findings from this model were related to alterations of differentiation markers, where p63 is required to suppress expression of luminal markers and maintain the basal epithelial phenotype, but p63 loss is insufficient to induce full luminal-type differentiation.…”

Oncogenic Intra-p53 Family Member Interactions in Human Cancers

“…The same mechanisms occur for p73 and p63: p73 displays 14 isoforms and p63 exhibits 10 isoforms ( 59 , 90 , 109 – 117 ) (Figure 1 ). The N-terminal truncated isoforms ΔNp73 and ΔNp63 are highly expressed in the development and display an oncogenic dominant-negative function to p73 and p63 full-length (TAp73 and TAp63, respectively) and wt-p53 ( 39 , 114 , 118 – 124 ).…”

Epigenomic Profiling Discovers Trans-lineage SOX2 Partnerships Driving Tumor Heterogeneity in Lung Squamous Cell Carcinoma

“…The knockout efficiency was confirmed in the bulk populations via immunoblotting. sgRNA target sequences were selected from Brunello library (29) and Orzol and colleagues (30). Nontarget sgRNAs from the Gecko library v2 (31) were used as scramble sgRNAs.…”