∆Np63/p40 correlates with the location and phenotype of basal/mesenchymal cancer stem-like cells in human ER+ and HER2+ breast cancers

ΔNp63, also known as p40, regulates stemness of normal mammary gland epithelium and provides stem cell characteristics in basal and HER2‐driven murine breast cancer models. Whilst ΔNp63/p40 is a characteristic feature of normal basal cells and basal‐type triple‐negative breast cancer, some receptor‐positive breast cancers express ΔNp63/p40 and its overexpression imparts cancer stem cell‐like properties in ER+ cell lines. However, the incidence of ER+ and HER2+ tumours that express ΔNp63/p40 is unclear and the phenotype of ΔNp63/p40+ cells in these tumours remains uncertain. Using immunohistochemistry with p63 isoform‐specific antibodies, we identified a ΔNp63/p40+ tumour cell subpopulation in 100 of 173 (58%) non‐triple negative breast cancers and the presence of this population associated with improved survival in patients with ER−/HER2+ tumours (p = 0.006). Furthermore, 41% of ER+/PR+ and/or HER2+ locally metastatic breast cancers expressed ΔNp63/p40, and these cells commonly accounted for <1% of the metastatic tumour cell population that localised to the tumour/stroma interface, exhibited an undifferentiated phenotype and were CD44+/ALDH−. In vitro studies revealed that MCF7 and T47D (ER+) and BT‐474 (HER2+) breast cancer cell lines similarly contained a small subpopulation of ΔNp63/p40+ cells that increased in mammospheres. In vivo, MCF7 xenografts contained ΔNp63/p40+ cells with a similar phenotype to primary ER+ cancers. Consistent with tumour samples, these cells also showed a distinct location at the tumour/stroma interface, suggesting a role for paracrine factors in the induction or maintenance of ΔNp63/p40. Thus, ΔNp63/p40 is commonly present in a small population of tumour cells with a distinct phenotype and location in ER+ and/or HER2+ human breast cancers.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153532/1/cjp2149_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153532/2/cjp2149.pd

P73 regulates cisplatin-induced apoptosis in ovarian cancer cells via a calcium/calpain-dependent mechanism

P73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in ovarian cancer (OVCA) is poorly understood. Furthermore, if and how the deregulation of p73-mediated apoptosis confers resistance to cisplatin (CDDP) treatment is unclear. Here we demonstrate that TAp73α over-expression enhanced CDDP-induced PARP cleavage and apoptosis in both chemosensitive (OV2008 and A2780s) and their resistant counterparts (C13* and A2780cp) and another chemoresistant OVCA cells (Hey); in contrast, the effect of ΔNp73α over-expression was variable. P73α downregulation attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008 cells. CDDP decreased p73α steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced p73α downregulation was mediated by a calpain-dependent pathway. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73α in OV2008, but not C13* cells. CDDP increased the intracellular calcium concentration ([Ca2+]i) in OV2008 but not C13* whereas cyclopiazonic acid (CPA), a Ca2+-ATPase inhibitor, caused this response and calpain activation, p73α processing and apoptosis in both cell types. CDDP-induced [Ca2+]i increase in OV2008 cells was not effected by the elimination of extracellular Ca2+, but this was attenuated by the depletion of internal Ca2+ store, indicating that mobilization of intracellular Ca2+] stores was potentially involved. These findings demonstrate that p73α and its regulation by the Ca2+-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that dysregulation of Ca2+/calpain/p73 signaling may in part be the pathophysiology of CDDP resistance. Understanding the cellular and molecular mechanisms of chemoresistance will direct the development of effective strategies for the treatment of chemoresistant OVCA

The diverse oncogenic and tumour suppressor roles of p63 and p73 in cancer: a review by cancer site

p63 and p73, the two other members of the p53 family, were identified almost 15 years ago. Here, we review their potential use for diagnosis, prognosis and prediction of response to therapy in various cancers. The two genes show distinct expression patterns in both normal and cancer tissues and each gene gives rise to multiple protein isoforms with different activities, including those with tumour-suppressor or oncogenic effects. Despite such complexity, some common themes emerge; p63 is commonly overexpressed as the ΔNp63 isoform and sometimes associated with TP63 amplification, whereas p73 is often reduced (by methylation or gene loss), or there is an increase in the ratio of ΔNp73 to TAp73. These generalisations do not apply universally; TAp63 is overexpressed in haematological malignancies, TP63 mis-sense mutations have been reported in squamous cancers and TP63 translocations occur in lymphomas and some lung adenocarcinomas. There are associations with disease prognosis and response to specific therapies in individual cancer types for both p63 and p73, making their analysis of clinical relevance. We also discuss their utility for aiding in differential diagnosis, which has been demonstrated for p63, but not yet for p73. Throughout, we highlight the discrepant nature of many studies due to the variable methodologies employed, the lack of systematic evaluation of isoforms and the problems of poor antibody characterization and crossreactions within the p63/p73 family. Finally, we emphasize the value of recently developed isoformspecific reagents that have clear advantages for the study of p63 and p73 experimentally and clinically

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

Background: p63, a member of the p53 protein family, plays key roles in epithelial development and carcinogenesis. In breast cancer, p63 expression has been found predominantly in basal-A (epithelial-type) triple-negative breast carcinomas (TNBC). To investigate the functional role of p63 in basal-A TNBC, we created MDA-MB-468 cell lines with inducible expression of the two major N-terminal p63 isoforms, TAp63α and ΔNp63α. Results: TAp63α did not have significant effect on gene expression profile and cell phenotype, whilst the main effect of ΔNp63α was reduction of cell adhesion. Gene expression profiling revealed genes involved in cell adhesion and migration whose expression relies on overexpression of ΔNp63α. Reduced cell adhesion also led to decreased cell proliferation in vitro and in vivo. Similar data were obtained in another basal-A cell line, BT-20, but not in BT-549 basal-B (mesenchymal-like) TNBC cells. Conclusions: In basal-A TNBC cells, ΔNp63α has much stronger effects on gene expression than TAp63α. Although p63 is mentioned mostly in connection with breast cell differentiation and stem cell regulation, we showed that a major effect of p63 is regulation of cell adhesion, a process important in metastasis and invasion of tumour cells. That this effect is not seen in mesenchymal-type TNBC cells suggests lineage-dependent functions, mirroring the expression of ΔNp63α in primary human breast cancers. © 2016 The Author(s)

Delta Np63 alpha expression induces loss of cell adhesion in triple-negative breast cancer cells

Background: p63, a member of the p53 protein family, plays key roles in epithelial development and carcinogenesis. In breast cancer, p63 expression has been found predominantly in basal-A (epithelial-type) triple-negative breast carcinomas (TNBC). To investigate the functional role of p63 in basal-A TNBC, we created MDA-MB-468 cell lines with inducible expression of the two major N-terminal p63 isoforms, TAp63 alpha and Delta Np63 alpha. Results: TAp63 alpha did not have significant effect on gene expression profile and cell phenotype, whilst the main effect of Delta Np63 alpha was reduction of cell adhesion. Gene expression profiling revealed genes involved in cell adhesion and migration whose expression relies on overexpression of Delta Np63 alpha. Reduced cell adhesion also led to decreased cell proliferation in vitro and in vivo. Similar data were obtained in another basal-A cell line, BT-20, but not in BT-549 basal-B (mesenchymal-like) TNBC cells. Conclusions: In basal-A TNBC cells, Delta Np63 alpha has much stronger effects on gene expression than TAp63 alpha. Although p63 is mentioned mostly in connection with breast cell differentiation and stem cell regulation, we showed that a major effect of p63 is regulation of cell adhesion, a process important in metastasis and invasion of tumour cells. That this effect is not seen in mesenchymal-type TNBC cells suggests lineage-dependent functions, mirroring the expression of Delta Np63 alpha in primary human breast cancers