Biochemical evidence for conformational variants in the anti-viral and pro-metastatic protein IFITM1.

Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.

ΔNp63 is regulated by insulin/IGF-1 signaling in normal basal/progenitor mammary cells and in luminal-type breast cancer cells.

Breast cancers are a heterogeneous group of tumors classified according to their histological growth patterns and receptor expression characteristics. Intratumor heterogeneity also exists, with subpopulations of cells with different phenotypes found in individual cancers, including cells with stem or progenitor cell properties. At least two types of breast cancer stem cells (CSCs) exist, the epithelial and the basal/mesenchymal subtypes, although how these phenotypes are controlled is unknown. ΔNp63 is a basal cell marker and regulator of stem/progenitor cell activities in the normal mammary gland and is expressed in the basal-like CSC subpopulation in some estrogen receptor-positive (ER+) and/or human epidermal growth factor receptor 2-positive (HER2+) breast adenocarcinomas. Whilst p63 is known to directly impart CSC properties in luminal breast cancer cells, how p63 is regulated and induced in these cells is unknown. We initially confirmed the existence of a small subpopulation of ΔNp63+ cells in lymph node metastases of ER+ human ductal adenocarcinomas, indicating together with previous reports that ΔNp63+ tumor cells are present in approximately 40% of these metastases. Notably, ΔNp63+ cells show a preferential location at the edge of tumor areas, suggesting possible regulation of ΔNp63 by the tumor microenvironment. Subsequently, we showed that the high levels of ΔNp63 in basal non-transformed MCF-10A mammary epithelial cells rely on insulin in their culture medium, whilst ΔNp63 levels are increased in MCF-7 ER+ luminal-type breast cancer cells treated with insulin or insulin-like growth factor 1 (IGF-1). Mechanistically, small molecule inhibitors and siRNA gene knockdown demonstrated that induction of ΔNp63 by IGF-1 requires PI3K, ERK1/2, and p38 MAPK activation, and acts through FOXO transcriptional inactivation. We also show that metformin inhibits ΔNp63 induction. These data reveal an IGF-mediated mechanism to control basal-type breast CSCs, with therapeutic implications to modify intratumor breast cancer cell heterogeneity and plasticity.

IFITM protein regulation and functions: Far beyond the fight against viruses.

Interferons (IFNs) are important cytokines that regulate immune responses through the activation of hundreds of genes, including interferon-induced transmembrane proteins (IFITMs). This evolutionarily conserved protein family includes five functionally active homologs in humans. Despite the high sequence homology, IFITMs vary in expression, subcellular localization and function. The initially described adhesive and antiproliferative or pro-oncogenic functions of IFITM proteins were diluted by the discovery of their antiviral properties. The large set of viruses that is inhibited by these proteins is constantly expanding, as are the possible mechanisms of action. In addition to their beneficial antiviral effects, IFITM proteins are often upregulated in a broad spectrum of cancers. IFITM proteins have been linked to most hallmarks of cancer, including tumor cell proliferation, therapeutic resistance, angiogenesis, invasion, and metastasis. Recent studies have described the involvement of IFITM proteins in antitumor immunity. This review summarizes various levels of IFITM protein regulation and the physiological and pathological functions of these proteins, with an emphasis on tumorigenesis and antitumor immunity.

Emergent Role of IFITM1/3 towards Splicing Factor (SRSF1) and Antigen-Presenting Molecule (HLA-B) in Cervical Cancer.

The IFITM restriction factors play a role in cancer cell progression through undefined mechanisms. We investigate new protein-protein interactions for IFITM1/3 in the context of cancer that would shed some light on how IFITM1/3 attenuate the expression of targeted proteins such as HLA-B. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the SRSF1 splicing factor and transporter of mRNA to the ribosome. Using in situ proximity ligation assays, we confirmed a predominant cytosolic protein-protein association for SRSF1 and IFITM1/3. Accordingly, IFITM1/3 interacted with HLA-B mRNA in response to IFNγ stimulation using RNA-protein proximity ligation assays. In addition, RT-qPCR assays in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that HLA-B gene expression at the mRNA level does not account for lowered HLA-B protein synthesis in response to IFNγ. Complementary, shotgun RNA sequencing did not show major transcript differences between IFITM1/IFITM3 null cells and wt-SiHa cells. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to wild type. It was partially reverted by IFITM1/3 complementation. Our data link IFITM1/3 proteins to HLA-B mRNA and SRSF1 and, all together, our results begin to elucidate how IFITM1/3 catalyze the synthesis of target proteins. IFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. Our study has identified new proteins associated with IFITMs which support their role in mediating protein expression; a pivotal function that is highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 affect the expression of targeted proteins; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs in mediating protein abundance is relevant, as it has the potential for regulating the expression of viral and oncogenic proteins.

The Role of IFITM Proteins in Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR-Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. IMPORTANCE TBEV infection may result in encephalitis, chronic illness, or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new centers of endemicity have arisen. Although effective TBEV vaccines have been approved, vaccination coverage is low, and due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies on the role of IFITM proteins in TBEV infection have been published thus far. Understanding antiviral innate immune responses is crucial for the future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both interferon- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

TAp73ß Can Promote Hepatocellular Carcinoma Dedifferentiation.

Hepatocyte dedifferentiation is a major source of hepatocellular carcinoma (HCC), but its mechanisms are unknown. We explored the p73 expression in HCC tumors and studied the effects of transcriptionally active p73ß (TAp73ß) in HCC cells. Expression profiles of p73 and patient clinical data were collected from the Genomic Data Commons (GDC) data portal and the TSVdb database, respectively. Global gene expression profiles were determined by pan-genomic 54K microarrays. The Gene Set Enrichment Analysis method was used to identify TAp73ß-regulated gene sets. The effects of TAp73 isoforms were analyzed in monolayer cell culture, 3D-cell culture and xenograft models in zebrafish using western blot, flow cytometry, fluorescence imaging, real-time polymerase chain reaction (RT-PCR), immunohistochemistry and morphological examination. TAp73 isoforms were significantly upregulated in HCC, and high p73 expression correlated with poor patient survival. The induced expression of TAp73ß caused landscape expression changes in genes involved in growth signaling, cell cycle, stress response, immunity, metabolism and development. Hep3B cells overexpressing TAp73ß had lost hepatocyte lineage biomarkers including ALB, CYP3A4, AFP, HNF4α. In contrast, TAp73ß upregulated genes promoting cholangiocyte lineage such as YAP, JAG1 and ZO-1, accompanied with an increase in metastatic ability. Our findings suggest that TAp73ß may promote malignant dedifferentiation of HCC cells.

Hydrogen deuterium exchange mass spectrometry identifies the dominant paratope in CD20 antigen binding to the NCD1.2 monoclonal antibody.

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.

∆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.

The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.

Interferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFITM1/3 signalling. A pulse SILAC methodology identified IRF1, HLA-B, and ISG15 as the most dominating IFNγ inducible proteins whose synthesis was attenuated in the IFITM1/IFITM3 double-null cells. Conversely, SWATH-IP mass spectrometry of ectopically expressed SBP-tagged IFITM1 identified ISG15 and HLA-B as dominant co-associated proteins. ISG15ylation was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. Proximity ligation assays indicated that HLA-B can interact with IFITM1/3 proteins in parental SiHa cells. Cell surface expression of HLA-B was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. SWATH-MS proteomic screens in cells treated with IFITM1-targeted siRNA cells resulted in the attenuation of an interferon regulated protein subpopulation including MHC Class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1/3 in mediating IFNγ stimulated protein synthesis including ISG15ylation and MHC Class I production in cancer cells. The data together suggest that pro-metastatic growth associated with IFITM1/3 negative cervical cancers relates to attenuated expression of MHC Class I molecules that would support tumor immune escape.

Publisher Correction: A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage.

The original PDF version of this Article listed the authors as "Marcus J.G.W. Ladds," where it should have read "Marcus J. G. W. Ladds, Ingeborg M. M. van Leeuwen, Catherine J. Drummond et al.#".Also in the PDF version, it was incorrectly stated that "Correspondence and requests for materials should be addressed to S. Lín.", instead of the correct "Correspondence and requests for materials should be addressed to S. Laín."This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication.

A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage.

The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.

p63 isoforms in triple-negative breast cancer: ΔNp63 associates with the basal phenotype whereas TAp63 associates with androgen receptor, lack of BRCA mutation, PTEN and improved survival.

The TP63 gene encodes two major protein variants that differ in their N-terminal sequences and have opposing effects. In breast, ΔNp63 is expressed by immature stem/progenitor cells and mature myoepithelial/basal cells and is a characteristic feature of basal-like triple-negative breast cancers (TNBCs). The expression and potential role of TAp63 in the mammary gland and breast cancers is less clear, partly due to the lack of studies that employ p63 isoform-specific antibodies. We used immunohistochemistry with ΔNp63-specific or TAp63-specific monoclonal antibodies to investigate p63 isoforms in 236 TNBCs. TAp63, but not ΔNp63, was seen in tumour-associated lymphocytes and other stromal cells. Tumour cells showed nuclear staining for ΔNp63 in 17% of TNBCs compared to 7.3% that were positive for TAp63. Whilst most TAp63+ tumours also contained ΔNp63+ cells, the levels of the two isoforms were independent of each other. ΔNp63 associated with metaplastic and medullary cancers, and with a basal phenotype, whereas TAp63 associated with androgen receptor, BRCA1/2 wild-type status and PTEN positivity. Despite the proposed effects of p63 on proliferation, Ki67 did not correlate with either p63 isoform, nor did they associate with p53 mutation status. ΔNp63 showed no association with patient outcomes, whereas TAp63+ patients showed fewer recurrences and improved overall survival. These findings indicate that both major p63 protein isoforms are expressed in TNBCs with different tumour characteristics, indicating distinct functional activities of p63 variants in breast cancer. Analysis of individual p63 isoforms provides additional information into TNBC biology, with TAp63 expression indicating improved prognosis.

Importance of Membrane Proteins in the Treatment of Tumor Diseases and the Possibilities of Their Further Study.

BACKGROUND: The proteins of the cellular cytoplasmic membrane represent a heterogeneous group of proteins with different structures, localizations, and functions. They participate in many cellular processes including cellular signaling and communication with the external environment and communication between cells. Mutations and post-translational modifications alter the chemical-physical properties of membrane proteins and thus significantly affect the process of carcinogenesis. Therefore, membrane proteins represent important targets for the diagnosis and treatment of cancer. Nowadays, treatment in the form of monoclonal antibodies or low molecular weight inhibitors targets mainly receptors of growth factors on the surface of tumor cells and various types of molecules including the targets of the so-called checkpoint inhibitors on the surface of the cells of the immune system. In order to better understand the properties and functions of membrane proteins, especially with the perspective of developing new targeted approaches in therapy, mainly proteomic and molecular biological approaches are currently being used. AIM: The aim of this article is to describe the properties and functions of different groups of membrane proteins and to summarize their current relevance and potential for use in oncology. Attention is focused on those groups that regulate the proliferation of tumor cells, affect the immune response, cause drug resistance and metastasis, and are already used or accepted as potential targets of biological therapy. Glycosylation and phosphorylation are described in detail as the most studied post-translational modification of membrane proteins, and mass spectrometry is presented as an effective tool for the identification and quantification of membrane proteins. Key words: membrane proteins - glycosylation - phosphorylation - proteomic analysis - targeted therapy This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 3. 8. 2018.

Distinctive behaviour of live biopsy-derived carcinoma cells unveiled using coherence-controlled holographic microscopy.

Head and neck squamous cell carcinoma is one of the most aggressive tumours and is typically diagnosed too late. Late diagnosis requires an urgent decision on an effective therapy. An individualized test of chemosensitivity should quickly indicate the suitability of chemotherapy and radiotherapy. No ex vivo chemosensitivity assessment developed thus far has become a part of general clinical practice. Therefore, we attempted to explore the new technique of coherence-controlled holographic microscopy to investigate the motility and growth of live cells from a head and neck squamous cell carcinoma biopsy. We expected to reveal behavioural patterns characteristic for malignant cells that can be used to imrove future predictive evaluation of chemotherapy. We managed to cultivate primary SACR2 carcinoma cells from head and neck squamous cell carcinoma biopsy verified through histopathology. The cells grew as a cohesive sheet of suspected carcinoma origin, and western blots showed positivity for the tumour marker p63 confirming cancerous origin. Unlike the roundish colonies of the established FaDu carcinoma cell line, the SACR2 cells formed irregularly shaped colonies, eliciting the impression of the collective invasion of carcinoma cells. Time-lapse recordings of the cohesive sheet activity revealed the rapid migration and high plasticity of these epithelial-like cells. Individual cells frequently abandoned the swiftly migrating crowd by moving aside and crawling faster. The increasing mass of fast migrating epithelial-like cells before and after mitosis confirmed the continuation of the cell cycle. In immunofluorescence, analogously shaped cells expressed the p63 tumour marker, considered proof of their origin from a carcinoma. These behavioural traits indicate the feasible identification of carcinoma cells in culture according to the proposed concept of the carcinoma cell dynamic phenotype. If further developed, this approach could later serve in a new functional online analysis of reactions of carcinoma cells to therapy. Such efforts conform to current trends in precision medicine.

ΔNp63 activates EGFR signaling to induce loss of adhesion in triple-negative basal-like breast cancer cells.

PURPOSE: The basal-A subtype of triple-negative breast cancer is characterized by high levels of ΔNp63. Various functions have been proposed for p63 in breast cancer initiation and growth, and p63 mediates chemotherapeutic response in a subset of triple-negative breast cancers. We investigated the signaling pathways that are controlled by ΔNp63 in basal-A triple-negative breast cancer. METHODS: Human basal-A triple-negative breast cancer cell lines with ΔNp63α induction or inhibition were studied, along with primary human triple-negative breast cancer tissues. Proteomic, phospho-kinase array, mRNA measurements, and immunohistochemistry were employed. RESULTS: Global phosphoproteomics identified increased EGFR phosphorylation in MDA-MB-468 cells expressing ΔNp63α. ΔNp63α expression increased EGFR mRNA, total EGFR protein, and phospho-EGFR(Y1086), whereas silencing endogenous ΔNp63 in HCC1806 cells reduced both total and phospho-EGFR levels and inhibited the ability of EGF to activate EGFR. EGFR pathway gene expression analysis indicated that ΔNp63 alters EGFR-regulated genes involved in cell adhesion, migration, and angiogenesis. Addition of EGF or neutralizing EGFR antibodies demonstrated that EGFR activation is responsible for ΔNp63-mediated loss of cellular adhesion. Finally, immunohistochemical staining showed that p63-positive triple-negative breast cancers were more likely to express high levels of EGFR than p63-negative cancers, corroborated by in silico analysis of gene expression profiling data. CONCLUSIONS: These data identify EGFR as a major target for ΔNp63 regulation that influences cancer cell adhesion in basal-like triple-negative breast cancer.

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 ∆Np63α. 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, ∆Np63α 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 ∆Np63α in primary human breast cancers.

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

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.

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 cross-reactions within the p63/p73 family. Finally, we emphasize the value of recently developed isoform-specific reagents that have clear advantages for the study of p63 and p73 experimentally and clinically.