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.

• Keywords
• CAS9 gene editing, Cervical cancer, IFITM1, Interferon, MHC Class I molecule, SILAC mass spectrometry,
• MeSH
• Cell Line MeSH
• Antigens, Differentiation physiology   MeSH
• Humans MeSH
• Membrane Proteins physiology   MeSH
• Histocompatibility Antigens Class I metabolism   MeSH
• Uterine Cervical Neoplasms metabolism   MeSH
• RNA-Binding Proteins physiology   MeSH
• Protein Biosynthesis physiology   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Differentiation MeSH
• IFITM3 protein, human MeSH Browser
• leu-13 antigen MeSH Browser
• Membrane Proteins MeSH
• Histocompatibility Antigens Class I MeSH
• RNA-Binding Proteins MeSH

The incidence of cutaneous squamous cell carcinoma (cSCC) is rising. Whilst the majority are cured surgically, aggressive metastatic cSCC carry a poor prognosis. Inactivating mutations in transforming growth factor beta (TGF-β) receptors have been identified amongst genetic drivers of sporadic tumours and murine models of cSCC, suggesting a tumour suppressor function for TGF-β in normal skin. However, paradoxically, TGF-β acts as a tumour promoter in some murine model systems. Few studies have analysed the role of TGF-β/activin signalling in human normal skin, hyper-proliferative skin disorders and cSCC. Antibodies recognising phospho-SMAD proteins which are activated during canonical TGF-β/activin signalling were validated for use in immunohistochemistry. A tissue microarray comprising FFPE lesional and perilesional tissue from human primary invasive cSCC (n=238), cSCC in-situ (n=2) and keratocanthoma (n=9) were analysed in comparison with tissues from normal human scalp (n=10). Phosphorylated SMAD2 and SMAD3 were detected in normal interfollicular epidermal keratinocytes and were also highly localised to inner root sheath, matrix cells and Keratin 15 positive cells. Lesional cSCC tissue had significantly reduced activated SMAD2/3 compared to perilesional tissue, consistent with a tumour suppressor role for SMAD2/3 activators in cSCC. Increased cSCC tumour thickness inversely correlated with the presence of phospho-SMADs in tumour tissue suggesting that a reduction in canonical TGF-β/activin signalling may be associated with disease progression.

• Keywords
• FFPE, IHC, SMAD, TGF-β, carcinoma,
• Publication type
• Journal Article MeSH

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.

• Keywords
• Androgen receptor, BRCA1, BRCA2, PTEN, TAp63, Triple-negative breast cancer, ΔNp63,
• MeSH
• Phenotype MeSH
• PTEN Phosphohydrolase genetics   MeSH
• Humans MeSH
• Neoplasm Recurrence, Local genetics   MeSH
• Mutation genetics   MeSH
• Tumor Suppressor Proteins genetics   MeSH
• Protein Isoforms genetics   MeSH
• BRCA1 Protein genetics   MeSH
• BRCA2 Protein genetics   MeSH
• Gene Expression Regulation, Neoplastic genetics   MeSH
• Transcription Factors genetics   MeSH
• Triple Negative Breast Neoplasms genetics   metabolism   mortality   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• BRCA1 protein, human MeSH Browser
• BRCA2 protein, human MeSH Browser
• PTEN Phosphohydrolase MeSH
• Tumor Suppressor Proteins MeSH
• Protein Isoforms MeSH
• BRCA1 Protein MeSH
• BRCA2 Protein MeSH
• PTEN protein, human MeSH Browser
• TP63 protein, human MeSH Browser
• Transcription Factors MeSH

Plasticity of cancer cells, manifested by transitions between epithelial and mesenchymal phenotypes, represents a challenging issue in the treatment of neoplasias. Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are implicated in the processes of metastasis formation and acquisition of stem cell-like properties. Mouse double minute (MDM) 2 and MDMX are important players in cancer progression, as they act as regulators of p53, but their function in EMT and metastasis may be contradictory. Here, we show that the EMT phenotype in multiple cellular models and in clinical prostate and breast cancer samples is associated with a decrease in MDM2 and increase in MDMX expression. Modulation of EMT-accompanying changes in MDM2 expression in benign and transformed prostate epithelial cells influences their migration capacity and sensitivity to docetaxel. Analysis of putative mechanisms of MDM2 expression control demonstrates that in the context of defective p53 function, MDM2 expression is regulated by EMT-inducing transcription factors Slug and Twist. These results provide an alternative context-specific role of MDM2 in EMT, cell migration, metastasis, and therapy resistance.

• Keywords
• MDM2/MDMX, SNAI2/SLUG, TWIST, epithelial-mesenchymal transition, prostate/breast cancer,
• MeSH
• Epithelial-Mesenchymal Transition physiology   MeSH
• Phenotype MeSH
• Heterografts MeSH
• Nuclear Proteins biosynthesis   MeSH
• Humans MeSH
• Mice, Nude MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms genetics   metabolism   pathology   MeSH
• Breast Neoplasms genetics   metabolism   pathology   MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 biosynthesis   MeSH
• Proto-Oncogene Proteins biosynthesis   MeSH
• Transfection MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nuclear Proteins MeSH
• MDM2 protein, human MeSH Browser
• MDM4 protein, human MeSH Browser
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• Proto-Oncogene Proteins MeSH

Glioblastoma is the most common and the most aggressive type of brain cancer. Aberrations of the RTK/RAS/PI3K-, p53-, and RB cell signaling pathways were recognized as a core requirement for pathogenesis of glioblastoma. The p53 tumor suppressor functions as a transcription factor transactivating expression of its target genes in response to various stress stimuli. We determined the p53 status in 36 samples of glioblastoma by functional analyses FASAY and split assay. Seventeen p53 mutations were detected and further analyzed by cDNA and gDNA sequencing in 17 patients (47.2 %). Fifteen (88.2 %) of the mutations were missense mutations causing amino acid substitutions, seven of them exhibited temperature-sensitivity. Two mutations were determined as short deletions, one of them causing formation of premature termination codon in position 247. Fluorescent in situ hybridization revealed the loss of the p53-specific 17p13.3 locus in four of 33 analyzed samples (12 %). In 12 out of 30 samples (40 %), the p53 protein accumulation was shown by immunoblotting. There was high (80 %) concordance between the presence of the clonal p53 mutation and the p53 protein accumulation.

• MeSH
• Gene Deletion MeSH
• Genes, p53 * MeSH
• Glioblastoma genetics   metabolism   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Yeasts MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation * MeSH
• DNA Mutational Analysis MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Brain Neoplasms genetics   metabolism   MeSH
• Organ Specificity MeSH
• Sequence Analysis, DNA MeSH
• Aged MeSH
• Temperature MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH
• TP53 protein, human MeSH Browser