BACKGROUND: Cancer stem-like cells (CSCs) represent a subset of tumor cells that have the ability to self-renew, a long lifespan and a relatively quiescent phenotype, and show resistance to conventional therapies. Various markers are used to identify CSCs, and have shown that different CSC subtypes may be present within a tumor. One functional property of CSCs is their relative lack of proteasomal activity compared to the tumor bulk. METHODS: We introduced an unstable fluorescent molecule into FaDu oropharyngeal squamous cell carcinoma cells and analyzed the association of proteasome activity with aldehydehyde dehydrogenase (ALDH) activity as another common CSC marker, and with other stem-cell related properties of glucose metabolism. We also analyzed publicly available gene expression profiling data of ALDH+ CSCs for alterations in mRNAs associated with proteostasis. RESULTS: We show that FaDu CSCs identified by low proteasome activity are associated with the population identified by high ALDH activity. Futher characterization shows that these CSCs have a relatively high mitochondrial membrane potential and low levels of glucose transporter, indicating a non-Warburg metabolic phenotype. We also show that proteasome-low FaDu CSCs exhibit decreased rates of protein synthesis. Gene expression profiling of other cancer cell lines reveal common statistically significant differences in proteostasis in ALDH+ CSCs compared to the bulk of the tumor cells, including reduced levels of Hsp70 and/or Hsp90 in CSCs defined by ALDH, together with reduced levels of UCHL5 mRNA. CONCLUSIONS: These data provide additional insights into the functional characteristics of proteasome-low/ALDH-high CSCs, indicating a metabolic phenotype of reduced reliance on aerobic glycolysis and a decreased protein synthesis rate. We also identify specific chaperone and ubiquitin ligase activities that can be used to identify CSCs, with corresponding implications for therapeutic strategies that target CSCs through their altered metabolic properties.

• Klíčová slova
• Cancer stem cells, Glucose transporter, Mitochondrial membrane potential, Protein degradation, Proteosynthesis, Squamous cell carcinoma,
• MeSH
• aldehyddehydrogenasa * metabolismus   genetika   MeSH
• glukosa metabolismus   MeSH
• lidé MeSH
• membránový potenciál mitochondrií MeSH
• mitochondrie * metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky * metabolismus   patologie   MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• proteiny usnadňující transport glukosy * metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• spinocelulární karcinom * metabolismus   patologie   genetika   MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aldehyddehydrogenasa * MeSH
• glukosa MeSH
• proteasomový endopeptidasový komplex MeSH
• proteiny usnadňující transport glukosy * MeSH

Signal transducer and activator of transcription 3 (Stat3) is responsible for many aspects of normal development and contributes to the development and progression of cancer through regulating epithelial cell identity and cancer stem cells. In breast cancer, Stat3 is associated with triple-negative breast cancers (TNBC) and its function has been related to the activation of p63, itself a marker of basal-like TNBC and a master regulator of stem cell activities. Stat3 activation is controlled by dual phosphorylation at tyrosine 705 (pTyr705) and serine 727 (pSer727), although it is unclear whether these have equivalent effects, and whether they are related or independent events. To address these issues, we investigated Stat3 phosphorylation at the two sites by immunohistochemistry in 173 patients with TNBC. Stat3 phosphorylation was assessed by automated quantitative measurements of digitized scanned images and classified into four categories based on histoscore. The results were analyzed for associations with multiple markers of tumor phenotype, proliferation, BRCA status, and clinicopathological characteristics. We show that the levels of pTyr705- and pSer727-Stat3 were independent in 34% of tumors. High pTyr705-Stat3 levels were associated with the luminal differentiation markers ERβ/AR and MUC1, whereas tumors with high levels of pSer727-Stat3 were more likely to be positive for the basal marker CK5/6, but were independent of p63 and were EGFR negative. Combined high pSer727- and low Tyr705-Stat3 phosphorylation associated with basal-like cancer. Although high Stat3 phosphorylation levels were associated with less aggressive tumor characteristics, they did not associate with improved survival, indicating that Stat3 phosphorylation is an unfavorable indicator for tumors with an otherwise good prognosis according to clinicopathological characteristics. These findings also show that pTyr705-Stat3 and pSer727-Stat3 associate with specific breast tumor phenotypes, implying that they exert distinct functional activities in breast cancer.

• Klíčová slova
• Stat3 serine phosphorylation, Stat3 tyrosine phosphorylation, clinicopathological characteristics, triple-negative breast cancer, tumor cell phenotypes,
• MeSH
• fenotyp MeSH
• fosforylace MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• serin genetika   MeSH
• transkripční faktor STAT3 metabolismus   MeSH
• triple-negativní karcinom prsu * patologie   MeSH
• tyrosin genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• serin MeSH
• STAT3 protein, human MeSH Prohlížeč
• transkripční faktor STAT3 MeSH
• tyrosin MeSH

The TP63 gene encodes two major protein variants; TAp63 contains a p53-like transcription domain and consequently has tumor suppressor activities whereas ΔNp63 lacks this domain and acts as an oncogene. The two variants show distinct expression patterns in normal tissues and tumors, with lymphocytes and lymphomas/leukemias expressing TAp63, and basal epithelial cells and some carcinomas expressing high levels of ΔNp63, most notably squamous cell carcinomas (SCC). Whilst the transcriptional functions of TAp63 and ΔNp63 isoforms are known, the mechanisms involved in their regulation are poorly understood. Using squamous epithelial cells that contain high levels of ΔNp63 and low/undetectable TAp63, the DNA demethylating agent decitabine (5-aza-2'-deoxycytidine, 5-dAza) caused a dose-dependent increase in TAp63, with a simultaneous reduction in ΔNp63, indicating DNA methylation-dependent regulation at the isoform-specific promoters. The basal cytokeratin KRT5, a direct ΔNp63 transcriptional target, was also reduced, confirming functional alteration of p63 activity after DNA demethylation. We also showed high level methylation of three CpG sites in the TAP63 promoter in these cells, which was reduced by decitabine. DNMT1 depletion using inducible shRNAs partially replicated these effects, including an increase in the ratio of TAP63:ΔNP63 mRNAs, a reduction in ΔNp63 protein and reduced KRT5 mRNA levels. Finally, high DNA methylation levels were found at the TAP63 promoter in clinical SCC samples and matched normal tissues. We conclude that DNA methylation at the TAP63 promoter normally silences transcription in squamous epithelial cells, indicating DNA methylation as a therapeutic approach to induce this tumor suppressor in cancer. That decitabine simultaneously reduced the oncogenic activity of ΔNp63 provides a "double whammy" for SCC and other p63-positive carcinomas. Whilst a variety of mechanisms may be involved in producing the opposite effects of DNA demethylation on TAp63 and ΔNp63, we propose an "either or" mechanism in which TAP63 transcription physically interferes with the ability to initiate transcription from the downstream ΔNP63 promoter on the same DNA strand. This mechanism can explain the observed inverse expression of p63 isoforms in normal cells and cancer.

• Klíčová slova
• DNA methylation, TAp63, alternative promoter usage, decitabine, keratinocytes, squamous cell carcinoma, ΔNp63,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: ΔNp63 overexpression is a common event in squamous cell carcinoma (SCC) that contributes to tumorigenesis, making ΔNp63 a potential target for therapy. METHODS: We created inducible TP63-shRNA cells to study the effects of p63-depletion in SCC cell lines and non-malignant HaCaT keratinocytes. DNA damaging agents, growth factors, signaling pathway inhibitors, histone deacetylase inhibitors, and metabolism-modifying drugs were also investigated for their ability to influence ΔNp63 protein and mRNA levels. RESULTS: HaCaT keratinocytes, FaDu and SCC-25 cells express high levels of ΔNp63. HaCaT and FaDu inducible TP63-shRNA cells showed reduced proliferation after p63 depletion, with greater effects on FaDu than HaCaT cells, compatible with oncogene addiction in SCC. Genotoxic insults and histone deacetylase inhibitors variably reduced ΔNp63 levels in keratinocytes and SCC cells. Growth factors that regulate proliferation/survival of squamous cells (IGF-1, EGF, amphiregulin, KGF, and HGF) and PI3K, mTOR, MAPK/ERK or EGFR inhibitors showed lesser and inconsistent effects, with dual inhibition of PI3K and mTOR or EGFR inhibition selectively reducing ΔNp63 levels in HaCaT cells. In contrast, the antihyperlipidemic drug lovastatin selectively increased ΔNp63 in HaCaT cells. CONCLUSIONS: These data confirm that ΔNp63-positive SCC cells require p63 for continued growth and provide proof of concept that p63 reduction is a therapeutic option for these tumors. Investigations of ΔNp63 regulation identified agent-specific and cell-specific pathways. In particular, dual inhibition of the PI3K and mTOR pathways reduced ΔNp63 more effectively than single pathway inhibition, and broad-spectrum histone deacetylase inhibitors showed a time-dependent biphasic response, with high level downregulation at the transcriptional level within 24 h. In addition to furthering our understanding of ΔNp63 regulation in squamous cells, these data identify novel drug combinations that may be useful for p63-based therapy of SCC.

• Klíčová slova
• DNA damage, Growth factor signaling, Histone deacetylase inhibitors, Oncogene addiction, Squamous cell carcinoma, ΔNp63,
• MeSH
• inhibitory histondeacetylas MeSH
• karcinogeneze MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• nádorový supresorový protein p53 * genetika   MeSH
• rodina MeSH
• spinocelulární karcinom * farmakoterapie   genetika   metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inhibitory histondeacetylas MeSH
• nádorové supresorové proteiny MeSH
• nádorový supresorový protein p53 * MeSH
• TP63 protein, human MeSH Prohlížeč
• transkripční faktory MeSH

Like other malignancies, prostate tumors are thought to contain cancer stem-like cells (CSCs) that are responsible for growth, metastasis, and therapy resistance. ΔNp63 (also called p40) is a regulator of normal prostate stem/progenitor cell activities and a marker of normal basal epithelial cells. The levels of ΔNp63 are reduced in prostate adenocarcinomas, although there is also evidence that ΔNp63 is involved in CSC regulation and drives metastasis to the bone. We studied metastatic deposits of prostate cancers with isoform-specific ΔNp63 and TAp63 antibodies. We identified p63-positive cells in only 3 of 42 metastatic prostate tumors (7%), including 2/38 (5.3%) "usual-type" adenocarcinomas. ΔNp63 and TAp63 isoforms were present in the nuclei of a small subpopulation (< 1%) of tumor cells in these metastases. ΔNp63-positive cells showed a basal-like cell phenotype (cytokeratin 8- and androgen receptor-negative, high molecular weight cytokeratin- and cytokeratin 19-positive), distinct from the tumor bulk. TAp63-positive cells were similar but were sometimes cytokeratin 8-positive. A subset of ΔNp63-positive tumor cells were CD44-positive, a marker of "basal" CSCs but were not positive for the "epithelial" CSC marker ALDH1. TAp63 was not associated with either of these CSC markers. None of the tumors containing p63-positive cells showed evidence of bone metastasis, compared with 28% of the p63-negative tumors. These data show that both ΔNp63 and TAp63 are present in only a small proportion of prostate adenocarcinomas and do not associate with metastasis. The data suggest heterogeneity of CSCs in prostate cancer, similar to other cancer types.

• Klíčová slova
• CD44, Cancer stem cells, Metastasis, Prostate cancer, p40, p63,
• MeSH
• adenokarcinom metabolismus   sekundární   MeSH
• dospělí MeSH
• fenotyp MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové kmenové buňky metabolismus   patologie   MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• nádory kostí metabolismus   sekundární   MeSH
• nádory prostaty metabolismus   patologie   MeSH
• retrospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• transkripční faktory metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nádorové biomarkery MeSH
• nádorové supresorové proteiny MeSH
• TP63 protein, human MeSH Prohlížeč
• transkripční faktory MeSH