DeltaNp63 isoforms regulate CD44 and keratins 4, 6, 14 and 19 in squamous cell carcinoma of head and neck.

DeltaNp63 isoforms regulate CD44 and keratins 4, 6, 14 and 19 in squamous cell carcinoma of head and neck

Heterozygous mutations of p63, a key transcription factor in epithelial development, are causative in a variety of human ectodermal dysplasia disorders. Although the mutation spectrum of these disorders displays a striking genotype-phenotype association, the molecular basis for this association is only superficially known. Here, we characterize the transcriptional activity and protein stability of ΔNp63 mutants (that is, mutants of a p63 isoform that lacks the N-terminal transactivation domain) that are found in ectrodactyly-ectodermal dysplasia-cleft syndrome (EEC), ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC) and nonsyndromic split-hand/split-foot malformation (SHFM). DNA-binding and sterile alpha motif (SAM) domain mutants accumulate in the skin of EEC and AEC syndrome patients, respectively, and show extended half lives in vitro. By contrast, C-terminal mutations found in SHFM patients have half-lives similar to that of the wild-type protein. The increased half-life of EEC and AEC mutant proteins was reverted by overexpression of wild-type ΔNp63. Interestingly, the mutant proteins exhibit normal binding to and degradation by the E3 ubiquitin ligase Itch. Finally, EEC and AEC mutant proteins have reduced transcriptional activity on several skin-specific gene promoters, whereas SHFM mutant proteins are transcriptionally active. Our results, therefore, provide evidence for a regulatory feedback mechanism for p63 that links transcriptional activity to regulation of protein homeostasis by an unknown mechanism. Disruption of this regulatory mechanism might contribute to the pathology of p63-related developmental disorders.

Differential altered stability and transcriptional activity of ΔNp63 mutants in distinct ectodermal dysplasias

Missense mutations in the 3' end of the p63 gene are associated with either RHS (Rapp-Hodgkin syndrome) or AEC (Ankyloblepharon Ectodermal defects Cleft lip/palate) syndrome. These mutations give rise to mutant p63alpha protein isoforms with dominant effects towards their wild-type counterparts. Here we report four RHS/AEC-like patients with mutations (p.Gln9fsX23, p.Gln11X, p.Gln16X), that introduce premature termination codons in the N-terminal part of the p63 protein. These mutations appear to be incompatible with the current paradigms of dominant-negative/gain-of-function outcomes for other p63 mutations. Moreover it is difficult to envisage how the remaining small N-terminal polypeptide contributes to a dominant disease mechanism. Primary keratinocytes from a patient containing the p.Gln11X mutation revealed a normal and aberrant p63-related protein that was just slightly smaller than the wild-type p63. We show that the smaller p63 protein is produced by translation re-initiation at the next downstream methionine, causing truncation of a non-canonical transactivation domain in the DeltaN-specific isoforms. Interestingly, this new DeltaDeltaNp63 isoform is also present in the wild-type keratinocytes albeit in small amounts compared with the p.Gln11X patient. These data establish that the p.Gln11X-mutation does not represent a null-allele leading to haploinsufficiency, but instead gives rise to a truncated DeltaNp63 protein with dominant effects. Given the nature of other RHS/AEC-like syndrome mutations, we conclude that these mutations affect only the DeltaNp63alpha isoform and that this disruption is fundamental to explaining the clinical characteristics of these particular ectodermal dysplasia syndromes.

/pdf/a-novel-translation-re-initiation-mechanism-for-the-p63-gene-4k3oyu8g4y.pdf

A novel translation re-initiation mechanism for the p63 gene revealed by amino-terminal truncating mutations in Rapp-Hodgkin/Hay-Wells-like syndromes

Strategies to address resistance to platin drugs are greatly needed in human epithelial cancers (e.g., ovarian, head/neck, and lung) where platins are used widely and resistance occurs commonly. We found that upon ΔNp63α overexpression, AKT1 and phospho-AKT1 levels are upregulated in cancer cells. Investigations using gel-shift, chromatin immunoprecipitation and functional reporter assays implicated ΔNp63α in positive regulation of AKT1 transcription. Importantly, we found that ΔNp63α, AKT1, and phospho-AKT levels are greater in 2008CI3 CDDP-resistant ovarian cancer cells than in 2008 CDDP-sensitive cells. siRNA-mediated knockdown of ΔNp63α expression dramatically decreased AKT1 expression, whereas knockdown of either ΔNp63α or AKT1 decreased cell proliferation and increased death of ovarian and head/neck cancer cells. Conversely, enforced expression of ΔNp63α increased cancer cell proliferation and reduced apoptosis. Together, our findings define a novel ΔNp63α-dependent regulatory mechanism for AKT1 expression and its role in chemotherapeutic resistance of ovarian and head/neck cancer cells.

https://cancerres.aacrjournals.org/content/canres/71/3/1167.full.pdf

ΔNp63α Confers Tumor Cell Resistance to Cisplatin through the AKT1 Transcriptional Regulation

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of Delta Np63 alpha associated with its increased phosphorylated status. Since Delta Np63 alpha usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of Delta Np63 alpha in HNSCC cells upon cisplatin exposure. We found that Delta Np63 alpha is phosphorylated in the time-dependent fashion at the following positions: S385, T397 and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of Delta Np63 alpha in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of Delta Np63 alpha residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of Delta Np63 alpha degradation. Finally, we demonstrated that the Delta Np63 alpha protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of Delta Np63 alpha following DNA damage.

ATM kinase is a master switch for the ΔNp63α phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage

P63, the major regulator of epithelial development/differentiation, is mutated in humanectodermal dysplasias, such as ankyloblepharon, ectodermal dysplasia and clefting (AEC). Werecently identified that p63? physically associated with mRNA processing/splicing proteins. Wepreviously showed that p63 mutations mapped to the sterile ?-motif led to disruption of theseinteractions and modulated an aberrant splicing of keratinocyte growth factor receptorcontributing into molecular mechanism underlying AEC phenotype.To further investigate the molecular mechanisms associated with AEC syndrome weestablished the cellular model for this disorder by stable introduction of mutated allele [L514F]of p63? into immortalized keratinocyte cells. We showed that mutated ?Np63? mediated anaberrant splicing of its own p63 mRNA transcript, which in turn led to accumulation ofproteasome-resistant C- terminal truncated p63. The truncated p63 failed to associate with theC-terminal domain of RNA polymerase II through SRA4 protein and...

AEC-associated p63 mutations lead to alternative splicing/protein stabilization of p63 and modulation of Notch signaling.

Of the 37.9 million individuals infected with human immunodeficiency virus type 1 (HIV-1), approximately 50% exhibit HIV-associated neurocognitive disorders (HAND). We and others previously showed that HIV-1 viral RNAs, such as trans-activating response (TAR) RNA, are incorporated into extracellular vesicles (EVs) and elicit an inflammatory response in recipient naïve cells. Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the primary cannabinoids present in cannabis, are effective in reducing inflammation. Studies show that cannabis use in people living with HIV-1 is associated with lower viral load, lower circulating CD16+ monocytes and high CD4+ T-cell counts, suggesting a potentially therapeutic application. Here, HIV-1 infected U1 monocytes and primary macrophages were used to assess the effects of CBD. Post-CBD treatment, EV concentrations were analyzed using nanoparticle tracking analysis. Changes in intracellular and EV-associated viral RNA were quantified using RT-qPCR, and changes in viral proteins, EV markers, and autophagy proteins were assessed by Western blot. Our data suggest that CBD significantly reduces the number of EVs released from infected cells and that this may be mediated by reducing viral transcription and autophagy activation. Therefore, CBD may exert a protective effect by alleviating the pathogenic effects of EVs in HIV-1 and CNS-related infections.

/pdf/cannabinoids-reduce-extracellular-vesicle-release-from-hiv-1-habn81q0.pdf

Cannabinoids Reduce Extracellular Vesicle Release from HIV-1 Infected Myeloid Cells and Inhibit Viral Transcription

HIV-1 remains an incurable infection that is associated with substantial economic and epidemiologic impacts. HIV-associated neurocognitive disorders (HAND) are commonly linked with HIV-1 infection; despite the development of combination antiretroviral therapy (cART), HAND is still reported to affect at least 50% of HIV-1 infected individuals. It is believed that the over-amplification of inflammatory pathways, along with release of toxic viral proteins from infected cells, are primarily responsible for the neurological damage that is observed in HAND; however, the underlying mechanisms are not well-defined. Therefore, there is an unmet need to develop more physiologically relevant and reliable platforms for studying these pathologies. In recent years, neurospheres derived from induced pluripotent stem cells (iPSCs) have been utilized to model the effects of different neurotropic viruses. Here, we report the generation of neurospheres from iPSC-derived neural progenitor cells (NPCs) and we show that these cultures are permissive to retroviral (e.g. HIV-1, HTLV-1) replication. In addition, we also examine the potential effects of stem cell derived extracellular vesicles (EVs) on HIV-1 damaged cells as there is abundant literature supporting the reparative and regenerative properties of stem cell EVs in the context of various CNS pathologies. Consistent with the literature, our data suggests that stem cell EVs may modulate neuroprotective and anti-inflammatory properties in damaged cells. Collectively, this study demonstrates the feasibility of NPC-derived neurospheres for modeling HIV-1 infection and, subsequently, highlights the potential of stem cell EVs for rescuing cellular damage induced by HIV-1 infection. 

/pdf/retroviral-infection-of-human-neurospheres-and-use-of-stem-3q8c4nfn.pdf

Retroviral infection of human neurospheres and use of stem Cell EVs to repair cellular damage

Abstract We characterized the in vivo interstitial fluid (IF) content of extracellular vesicles (EVs) using the GFP‐4T1 syngeneic murine cancer model to study EVs in‐transit to the draining lymph node. GFP labelling confirmed the IF EV tumour cell origin. Molecular analysis revealed an abundance of IF EV‐associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission‐induced mitophagy preferentially populated the CD81+/PD‐L1+ IF EVs; PINK1, TOM20, and ARIH1 E3 ubiquitin ligase (required for Parkin‐independent mitophagy), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC‐1 (ubiquitination state triggers mitophagy away from apoptosis), VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti‐CD81 immunoprecipitation, molecular sieve chromatography, and MitoTracker export into CD81+ EVs. We propose the 4T1 in vivo model as a versatile tool to functionally characterize IF EVs. IF EV export of fission mitophagy proteins has broad implications for mitochondrial function and cellular immunology.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/jev2.12244

A secretory form of Parkin‐independent mitophagy contributes to the repertoire of extracellular vesicles released into the tumour interstitial fluid in vivo

Yuriy Kim

Papers

AEC-associated p63 mutations lead to alternative splicing/protein stabilization of p63 and modulation of Notch signaling.

Cannabinoids Reduce Extracellular Vesicle Release from HIV-1 Infected Myeloid Cells and Inhibit Viral Transcription

Retroviral infection of human neurospheres and use of stem Cell EVs to repair cellular damage

A secretory form of Parkin‐independent mitophagy contributes to the repertoire of extracellular vesicles released into the tumour interstitial fluid in vivo