A novel DLX3–PKC integrated signaling network drives keratinocyte differentiation

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3- dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3–PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis

Inflammasome Sensor Nlrp1b-Dependent Resistance to Anthrax Is Mediated by Caspase-1, IL-1 Signaling and Neutrophil Recruitment

Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1bS/S or Nlrp1bR/R, respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1bS/S alleles (which allow activation of caspase-1 and IL-1β release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1bR/R alleles (which cannot activate caspase-1 in response to toxin). Nlrp1bS-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1bS/S mice. Resistance to infection required the actions of both caspase-1 and IL-1β as Nlrp1bS/S mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1β responses in Nlrp1bS/S,Nlrp1bR/R and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1bS, caspase-1, and IL-1β in countering anthrax infection

MECANISMES ET ROLES DE L'HYPEREXPRESSION DE LA PROTEINE APPARENTEE A L'HORMONE PARATHYROIDIENNE (PTHRP) AU COURS DE LA TUMORIGENESE MAMMAIRE

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Abstract 5165: ADAM17: a common effector for RAS and MET-driven transformation of primary keratinocytes

Abstract The purpose of the current study is to determine the contribution of the metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) to the transformation driven by activated MET or oncogenic RAS in primary keratinocytes. Transgenic keratinocytes overexpressing HGF (MT-HGF) to activate MET and keratinocytes transduced with an oncogenic RAS share identical phenotypic and biochemical features of transformation and produce squamous tumors in vivo. In both keratinocyte populations, these common features arise from autocrine activation of EGFR through elevated expression and release of EGFR ligands. Amphiregulin (AREG) blockade in MT-HGF keratinocytes decreases MET-mediated EGFR transactivation. Inhibition of EGFR ablates the initiated signature of MT-HGF keratinocytes in vitro and causes regression of tumors from MT-HGF keratinocytes tranplanted in vivo. Deletion or knock-down of ADAM17 decreases EGFR activation in both RAS and MT-HGF keratinocytes and reverses the transformation associated gene signature in RAS keratinocytes. Using AREG release as an indicator of ADAM17 activity, we determined that knock-down of either the SRC kinase or inactive Rhomboid 2 (iRhom2) reduces the ability of keratinocytes to release AREG upon HGF stimulation. Collectively our data suggest that MET-mediated transformation of keratinocytes occurs through EGFR transactivation and that ADAM17 is a necessary effector for RAS and MET-driven transformation. Citation Format: Christophe Cataisson, Mary Klosterman, Kelly Shibuya, Glenn Merlino, Stuart H. Yuspa. ADAM17: a common effector for RAS and MET-driven transformation of primary keratinocytes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5165.</jats:p

Abstract 1064: The combined activation of MET signaling and PKCα is sufficient to induce tumors in mouse skin.

Abstract Aberrant activation of MET signaling has been identified in multiple cancers but its relation to cancer-associated inflammation has not been explored. To address this issue in the context of skin carcinogenesis, a double transgenic mouse model (MT1-HGF/ K5-PKCα or DT) was generated. K5-PKCα mice that overexpress PKCα in basal keratinocytes develop a strong neutrophilic cutaneous inflammatory response upon topical TPA (12-tetradecanoylphorbol-13-acetate) application. These mice were crossed with MT1-HGF mice that overexpress HGF under a metallothionein promoter to create MT1-HGF/ K5-PKCα double transgenic (DT) mice and their respective controls. We observed that DT animals were very sensitive to squamous carcinogenesis. Using a low promoting dose of TPA that primarily activates PKCα in K5-PKCα or DT mice yielded an average of 6 tumors per mouse in the DT group; single transgenic K5-PKCα or MT-HGF mice developed an average of 1 tumor while WT developed none at this TPA dose. We hypothesize that in DT mice, keratinocyte-derived HGF synergizes with PKCα to drive tumor promotion and increase tumor growth. Primary keratinocytes derived from MT1-HGF or DT mice display a phenotype reminiscent of EGFR activated cells: increased expression of pro-inflammatory factors, upregulation of Keratin 8 (K8) and downregulation of K1 and K10 mRNAs. Indeed western blot analysis shows that EGFR is transactivated in MT1-HGF and DT keratinocytes and cell proliferation is elevated as well but not in K5-PKCα or WT keratinocytes. The release of CXCL1,a hallmark of RAS transformation, is augmented in RAS-keratinocyte cell culture supernatants with MT1-HGF and DT keratinocytes producing the most in the absence of RAS-transduction. Blockade of EGFR or IL-1 activity can mitigate the HGF-induced RAS-phenotype. K5-PKCα and DT mice exhibit the same acute inflammatory response and regenerative epidermal hyperplasia following low dose TPA treatment. When promoted for 10 weeks with TPA, 100% of DT mice but none of the single transgenic controls developed squamous papillomas in the absence of DMBA-mediated initiation. Our data suggest that the synergistic activity of Met and PKCα can substitute for the lack of RAS mutation during skin carcinogenesis. It remains to be determined if that mechanism could provide an alternate route to transformation in human skin tumors lacking RAS mutations. Citation Format: Christophe Cataisson, Fan Liu, Lisa N. Wright, Anne Zhuang, Glenn Merlino, Stuart H. Yuspa. The combined activation of MET signaling and PKCα is sufficient to induce tumors in mouse skin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1064. doi:10.1158/1538-7445.AM2013-1064</jats:p

Abstract 1482: Autocrine IL-1α mediates NF-κB activation by oncogenic ras in keratinocytes

Abstract Inflammation is a complex process evolved to protect us against pathogens. There is increasing evidence that chronic inflammation can be a major cancer risk. Clinical and epidemiological evidence link inflammation with epithelial skin cancer. One of the best established models linking inflammation with the development of skin cancer is the classical two stage skin carcinogenesis in mice. MyD88 is a crucial intermediate in the signaling from pro-inflammatory receptors. Mice ablated for MyD88 or the IL-1R are resistant to topical skin carcinogenesis and MyD88 deficient ras -transduced keratinocytes form only small tumors in orthotopic grafts. In the skin model the clonal benign tumor defines the phenotype of the initiated cell. High proliferation, resistance to terminal differentiation and altered gene expression are hallmarks of that phenotype. Our previous studies have revealed that Ras activation is sufficient to produce this complete phenotype and fully initiate tumor formation. We recently reported that Ras induction of CXCR2 ligands was mediated by autocrine activation of EGFR and NF-κB and that activation of CXCR2 on ras transformed keratinocytes has both pro-migratory and pro-tumorigenic functions. In MyD88 deficient cells, transduction of ras activates the EGFR loop, but these keratinocytes do not upregulate CXCR2 ligands. Using both genetic and pharmacological approaches we have demonstrated that IL-1α autocrine signaling is activated downstream of EGFR in ras transformed keratinocytes. We further demonstrate that IL-1α is responsible for NF-κB mediated activation of CXCR2 ligands in those keratinocytes. Collectively, those results demonstrate that ras activation converts normal keratinocytes to a tumor phenotype through a series of feedback signals that provide therapeutic opportunities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1482.</jats:p

Crosstalk between tumor and stroma modifies CLIC4 cargo in extracellular vesicles.

Mouse models of breast cancer have revealed that tumor-bearing hosts must express the oxidoreductase CLIC4 to develop lung metastases. In the absence of host CLIC4, primary tumors grow but the lung premetastatic niche is defective for metastatic seeding. Primary breast cancer cells release EVs that incorporate CLIC4 as cargo and circulate in plasma of wildtype tumor-bearing hosts. CLIC4-deficient breast cancer cells also form tumors in wildtype hosts and release EVs in plasma, but these EVs lack CLIC4, suggesting that the tumor is the source of the plasma-derived EVs that carry CLIC4 as cargo. Paradoxically, circulating EVs are also devoid of CLIC4 when CLIC4-expressing primary tumors are grown in CLIC4 knockout hosts. Thus, the incorporation of CLIC4 (and perhaps other factors) as EV cargo released from tumors involves specific signals from the surrounding stroma determined by its genetic composition. Since CLIC4 is also detected in circulating EVs from human breast cancer patients, future studies will address its association with disease

Abstract 1642: Bryostatin 1 and the simplified analog Merle 23 have similar and opposing properties on mouse epidermal cells and mouse skin

Abstract Protein kinase C (PKC) is differentially regulated in a range of cancers and has become an attractive therapeutic target. A number of natural compounds exist that have been found to regulate PKCs, and the biological responses of each compound can vary. The PKC activator PMA is an established tumor promoter, while others, such as the bryostatins, are non-tumor promoting and have inhibitory effects on the PKC pathway. Because of its effects on the PKC pathway, bryostatin 1 is currently in clinical trials as an anti-cancer agent. Limited availability and the difficulty involved in synthesizing bryostatin 1 make its use as an anti-cancer agent less attractive. Merle 23, a simplified synthetic analog of bryostatin 1, could be an alternative to bryostatin 1 if it acted similarly. Initial investigations of Merle 23 have revealed biological responses both similar to bryostatin 1 and similar to PMA, depending on the system. In U937 leukemia cells Merle 23 acts similar to PMA, and in the LNCaP prostate cancer cell line it behaves more like bryostatin 1. Its behavior in mouse skin is of particular interest, since this is the system in which the tumor promoting activity of PMA and the anti-tumor promoting activity of bryostatin 1 have been characterized. The activity of Merle 23 was compared to PMA and bryostatin 1 in mouse epidermal cells and mouse skin. In mouse primary keratinocytes PMA produces a prolonged morphological response, while the morphological response to bryostatin 1 is transient. The response to Merle 23 is intermediate, but more similar to bryostatin 1. When applied simultaneously, Merle 23, like bryostatin 1, is able to protect the cells from the morphological response induced by PMA. In the PKC pathway, all three compounds promote similar levels of initial PKCδ activation and ERK1/2 phosphorylation. By 24 hrs, however, the responses differ. At high concentrations, bryostatin 1 protects PKCδ from down regulation whereas Merle 23 does not. Bryostatin 1 is also much more potent for down regulation of PKCα, whereas Merle 23 and PMA are similar. The inflammatory response was measured by monitoring the transcript levels of various genes involved in inflammation. PMA induced the largest increase in transcript levels, while Merle 23 was more similar to bryostatin 1. Short-term in vivo analysis reveals no epidermal thickening in response to low doses of Merle 23 whereas the corresponding dose of PMA induced significant hyperplasia. The in vivo analysis of Merle 23 is ongoing. The results presented suggest that Merle 23 behaves more similarly to bryostatin 1 in the mouse epidermal cells at the level of biological response, raising the possibility that it may likewise be non-promoting. The results emphasize that the structure activity relations responsible for the distinct behavior between PMA and bryostatin 1 are distinct for different cellular systems and different biological endpoints. Citation Format: Jessica Kelsey, Noemi Kedei, Christophe Cataisson, Mark Petersen, Stuart Yuspa, Gary Keck, Peter Blumberg. Bryostatin 1 and the simplified analog Merle 23 have similar and opposing properties on mouse epidermal cells and mouse skin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1642. doi:10.1158/1538-7445.AM2014-1642</jats:p

Abstract 4923: MyD88 and IL-17 have essential local and systemic functions in promoting tumors on mouse skin

Abstract NF-kB has been causally implicated with cancer-associated inflammation. In the skin, ligands of the Toll-like receptor/IL-1 receptor superfamily induce the recruitment of MyD88 (Myeloid differentiation primary response gene 88), ultimately leading to NF-kB activation. We previously reported that MyD88 is required for two stage chemical carcinogenesis on mouse skin, and that MyD88 exerts a cell-intrinsic function in RAS-mediated transformation of keratinocytes (Ras-keratinocytes). Now, we explore the role of MyD88 during tumor promotion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Epidermal hyperplasia is reduced in MyD88 deficient mice relative to controls after multiple applications of TPA. Furthermore, the upregulation of the chemokines Cxcl1 and S100a8 in response to TPA was deficient in MyD88 null cultured keratinocytes revealing a necessary role for MyD88 in the intrinsic response of keratinocytes to phorbol ester. We also uncovered a systemic requirement for MyD88 in carcinogenesis since lineage specific ablation of MyD88 in T cells conferred significant resistance to skin tumorigenesis and reduced infiltration of IL-17 positive T cells in TPA treated MyD88 null mouse skin. IL-17 treatment of normal primary keratinocytes increased their proliferation while reducing their response to calcium-induced differentiation but had no effect on the proliferation of RAS-keratinocytes. The atypical nuclear IkBzeta protein can act as an activator of so-called secondary NF-kB response genes. IkBzeta deficiency blocked the keratinocyte proliferative response to IL-17 but had no effect on altered differentiation. Finally, both IL-17 and oncogenic RAS required IkBzeta to upregulate the expression of S100a8, lipocalin2 and Steap4 mRNAs. Collectively, these results demonstrate that MyD88 plays a pro-tumorigenic function during tumor promotion in both epithelial and T-cell compartments and that IL-17/IkBzeta could play a facilitating role on keratinocytes during tumor promotion beyond its pro-inflammatory activity. Citation Format: Mary Klosterman, Christophe Cataisson, Rosalba Salcedo, Kelly Shibuya, Jennifer Waters, Giorgio Trinchieri, Stuart H. Yuspa. MyD88 and IL-17 have essential local and systemic functions in promoting tumors on mouse skin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4923. doi:10.1158/1538-7445.AM2017-4923</jats:p