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Supplemental table 2: List of genes from RNA profiling, the expression of which is significantly affected (by at least 1.5-fold change and FDR

Cancer development requires a favorable tissue microenvironment. By deleting Myd88 in keratinocytes or specific bone marrow subpopulations in oncogenic RAS-mediated skin carcinogenesis, we show that IL17 from infiltrating T cells and IκBζ signaling in keratinocytes are essential to produce a permissive microenvironment and tumor formation. Both normal and RAS-transformed keratinocytes respond to tumor promoters by activating canonical NF-κB and IκBζ signaling, releasing specific cytokines and chemokines that attract Th17 cells through MyD88-dependent signaling in T cells. The release of IL17 into the microenvironment elevates IκBζ in normal and RAS-transformed keratinocytes. Activation of IκBζ signaling is required for the expression of specific promoting factors induced by IL17 in normal keratinocytes and constitutively expressed in RAS-initiated keratinocytes. Deletion of Nfkbiz in keratinocytes impairs RAS-mediated benign tumor formation. Transcriptional profiling and gene set enrichment analysis of IκBζ−deficient RAS-initiated keratinocytes indicate that IκBζ signaling is common for RAS transformation of multiple epithelial cancers. Probing The Cancer Genome Atlas datasets using this transcriptional profile indicates that reduction of IκBζ signaling during cancer progression associates with poor prognosis in RAS-driven human cancers.Implications:The paradox that elevation of IκBζ and stimulation of IκBζ signaling through tumor extrinsic factors is required for RAS-mediated benign tumor formation while relative IκBζ expression is reduced in advanced cancers with poor prognosis implies that tumor cells switch from microenvironmental dependency early in carcinogenesis to cell-autonomous pathways during cancer progression.

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Purpose: Epidermal growth factor receptor (EGFR)–targeted therapy is in clinical use to treat squamous cell carcinoma of the head and neck and other cancers of lining epithelium. RAS mutations in these tumors are a negative prognostic factor for response, and skin inflammation is an adverse reaction to therapy. We investigated transcriptional and biochemical changes that could account for the confounding effects of RAS activation and inflammation in a squamous tissue.Experimental Design: We carried out gene expression profiling on oncogenic Ras-transformed and wild-type mouse and human keratinocytes with EGFR ablated chronically by genetic deletion or acutely by drug treatment and followed leads provided by pathway analysis with biochemical studies.Results: We identified a 25-gene signature specific to the Ras–EGFR ablation interaction and a distinct 19-gene EGFR ablation signature on normal keratinocytes. EGFR ablation in the context of wild-type Ras reduces ontologies favoring cell-cycle control and transcription, whereas oncogenic Ras enriches ontologies for ion channels and membrane transporters, particularly focused on calcium homeostasis. Ontologies between chronic EGFR ablation and acute pharmacologic ablation were unique, both with and without Ras activation. p38α is activated in response to abrogation of EGFR signaling under conditions of Ras activation in both mouse and human keratinocytes and in RAS-transformed tumor orthografts of EGFR-ablated mouse keratinocytes. EGFR ablation in the absence of oncogenic Ras revealed Erk and interleukin-1β–related pathways.Conclusion: These findings reveal unrecognized interactions between Ras and EGFR signaling in squamous tumor cells that could influence the therapeutic response to EGFR ablation therapy. Clin Cancer Res; 18(1); 170–83. ©2011 AACR.

Supplementary Figure 6 from RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

Supplementary Figure 1 from RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

Ingenol-3-angelate (Ing3A), extracted from Euphorbia peplus, is currently in clinical trials for eradicating basal cell carcinoma, actinic keratosis, and squamous cell carcinoma (SCC) in situ by topical application. Although structurally related to phorbol esters and a protein kinase C activator, topical Ing3A, but not phorbol 12-myristate 13-acetate (PMA), inhibited the growth of subcutaneous tumors derived from PAM212 (mouse SCC) and B16 (mouse melanoma). Ing3A and PMA both induced acute neutrophilic inflammation on mouse skin, but only Ing3A caused subcutaneous hemorrhage and vascular damage. Both Ing3A and PMA activated extracellular signal-regulated kinase 1/2 (ERK1/2) in epidermis, but Ing3A also activated ERK1/2 in skin dermal fibroblasts and endothelial cells. Pretreatment with topical cyclosporin A (CsA), verapamil, or XR9576, modulators of P-glycoprotein (P-gp), prevented Ing3A-induced hemorrhage but not neutrophil infiltration. CsA also impaired the anticancer activity of Ing3A, whereas the anti-inflammatory dexamethasone did not. Ing3A, but not PMA, blocked photoaffinity labeling of human P-gp with [125I]iodoaryazidoprazosin and inhibited P-gp–mediated drug resistance to HCT-15 cells. The intracellular levels of Ing3A were significantly lower in P-gp–expressing cells, and treatment with XR9576 increased the levels to those of cells that do not express P-gp, showing that Ing3A binds to and is transported by P-gp. Taken together, our results suggest that P-gp–mediated absorptive transport, dermal penetration, and vascular damage contribute to the anticancer activity of Ing3A in vivo. Cancer Res; 70(11); 4509–19. ©2010 AACR.

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Supplementary Figure 2 from RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

S100A7/psoriasin, a member of the epidermal differentiation complex, is widely overexpressed in invasive estrogen receptor (ER)α-negative breast cancers. However, it has not been established whether S100A7 contributes to breast cancer growth or metastasis. Here, we report the consequences of its expression on inflammatory pathways that impact breast cancer growth. Overexpression of human S100A7 or its murine homologue mS100a7a15 enhanced cell proliferation and upregulated various proinflammatory molecules in ERα-negative breast cancer cells. To examine in vivo effects, we generated mice with an inducible form of mS100a7a15 (MMTV-mS100a7a15 mice). Orthotopic implantation of MVT-1 breast tumor cells into the mammary glands of these mice enhanced tumor growth and metastasis. Compared with uninduced transgenic control mice, the mammary glands of mice where mS100a7a15 was induced exhibited increased ductal hyperplasia and expression of molecules involved in proliferation, signaling, tissue remodeling, and macrophage recruitment. Furthermore, tumors and lung tissues obtained from these mice showed further increases in prometastatic gene expression and recruitment of tumor-associated macrophages (TAM). Notably, in vivo depletion of TAM inhibited the effects of mS100a7a15 induction on tumor growth and angiogenesis. Furthermore, introduction of soluble hS100A7 or mS100a7a15 enhanced chemotaxis of macrophages via activation of RAGE receptors. In summary, our work used a powerful new model system to show that S100A7 enhances breast tumor growth and metastasis by activating proinflammatory and metastatic pathways. Cancer Res; 72(3); 604–15. ©2011 AACR.

Supplementary Legends for Tables 1-2, Figures 1-7, Methods from RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

Supplementary Figure 4 from RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

Supplementary Figure 7 from RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

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Supplementary Figure 5 from RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

Supplementary Figures 1-3 from Inducible Cutaneous Inflammation Reveals a Protumorigenic Role for Keratinocyte CXCR2 in Skin Carcinogenesis

Supplementary Methods and Materials from Activator Protein-1 Activity Regulates Epithelial Tumor Cell Identity

To examine the consequences of inhibiting activator protein-1 (AP-1) transcription factors in skin, transgenic mice were generated, which use the tetracycline system to conditionally express A-FOS, a dominant negative that inhibits AP-1 DNA binding. Older mice develop mild alopecia and hyperplasia of sebaceous glands, particularly around the eyes. When A-FOS was expressed during chemical-induced skin carcinogenesis, mice do not develop characteristic benign and malignant squamous lesions but instead develop benign sebaceous adenomas containing a signature mutation in the H-ras proto-oncogene. Inhibiting AP-1 activity after tumor formation caused squamous tumors to transdifferentiate into sebaceous tumors. Furthermore, reactivating AP-1 in sebaceous tumors results in a reciprocal transdifferentiation into squamous tumors. In both cases of transdifferentiation, individual cells express molecular markers for both cell types, indicating individual tumor cells have the capacity to express multiple lineages. Molecular characterization of cultured keratinocytes and tumor material indicates that AP-1 regulates the balance between the wnt/β-catenin and hedgehog signaling pathways that determine squamous and sebaceous lineages, respectively. Chromatin immunoprecipitation analysis indicates that c-Jun binds several wnt promoters, which are misregulated by A-FOS expression, suggesting that members of the wnt pathway can be a primary targets of AP-1 transcriptional regulation. Thus, AP-1 activity regulates tumor cell lineage and is essential to maintain the squamous tumor cell identity. (Cancer Res 2006; 66(15): 7578-88)

Supplementary Figure 3 from RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

Supplementary Figures 1-5, Tables 1-2 from The Skin Cancer Chemotherapeutic Agent Ingenol-3-Angelate (PEP005) Is a Substrate for the Epidermal Multidrug Transporter (ABCB1) and Targets Tumor Vasculature

Cellular senescence is a well-documented response to oncogene activation in many tissues. Multiple pathways are invoked to achieve senescence indicating its importance to counteract the transforming activities of oncogenic stimulation. We now report that the Rho-associated protein kinase (ROCK) signaling pathway is a critical regulator of oncogene-induced senescence in skin carcinogenesis. Transformation of mouse keratinocytes with oncogenic RAS upregulates ROCK activity and initiates a senescence response characterized by cell enlargement, growth inhibition, upregulation of senescence associated β-galactosidase (SAβgal) expression, and release of multiple pro-inflammatory factors comprising the senescence-associated secretory phenotype (SASP). The addition of the ROCK inhibitor Y-27632 and others prevents these senescence responses and maintains proliferating confluent RAS transformed keratinocyte cultures indefinitely. Mechanistically, oncogenic RAS transformation is associated with upregulation of cell cycle inhibitors p15Ink4b , p16Ink4a , and p19Arf and downregulation of p-AKT, all of which are reversed by Y-27632. RNA-seq analysis of Y-27632 treated RAS-transformed keratinocytes indicated that the inhibitor reduced growth-inhibitory gene expression profiles and maintained expression of proliferative pathways. Y-27632 also reduced the expression of NF-κB effector genes and the expression of IκBζ downstream mediators. The senescence inhibition from Y-27632 was reversible, and upon its removal, senescence reoccurred in vitro with rapid upregulation of cell cycle inhibitors, SASP expression, and cell detachment. Y-27632 treated cultured RAS-keratinocytes formed tumors in the absence of the inhibitor when placed in skin orthografts suggesting that factors in the tumor microenvironment can overcome the drive to senescence imparted by overactive ROCK activity.

Environmental and molecular carcinogenesis are linked by the discovery that chemical carcinogen induced-mutations in the Hras or Kras genes drives tumor development in mouse skin. Importantly, enhanced expression or allele amplification of the mutant Ras gene contributes to selection of initiated cells, tumor persistence, and progression. To explore the consequences of Ras oncogene signal strength, primary keratinocytes were isolated and cultured from the LSL-HrasG12D and LSL-KrasG12D C57BL/6J mouse models and the mutant allele was activated by adeno-Cre recombinase. Keratinocytes expressing one (H) or two (HH) mutant alleles of HrasG12D, one KrasG12D allele (K), or one of each (HK) were studied. All combinations of activated Ras alleles stimulated proliferation and drove transformation marker expression, but only HH and HK formed tumors. HH, HK, and K sustained long-term keratinocyte growth in vitro, while H and WT could not. RNA-Seq yielded two distinct gene expression profiles; HH, HK, and K formed one cluster while H clustered with WT. Weak MAPK activation was seen in H keratinocytes but treatment with a BRAF inhibitor enhanced MAPK signaling and facilitated tumor formation. K keratinocytes became tumorigenic when they were isolated from mice where the LSL-KrasG12D allele was backcrossed from the C57BL/6 onto the FVB/N background. All tumorigenic keratinocytes but not the non-tumorigenic precursors shared a common remodeling of matrisomal gene expression that is associated with tumor formation. Thus, RAS oncogene signal strength determines cell-autonomous changes in initiated cells that are critical for their tumor-forming potential.

The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H

Chloride intracellular channel 4 (CLIC4) is a tumor suppressor implicated in processes including growth arrest, differentiation, and apoptosis. CLIC4 protein expression is diminished in the tumor parenchyma during progression in squamous cell carcinoma (SCC) and other neoplasms, but the underlying mechanisms have not been identified. Data from The Cancer Genome Atlas suggest this is not driven by genomic alterations. However, screening and functional assays identified miR-142-3p as a regulator of CLIC4. CLIC4 and miR-142-3p expression are inversely correlated in head and neck (HN) SCC and cervical SCC, particularly in advanced stage cancers. In situ localization revealed that stromal immune cells, not tumor cells, are the predominant source of miR-142-3p in HNSCC. Furthermore, HNSCC single-cell expression data demonstrated that CLIC4 is lower in tumor epithelial cells than in stromal fibroblasts and endothelial cells. Tumor-specific downregulation of CLIC4 was confirmed in an SCC xenograft model concurrent with immune cell infiltration and miR-142-3p upregulation. These findings provide the first evidence of CLIC4 regulation by miRNA. Furthermore, the distinct localization of CLIC4 and miR-142-3p within the HNSCC tumor milieu highlight the limitations of bulk tumor analysis and provide critical considerations for both future mechanistic studies and use of miR-142-3p as a HNSCC biomarker.

Summary DNA-damaging compounds, commonly used as chemotherapeutic drugs, are known to trigger cells to undergo programmed cell death such as apoptosis and necroptosis. However, the molecular mechanism of DNA damage-induced cell death is not fully understood. Here, we report that RARγ has a critical role in DNA damage-induced programmed cell death, specifically in necroptosis. The loss of RARγ abolishes the necroptosis induced by DNA damage. In addition, cells that lack RARγ are less susceptible to extrinsic apoptotic pathway activated by DNA-damaging agents whereas the intrinsic apoptotic pathway is not affected. We demonstrate that RARγ is essential for the formation of RIPK1/RIPK3 death complex, known as Ripoptosome, in response to DNA damage. Furthermore, we show that RARγ plays a role in skin cancer development by using RARγ1 knockout mice and human squamous cell carcinoma biopsies. Hence, our study reveals that RARγ is a critical component of DNA damage-induced cell death., Graphical Abstract, Highlights • RARγ plays a key role in DNA damage-induced cell death • RARγ is essential for RIPK1-mediated necroptosis and apoptosis following DNA damage • RARγ is required for the formation of Ripoptosome in response to DNA damage • Loss of RARγ correlates with skin cancer development, Biological Sciences; Biochemistry; Molecular Biology; Cell Biology

Translation and transcription are frequently dysregulated in cancer. These two processes are generally regulated by distinct sets of factors. The CBFB gene, which encodes a transcription factor, has recently emerged as a highly mutated driver in a variety of human cancers including breast cancer. Here we report a noncanonical role of CBFB in translation regulation. RNA immunoprecipitation followed by deep sequencing (RIP-seq) reveals that cytoplasmic CBFB binds to hundreds of transcripts and regulates their translation. CBFB binds to mRNAs via hnRNPK and enhances translation through eIF4B, a general translation initiation factor. Interestingly, the RUNX1 mRNA, which encodes the transcriptional partner of CBFB, is bound and translationally regulated by CBFB. Furthermore, nuclear CBFB/RUNX1 complex transcriptionally represses the oncogenic NOTCH signaling pathway in breast cancer. Thus, our data reveal an unexpected function of CBFB in translation regulation and propose that breast cancer cells evade translation and transcription surveillance simultaneously through downregulating CBFB., CBFB is highly mutated in breast cancers and is known to interact with RUNX proteins to regulate transcription. Here, the authors describe a non-canonical role of CBFB in translation regulation in which it binds to mRNAs through hnRNPK, facilitating translation by eIF4B.

Cutaneous squamous cell carcinoma (cSCC) ranks second in the frequency of all skin cancers. The balance between keratinocyte proliferation and differentiation is disrupted in the pathological development of cSCC. DLX3 is a homeobox transcription factor which plays pivotal roles in embryonic development and epidermal homeostasis. To investigate the impact of DLX3 expression on cSCC prognosis, we carried out clinicopathologic analysis of DLX3 expression which showed statistical correlation between tumors of higher pathologic grade and levels of DLX3 protein expression. Further, Kaplan-Meier survival curve analysis demonstrated that low DLX3 expression correlated with poor patient survival. To model the function of Dlx3 in skin tumorigenesis, a two-stage dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA) study was performed on mice genetically depleted of Dlx3 in skin epithelium (Dlx3cKO). Dlx3cKO mice developed significantly more tumors, with more rapid tumorigenesis compared to control mice. In Dlx3cKO mice treated only with DMBA, tumors developed after ~16 weeks suggesting that loss of Dlx3 has a tumor promoting effect. Whole transcriptome analysis of tumor and skin tissue from our mouse model revealed spontaneous activation of the EGFR-ERBB2 pathway in the absence of Dlx3. Together, our findings from human and mouse model system support a tumor suppressive function for DLX3 in skin and underscore the efficacy of therapeutic approaches that target EGFR-ERBB2 pathway.

Summary The microbiota plays a fundamental role in regulating host immunity. However, the processes involved in the initiation and regulation of immunity to the microbiota remain largely unknown. Here, we show that the skin microbiota promotes the discrete expression of defined endogenous retroviruses (ERVs). Keratinocyte-intrinsic responses to ERVs depended on cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING) signaling and promoted the induction of commensal-specific T cells. Inhibition of ERV reverse transcription significantly impacted these responses, resulting in impaired immunity to the microbiota and its associated tissue repair function. Conversely, a lipid-enriched diet primed the skin for heightened ERV- expression in response to commensal colonization, leading to increased immune responses and tissue inflammation. Together, our results support the idea that the host may have co-opted its endogenous virome as a means to communicate with the exogenous microbiota, resulting in a multi-kingdom dialog that controls both tissue homeostasis and inflammation., Graphical abstract, Highlights • Endogenous retrovirus sensing is required for commensal-specific immunity • Reverse transcription inhibition impairs commensal-induced immunity in the skin • cGAS/STING signaling in keratinocytes is required for skin immunity to the microbiota • Enhanced endogenous retrovirus expression promotes microbiota-induced inflammation, The host immune system uses the endogenous virome to communicate with the exogenous microbiota in a multi-kingdom interaction that modulates tissue repair or inflammation.

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-phorbol-13-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.

Cancer development requires a favorable tissue microenvironment. By deleting Myd88 in keratinocytes or specific bone marrow subpopulations in oncogenic RAS-mediated skin carcinogenesis, we show that IL17 from infiltrating T cells and IκBζ signaling in keratinocytes are essential to produce a permissive microenvironment and tumor formation. Both normal and RAS-transformed keratinocytes respond to tumor promoters by activating canonical NF-κB and IκBζ signaling, releasing specific cytokines and chemokines that attract Th17 cells through MyD88-dependent signaling in T cells. The release of IL17 into the microenvironment elevates IκBζ in normal and RAS-transformed keratinocytes. Activation of IκBζ signaling is required for the expression of specific promoting factors induced by IL17 in normal keratinocytes and constitutively expressed in RAS-initiated keratinocytes. Deletion of Nfkbiz in keratinocytes impairs RAS-mediated benign tumor formation. Transcriptional profiling and gene set enrichment analysis of IκBζ−deficient RAS-initiated keratinocytes indicate that IκBζ signaling is common for RAS transformation of multiple epithelial cancers. Probing The Cancer Genome Atlas datasets using this transcriptional profile indicates that reduction of IκBζ signaling during cancer progression associates with poor prognosis in RAS-driven human cancers. Implications: The paradox that elevation of IκBζ and stimulation of IκBζ signaling through tumor extrinsic factors is required for RAS-mediated benign tumor formation while relative IκBζ expression is reduced in advanced cancers with poor prognosis implies that tumor cells switch from microenvironmental dependency early in carcinogenesis to cell-autonomous pathways during cancer progression.

Targeting mutations and amplifications in the EGFR has been successful precision therapy for cancers of the lung, oral cavity and gastrointestinal track. However, a systemic immune reaction manifested by dose-limiting inflammation in the skin and gut has been a consistent adverse effect. To address the possibility that intra-tumoral immune changes contribute to the anti-cancer activity of EGFR inhibition, squamous cancers were produced by syngeneic orthografts of either EGFR null or wildtype mouse primary keratinocytes transduced with an oncogenic H-ras retrovirus. Flow cytometric, RNA and Bioplex immunoassay analyses of the tumor immune milieu were performed. Cancers forming from keratinocytes genetically depleted of EGFR were smaller than wildtype cancers and had fewer infiltrating FoxP3 Treg cells, lower Foxp3 RNA and a lower percentage of CD4 PD1 positive cells indicating a tumor cell autonomous regulation of its microenvironment. Hosts bearing wildtype cancers treated with gefitinib for 1 week showed a trend for smaller tumors. In this short term pharmacological model, there was also a trend to reduced FoxP3 cells and FoxP3 RNA in the tumors of treated mice as well as a substantial increase in the ratio of IL-1A/IL-1RA transcripts. These results suggest that relatively brief systemic inhibition of EGFR signaling alters the immune environment of the targeted cancer. Together these data imply that an EGFR dependent Treg function supports the growth of squamous cancers and is a target for the therapeutic activity of EGFR inhibition.

Bryostatin 1, a complex macrocyclic lactone, is the subject of multiple clinical trials for cancer chemotherapy. Although bryostatin 1 biochemically functions like the classic mouse skin tumor promoter phorbol 12-myristate 13-acetate (PMA) to bind to and activate protein kinase C, paradoxically, it fails to induce many of the typical phorbol ester responses, including tumor promotion. Intense synthetic efforts are currently underway to develop simplified bryostatin analogs that preserve the critical functional features of bryostatin 1, including its lack of tumor promoting activity. The degree to which bryostatin analogs maintain the unique pattern of biological behavior of bryostatin 1 depends on the specific cellular system and the specific response. Merle 23 is a significantly simplified bryostatin analog that retains bryostatin like activity only to a limited extent. Here, we show that in mouse epidermal cells the activity of Merle 23 was either similar to bryostatin 1 or intermediate between bryostatin 1 and PMA, depending on the specific parameter examined. We then examined the hyperplastic and tumor promoting activity of Merle 23 on mouse skin. Merle 23 showed substantially reduced hyperplasia and was not tumor promoting at a dose comparable to that for PMA. These results suggest that there may be substantial flexibility in the design of bryostatin analogs that retain its lack of tumor promoting activity. © 2016 Wiley Periodicals, Inc.

Epidermal homeostasis depends on the coordinated control of keratinocyte cell cycle. Differentiation and the alteration of this balance can result in neoplastic development. Here we report on a novel DLX3-dependent network that constrains epidermal hyperplasia and squamous tumorigenesis. By integrating genetic and transcriptomic approaches, we demonstrate that DLX3 operates through a p53-regulated network. DLX3 and p53 physically interact on the p21 promoter to enhance p21 expression. Elevating DLX3 in keratinocytes produces a G1-S blockade associated with p53 signature transcriptional profiles. In contrast, DLX3 loss promotes a mitogenic phenotype associated with constitutive activation of ERK. DLX3 expression is lost in human skin cancers and is extinguished during progression of experimentally induced mouse squamous cell carcinoma (SCC). Reinstatement of DLX3 function is sufficient to attenuate the migration of SCC cells, leading to decreased wound closure. Our data establish the DLX3-p53 interplay as a major regulatory axis in epidermal differentiation and suggest that DLX3 is a modulator of skin carcinogenesis.

p38δ expression and/or activity are increased in human cutaneous malignancies, including invasive squamous cell carcinoma (SCC) and head and neck SCC, but the role of p38δ in cutaneous carcinogenesis has not been well-defined. We have reported that mice with germline loss of p38δ exhibited a reduced susceptibility to skin tumor development compared with wild-type mice in the two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical skin carcinogenesis model. Here, we report that p38δ gene ablation inhibited the growth of tumors generated from v-ras(Ha) -transformed keratinocytes in skin orthografts to nude mice, indicating that keratinocyte-intrinsic p38δ is required for Ras-induced tumorigenesis. Gene expression profiling of v-ras(Ha) -transformed p38δ-null keratinocytes revealed transcriptional changes associated with cellular responses linked to tumor suppression, such as reduced proliferation and increased differentiation, cell adhesion, and cell communications. Notably, a short-term DMBA/TPA challenge, modeling the initial stages of chemical skin carcinogenesis treatment, elicited an enhanced inflammation in p38δ-null skin compared with skin of wild-type mice, as assessed by measuring the expression of pro-inflammatory cytokines, including IL-1β, IL-6, IL-17, and TNFα. Additionally, p38δ-null skin and p38δ-null keratinocytes exhibited increased p38α activation and signaling in response to acute inflammatory challenges, suggesting a role for p38α in stimulating the elevated inflammatory response in p38δ-null skin during the initial phases of the DMBA/TPA treatment compared with similarly treated p38δ(+/+) skin. Altogether, our results indicate that p38δ signaling regulates skin carcinogenesis not only by keratinocyte cell-autonomous mechanisms, but also by influencing the interaction between between the epithelial compartment of the developing skin tumor and its stromal microenvironment.

Centuries ago, an obscure report on an unusual skin cancer launched the modern era of cancer research. Ever since, skin research has made key contributions to our understanding of the biology and biochemistry of cancer pathogenesis. In 1775, Percival Pott published his essay entitled Chirurgical observations Relative to the Cataract, the Polypus of the Nose, the Cancer of the Scrotum, [etc.] describing the origin of the ‘‘soot-wart’’, a cancer of the inferior surface of the scrotum occurring with high frequency in English chimney sweeps (Brown and Thornton 1957). Pott attributed the cause of this tumor to ‘‘the lodgement of soot in the rugae of the scrotum’’, thus providing in one description the first recognition of environmental and occupational cancer, the first identification of a human carcinogen, the first opportunity for cancer prevention, and a protocol for surgical treatment. Included in that 797 word essay was a remarkably detailed description of the course of the disease from a local confined lesion to invasive and then disseminated lethal cancer. These insights were little noticed for almost 150 years until (Yamagiwa and Ichikawa, 1918) reported that the chronic topical application of coal tar on rabbit ears produced cutaneous squamous cell carcinomas, the first chemical carcinogenesis experiment. By describing the evolution Figure 1 of individual lesions, Yamagiwa and Ichikawa concluded that cancer developed through multiple phenotypic stages and progressed even after the carcinogen was removed. Over the ensuing 20 years, many biologists and chemists sought to identify the active carcinogen in coal tar, culminating in 1932 with the report by a group led by Ernest Kennaway that 3,4 benzpyrene was the potent polycyclic aromatic hydrocarbon in coal tar that produced cancers when topically applied to mouse skin. Incorporated into this project was the isolation or synthesis of multiple carcinogenic hydrocarbons, all recognized by their action as skin carcinogens (summarized in Kennaway, 1955). At the same time, the availability of synthetic carcinogens and a reliable squamous cancer model emboldened a group of pathologists and biologists to methodically explore the pathogenesis of the neoplastic skin lesions. For the next two decades, these investigators discovered concepts regarding skin cancer development that dominate our current understanding of cancer development in almost all epithelial tissues. In particular, the work of Cecil Mottram, Friedewald and Rous (Rous later won the Nobel Prize for his work on the viral etiology of skin cancer), and Berenblum and Shubik led to the following conclusions on the biology of cancer: cancer occurs through multiple stages with distinct operational mechanisms and required sequence; cancer is more frequent when proliferating tissues are exposed to carcinogens; the extent of exposure or potency of the carcinogen determines the latent period for tumor development; tumor development does not require continuous exposure to carcinogenic agents and the effect of a subtumorigenic carcinogen exposure is permanent; tumors will develop after a subtumorigenic carcinogen exposure if the target tissue is subjected to regenerative hyperplasia as caused by repeated wounding or application of an irritant (Berenblum and Shubik, 1947, 1949; Mottram, 1944a, b; Berenblum and Haran, 1955; Berenblum, 1957; Friedewald and Rous, 1944). It was Friedewald and Rous who coined the terms ‘‘initiation’’ and ‘‘promotion’’ to distinguish the irreversible action of a carcinogen and the non-carcinogenic reversible activity of an irritant, still a major part of the cancer lexicon today. One of the important additions to the skin cancer protocols provided by Berenblum was the use of topical croton oil, a non-carcinogenic skin irritant that induced the outgrowth of tumors from skin that had been treated with a subtumorigenic dose of carcinogen (initiated). Croton oil (Crotonis Oleum) is prepared from the seeds of Croton tiglium, a tree belonging to the natural order Euphorbiales and family Euphorbiaceae. Croton oil had been used for centuries as folk medicine and was known to induce inflammation and peeling of skin when applied topically and diarrhea when taken internally. Its potency to promote tumors on skin catalyzed interest among cancer biologists to identify the active compounds contained within the complex mixture. In a series of brilliant chemical purifications and analyses, Van Duuren and Hecker almost simultaneously identified phorbol myristate acetate (12-O-tetradecanoyl-phorbol-13-acetate), commonly

Background— Chloride intracellular channel 4 (CLIC4) is highly expressed in the endothelium of remodeled pulmonary vessels and plexiform lesions of patients with pulmonary arterial hypertension. CLIC4 regulates vasculogenesis through endothelial tube formation. Aberrant CLIC4 expression may contribute to the vascular pathology of pulmonary arterial hypertension. Methods and Results— CLIC4 protein expression was increased in plasma and blood-derived endothelial cells from patients with idiopathic pulmonary arterial hypertension and in the pulmonary vascular endothelium of 3 rat models of pulmonary hypertension. CLIC4 gene deletion markedly attenuated the development of chronic hypoxia-induced pulmonary hypertension in mice. Adenoviral overexpression of CLIC4 in cultured human pulmonary artery endothelial cells compromised pulmonary endothelial barrier function and enhanced their survival and angiogenic capacity, whereas CLIC4 shRNA had an inhibitory effect. Similarly, inhibition of CLIC4 expression in blood-derived endothelial cells from patients with idiopathic pulmonary arterial hypertension attenuated the abnormal angiogenic behavior that characterizes these cells. The mechanism of CLIC4 effects involves p65-mediated activation of nuclear factor-κB, followed by stabilization of hypoxia-inducible factor-1α and increased downstream production of vascular endothelial growth factor and endothelin-1. Conclusion— Increased CLIC4 expression is an early manifestation and mediator of endothelial dysfunction in pulmonary hypertension.

Among the molecular signals underlying cutaneous inflammation is the transcription complex NF-κB, its upstream modulators, and cytokines and chemokines that are the downstream proinflammatory effectors. Central to NF-κB activation is IκB kinase (IKK), which phosphorylates IκBα, releasing NF-κB to the nucleus. In a screening of a kinase inhibitor library, we identified two IKK inhibitors that were high-affinity substrates for p-glycoprotein (ABCB1), the multidrug resistance protein known to facilitate transdermal drug delivery. ACHP (2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3-pyridinecarbonitrile) and IKK 16 prevented both nuclear translocation of NF-κB and activation of a NF-κB reporter and reduced the induction of cytokine and chemokine transcripts in human or mouse keratinocytes by IL-1α, tumor necrosis factor-α, and phorbol myristate acetate. ACHP, but not IKK 16, was nontoxic to mouse or human keratinocytes at any dose tested. In mice, topical ACHP prevented the cutaneous inflammation induced by topical phorbol myristate acetate or imiquimod, reduced the inflammation from erythema doses of artificial sunlight, and lowered the tumor incidence of mice treated with 7,12-dimethyl benzanthracene when applied before phorbol myristate acetate. Topical ACHP also reduced the NF-κB and IL-17 inflammatory signature after multiple doses of imiquimod. Thus, ACHP and IKK 16 hit their NF-κB target in mouse and human keratinocytes, and ACHP is an effective topical nonsteroidal anti-inflammatory in mice.