Protein phosphatase 6 activates NF-κB to confer sensitivity to MAPK pathway inhibitors in KRAS- and BRAF-mutant cancer cells.

The Ras-mitogen-activated protein kinase (MAPK) pathway is a major target for cancer treatment. To better understand the genetic pathways that modulate cancer cell sensitivity to MAPK pathway inhibitors, we performed a CRISPR knockout screen with MAPK pathway inhibitors on a colorectal cancer (CRC) cell line carrying mutant KRAS. Genetic deletion of the catalytic subunit of protein phosphatase 6 (PP6), encoded by PPP6C, rendered KRAS- and BRAF-mutant CRC and BRAF-mutant melanoma cells more resistant to these inhibitors. In the absence of MAPK pathway inhibition, PPP6C deletion in CRC cells decreased cell proliferation in two-dimensional (2D) adherent cultures but accelerated the growth of tumor spheroids in 3D culture and tumor xenografts in vivo. PPP6C deletion enhanced the activation of nuclear factor κB (NF-κB) signaling in CRC and melanoma cells and circumvented the cell cycle arrest and decreased cyclin D1 abundance induced by MAPK pathway blockade in CRC cells. Inhibiting NF-κB activity by genetic and pharmacological means restored the sensitivity of PPP6C-deficient cells to MAPK pathway inhibition in CRC and melanoma cells in vitro and in CRC cells in vivo. Furthermore, a R264 point mutation in PPP6C conferred loss of function in CRC cells, phenocopying the enhanced NF-κB activation and resistance to MAPK pathway inhibition observed for PPP6C deletion. These findings demonstrate that PP6 constrains the growth of KRAS- and BRAF-mutant cancer cells, implicates the PP6-NF-κB axis as a modulator of MAPK pathway output, and presents a rationale for cotargeting the NF-κB pathway in PPP6C-mutant cancer cells.

Murine models of HRAS-mediated cutaneous skeletal hypophosphatemia syndrome suggest bone as the FGF23 excess source.

Cutaneous skeletal hypophosphatemia syndrome (CSHS) is a mosaic RASopathy characterized by the association of dysplastic skeletal lesions, congenital skin nevi of epidermal and/or melanocytic origin, and FGF23-mediated hypophosphatemia. The primary physiological source of circulating FGF23 is bone cells. However, several reports have suggested skin lesions as the source of excess FGF23 in CSHS. Consequently, without convincing evidence of efficacy, many patients with CSHS have undergone painful removal of cutaneous lesions in an effort to normalize blood phosphate levels. This study aims to elucidate whether the source of FGF23 excess in CSHS is RAS mutation-bearing bone or skin lesions. Toward this end, we analyzed the expression and activity of Fgf23 in two mouse models expressing similar HRAS/Hras activating mutations in a mosaic-like fashion in either bone or epidermal tissue. We found that HRAS hyperactivity in bone, not skin, caused excess of bioactive intact FGF23, hypophosphatemia, and osteomalacia. Our findings support RAS-mutated dysplastic bone as the primary source of physiologically active FGF23 excess in patients with CSHS. This evidence informs the care of patients with CSHS, arguing against the practice of nevi removal to decrease circulating, physiologically active FGF23.

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.

RAS oncogene signal strength regulates matrisomal gene expression and tumorigenicity of mouse keratinocytes.

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 oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells.

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 H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells.

Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence.

The TGF-ß-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-ß pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.

RAS induced senescence of skin keratinocytes is mediated through Rho-associated protein kinase (ROCK).

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 ß-galactosidase (SAßgal) 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.

Endogenous retroviruses promote homeostatic and inflammatory responses to the microbiota.

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.

Loss of DLX3 tumor suppressive function promotes progression of SCC through EGFR-ERBB2 pathway.

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.

Role of Retinoic Acid Receptor-γ in DNA Damage-Induced Necroptosis.

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.

T-Cell Deletion of MyD88 Connects IL17 and IκBζ to RAS Oncogenesis.

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.

The transcription factor CBFB suppresses breast cancer through orchestrating translation and transcription.

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.

Topical Application of a Dual ABC Transporter Substrate and NF-κB Inhibitor Blocks Multiple Sources of Cutaneous Inflammation in Mouse Skin.

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.

Head and neck squamous cancer progression is marked by CLIC4 attenuation in tumor epithelium and reciprocal stromal upregulation of miR-142-3p, a novel post-transcriptional regulator of CLIC4.

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.

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-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.

Cell autonomous or systemic EGFR blockade alters the immune-environment in squamous cell carcinomas.

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.

Elevating CLIC4 in Multiple Cell Types Reveals a TGF- Dependent Induction of a Dominant Negative Smad7 Splice Variant.

CLIC4 (Chloride intracellular channel 4) belongs to a family of putative intracellular chloride channel proteins expressed ubiquitously in multiple tissues. CLIC4 is predominantly soluble and traffics between the cytoplasm and nucleus and participates in cell cycle control and differentiation. Transforming growth factor beta (TGF-ß) elevates CLIC4, which enhances TGF-ß signaling through CLIC4 mediated stabilization of phospho-Smad2/3. CLIC4 is essential for TGF-ß induced conversion of fibroblasts to myofibroblasts and expression of matrix proteins, signaling via the p38MAPK pathway. Therefore, regulation of TGF-ß signaling is a major mechanism by which CLIC4 modifies normal growth and differentiation. We now report that elevated CLIC4 alters Smad7 function, a feedback inhibitor of the TGF-ß pathway. Overexpression of CLIC4 in keratinocytes, mouse embryonic fibroblasts and other mouse and human cell types increases the expression of Smad7Δ, a novel truncated form of Smad7. The alternatively spliced Smad7Δ variant is missing 94bp in exon 4 of Smad 7 and is conserved between mouse and human cells. The deletion is predicted to lack the TGF-ß signaling inhibitory MH2 domain of Smad7. Treatment with exogenous TGF-ß1 also enhances expression of Smad7Δ that is amplified in the presence of CLIC4. While Smad7 expression inhibits TGF-ß signaling, exogenously expressed Smad7Δ does not inhibit TGF-ß signaling as determined by TGF-ß dependent proliferation, reporter assays and phosphorylation of Smad proteins. Instead, exogenous Smad7Δ acts as a dominant negative inhibitor of Smad7, thus increasing TGF-ß signaling. This discovery adds another dimension to the myriad ways by which CLIC4 modifies TGF-ß signaling.

Science Signaling Podcast for 21 June 2016: MET and skin cancer.

This Podcast features an interview with Stuart Yuspa, senior author of a Research Article that appears in the 21 June 2016 issue of Science Signaling, about how activation of the receptor tyrosine kinase MET stimulates the formation of squamous cell carcinoma in the skin. Hepatocyte growth factor (HGF) is produced by mesenchymal cells and stimulates MET, which is present on the surface of epithelial cells. HGF-MET signaling directs the proliferation and migration of epithelial cells during the development of various organs and is important during wound healing. Aberrant MET activation has been implicated in several types of cancer, including squamous cell carcinoma. Using a model in which mice overexpressing HGF develop spontaneous squamous cell carcinomas in the skin, Cataisson et al found that MET promoted the development of squamous tumors by stimulating the synthesis and release of ligands that activate the epidermal growth factor receptor (EGFR). This mechanism was similar to that through which oncogenic RAS promotes skin tumors. Blocking EGFR signaling caused HGF-induced squamous tumors to regress, suggesting that EGFR inhibitors might be useful for treating squamous cell carcinomas.Listen to Podcast.

MET signaling in keratinocytes activates EGFR and initiates squamous carcinogenesis.

The receptor tyrosine kinase MET is abundant in many human squamous cell carcinomas (SCCs), but its functional significance in tumorigenesis is not clear. We found that the incidence of carcinogen-induced skin squamous tumors was substantially increased in transgenic MT-HGF (mouse metallothionein-hepatocyte growth factor) mice, which have increased abundance of the MET ligand HGF. Squamous tumors also erupted spontaneously on the skin of MT-HGF mice that were promoted by wounding or the application of 12-O-tetradecanoylphorbol 13-acetate, an activator of protein kinase C. Carcinogen-initiated tumors had Ras mutations, but spontaneous tumors did not. Cultured keratinocytes from MT-HGF mice and oncogenic RAS-transduced keratinocytes shared phenotypic and biochemical features of initiation that were dependent on autocrine activation of epidermal growth factor receptor (EGFR) through increased synthesis and release of EGFR ligands, which was mediated by the kinase SRC, the pseudoproteases iRhom1 and iRhom2, and the metallopeptidase ADAM17. Pharmacological inhibition of EGFR caused the regression of MT-HGF squamous tumors that developed spontaneously in orthografts of MT-HGF keratinocytes combined with dermal fibroblasts and implanted onto syngeneic mice. The global gene expression profile in MET-transformed keratinocytes was highly concordant with that in RAS-transformed keratinocytes, and a core RAS/MET coexpression network was activated in precancerous and cancerous human skin lesions. Tissue arrays revealed that many human skin SCCs have abundant HGF at both the transcript and protein levels. Thus, through the activation of EGFR, MET activation parallels a RAS pathway to contribute to human and mouse cutaneous cancers.

Biological activity of the bryostatin analog Merle 23 on mouse epidermal cells and mouse skin.

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.