NRF3 suppresses squamous carcinogenesis, involving the unfolded protein response regulator HSPA5.

Epithelial skin cancers are extremely common, but the mechanisms underlying their malignant progression are still poorly defined. Here, we identify the NRF3 transcription factor as a tumor suppressor in the skin. NRF3 protein expression is strongly downregulated or even absent in invasively growing cancer cells of patients with basal and squamous cell carcinomas (BCC and SCC). NRF3 deficiency promoted malignant conversion of chemically induced skin tumors in immunocompetent mice, clonogenic growth and migration of human SCC cells, their invasiveness in 3D cultures, and xenograft tumor formation. Mechanistically, the tumor-suppressive effect of NRF3 involves HSPA5, a key regulator of the unfolded protein response, which we identified as a potential NRF3 interactor. HSPA5 levels increased in the absence of NRF3, thereby promoting cancer cell survival and migration. Pharmacological inhibition or knock-down of HSPA5 rescued the malignant features of NRF3-deficient SCC cells in vitro and in preclinical mouse models. Together with the strong expression of HSPA5 in NRF3-deficient cancer cells of SCC patients, these results suggest HSPA5 inhibition as a treatment strategy for these malignancies in stratified cancer patients.

Phase-specific signatures of wound fibroblasts and matrix patterns define cancer-associated fibroblast subtypes.

Healing wounds and cancers present remarkable cellular and molecular parallels, but the specific roles of the healing phases are largely unknown. We developed a bioinformatics pipeline to identify genes and pathways that define distinct phases across the time-course of healing. Their comparison to cancer transcriptomes revealed that a resolution phase wound signature is associated with increased severity in skin cancer and enriches for extracellular matrix-related pathways. Comparisons of transcriptomes of early- and late-phase wound fibroblasts vs skin cancer-associated fibroblasts (CAFs) identified an "early wound" CAF subtype, which localizes to the inner tumor stroma and expresses collagen-related genes that are controlled by the RUNX2 transcription factor. A "late wound" CAF subtype localizes to the outer tumor stroma and expresses elastin-related genes. Matrix imaging of primary melanoma tissue microarrays validated these matrix signatures and identified collagen- vs elastin-rich niches within the tumor microenvironment, whose spatial organization predicts survival and recurrence. These results identify wound-regulated genes and matrix patterns with prognostic potential in skin cancer.

Integrated multi-omics reveals cellular and molecular interactions governing the invasive niche of basal cell carcinoma.

Tumors invade the surrounding tissues to progress, but the heterogeneity of cell types at the tumor-stroma interface and the complexity of their potential interactions hampered mechanistic insight required for efficient therapeutic targeting. Here, combining single-cell and spatial transcriptomics on human basal cell carcinomas, we define the cellular contributors of tumor progression. In the invasive niche, tumor cells exhibit a collective migration phenotype, characterized by the expression of cell-cell junction complexes. In physical proximity, we identify cancer-associated fibroblasts with extracellular matrix-remodeling features. Tumor cells strongly express the cytokine Activin A, and increased Activin A-induced gene signature is found in adjacent cancer-associated fibroblast subpopulations. Altogether, our data identify the cell populations and their transcriptional reprogramming contributing to the spatial organization of the basal cell carcinoma invasive niche. They also demonstrate the power of integrated spatial and single-cell multi-omics to decipher cancer-specific invasive properties and develop targeted therapies.

A Protumorigenic mDia2-MIRO1 Axis Controls Mitochondrial Positioning and Function in Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAF) are key regulators of tumorigenesis. Further insights into the tumor-promoting mechanisms of action of CAFs could help improve cancer diagnosis and treatment. Here we show that the formin mDia2 regulates the positioning and function of mitochondria in dermal fibroblasts, thereby promoting a protumorigenic CAF phenotype. Mechanistically, mDia2 stabilized the mitochondrial trafficking protein MIRO1. Loss of mDia2 or MIRO1 in fibroblasts or CAFs reduced the presence of mitochondria and ATP levels near the plasma membrane and at CAF-tumor cell contact sites, caused metabolic alterations characteristic of mitochondrial dysfunction, and suppressed the secretion of protumorigenic proteins. In mouse models of squamous carcinogenesis, genetic or pharmacologic inhibition of mDia2, MIRO1, or their common upstream regulator activin A inhibited tumor formation. Consistently, co-upregulation of mDia2 and MIRO1 in the stroma of various human cancers negatively correlated with survival. This work unveils a key role of mitochondria in the protumorigenic CAF phenotype and identifies an activin A-mDia2-MIRO1 signaling axis in CAFs with diagnostic and therapeutic potential. SIGNIFICANCE: Inhibition of mDia2/MIRO1-mediated mitochondrial positioning in CAFs induces mitochondrial dysfunction and suppresses tumor growth, revealing a promising therapeutic strategy to target tumor-stroma cross-talk.

Grainyhead-like transcription factors: guardians of the skin barrier.

There has been selective pressure to maintain a skin barrier since terrestrial animals evolved 360 million years ago. These animals acquired an unique integumentary system with a keratinized, stratified, squamous epithelium surface barrier. The barrier protects against dehydration and entry of microbes and toxins. The skin barrier centres on the stratum corneum layer of the epidermis and consists of cornified envelopes cemented by the intercorneocyte lipid matrix. Multiple components of the barrier undergo cross-linking by transglutaminase (TGM) enzymes, while keratins provide additional mechanical strength. Cellular tight junctions also are crucial for barrier integrity. The grainyhead-like (GRHL) transcription factors regulate the formation and maintenance of the integument in diverse species. GRHL3 is essential for formation of the skin barrier during embryonic development, whereas GRHL1 maintains the skin barrier postnatally. This is achieved by transactivation of Tgm1 and Tgm5, respectively. In addition to its barrier function, GRHL3 plays key roles in wound repair and as an epidermal tumour suppressor. In its former role, GRHL3 activates the planar cell polarity signalling pathway to mediate wound healing by providing directional migration cues. In squamous epithelium, GRHL3 regulates the balance between proliferation and differentiation, and its loss induces squamous cell carcinoma (SCC). In the skin, this is mediated through increased expression of MIR21, which reduces the expression levels of GRHL3 and its direct target, PTEN, leading to activation of the PI3K-AKT signalling pathway. These data position the GRHL family as master regulators of epidermal homeostasis across a vast gulf of evolutionary history.

Il y a eu une pression de sélection pour maintenir la barrière cutanée depuis l'évolution des animaux terrestres pendant 360 millions d'années. Ces animaux ont acquis un système tégumentaire unique avec un épithélium squameux, stratifié, kératinisé comme barrière de surface. La barrière protège contre la déshydratation et l'entée de microbes et de toxines. La barrière cutanée est centrée sur la couche du stratum corneum de l'épiderme et consiste en des enveloppes cimentées par une matrice lipidique intercornéocytaire. Les composants multiples de la barrière subissent des remaniements par les enzymes transglutaminases (TGM) tandis que la kératine fournit un soutien mécanique supplémentaire. Les jonctions serrées cellulaires jouent aussi un rôle crucial pour l'intégrité de barrière. Les facteurs de transcriptions GRHL (grainyhead-like) régulent la formation et le maintien du tégument dans différentes espèces. GRHL3 est essentielle pour la formation de la barrière cutanée au cours du développement embryonnaire tandis que GRHL1 maintient la barrière cutanée après la naissance. Ceci est permis respectivement par transactivation de Tgm1 et Tgm5. En plus de cette fonction barrière, GRHL3 joue un rôle clé dans la cicatrisation et en tant que suppresseur de tumeur épidermique. Dans ses rôles principaux, GRHL3 active la voie de signal de polarité cellulaire plane pour soutenir la cicatrisation en fournissant des repaires directionnels de migration. Dans les épithéliums squameux, GRHL3 régule la balance entre prolifération et différentiation, et sa perte induit le carcinome épidermoïde (SCC). Dans la peau ceci est médié par une augmentation de l'expression de MIR21, qui réduit le niveau d'expression de GRHL3 et sa cible directe, PTEN, menant à l'activation de la voie de signal PI3K-AKT. Ces données positionnent la famille GRHL comme régulatrice majeure de l'homéostasie épidermique à travers le vaste gouffre de l'histoire de l'évolution.

Ha habido una presión selectiva para mantener una barrera cutánea desde que los animales terrestres evolucionaron hace 360 ​​millones de años. Estos animales adquirieron un sistema tegumentario único con una barrera superficial de epitelio escamoso estratificado queratinizado. La barrera protege contra la deshidratación y la entrada de microbios y toxinas. La barrera cutánea se centra en la capa de estrato córneo de la epidermis y consta de membranas cornificadas cementadas por una matriz lipídica intercorneocitaria. Múltiples componentes de la barrera se unen por la actividad de enzimas transglutaminasas (TGM), mientras que las queratinas proporcionan resistencia mecánica adicional. Las uniones celulares estrechas también son cruciales para la integridad de la barrera. Los factores de transcripción similares a grainyhead (cabeza granulada) (GRHL) regulan la formación y mantenimiento del tegumento en diversas especies. GRHL3 es esencial para la formación de la barrera cutánea durante el desarrollo embrionario, mientras que GRHL1 mantiene la barrera cutánea postnatal. Esto se logra mediante la transactivación de Tgm1 y Tgm5, respectivamente. Además de su función de barrera, GRHL3 juega un papel clave en la reparación de heridas y como supresor de tumores epidérmicos. En su función de cicatrización, GRHL3 activa la vía de señalización de la polaridad celular plana para mediar en la cicatrización de heridas proporcionando señales de migración direccional. En el epitelio escamoso, GRHL3 regula el equilibrio entre la proliferación y la diferenciación, y su pérdida induce el carcinoma de células escamosas (SCC). En la piel, esto está mediado por una mayor expresión de MIR21, que reduce los niveles de expresión de GRHL3 y su sustrato directo, PTEN, lo que lleva a la activación de la vía de señal intracelular PI3K-AKT. Estos datos colocan la familia de factores de transcripción GRHL como reguladores críticos de la homeostasis epidérmica a través de una extensa historia evolutiva.

Tem havido uma pressão seletiva para manter a barreira cutânea desde a evolução dos animais terrestres há 360 milhões de anos. Estes animais adquiriram um sistema tegumentar único com uma barreira de superfície escamosa, estratificada e queratinizada. A barreira protege contra a desidratação e entrada de micróbios e toxinas. A barreira cutânea é centrada na camada do estrato córneo da epiderme e consiste em envelopes cornificados revestidos pela matriz lipídica intercorneocítica. Vários componentes da barreira sofrem ligação cruzada por enzimas transglutaminase (TGM), enquanto as queratinas fornecem resistência mecânica adicional. As junções celulares também são cruciais para a integridade da barreira. Os fatores de transcrição do tipo grainyhead (GRHL) regulam a formação e manutenção do tegumento em diversas espécies. GRHL3 é essencial para a formação da barreira cutânea durante o desenvolvimento embrionário, enquanto GRHL1 mantém a barreira cutânea pós-natal. Isso é obtido pela transativação de Tgm1 e Tgm5, respectivamente. Além de sua função de barreira, GRHL3 desempenha papéis importantes no reparo de feridas e como supressor de tumor epidérmico. Em sua função anterior, GRHL3 ativa a via de sinalização de polaridade celular planar para mediar a cicatrização de feridas, fornecendo pistas de migração direcional. No epitélio escamoso, o GRHL3 regula o equilíbrio entre a proliferação e a diferenciação, e sua perda induz o carcinoma de células escamosas (CCE). Na pele, isso é mediado pelo aumento da expressão de MIR21, que reduz os níveis de expressão de GRHL3 e seu alvo direto, PTEN, levando à ativação da via de sinalização PI3K-AKT. Esses dados posicionam a família GRHL como reguladores mestres da homeostase epidérmica em um vasto abismo da história evolutiva.

Genotoxic Agents: An Unexpected Effect on Healthy Epithelia.

DNA cross-linking agents are common chemotherapeutics for cancer treatment, but their effect on normal cells is largely unknown. In this issue of Developmental Cell, Seldin and Macara (2020) show that such compounds induce epithelial hyperplasia and stem cell fate mis-specification in a non-cell-autonomous manner via inflammasome activation in dermal fibroblasts.

Wound Repair, Scar Formation, and Cancer: Converging on Activin.

Wound repair is a highly regulated process that requires the interaction of various cell types. It has been shown that cancers use the mechanisms of wound healing to promote their own growth. Therefore, it is of importance to identify common regulators of wound repair and tumor formation and to unravel their functions and mechanisms of action. An exciting example is activin, which acts on multiple cell types in wounds and tumors, thereby promoting healing, but also scar formation and tumorigenesis. Here, we summarize current knowledge on the role of activin in these processes and highlight the therapeutic potential of activin or activin antagonists for the treatment of impaired healing or excessive scarring and cancer, respectively.

Activin-mediated alterations of the fibroblast transcriptome and matrisome control the biomechanical properties of skin wounds.

Matrix deposition is essential for wound repair, but when excessive, leads to hypertrophic scars and fibrosis. The factors that control matrix deposition in skin wounds have only partially been identified and the consequences of matrix alterations for the mechanical properties of wounds are largely unknown. Here, we report how a single diffusible factor, activin A, affects the healing process across scales. Bioinformatics analysis of wound fibroblast transcriptome data combined with biochemical and histopathological analyses of wounds and functional in vitro studies identify that activin promotes pro-fibrotic gene expression signatures and processes, including glycoprotein and proteoglycan biosynthesis, collagen deposition, and altered collagen cross-linking. As a consequence, activin strongly reduces the wound and scar deformability, as identified by a non-invasive in vivo method for biomechanical analysis. These results provide mechanistic insight into the roles of activin in wound repair and fibrosis and identify the functional consequences of alterations in the wound matrisome at the biomechanical level.

A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming.

Cancer-associated fibroblasts (CAFs) are key regulators of tumorigenesis and promising targets for next-generation therapies. We discovered that cancer cell-derived activin A reprograms fibroblasts into pro-tumorigenic CAFs. Mechanistically, this occurs via Smad2-mediated transcriptional regulation of the formin mDia2, which directly promotes filopodia formation and cell migration. mDia2 also induces expression of CAF marker genes through prevention of p53 nuclear accumulation, resulting in the production of a pro-tumorigenic matrisome and secretome. The translational relevance of this finding is reflected by activin A overexpression in tumor cells and of mDia2 in the stroma of skin cancer and other malignancies and the correlation of high activin A/mDia2 levels with poor patient survival. Blockade of this signaling axis using inhibitors of activin, activin receptors, or mDia2 suppressed cancer cell malignancy and squamous carcinogenesis in 3D organotypic cultures, ex vivo, and in vivo, providing a rationale for pharmacological inhibition of activin A-mDia2 signaling in stratified cancer patients.

Nrf2-Mediated Fibroblast Reprogramming Drives Cellular Senescence by Targeting the Matrisome.

Nrf2 is a key regulator of the antioxidant defense system, and pharmacological Nrf2 activation is a promising strategy for cancer prevention and promotion of tissue repair. Here we show, however, that activation of Nrf2 in fibroblasts induces cellular senescence. Using a combination of transcriptomics, matrix proteomics, chromatin immunoprecipitation and bioinformatics we demonstrate that fibroblasts with activated Nrf2 deposit a senescence-promoting matrix, with plasminogen activator inhibitor-1 being a key inducer of the senescence program. In vivo, activation of Nrf2 in fibroblasts promoted re-epithelialization of skin wounds, but also skin tumorigenesis. The pro-tumorigenic activity is of general relevance, since Nrf2 activation in skin fibroblasts induced the expression of genes characteristic for cancer-associated fibroblasts from different mouse and human tumors. Therefore, activated Nrf2 qualifies as a marker of the cancer-associated fibroblast phenotype. These data highlight the bright and the dark sides of Nrf2 and the need for time-controlled activation of this transcription factor.

Activin promotes skin carcinogenesis by attraction and reprogramming of macrophages.

Activin has emerged as an important player in different types of cancer, but the underlying mechanisms are largely unknown. We show here that activin overexpression is an early event in murine and human skin tumorigenesis. This is functionally important, since activin promoted skin tumorigenesis in mice induced by the human papillomavirus 8 oncogenes. This was accompanied by depletion of epidermal γδ T cells and accumulation of regulatory T cells. Most importantly, activin increased the number of skin macrophages via attraction of blood monocytes, which was prevented by depletion of CCR2-positive monocytes. Gene expression profiling of macrophages from pre-tumorigenic skin and bioinformatics analysis demonstrated that activin induces a gene expression pattern in skin macrophages that resembles the phenotype of tumor-associated macrophages in different malignancies, thereby promoting angiogenesis, cell migration and proteolysis. The functional relevance of this finding was demonstrated by antibody-mediated depletion of macrophages, which strongly suppressed activin-induced skin tumor formation. These results demonstrate that activin induces skin carcinogenesis via attraction and reprogramming of macrophages and identify novel activin targets involved in tumor formation.

Two Ancient Gene Families Are Critical for Maintenance of the Mammalian Skin Barrier in Postnatal Life.

The skin barrier is critical for mammalian survival in the terrestrial environment, affording protection against fluid loss, microbes, toxins, and UV exposure. Many genes indispensable for barrier formation in the embryo have been identified, but loss of these genes in adult mice does not induce barrier regression. We describe a complex regulatory network centered on two ancient gene families, the grainyhead-like (Grhl) transcription factors and the protein cross-linking enzymes (tissue transglutaminases [Tgms]), which are essential for skin permeability barrier maintenance in adult mice. Embryonic deletion of Grhl3 induces loss of Tgm1 expression, which disrupts the cornified envelope, thus preventing permeability barrier formation leading to neonatal death. However, gene deletion of Grhl3 in adult mice does not disrupt the preformed barrier, with cornified envelope integrity maintained by Grhl1 and Tgm5, which are up-regulated in response to postnatal loss of Grhl3. Concomitant deletion of both Grhl factors in adult mice induced loss of Tgm1 and Tgm5 expression, perturbation of the cornified envelope, and complete permeability barrier regression that was incompatible with life. These findings define the molecular safeguards for barrier function that accompany the transition from intrauterine to terrestrial life.

Identification of a Novel Proto-oncogenic Network in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: The developmental transcription factor Grainyhead-like 3 (GRHL3) plays a critical tumor suppressor role in the mammalian epidermis through direct regulation of PTEN and the PI3K/AKT/mTOR signaling pathway. GRHL3 is highly expressed in all tissues derived from the surface ectoderm, including the oral cavity, raising a question about its potential role in suppression of head and neck squamous cell carcinoma (HNSCC). METHODS: We explored the tumor suppressor role of Grhl3 in HNSCC using a conditional knockout (Grhl3 (∆/-) /K14Cre (+) ) mouse line (n = 26) exposed to an oral chemical carcinogen. We defined the proto-oncogenic pathway activated in the HNSCC derived from these mice and assessed it in primary human HNSCC samples, normal oral epithelial cell lines carrying shRNA to GRHL3, and human HNSCC cell lines. Data were analyzed with two-sided chi square and Student's t tests. RESULTS: Deletion of Grhl3 in oral epithelium in mice did not perturb PTEN/PI3K/AKT/mTOR signaling, but instead evoked loss of GSK3B expression, resulting in stabilization and accumulation of c-MYC and aggressive HNSCC. This molecular signature was also evident in a subset of primary human HNSCC and HNSCC cell lines. Loss of Gsk3b in mice, independent of Grhl3, predisposed to chemical-induced HNSCC. Restoration of GSK3B expression blocked proliferation of normal oral epithelial cell lines carrying shRNA to GRHL3 (cell no., Day 8: Scramble ctl, 616±21.8 x 10(3) vs GRHL3-kd, 1194±44 X 10(3), P < .001; GRHL3-kd vs GRHL3-kd + GSK3B, 800±98.84 X 10(3), P = .003) and human HNSCC cells. CONCLUSIONS: We defined a novel molecular signature in mammalian HNSCC, suggesting new treatment strategies targeting the GRHL3/GSK3B/c-MYC proto-oncogenic network.

Stem cells behind the barrier.

Epidermal stem cells sustain the adult skin for a lifetime through self-renewal and the production of committed progenitors. These stem cells generate progeny that will undergo terminal differentiation leading to the development of a protective epidermal barrier. Whereas the molecular mechanisms that govern epidermal barrier repair and renewal have been extensively studied, pathways controlling stem cell differentiation remain poorly understood. Asymmetric cell divisions, small non-coding RNAs (microRNAs), chromatin remodeling complexes, and multiple differentiation factors tightly control the balance of stem and progenitor cell proliferation and differentiation, and disruption of this balance leads to skin diseases. In this review, we summarize and discuss current advances in our understanding of the mechanisms regulating epidermal stem and progenitor cell differentiation, and explore new relationships for maintenance of skin barrier function.

The unique and cooperative roles of the Grainy head-like transcription factors in epidermal development reflect unexpected target gene specificity.

The Grainy head-like 3 (Grhl3) gene encodes a transcription factor that plays essential roles in epidermal morphogenesis during embryonic development, with deficient mice exhibiting failed skin barrier formation, defective wound repair, and loss of eyelid fusion. Despite sharing significant sequence homology, overlapping expression patterns, and an identical core consensus DNA binding site, the other members of the Grhl family (Grhl1 and -2) fail to compensate for the loss of Grhl3 in these processes. Here, we have employed diverse genetic models, coupled with biochemical studies, to define the inter-relationships of the Grhl factors in epidermal development. We show that Grhl1 and Grhl3 have evolved complete functional independence, as evidenced by a lack of genetic interactions in embryos carrying combinations of targeted alleles of these genes. In contrast, compound heterozygous Grhl2/Grhl3 embryos displayed failed wound repair, and loss of a single Grhl2 allele in Grhl3-null embryos results in fully penetrant eyes open at birth. Expression of Grhl2 from the Grhl3 locus in homozygous knock-in mice corrects the wound repair defect, but these embryos still display a complete failure of skin barrier formation. This functional dissociation is due to unexpected differences in target gene specificity, as both GRHL2 and GRHL3 bind to and regulate expression of the wound repair gene Rho GEF 19, but regulation of the barrier forming gene, Transglutaminase 1 (TGase1), is unique to GRHL3. Our findings define the mechanisms underpinning the unique and cooperative roles of the Grhl genes in epidermal development.