Informing a Canadian Skin Science Trainee Program Based on the State of Trainee Programs Offered by International Academic Societies.

BACKGROUND: For dermatology to effectively address the ever-growing medical needs, longstanding communication barriers across investigators working in different research pillars and practicing clinicians must be improved. To address this problem, trainee-specific programs are now evolving to align their educational landscape across basic science, translational and clinical research programs. OBJECTIVES: To establish a Skin Investigation Network of Canada (SkIN Canada) training roadmap for the career and skill development of future clinicians, clinican scientists and basic scientists in Canada. This Working Group aims to strengthen and harmonize collaborations and capacity across the skin research community. METHODS: The Working Group conducted a search of established international academic societies which offered trainee programs with mandates similar to SkIN Canada. Societies' program items and meetings were evaluated by use of an interview survey and/or the collection of publicly available data. Program logistics, objectives and feedback were assessed for commonalities and factors reported or determined to improve trainee experience. RESULTS: Through the various factors explored, the Working Group discovered the need for increasing program accessibility, creating opportunities for soft skill development, emphasizing the importance of current challenges, collecting and responding to feedback, and improving knowledge sharing to bridge pillars of skin research. CONCLUSIONS: Although improvements have been made to trainee education in recent years, a plurality of approaches exist and many of the underlying roadblocks remain unresolved. To establish fundamental clinician-basic scientist collaboration and training efforts, this Working Group highlights important factors to include and consider in building a trainee program and emphasizes the importance of trainee education.

Top Ten Research Priorities for Psoriasis, Atopic Dermatitis and Hidradenitis Suppurativa: The SkIN Canada Priority Setting Initiative.

BACKGROUND: The Skin Investigation Network of Canada (SkIN Canada) is a new national skin research network. To shape the research landscape and ensure its value to patient care, research priorities that are important to patients, caregivers, and health care providers must be identified. OBJECTIVES: To identify the Top Ten research priorities for 9 key skin conditions. METHODS: We first surveyed health care providers and researchers to select the top skin conditions for future research within the categories of inflammatory skin disease, skin cancers (other than melanoma), and wound healing. For those selected skin conditions, we conducted scoping reviews to identify previous priority setting exercises. We combined the results of those scoping reviews with a survey of patients, health care providers, and researchers to generate lists of knowledge gaps for each condition. We then surveyed patients and health care providers to create preliminary rankings to prioritize those knowledge gaps. Finally, we conducted workshops of patients and health care providers to create the final Top Ten lists of research priorities for each condition. RESULTS: Overall, 538 patients, health care providers, and researchers participated in at least one survey or workshop. Psoriasis, atopic dermatitis and hidradenitis suppurativa (inflammatory skin disease); chronic wounds, burns and scars (wound healing); and basal cell, squamous cell and Merkel cell carcinoma (skin cancer) were selected as priority skin conditions. Top Ten lists of knowledge gaps for inflammatory skin conditions encompassed a range of issues relevant to patient care, including questions on pathogenesis, prevention, non-pharmacologic and pharmacologic management. CONCLUSIONS: Research priorities derived from patients and health care providers should be used to guide multidisciplinary research networks, funders, and policymakers in Canada and internationally.

Non-canonical (non-SMAD2/3) TGF-ß signaling in fibrosis: Mechanisms and targets.

Transforming growth factor (TGF)-ß uses several intracellular signaling pathways besides canonical ALK5-Smad2/3 signaling to regulate a diverse array of cellular functions. Several of these so-called non-canonical (non-Smad2/3) pathways have been implicated in the pathogenesis of fibrosis and may therefore represent targets for therapeutic intervention. This review summarizes our current knowledge on the mechanisms of non-canonical TGF-ß signaling in fibrosis, the potential molecular targets and the use of agonists/antagonists for therapeutic intervention.

The Deubiquitinating Enzyme Ubiquitin-Specific Peptidase 11 Potentiates TGF-ß Signaling in CD4+ T Cells to Facilitate Foxp3+ Regulatory T and TH17 Cell Differentiation.

Foxp3+ regulatory T (TREG) cells are central mediators in the control of peripheral immune responses. Genome-wide transcriptional profiles show canonical signatures for Foxp3+ TREG cells, distinguishing them from Foxp3- effector T (TEFF) cells. We previously uncovered distinct mRNA translational signatures differentiating CD4+ TEFF and TREG cells through parallel measurements of cytosolic (global) and polysome-associated (translationally enhanced) mRNA levels in both subsets. We show that the mRNA encoding for the ubiquitin-specific peptidase 11 (USP11), a known modulator of TGF-ß signaling, was preferentially translated in TCR-activated TREG cells compared with conventional, murine CD4+ T cells. TGF-ß is a key cytokine driving the induction and maintenance of Foxp3 expression in T cells. We hypothesized that differential translation of USP11 mRNA endows TREG cells with an advantage to respond to TGF-ß signals. In an in vivo mouse model promoting TREG cells plasticity, we found that USP11 protein was expressed at elevated levels in stable TREG cells, whereas ectopic USP11 expression enhanced the suppressive capacity and lineage commitment of these cells in vitro and in vivo. USP11 overexpression in TEFF cells enhanced the activation of the TGF-ß pathway and promoted TREG or TH17, but not Th1, cell differentiation in vitro and in vivo, an effect abrogated by USP11 gene silencing or the inhibition of enzymatic activity. Thus, USP11 potentiates TGF-ß signaling in both TREG and TEFF cells, in turn driving increased suppressive function and lineage commitment in thymic-derived TREG cells and potentiating the TGF-ß-dependent differentiation of TEFF cells to peripherally induced TREG and TH17 cells.

Activation of Smad2 but not Smad3 is required to mediate TGF-ß signaling during axolotl limb regeneration.

Axolotls are unique among vertebrates in their ability to regenerate tissues, such as limbs, tail and skin. The axolotl limb is the most studied regenerating structure. The process is well characterized morphologically; however, it is not well understood at the molecular level. We demonstrate that TGF-ß1 is highly upregulated during regeneration and that TGF-ß signaling is necessary for the regenerative process. We show that the basement membrane is not prematurely formed in animals treated with the TGF-ß antagonist SB-431542. More importantly, Smad2 and Smad3 are differentially regulated post-translationally during the preparation phase of limb regeneration. Using specific antagonists for Smad2 and Smad3 we demonstrate that Smad2 is responsible for the action of TGF-ß during regeneration, whereas Smad3 is not required. Smad2 target genes (Mmp2 and Mmp9) are inhibited in SB-431542-treated limbs, whereas non-canonical TGF-ß targets (e.g. Mmp13) are unaffected. This is the first study to show that Smad2 and Smad3 are differentially regulated during regeneration and places Smad2 at the heart of TGF-ß signaling supporting the regenerative process.

Soluble CD109 binds TGF-ß and antagonizes TGF-ß signalling and responses.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine implicated in many diseases, including tissue fibrosis and cancer. TGF-ß mediates diverse biological responses by signalling through type I and II TGF-ß receptors (TßRI and TßRII). We have previously identified CD109, a glycosylphosphatidylinositol (GPI)-anchored protein, as a novel TGF-ß co-receptor that negatively regulates TGF-ß signalling and responses and demonstrated that membrane-anchored CD109 promotes TGF-ß receptor degradation via a SMAD7/Smurf2-mediated mechanism. To determine whether CD109 released from the cell surface (soluble CD109 or sCD109) also acts as a TGF-ß antagonist, we determined the efficacy of recombinant sCD109 to interact with TGF-ß and inhibit TGF-ß signalling and responses. Our results demonstrate that sCD109 binds TGF-ß with high affinity as determined by surface plasmon resonance (SPR) and cell-based radioligand binding and affinity labelling competition assays. SPR detected slow dissociation kinetics between sCD109 and TGF-ß at low concentrations, indicating a stable and effective interaction. In addition, sCD109 antagonizes TGF-ß-induced Smad2/3 phosphorylation, transcription and cell migration. Together, our results suggest that sCD109 can bind TGF-ß, inhibit TGF-ß binding to its receptors and decrease TGF-ß signalling and TGF-ß-induced cellular responses.

CD109, a novel TGF-ß antagonist, decreases fibrotic responses in a hypoxic wound model.

Excessive extracellular matrix deposition that occurs in many fibrotic skin disorders such as hypertrophic scarring and scleroderma is often associated with hypoxia. CD109 is a novel TGF-ß co-receptor and TGF-ß antagonist shown to inhibit TGF-ß-induced extracellular matrix protein production in vitro. We examined whether CD109 is able to regulate extracellular matrix deposition under low oxygen tension in vivo using transgenic mice overexpressing CD109 in the epidermis. By creating dorsal bipedicle skin flaps with centrally located excisional wounds in these mice and their wild-type littermates, we generated a novel murine hypoxic wound model. Mice were sacrificed on 7 or 14 days post-wounding, and tissues were harvested for histological and biochemical analysis. Hypoxic wounds in both transgenic and wild-type mice showed increased levels of HIF-1α and delayed wound closure, validating this model in mice. Hypoxic wounds in CD109 transgenic mice demonstrated decreased collagen type 1 and fibronectin expression, and reduced dermal thickness on day 7 post-wounding as compared to those in wild-type mice and to non-hypoxic control wounds. These results suggest that CD109 decreases extracellular matrix production and fibrotic responses during hypoxic wound healing. Manipulating CD109 levels may have potential therapeutic value for the treatment of fibrotic skin disorders associated with poor oxygen delivery.

CD109 overexpression ameliorates skin fibrosis in a mouse model of bleomycin-induced scleroderma.

OBJECTIVE: Transforming growth factor ß (TGFß) is a profibrotic cytokine, and its aberrant function is implicated in several types of fibrotic pathologies including scleroderma (systemic sclerosis [SSc]). Multiple lines of evidence show that increased TGFß signaling contributes to progressive fibrosis in SSc by promoting fibroblast activation, excessive extracellular matrix (ECM) deposition, and dermal thickening. We have previously identified CD109 as a TGFß coreceptor and have shown that it antagonizes TGFß signaling and TGFß-induced ECM expression in vitro in human keratinocytes and fibroblasts. The aim of the present study was to examine the ability of CD109 to prevent skin fibrosis in a mouse model of bleomycin-induced SSc. METHODS: Transgenic mice overexpressing CD109 in the epidermis and their wild-type (WT) littermates were injected with bleomycin in phosphate buffered saline (PBS) or with PBS alone every other day for 21 days or 28 days. Dermal thickness and collagen deposition were determined histologically using Masson's trichrome and picrosirius red staining. In addition, collagen and fibronectin content was analyzed using Western blotting, and activation of TGFß signaling was examined by determining phospho-Smad2 and phospho-Smad3 levels using Western blotting and immunohistochemistry. RESULTS: Transgenic mice overexpressing CD109 in the epidermis showed resistance to bleomycin-induced skin fibrosis, as evidenced by a significant decrease in dermal thickness, collagen crosslinking, collagen and fibronectin content, and phospho-Smad2/3 levels, as compared to their WT littermates. CONCLUSION: Our findings suggest that CD109 inhibits TGFß signaling and fibrotic responses in experimental murine scleroderma. They also suggest that CD109 regulates dermal-epidermal interactions to decrease extracellular matrix synthesis in the dermis. Thus, CD109 is a potential molecular target for therapeutic intervention in scleroderma.

Transgenic mice overexpressing CD109 in the epidermis display decreased inflammation and granulation tissue and improved collagen architecture during wound healing.

Transforming growth factor-ß (TGF-ß) is a multifunctional growth factor involved in all aspects of wound healing. TGF-ß accelerates wound healing, but an excess of its presence at the wound site has been implicated in pathological scar formation. Our group has recently identified CD109, a glycophosphatidylinositol-anchored protein, as a novel TGF-ß coreceptor and inhibitor of TGF-ß signaling in vitro. To determine the effects of CD109 in vivo on wound healing, we generated transgenic mice overexpressing CD109 in the epidermis. In excisional wounds, we show that CD109 transgenic mice display markedly reduced macrophage and neutrophil recruitment, granulation tissue area, and decreased Smad2 and Smad3 phosphorylation, whereas wound closure remains unaffected as compared with wild-type littermates. Futhermore, we demonstrate that the expression of the proinflammatory cytokines interleukin-1α and monocyte chemoattractant protein-1, and extracellular matrix components is markedly decreased during wound healing in CD109 transgenic mice. In incisional wounds, CD109 transgenic mice show improved dermal architecture, whereas the tensile strength of the wound remains unchanged. Taken together, our findings demonstrate that CD109 overexpression in the epidermis reduces inflammation and granulation tissue area and improves collagen organization in vivo.

The TGF-ß co-receptor, CD109, promotes internalization and degradation of TGF-ß receptors.

Transforming growth factor-ß (TGF-ß) is implicated in numerous pathological disorders, including cancer and mediates a broad range of biological responses by signaling through the type I and II TGF-ß receptors. Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling, whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules that modulate receptor endocytosis are likely to play a critical role in regulating TGF-ß action. We previously identified CD109, a GPI-anchored protein, as a TGF-ß co-receptor and a negative regulator of TGF-ß signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-ß to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-ß receptors into the caveolar compartment in the presence of ligand and facilitates TGF-ß-receptor degradation. Thus, CD109 regulates TGF-ß receptor endocytosis and degradation to inhibit TGF-ß signaling. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

CD109-mediated degradation of TGF-ß receptors and inhibition of TGF-ß responses involve regulation of SMAD7 and Smurf2 localization and function.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-ß signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-ß signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-ß co-receptors can modulate TGF-ß signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-ß co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-ß signaling. Despite CD109's potential relevance as a regulator of TGF-ß action in vivo, the mechanisms by which CD109 regulates TGF-ß signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-ß signaling by promoting TGF-ß receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-ß receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-ß signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.

CD109 Is a Critical Determinant of EGFR Expression and Signaling, and Tumorigenicity in Squamous Cell Carcinoma Cells.

(1) Background: Squamous cell carcinoma (SCC) is one of the leading causes of cancer-related deaths worldwide. CD109 is overexpressed in many cancers including SCC. Although a pro-tumorigenic role for CD109 has been shown in non-SCC cancers, and in one type of SCC, the mechanisms and signaling pathways reported are discrepant. (2) Methods: The CD109-EGFR interaction and CD109-mediated regulation of EGFR expression, signaling, and stemness were studied using microarray, immunoblot, immunoprecipitation, qPCR, immunofluorescence, and/or spheroid formation assays. The role of CD109 in tumor progression and metastasis was studied using xenograft tumor growth and metastatic models. (3) Results: We establish the in vivo tumorigenicity of CD109 in vulvar SCC cells and demonstrate that CD109 is an essential regulator of EGFR expression at the mRNA and protein levels and of EGFR/AKT signaling in vulvar and hypopharyngeal SCC cells. Furthermore, we show that the mechanism involves EGFR-CD109 heteromerization and colocalization, leading to the stabilization of EGFR levels. Additionally, we demonstrate that the maintenance of epithelial morphology and in vitro tumorigenicity of SCC cells require CD109 localization to the cell surface. (4) Conclusions: Our study identifies an essential role for CD109 in vulvar SCC progression. We demonstrate that CD109 regulates SCC cellular stemness and epithelial morphology via a cell-surface CD109-EGFR interaction, stabilization of EGFR levels and EGFR/AKT signaling.

CD109 release from the cell surface in human keratinocytes regulates TGF-ß receptor expression, TGF-ß signalling and STAT3 activation: relevance to psoriasis.

Transforming growth factor (TGF)-ß is an important cytokine that negatively regulates keratinocyte proliferation. Deregulation of TGF-ß signalling has been reported in psoriasis, where despite increased expression of TGF-ß, psoriatic keratinocytes continue to hyperproliferate. Recently, we have identified CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein, as a novel co-receptor and negative regulator of TGF-ß signalling. In the current work, we demonstrate that release of CD109 from the cell surface or the addition of CD109 protein results in downregulation of TGF-ß signalling and TGF-ß receptor expression in human keratinocytes. Moreover, these effects are associated with an increase in phospho-STAT3 levels, enhanced total STAT3 and Bcl-2 expression and an increase in cell growth and survival, suggesting that released/soluble CD109 is able to induce molecular changes that are known to occur in psoriasis. Analysis of CD109 expression in psoriasis patients reveals that CD109 protein expression is markedly decreased in psoriatic epidermis as compared to adjacent uninvolved skin. In contrast, CD109 mRNA expression is unchanged in psoriatic plaques in comparison with normal skin. This raises a possibility that CD109 protein release is enhanced in psoriatic keratinocytes. Furthermore, psoriatic epidermis displays decreased expression of TGF-ß receptors, consistent with the results obtained in vitro in keratinocytes with CD109 release or addition of CD109 recombinant protein. Together our findings suggest that aberrant CD109 release from the cell surface in human keratinocytes may induce molecular changes that are usually observed in psoriasis and may explain TGF-ß receptor downregulation and decrease in TGF-ß signalling in psoriasis.

Cholesterol-Induced Metabolic Reprogramming in Breast Cancer Cells Is Mediated via the ERRα Pathway.

The molecular mechanism underlying the metabolic reprogramming associated with obesity and high blood cholesterol levels is poorly understood. We previously reported that cholesterol is an endogenous ligand of the estrogen-related receptor alpha (ERRα). Using functional assays, metabolomics, and genomics, here we show that exogenous cholesterol alters the metabolic pathways in estrogen receptor-positive (ER+) and triple-negative breast cancer (TNBC) cells, and that this involves increased oxidative phosphorylation (OXPHOS) and TCA cycle intermediate levels. In addition, cholesterol augments aerobic glycolysis in TNBC cells although it remains unaltered in ER+ cells. Interestingly, cholesterol does not alter the metabolite levels of glutaminolysis, one-carbon metabolism, or the pentose phosphate pathway, but increases the NADPH levels and cellular proliferation, in both cell types. Importantly, we show that the above cholesterol-induced modulations of the metabolic pathways in breast cancer cells are mediated via ERRα. Furthermore, analysis of the ERRα metabolic gene signature of basal-like breast tumours of overweight/obese versus lean patients, using the GEO database, shows that obesity may modulate ERRα gene signature in a manner consistent with our in vitro findings with exogenous cholesterol. Given the close link between high cholesterol levels and obesity, our findings provide a mechanistic explanation for the association between cholesterol/obesity and metabolic reprogramming in breast cancer patients.

BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs.

BACKGROUND: Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas) in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration. RESULTS: The expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration) but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor) in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division. CONCLUSION: The expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has described in other tetrapods. Overexpression of BMP-2 did not cause the formation of extra digits, which is consistent with the hypothesis that it is not the secondary signal of sonic hedgehog. However, it did cause the formation of hypomorphic limbs as a result of increased cellular condensation and apoptosis. Taken together, these results suggest that BMP-2 does not have a direct role in patterning regenerating limbs but may be important to trigger condensation prior to ossification and to mediate apoptosis.

Cholesterol as an Endogenous Ligand of ERRα Promotes ERRα-Mediated Cellular Proliferation and Metabolic Target Gene Expression in Breast Cancer Cells.

Breast cancer is the 2nd leading cause of cancer-related death among women. Increased risk of breast cancer has been associated with high dietary cholesterol intake. However, the underlying mechanisms are not known. The nuclear receptor, estrogen-related receptor alpha (ERRα), plays an important role in breast cancer cell metabolism, and its overexpression has been linked to poor survival. Here we identified cholesterol as an endogenous ligand of ERRα by purification from human pregnancy serum using a GST-ERRα affinity column and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We show that cholesterol interacts with ERRα and induces its transcriptional activity in estrogen receptor positive (ER+) and triple negative breast cancer (TNBC) cells. In addition, we show that cholesterol enhances ERRα-PGC-1α interaction, induces ERRα expression itself, augments several metabolic target genes of ERRα, and increases cell proliferation and migration in both ER+ and TNBC cells. Furthermore, the stimulatory effect of cholesterol on metabolic gene expression, cell proliferation, and migration requires the ERRα pathway. These findings provide a mechanistic explanation for the increased breast cancer risk associated with high dietary cholesterol and possibly the pro-survival effect of statins in breast cancer patients, highlighting the clinical relevance of lowering cholesterol levels in breast cancer patients overexpressing ERRα.

CD109 acts as a gatekeeper of the epithelial trait by suppressing epithelial to mesenchymal transition in squamous cell carcinoma cells in vitro.

There is increasing evidence that the expression of CD109, a GPI-anchored cell surface protein is dysregulated in squamous cell carcinoma (SCC). However, the functional role of CD109 in SCC progression is poorly understood. In current study, we demonstrate that CD109 is a critical regulator of epithelial phenotype in SSC cells. CD109 levels inversely correlate with TGF-ß signaling, EMT, migration, and invasion in cultured SCC cells. CRISPR/Cas9-mediated knockout CD109 (CD109 KO) in SCC cells represses epithelial traits and promotes the mesenchymal phenotype, as evidenced by elevated expression of mesenchymal proteins and markers of epithelial to mesenchymal transition. Treatment with recombinant CD109 protein causes CD109 KO cells to regain their epithelial traits. CD109 loss results in pronounced alterations of gene expression as detected by microarray analysis and in dysregulation of 15 important signalling pathways as shown by KEGG pathway cluster analysis. Validation using 52 human oral SCC tumor samples show that CD109 levels inversely correlate with tumor grade and the activation state of one such pathway, the TGF-ß signaling pathway. Taken together, our findings highlight a novel role for CD109 as a gatekeeper of the epithelial phenotype by regulating TGF-ß pathway in SCC cells.