• Cancer Stem Cell-Induced Vascularization of Skin Cancer Tumors is Driven by Novel Signaling Mechanism Specific to VEGF-A/NRP-1 Interaction

      Grun, Daniel; Eckert, Richard (Richard L.) (2017)
      Epidermal squamous cell carcinoma is among the most common cancers. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation, in order to therapeutically target and eliminate this population of cells. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including ALDH1, keratin 15, CD200, and keratin 19. This limited subpopulation of epidermal cancer stem cells (ECS cells), in squamous cell carcinoma, form rapidly growing, invasive and highly vascularized tumors, as compared with non stem cancer cells. We show that ECS cell-produced vascular endothelial growth factor (VEGF)-A is required for the maintenance of this phenotype, as knockdown of VEGF-A gene expression or treatment with VEGF-A inactivating antibody reduces these responses. Surprisingly, the classical mechanism of VEGF-A action via interaction with VEGF receptors does not mediate these events, as these cells lack VEGFR1 and VEGFR2. Instead, VEGF-A acts via the Neuropilin-1 (NRP-1) co-receptor to trigger intracellular events leading to ECS cell survival and formation of aggressive, invasive and highly vascularized tumors. We further identify a novel signaling cascade downstream of this VEGF-A/NRP-1 interaction that shows NRP-1 forms a complex with GIPC1 and α6/4-integrin to activate FAK/Src signaling which leads to stabilization of a YAP1/∆Np63α to enhance ECS cell survival, invasion and angiogenesis.
    • A Possible Role for AP-1 Transcription Factors in Development of Epidermal Ichthyoses

      Young, Christina; Eckert, Richard (Richard L.) (2017)
      Ichthyoses are highly debilitating and painful disorders characterized by thickening of the skin with marked hyperkeratosis and hyperplasia. Some ichthyosis-related skin disorders are caused by genetic mutation while other forms are acquired. AP-1 transcription factors are known regulators proliferation and differentiation in the epidermis and their activity is necessary in achieving homeostasis of the epidermal barrier. We have previously described a mouse model where inactivation of AP-1 transcription factor function in the suprabasal epidermis, via targeted expression of a dominant negative c-jun (TAM67), produces an ichthyosis-related phenotype. The observed phenotype includes keratinocyte hyperproliferation, delayed differentiation, hyperkeratosis, parakeratosis, extensive vasodilation/erythroderma, tail and digit pseudoainhum, reduced barrier integrity, reduced filaggrin level, and nuclear accumulation of loricrin. To further characterize this mouse model, we sought out to follow phenotype development during embryogenesis. TAM67 expression was turned on during embryogenesis at embryonic day (E) E13.5, E15.5, and E17.5. The E13.5 and E15.5 treated embryos had severely decreased filaggrin levels, nuclear loricrin, and were born with a collodion baby-like phenotype as seen in many human ichthyosis patients. These data suggest a role for AP-1 transcription factors in epidermal development. Next, we focused on the downstream signaling effects of suprabasal AP-1 transcription factor inhibition in the adult mouse by assessing the role of specific chemokine mediators in phenotype development. We monitored impact on chemokine production and used knockout methods to study the role of the most highly induced chemokines in this process. Suprabasal AP-1 factor inactivation results in increased levels of Th1 (IFN?, CCL3, CCL5, CXCL9, CXCL10, and CXCL11) and Th2 (CCL1, CCL2, CCL5, and CCL11) chemokines in epidermis and serum. S100A8 and S100A9 levels are also elevated. Interestingly, we observe no attenuation of phenotype when TAM67 is expressed in the epidermis of CXCR3 (CXCL9, CXCL10, and CXCL11 receptor) or S100A8/A9 knockout mice indicating lack of a role of these regulators in phenotype development. We propose that loss of AP-1 transcription factor function leads to impaired barrier function and enhanced epidermal chemokine production. These findings suggest that reduced AP-1 transcription factor activity may play a role in the pathogenesis of ichthyosis related skin disorders.
    • Potential Role of Tazarotene-Induced Gene 3 and Transglutaminase 1 in Tauopathies

      Kizilyer, Yasin; Eckert, Richard (Richard L.) (2013)
      The family of neurodegenerative diseases, called tauopathies, includes Alzheimer's disease, progressive supranuclear palsy, and Pick's disease. These diseases are associated with the formation of tau inclusions and neuronal cell death. Tau inclusions are characterized by the presence of neurofibrillary tangles (NFTs), where protein Tau, a member of microtubule-associated proteins (MAPs), is found to be, hyperphosphorylated and aggregated. The formation of these aggregates leads to microtubule instability and cellular toxicity. There are several lines of evidence that demonstrate this aggregation is due to transglutaminase mediated cross-linking of Tau protein. However, little is known about the mechanism of the hyperphosphorylation, aggregation and tau-associated toxicity. Analysis of brains with tau pathologies showed the colocalized expression of transglutaminase 1 (TG1) and its activator, tazarotene-induced gene 3 (TIG3) in NFTs, suggesting functional relevancy. Hereby, we hypothesized that TIG3 regulates TG1-catalysed cross-linking of Tau, and therefore the formation of Tau inclusions in neurons. To test this hypothesis, neurons differentiated from human embryonic stem cells used to develop an in-vitro tauopathy model. Endogenous expression analyses of TIG3, TG1 and Tau were performed and induction of Tau occlusions via overexpression of TIG3 studied. Results have shown endogenous TIG3 mRNA and protein were expressed in neurons derived from embryonic stem cells. Endogenous TIG3, TG1 and TAU have shown colocalization. Cells overexpressing TIG3 have shown a higher level of colocalization and increased Tau aggregation, which confirmed our hypothesis. The results of this study provide new mechanistic insights into the formation of neurofibrillary tangles and the resulting pathology.
    • Role of p38δ and MEP50 in Epidermal Keratinocyte Homeostasis

      Saha, Kamalika; Eckert, Richard (Richard L.) (2015)
      Keratinocytes are the major cell type in the human epidermis, the outer layer of skin. These cells undergo a tightly regulated terminal differentiation program which results in the formation of the cornified envelope. This envelope is essentially the barrier which protects us from mechanical stress, extreme climate conditions and infectious agents. A fully functional epidermal barrier dictates a perfect balance between proliferation and differentiation. PKCδ, a novel PKC isoform and a key controller of epidermal differentiation regulates proliferation by increasing p21Cip1 expression. However, little is known about the mediators involved in this regulation. We hypothesize that p38δ MAPK, a downstream kinase is a mediator in this process. We observe that p38δ regulates p21Cip1 mRNA/protein levels in a p53 dependent manner. Additionally, PKCδ selectively activates p38δ and treatment with p38δ-siRNA or dominant negative p38 results in attenuation of the PKCδ induced p21Cip1 response. Furthermore, p53 is identified as a novel target of p38δ and is a key mediator in the p38δ -p21Cip1 signaling cascade. Moreover, the physiological relevance of this pathway is confirmed in the PKCδ and p38δ knockdown organotypic cultures which are thicker, have additional layers and reduced cornified envelope formation as compared to the controls. We have also identified the Protein Arginine Methyltransferase (PRMT5) and Methylosome Protein 50 (MEP50) as novel targets of PKCδ and p38δ. MEP50 enhances keratinocyte proliferation and opposes differentiation by mechanisms involving silencing p21Cip1 and involucrin. We found that symmetric demethylation of arginines in histones H3 and H4 plays a key role in this regulation. Additionally, the pro-proliferation role of MEP50 is physiologically relevant as MEP50 knockdown rafts are significantly thinner and have fewer number of Ki67 positive cells than the control rafts. These studies highlight p38δ as a common kinase regulating the dual processes of proliferation and differentiation and characterize the role of MEP50/PRMT5 as novel players in controlling epidermal homeostasis.
    • TIG3: A multifunctional regulator of cell proliferation and survival

      Scharadin, Tiffany M.; Eckert, Richard (Richard L.) (2012)
      The epidermis is a multi-layered organ which functions as a protective barrier from the environment. Keratinocytes form the epidermis in a tightly regulated process of terminal differentiation. Dysregulation of this process leads to hyperproliferative disease. TIG3 is a tumor suppressor in the H-rev107 family, which was identified as having elevated expression following treatment with the psoriasis drug, tazarotene. Levels of TIG3 are decreased in diseases associated with hyperproliferation like psoriasis and skin cancer. Restoration of TIG3 expression in these diseases results in a reduction of cell proliferation rate and normalization of the disease phenotype. In normal epidermis, TIG3 is expressed in the suprabasal epidermal layers and is associated with cessation of cell proliferation and activation of type I transglutaminase. Here we characterize the impact of restoring TIG3 expression in skin cancer cell lines and in normal keratinocytes. We observe that in skin cancer cells, TIG3 halts cell cycle progression at the G1/S phase and induces apoptosis. This is associated with the novel finding that TIG3 associates at the centrosome, which is also observed in normal keratinocytes. TIG3 distribution to the centrosome is essential for its function and it acts by altering the function of the centrosome. We observe a dramatic reorganization of the microtubule network and a reduction in centrosome separation and cell division. We have also identified the region of TIG3 (amino acids 102 - 125) responsible for TIG3 centrosome localization. This region contains the highly conserved NC and LRYG motifs and we show that mutation of these conserved elements prevent distribution to the centrosome. Tazarotene has been successfully used clinically to treat psoriasis and nonmelanoma skin cancers, indicating the potential role for TIG3 as a mediator of drug action. These studies support this concept by identifying the mechanisms of TIG3 action.
    • Transglutaminase as a Driver of Aggressive Squamous Cell Carcinoma

      Fisher, Matthew Lawrence; Eckert, Richard (Richard L.) (2017)
      Non-melanoma skin cancer is the most common cancer in human populations. Cutaneous squamous cell carcinoma (cSCC) is extremely frequent and is the most common cancer capable of metastasis. cSCC is primarily linked to exposure to ultraviolet (UV) radiation. Immunosuppressed patients are two-hundred times more likely to get metastatic SCC. Moreover, environmental irritants and increased exposure to UV irradiation with indoor tanning have led to increased skin cancer incidence. Thus, skin cancer is an important health concern. It is thought that a small fraction of tumor stem cells possess the ability to initiate and sustain tumor growth. These cells, referred to as cancer stem cells (CSC) possess traits of normal stem cells and are slow-cycling, capable of self-renewal through asymmetrical division, and are able to give rise to all the cell types in the tumor population. Bulk tumor cells represent the majority of the tumor mass, but in contrast to cancer stem cells, are dispensable for tumor propagation. Thus, therapeutic targeting of cancer stem cell survival mechanisms is an important cancer therapy strategy. Herein we provide evidence that transglutaminase type 2 (TG2) is a key controller of epidermal cancer stem cells (ECS cell) survival and an important therapeutic target. TG2 is a multifunctional member of the transglutaminase family of proteins. In the closed conformation, which exists in cells, TG2 functions as a GTP binding/G protein-related signaling protein. A rise in intracellular calcium concentration shifts TG2 to an open conformation that functions in cell and matrix remodeling. TG2 has been shown to be elevated in a number of metastatic cancers, but its role in disease progression and survival is not well characterized. We show that TG2 is constitutively expressed in ECS cells where it facilitates migration, invasion, spheroid formation and survival of ECS cells. Mechanistic studies, using TG2 mutants, revealed that the GTP-binding activity is required for maintenance of ECS cell growth and survival. Furthermore, signaling analysis showed that TG2 is involved in regulating several key pathways involved in stemness and metastasis, including EMT and Hippo signaling. These studies suggest TG2 is an important therapeutic target for invasive and metastatic cSCC.