Ellen A. Rorke

Epigallocatechin-3-gallate Inhibits Epidermal Growth Factor Receptor Signaling Pathway EVIDENCE FOR DIRECT INHIBITION OF ERK1/2 AND AKT KINASES

Epidermal growth factor receptor (EGFR) activation is absolutely required for cervical cell proliferation. This suggests that EGFR-inhibitory agents may be of therapeutic value. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a bioactive green tea polyphenol, on EGFR signaling in cervical cells. EGCG inhibits epidermal growth factor-dependent activation of EGFR, and EGFR-dependent activation of the mitogen-activated protein kinases ERK1/2. EGCG also inhibits EGFR-dependent AKT activity. The EGCG-dependent reduction in ERK and AKT activity is associated with reduced phosphorylation of downstream substrates, including p90RSK, FKHR, and BAD. These changes are associated with increased p53, p21WAF-1, and p27KIP-1 levels, reduced cyclin E level, and reduced CDK2 kinase activity. Consistent with these findings, flow cytometry and TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling) staining revealed EGCG-dependent G1 arrest. Moreover, sustained EGCG treatment caused apoptotic cell death. In addition to inhibiting EGFR, cell-free studies demonstrated that EGCG directly inhibits ERK1/2 and AKT, suggesting that EGCG acts simultaneously at multiple levels to inhibit EGF-dependent signaling. Importantly, the EGCG inhibition is selective, as EGCG does not effect the EGFR-dependent activation of JNK. These results suggest that EGCG acts to selectively inhibit multiple EGF-dependent kinases to inhibit cell proliferation.

Regulation of the Matriptase-Prostasin Cell Surface Proteolytic Cascade by Hepatocyte Growth Factor Activator Inhibitor-1 during Epidermal Differentiation

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.

Polycomb Group Proteins Are Key Regulators of Keratinocyte Function

The Polycomb group (PcG) proteins are epigenetic suppressors of gene expression that function through modification of histones to change chromatin structure and modulate gene expression and cell behavior. Recent studies show that PcG proteins are expressed in epidermis, that their levels change during differentiation and in disease states, and that PcG expression is regulated by agents that influence cell proliferation and survival. The results indicate that PcG proteins regulate keratinocyte cell-cycle progression, apoptosis, senescence, and differentiation. These proteins are expressed in progenitor cells, in the basal layer, and in suprabasal keratinocytes, and the level, timing, and distribution of expression suggest that the PcG proteins have a central role in maintaining the balance between cell survival and death in multiple epidermal compartments. Additional studies indicate an important role in skin cancer progression.

AP1 transcription factors in epidermal differentiation and skin cancer.

AP1 (jun/fos) transcription factors (c-jun, junB, junD, c-fos, FosB, Fra-1, and Fra-2) are key regulators of epidermal keratinocyte survival and differentiation and important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each protein is expressed, at different levels, in multiple cells layers in differentiating epidermis, and because AP1 transcription factors regulate competing processes (i.e., proliferation, apoptosis, and differentiation). Various in vivo genetic approaches have been used to study these proteins including targeted and conditional knockdown, overexpression, and expression of dominant-negative inactivating AP1 transcription factors in epidermis. Taken together, these studies suggest that individual AP1 transcription factors have different functions in the epidermis and in cancer development and that altering AP1 transcription factor function in the basal versus suprabasal layers differentially influences the epidermal differentiation response and disease and cancer development.

The Bmi-1 polycomb group gene in skin cancer: regulation of function by (−)–epigallocatechin-3-gallate

The PcG genes encode a family of evolutionarily conserved regulators that were discovered in Drosophila as repressors of Homoeotic gene expression. Homeotic genes are required to establish body segmentation patterns during development. In mammalian systems, PcG proteins regulate genes involved in development and differentiation, via epigenetic (e.g., chromatin modification) mechanisms. They are intimately involved as silencers of gene expression during cell lineage determination, and they play an important role in promoting cell survival. Proteins encoded by PcG genes comprise two protein complexes that act coordinately to regulate gene expression – the Bmi-1 complex and the Eed complex. The Bmi-1 complex includes Bmi-1, Mel-18, Mph1/Rae28, M33, Scmh1, Ring1A, and Ring1B, while the Eed complex includes Eed, EzH1, and EzH2. The first step in gene silencing is mediated by the Eed complex. Eed-containing complexes modify chromatin by recruiting histone deacetylase, which leads to local chromatin deacetylation. The Eed complex also catalyzes the methylation of Lys27 of histone H3. In the second step, the Bmi-1 complex binds to the methylated Lys27 of histone H3 and then catalyzes the ubiquitinylation of histone H2A. This cooperation between the Eed and Bmi-1 complexes leads to silencing of gene expression. The Bmi-1 complex appears to remain anchored to the chromatin after these events are completed. The Bmi-1 PcG protein has a particularly important role in gene silencing due to its ability to enhance the rate and extent of histone ubiquitinylation. This is accomplished through activation of Ring 1B, a ubiquitin ligase (and PcG protein) present in the Bmi-1 complex.1,2 Bmi-1 also functions in normal adult tissues. For example, aging Bmi-1−/− mice progressively lose stem cells in the leukemic, neuronal, and cerebellar granule cell lineages3–5 and Bmi-1-deficient fibroblasts display enhanced senescence and slow proliferation rates.6 In contrast, Bmi-1 …

Identification of a Population of Epidermal Squamous Cell Carcinoma Cells with Enhanced Potential for Tumor Formation

Epidermal squamous cell carcinoma is among the most common cancers in humans. 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. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation.

AP1 Factor Inactivation in the Suprabasal Epidermis Causes Increased Epidermal Hyperproliferation and Hyperkeratosis but Reduced Carcinogen-Dependent Tumor Formation

Activator protein one (AP1) (jun/fos) factors comprise a family of transcriptional regulators (c-jun, junB, junD, c-fos, FosB, Fra-1 and Fra-2) that are key controllers of epidermal keratinocyte survival and differentiation, and are important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each member is expressed in defined cell layers during epidermal differentiation, and because AP1 factors regulate competing processes (that is, proliferation, apoptosis and differentiation). We have proposed that AP1 factors function differently in basal versus suprabasal epidermis. To test this, we inactivated suprabasal AP1 factor function in mouse epidermis by targeted expression of dominant-negative c-jun (TAM67), which inactivates function of all AP1 factors. This produces increased basal keratinocyte proliferation, delayed differentiation and extensive hyperkeratosis. These findings contrast with previous studies showing that basal layer AP1 factor inactivation does not perturb resting epidermis. It is interesting that in spite of extensive keratinocyte hyperproliferation, susceptibility to carcinogen-dependent tumor induction is markedly attenuated. These novel observations strongly suggest that AP1 factors have distinct roles in the basal versus suprabasal epidermis, confirm that AP1 factor function is required for normal terminal differentiation, and suggest that AP1 factors have a different role in normal epidermis versus cancer progression.

Biochemistry of epidermal stem cells.

BACKGROUND The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue. SCOPE OF REVIEW A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer. MAJOR CONCLUSIONS An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis. GENERAL SIGNIFICANCE Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Suppression of AP1 Transcription Factor Function in Keratinocyte Suppresses Differentiation

Our previous study shows that inhibiting activator protein one (AP1) transcription factor function in murine epidermis, using dominant-negative c-jun (TAM67), increases cell proliferation and delays differentiation. To understand the mechanism of action, we compare TAM67 impact in mouse epidermis and in cultured normal human keratinocytes. We show that TAM67 localizes in the nucleus where it forms TAM67 homodimers that competitively interact with AP1 transcription factor DNA binding sites to reduce endogenous jun and fos factor binding. Involucrin is a marker of keratinocyte differentiation that is expressed in the suprabasal epidermis and this expression requires AP1 factor interaction at the AP1-5 site in the promoter. TAM67 interacts competitively at this site to reduce involucrin expression. TAM67 also reduces endogenous c-jun, junB and junD mRNA and protein level. Studies with c-jun promoter suggest that this is due to reduced transcription of the c-jun gene. We propose that TAM67 suppresses keratinocyte differentiation by interfering with endogenous AP1 factor binding to regulator elements in differentiation-associated target genes, and by reducing endogenous c-jun factor expression.

Differentiation-independent constitutive expression of the human papillomavirus type 16 E6 and E7 oncogenes in the CaSki cervical tumour cell line.

CaSki cells are a human papillomavirus type 16 (HPV-16)-positive cell line that serves as a model for the study of advanced cervical carcinoma. Calcium is an important regulator of normal ectocervical epithelial cell differentiation. HPV E6 and E7 gene products are thought to be important in the process of cervical cell immortalization and hence important in the development of cervical cancer. In the present study we examine the relationship between CaSki cell differentiation and expression of the papillomavirus oncogenes. Shifting CaSki cells from medium containing low (0.06 mM) to high (1.4 mM) calcium results in an increase in cell-cell contact and increased differentiation as measured by an increase in the level of mRNA encoding cytokeratin K13, involucrin and type 1 transglutaminase, which are markers of differentiation in the cervical epithelium. In contrast, E6/E7 transcripts are produced in a differentiation-independent constitutive manner. These results and those from our previous experiments with HPV-16-immortalized but non-tumorigenic cell lines suggest that the constitutive, differentiation-independent expression of E6/E7 levels is a property of both tumorigenic and non-tumorigenic HPV-16-positive cancer cells.