Evaluating the efficacy of enzalutamide and the development of resistance in a preclinical mouse model of type-I endometrial carcinoma.

Androgen Receptor (AR) signaling is a critical driver of hormone-dependent prostate cancer and has also been proposed to have biological activity in female hormone-dependent cancers, including type I endometrial carcinoma (EMC). In this study, we evaluated the preclinical efficacy of a third-generation AR antagonist, enzalutamide, in a genetic mouse model of EMC, Sprr2f-Cre;Ptenfl/fl. In this model, ablation of Pten in the uterine epithelium leads to localized and distant malignant disease as observed in human EMC. We hypothesized that administering enzalutamide through the diet would temporarily decrease the incidence of invasive and metastatic carcinoma, while prolonged administration would result in development of resistance and loss of efficacy. Short-term treatment with enzalutamide reduced overall tumor burden through increased apoptosis but failed to prevent progression of invasive and metastatic disease. These results suggest that AR signaling may have biphasic, oncogenic and tumor suppressive roles in EMC that are dependent on disease stage. Enzalutamide treatment increased Progesterone Receptor (PR) expression within both stromal and tumor cell compartments. Prolonged administration of enzalutamide decreased apoptosis, increased tumor burden and resulted in the clonal expansion of tumor cells expressing high levels of p53 protein, suggestive of acquired Trp53 mutations. In conclusion, we show that enzalutamide induces apoptosis in EMC but has limited efficacy overall as a single agent. Induction of PR, a negative regulator of endometrial proliferation, suggests that adding progestin therapy to enzalutamide administration may further decrease tumor burden and result in a prolonged response.

RNA sequencing and pathway analysis identify tumor necrosis factor alpha driven small proline-rich protein dysregulation in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory disorder in which many pathways contribute to end-organ disease. Small proline-rich proteins (SPRR) are polypeptides that have recently been shown to contribute to epithelial biomechanical properties relevant in T-helper type 2 inflammation. There is evidence that genetic polymorphism in SPRR genes may predict the development of asthma in children with atopy and, correlatively, that expression of SPRRs is increased under allergic conditions, which leads to epithelial barrier dysfunction in atopic disease. METHODS: RNAs from uncinate tissue specimens from patients with CRS and control subjects were compared by RNA sequencing by using Ingenuity Pathway Analysis (n = 4 each), and quantitative polymerase chain reaction (PCR) (n = 15). A separate cohort of archived sinus tissue was examined by immunohistochemistry (n = 19). RESULTS: A statistically significant increase of SPRR expression in CRS sinus tissue was identified that was not a result of atopic presence. SPRR1 and SPRR2A expressions were markedly increased in patients with CRS (p < 0.01) on RNA sequencing, with confirmation by using real-time PCR. Immunohistochemistry of archived surgical samples demonstrated staining of SPRR proteins within squamous epithelium of both groups. Pathway analysis indicated tumor necrosis factor (TNF) alpha as a master regulator of the SPRR gene products. CONCLUSION: Expression of SPRR1 and of SPRR2A is increased in mucosal samples from patients with CRS and appeared as a downstream result of TNF alpha modulation, which possibly resulted in epithelial barrier dysfunction.

Functional Analysis of Periplakin and Envoplakin, Cytoskeletal Linkers, and Cornified Envelope Precursor Proteins.

Envoplakin and periplakin are the two smallest plakin family cytoskeletal linker proteins that connect intermediate filaments to cellular junctions and other membrane locations. These two plakins have a structural role in the assembly of the cornified envelope (CE), the terminal stage of epidermal differentiation. Analysis of gene-targeted mice lacking both these plakins and the third initial CE scaffold protein, involucrin, demonstrate the importance of the structural integrity of CE for a proper epidermal barrier function. It has emerged that periplakin, which also has a wider tissue distribution than envoplakin, has additional, independent roles. Periplakin participates in the cytoskeletal organization also in other tissues and interacts with a wide range of membrane-associated proteins such as kazrin and butyrophilin BTN3A1. This review covers methods used to understand periplakin and envoplakin functions in cell culture models, including siRNA ablation of periplakin expression and the use of tagged protein domain constructs to study localization and interactions. In addition, assays that can be used to analyze CEs and epidermal barrier function in gene-targeted mice are described and discussed.

Skin aging, gene expression and calcium.

The human epidermis provides a very effective barrier function against chemical, physical and microbial insults from the environment. This is only possible as the epidermis renews itself constantly. Stem cells located at the basal lamina which forms the dermoepidermal junction provide an almost inexhaustible source of keratinocytes which differentiate and die during their journey to the surface where they are shed off as scales. Despite the continuous renewal of the epidermis it nevertheless succumbs to aging as the turnover rate of the keratinocytes is slowing down dramatically. Aging is associated with such hallmarks as thinning of the epidermis, elastosis, loss of melanocytes associated with an increased paleness and lucency of the skin and a decreased barrier function. As the differentiation of keratinocytes is strictly calcium dependent, calcium also plays an important role in the aging epidermis. Just recently it was shown that the epidermal calcium gradient in the skin that facilitates the proliferation of keratinocytes in the stratum basale and enables differentiation in the stratum granulosum is lost in the process of skin aging. In the course of this review we try to explain how this calcium gradient is built up on the one hand and is lost during aging on the other hand. How this disturbed calcium homeostasis is affecting the gene expression in aged skin and is leading to dramatic changes in the composition of the cornified envelope will also be discussed. This loss of the epidermal calcium gradient is not only specific for skin aging but can also be found in skin diseases such as Darier disease, Hailey-Hailey disease, psoriasis and atopic dermatitis, which might be very helpful to get a deeper insight in skin aging.

Proteomic identification of in vivo interactors reveals novel function of skin cornification proteins.

Protection against injurious external insults and loss of vital fluids is essential for life and is in all organisms, from bacteria to plants and humans, provided by some form of barrier. Members of the small proline-rich (SPRR) protein family are major components of the cornified cell envelope (CE), a structure responsible for the barrier properties of our skin. These proteins are efficient reactive oxygen species (ROS) quenchers involved not only in the establishment of the skin's barrier function but also in cell migration and wound healing. Here, a proteomic analysis of in vivo SPRR-interacting proteins confirmed their function in CE-formation and ROS-quenching and also revealed a novel unexpected role in DNA-binding. Direct in vitro and in vivo evidence proved that the DNA-binding capacity of SPRRs is regulated by the oxidation state of the proteins. At low ROS levels, nuclear SPRR is able to bind DNA and prevent ROS-induced DNA damage. When ROS levels increase, SPRR proteins multimerize and form an effective antioxidant barrier at the cell periphery, possibly to prevent the production or infiltration of ROS. At even higher ROS exposure, DNA-binding is restituted. A molecular model explaining how the intracellular oxidation state of SPRRs likely influences their selective protective function is provided.

The cornified envelope: a first line of defense against reactive oxygen species.

Human skin serves as a barrier against multiple environmental insults, including pathogenic microorganisms, pollutants, toxic chemicals, and UV radiation. In the outermost layer of the epidermis, the cornified envelope functions as a mechanical and permeability barrier. In this issue, Vermeij et al. report a novel function of cornified envelope proteins as a first-line antioxidant barrier to protect the body from damage induced by reactive oxygen species.

ROS quenching potential of the epidermal cornified cell envelope.

The cornified cell envelope (CE) is a specialized structure assembled beneath the plasma membrane of keratinocytes in the outermost layers of the epidermis. It is essential for the physical and permeability properties of the barrier function of the skin. Our skin is continuously exposed to atmospheric oxygen and threatened by reactive oxygen species (ROS). Here, we identify the CE as a first line of antioxidant defense and show that the small proline-rich (SPRR) family of CE precursor proteins have a major role in ROS detoxification. Cysteine residues within these proteins are responsible for ROS quenching, resulting in inter- and intramolecular S-S bond formation, both in isolated proteins and purified CEs. The related keratinocyte proline-rich protein is also oxidized on several cysteine residues within the CE. Differences in antioxidant potential between various SPRR family members are likely determined by structural differences rather than by the amount of cysteine residues per protein. Loricrin, a major component of the CE with a higher cysteine content than SPRRs, is a weak ROS quencher and oxidized on a single cysteine residue within the CE. It is inferred that SPRR proteins provide the outermost layer of our skin with a highly adaptive and protective antioxidant shield.