Christine Marshall

Srcasm Inhibits Fyn-Induced Cutaneous Carcinogenesis with Modulation of Notch1 and p53

Src family tyrosine kinases (SFK) regulate cell proliferation, and increased SFK activity is common in human carcinomas, including cutaneous squamous cell carcinomas (SCC) and its precursors. The elevated SFK activity in cutaneous SCCs was modeled using K14-Fyn Y528F transgenic mice, which spontaneously form punctate keratotic lesions, scaly plaques, and large tumors resembling actinic keratoses, SCC in situ, and SCCs, respectively. Lesional tissue showed increased levels of activated SFKs, PDK1, STAT3, and ERK1/2, whereas Notch1/NICD protein and transcript levels were decreased. p53 levels also were decreased in SCC in situ and SCCs. Increasing Srcasm levels using a K14-Fyn Y528F/K14-Srcasm double transgenic model markedly inhibited cutaneous neoplasia. In contrast, increased expression of a nonphosphorylatable Srcasm mutant maintained the neoplastic phenotype. Increasing Srcasm levels decreased levels of Fyn, activated SFKs, ERK1/2, PDK1, and phospho-STAT3, and increased Notch1/NICD and p53 levels. Analysis of human specimens revealed that levels of Fyn and activated SFKs were elevated in SCCs compared with adjacent nonlesional epidermis. In addition, Notch1 and Srcasm protein and transcript levels were decreased in human SCCs compared with nonlesional epidermis. Therefore, the SCCs produced by the Fyn Y528F mice resemble their human counterparts at the molecular level. K14-Fyn Y528F mice represent a robust model of cutaneous carcinogenesis that manifests precancerous lesions and SCCs resembling human disease. The Fyn/Srcasm signaling nexus modulates activity of STAT3, PDK1, ERK1/2, Notch1, and p53. Further study of Fyn and Srcasm should provide insights into the mechanisms regulating keratinocyte proliferation and skin carcinogenesis.

Srcasm Corrects Fyn-induced Epidermal Hyperplasia by Kinase Down-regulation *

Src family tyrosine kinases (SFKs) are important regulators of epithelial cell growth and differentiation. Characterization of cellular mechanisms that regulate SFK activity will provide insights into the pathogenesis of diseases associated with increased SFK activity. Keratin 14-Fyn (K14) transgenic mice were derived to characterize the effect of Fyn on epidermal growth and differentiation in vivo. The epidermis of K14-Fyn mice is thickened, manifests prominent scale, and exhibits features consistent with hyperproliferation. Increased epidermal Fyn levels correlate with activation of p44/42 MAP kinases, STAT-3, and PDK-1, key signaling molecules that promote epithelial cell growth. The Src-activating and signaling molecule (Srcasm) is a substrate of SFKs that becomes tyrosine-phosphorylated downstream of the EGF receptor. In vitro, increased Srcasm levels promote activation of endogenous Fyn and keratinocyte differentiation. To study the in vivo effect of Srcasm upon Fyn, double transgenic lines were derived. K14-Fyn/Srcasm transgenic mice did not manifest the hyperproliferative phenotype. In contrast, K14-Fyn/Srcasm-P transgenic mice, which express a nonphosphorylatable Srcasm mutant, maintained the hyperproliferative phenotype. Resolution of the hyperproliferative phenotype correlated with reduced Fyn levels in vivo in three experimental systems: transgenic mice, primary keratinocytes, and cell lines. Biochemical studies revealed that Srcasm-dependent Fyn down-regulation requires Fyn kinase activity, phosphorylation of Srcasm, and the Srcasm GAT domain. Therefore, Srcasm is a novel regulator of Fyn promoting kinase down-regulation in a phosphorylation-dependent manner. Srcasm may act as a molecular “rheostat” for activated SFKs, and cellular levels of Srcasm may be important for regulating epithelial hyperproliferation associated with increased SFK activity.

Cytoplasmic and nuclear interaction between Rb family proteins and PAI-2: a physiological crosstalk in human corneal and conjunctival epithelial cells.

Extracellular plasminogen activator inhibitor type-2 (PAI-2) is a potent inhibitor of urokinase-type plasminogen activator (u-PA) and also acts as a multifunctional protein. However, the biological activity of intracellular PAI-2, as well as its intracellular targets, until now remain an enigma. Here, we show that pRb2/p130 and Rb1/p105, but not p107, interact with PAI-2 in both the cytoplasm and nucleus of normal primary human corneal and conjunctival epithelial cells. We provided the first in vivo evidence that a specific fragment of the PAI-2 promoter is bound simultaneously by pRb2/ p130, PAI-2, E2F5, histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1), and histone methyltransferase (SUV39H1), in normal primary human corneal epithelial cells, and by pRb2/p130, PAI-2, E2F5, HDAC1, and DNMT1, in normal primary human conjunctiva epithelial cells. Our results strongly indicate a physiological interaction between pRb family members and PAI-2, suggesting the hypothesis that pRb2/p130 and PAI-2 may cooperate in modulating PAI-2 gene expression by chromatin remodeling, in normal corneal and conjunctival cells.

Curcuminoids activate p38 MAP kinases and promote UVB-dependent signalling in keratinocytes

Please cite this paper as: Curcuminoids activate P38 map kinases and promote UVB‐dependent signalling in keratinocytes. Experimental Dermatology 2010; 19: 493–500.

Therapeutic Elimination of the Type 1 Interferon Receptor for Treating Psoriatic Skin Inflammation

Phototherapy with UV light is a standard treatment for psoriasis, yet the mechanisms underlying the therapeutic effects are not well understood. Studies in human and mouse keratinocytes and in the skin tissues from human patients and mice showed that UV treatment triggers ubiquitination and downregulation of the type I IFN receptor chain IFNAR1, leading to suppression of IFN signaling and an ensuing decrease in the expression of inflammatory cytokines and chemokines. The severity of imiquimod-induced psoriasiform inflammation was greatly exacerbated in skin of mice deficient in IFNAR1 ubiquitination (Ifnar1(SA)). Furthermore, these mice did not benefit from UV phototherapy. Pharmacologic induction of IFNAR1 ubiquitination and degradation by an antiprotozoal agent halofuginone also relieved psoriasiform inflammation in wild-type but not in Ifnar1(SA) mice. These data identify downregulation of IFNAR1 by UV as a major mechanism of the UV therapeutic effects against the psoriatic inflammation and provide a proof of principle for future development of agents capable of inducing IFNAR1 ubiquitination and downregulation for the treatment of psoriasis.

Plasminogen activator inhibitor type 2 (PAI-2) is present in normal human conjunctiva.

The purpose was to characterize plasminogen activator inhibitor type 2 (PAI‐2) expression in normal human conjunctiva in vivo and in vitro. PAI‐2 antigen was assayed by immunostaining and immunoblotting of extracts from normal human conjunctival epithelial lysates and conditioned media (CM) of cultured human conjunctival keratinocytes. Immunostaining of normal human conjunctival epithelia revealed that PAI‐2 was found consistently in the superficial keratinocytes and, in some biopsies, also in the lower keratinocyte layers. In all cases, PAI‐2 was concentrated around the cell periphery. In extracts of conjunctival epithelia and cultured conjunctival keratinocytes, PAI‐2 had an apparent molecular weight of 45 kDa, consistent with the non‐glycosylated form. The majority of PAI‐2, approximately 90%, was cell associated, however, a small percentage of PAI‐2 was released into the CM in a linear manner with time. PAI‐2 in the conditioned medium had a higher molecular weight, consistent with a glycosylated form. Conjunctival PAI‐2 was active, as shown by its ability to complex with a target enzyme, urokinase plasminogen activator (uPA). Although PAI‐2 was detectable both in monolayer (i.e., relatively undifferentiated) conjunctival keratinocyte cultures as well as in stratified (i.e., more differentiated) cultures, steady state levels of PAI‐2 were greater in the latter. PAI‐2 is constitutively expressed by normal human conjunctival epithelial cells. The expression of PAI‐2 throughout all epithelial layers in some biopsies of conjunctiva in vivo contrasts with the previously established distribution of PAI‐2 in corneal epithelia, where it is present exclusively in the most superficial (i.e. most highly differentiated) cells. The role of PAI‐2 in either tissue is unclear. However, we speculate that its distinct distribution in conjunctival versus corneal epithelia underscores inherent differences between these tissues, and may reflect specific functions of this proteinase inhibitor in both conjunctival and corneal epithelial cells.

Abstract 806: Modeling human UV-induced skin cancer through genetic manipulation of Src-kinases and Srcasm

UV-induced skin cancer is the most common form of cancer and its incidence continues to increase. Novel in vivo models that mimic human UV-induced skin cancer are needed to further studies and evaluate new therapies. Keratinocyte growth is regulated through oncogenic signals from EGFR and Src-family tyrosine kinases (SFKs); these tyrosine kinases are primary drivers of keratinocyte growth in UV-induced skin cancer. At the proteomic level, activated EGFR and SFKs are negatively regulated by Srcasm which targets activated tyrosine kinases for lysosomal degradation. Studies have shown that SFK activity is increased and Srcasm levels are decreased in human cutaneous squamous cell carcinomas and related precancerous lesions. We hypothesized that genetic downregulation of Srcasm by gene deletion or lentivirally mediated shRNA knockdown would promote oncogenic signaling and keratinocyte growth thereby promoting UV-induced skin cancer. To test this hypothesis Srcasm null mice and Srcasm lentiviral knockdown in human keratinocytes were generated and studied. Western blotting of epidermal lysates from Srcasm null mice demonstrates that loss of Srcasm promotes activation of EGFR and Src kinases and decreases levels of p53, NICD and p21. Activation of the canonical Erk1/2, STAT-3 and PDK-1 oncogenic pathways also was seen in Srcasm null keratinocytes. The epidermis of Srcasm null mice exhibits a higher Ki-67 index of 8.7 +/- 2.0 compared to 1.5 +/- 1.1 in controls (p 2 × 15 doses) produced precancerous lesions in Srcasm null mice resembling human actinic keratoses but not in controls; the precancerous lesions show an increased Ki-67 index and activation of Src kinases, STAT-3 and PDK-1. Acute UVB irradiation (1200 mJ/cm 2 × 1 dose) of Srcasm null mice induced prominent erythema, epidermal necrosis and a prominent increase in the TUNEL-positive index (14.0 vs 1.3). Srcasm knockdown in human keratinocytes accelerated keratinocyte growth 130%; it also increased levels of EGFR and accelerated the kinetics of EGFR and MAPK activation in response to EGF stimulation. In human keratinocytes, Srcasm knockdown accelerated cell growth post-UV exposure which could promote neoplasia. These data show that loss of Srcasm is associated with increased oncogenic signaling, impaired p53 function and keratinocyte proliferation. Loss of Srcasm promotes keratinocyte proliferation post-UV exposure leading to the early stages of UVB-induced cutaneous neoplasia. Citation Format: Xiaoping Yang, Sheila Shankar, Hiroshi Maeno, Micheal Gober, Christine Marshall, Tzvete Dentchev, John Seykora. Modeling human UV-induced skin cancer through genetic manipulation of Src-kinases and Srcasm. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2015-806