Jennifer L. Koetsier

Desmoglein 1 deficiency results in severe dermatitis, multiple allergies and metabolic wasting

The relative contribution of immunological dysregulation and impaired epithelial barrier function to allergic diseases is still a matter of debate. Here we describe a new syndrome featuring severe dermatitis, multiple allergies and metabolic wasting (SAM syndrome) caused by homozygous mutations in DSG1. DSG1 encodes desmoglein 1, a major constituent of desmosomes, which connect the cell surface to the keratin cytoskeleton and have a crucial role in maintaining epidermal integrity and barrier function. Mutations causing SAM syndrome resulted in lack of membrane expression of DSG1, leading to loss of cell-cell adhesion. In addition, DSG1 deficiency was associated with increased expression of a number of genes encoding allergy-related cytokines. Our deciphering of the pathogenesis of SAM syndrome substantiates the notion that allergy may result from a primary structural epidermal defect.

The transcriptional repressor SNAIL is overexpressed in human colon cancer.

Overexpression of the transcriptional repressor, SNAIL, has been implicated in the pathogenesis of a number of malignancies; however, there are no previous reports on the role of SNAIL in colorectal cancers (CRCs). We, therefore, evaluated human CRC specimens for the presence of the SNAIL protein. Immunohistochemical studies were performed using samples obtained from archived CRC paraffin blocks and a tissue array. Tissue sections were probed with a polyclonal antibody to human SNAIL and scored by a gastrointestinal pathologist. SNAIL was not detectable in uninvolved mucosa, but immunoreactivity was evident in 78% of tumors. SNAIL protein expression did not correlate with subsite location or gender, however, SNAIL-positive tumors had an older mean age (58.9 ± 12.7 versus 49.8 ± 127; P = 0.028). Furthermore, there was a trend that CRCs with metastatic ability more frequently overexpressed SNAIL (100 versus 65%; P = 0.11). In conclusion, we demonstrate, for the first time, that SNAIL is upregulated in human colon cancer, which potentially may have significance in control of metastasis and possibly serve as a target for chemopreventive agents.

Desmoglein-1/Erbin interaction suppresses ERK activation to support epidermal differentiation

Genetic disorders of the Ras/MAPK pathway, termed RASopathies, produce numerous abnormalities, including cutaneous keratodermas. The desmosomal cadherin, desmoglein-1 (DSG1), promotes keratinocyte differentiation by attenuating MAPK/ERK signaling and is linked to striate palmoplantar keratoderma (SPPK). This raises the possibility that cutaneous defects associated with SPPK and RASopathies share certain molecular faults. To identify intermediates responsible for executing the inhibition of ERK by DSG1, we conducted a yeast 2-hybrid screen. The screen revealed that Erbin (also known as ERBB2IP), a known ERK regulator, binds DSG1. Erbin silencing disrupted keratinocyte differentiation in culture, mimicking aspects of DSG1 deficiency. Furthermore, ERK inhibition and the induction of differentiation markers by DSG1 required both Erbin and DSG1 domains that participate in binding Erbin. Erbin blocks ERK signaling by interacting with and disrupting Ras-Raf scaffolds mediated by SHOC2, a protein genetically linked to the RASopathy, Noonan-like syndrome with loose anagen hair (NS/LAH). DSG1 overexpression enhanced this inhibitory function, increasing Erbin-SHOC2 interactions and decreasing Ras-SHOC2 interactions. Conversely, analysis of epidermis from DSG1-deficient patients with SPPK demonstrated increased Ras-SHOC2 colocalization and decreased Erbin-SHOC2 colocalization, offering a possible explanation for the observed epidermal defects. These findings suggest a mechanism by which DSG1 and Erbin cooperate to repress MAPK signaling and promote keratinocyte differentiation.

Increased microvascular blood content is an early event in colon carcinogenesis

Background: Increased premalignant epithelial microvascular blood content is a common theme in neoplastic transformation; however, demonstration of this phenomenon in colon carcinogenesis has been stymied by methodological limitations. Our group has recently developed a novel optics technology, four dimensional elastic light scattering fingerprinting (4D-ELF), which allows examination of the colonic mucosal architecture with unprecedented accuracy. In this study, we utilised 4D-ELF to probe the preneoplastic colonic microvasculature. Methods: Colonic mucosal blood content was assessed by 4D-ELF at serial preneoplastic time points from azoxymethane (AOM) treated Fisher 344 rats and age matched control animals. We also examined the pretumorigenic intestinal mucosa of the MIN mouse, and compared with wild-type mice. Finally, in a pilot study, we examined superficial blood content from the endoscopically normal mid transverse colon in 37 patients undergoing screening colonoscopy. Results: In the AOM treated rat model, augmentation of superficial mucosal and total mucosal/superficial submucosal blood supply preceded the appearance of aberrant crypt foci (ACF) and temporally and spatially correlated with future ACF occurrence. These findings were replicated in MIN mice. The 4D-ELF based results were corroborated with immunoblot analysis for haemoglobin on mucosal scrapings from AOM treated rats. Moreover, 4D-ELF analysis of normal human colonic mucosa indicated that there was a threefold increase in superficial blood in patients who harboured advanced adenomas. Conclusion: We report, for the first time, that blood content is increased in the colonic microvasculature at the earliest stages of colon carcinogenesis. These findings may provide novel insights into early biological events in colorectal carcinogenesis and have potential applicability for screening.

Down-regulation of the tumor suppressor gene C-terminal Src kinase: an early event during premalignant colonic epithelial hyperproliferation.

Hyperproliferation of the premalignant epithelium is critical for colonic carcinogenesis; however the mechanisms remain largely unexplored. We report herein that prior to occurrence of neoplastic lesions in the azoxymethane‐rat model of colon carcinogenesis; the tumor suppressor gene C‐terminal Src kinase (Csk) was down‐regulated with a concomitant increase in Src activity. Furthermore, pharmacological or genetic (RNA interference) inhibition of Csk resulted in increased proliferation in colon cancer cell lines through the mitogen‐activated protein kinase dependent pathway. Thus, we demonstrate, for the first time, that Csk suppression is an important early event in colorectal cancer pathogenesis.

Spectral Markers in Preneoplastic Intestinal Mucosa: An Accurate Predictor of Tumor Risk in the MIN Mouse

Background: We have reported recently that microarchitectural analysis of the histologically normal mucosa using a novel optics technology, four-dimensional elastic light scattering fingerprinting (ELF), provided unprecedented sensitivity for early detection of colon carcinogenesis. In the present study, we explored the ability of four-dimensional ELF to identify an inherited predisposition to colorectal cancer, an issue of considerable importance for optimizing population screening strategies. Methods: We used the MIN mouse, a model whose germ line adenomatous polyposis coli truncation leads to spontaneous intestinal tumorigenesis, thus replicating the human syndrome, familial adenomatous polyposis. Spectral markers were assessed by four-dimensional ELF analysis in MIN mice at preneoplastic time points and compared with age-matched controls (C57BL6 mice with wild-type adenomatous polyposis coli). To assess the responsiveness of spectral markers to chemopreventive agents, a subset of MIN mice was supplemented with celecoxib 1,500 ppm. Results: Spectral slope, fractal dimension, and principal component 3 were dramatically altered in the uninvolved MIN mouse mucosa at the earliest time points. Furthermore, alteration in spectral variables increased over time, consonant with the microarchitectural underpinnings of subsequent tumorigenesis. Additionally, these markers spatially correlated with future adenoma development (small intestine > colon). Short-term treatment with the potent chemopreventive agent, celecoxib, resulted in near normalization of fractal dimension and principal component 3. Conclusions: We report, for the first time, that spectral markers, assayed by four-dimensional ELF, were able to sensitively identify a genetic predisposition for intestinal tumorigenesis before the occurrence of phenotypic manifestations. Moreover, the reversal of spectral markers by celecoxib treatment supports the neoplastic relevance.

The C-terminal unique region of desmoglein 2 inhibits its internalization via tail-tail interactions

Tail–tail interactions of desmoglein 2, promoted by its C-terminal unique region, inhibit its internalization, stabilizing it at the cell surface and promoting intercellular adhesion.

Inducible nitric oxide synthase (iNOS) mediates the early increase of blood supply (EIBS) in colon carcinogenesis

We have recently demonstrated that dramatic alteration in mucosal microvascular blood content termed early increase in blood supply (EIBS) is a hallmark of early colon carcinogenesis. In the current study, we elucidate the mechanism of EIBS by assessing iNOS/nitric oxide axis in the histologically normal colonic mucosa of rats treated with the colon‐specific carcinogen, azoxymethane. We demonstrate that there was a strong temporal correlation between EIBS and iNOS expression/activity. Importantly, we also observed that short‐term treatment with nitric oxide inhibitor abrogated EIBS. These data indicate that iNOS induction may have a critical role in augmenting the predysplastic mucosal blood supply and thereby fostering colon carcinogenesis.

Chemoprevention of colon carcinogenesis by polyethylene glycol: suppression of epithelial proliferation via modulation of SNAIL/β-catenin signaling

Polyethylene glycol (PEG) is one of the most potent chemopreventive agents against colorectal cancer; however, the mechanisms remain largely unexplored. In this study, we assessed the ability of PEG to target cyclin D1–β-catenin–mediated hyperproliferation in the azoxymethane-treated rat model and the human colorectal cancer cell line, HT-29. Azoxymethane-treated rats were randomized to AIN-76A diet alone or supplemented with 5% PEG-8000. After 30 weeks, animals were euthanized and biopsies of aberrant crypt foci and uninvolved crypts were subjected to immunohistochemical and immunoblot analyses. PEG markedly suppressed both early and late markers of azoxymethane-induced colon carcinogenesis (fractal dimension by 80%, aberrant crypt foci by 64%, and tumors by 74%). In both azoxymethane-treated rats and HT-29 cells treated with 5% PEG-3350 for 24 hours, PEG decreased proliferation (45% and 52%, respectively) and cyclin D1 (78% and 56%, respectively). Because β-catenin is the major regulator of cyclin D1 in colorectal cancer, we used the T-cell factor (Tcf)–TOPFLASH reporter assay to show that PEG markedly inhibited β-catenin transcriptional activity. PEG did not alter total β-catenin expression but rather its nuclear localization, leading us to assess E-cadherin expression (a major determinant of β-catenin subcellular localization), which was increased by 73% and 71% in the azoxymethane-rat and HT-29 cells, respectively. We therefore investigated the effect of PEG treatment on levels of the negative regulator of E-cadherin, SNAIL, and observed a 50% and 75% decrease, respectively. In conclusion, we show, for the first time, a molecular mechanism through which PEG imparts its antiproliferative and hence profound chemopreventive effect. [Mol Cancer Ther 2006;5(8):2060–9]

Polyethylene glycol-mediated colorectal cancer chemoprevention: roles of epidermal growth factor receptor and Snail

Polyethylene glycol (PEG) is a clinically widely used agent with profound chemopreventive properties in experimental colon carcinogenesis. We reported previously that Snail/β-catenin signaling may mediate the suppression of epithelial proliferation by PEG, although the upstream events remain unclear. We report herein the role of epidermal growth factor receptor (EGFR), a known mediator of Snail and overepressed in ∼80% of human colorectal cancers, on PEG-mediated antiproliferative and hence antineoplastic effects in azoxymethane (AOM) rats and HT-29 colon cancer cells. AOM rats were randomized to either standard diet or one with 10% PEG-3350 and euthanized 8 weeks later. The colonic samples were subjected to immunohistochemical or Western blot analyses. PEG decreased mucosal EGFR by 60% (P < 0.001). Similar PEG effects were obtained in HT-29 cells. PEG suppressed EGFR protein via lysosmal degradation with no change in mRNA levels. To show that EGFR antagonism per se was responsible for the antiproliferative effect, we inhibited EGFR by either pretreating cells with gefitinib or stably transfecting with EGFR-short hairpin RNA and measured the effect of PEG on proliferation. In either case, PEG effect was blunted, suggesting a vital role of EGFR. Flow cytometric analysis revealed that EGFR-short hairpin RNA cells, besides having reduced membrane EGFR, also expressed low Snail levels (40%), corroborating a strong association. Furthermore, in EGFR silenced cells, PEG effect on EGFR or Snail was muted, similar to that on proliferation. In conclusion, we show that EGFR is the proximate membrane signaling molecule through which PEG initiates antiproliferative activity with Snail/β-catenin pathway playing the central intermediary function. [Mol Cancer Ther 2008;7(9):3103–11]