Kras^G12D mutation led to mucinous metaplasia in pancreatic ducts.

<p>H&E staining of a typical lesion in mouse pancreas (A) and its connection to normal ductal epithelium in a nearby serial section (B) with alcian blue staining to denote mucin-producing cells. Arrowhead, junction between nonmucinous and mucinous cells; arrows, junction between cuboidal and columnar cells. (C) Mucinous lesions were all strongly positive for claudin-18 (brown) with highest concentrations along lateral cell borders. Size bars, 50 µm.</p

Kras^G12D mutation led to adenoma formation in lungs.

<p>A, B. PAS and hematoxylin staining of lung adenomas typical of CK19^CreERT; LSL-Kras^G12D mice. Size bars, 50 µm.</p

CK19^CreERT recombined an EYFP reporter in lung and oral cavity.

<p>Bronchus (A), bronchiole (B), lingual epithelium (C), and buccal epithelium (D) from CK19^CreERT; R26R^EYFP mice immunolabeled for EYFP (brown). Arrows in C and D, labeled cells in basal layer of epithelia. Size bars, 50 µm.</p

Cre-mediated recombination of the LSL-Kras^G12D allele in different tissues.

<p>A. DNA was extracted from the indicated tissues of three different mice and subjected to 35 cycles of PCR that would detect both the wildtype Kras allele (271 bp) and the recombined LSL-Kras^G12D allele (310 bp) but not the unrecombined LSL-Kras^G12D allele. Tissues examined: 1) tail, 2) small intestine, 3) liver, 4) pancreas, 5) kidney, 6) stomach, and 7) colon. Strong recombined bands were detected for small intestine, pancreas and colon and weaker bands for stomach, kidney and liver, while recombination was never detected in tail DNA. B. The amount of recombination of an unaffected tissue, the small intestine, was compared to the amount in an oral papilloma in which all or nearly all of the epithelium should have a recombined Kras allele. For three different mice, DNA was prepared from total oral tissue (lanes 1–3), isolated papilloma tissue (lanes 4–6) and total small intestine tissue (lanes 7–9) and subjected to 30 cycles of PCR. Little recombination can be detected in total oral tissue while the recombination is abundant in the isolated papilloma (upper band in each lane). The amount of recombination in the small intestine was intermediate between these two populations.</p

Kras^G12D led to squamous papillomas in the oral cavity.

<p>A.H&E staining of papillomas on back of tongue. B. H&E staining of buccal papillomas. C. Immunolabeling for phosphohistone H3 (brown), an M phase marker, revealed that the proliferative zone in papillomas remained in the basal layer. Size bars, 50 µm.</p

Kras^G12D mutation led to foveolar hyperplasia in the gastric fundus.

<p>A. PAS (pink) showed extensive mucin production deep into affected glands while surrounding normal glands only had staining at the tops of glands. B. Immunolabeling for phosphohistone H3 as an M phase marker. The proliferative zone in affected glands was shifted toward the base of the glands. C. Immunolabeling for TFF2 (brown) showed positive cells near base of affected fundic glands while normal glands had expression in the normal mucous neck region. Arrows, staining in affected glands; arrowheads, staining in normal glands. Size bars, 50 µm.</p

Morbidity analysis: epithelial expression of Kras^G12D led to weight loss.

<p>Mice were monitored weekly for weight and other indications of overall health. Number maintaining at least 80% of highest body weight is plotted as a function of time for CK19^CreERT; LSL-Kras^G12D (solid line) and for littermates also injected with tamoxifen (dashed line). An additional 3 control mice were also injected with tamoxifen but were only followed for 3–5 months as controls for earlier timepoints and did not lose weight during that time (data not shown).</p

Kras^G12D mutation was associated with patches of villus-like structures in the ascending colon.

<p>A. H&E staining shows villus-like architecture in an area of the ascending colon. Normal colonic crypt structure resumes on upper right side of each panel (arrowhead). B. Immunolabeling for Ki67 (brown) indicates that the proliferative zones associated with villus-like structures are similar in cell number to normal colonic crypts. Size bars, 50 µm.</p

GLI1 and CTNNB1 expression in TCGA.

<p>Boxplots of (a) GLI1 and (b) CTNNB1 expression in TCGA RNA-seq data.</p

Preferential Activation of the Hedgehog Pathway by Epigenetic Modulations in HPV Negative HNSCC Identified with Meta-Pathway Analysis

<div><p>Head and neck squamous cell carcinoma (HNSCC) is largely divided into two groups based on their etiology, human papillomavirus (HPV)-positive and –negative. Global DNA methylation changes are known to drive oncogene and tumor suppressor expression in primary HNSCC of both types. However, significant heterogeneity in DNA methylation within the groups results in different transcriptional profiles and clinical outcomes. We applied a meta-pathway analysis to link gene expression changes to DNA methylation in distinguishing HNSCC subtypes. This approach isolated specific epigenetic changes controlling expression in HPV− HNSCC that distinguish it from HPV+ HNSCC. Analysis of genes identified Hedgehog pathway activation specific to HPV− HNSCC. We confirmed that <i>GLI1</i>, the primary Hedgehog target, showed higher expression in tumors compared to normal samples with HPV− tumors having the highest <i>GLI1</i> expression, suggesting that increased expression of <i>GLI1</i> is a potential driver in HPV− HNSCC. Our algorithm for integration of DNA methylation and gene expression can infer biologically significant molecular pathways that may be exploited as therapeutics targets. Our results suggest that therapeutics targeting the Hedgehog pathway may be of benefit in HPV− HNSCC. Similar integrative analysis of high-throughput coupled DNA methylation and expression datasets may yield novel insights into deregulated pathways in other cancers.</p></div