Project description:Alternative splicing analysis of laser micro-dissected lung dysplasia RNA samples Lung dysplasia is a precancerous condition with high risk of malignant transformation. Little is known about alternative spliced (AS) genes in dysplasia. We therefore investigated laser micro-dissected microscopic foci of non-contaminant dysplasia and/or lung adenocarcinoma of c-Raf transgenic mice and searched genome wide for AS genes using exon arrays. Notably, bioinformatics defined 34 and 36 AS genes in the comparison dysplasia versus transgenic unaltered and non-transgenic lung tissue while the comparison adenocarcinoma versus transgenic unaltered and non-transgenic lung tissue revealed 54 and 56 genes, respectively. So far only 6 of these were reported for lung cancer. Importantly, dysplasia related AS genes were also regulated in lung cancer to suggest a role in disease onset. Next to exon skipping/inclusion alternative splicing at the 3M-bM-^@M-^Y and 5M-bM-^@M-^Y was common and included genes of the splicing regulatory pathway. Disease dependent changes of variant transcripts were confirmed by RT-PCR while Western blotting identified alternative splicing to modulate protein levels of AS genes. For the AS genes Add3, Cast, Osbpl6, Nedd4l, Numb, Picalm and Slk transcript and protein level agreed well, whereas for Arhgef11, Clstn1, Dlg1, Dock9, Mbnl2, Mfge8, Npnt, Pdlim5, Ppp2r5c, Tjp1 notable differences in the abundance of variant transcripts were observed by RT-PCR and gel electrophoresis. Moreover, expression of individual variants differed between dysplasia and carcinoma to suggest their disease dependent regulation. Western blotting of IQGAP1, MYO6, PTPRM, RABGAP1L and RAD50 confirmed significant regulation of isoforms in lung cancer as compared to non-transgenic, transgenic unaltered and dysplastic lung tissue. For 46 AS genes expression of the non-variant protein was reported in human lung cancer (www.proteinatlas.org) and for 13 of these, cancer related AS events are known. Overall, new insight into lung dysplasia was obtained with 43 new cancer related AS genes to aid diagnosis and molecular intervention strategies. We analyzed and compared dysplastic (n=4) and adenocarcinoma (n=4) lesions and with transgenic but unaltered (n=4) and non-transgenic (n=5) lung tissue obtained from SPC/c-Raf transgenic mice with aid of laser micro dissection pressure catapulted (LMPC) technique. Dysplasia is a pre-neoplastic condition and developed at 5 month of age in the proposed mouse model. LMPC provides precise microscopic lesion collection without contamination. The RNA samples were analyzed using the Affymetrix Mouse Exon 1.0 ST platform. Alternative splicing was analyzed using Biotique XRAY tool. No technical replicates were performed.

Project description:Alternative splicing analysis of laser micro-dissected lung dysplasia RNA samples Lung dysplasia is a precancerous condition with high risk of malignant transformation. Little is known about alternative spliced (AS) genes in dysplasia. We therefore investigated laser micro-dissected microscopic foci of non-contaminant dysplasia and/or lung adenocarcinoma of c-Raf transgenic mice and searched genome wide for AS genes using exon arrays. Notably, bioinformatics defined 34 and 36 AS genes in the comparison dysplasia versus transgenic unaltered and non-transgenic lung tissue while the comparison adenocarcinoma versus transgenic unaltered and non-transgenic lung tissue revealed 54 and 56 genes, respectively. So far only 6 of these were reported for lung cancer. Importantly, dysplasia related AS genes were also regulated in lung cancer to suggest a role in disease onset. Next to exon skipping/inclusion alternative splicing at the 3’ and 5’ was common and included genes of the splicing regulatory pathway. Disease dependent changes of variant transcripts were confirmed by RT-PCR while Western blotting identified alternative splicing to modulate protein levels of AS genes. For the AS genes Add3, Cast, Osbpl6, Nedd4l, Numb, Picalm and Slk transcript and protein level agreed well, whereas for Arhgef11, Clstn1, Dlg1, Dock9, Mbnl2, Mfge8, Npnt, Pdlim5, Ppp2r5c, Tjp1 notable differences in the abundance of variant transcripts were observed by RT-PCR and gel electrophoresis. Moreover, expression of individual variants differed between dysplasia and carcinoma to suggest their disease dependent regulation. Western blotting of IQGAP1, MYO6, PTPRM, RABGAP1L and RAD50 confirmed significant regulation of isoforms in lung cancer as compared to non-transgenic, transgenic unaltered and dysplastic lung tissue. For 46 AS genes expression of the non-variant protein was reported in human lung cancer (www.proteinatlas.org) and for 13 of these, cancer related AS events are known. Overall, new insight into lung dysplasia was obtained with 43 new cancer related AS genes to aid diagnosis and molecular intervention strategies.

Project description:Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histologic analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal appearing p53-positive epithelium that are similar to “p53 signatures” in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these noninvasive precursor lesions, we also identified invasive adenocarcinoma in the ovary of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and WT C57BL/6. One of these genes, Top2a, which encodes topoisomerase II-alpha, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and specifically elevated in mouse STICs, but not in surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade, and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumorigenesis, as well as for developing novel approaches for the prevention, diagnosis, and therapy of this disease. Keywords: transgenic mouse model, ovarian cancer, fallopian tube, intraepithelial carcinoma 6 mouse fallopian tubes (FT) were analyzed with experimental repeats; 3 wildtype C57BL6 mice (FT) and 3 transgenic mogp-TAg (FT), with one set of each at 7, 8 and 9 weeks of age.

Project description:Lung cancer is a leading cause of deaths in the world. There is a need to improve an understanding of mechanisms of malignant transformation and to develop genetic markers of disease for better and targeted therapies. Here, we report findings with a transgenic disease model where targeted expression of c-raf to respiratory epithelium induced adenocarcinomas. Specifically, by use of laser microdissection we harvested either tumor or transgenic and non-transgenic but otherwise morphologically unaltered cells. We then searched genome wide for regulated genes and validated results by quantitative real-time PCR. Overall, 473 and 541 genes were significantly regulated when cancer versus transgenic and cancer versus non-transgenic cells were compared. Principal component analysis and hierarchical clustering of the data clearly separated the cancer cells from transgenic and non-transgenics and we observed predominately repression of gene expression at advanced stages of tumor growth. Nonetheless genes up-regulated in dysplasia were also up-regulated in solid tumors. We observed groups of genes acting in concert either linked to development with an unexpected high number of genes coding either for the epithelial mesenchymal or mesenchymal endothelial transition. Additionally, genes coding for cell adhesion including the integrins and the tight and gap junction proteins were repressed (integrin alpha 1, integrin alpha 8, claudin 2, claudin 5, gap junction membrane channel protein alpha 5 and cadherin 5) but ligands for the membrane bound epidermal growth factor tyrosine kinase i.e. epi- and amphiregulin were up-regulated. Moreover, molecules in the signalling of vascular endothelial growth factor receptor- 2 and VEGFD, Notch and WNT were regulated as were glycosylases that facilitate cellular recognition. Other regulated signalling molecules included exchange factor such as RAP guanine nucleotide exchange factor 3, RHO guanine nucleotide exchange factor 10, RAS guanine releasing protein 2 and 3, and RAS guanine nucleotide-releasing factor 1 that play a role in an activation of the MAP kinases. Notably, we found the tumor suppressors MCC (mutated in colorectal cancers), HEY1 (hairy/enhancer-of-split related with YRPW motif 1), FAT3 (FAT tumor suppressor homolog 3), ARMCX1 (armadillo repeat containing, X-linked 1) and RECK (reversion-inducing-cysteine-rich protein with kazal motifs) to be significantly repressed. Taken collectively, our study provides valuable information for new candidate genes in lung adenocarcinoma induced by exaggerted c-raf kinase activity.

Project description:Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histologic analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal appearing p53-positive epithelium that are similar to “p53 signatures” in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these noninvasive precursor lesions, we also identified invasive adenocarcinoma in the ovary of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and WT C57BL/6. One of these genes, Top2a, which encodes topoisomerase II-alpha, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and specifically elevated in mouse STICs, but not in surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade, and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumorigenesis, as well as for developing novel approaches for the prevention, diagnosis, and therapy of this disease. Keywords: transgenic mouse model, ovarian cancer, fallopian tube, intraepithelial carcinoma

Project description:Background & Aims: Previous studies have suggested that dietary folic acid (FA) can protect against certain types of cancers. However, the findings have varied and the mechanisms by which this vitamin exerts chemopreventive effects remain to be clarified. We examined the effects of FA supplementation on DNA methylation, gene expression and gastric dysplasia in a transgenic mouse model that is etiologically and histologically well matched with human gastric cancers. Methods: Hypergastrinemic mice (INS-GAS) infected with Helicobacter felis were studied at multiple stages of gastric dysplasia and early cancer, with FA supplementation initiated both at weaning and later in life. Global DNA methylation was assessed by a methylation-sensitive cytosine incorporation assay, bisulfite pyrosequencing of B1 repetitive elements and immunohistochemistry (IHC) with anti-5-methylcytosine. We also profiled gene expression in the same tissues. Results: We found a decrease in global DNA methylation and tissue folate and an increase in serum homocysteine with progression of gastric dysplasia. FA supplementation prevented this loss of global DNA methylation and markedly reduced gastric dysplasia and mucosal inflammation. FA protected against the loss of global DNA methylation both in the dysplastic gastric epithelial cells and in gastric stromal myofibroblasts. In addition, FA supplementation had an anti-inflammatory effect, as indicated by expression profiling and IHC for lymphocyte markers. Conclusions: We conclude that FA supplementation is chemopreventive in this model of Helicobacter-associated gastric cancer. The beneficial effect of FA is likely due to its ability to reverse global loss of methylation and suppress inflammation. Study the difference in gene expression between transgenic hypergastrinemic (INS-GAS) mice fed a folate-supplemented diet and a diet with normal folate content. Because folate had a substantial protective effect, we matched the animals not only by folate status but also by dysplasia score (DYS). The degree of dysplasia was scored on a 1-4 scale. True replicates are not included. The first five samples are from folate supplemented animals, and the latter five are from controls on regular chow.

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Molecular characterization of lung dysplasia induced by c-raf-1

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