rats. These results were supported by significantly higher increase in proliferation markers(MCM2 positivity and number of cells/crypt; increased nuclear atypia, mitotic figures andmucin production) in the pre-malignant mucosa of males as compared to matched females.Interestingly, significantly increased expression of CDX2 and VEGF (figure1) was noted inthe pre-malignant mucosa of males whereas decreased expression of CDX2 and no changein VEGF expression was noted in in females. Conclusions: We, herein, provide compellingevidence of the gender paradox being related to alterations in the initiation→progressiontransition. This paradox may be related to CDX2 and VEGF augmentation in males. The latteris consistent with our recent report on gender specificity in micro-circulatory abnormalities inearly colon carcinogenesis (Cancer Prev Res, 2010). Our work underscores the need toconsider colon carcinogenesis in males and females as being distinct and therefore, tailoringchemoprevention strategies accordingly.The gender paradox in colorectal carcinogenesis

Gender differential expresison of CDX2 and VEGF during colorectal carcinogenesis

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Functionality of EGFR/IRS-4 System in Rat Colonocytes and Its Involvementin the Human Colon CancerI. Román-Curto, Dolores Fernández-Moreno, Borja Hernandez Breijo, Maria EncarnacionFernandez Contreras, Mercedes Guijarro Rojas, Fernando Noguerales-Fraguas, Javier P.Gisbert, Luis González Guijarro

Background: Insulin receptor substrate proteins (IRSs), cytoplasmic adaptors that organizesignaling complexes downstream of activated cell surface receptors, are implicated in mediat-ing signals of tumor cells. Recently, it has been shown that IRS-1, IRS-2, and IRS-4 wereeach overexpressed in 80% of the hepatocellular carcinoma samples, and we have previouslydemonstrated that IRS-4 is an essential protein for the proliferation/differentiation of HepG2human hepatoblastoma cells. On the other hand, colorectal cancer is one of the most frequentin developed countries, although its treatment remains poor and systemic therapies havebeen relative ineffective. Aim: To study IRS-4 expression in several experimental models ofcolon cancer and human samples. Methods: We employed: i) several tumor colon lines(HCT116, HT29, LOVO, MAWI, COLO205, RKO16), ii) rat colonocytes stimulated In Vivo(for 30 min) with epidermal growth factor (EGF), iii) samples of human polyps, and iv)samples of human colorectal tumor and normal adjacent mucosa. IRS-4 and EGFR wereanalyzed by Western blotting using specific antibodies. Immunoprecipitation assays werecarried out with anti-pTyr. Immunohistochemistry experiments were performed using anti-IRS-4 and anti-EGFR. Results and conclusions: Immunohistochemistry results in humancolon mucosa showed that in healthy tissue adjacent to tumor, IRS-4 is located in coloncrypts, mainly in the nuclei of crypt colonocytes; however, in tumor tissue, IRS-4 expressionis even higher and the protein is located in crypt colonocytes cytoplasm, mainly in apicalside. In both kinds of tissues, EGFR is located in the cytoplasm. Interesting, IRS-4 isdramatically expressed in human polyp samples and in all tumor colon lines. Moreover, inhuman colon samples, IRS-4 is constitutively phosphorylated in tyrosine residues which areconsidered a hallmark of their activation. Rat colonocytes stimulated In Vivo with EGFshowed an increase in p-Tyr (PY99) phosphorylation profile, a decrease in EGFR expressionand an increase in IRS-4 (130 kDa) proteolysis, which suggest an activation of the EGFReceptor/IRS-4 signaling system after EGF stimulus. Given the relevance of EGF in mosthuman tumors, we suggest the importance of the EGFR/IRS-4 system in rat colonocytesand its involvement in the human colon cancer

S-817 AGA Abstracts

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MiR196a Plays a Critical Role in Regulating Small Intestinal NeuroendocrineTumor Proliferation and Metastasis via HOX/AKT Pathway ActivationMark Kidd, Ben J. Lawrence, Daniele Alaimo, Bernhard Svejda, Davinderpal Singh,Roswitha Pfragner, Irvin Modlin

Background: MicroRNAs (miRNAs) regulate cell proliferation, differentiation, and apoptosisand can function as tumor suppressor genes or oncogenes. In neuroendocrine tumors, themuscle-specific miRNA-133a, which regulates myocyte proliferation, was identified to bedownregulated in metastasis. We used a global approach to identify miRNAs and theircorresponding mRNA targets that were specifically up-regulated during proliferation andmetastasis in small intestinal neuroendocrine tumors and examined the effect of knock-down of specific miRNAs on neuroendocrine tumor cell line proliferation and signalingpathway activation. Methods: Seven normal mucosa, 4 localized small intestinal neuroendoc-rine tumors and 7 carcinoid metastases to the liver as well as 4 neuroendocrine tumor celllines (KRJ-I, P-STS (both localized) and L-STS/H-STS (metastatic)) were examined using amiRNA _v1 (Afymmetrix) approach (847 miRNAs). Differences in miRNA expression werequantitated using PARTEK (cluster analysis based on p<0.05; f value (fold value) rangingfrom -2 to +2). Real-time PCR was undertaken to confirm miRNA levels in tumor samples(normalized to RNU24 and HY3). For physiological studies, miR196a was knocked downin KRJ-I andH-STS using ExiqonmiRNA antisense and proliferation (WST-1),miR196a targettranscript alterations (HOXB6,B7,C8) and AKT activation (pSER473/THR308) determined(western blot). Results: Localized carcinoids expressed 141 miRNAs, none of which wereexclusively present in these tumors. Comparing primary tumors with metastases identified12 miRNAs with significantly increased expression (f-value >2, p<0.05). Real-time PCRconfirmed over-expression of miR196a in malignant samples (300-3000-fold in lymph nodeand liver metastases). This miRNA was also differentially expressed between the localizedand metastatic cell lines (75-fold in L-/H-STS cell lines). Knock-down of miR196a (to <2%of metastases levels) decreased cell proliferation in both KRJ-1 and H-STS but the effect wasmore pronounced in the latter, 23±5%. A combination of RAD001 and knockdown inhibitedproliferation even more significantly (41%, p<0.05). Knockdown was associated with reversalof expression of (HOXB6, B7 and C8) (by real-time PCR). Western blot confirmed decreasedpSER473 in both cell lines in response to knockdown. Conclusion: These results identifieda small focus of miRNA (miR196a) that are upregulated in malignant small intestinal neuroen-docrine tumors. This microRNA functions to alter neuroendocrine tumor cell line prolifera-tion via the AKT pathway and synthesis of HOXB/C genes. Dual targeting both miR196 andmTOR appears to be an effective mechanism to inhibit small intestinal neuroendocrinetumor/carcinoid proliferation.

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MicroRNA-200c/141 Cluster as a Control Switch Between Epithelial-to-Mesenchymal Transition (EMT) and Mesenchymal-to-Epithelial Transition(MET) in Human Colorectal Cancer MetastasisKeun Hur, Masanobu Takahashi, Francesc Balaguer, Takeshi Nagasaka, Hiromichi Hemmi,Minoru Koi, C. Richard Boland, Ajay Goel

Background: The development of metastases is one of the main causes of the cancer-relateddeath in colorectal cancer (CRC). The epithelial-to-mesenchymal transition (EMT) occursduring tumor progression, which provides cancer cells with invasive and metastatic proper-ties. The molecular mechanisms by which colorectal cancer cells exploit the hepatic micro-environment for selective growth and survival remain obscure. Recently, loss of expressionof the miR-200 family of microRNAs has been linked to an aggressive cancer phenotype inbreast cancers. In this context, miR-200 families of miRNAs are proposed to inhibit EMTprocess by targeting the E-cadherin transcriptional repressors, ZEB1 and ZEB2. AlthoughEMT plays a central role in metastasis, the contribution of miR-200 family members in thedevelopment of distant metastasis in CRC remains unclear. Aim: This study aimed todetermine the role of miR-200 family members (miR-200b, miR-200c, miR-141 and miR-429) in CRC metastasis development. Materials and Methods: We analyzed a panel ofCRC cell lines with different metastatic potential (HCT116, SW480 and SW620), as wellas clinical specimens from 55 patients with primary CRC and matched liver metastasis tissues.MicroRNAs expression of miR-200b, miR-200c, miR-141 and miR-429 was determined byquantitative real-time PCR and the data were normalized relative to miR-16 expression.Results: Lower levels of miR-200c/141 cluster expression were observed in SW620 (derivedfrom lymph node metastasis) compared to HCT116 and SW480 cell lines (derived fromprimary CRCs), but no significant changes in miR-200b/429 expression were observed inany of the cell lines.Whenwe analyzedmiR-200 family expression levels in clinical specimens,the relative levels of miR-200c expression were progressively lower with higher tumor stagesin primary CRC tissues (P<0.05). However, miR-200c expression was significantly up-regulated in liver metastasis compared to the correspondingmatched primary CRC (P<0.001).Similar observations were made for miR-141 expression in metastatic liver foci comparedto the correspondingmatched primary CRC. Conclusions: This study provides novel insightsinto the involvement of miR-200/141 family members in the development of CRC metastasis.The decreased expression of the miR-200/141 cluster in primary CRCs supports the participa-tion of these miRNAs in the EMT process. In contrast, the increased expression of the miR-200/141 cluster in liver metastasis suggests a potential role in the initiation of mesenchymal-to-epithelial transition (MET) process, in which increased expression of these miRNAsfacilitates the enhanced proliferation of these metastatic tumor cells following their settlementin the liver.

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