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M1621 Gene Expression Profile in Colon Cancer Cells in Response to Troglitazone: Impact of XIAP Expression
AGA Abstracts
M1620
of PTEN is its role on cell motility and adhesion regulation. E-cadherin is an adhesion molecule in epithelial cells and its alternation can contribute to tumor invasiveness and metastasis. Aims : To determine the role that PTEN gene alterations play in E-cadherin expression, we analyzed 48 primary gastric carcinomas for LOH on 10q23 and for mutation in all 9 coding exons and compared them with the immunohistochemical profile of Ecadherin. We also analyzed the clinicopathologic significance of allelic imbalance or mutation of PTEN tumor suppressor genes and E-cadherin expression in sporadic gastric carcinomas. Methods : Genomic DNA from 48 gastric carcinoma cases was extracted from paraffin blocks, and PCR was performed to determine the loss of heterozygosity for DNA markers around the PTEN genes and all 9 coding axons of PTEN. Immunohistochemical staining for Ecadherin using tissue microarray was also performed. Results : LOH for DNA markes surrounding PTEN was observed in 19 (40.4%) of the 48 cases. Five cases (10.4%) showed somatic PTEN mutations. Tumors with LOH on 10q23 exhibited loss of E-cadherin immunoreactivity (p<0.05). Tumors with loss of E-cadherin immunoreactivity had a tendency to show single cell infiltrative pattern and smaller size (p<0.05). Conclusions : Our results indicate that tumors with LOH at 10q23 had a higher tendency for loss of E-cadherin immunoreactivity.
Caldesmon Suppresses Cancer Cell Invasion By Regulating Podosome/ Invadopodium Formation Toshiyuki Yoshio, Tsuyoshi Morita, Masahiko Tsujii, Jumpei Kondo, Sachiko Nakajima, Shuji Ishii, Shinichiro Shinzaki, Satoshi Egawa, Tsutomu Nishida, Yoshimi Kakiuchi, Hideki Iijima, Shingo Tsuji, Kenji Sobue, Norio Hayashi Cell invasion, one of the hallmarks of cancer progression, is mediated, at least in part, by podosomes and invadopodia. These protrusions from the ventral surface of the plasma membrane are highly dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) by secreting matrix metalloproteases. Thus, podosomes/invadopodia permit cells to cross the basement membrane and invade tissues. These adhesive structures are referred to as “podosomes” in monocyte-derived cells, osteoclasts, smooth muscle cells, and Src- or Rous sarcoma virus (RSV)-transformed cells and as “invadopodia” in cancer cells. Because podosomes and invadopodia appear to have similar morphologies, functions, and molecular compositions, they are considered related structures. We recently reported that the actinbinding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon's involvement in podosome/invadopodium-mediated invasion in transformed and cancer cells. We first analyzed the relationship between the expression levels of caldesmon and podosome/invadopodium formation in rat fibroblast (3Y1), RSV-transformed 3Y1 (BY1), human colon carcinoma (HCA7), murine melanoma (B16F10), human breast cancer (MB435s), and rat breast cancer (MTC) cell lines. The caldesmon expression level in BY1 cells was markedly lower than in 3Y1 cells. In BY1 cells, podosomes containing dot-shaped F-actin clusters were predominantly observed in place of stress fibers. About the cancer cells, both HCA7 and B16F10 cells expressed extremely low levels of caldesmon and formed invadopodia, whereas MB435s and MTC cells, expressing higher amounts of caldesmon, exhibited fully extended stress fibers without invadopodia. These results suggest that the expression level of caldesmon inversely correlated with podosomes/invadopodium formation in transformed and cancer cell lines. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and led to the formation of new cortical actin bundles with thin stress fibers in the BY1, HCA7 and B16F10. In the consequence, their ECM degradation activities and the invasion activities were decreased. Conversely, the depletion of caldesmon induced a neumorous number of small podosomes/invadopodia, facilitated the degradation activity and facilitated the cell invasion. On the contrary, cell migration activity was not infuluenced ether in the caldesmon overexpressed cells nor the caldesmon depleted cells. Taken together, the expression levels of caldesmon are inversely associated with cell invasiveness, and our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.
M1623 Differential Expression of iL10r2, STAT1 and STAT3 in Colorectal Cancer Oliver Movadat, Sabine G. Gruber, Chukwuma A. Agu, Brigitte Marian, Friedrich Wrba, Christoph Gasche BACKGROUND Patients with IBD carry an increased risk for the development of colorectal cancer (CRC). IL-10-deficient mice exhibit enterocolitis and CRC, similar to IBD-associated cancer in humans; this suggests an involvement of IL-10 and its signaling cascade in the development of inflammation-driven CRC. In fact, excess expression of STAT3 has tumor promoting effects (Bromberg J, Cell 1999) whereas SOCS1 and SOCS3 are considered as tumor suppressor genes (Hanada T, JEM 2006 and Rigby RJ, Oncogene 2007). We have recently identified IL10R1 variants that protect from CRC (Gasche C, DDW 2007). To improve our understanding of the role of IL-10 signaling in colorectal carcinogenesis, we analyzed the differential expression of IL-10 pathway genes. METHODS Colonic normal/ tumor tissue pairs were sampled from 25 patients (66±11years) with CRC upon surgery, frozen in OCT and stored at -80°C. Blocks were cut into 10µm sections, RNA was extracted with TRIzol and 1µg was reverse transcribed using oligo (dt) primers (Invitrogen, ThermoScript). Expression levels of IL-10 pathway genes and c-myc (as positive control) were analyzed by real time PCR (ABI Prism 7700), blotted for ct (cycle threshold) values and compared by paired t-test (*p<0.01; **p<0.001). RESULTS: (table, ct values): In normal colonic tissue, c-myc mRNA was strongly expressed, followed by STAT1, SOCS1, IL10R2, STAT3, and SOCS3. IL-10 and IL10R1 mRNA was detected at a low level. An increase in mRNA expression in tumor tissue was detected for c-myc, STAT1, STAT3 and IL10R2, but not for IL-10, IL-10R1, or both SOCS. CONCLUSION: The overexpression of STAT1, STAT3 and IL10R2 mRNA in colorectal tumors is indicative for a tumor promoting effect of these genes in CRC thereby linking the IL-10 pathway to human colon carcinogenesis. As the signal cascade seems to act independent of IL10R1 or its ligand, alternative IL10R2 activation, e.g. through IL-22, IL-26,IL-28A, IL-28B, or IL-29, may play a role.
M1621 Gene Expression Profile in Colon Cancer Cells in Response to Troglitazone: Impact of XIAP Expression Liang Qiao, Gloria HY Li, Yun Dai, Zesong Li, Bing Zou, Juan Ma, Marie CM Lin, Benjamin C.Y. Wong BACKGROUND AND OBJECTIVES We previously observed a marked synergism between PPARγ ligands and XIAP down-regulation in colon cancer. In order to explore the possible molecular mechanisms, we performed a microarray study in HCT116 cells treated with or without PPARγ ligand troglitazone. MATERIALS AND METHODS HCT116-XIAP+/+ and XIAP-/- cells were treated with or without 50 µM troglitazone for 48 h. Gene expression changes were detected by Human Genome U133 Plus 2.0 microarray and selected genes were validated by RT-PCR and Western blot. RESULTS Using the gene expression profile in HCT116-XIAP+/+ cells as the base line, 74 genes were found to be up-regulated and 43 genes down-regulated in HCT116-XIAP-/- cells, all by ≥4-folds. These genes can be classified into a wide range of functional classes, but we focused on genes related to angiogenesis, apoptosis, and proliferation. Among the up-regulated genes are two pro-apoptotic genes (PTEN, IGFBP3) and one pro-proliferation gene (ENC1). Five genes were down-regulated including two pro-proliferation genes (MIA, Gas6), one pro-angiogenesis gene (IL-8), one anti-angiogenesis gene (THBS1), and one anti-apoptosis gene (BIRC4). In HCT116-XIAP+/ + cells treated with troglitazone, 38 genes were up-regulated and 42 genes were downregulated by ≥8-folds. In contrast, in the HCT116-XIAP-/- cells treated with troglitazone, 70 genes were up-regulated and 64 genes were down-regulated by ≥8-fold. Relative to XIAP+/+ cells treated with troglitazone, 19 genes were differentially expressed in HCT116XIAP-/- cells treated with troglitazone, of which 13 genes were up-regulated and 6 genes were down-regulated. Among the up-regulated genes are 2 anti-angiogenesis genes (TNFSF15, Jagged1), 7 pro-apoptosis genes (RHOT1, FOXO3A, AD7C-NTP, RAF1, ERC1, IGFBP3, ATXN1), and 4 anti-proliferation genes (EVI5, JAG1, SPEG, DIRAS3, GLMN). Two upregulated genes (TNFSF1 and Jagged1) were also functionally classified as anti-angiogenesis, pro-apoptosis, and anti-proliferation. Among the down-regulated genes are 1 anti-angiogenesis gene (THBS1), 1 pro-angiogenesis gene (IL-8), 1 anti-apoptosis gene (BIRC4), 1 antiproliferation gene (DAB2), and 2 pro-proliferation genes (FGF9, CSF2). CONCLUSION XIAP is involved in the maintenance of homeostasis by controlling apoptosis, angiogenesis and cell proliferation. Down-regulation of XIAP results in a state that favors an increased propensity to apoptosis and a decreased proliferation potential. Down-regulation of XIAP coupled with troglitazone treatment is associated with changes of genes expression that favor increased tendency to apoptosis, decreased cell proliferation and angiogenesis potential.
M1624 Identification of Oncogene By Retroviral Expression Screening in Gallbladder Cancer Hisashi Hatanaka, Kiichi Tamada, Kentaro Sugano, Hiroyuki Mano Background: Gallbladder cancer (GBC) is an uncommon but highly fatal malignancy in humans. GBC is prevalent in South American and Asian countries such as Japan and Korea. In the United States, GBC is the fifth most common gastrointestinal cancer, and fewer than 5000 new cases are diagnosed each year. Although genetic alternations in TP53 or KRAS may contribute to the development of a part of GBC, only a little is yet understood for the genes proved to be involved in its carcinogenesis. Objective: To identify novel cancerpromoting genes in GBC, we here took an advantage of functional screening of oncogenes with a cDNA expression library. Methods, Results: From a surgically operated clinical specimens of GBC, more than 3.2 x 105 of independent cDNA clones were generated and inserted into a retroviral plasmid, pMXS. A recombinant ecotropic retroviral library was generated based on such plasmid library, which was then used to infect 3T3 fibroblasts for the focus formation assay. A number of transformed foci were identified, and we could recover retroviral inserts by PCR from such foci. One of these cDNAs was shown to encode a G-protein coupled transmembrane receptor which had not been known to carry oncogenic activity. The oncogenic potential of this gene was confirmed In Vitro with the focus formation assay as well as soft agar-growth assay, and also In Vivo with a tumorigenicity assay in nude mice. Since our identified cDNA sequence has no point mutations, we suppose that an overexpression of this receptor may contribute to transformation. Measuring the mRNA level of this receptor gene among seven cases of biliary tract cancer, 80 cases of colorectal cancer and 89 cases of gastric cancer has revealed a significantly higher expression of the gene in the cancer tissues compared to the control ones among colorectal (p=0.004) and
M1622 Loss of Heterozygosity of PTEN Tumor Supressor Gene and Expression Pattern of E-Cadherin in Sporadic Gastric Carcinoma Suck Chei Choi, Hun Soo Kim, Kyu Sang Song, Soo Cheon Chae, Young-Mi Lee, Tae Hyeon Kim, Yong Sung Kim, Chang Soo Choi, Ki Hoon Kim, Ki Jung Yun Background : The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) on chromosome 10q23 has been known to be somatically mutated in association with frequent LOH at 10q23.3 in various tumors. One of the major function
AGA Abstracts
A-384
M1620
of PTEN is its role on cell motility and adhesion regulation. E-cadherin is an adhesion molecule in epithelial cells and its alternation can contribute to tumor invasiveness and metastasis. Aims : To determine the role that PTEN gene alterations play in E-cadherin expression, we analyzed 48 primary gastric carcinomas for LOH on 10q23 and for mutation in all 9 coding exons and compared them with the immunohistochemical profile of Ecadherin. We also analyzed the clinicopathologic significance of allelic imbalance or mutation of PTEN tumor suppressor genes and E-cadherin expression in sporadic gastric carcinomas. Methods : Genomic DNA from 48 gastric carcinoma cases was extracted from paraffin blocks, and PCR was performed to determine the loss of heterozygosity for DNA markers around the PTEN genes and all 9 coding axons of PTEN. Immunohistochemical staining for Ecadherin using tissue microarray was also performed. Results : LOH for DNA markes surrounding PTEN was observed in 19 (40.4%) of the 48 cases. Five cases (10.4%) showed somatic PTEN mutations. Tumors with LOH on 10q23 exhibited loss of E-cadherin immunoreactivity (p<0.05). Tumors with loss of E-cadherin immunoreactivity had a tendency to show single cell infiltrative pattern and smaller size (p<0.05). Conclusions : Our results indicate that tumors with LOH at 10q23 had a higher tendency for loss of E-cadherin immunoreactivity.
Caldesmon Suppresses Cancer Cell Invasion By Regulating Podosome/ Invadopodium Formation Toshiyuki Yoshio, Tsuyoshi Morita, Masahiko Tsujii, Jumpei Kondo, Sachiko Nakajima, Shuji Ishii, Shinichiro Shinzaki, Satoshi Egawa, Tsutomu Nishida, Yoshimi Kakiuchi, Hideki Iijima, Shingo Tsuji, Kenji Sobue, Norio Hayashi Cell invasion, one of the hallmarks of cancer progression, is mediated, at least in part, by podosomes and invadopodia. These protrusions from the ventral surface of the plasma membrane are highly dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) by secreting matrix metalloproteases. Thus, podosomes/invadopodia permit cells to cross the basement membrane and invade tissues. These adhesive structures are referred to as “podosomes” in monocyte-derived cells, osteoclasts, smooth muscle cells, and Src- or Rous sarcoma virus (RSV)-transformed cells and as “invadopodia” in cancer cells. Because podosomes and invadopodia appear to have similar morphologies, functions, and molecular compositions, they are considered related structures. We recently reported that the actinbinding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon's involvement in podosome/invadopodium-mediated invasion in transformed and cancer cells. We first analyzed the relationship between the expression levels of caldesmon and podosome/invadopodium formation in rat fibroblast (3Y1), RSV-transformed 3Y1 (BY1), human colon carcinoma (HCA7), murine melanoma (B16F10), human breast cancer (MB435s), and rat breast cancer (MTC) cell lines. The caldesmon expression level in BY1 cells was markedly lower than in 3Y1 cells. In BY1 cells, podosomes containing dot-shaped F-actin clusters were predominantly observed in place of stress fibers. About the cancer cells, both HCA7 and B16F10 cells expressed extremely low levels of caldesmon and formed invadopodia, whereas MB435s and MTC cells, expressing higher amounts of caldesmon, exhibited fully extended stress fibers without invadopodia. These results suggest that the expression level of caldesmon inversely correlated with podosomes/invadopodium formation in transformed and cancer cell lines. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and led to the formation of new cortical actin bundles with thin stress fibers in the BY1, HCA7 and B16F10. In the consequence, their ECM degradation activities and the invasion activities were decreased. Conversely, the depletion of caldesmon induced a neumorous number of small podosomes/invadopodia, facilitated the degradation activity and facilitated the cell invasion. On the contrary, cell migration activity was not infuluenced ether in the caldesmon overexpressed cells nor the caldesmon depleted cells. Taken together, the expression levels of caldesmon are inversely associated with cell invasiveness, and our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.
M1623 Differential Expression of iL10r2, STAT1 and STAT3 in Colorectal Cancer Oliver Movadat, Sabine G. Gruber, Chukwuma A. Agu, Brigitte Marian, Friedrich Wrba, Christoph Gasche BACKGROUND Patients with IBD carry an increased risk for the development of colorectal cancer (CRC). IL-10-deficient mice exhibit enterocolitis and CRC, similar to IBD-associated cancer in humans; this suggests an involvement of IL-10 and its signaling cascade in the development of inflammation-driven CRC. In fact, excess expression of STAT3 has tumor promoting effects (Bromberg J, Cell 1999) whereas SOCS1 and SOCS3 are considered as tumor suppressor genes (Hanada T, JEM 2006 and Rigby RJ, Oncogene 2007). We have recently identified IL10R1 variants that protect from CRC (Gasche C, DDW 2007). To improve our understanding of the role of IL-10 signaling in colorectal carcinogenesis, we analyzed the differential expression of IL-10 pathway genes. METHODS Colonic normal/ tumor tissue pairs were sampled from 25 patients (66±11years) with CRC upon surgery, frozen in OCT and stored at -80°C. Blocks were cut into 10µm sections, RNA was extracted with TRIzol and 1µg was reverse transcribed using oligo (dt) primers (Invitrogen, ThermoScript). Expression levels of IL-10 pathway genes and c-myc (as positive control) were analyzed by real time PCR (ABI Prism 7700), blotted for ct (cycle threshold) values and compared by paired t-test (*p<0.01; **p<0.001). RESULTS: (table, ct values): In normal colonic tissue, c-myc mRNA was strongly expressed, followed by STAT1, SOCS1, IL10R2, STAT3, and SOCS3. IL-10 and IL10R1 mRNA was detected at a low level. An increase in mRNA expression in tumor tissue was detected for c-myc, STAT1, STAT3 and IL10R2, but not for IL-10, IL-10R1, or both SOCS. CONCLUSION: The overexpression of STAT1, STAT3 and IL10R2 mRNA in colorectal tumors is indicative for a tumor promoting effect of these genes in CRC thereby linking the IL-10 pathway to human colon carcinogenesis. As the signal cascade seems to act independent of IL10R1 or its ligand, alternative IL10R2 activation, e.g. through IL-22, IL-26,IL-28A, IL-28B, or IL-29, may play a role.
M1621 Gene Expression Profile in Colon Cancer Cells in Response to Troglitazone: Impact of XIAP Expression Liang Qiao, Gloria HY Li, Yun Dai, Zesong Li, Bing Zou, Juan Ma, Marie CM Lin, Benjamin C.Y. Wong BACKGROUND AND OBJECTIVES We previously observed a marked synergism between PPARγ ligands and XIAP down-regulation in colon cancer. In order to explore the possible molecular mechanisms, we performed a microarray study in HCT116 cells treated with or without PPARγ ligand troglitazone. MATERIALS AND METHODS HCT116-XIAP+/+ and XIAP-/- cells were treated with or without 50 µM troglitazone for 48 h. Gene expression changes were detected by Human Genome U133 Plus 2.0 microarray and selected genes were validated by RT-PCR and Western blot. RESULTS Using the gene expression profile in HCT116-XIAP+/+ cells as the base line, 74 genes were found to be up-regulated and 43 genes down-regulated in HCT116-XIAP-/- cells, all by ≥4-folds. These genes can be classified into a wide range of functional classes, but we focused on genes related to angiogenesis, apoptosis, and proliferation. Among the up-regulated genes are two pro-apoptotic genes (PTEN, IGFBP3) and one pro-proliferation gene (ENC1). Five genes were down-regulated including two pro-proliferation genes (MIA, Gas6), one pro-angiogenesis gene (IL-8), one anti-angiogenesis gene (THBS1), and one anti-apoptosis gene (BIRC4). In HCT116-XIAP+/ + cells treated with troglitazone, 38 genes were up-regulated and 42 genes were downregulated by ≥8-folds. In contrast, in the HCT116-XIAP-/- cells treated with troglitazone, 70 genes were up-regulated and 64 genes were down-regulated by ≥8-fold. Relative to XIAP+/+ cells treated with troglitazone, 19 genes were differentially expressed in HCT116XIAP-/- cells treated with troglitazone, of which 13 genes were up-regulated and 6 genes were down-regulated. Among the up-regulated genes are 2 anti-angiogenesis genes (TNFSF15, Jagged1), 7 pro-apoptosis genes (RHOT1, FOXO3A, AD7C-NTP, RAF1, ERC1, IGFBP3, ATXN1), and 4 anti-proliferation genes (EVI5, JAG1, SPEG, DIRAS3, GLMN). Two upregulated genes (TNFSF1 and Jagged1) were also functionally classified as anti-angiogenesis, pro-apoptosis, and anti-proliferation. Among the down-regulated genes are 1 anti-angiogenesis gene (THBS1), 1 pro-angiogenesis gene (IL-8), 1 anti-apoptosis gene (BIRC4), 1 antiproliferation gene (DAB2), and 2 pro-proliferation genes (FGF9, CSF2). CONCLUSION XIAP is involved in the maintenance of homeostasis by controlling apoptosis, angiogenesis and cell proliferation. Down-regulation of XIAP results in a state that favors an increased propensity to apoptosis and a decreased proliferation potential. Down-regulation of XIAP coupled with troglitazone treatment is associated with changes of genes expression that favor increased tendency to apoptosis, decreased cell proliferation and angiogenesis potential.
M1624 Identification of Oncogene By Retroviral Expression Screening in Gallbladder Cancer Hisashi Hatanaka, Kiichi Tamada, Kentaro Sugano, Hiroyuki Mano Background: Gallbladder cancer (GBC) is an uncommon but highly fatal malignancy in humans. GBC is prevalent in South American and Asian countries such as Japan and Korea. In the United States, GBC is the fifth most common gastrointestinal cancer, and fewer than 5000 new cases are diagnosed each year. Although genetic alternations in TP53 or KRAS may contribute to the development of a part of GBC, only a little is yet understood for the genes proved to be involved in its carcinogenesis. Objective: To identify novel cancerpromoting genes in GBC, we here took an advantage of functional screening of oncogenes with a cDNA expression library. Methods, Results: From a surgically operated clinical specimens of GBC, more than 3.2 x 105 of independent cDNA clones were generated and inserted into a retroviral plasmid, pMXS. A recombinant ecotropic retroviral library was generated based on such plasmid library, which was then used to infect 3T3 fibroblasts for the focus formation assay. A number of transformed foci were identified, and we could recover retroviral inserts by PCR from such foci. One of these cDNAs was shown to encode a G-protein coupled transmembrane receptor which had not been known to carry oncogenic activity. The oncogenic potential of this gene was confirmed In Vitro with the focus formation assay as well as soft agar-growth assay, and also In Vivo with a tumorigenicity assay in nude mice. Since our identified cDNA sequence has no point mutations, we suppose that an overexpression of this receptor may contribute to transformation. Measuring the mRNA level of this receptor gene among seven cases of biliary tract cancer, 80 cases of colorectal cancer and 89 cases of gastric cancer has revealed a significantly higher expression of the gene in the cancer tissues compared to the control ones among colorectal (p=0.004) and
M1622 Loss of Heterozygosity of PTEN Tumor Supressor Gene and Expression Pattern of E-Cadherin in Sporadic Gastric Carcinoma Suck Chei Choi, Hun Soo Kim, Kyu Sang Song, Soo Cheon Chae, Young-Mi Lee, Tae Hyeon Kim, Yong Sung Kim, Chang Soo Choi, Ki Hoon Kim, Ki Jung Yun Background : The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) on chromosome 10q23 has been known to be somatically mutated in association with frequent LOH at 10q23.3 in various tumors. One of the major function
AGA Abstracts
A-384