Shuiping Tu

Identification of gastric cancer stem cells using the cell surface marker CD44.

Cancer stem cells (CSCs) have been defined as a unique subpopulation in tumors that possess the ability to initiate tumor growth and sustain tumor self‐renewal. Although the evidence has been provided to support the existence of CSCs in various solid tumors, the identity of gastric CSCs has not been reported. In this study, we have identified gastric cancer‐initiating cells from a panel of human gastric cancer cell lines using cell surface marker CD44. Among six gastric cancer cell lines, three lines MKN‐45, MKN‐74, and NCI‐N87 had a sizeable subpopulation of CD44(+) cells, and these cells showed spheroid colony formation in serum‐free media in vitro as well as tumorigenic ability when injected into stomach and skin of severe combined immunodeficient (SCID) mice in vivo. The CD44(+) gastric cancer cells showed the stem cell properties of self‐renewal and the ability to form differentiated progeny and gave rise to CD44(−) cells. CD44 knockdown by short hairpin RNA resulted in much reduced spheroid colony formation and smaller tumor production in SCID mice, and the CD44(−) populations had significantly reduced tumorigenic ability in vitro and in vivo. Other potential CSC markers, such as CD24, CD133, CD166, stage‐specific embryonic antigen‐1 (SSEA‐1), and SSEA‐4, or sorting for side population did not show any correlation with tumorigenicity in vitro or in vivo. The CD44(+) gastric cancer cells showed increased resistance for chemotherapy‐ or radiation‐induced cell death. These results support the existence of gastric CSCs and may provide novel approaches to the diagnosis and treatment of gastric cancer. Stem Cells 2009;27:1006–1020

Overexpression of Interleukin-1β Induces Gastric Inflammation and Cancer and Mobilizes Myeloid-Derived Suppressor Cells in Mice

Polymorphisms of interleukin-1beta (IL-1beta) are associated with an increased risk of solid malignancies. Here, we show that stomach-specific expression of human IL-1beta in transgenic mice leads to spontaneous gastric inflammation and cancer that correlate with early recruitment of myeloid-derived suppressor cells (MDSCs) to the stomach. IL-1beta activates MDSCs in vitro and in vivo through an IL-1RI/NF-kappaB pathway. IL-1beta transgenic mice deficient in T and B lymphocytes develop gastric dysplasia accompanied by a marked increase in MDSCs in the stomach. Antagonism of IL-1 receptor signaling inhibits the development of gastric preneoplasia and suppresses MDSC mobilization. These results demonstrate that pathologic elevation of a single proinflammatory cytokine may be sufficient to induce neoplasia and provide a direct link between IL-1beta, MDSCs, and carcinogenesis.

Global Hypomethylation of Genomic DNA in Cancer-Associated Myofibroblasts

Global hypomethylation has long been recognized as a feature of the malignant epithelial component in human carcinomas. Here we show evidence for this same type of epigenetic alteration in cancer-associated stromal myofibroblasts. We used methylation-sensitive SNP array analysis (MSNP) to profile DNA methylation in early-passage cultures of stromal myofibroblasts isolated from human gastric cancers. The MSNP data indicated widespread hypomethylation in these cells, with rare focal gains of methylation, conclusions that were independently validated by bisulfite sequencing and by a methylation-sensitive cytosine incorporation assay. Immunohistochemistry with anti-5-methylcytosine (anti-5-methyl-C) in a series of gastrectomy specimens showed frequent loss of methylation in nuclei of both the malignant epithelial cells and alpha-smooth muscle actin (ASMA)-positive stromal myofibroblasts of both intestinal-type and diffuse carcinomas. We confirmed this phenomenon and established its onset at the stage of noninvasive dysplastic lesions by immunohistochemistry for anti-5-methyl-C in a transgenic mouse model of multistage gastric carcinogenesis. These findings indicate similar general classes of epigenetic alterations in carcinoma cells and their accompanying reactive stromal cells and add to accumulating evidence for biological differences between normal and cancer-associated myofibroblasts.

IFN-γ Inhibits Gastric Carcinogenesis by Inducing Epithelial Cell Autophagy and T-Cell Apoptosis

IFN-γ mediates responses to bacterial infection and autoimmune disease, but it is also an important tumor suppressor. It is upregulated in the gastric mucosa by chronic Helicobacter infection; however, whether it plays a positive or negative role in inflammation-associated gastric carcinogenesis is unexplored. To study this question, we generated an H(+)/K(+)-ATPase-IFN-γ transgenic mouse that overexpresses murine IFN-γ in the stomach mucosa. In contrast to the expected proinflammatory role during infection, we found that IFN-γ overexpression failed to induce gastritis and instead inhibited gastric carcinogenesis induced by interleukin-1beta (IL-1β) and/or Helicobacter infection. Helper T cell (Th) 1 and Th17 immune responses were inhibited by IFN-γ through Fas induction and apoptosis in CD4 T cells. IFN-γ also induced autophagy in gastric epithelial cells through increased expression of Beclin-1. Finally, in the gastric epithelium, IFN-γ also inhibited IL-1β- and Helicobacter-induced epithelial apoptosis, proliferation, and Dckl1(+) cell expansion. Taken together, our results suggest that IFN-γ coordinately inhibits bacterial infection and carcinogenesis in the gastric mucosa by suppressing putative gastric progenitor cell expansion and reducing epithelial cell apoptosis via induction of an autophagic program.

Inactivating cholecystokinin-2 receptor inhibits progastrin-dependent colonic crypt fission, proliferation, and colorectal cancer in mice

Hyperproliferation of the colonic epithelium, leading to expansion of colonic crypt progenitors, is a recognized risk factor for colorectal cancer. Overexpression of progastrin, a nonamidated and incompletely processed product of the gastrin gene, has been shown to induce colonic hyperproliferation and promote colorectal cancer in mice, but the mechanism of pathogenesis has not been defined. Cholecystokinin-2 receptor (CCK2R) is the primary receptor for cholecystokinin (CCK) and amidated gastrin. Here, we show that Cck2r was expressed in murine colonic crypts and upregulated in the transgenic mice that overexpress human progastrin. Murine deletion of Cck2r abrogated progastrin-dependent increases in colonic proliferation, mucosal thickness, and beta-catenin and CD44 expression in the colon tumor. In addition, either deletion or antagonism of Cck2r resulted in the inhibition of progastrin-dependent increases in progenitors expressing doublecortin and CaM kinase-like-1 (DCAMKL1), stem cells expressing leucine rich repeat-containing G protein-coupled receptor 5 (LgR5), and colonic crypt fission. Furthermore, in the azoxymethane mouse model of colorectal carcinogenesis, Cck2r deletion in human progastrin-overexpressing mice resulted in markedly decreased aberrant crypt foci formation and substantially reduced tumor size and multiplicity. Taken together, these observations indicate that progastrin induces proliferative effects, primarily in colonic progenitor cells, through a CCK2R-dependent pathway. Moreover, our data suggest that CCK2R may be a potential target in the treatment or prevention of colorectal cancer.

Gastrin Is an Essential Cofactor for Helicobacter-Associated Gastric Corpus Carcinogenesis in C57BL/6 Mice

We have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in H. felis-infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice.

Fibroblastic Colony‐Forming Unit Bone Marrow Cells Delay Progression to Gastric Dysplasia in a Helicobacter Model of Gastric Tumorigenesis

Bone marrow mesenchymal stem cells (MSCs) have been shown to have immune modulatory effects. Despite efforts to identify these cells in vivo, to date, MSCs have been defined mainly by their in vitro cell characteristics. Here, we show that Lin−CD44hiSca1−cKit+CD34− cells make up ∼0.5%–1% of murine whole bone marrow cells and yield nearly an equal amount of fibroblastic colony‐forming units (CFU‐F) as whole bone marrow. After transplantation into lethally irradiated recipients, Lin−CD44hiSca1−cKit+CD34− cells engrafted in the bone marrow long‐term and demonstrated characteristics of MSCs, including capacity to differentiate into osteoblasts and adipocytes. To examine whether Lin−CD44hiSca1−cKit+CD34− cells have immune modulatory effects, in vitro coculture with activated CD4+ T‐cells resulted in decreased Th17 cell differentiation by Lin−CD44hiSca1−cKit+CD34− cells. Furthermore, serial infusions with Lin−CD44hiSca1−cKit+CD34− cells reduced the progression to low‐grade gastric dysplasia in mice infected with chronic Helicobacter felis (p = .038). This correlated with reduced gastric interleukin (IL)‐17F, IL‐22, and ROR‐γt gene expression in responding mice (p < .05). These data suggest that bone marrow derived Lin−CD44hiSca1−cKit+CD34− cells have characteristics of MSCs and reduce progression of early gastric tumorigenesis induced by chronic H. felis infection. The prevention of dysplastic changes may occur through inhibition of Th17‐dependent pathways. STEM CELLS 2009;27:2301–2311

Tu1893 Tumor Suppressor XAF1 and TRAIL Cooperate to Inhibit Migration and Invasion of Colon Cancer Cells

Aim: The aim of the present study was to evaluate whether chronomodulated administration of capecitabine would reduce toxicity of the drug in patients with metastatic colorectal cancer. Patients & Methods: 27 patients with advanced colorectal cancer were randomized to receive capecitabine (2500mg/KOF daily) either in standard administration with 50% of the dose in themorning and 50% in the evening separated by 12 hours or as chronomodulated dose-regime with 25% morning-dose and 75% late evening-dose. Treatment was continued until thirteen treatment-cycles were finished or progress of cancer or limitation of therapy due to side effects occurred. Results: Overall, response rates to chemotherapy were similar in both treatment groups (p= 0,296). However, within the group of patients with chronomodulated application of capecitabine, reduction of drug-doses due to side effects was less frequently necessary (14.8 vs. 19.3%, p=0,026) and more patients were able to finish all thirteen chemotherapy-cycles (41.7 vs. 7.1%, p=0,007). While there was no statistically significant difference in the frequency of hand-foot-syndrome in both treatment-arms (51.7 vs. 41.5%, p=0,119) other side effects as nightly nausea, tiredness and sleeplessness were significantly reduced in patients receiving chronomodulated therapy (p=0,035, p=0,001 and 0,009, respectively). Additionally, there was a trend to lower frequency of nausea, diarrhea and stomatitis within this group compared to standard treatment (9.0 vs. 19.5%, 19.1 vs. 25.6%, and 4.5 vs. 8.5%) Conclusion: The chronomodulated capecitabine schedule achieved similar response rate and better tolerability regarding various side effects compared with standard application of the drug in patients with colorectal cancer. This schedule could enable patients to sustain on capecitabine therapy for a longer time-period.

Mo1938 Survivin Inhibitor YM155 Inhibits Cell Miration and Invasion in Gastric and Colon Cancer Cells Through Inhibition of Metastasis-Related Genes

Background: Aurora kinase A (AURKA) is a serine-threonine kinase that is amplified and/ or overexpressed in several cancers. STAT3 is an important transcription factor that regulates several cytokines, growth factors, and proteins that modulate various cellular events; including survival, cell cycle, invasion, and angiogenesis. Methods and Results: In this study, by using in vitro human cell models of upper gastrointestinal adenocarcinomas (UGCs), we investigated whether AURKA can regulate STAT3.WB analysis show that AURKA overexpression led to increase in p.STAT3 (Tyr705) in FLO-1 and AGS cells. On the other hand, AURKA knockdown by siRNA resulted in decrease in p.STAT3 (Tyr705) in FLO-1 and MKN45 cells. Further, STAT3 transcriptional activity was significantly (P=0.01) up regulated in response to AURKA overexpression in the three cell lines. Consistently, AURKA overexpression significantly (P=0.01) enhanced STAT3 nuclear translocation in AGS cells, whereas AURKA knockdown significantly (P=0.01) decreased the nuclear positive stating of STAT3 in FLO-1 cells. By using alisertib (0.5 μM), an investigational AURKA specific inhibitor, we demonstrate that AURKA inhibition results in decreased phosphorylation of STAT3 (Tyr705) as early as 30 min after treatment in all cell lines. Additionally, extended treatment time points of 72 h reduced the phosphorylation of STAT3 and importantly reduced the expression of its pro-survival targets BCL2 and Mcl1 in AGS and FLO-1 cells. Clonogenic survival assay data showed that single dose treatment of alisertib for 24 h (0.5 μM), led to significant decrease in colonies intensity in AGS and FLO-1 cells. To elucidate how AURKA regulates STAT3, we investigated whether JAK2 kinases expressions changed in response to AURKA

937 Survivin Inhibitor YM155 Induces Apoptosis and Inhibits Tumor Growth of Human Gastric Carcinoma

G A A b st ra ct s size (from 6,4±0,4 mm to 2,3 ±0,1 mm, p,0,01), in comparison with untreated mice. In contrast, tumor density and growth was enhanced by systemic delivery of the lymphangiogenic factor VEGF-C, which in turn increased lymphangiogenesis within the same regions. HILEC isolated from colorectal cancer biopsies showed increased growth rate and capacity to undergo tubulogenesis in vitro (38 ±1,5 number of tumoral tubes vs 14 ±0,5 number of NL tubes, p,0,01) compared to healthy control tissues. Conclusion: Our findings demonstrate that VEGFC/VEGFR-3-dependent lymphangiogenesis plays a role not only in metastasis dissemination, but also in colitis-associated tumor growth. Therefore, the lymphatic vasculature may be a promising target for the prevention and the treatment of IBD-related Colorectal Cancer.