Timothy C. Wang

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

Gastric Cancer Stem Cells

Cancer stem cells are defined as the unique subpopulation in the tumors that possess the ability to initiate tumor growth and sustain self-renewal as well as metastatic potential. Accumulating evidence in recent years strongly indicate the existence of cancer stem cells in solid tumors of a wide variety of organs. In this review, we will discuss the possible existence of a gastric cancer stem cell. Our recent data suggest that a subpopulation with a defined marker shows spheroid colony formation in serum-free media in vitro, as well as tumorigenic ability in immunodeficient mice in vivo. We will also discuss the possible origins of the gastric cancer stem cell from an organ-specific stem cell versus a recently recognized new candidate bone marrow-derived cell (BMDC). We have previously shown that BMDC contributed to malignant epithelial cells in the mouse model of Helicobacter-associated gastric cancer. On the basis of these findings from animal model, we propose that a similar phenomenon may also occur in human cancer biology, particularly in the cancer origin of other inflammation-associated cancers. The expanding research field of cancer stem-cell biology may offer a novel clinical apparatus to the diagnosis and treatment of cancer.

Gastrin-mediated activation of cyclin D1 transcription involves β-catenin and CREB pathways in gastric cancer cells

Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G1-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho β-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of β-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of β-catenin and CREB pathways.

Kruppel-like factor 4 (KLF4) represses histidine decarboxylase gene expression through an upstream Sp1 site and downstream gastrin responsive elements

Histidine decarboxylase (HDC) is the enzyme that catalyzes the conversion of histidine to histamine, a bioamine that plays an important role in allergic responses, inflammation, neurotransmission, and gastric acid secretion. Previously, we demonstrated that gastrin activates HDC promoter activity in a gastric cancer (AGS-E) cell line through three overlapping downstream promoter elements. In the current study, we used the yeast one-hybrid strategy to identify nuclear factors that bind to these three elements. Among eight positives from the one-hybrid screen, we identified Kruppel-like factor 4 (KLF4) (previously known as gut-enriched Kruppel-like factor (GKLF)) as one factor that binds to the gastrin responsive elements in the HDC promoter. Electrophoretic mobility shift assays confirmed that KLF4 is able to bind all three gastrin responsive elements. In addition, transient cotransfection experiments showed that overexpression of KLF4 dose dependently and specifically inhibited HDC promoter activity. Regulation of HDC transcription by KLF4 was confirmed by changes in the endogenous HDC messenger RNA by KLF4 small interfering RNA and KLF4 overexpression. We further showed that KLF4 inhibits HDC promoter activity by competing with Sp1 at the upstream GC box and also independently by binding the three downstream gastrin responsive elements. Taken together, these results indicate that KLF4 can act to repress HDC gene expression by Sp1-dependent and -independent mechanisms.

K-ras Mutation Targeted to Gastric Tissue Progenitor Cells Results in Chronic Inflammation, an Altered Microenvironment, and Progression to Intraepithelial Neoplasia

Chronic infectious diseases, such as Helicobacter pylori infection, can promote cancer in a large part through induction of chronic inflammation. Oncogenic K-ras mutation in epithelial cells activates inflammatory pathways, which could compensate for a lack of infectious stimulus. Gastric histopathology and putative progenitor markers [doublecortin and calcium/calmodulin-dependent protein kinase-like 1 (Dcamkl1) and keratin 19 (K19)] in K19-K-ras-V12 (K19-kras) transgenic mice were assessed at 3, 6, 12, and 18 months of age, in comparison with Helicobacter felis-infected wild-type littermates. Inflammation was evaluated by reverse transcription-PCR of proinflammatory cytokines, and K19-kras mice were transplanted with green fluorescent protein (GFP)-labeled bone marrow. Both H. felis infection and K-ras mutation induced upregulation of proinflammatory cytokines, expansion of Dcamkl1(+) cells, and progression to oxyntic atrophy, metaplasia, hyperplasia, and high-grade dysplasia. K19-kras transgenic mice uniquely displayed mucous metaplasia as early as 3 months and progressed to high-grade dysplasia and invasive intramucosal carcinoma by 20 months. In bone marrow-transplanted K19-kras mice that progressed to dysplasia, a large proportion of stromal cells were GFP(+) and bone marrow-derived, but only rare GFP(+) epithelial cells were observed. GFP(+) bone marrow-derived cells included leukocytes and CD45(-) stromal cells that expressed vimentin or α smooth muscle actin and were often found surrounding clusters of Dcamkl1(+) cells at the base of gastric glands. In conclusion, the expression of mutant K-ras in K19(+) gastric epithelial cells can induce chronic inflammation and promote the development of dysplasia.

Identification of a bone marrow-derived mesenchymal progenitor cell subset that can contribute to the gastric epithelium

Recent studies with Helicobacter-infected mice have shown that bone marrow-derived cells can repopulate the gastric epithelium and progress to cancer. However, it has not been established which cellular subset can potentially contribute to the epithelium. The aim of this study was to investigate the ability of bone marrow-derived mesenchymal stem cells (MSCs) that express cytokeratin 19 (K19) to contribute to the gastric epithelium. MSCs cultures were established from whole bone marrow and expression of K19 was detected in a minority (1 of 13) of clones by real-time PCR and immunostaining. Transfection of a K19-green fluorescent protein (GFP) vector and isolation of GFP-expressing colonies generated high K19-expressing MSC clones (K19GFPMSC). Incubation of MSCs with gastric tissue extract markedly induced mRNA expression of gastric phenotypic markers and was observed to a greater extent in K19GFPMSCs compared with parental MSCs and mock transfectants. Both K19GFPMSCs and GFP-labeled control MSCs gave rise to gastric epithelial cells after injection into the murine stomach. In addition, after blastocyst injections, K19GFPMSCs gave rise to GFP-positive gastric epithelial cells in all 13 pups, whereas only 3 of 10 offspring showed GFP-positive gastric epithelial cells after injection of GFP-labeled control MSCs. Although K19 expression could not be detected in murine whole bone marrow, H. felis infection increased K19-expressing MSCs in the circulation. Taken together, our results show that bone marrow-derived MSCs can contribute to the gastric epithelium. The K19-positive MSC fraction that is induced by chronic H. felis infection appears to be the important subset in this process.

Mouse Models of Gastric Cancer

Animal models have greatly enriched our understanding of the molecular mechanisms of numerous types of cancers. Gastric cancer is one of the most common cancers worldwide, with a poor prognosis and high incidence of drug-resistance. However, most inbred strains of mice have proven resistant to gastric carcinogenesis. To establish useful models which mimic human gastric cancer phenotypes, investigators have utilized animals infected with Helicobacter species and treated with carcinogens. In addition, by exploiting genetic engineering, a variety of transgenic and knockout mouse models of gastric cancer have emerged, such as INS-GAS mice and TFF1 knockout mice. Investigators have used the combination of carcinogens and gene alteration to accelerate gastric cancer development, but rarely do mouse models show an aggressive and metastatic gastric cancer phenotype that could be relevant to preclinical studies, which may require more specific targeting of gastric progenitor cells. Here, we review current gastric carcinogenesis mouse models and provide our future perspectives on this field.

The C-terminus of rat L-histidine decarboxylase specifically inhibits enzymic activity and disrupts pyridoxal phosphate-dependent interactions with L-histidine substrate analogues

Full-length rat HDC (L-histidine decarboxylase) translated in reticulocyte cell lysate reactions is inactive, whereas C-terminally truncated isoforms are capable of histamine biosynthesis. C-terminal processing of the approximately 74 kDa full-length protein occurs naturally in vivo, with the production of multiple truncated isoforms. The minimal C-terminal truncation required for the acquisition of catalytic competence has yet to be defined, however, and it remains unclear as to why truncation is needed. Here we show that approximately 74 kDa HDC monomers can form dimers, which is the conformation in which the enzyme is thought to be catalytically active. Nevertheless, the resulting dimer is unable to establish pyridoxal phosphate-dependent interactions with an L-histidine substrate analogue. Protein sequences localized to between amino acids 617 and 633 specifically mediate this inhibition. Removing this region or replacing the entire C-terminus with non-HDC protein sequences permitted interactions with the substrate analogue to be re-established. This corresponded exactly with the acquisition of catalytic competence, and the ability to decarboxylate natural L-histidine substrate. These studies suggested that the approximately 74 kDa full-length isoform is deficient in substrate binding, and demonstrated that C-terminally truncated isoforms with molecular masses between approximately 70 kDa and approximately 58 kDa have gradually increasing specific activities. The physiological relevance of our results is discussed in the context of differential expression of HDC isoforms in vivo.

Differential protein analysis of spasomolytic polypeptide expressing metaplasia using laser capture microdissection and two-dimensional difference gel electrophoresis.

Full analysis of cellular protein constituents is a valuable tool in the evaluation of tissues. Traditional methods of evaluation, however, are time-consuming and difficult to reproduce. Two-dimensional difference gel electrophoresis (2D-DIGE), a recently developed proteomic system, affords the ability to compare and evaluate protein extracts from multiple sources. Coupled with laser capture microdissection (LCM), this technology is a powerful tool in comparing the protein profiles of separate pure cell populations. Proteins are labeled in vitro with reactive cyanine dyes that fluoresce at differential wavelengths, and after comigration on two-dimensional gels, differing protein populations become apparent. The unique aspect of this technology is the ability to identify and quantify proteins from separate preparations without issues of gel-to-gel differences. These techniques coupled with the systems for robotic acquisition of specific spots on the gel, tryptic digestion, and MALDI mass spectrometry permit identification of proteins differentially expressed in two pure cell populations. The authors used these new technologies to analyze the protein constituents of spasmolytic polypeptide expressing metaplasia (SPEM), a gastric mucosal metaplasia (fundic antralization or pseudopyloric metaplasia) that develops in the atrophic fundus mucosa of mice infected with Helicobacter felis and in humans infected with Helicobacter pylori. In addition, SPEM has been identified in the atrophic mucosa surrounding a high percentage of gastric adenocarcinomas and may represent a precursor lineage of malignancy. This technology recognized 28 differentially expressed proteins between SPEM and surface cells. Identification of novel SPEM-related proteins would allow the development of new immunohistochemical antibodies to further study this important metaplasia.

Mapping of catalytically important residues in the rat L-histidine decarboxylase enzyme using bioinformatic and site-directed mutagenesis approaches.

HDC (L-histidine decarboxylase), the enzyme responsible for the catalytic production of histamine from L-histidine, belongs to an evolutionarily conserved family of vitamin B6-dependent enzymes known as the group II decarboxylases. Yet despite the obvious importance of histamine, mammalian HDC enzymes remain poorly characterized at both the biochemical and structural levels. By comparison with the recently described crystal structure of the homologous enzyme L-DOPA decarboxylase, we have been able to identify a number of conserved domains and motifs that are important also for HDC catalysis. This includes residues that were proposed to mediate events within the active site, and HDC proteins carrying mutations in these residues were inactive when expressed in reticulocyte cell lysates reactions. Our studies also suggest that a significant change in quartenary structure occurs during catalysis. This involves a protease sensitive loop, and incubating recombinant HDC with an L-histidine substrate analogue altered enzyme structure so that the loop was no longer exposed for tryptic proteolysis. In total, 27 mutant proteins were used to test the proposed importance of 34 different amino acid residues. This is the most extensive mutagenesis study yet to identify catalytically important residues in a mammalian HDC protein sequence and it provides a number of novel insights into the mechanism of histamine biosynthesis.