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Background & Aim Under the regulation of various oncogenic pathways, cancer cells undergo adaptive metabolic programming to maintain specific metabolic states that support their uncontrolled proliferation. As it has been difficult to directly and effectively inhibit oncogenic signaling cascades with pharmaceutical compounds, focusing on the downstream metabolic pathways that enable indefinite growth may provide therapeutic opportunities. Thus, we sought to characterize metabolic changes in hepatocellular carcinoma (HCC) development and identify metabolic targets required for tumorigenesis. Methods We compared gene expression profiles of Morris Hepatoma (MH3924a) and DEN (diethylnitrosamine)-induced HCC models to those of liver tissues from normal and rapidly regenerating liver models, and performed gain- and loss-of-function studies of the identified gene targets for their roles in cancer cell proliferation in vitro and in vivo. Results The proline biosynthetic enzyme PYCR1 (pyrroline-5-carboxylate reductase 1) was identified as one of the most upregulated genes in the HCC models. Knockdown of PYCR1 potently reduced cell proliferation of multiple HCC cell lines in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of the HCC cell lines. Importantly, PYCR1 expression was not elevated in the regenerating liver, and KD or overexpression of PYCR1 had no effect on proliferation of non-cancerous cells. Besides PYCR1, we found that additional proline biosynthetic enzymes, such as ALDH18A1, were upregulated in HCC models and also regulated HCC cell proliferation. Clinical data demonstrated that PYCR1 expression was increased in HCC, correlated with tumor grade, and was an independent predictor of clinical outcome. Conclusion Enhanced expression of proline biosynthetic enzymes promotes HCC cell proliferation. Inhibition of PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC. Lay summary Even with the recently approved immunotherapies against liver cancer, currently available medications show limited clinical benefits or efficacy in the majority of patients. As such, it remains a top priority to discover new targets for effective liver cancer treatment. Here, we identify a critical role for the proline biosynthetic pathway in liver cancer development, and demonstrate that targeting key proteins in the pathway, namely PYCR1 and ALDH18A1, may be a novel therapeutic strategy for liver cancer.

Dysregulated cellular energetics has recently been recognized as a hallmark of cancer and garnered attention as a potential targeting strategy for cancer therapeutics. Cancer cells reprogram metabolic activities to meet bio-energetic, biosynthetic and redox requirements needed to sustain indefinite proliferation. In many cases, metabolic reprogramming is the result of complex interactions between genetic alterations in well-known oncogenes and tumor suppressors and epigenetic changes. While the metabolism of the two most abundant nutrients, glucose and glutamine, is reprogrammed in a wide range of cancers, accumulating evidence demonstrates that additional metabolic pathways are also critical for cell survival and growth. Proline metabolism is one such metabolic pathway that promotes tumorigenesis in multiple cancer types, including liver cancer, which is the fourth main cause of cancer mortality in the world. Despite the recent spate of approved treatments, including targeted therapy and combined immunotherapies, there has been no significant gain in clinical benefits in the majority of liver cancer patients. Thus, exploring novel therapeutic strategies and identifying new molecular targets remains a top priority for liver cancer. Two of the enzymes in the proline biosynthetic pathway, pyrroline-5-carboxylate reductase (PYCR1) and Aldehyde Dehydrogenase 18 Family Member A1 (ALDH18A1), are upregulated in liver cancer of both human and animal models, while proline catabolic enzymes, such as proline dehydrogenase (PRODH) are downregulated. Here we review the latest evidence linking proline metabolism to liver and other cancers and potential mechanisms of action for the proline pathway in cancer development.

Tumor cells have devised unique metabolic strategies to garner enough nutrients to sustain continuous growth and cell division. Oncogenic mutations may alter metabolic pathways to unlock new sources of energy, and cells take the advantage of various scavenging pathways to ingest material from their environment. These changes in metabolism result in a metabolic profile that, in addition to providing the building blocks for macromolecules, can also influence cell signaling pathways to promote tumor initiation and progression. Understanding what pathways tumor cells use to synthesize the materials necessary to support metabolic growth can pave the way for new cancer therapeutics. Potential strategies include depriving tumors of the materials needed to grow or targeting pathways involved in dependencies that arise by virtue of their altered metabolis.

We recently demonstrated that retinoic acid receptor-γ (RARγ) is overexpressed and acts as a tumor promoter in hepatocellular carcinoma (HCC). The oncogenic activity of RARγ is mainly attributed to its physiological interaction with p85α regulatory subunit of PI3K leading to constitutive activation of AKT. Here we report RARγ as a negative regulator of p53 signaling and thus extend the oncogenic potential of RARγ to a new role in controlling the balance between AKT and p53. A natural flavonoid acacetin is then identified to be capable of modulating RARγ-dependent AKT-p53 network. It specifically binds to RARγ and inhibits all-trans retinoic acid (atRA) stimulation of RARγ transactivation. However, the anticancer action of acacetin is independent on its modulation of RARγ-driven transcriptional activity. Acacetin induces cancer cell apoptosis through antagonizing the non-genomic effect of RARγ on AKT and p53. When bound to RARγ, acacetin prevents RARγ from its activation of AKT followed by recovery of the normal p53 signaling. Given the implication of AKT-p53 dysregulation in most HCC, targeting the non-genomic signaling of RARγ that switches AKT-p53 from a pro-survival to a pro-apoptotic program in cancer cells should be a promising strategy for developing novel anti-HCC drugs.

Summary Tumors display profound changes in cellular metabolism, yet how these changes aid the development and growth of tumors is not fully understood. Here we use a multi-omic approach to examine liver carcinogenesis and regeneration, and find that progressive loss of branched-chain amino acid (BCAA) catabolism promotes tumor development and growth. In human hepatocellular carcinomas and animal models of liver cancer, suppression of BCAA catabolic enzyme expression led to BCAA accumulation in tumors, though this was not observed in regenerating liver tissues. The degree of enzyme suppression strongly correlated with tumor aggressiveness, and was an independent predictor of clinical outcome. Moreover, modulating BCAA accumulation regulated cancer cell proliferation in vitro, and tumor burden and overall survival in vivo. Dietary BCAA intake in humans also correlated with cancer mortality risk. In summary, loss of BCAA catabolism in tumors confers functional advantages, which could be exploited by therapeutic interventions in certain cancers.

The mammalian target of rapamycin complex 1 (mTORC1) plays an important role in the development and progression of multiple cancers. Its activity is regulated by both growth factor and nutrient signals, and the branched-chain amino acid (BCAA) leucine plays an important and unique role in this process. Recently we found that cancers of the liver and multiple other tissues suppress the catabolism of BCAAs, thereby facilitating the chronic activation of mTORC1. Our results unveil how mTORC1’s nutrient-sensing arm can be manipulated by tumors, and suggest that restoring BCAA catabolism may help control mTORC1 activity in cancer cells.

Objective: To investigate the role of obesity-associated inflammation and immune modulation in gastric carcinogenesis during Helicobacter-induced chronic gastric inflammation. Design: C57BL/6 male mice were infected with H felis and placed on a high-fat diet (45% calories from fat). Study animals were analysed for gastric and adipose pathology, inflammatory markers in serum, stomach and adipose tissue, and immune responses in blood, spleen, stomach and adipose tissue. Results: H felis-induced gastric carcinogenesis was accelerated in diet-induced obese mice compared with lean controls. Obesity increased bone marrow-derived immature myeloid cells in blood and gastric tissue of H felis-infected mice. Obesity also led to elevations in CD4 T cells, IL-17A, granulocyte macrophage colony-stimulating factor, phosphorylated STAT3 and prosurvival gene expression in gastric tissue of H felis-infected mice. Conversely, in adipose tissue of obese mice, H felis infection increased macrophage accumulation and expression of IL-6, C-C motif ligand 7 (CCL7) and leptin. Finally, the combination of obesity and gastric inflammation synergistically increased serum proinflammatory cytokines, including IL-6. Conclusions: Here, we have established a model to study the molecular mechanism by which obesity predisposes individuals to gastric cancer. In H felis-infected mice, obesity increased proinflammatory immune responses and accelerated gastric carcinogenesis. Interestingly, gastric inflammation augmented obesity-induced adipose inflammation and production of adipose-derived factors in obese, but not lean, mice. Our findings suggest that obesity accelerates Helicobacter-associated gastric cancer through cytokine-mediated cross-talk between inflamed gastric and adipose tissues, augmenting immune responses at both tissue sites, and thereby contributing to a protumorigenic gastric microenvironment., National Institutes of Health (U.S.) (grant 5R01CA093405-11), Columbia University Medical Center (Naomi Berrie Diabetes Center, grant P30DK063608)

Background & Aims Interleukin (IL)-8 has an important role in initiating inflammation in humans, attracting immune cells such as neutrophils through their receptors CXCR1 and CXCR2. IL-8 has been proposed to contribute to chronic inflammation and cancer. However, mice do not have the IL-8 gene, so human cancer cell lines and xenograft studies have been used to study the role of IL-8 in colon and gastric carcinogenesis. We generated mice that carry a bacterial artificial chromosome that encompasses the entire human IL-8 gene, including its regulatory elements (IL-8Tg mice). Methods We studied the effects of IL-8 expression in APCmin[superscript +/−] mice and IL-8Tg mice given azoxymethane and dextran sodium sulfate (DSS). We also examined the effects of IL-8 expression in gastric cancer in INS-GAS mice that overexpress gastrin and IL-8Tg mice infected with Helicobacter felis. Results In IL-8Tg mice, expression of human IL-8 was controlled by its own regulatory elements, with virtually no messenger RNA or protein detectable under basal conditions. IL-8 was strongly up-regulated on systemic or local inflammatory stimulation, increasing mobilization of immature CD11b[superscript +]Gr-1[superscript +] myeloid cells (IMCs) with thioglycolate-induced peritonitis, DSS-induced colitis, and H. felis–induced gastritis. IL-8 was increased in colorectal tumors from patients and IL-8Tg mice compared with nontumor tissues. IL-8Tg mice developed more tumors than wild-type mice following administration of azoxymethane and DSS. Expression of IL-8 increased tumorigenesis in APCmin[superscript +/−] mice compared with APCmin[superscript +/−] mice that lack IL-8; this was associated with increased numbers of IMCs and angiogenesis in the tumors. Conclusions IL-8 contributes to gastrointestinal carcinogenesis by mobilizing IMCs and might be a therapeutic target for gastrointestinal cancers.

Cancer cells undergo dramatic metabolic reprogramming, largely to meet the requirements of sustained proliferation. These metabolic pathways are tightly regulated by various oncogenes, such as Myc and Ras. However, it has been difficult to directly and effectively inhibit these oncogenic signaling cascades with pharmacological compounds. Therefore, focusing on the downstream metabolic pathways that enable indefinite growth may provide a therapeutic opportunity. To identify metabolic enzymes required for hepatocellular carcinoma (HCC) tumorigenesis, we compared gene expression profiles of normal liver tissue to the Morris Hepatoma and DEN (Diethylnitrosamine)-induced HCC models as well as a liver regeneration model. PYCR1 (Pyrroline-5-Carboxylate Reductase 1), an enzyme in the proline biosynthesis pathway, was identified as one of the top up-regulated genes in the HCC models. An increase in PYCR1 protein levels in tumor samples versus normal liver tissue was confirmed by Western blot. shRNA-mediated knockdown of PYCR1 in HCC cell lines (Huh-7, Hep3B, HepG2 SNU-398, and MH3924a) potently reduced cell proliferation in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of Hep3B and AML12 cells. Importantly, PYCR1 expression was not elevated in regenerating tissues, and knockdown in a non-tumorigenic cell line did not influence proliferation. Additional enzymes in the proline biosynthesis pathway were also altered in tumors. For instance, ALDH18A1 was up-regulated and knockdown by shRNA decreased proliferation of human HCC cell lines. Metabolomic analyses suggested that the metabolism of glucose was disrupted in PYCR1 knockdown cells, with reduced fluxes through glycolytic and pentose phosphate pathways. Clinical data demonstrated that PYCR1 expression was increased in HCC, and expression levels correlated with increasing tumor grades and were independent predictors of clinical outcome. Overall, our data suggest that enhanced expression of proline biosynthetic enzymes promotes cell proliferation, and that inhibiting PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC. Citation Format: Zhaobing Ding, Russell E. Ericksen, Michael Steckel, Andrea Haegebarth, Sylvia Gruenewald, Weiping Han. Metabolic pathway analyses identify proline biosynthesis as a promoter of liver carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3477.

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.

Epithelium of the colon and intestine are renewed every 3 days. In the intestine there are at least two principal stem cell pools. The first contains rapid cycling crypt-based columnar (CBC) Lgr5(+) cells, and the second is composed of slower cycling Bmi1-expressing cells at the +4 position above the crypt base. In the colon, however, the identification of Lgr5(-) stem cell pools has proven more challenging. Here, we demonstrate that the intermediate filament keratin-19 (Krt19) marks long-lived, radiation-resistant cells above the crypt base that generate Lgr5(+) CBCs in the colon and intestine. In colorectal cancer models, Krt19(+) cancer-initiating cells are also radioresistant, while Lgr5(+) stem cells are radiosensitive. Moreover, Lgr5(+) stem cells are dispensable in both the normal and neoplastic colonic epithelium, as ablation of Lgr5(+) stem cells results in their regeneration from Krt19-expressing cells. Thus, Krt19(+) stem cells are a discrete target relevant for cancer therapy.

While molecular regulation of insulin granule exocytosis is relatively well understood, insulin granule biogenesis and maturation and its influence on glucose homeostasis are relatively unclear. Here, we identify a novel protein highly expressed in insulin-secreting cells and name it BIG3 due to its similarity to BIG/GBF of the Arf-GTP exchange factor (GEF) family. BIG3 is predominantly localized to insulin- and clathrin-positive trans-Golgi network (TGN) compartments. BIG3-deficient insulin-secreting cells display increased insulin content and granule number and elevated insulin secretion upon stimulation. Moreover, BIG3 deficiency results in faster processing of proinsulin to insulin and chromogranin A to β-granin in β-cells. BIG3-knockout mice exhibit postprandial hyperinsulinemia, hyperglycemia, impaired glucose tolerance, and insulin resistance. Collectively, these results demonstrate that BIG3 negatively modulates insulin granule biogenesis and insulin secretion and participates in the regulation of systemic glucose homeostasis. © 2014 Singapore Bioimaging Consortium, A*STAR., link_to_OA_fulltext

Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (longlasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene. Copyright © 2011 the American Physiological Society.

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., National Institutes of Health (U.S.) (Grant NIH 5R01 CA120979-02), National Institutes of Health (U.S.) (Grant R01 DK060694), National Institutes of Health (U.S.) (Grant U01 CA143056), National Institutes of Health (U.S.) (Grant P30 DK050306), Uehara Memorial Foundation

There is considerable evidence that co-infection with the sexually transmitted pathogen Neisseria gonorrhoeae (Gc) can increase the likelihood of both transmitting and acquiring HIV-1 worldwide. However, less information is available on how host immune response to co-infection differs with immune response to HIV-1 infection alone. To evaluate HIV-1 burden effects on host response to co-infection with Gc, we performed gene-expression profiling of human PBMCs infected over a broad range of viral titers (HIV-1 series) and upon exposure to a single infectious dose of Gc (HIV-1/Gc series). The transcriptional profiles differed substantially between each series (P < 0.0001). Major shifts in the transcriptional landscape were identified in contour plots based on fold stimulation and hierarchical clustering. Prominent regions of transcriptional activity were evaluated for statistical enrichment to identify up-regulated pathways associated with immune response, infection and T-cell stimulation. Notably, gene enrichment was dependent on HIV-1 burden and shifted during co-infection to reveal a disproportionate effect on lymphocyte signaling, apoptosis and proteasome activity. Further evaluation of these findings may help to better understand the role of viral burden in defining cellular contribution to host immune response upon co-infection with secondary sexually transmitted pathogens.

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

Tumors are postulated to arise from tissue stem or progenitor cells. In the stomach, keratin 19 (K19) is thought to mark a potential progenitor cell within the gastric isthmus. To examine the contribution of tissue stem cells to the origin of gastric cancer, we generated a new K19-BAC-CreER transgenic line to study the lineage of K19+ cells and determine whether K19+ might mark both normal and cancer initiating gastric progenitors. K19-BAC-CreER mice were crossed to a ROSA26r(LacZ or GFP) reporter line. The offspring were induced with tamoxifen and studied in a MNU model of antral carcinogenesis. In separate experiments, K19CreER/ROSA26r mice were crossed to LSL-Kras mice to examine the contribution of the K19 lineage to a genetic model of gastric dysplasia. Tamoxifen-induced X-gal labeling occurred in ~10-30% of corpus pit and antral glands. The labeling persisted for greater than 52 weeks, consistent with K19 marking long-lived gastric stem cells. In K19CreER/ROSA26r mice treated with MNU there was a dramatic increase in the labeling of contiguous GFP/X-gal positive glands, consistent with K19+ stem cell expansion and crypt fission. To test whether K19+ progenitor cells labeled by tamoxifen during MNU treatment contribute to the origin of gastric tumors, we examined the pattern of X-gal lineage tracing in antral tumors. Interestingly, when K19+ cells were labeled by tamoxifen during MNU carcinogenesis, tumors originated from both K19-Cre recombined and non-recombined cells, suggesting that MNU stimulated the K19+ progenitor cell to give rise to multiple K19+ daughter cells that contribute to the tumor. In a genetic model of cancer, when oncogenic Kras was expressed in K19+ progenitors, gastric dysplasia was detected as early as 3 months following tamoxifen induction. In summary, K19 marks corpus, as well as, antral stem cells located in the isthmus (above the crypt base), and these K19+ cells serve as cancer initiating cells in both carcinogen and genetic models of gastric tumorigenesis. Kras or MNU-induced division of K19+ cells, evidenced by contiguous crypt labeling and suggestive of symmetric cell division, appears to be an initiating event. Gastric tumors are polyclonal with multiple K19+ cancer stem cells contributing to antral tumors. Thus K19+ stem cells appear relevant for normal homeostasis as well as the development of gastric cancer. Citation Format: Samuel Asfaha, Yoku Hayakawa, Dana Lukin, Russell Ericksen, Ashlesha Muley, Rain Yu, Daniel L. Worthley, Christoph B. Westphalen, Sarah Stokes, Johannes von Burstin, Anil K. Rustgi, Timothy C. Wang. Role of keratin-19 positive stem cells in gastric tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-267. doi:10.1158/1538-7445.AM2013-LB-267

Introduction: The clonal origin of tumors is not well understood. Previous studies using chimeric mice have suggested that the origin of tumors in the mammalian small intestine may be polyclonal, with 2 or more progenitor cells contributing to intestinal adenomas. In the intestine, tissue stem cells are believed to be present in small numbers, and either quiescent or actively dividing, as previously shown with Lgr-5+ cells. Cytokeratin 19 (K19) is a potential progenitor marker expressed in the intestinal isthmus. Using the K19 gene promoter to drive Cre recombinase expression, we tracked the lineage of K19+ cells in an inducible fashion. Here, we used K19-CreERT2;ROSA26rLacZ mice to examine whether K19+ cells contribute to colonic tumor formation. Methods: In order to recapitulate the endogenous expression pattern of K19, we used a BAC strategy and generated a transgenic mouse with a Tamoxifen inducible Cre under the control of the K19 promoter (K19-BAC-Cre-ERT2). K19CreERT2 mice were crossed to ROSA26rLacZ (or GFP) reporter mice and treated with tamoxifen (6 mg/ day p.o. for 3 days) to assess K19+ lineage tracing by X-gal or GFP staining. To examine the contribution of the K19+ cells to colonic tumorigenesis, we used the AOM/DSS inflammation-associated model of colonic carcinogenesis. Mice were sacrificed 20 weeks following AOM induction and X-gal staining performed to stain for the K19+ cell lineage. Results: Treatment of control K19CreERT2/Rosa26r mice with tamoxifen resulted in the stochastic labelling of about 20-50% of colonic and intestinal glands, respectively. Consistent with the labeling of stem cells, marked glands stained positive beyond 52 weeks following tamoxifen induction. K19CreERT2/Rosa26r mice treated with AOM or DSS alone following tamoxifen labelling of K19+ glands displayed evidence of K19+ cell expansion and crypt fission, as determined by increased labelling of contiguous X-gal positive glands. K19CreERT2/Rosa26r mice were then treated with a single dose of AOM followed by DSS. When K19+ cells were labelled by tamoxifen administration prior to AOM, we observed that ∼10% tumors were entirely X-gal positive, suggesting derivation from a single K19+ cell. Interestingly, when K19+ cells were labelled by tamoxifen shortly after tumor initiation with AOM, some tumors were derived from both K19 recombined and non-recombined cells in a clonal fashion, suggesting a model whereby a K19+ cell gives rise to multiple K19+ daughter cells that each contribute to the tumor. Conclusions. Using K19CreERT2/Rosa26r mice, we show that K19 marks stem cells within the intestinal isthmus of the small intestine and colon. Using a carcinogen induced model of inflammation-associated carcinogenesis, we demonstrate that K19 expressing cells contribute to tumor formation in a manner consistent with tumor initiation following division of K19+ cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3301. doi:1538-7445.AM2012-3301

Introduction: Tissue stem cells, characterized by multipotentiality and self renewing ability, are not readily distinguishable from other resident cells. Tissue stem cells are believed to be present in small numbers that may remain quiescent, or divide frequently as previously shown with Lgr-5+ cells. In the gastric antrum, intestine and colon, epithelial cells derived from the stem cells at the crypt base rapidly migrate up towards to the mucosal surface, whereas in the gastric corpus, stem cells located in the isthmus are believed to give rise to epithelial progenitors that undergo bipolar migration and differentiation into pit and oxyntic cell lineages. Cytokeratin 19 (K19) has been shown to be expressed in cells in the gastric isthmus and colonic and intestinal crypts. Therefore, we aimed to use the K19 gene promoter to drive Cre recombinase expression and to track the lineage of K19+ cells in an inducible fashion. Methods: In order to recapitulate the endogenous expression pattern of K19, we used a BAC strategy and generated a transgenic mouse with a Tamoxifen inducible Cre under the control of the K19 promoter (K19-BAC-Cre-ERTM). K19CreERT2 mice were crossed to ROSA26rLacZ (or GFP) reporter mice, treated the mice with tamoxifen (4 mg/ day p.o. for 3 days) and sacrificed at various time points (3 days, 7 days, 26 weeks and 52 weeks) following tamoxifen induction. Results: Treatment of K19CreERT2/Rosa26r mice with tamoxifen resulted in the selective marking of about 30% of gastric and intestinal glands. Following tamoxifen induction, in the corpus a few gastric pit cells near the isthmus were labeled at 3 days, whereas, all of pit cells within the labeled glands stained positively by 7 days. Consistent with the labeling of progenitor or stem cells, all pit cells within marked glands stained positively even up to 52 weeks following tamoxifen induction. In contrast, in the antrum, intestine and colon nearly all epithelial cells were labeled within 3 days following tamoxifen induction. Once again, lineage tracing of all the epithelial cells within marked glands was observed up to 52 weeks following tamoxifen induction, suggesting that K19 likely marks a common progenitor or stem cell in the antrum, intestine and colon. Interestingly, in the small intestine and antrum K19 positive cells do not overlap with Lgr5-GFP+ cells yet clearly demonstrate stem cell properties. Conclusions. Using K19CreERT2/Rosa26r mice, we show that K19 marks pit progenitors in the gastric corpus, as well as stem cells within the antrum, intestine and colon. These findings identify a novel population of stem cells for the lineage tracing of gastric pit cells following gastric injury or cancer, and for tracing epithelial cells in the development of intestinal or colonic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5186. doi:10.1158/1538-7445.AM2011-5186

overexpressed Plexin B1 compared to normal pancreas tissues. Endogenous Plexin B1 and Met protein were detected in eight pancreatic cancer cell lines, with a variety of expression levels. Sema 4D stimulation activated Met protein, and significantly induced cell migration. Discussion: Here we show that Sema 4D involves in the regulation of cell motility through its receptor Plexin B1. And the results of immunostaining suggest that Sema 4D could play a role in the interaction between tumor cells and tumor microenvironment in pancreatic cancer tissues. As many attempts are now ongoing to target the tumor stroma, these findings provide a new insight into molecular targeted therapies for pancreatic cancer.