Yoshihiro Takemoto

Long-lived intestinal tuft cells serve as colon cancer–initiating cells

Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth-sustaining stem cells. DCLK1⁺ tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT-dependent genetic lineage-tracing strategy, we determined that a subpopulation of DCLK1⁺ cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that DCLK1⁺ tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in DCLK1⁺ cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate-induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in DCLK1⁺ cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in DCLK1⁺ cells. Thus, our data define an intestinal DCLK1⁺ tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived DCLK1⁺ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer.

Dclk1 Defines Quiescent Pancreatic Progenitors that Promote Injury-Induced Regeneration and Tumorigenesis

The existence of adult pancreatic progenitor cells has been debated. While some favor the concept of facultative progenitors involved in homeostasis and repair, neither a location nor markers for such cells have been defined. Using genetic lineage tracing, we show that Doublecortin-like kinase-1 (Dclk1) labels a rare population of long-lived, quiescent pancreatic cells. In vitro, Dclk1+ cells proliferate readily and sustain pancreatic organoid growth. In vivo, Dclk1+ cells are necessary for pancreatic regeneration following injury and chronic inflammation. Accordingly, their loss has detrimental effects after cerulein-induced pancreatitis. Expression of mutant Kras in Dclk1+ cells does not affect their quiescence or longevity. However, experimental pancreatitis converts Kras mutant Dclk1+ cells into potent cancer-initiating cells. As a potential effector of Kras, Dclk1 contributes functionally to the pathogenesis of pancreatic cancer. Taken together, these observations indicate that Dclk1 marks quiescent pancreatic progenitors that are candidates for the origin of pancreatic cancer.

CCK2R identifies and regulates gastric antral stem cell states and carcinogenesis

Objective Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. Design We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. Results Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5neg or low cell population but was distinct from typical antral Lgr5high stem cells. Treatment with progastrin interconverts Lgr5neg or low CCK2R+ cells into Lgr5high cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. Conclusions CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

Histidine decarboxylase (HDC)-expressing granulocytic myeloid cells induce and recruit Foxp3+ regulatory T cells in murine colon cancer

ABSTRACT The colorectal tumor microenvironment contains a diverse population of myeloid cells that are recruited and converted to immunosuppressive cells, thus facilitating tumor escape from immunoediting. We have identified a genetically and functionally distinct subset of dynamic bone marrow myeloid cells that are characterized by histidine decarboxylase (HDC) expression. Lineage tracing in Hdc-CreERT2;R26-LSL-tdTomato mice revealed that in homeostasis, there is a strong bias by HDC+ myeloid cells toward the CD11b+Ly6Ghi granulocytic lineage, which was accelerated during azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colonic carcinogenesis. More importantly, HDC+ myeloid cells strongly promoted colonic tumorigenesis, and colon tumor progression was profoundly suppressed by diphtheria toxin A (DTA)-mediated depletion of HDC+ granulocytic myeloid cells. In addition, tumor infiltration by Foxp3+ regulatory T cells (Tregs) was markedly impaired following HDC+ myeloid cell depletion. We identified an HDC+ myeloid-derived Cxcl13/Cxcr5 axis that mediated Foxp3 expression and Treg proliferation. Ablation of HDC+ myeloid cells or disruption of the Cxcl13/Cxcr5 axis by gene knockdown impaired the production and recruitment of Tregs. Cxcl13 induction of Foxp3 expression in Tregs during tumorigenesis was associated with Stat3 phosphorylation. Overall, HDC+ granulocytic myeloid cells affect CD8+ T cells directly and indirectly through the modulation of Tregs and thus appear to play key roles in suppressing tumoricidal immunity.

Abstract LB-272: Histidine decarboxylase (Hdc)-expressing myeloid cells support Foxp3+ Treg cells and promote colorectal cancer progression

The tumor microenvironment contains a diverse population of myeloid cells that are recruited from bone marrow and converted to immunosuppressive cells, thus mediating tumor cell escape from immune checkpoint. We have identified a subset of dynamic bone marrow myeloid cells, which can be identified by histidine decarboxylase (Hdc) mRNA and GFP expression in Hdc-GFP transgenic mice. Gene expression profiling showed that Hdc-GFP+ CD11b+Gr1+ myeloid cells express higher levels of cell cycle promoting genes such as Ccnd2, Ccnd3 and Cdc14a, while cell proliferation repressors including Cdc14b, Cdc16 and Cdk4 were downregulated in Hdc-GFP+ myeloid cells. To further elucidate the role of Hdc+ myeloid cells in tumor progression, we performed lineage-tracing studies using Hdc-creERT2;R26-LSL-TdTomato reporter mice. Pulsed with tamoxifen, the majority of TdTomato+ cells were localized initially to a group of CD11b+Gr1+ myeloid cells representing the highest Hdc mRNA expression in bone marrow, spleen and blood. Later on, TdTomato+CD11b+Gr1- F4/80+ macrophages can be detected, indicating a hierarchy of Hdc+ myeloid cells in which Hdc+CD11b+Gr1+ myeloid cells reside at the apex. In general, CD11b+ myeloid cells have a relative short lifespan ( In a mouse model of azoxymethane (AOM)/DSS colorectal carcinogenesis, Hdc-creERT2;R26-LSL-TdTomato;R26-LSL-DTA mice were injected with 10 mg/kg AOM and followed by 3 circles of 10 days 1.5% DSS ad libitum in drinking water. We found that Hdc-TdTomato labeled a proportion of tumor infiltrating CD11b+Gr1+ myeloid cells that expressed higher levels of Arg-1, Cox2, and Pdl1 transcripts. Continuous depletion of Hdc+ myeloid cells by administration of tamoxifen chow to induce DTA (diphtheria toxin subunit A) killing in Hdc-expressing myeloid cells abrogated half of the tumor-infiltrating MDSCs and released tumoricidal CD8+ T cells (> 15-fold), leading to decreased tumor number. This tumor suppression could be rescued by Hdc+ CD11b+Gr1+ cell adoptive transfer. Serum chemokine profiling revealed that Hdc+ DTA mediated myeloid depletion also decreased serum chemokine levels, among which Cxcl13 decreased the most (>30-fold). Cxcl13 protein and Cxcl13 mRNA also decreased in the colonic tumor tissue in the Hdc+ myeloid cell depleted AOM/DSS treatment group. Along with reduction in Cxcl13 levels, we also detected a significant reduction of Foxp3+ Treg cells in the tumor frozen sections stained with antibody against Foxp3 compared to R26-LSL-TdTomato;R26-LSL-DTA controls received the same treatments. Pre-knockdown of Cxcl13 by Dicer-substrate SiRNA (DsiRNAs) in a co-culture of splenic Hdc+ CD11b+Gr1+ myeloid cells from colon tumor-bearing mice with splenic Foxp3-GFP+ Treg cells induced apoptosis and decreased numbers of GFP+ cells compared to the scramble knockdown control group. Taken together, our data suggest that Hdc marks a distinct subset of myeloid cells with greater potency for promoting tumorigenicity, in part through supporting Tregs and suppressing CD8+ Tcells in the tumor microenvironment. Citation Format: Xiaowei Chen, Yoshihiro Takemoto, Karan K. Nagar, Timothy H. Chu, Zhengyu Jiang, Wenju Chang, Richard A. Friedman, Yagnesh H. Tailor, Daniel L. Worthley, Timothy C. Wang. Histidine decarboxylase (Hdc)-expressing myeloid cells support Foxp3+ Treg cells and promote colorectal cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-272.

Abstract B73: Adrenergic signaling promotes pancreatic tumor initiation and progression

Background/Aims: There is increasing evidence that chronic sympathetic nervous system activation can lead to increased noradrenaline levels in the tumor microenvironment. This process has been associated with proliferative signals largely mediated by beta-adrenergic signaling. However, the mechanisms by which adrenergic neurotransmitters are delivered to the tumor microenvironment are not well understood. In this study we aimed to investigate the role of locally and systemically delivered catecholamines to the tumor microenvironment in a well-established genetically engineered pancreatic cancer mouse model (PDx1-Cre/KRasG12D (KC) and PDx1-Cre/KRasG12D/Trp53R172H (KPC)). Methods: Adrenergic signaling was induced or inhibited in KC or KPC mice or 3D pancreatic spheres in culture using specific non-selective and selective agonists or antagonists. Adrenergic receptor expression was assessed by RT-PCR, Western blot and immunohistochemistry. Adrenergic signaling was also modeled in vivo applying restraint stress. To elucidate the crosstalk between nerve and cancer cells, pancreatic spheres and pancreatic cancer cells were co-cultured with dorsal root ganglia and neuronal plasticity was quantified by evaluating neurite outgrowth and number of branches. Denervation of the pancreas was performed using surgical or chemical neural ablation. Results: Adrenergic signaling receptors, in particular ADRB2, is upregulated during pancreatic cancer development. Furthermore, in vitro stimulation of cells harboring an oncogenic KRas mutation displayed a significantly increased sphere forming efficiency. This effect was blocked by the non-specific beta-blocker propranolol and the selective beta2-blocker ICI 188,551. Stimulation of pancreatic sphere formation from KC mice induced by adrenergic signaling from dorsal root ganglia in vitro was also prevented by selective blockage of the beta2-adrenergic signaling pathway. While surgical or chemical (Botulinum toxin A) denervation of the pancreas appeared not to retard the development precancerous lesions in KC mice, denervation significantly increased the survival when performed in mice after a tumor (3-5mm) was detected by high–resolution ultrasound imaging. In contrast, stress accelerated pancreatic cancer development in KC mice. The effects of stress were prevented by treatment with the selective ADRB2 antagonist ICI 188,551. Conclusions: Taken together, our data suggests that an increased level of stress and an increased level of systemic catecholamines may promote pancreatic carcinogenesis in its early stages. During this phase, patients might benefit from pharmacological inhibition of stress-induced signaling. The progression of pancreatic cancer seems to depend on the local delivery of catecholamines to the microenvironment, and thus patients might also benefit by additional targeting of the ADRB2 signaling pathway. Citation Format: Bernhard W. Renz, Christoph B. Westphalen, Xiaowei Chen, Yoku Hayakawa, Yoshihiro Takemoto, Marina Macchini, Daniel L. Worthley, Samuel Asfaha, Axel Kleespies, Helen Remotti, Kenneth P. Olive, Timothy C. Wang. Adrenergic signaling promotes pancreatic tumor initiation and progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B73.

Abstract 4092: Long-lived Dclk1+ cells serve as colon cancer initiating cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnIntroduction: In the rapidly proliferating gastrointestinal epithelium, long-lived tissue stem cells, characterized by multipotentiality and self-renewing ability, remain the most likely cellular origin for cancer. Previous studies have suggested actively cycling Lgr5+ stem cells are one cellular origin for intestinal adenomas. However, it has also recently been suggested that non-Lgr5+ cells may also contribute to the cellular origin of colorectal cancer. Doublecortin-like kinase 1 (Dclk1) protein is a gastrointestinal tuft cell marker that has been proposed to identify quiescent stem cells and cancer stem cells that sustain tumor growth. The role of Dclk1+ tuft cells within the gastrointestinal epithelium and their potential to function as cancer-initiating cells, however, remain poorly understood. Here, we used Dclk1(BAC)-CreERT;ROSA26rLacZ mice crossed to APCff mice to examine whether Dclk1+ cells contribute to colonic tumor formation.nnMethods: To recapitulate the endogenous expression pattern of Dclk1, we used a BAC strategy and generated a transgenic mouse with a Tamoxifen inducible Cre under the control of the Dclk1 promoter (Dclk1-BAC-Cre-ERT). Dclk1-CreERT mice were crossed to both ROSA26rLacZ and APCff mice and treated with tamoxifen (6 mg p.o.). Dclk1+ lineage tracing was assessed by X-gal staining. To examine the contribution of the Dclk1+ cells to colonic tumorigenesis, we treated Dclk1(BAC)-CreERT;ROSA26rLacZ; APCff mice with DSS (3% in drinking water) to induce colitis. Mice were sacrificed 3-4 months after DSS weeks to assess for tumor formation and X-gal staining performed to stain for the Dclk1+ cell lineage.nnResults: Dclk1-BAC-CreERT genetic lineage tracing demonstrated that a subpopulation of Dclk1+ cells is extremely long-lived and shows rare stem cell abilities. Moreover, genetic ablation reveals a pivotal role for Dclk1+ tuft cells in the response to intestinal and colonic injury. Surprisingly, conditional loss of APC in Dclk1+ cells is not sufficient to drive colonic carcinogenesis, whereas induction of DSS colitis in Dclk1-CreERT; APCflox/flox mice leads to the development of poorly differentiated colonic adenocarcinoma. Importantly, colonic tumor formation occurs even when the onset of colitis is delayed for up to 3 months after APC loss in Dclk1+ cells.nnConclusions. Thus, our data define a novel intestinal Dclk1+ tuft cell population that is long-lived, quiescent and important for intestinal homeostasis and regeneration. Long-lived Dclk1+ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve as a potent cellular origin of colon cancer.nnCitation Format: Samuel Asfaha, Christoph Benedikt Westphalen, Yoku Hayakawa, Yoshihiro Takemoto, Dana J. Lukin, Wanda Setlik, Helen Remotti, Ashlesha Muley, Xiaowei Chen, Randal May, Courtney W. Houchen, James G. Fox, Michael D. Gershon, Michael Quante, Timothy Wang. Long-lived Dclk1+ cells serve as colon cancer initiating cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4092. doi:10.1158/1538-7445.AM2014-4092

Abstract 4795: CCK2R marks gastric antral stem cell and mediates antral carcinogenesis

Gastrin is a hormone that binds to the CCK2 receptor and promotes proximal gastric cancer, but inhibits distal gastric cancer development. However, the precise roles of the CCK2 receptor, and its alternative ligand, progastrin, in gastric carcinogenesis have not been clarified. In this study, we found that progastrin accelerated antral proliferation and carcinogenesis through CCK2R+ antral stem cell expansion, using mouse gastric cancer models and transgenic mice lines including human progastrin-overexpressing (hGAS) mice, CCK2R knockout mice, Lgr5-CreERT-IRES-GFP knockin mice, and newly generated CCK2R-BAC-CreERT mice. Progastrin promoted gastric antral cancer development induced by MNU and/or H. felis. During carcinogenesis, progastrin increased the expression of Lgr5 and gland fission in response to the chemical carcinogen MNU. Genetic ablation of CCK2R diminished these progastrin-mediated effects. In vitro 3D culture experiments revealed that progastrin, but not amidated gastrin, significantly increased gastric organoid formation and growth in Noggin-free condition, effects that were ablated by a CCK2R inhibitor YF-476 or CCK2R gene deletion. In the antrum, CCK2R was expressed in an Lgr5low cell population that displayed stemness, which could be enhanced by progastrin. In the presence of progastrin, Lgr5lowCCK2R+ cells interconverted to Lgr5hi cells. Finally, we generated a new BAC-transgenic CCK2R-CreERT murine line, and lineage tracing experiments showed that CCK2R+ cells, which reside slightly above the base of the antrum, contained long-lived stem cells in vivo and in vitro. Chemical inhibition of CCK2R attenuated progastrin-dependent cancer development in mice. In conclusion, CCK2R labels Lgr5low antral stem cells that can be activated and expanded by progastrin. These findings may help the understanding of the underlying mechanism in gastric stem cell regulation by a CCK2R signal. Citation Format: Hayakawa Yoku, Guanchun Jin, Hongshan Wang, Xiaowei Chen, Christoph B. Westphalen, Samuel Asfaha, Daniel L. Worthley, Bernhard Renz, Hiroshi Ariyama, Zinaida A. Dubeykovskaya, Yoshihiro Takemoto, Ashlesha Mulay, Yagnesh Tailor, Duan Chen, Sureshkumar Muthupalani, James G. Fox, Shigeo Takaishi, Timothy C. Wang. CCK2R marks gastric antral stem cell and mediates antral carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4795. doi:10.1158/1538-7445.AM2014-4795