Richard A. Friedman

Bile Acid and Inflammation Activate Gastric Cardia Stem Cells in a Mouse Model of Barrett-Like Metaplasia

Esophageal adenocarcinoma (EAC) arises from Barrett esophagus (BE), intestinal-like columnar metaplasia linked to reflux esophagitis. In a transgenic mouse model of BE, esophageal overexpression of interleukin-1β phenocopies human pathology with evolution of esophagitis, Barrett-like metaplasia and EAC. Histopathology and gene signatures closely resembled human BE, with upregulation of TFF2, Bmp4, Cdx2, Notch1, and IL-6. The development of BE and EAC was accelerated by exposure to bile acids and/or nitrosamines, and inhibited by IL-6 deficiency. Lgr5(+) gastric cardia stem cells present in BE were able to lineage trace the early BE lesion. Our data suggest that BE and EAC arise from gastric progenitors due to a tumor-promoting IL-1β-IL-6 signaling cascade and Dll1-dependent Notch signaling.

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.

Progastrin Stimulates Colonic Cell Proliferation via CCK2R- and β-Arrestin–Dependent Suppression of BMP2

BACKGROUND & AIMSnProgastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R)-partly by increasing the number of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R.nnnMETHODSnWe performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene, gastrin knockout mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2; levels of β-arrestin 1 and 2 were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division.nnnRESULTSnThe BMP pathway was down-regulated in the colons of human progastrin mice compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by β-arrestin 1 and 2. In mouse colonic epithelial cells, down-regulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of inhibitor of DNA binding 4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44(+), bromodeoxyuridine+, and NUMB(+) cells, indicating an increase in symmetric divisions of putative cancer stem cells.nnnCONCLUSIONSnProgastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and β-arrestin 1 and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division.

Phase Ib Randomized, Double-Blinded, Placebo-Controlled, Dose Escalation Study of Polyphenon E in Patients with Barrett's Esophagus

This study was conducted to determine the safety and efficacy of the green tea–derived Polyphenon E (Poly E) in patients with Barretts Esophagus (BE). Subjects were randomized to a 6-month, twice daily (BID) oral treatment of placebo or Poly E (200, 400, or 600 mg). Endoscopic evaluation, including biopsies, was performed before and after treatment. The primary objective was to demonstrate safety; secondary objectives investigated catechin accumulation and effects in clinical specimens. Of the 44 enrolled subjects, 11 received placebo, and 33 received Poly E. No dose-limiting toxicities were encountered, and a maximum tolerated dose (MTD) was not reached. The recommended phase II dose was 600 mg twice daily. The most common treatment-related adverse events (AE) in Poly E–treated subjects were grade I and II nausea, grade I belching, and grade I lactate dehydrogenase (LDH) elevation. No treatment-related AEs were reported in placebo-treated subjects, aside from grade I laboratory abnormalities. Pill counts and subject diaries were not consistently collected, and compliance was difficult to determine. However, on the basis of an intention-to-treat analysis, there was a significant relationship between Poly E dose and esophageal EGCG level—mean changes (pmol/g) of 0.79 (placebo), 6.06 (200 mg), 35.67 (400 mg), and 34.95 (600 mg); P = 0.005. There was a possible relationship between Poly E dose and urine PGE-M concentration. In conclusion, Poly E was well-tolerated, and treatment with Poly E (400 and 600 mg) but not Poly E (200 mg) or placebo resulted in clinically relevant and detectable EGCG accumulation in the target organ, esophageal mucosa. Cancer Prev Res; 8(12); 1131–7. ©2015 AACR.

Lithium ion uptake associated with the stimulation of action potential ionophores of cultured human neuroblastoma cells

Abstract Cultured human neuroblastoma cell lines were tested for the action potential sodium ionophore utilizing the Li + ion rather than the 22 Na + ion. The cell lines studied included CHP-134, CHP-100, CHP-126, CHP-212 and LA-N-1. Veratridine-dependent uptake of Li + and 22 Na + and its inhibition by tetrodotoxin implies the presence of the action-potential sodium ionophore. CHP-165, and undifferentiated tumor and RAJI a lymphoblast had no veratridine-dependent Li + uptake. Thus, veratridine-dependent Li + uptake provides a convenient means of assaying human neural cells for the action-potential sodium ionophore without the use of the radioactive Na + ion.

Bone Marrow Myeloid Cells Regulate Myeloid-Biased Hematopoietic Stem Cells via a Histamine-Dependent Feedback Loop

Myeloid-biased hematopoietic stem cells (MB-HSCs) play critical roles in recovery from injury, butxa0little is known about how they are regulated within the bone marrow niche. Here we describe an auto-/paracrine physiologic circuit that controls quiescence of MB-HSCs and hematopoietic progenitors marked by histidine decarboxylase (Hdc). Committed Hdc+ myeloid cells lie in close anatomical proximity to MB-HSCs and produce histamine, which activates the H2 receptor on MB-HSCs to promote their quiescence and self-renewal. Depleting histamine-producing cells enforces cell cycle entry, induces loss of serial transplant capacity, and sensitizes animals to chemotherapeutic injury. Increasing demand for myeloid cells via lipopolysaccharide (LPS) treatment specifically recruits MB-HSCs and progenitors into the cell cycle; cycling MB-HSCs fail to revert into quiescence in the absence of histamine feedback, leading to their depletion, while an H2 agonist protects MB-HSCs from depletion after sepsis. Thus, histamine couples lineage-specific physiological demands to intrinsically primed MB-HSCs to enforce homeostasis.

β2 Adrenergic-Neurotrophin Feedforward Loop Promotes Pancreatic Cancer

Catecholamines stimulate epithelial proliferation, but the role of sympathetic nerve signaling in pancreatic ductal adenocarcinoma (PDAC) is poorly understood. Catecholamines promoted ADRB2-dependent PDAC development, nerve growth factor (NGF) secretion, and pancreatic nerve density. Pancreatic Ngf overexpression accelerated tumor development in LSL-Kras+/G12D;Pdx1-Cre (KC) mice. ADRB2 blockade together with gemcitabine reduced NGF expression and nerve density, and increased survival of LSL-Kras+/G12D;LSL-Trp53+/R172H;Pdx1-Cre (KPC) mice. Therapy with a Trk inhibitor together with gemcitabine also increased survival of KPC mice. Analysis of PDAC patient cohorts revealed a correlation between brain-derived neurotrophic factor (BDNF) expression, nerve density, and increased survival of patients on nonselective β-blockers. These findings suggest that catecholamines drive a feedforward loop, whereby upregulation of neurotrophins increases sympathetic innervation and local norepinephrine accumulation.

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.

Deletion of the Formin, Diaph1, Protects from Structural and Functional Abnormalities in the Murine Diabetic Kidney

Diaphanous 1 (DIAPH1), a member of the formin family, binds to the cytoplasmic domain of the receptor for advanced glycation end products (RAGE) and is required for RAGE signal transduction. Experiments employing genetic overexpression or deletion of Ager (the gene encoding RAGE) or its pharmacological antagonism implicate RAGE in the pathogenesis of diabetes-associated nephropathy. We hypothesized that DIAPH1 contributes to pathological and functional derangements in the kidneys of diabetic mice. We show that DIAPH1 is expressed in the human and murine diabetic kidney, at least in part in the tubulointerstitium and glomerular epithelial cells or podocytes. To test the premise that DIAPH1 is linked to diabetes-associated derangements in the kidney, we rendered male mice globally devoid of Diaph1 ( Diaph1-/-) or wild-type controls (C57BL/6 background) diabetic with streptozotocin. Control mice received equal volumes of citrate buffer. After 6 mo of hyperglycemia, diabetic Diaph1-/- mice displayed significantly reduced mesangial sclerosis, podocyte effacement, glomerular basement thickening, and urinary albumin-to-creatinine ratio compared with diabetic mice expressing Diaph1. Analysis of whole kidney cortex revealed that deletion of Diaph1 in diabetic mice significantly reduced expression of genes linked to fibrosis and inflammation. In glomerular isolates, expression of two genes linked to podocyte stress, growth arrest-specific 1 ( Gas1) and cluster of differentiation 36 ( Cd36), was significantly attenuated in diabetic Diaph1-/- mice compared with controls, in parallel with significantly higher levels of nestin (Nes) mRNA, a podocyte marker. Collectively, these data implicate DIAPH1 in the pathogenesis of diabetes-associated nephropathy and suggest that the RAGE-DIAPH1 axis is a logical target for therapeutic intervention in this disorder.

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.