Stephen P. Fink

15-Hydroxyprostaglandin dehydrogenase inactivation as a mechanism of resistance to celecoxib chemoprevention of colon tumors

Pharmacologic inhibitors of the prostaglandin-synthesizing COX-2 oncogene prevent the development of premalignant human colon adenomas. However, resistance to treatment is common. In this study, we show that the adenoma prevention activity of the COX-2 inhibitor celecoxib requires the concomitant presence of the 15-hydroxyprostaglandin dehydrogenase (15-PGDH) tumor suppressor gene, and that loss of 15-PGDH expression imparts resistance to celecoxibs anti-tumor effects. We first demonstrate that the adenoma-preventive activity of celecoxib is abrogated in mice genetically lacking 15-PGDH. In FVB mice, celecoxib prevents 85% of azoxymethane-induced tumors >1 mm in size, but is essentially inactive in preventing tumor induction in 15-PGDH-null animals. Indeed, celecoxib treated 15-PGDH null animals develop more tumors than do celecoxib naive WT mice. In parallel with the loss of tumor prevention activity, celecoxib-mediated suppression of colonic PGE2 levels is also markedly attenuated in 15-PGDH-null versus WT mice. Finally, as predicted by the murine models, humans with low colonic 15-PGDH levels also exhibit celecoxib resistance. Specifically, in a colon adenoma prevention trial, in all cases tested, individuals who developed new adenomas while receiving celecoxib treatment were also found as having low colonic 15-PGDH levels.

Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration

A shot in the arm for damaged tissue Tissue damage can be caused by injury, disease, and even certain medical treatments. There is great interest in identifying drugs that accelerate tissue regeneration and recovery, especially drugs that might benefit multiple organ systems. Zhang et al. describe a compound with this desired activity, at least in mice (see the Perspective by FitzGerald). SW033291 promotes recovery of the hematopoietic system after bone marrow transplantation, prevents the development of ulcerative colitis in the intestine, and accelerates liver regeneration after hepatic surgery. It acts by inhibiting an enzyme that degrades prostaglandins, lipid signaling molecules that have been implicated in tissue stem cell maintenance. Science, this issue 10.1126/science.aaa2340; see also p. 1208 A compound that inhibits prostaglandin degradation enhances tissue regeneration in multiple organs in mice. [Also see Perspective by FitzGerald] INTRODUCTION Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. To date, therapeutic interventions have largely focused on targeting two PGE2 biosynthetic enzymes, cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), with the aim of reducing PGE2 production. In this study, we take the converse approach: We examine the role of a prostaglandin-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), as a negative regulator of tissue repair, and we explore whether inhibition of this enzyme can potentiate tissue regeneration in mouse models. RATIONALE We used 15-PGDH knockout mice to elucidate the role of 15-PGDH in regulating tissue levels of PGE2 and tissue repair capacity in multiple organs. We then developed SW033291, a potent small-molecule inhibitor of 15-PGDH with activity in vivo. We used SW033291 to investigate the therapeutic potential of 15-PGDH inhibitors in tissue regeneration and to identify a 15-PGDH–regulated hematopoietic pathway within the bone marrow niche. RESULTS We found that in comparison with wild-type mice, 15-PGDH–deficient mice display a twofold increase in PGE2 levels across multiple tissues—including bone marrow, colon, and liver—and that they show increased fitness of these tissues in response to damage. The mutant mice also show enhanced hematopoietic capacity, with increased neutrophils, increased bone marrow SKL (Sca-1+ C-kit+ Lin−) cells (enriched for stem cells), and greater capacity to generate erythroid and myeloid colonies in cell culture. The 15-PGDH–deficient mice respond to colon injury from dextran sulfate sodium (DSS) with a twofold increase in cell proliferation in colon crypts, which confers resistance to DSS-induced colitis. The mutant mice also respond to partial hepatectomy with a greater than twofold increase in hepatocyte proliferation, which leads to accelerated and more extensive liver regeneration. SW033291, a potent small-molecule inhibitor of 15-PGDH (inhibitor dissociation constant Ki ~0.1 nM), recapitulates in mice the phenotypes of 15-PGDH gene knockout, inducing increased hematopoiesis, resistance to DSS colitis, and more rapid liver regeneration after partial hepatectomy. Moreover, SW033291-treated mice show a 6-day-faster reconstitution of hematopoiesis after bone marrow transplantation, with accelerated recovery of neutrophils, platelets, and erythrocytes, and greater recovery of bone marrow SKL cells. This effect is mediated by bone marrow CD45– cells, which respond to increased PGE2 with a fourfold increase in production of CXCL12 and SCF, two cytokines that play key roles in hematopoietic stem cell homing and maintenance. CONCLUSIONS Studying mouse models, we have shown that 15-PGDH negatively regulates tissue regeneration and repair in the bone marrow, colon, and liver. Of most direct utility, our observations identify 15-PGDH as a therapeutic target and provide a chemical formulation, SW033291, that is an active 15-PGDH inhibitor in vivo and that potentiates repair in multiple tissues. SW033291 or related compounds may merit clinical investigation as a strategy to accelerate recovery after bone marrow transplantation and other tissue injuries. Inhibiting 15-PGDH accelerates tissue repair. (A) The enzyme 15-PGDH degrades and negatively regulates PGE2. (B) SW033291 inhibits 15-PGDH, increases tissue levels of PGE2, and induces CXCL12 and SCF expression from CD45– bone marrow cells. This in turn accelerates homing of transplanted hematopoietic stem cells (HSC), generation of mature blood elements, and post-transplant recovery of normal blood counts. Inhibiting 15-PGDH similarly stimulates cell proliferation after injury to colon or liver, accelerating repair of these tissues. Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.

Aspirin and the Risk of Colorectal Cancer in Relation to the Expression of 15-Hydroxyprostaglandin Dehydrogenase (HPGD)

Aspirin use is associated with a lower risk of colorectal cancer arising in association with high expression of 15-PGDH (HPGD) in normal colon mucosa. An Aspirin a Day May Keep Colon Cancer Away Aspirin, the ubiquitous drug that people use for everything, ranging from fever and headache to prevention of heart disease and colon cancer, is not without its drawbacks. Especially at high doses, aspirin increases the risk of gastrointestinal problems, such as bleeding and ulcers, in addition to other side effects such as bruising. Thus, it would be nice to know which patients are most likely to derive a benefit from aspirin treatment, and to avoid exposing everyone else to unnecessary adverse effects. Fink and coauthors analyzed two large studies totaling more than 100,000 participants and discovered that patients who used aspirin for colon cancer prevention were less likely to develop colon cancer with a high expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Conversely, colon cancers expressing low amounts of 15-PGDH were equally common in patients who used aspirin and those who didn’t. The applicability of these results for preventative medicine will still need to be confirmed in prospective trials, to determine whether the concentration of 15-PGDH (determined in advance, perhaps during routine colonoscopy) can predict which patients would benefit from using aspirin to prevent colon cancer. The current paper lays the groundwork for such studies and provides hope that we may someday be able to maximize the benefits of aspirin and use it for targeted efforts at cancer prevention. Aspirin use reduces the risk of colorectal neoplasia, at least in part, through inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase 2)–related pathways. Hydroxyprostaglandin dehydrogenase 15-(nicotinamide adenine dinucleotide) (15-PGDH, HPGD) is down-regulated in colorectal cancers and functions as a metabolic antagonist of PTGS2. We hypothesized that the effect of aspirin may be antagonized by low 15-PGDH expression in the normal colon. In the Nurses’ Health Study and the Health Professionals Follow-Up Study, we collected data on aspirin use every 2 years and followed up participants for diagnoses of colorectal cancer. Duplication-method Cox proportional, multivariable-adjusted, cause-specific hazards regression for competing risks data was used to compute hazard ratios (HRs) for incident colorectal cancer according to 15-PGDH mRNA expression level measured in normal mucosa from colorectal cancer resections. Among 127,865 participants, we documented 270 colorectal cancer cases from which we could assess 15-PGDH expression. Compared with nonuse, regular aspirin use was associated with lower risk of colorectal cancer that developed within a background of colonic mucosa with high 15-PGDH expression [multivariable HR, 0.49; 95% confidence interval (CI), 0.34 to 0.71], but not with low 15-PGDH expression (multivariable HR, 0.90; 95% CI, 0.63 to 1.27) (P for heterogeneity = 0.018). Regular aspirin use was associated with lower incidence of colorectal cancers arising in association with high 15-PGDH expression, but not with low 15-PGDH expression in normal colon mucosa. This suggests that 15-PGDH expression level in normal colon mucosa may serve as a biomarker that may predict stronger benefit from aspirin chemoprevention.

Oncogenic PIK3CA mutations reprogram glutamine metabolism in colorectal cancer

Cancer cells often require glutamine for growth, thereby distinguishing them from most normal cells. Here we show that PIK3CA mutations reprogram glutamine metabolism by upregulating glutamate pyruvate transaminase 2 (GPT2) in colorectal cancer (CRC) cells, making them more dependent on glutamine. Compared with isogenic wild-type (WT) cells, PIK3CA mutant CRCs convert substantially more glutamine to α-ketoglutarate to replenish the tricarboxylic acid cycle and generate ATP. Mutant p110α upregulates GPT2 gene expression through an AKT-independent, PDK1–RSK2–ATF4 signalling axis. Moreover, aminooxyacetate, which inhibits the enzymatic activity of aminotransferases including GPT2, suppresses xenograft tumour growth of CRCs with PIK3CA mutations, but not with WT PIK3CA. Together, these data establish oncogenic PIK3CA mutations as a cause of glutamine dependency in CRCs and suggest that targeting glutamine metabolism may be an effective approach to treat CRC patients harbouring PIK3CA mutations.

Colon cancer secreted protein-2 (CCSP-2), a novel candidate serological marker of colon neoplasia

Cancers of the colon and rectum are the second leading cause of cancer death among adult Americans. When detected at early stages, colon cancer is highly curable. Colonoscopy, an effective but invasive screening test, has been limited in its public acceptance. The goal of this study was to identify novel serum markers of colon cancers and precancerous colon adenomas as potential candidates for noninvasive detection of early colon neoplasms. Employing expression microarrays, we identified colon cancer secreted protein-2 (CCSP-2) as a novel transcript whose expression is generally absent in normal colon and other normal body tissues, but that is induced an average of 78-fold in Stage II, III, and IV colon cancers, as well as in colon adenomas and colon cancer cell lines. These findings were validated by real-time PCR analysis in an independent panel of colon cancer cases. Moreover, CCSP-2 was shown to encode a secreted protein that circulates stably and is detectable in the blood of mice bearing human cancer xenografts transfected with epitope-tagged CCSP-2. As a novel secreted protein that is markedly induced in colon adenomas and cancers, CCSP-2 is a novel candidate for development as a diagnostic serum marker of early stage colon cancer.

Induction of KIAA1199/CEMIP is associated with colon cancer phenotype and poor patient survival

Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. We originally identified KIAA1199 (now officially called CEMIP) as a transcript highly induced in colon cancer: initially designating the transcript as Colon Cancer Secreted Protein 1. We molecularly characterized CEMIP expression both at the mRNA and protein level and found it is a secreted protein induced an average of 54-fold in colon cancer. Knockout of CEMIPreduced the ability of human colon cancer cells to form xenograft tumors in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. We find CEMIP mRNA overexpression correlates with poorer patient survival. In stage III only (n = 31) or in combined stage II plus stage III colon cancer cases (n = 73), 5-year overall survival was significantly better (p = 0.004 and p = 0.0003, respectively) among patients with low CEMIP expressing tumors than those with high CEMIP expressing tumors. These results demonstrate that CEMIP directly facilitates colon tumor growth, and high CEMIP expression correlates with poor outcome in stage III and in stages II+III combined cohorts. We present CEMIP as a candidate prognostic marker for colon cancer and a potential therapeutic target.

Inactivating Mutation in the Prostaglandin Transporter Gene, SLCO2A1, Associated with Familial Digital Clubbing, Colon Neoplasia, and NSAID Resistance

HPGDand SLCO2A1 genes encode components of the prostaglandin catabolic pathway, with HPGD encoding the degradative enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH), and SLCO2A1 encoding the prostaglandin transporter PGT that brings substrate to 15-PGDH. HPGD-null mice show increased prostaglandin E2 (PGE2), marked susceptibility to developing colon tumors, and resistance to colon tumor prevention by nonsteroidal anti-inflammatory drugs (NSAID). But in humans, HPGD and SLCO2A1 mutations have only been associated with familial digital clubbing. We, here, characterize a family with digital clubbing and early-onset colon neoplasia. Whole-exome sequencing identified a heterozygous nonsense mutation (G104X) in the SLCO2A1 gene segregating in 3 males with digital clubbing. Two of these males further demonstrated notably early-onset colon neoplasia, 1 with an early-onset colon cancer and another with an early-onset sessile serrated colon adenoma. Two females also carried the mutation, and both these women developed sessile serrated colon adenomas without any digital clubbing. Males with clubbing also showed marked elevations in the levels of urinary prostaglandin E2 metabolite, PGE-M, whereas, female mutation carriers were in the normal range. Furthermore, in the male proband, urinary PGE-M remained markedly elevated during NSAID treatment with either celecoxib or sulindac. Thus, in this human kindred, a null SLCO2A1 allele mimics the phenotype of the related HPGD-null mouse, with increased prostaglandin levels that cannot be normalized by NSAID therapy, plus with increased colon neoplasia. The development of early-onset colon neoplasia in male and female human SLCO2A1 mutation carriers suggests that disordered prostaglandin catabolism can mediate inherited susceptibility to colon neoplasia in man. Cancer Prev Res; 7(8); 805–12. ©2014 AACR.

Genetic Variation in 15-Hydroxyprostaglandin Dehydrogenase and Colon Cancer Susceptibility

Background 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a metabolic antagonist of COX-2, catalyzing the degradation of inflammation mediator prostaglandin E2 (PGE2) and other prostanoids. Recent studies have established the 15-PGDH gene as a colon cancer suppressor. Methods We evaluated 15-PDGH as a colon cancer susceptibility locus in a three-stage design. We first genotyped 102 single-nucleotide polymorphisms (SNPs) in the 15-PGDH gene, spanning ∼50 kb up and down-stream of the coding region, in 464 colon cancer cases and 393 population controls. We then genotyped the same SNPs, and also assayed the expression levels of 15-PGDH in colon tissues from 69 independent patients for whom colon tissue and paired germline DNA samples were available. In the final stage 3, we genotyped the 9 most promising SNPs from stages 1 and 2 in an independent sample of 525 cases and 816 controls (stage 3). Results In the first two stages, three SNPs (rs1365611, rs6844282 and rs2332897) were statistically significant (p<0.05) in combined analysis of association with risk of colon cancer and of association with 15-PGDH expression, after adjustment for multiple testing. For one additional SNP, rs2555639, the T allele showed increased cancer risk and decreased 15-PGDH expression, but just missed statistical significance (p-adjusted = 0.063). In stage 3, rs2555639 alone showed evidence of association with an odds ratio (TT compared to CC) of 1.50 (95% CI = 1.05–2.15, p = 0.026). Conclusions Our data suggest that the rs2555639 T allele is associated with increased risk of colon cancer, and that carriers of this risk allele exhibit decreased expression of 15-PGDH in the colon.

15-Hydroxyprostaglandin Dehydrogenase is Downregulated and Exhibits Tumor Suppressor Activity in Gastric Cancer

We investigated the tumor suppressor activity and regulatory mechanism of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in gastric cancer; 15-PGDH expression was lost in 70.1% of malignant human gastric tissues, but was preserved in normal and metaplastic gastritis. KATO III and SNU-719 cells were transfected with pcDNA3.1-empty vector or an expression vector encoding wild-type 15-PGDH. In TUNEL assays apoptotic cell numbers were increased in KATO-PGDH-WT cells compared with control. We found that EGFR and ERK1/2 inhibitors clearly increased the expression of 15-PGDH in KATO III cells. Our findings demonstrate both downregulation and a tumor suppressor activity of 15-PGDH in gastric cancer.

Ugene, a newly identified protein that is commonly overexpressed in cancer and binds uracil DNA glycosylase.

Expression microarrays identified a novel transcript, designated as Ugene, whose expression is absent in normal colon and colon adenomas, but that is commonly induced in malignant colon cancers. These findings were validated by real-time PCR and Northern blot analysis in an independent panel of colon cancer cases. In addition, Ugene expression was found to be elevated in many other common cancer types, including breast, lung, uterus, and ovary. Immunofluorescence of V5-tagged Ugene revealed it to have a nuclear localization. In a pull-down assay, uracil DNA glycosylase 2 (UNG2), an important enzyme in the base excision repair (BER) pathway, was identified as a partner protein that binds to Ugene. Coimmunoprecipitation and Western blot analysis confirmed the binding between the endogenous Ugene and UNG2 proteins. Using deletion constructs, we find that Ugene binds to the first 25 amino acids of the UNG2 NH(2) terminus. We suggest that Ugene induction in cancer may contribute to the cancer phenotype by interacting with the BER pathway.