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We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor , and the most up-regulated gene in SC was () in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.
Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ(4)-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (R)-2-(6-methoxynaphthalen-2-yl)butanoic acid (in which the methyl group of R-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. R-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer R-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease.
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.
Copyright © 2015, American Association for the Advancement of Science.
Castrate-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer. CRPC is characterized by reactivation of the androgen axis due to changes in androgen receptor signaling and/or adaptive intratumoral androgen biosynthesis. AKR1C3 is upregulated in CRPC where it catalyzes the formation of potent androgens. This makes AKR1C3 a target for the treatment of CRPC. AKR1C3 inhibitors should not inhibit AKR1C1/AKR1C2, which inactivate 5α-dihydrotestosterone. Indomethacin, used to inhibit cyclooxygenase, also inhibits AKR1C3 and displays selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies were used to generate libraries of indomethacin analogues, which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP(+)·2'-des-methyl-indomethacin crystal structure was determined, and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the development of therapeutics for CRPC.
Colorectal cancer (CRC) is the second leading cause of cancer-related death and usually arises from colorectal polyps. Screening and removal of polyps reduce mortality from CRC. Colorectal polyps are known to aggregate in families; however the genetic determinants for risk of polyps are unknown. In addition, it has been shown that nonsteroidal anti-inflammatory drug (NSAID) use decreases the risk of CRC and the incidence and size of polyps. In this study, we used data from the Tennessee Colorectal Polyp Study and the Tennessee-Indiana Adenoma Recurrence Study to evaluate selected genes from the prostaglandin (PG) metabolism and signaling pathways for association with risk of polyps and for interactions with NSAIDs. Our design consisted of discovery and replication phases for a total of 2,551 Caucasian polyp cases and 3,285 Caucasian controls. We carried out multivariable logistic regression to test for association in both the discovery and replication phase and further examined the results with meta-analysis. We detected association signals in the genes PGE receptor 3 (PTGER3) and 15-hydroxyprostaglandin dehydrogenase (HPGD), both strong biologic candidates for influence on polyp risk. We did not observe the previously reported effects and effect modification in PG-endoperoxide synthase 2 (PTGS2), PGE receptor 2 (PTGER2), or PGE receptor 4 (PTGER4), although we did observe a single nucleotide polymorphism in PTGER2 associated with risk of multiple adenomas. We also observed effect modification of the HPGD signal by NSAID exposure.
We have recently reported that adult male C57BL/6 mice exposed in utero to the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) confer an increased risk of preterm birth (PTB) to unexposed females. Risk of PTB was coincident with decreased placental progesterone receptor (Pgr) mRNA expression and increased toll-like receptor 4 (Tlr4) mRNA expression, suggesting that toxicant exposure induced a heightened inflammatory response at the maternal-fetal interface. Since omega-3 fatty acids exhibit anti-inflammatory activity, in this study, we provided TCDD-exposed males a fish oil-enriched diet prior to mating. Although PTB was common in control females mated to TCDD-exposed males on the standard diet, fish oil supplementation of TCDD-exposed males eliminated PTB in unexposed partners. We also determined the influence of preconception, paternal fish oil supplementation on the placental inflammatory response in late pregnancy (E18.5) by examining the expression of Pgr and Tlr4 mRNA as well as the expression of 15-hydroxyprostaglandin dehydrogenase (PGDH). PGDH catabolizes the inflammatory PGE2 to an inactive form; thus, reduced expression of this enzyme would promote tissue inflammation. Compared with control pregnancies, examination of E18.5 placentas arising from TCDD-exposed males on the standard diet revealed a significant increase in Tlr4 mRNA expression corresponding to a reduction in Pgr mRNA and PGDH protein expression. In contrast, fish oil supplementation of toxicant-exposed males led to normalization of placental expression of both Pgr and Tlr4 mRNA and a marked increase in PGDH expression. These studies suggest that a paternal preconception diet that includes omega-3 fatty acids prevents the toxicant-associated increase in the placental inflammatory response at late gestation, preventing PTB.
Hypoxia-inducible factors (HIFs), in particular HIF-1alpha, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expression profiling was performed upon hypoxia-exposed non-transformed immortalized human esophageal epithelial cells, EPC2-hTERT, and comparing with a gene signature of esophageal squamous cell carcinoma (ESCC). In addition to known HIF-1alpha target genes such as carbonic anhydrase 9, insulin-like growth factor binding protein-3 (IGFBP3) and cyclooxygenase (COX)-2, prostaglandin E synthase (PTGES) was identified as a novel target gene among the commonly upregulated genes in ESCC as well as the cells exposed to hypoxia. The PTGES induction was augmented upon stabilization of HIF-1alpha by hypoxia or cobalt chloride under normoxic conditions and suppressed by dominant-negative HIF-1alpha. Whereas PTGES messenger RNA (mRNA) was negatively regulated by normoxia, PTGES protein remained stable upon reoxygenation. Prostaglandin E(2) (PGE(2)) biosynthesis was documented in transformed human esophageal cells by ectopic expression of PTGES as well as RNA interference directed against PTGES. Moreover, hypoxia stimulated PGE(2) production in a HIF-1alpha-dependent manner. In ESCC, PTGES was overexpressed frequently at the mRNA and protein levels. Finally, COX-2 and PTGES were colocalized in primary tumors along with HIF-1alpha and IGFBP3. Activation of the COX-2-PTGES axis in primary tumors was further corroborated by concomitant upregulation of interleukin-1beta and downregulation of hydroxylprostaglandin dehydrogenase. Thus, PTGES is a novel HIF-1alpha target gene, involved in prostaglandin E biosynthesis in the esophageal tumor hypoxic microenvironment, and this has implications in diverse tumors types, especially of squamous origin.
Tissue prostaglandin levels are determined by both biosynthesis and catabolism. The current studies report the expression and localization of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin catabolism in the kidneys. We also investigated potential interactions between 15-PGDH and cyclooxygenase (COX), a key enzyme in prostaglandin biosynthesis. Both 15-PGDH mRNA and protein levels were significantly higher in kidney cortex than in papilla, which is opposite to the expression pattern of COX-2. In situ hybridization indicated that 15-PGDH mRNA was mainly localized to the tubular epithelial cells in kidney cortex and outer medulla but not in the glomerulus or papilla. Dual immunofluorescent staining indicated that 15-PGDH was expressed in the proximal tubule, cortical, and outer medullary thick ascending limb and collecting duct but not in the macula densa or papilla. 15-PGDH levels were significantly lower in a macula densa cell line (MMDD1) than in a proximal tubule cell line. Although a high-salt diet decreased COX-2 expression in macula densa, it increased macula densa 15-PGDH expression in both mouse and rat kidneys. In MMDD1 cells, a COX-2 inhibitor increased 15-PGDH, whereas a COX-1 inhibitor had no effect. Furthermore, intense 15-PGDH immunofluorescent staining was found in both macula densa and glomerulus in COX-2 knockout mice. The intrarenal distribution of 15-PGDH and its interactions with COX-2 suggest that differential regulation of COX-2 and 15-PGDH may play an important role in determining levels of prostaglandins involved in regulation of salt, volume, and blood pressure homeostasis.
Evidence indicates that the induction of cyclooxygenase-2 (COX-2) and high prostaglandin E2 (PGE2) levels contribute to the pathogenesis of non-small-cell lung cancer (NSCLC). In addition to overproduction by COX-2, PGE2 concentrations also depend upon the levels of the PGE2 catabolic enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). We find a dramatic down-regulation of PGDH protein in NSCLC cell lines and in resected human tumors when compared with matched normal lung. Affymetrix array analysis of 10 normal lung tissue samples and 49 resected lung tumors revealed a much lower expression of PGDH transcripts in all NSCLC histologic groups. In addition, treatment with the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) erlotinib increased the expression of 15-PGDH in a subset of NSCLC cell lines. This effect may be due in part to an inhibition of the extracellular signal-regulated kinase (ERK) pathway as treatment with mitogen-activated protein kinase kinase (MEK) inhibitor U0126 mimics the erlotinib results. We show by quantitative reverse transcription-PCR that the transcript levels of ZEB1 and Slug transcriptional repressors are dramatically reduced in a responsive cell line upon EGFR and MEK/ERK inhibition. In addition, the Slug protein, but not ZEB1, binds to the PGDH promoter and represses transcription. As these repressors function by recruiting histone deacetylases to promoters, it is likely that PGDH is repressed by an epigenetic mechanism involving histone deacetylation, resulting in increased PGE2 activity in tumors. This effect is reversible in a subset of NSCLC upon treatment with an EGFR TKI.
Prostaglandin E2 (PGE2) can stimulate tumor progression by modulating several proneoplastic pathways, including proliferation, angiogenesis, cell migration, invasion, and apoptosis. Although steady-state tissue levels of PGE2 stem from relative rates of biosynthesis and breakdown, most reports examining PGE2 have focused solely on the cyclooxygenase-dependent formation of this bioactive lipid. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The present study examined a range of normal tissues in the human and mouse and found high levels of 15-PGDH in the large intestine. By contrast, the expression of 15-PGDH is decreased in several colorectal carcinoma cell lines and in other human malignancies such as breast and lung carcinomas. Consistent with these findings, we observe diminished 15-Pgdh expression in ApcMin+/- mouse adenomas. Enzymatic activity of 15-PGDH correlates with expression levels and the genetic disruption of 15-Pgdh completely blocks production of the urinary PGE2 metabolite. Finally, 15-PGDH expression and activity are significantly down-regulated in human colorectal carcinomas relative to matched normal tissue. In summary, these results suggest a novel tumor suppressive role for 15-PGDH due to loss of expression during colorectal tumor progression.