Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia.

OBJECTIVE: NRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC. DESIGN: We performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. RESULTS: Using high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30 mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity. CONCLUSION: We have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic for NRF2high ESCC.

Lymphatic Drainage System and Lymphatic Metastasis of Cancer Cells in the Mouse Esophagus.

BACKGROUND: Lymphatic metastasis is commonly seen in patients with esophageal squamous cell carcinoma (ESCC). Both lymphatic metastasis and the number of involved nodes are prognostic for post-operative survival. To better understand lymphatic metastasis of ESCC, there is a need to develop proper animal models. AIMS: This study is aimed to characterize the morphology and function of the lymphatic drainage system in the mouse esophagus. METHODS: Immunostaining and fluorescence imaging were used to visualize the lymphatic drainage system in the mouse esophagus. Tracers and cancer cells were orthotopically inoculated into the submucosa of the mouse esophagus to mimic lymphatic metastasis of T1 ESCC. RESULTS: Using immunostaining of a lymphatic vessel marker (LYVE1), we found that lymphatic vessels were located in the submucosa and muscularis propria of the mouse esophagus, similar to the human esophagus. In the esophagus of ProxTom mice expressing tdTomato in the lymphatic vessels, we discovered a microscopic meshwork of lymphatic vessels. Functionally, orthotopically inoculated tracers (Indian ink and FITC-dextran) were drained from the submucosa into peri-esophageal lymph nodes via lymphatic vessels. Orthotopically inoculated mouse cancer cells (LLC-eGFP, MOC2) metastasized from the submucosa to lymphatic vessels, peri-esophageal lymph nodes, and distant organs (liver and lung) in immunocompetent mice. Similarly, in immunodeficient mice, orthotopically inoculated human ESCC cells (KYSE450-eGFP-Luc) metastasized via the same route. CONCLUSION: We have characterized the morphology and function of the lymphatic drainage system of the mouse esophagus. These observations lay a foundation for mechanistic and therapeutic studies on lymphatic metastasis of T1 ESCC.

PAX9 in Cancer Development.

Paired box 9 (PAX9) is a transcription factor of the PAX family functioning as both a transcriptional activator and repressor. Its functional roles in the embryonic development of various tissues and organs have been well studied. However, its roles and molecular mechanisms in cancer development are largely unknown. Here, we review the current understanding of PAX9 expression, upstream regulation of PAX9, and PAX9 downstream events in cancer development. Promoter hypermethylation, promoter SNP, microRNA, and inhibition of upstream pathways (e.g., NOTCH) result in PAX9 silencing or downregulation, whereas gene amplification and an epigenetic axis upregulate PAX9 expression. PAX9 may contribute to carcinogenesis through dysregulation of its transcriptional targets and related molecular pathways. In summary, extensive studies on PAX9 in its cellular and tissue contexts are warranted in various cancers, in particular, HNSCC, ESCC, lung cancer, and cervical SCC.

Development of targeted therapy of NRF2high esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a deadly disease and one of the most aggressive cancers of the gastrointestinal tract. As a master transcription factor regulating the stress response, NRF2 is often mutated and becomes hyperactive, and thus causes chemo-radioresistance and poor survival in human ESCC. There is a great need to develop NRF2 inhibitors for targeted therapy of NRF2high ESCC. In this review, we mainly focus on three aspects, NRF2 inhibitors and their mechanisms of action, screening novel drug targets, and evaluation of NRF2 activity in the esophagus. A research strategy has been proposed to develop NRF2 inhibitors using human ESCC cells and mouse models.

NRF2/ACSS2 axis mediates the metabolic effect of alcohol drinking on esophageal squamous cell carcinoma.

Alcohol drinking is a leading risk factor for the development of esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms of alcohol-associated ESCC remain poorly understood. One of the most commonly mutated genes in ESCC is nuclear factor erythroid 2 like 2 (NFE2L2 or NRF2), which is a critical transcription factor regulating oxidative stress response and drug detoxification. When NRF2 is hyperactive in cancer cells, however, it leads to metabolic reprogramming, cell proliferation, chemoradioresistance, and poor prognosis. In this study, hyperactive NRF2 was found to up-regulate acetyl-CoA synthetase short-chain family members 2 (ACSS2), an enzyme that converts acetate to acetyl-CoA, in ESCC cells and mouse esophagus. We also showed that knockdown of NRF2 or ACSS2 led to decreased ACSS2 expression, which in turn reduced the levels of acetyl-CoA and ATP with or without ethanol exposure. In addition, ethanol exposure enhanced lipid synthesis in ESCC cells. Moreover, we observed a change in the metabolic profile of ESCC cells exposed to ethanol as a result of their NRF2 or ACSS2 status. We further showed that ACSS2 contributed to the invasive capability of NRF2high ESCC cells exposed to ethanol. In conclusion, the NRF2/ACSS2 axis mediates the metabolic effect of alcohol drinking on ESCC.

Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2.

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2(-/-)) mice were used to investigate the role of this receptor in E. faecalis-induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2(-/-) mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2(-/-) mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2(-/-) mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.