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.

Exploring cell competition for the prevention and therapy of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is characterized by the development of cancer in the esophageal squamous epithelium through a step-by-step accumulation of genetic, epigenetic, and histopathological alterations. Recent studies have demonstrated that cancer-associated gene mutations exist in histologically normal or precancerous clones of the human esophageal epithelium. However, only a small proportion of such mutant clones will develop ESCC, and most ESCC patients develop only one cancer. This suggests that most of these mutant clones are kept in a histologically normal state by neighboring cells with higher competitive fitness. When some of the mutant cells evade cell competition, they become "super-competitors" and develop into clinical cancer. It is known that human ESCC is composed of a heterogeneous population of cancer cells that interact with and influence their environment and neighbors. During cancer therapy, these cancer cells not only respond to therapeutic agents but also compete with each other. Therefore, competition between ESCC cells within the same ESCC tumor is a constantly dynamic process. However, it remains challenging to fine-tune the competitive fitness of various clones for therapeutic benefits. In this review, we will explore the role of cell competition in carcinogenesis, cancer prevention, and therapy, using NRF2, NOTCH pathway, and TP53 as examples. We believe that cell competition is a research area with promising targets for clinical translation. Manipulating cell competition may help improve the prevention and therapy of 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.

Canopy Fibroblast Growth Factor Signaling Regulator 2 (CNPY2) Inhibits Neuron Apoptosis in Parkinson's Disease via the AKT/GSK3ß Pathway.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic neurons. Canopy fibroblast growth factor signaling regulator 2 (CNPY2) is down-regulated in this disease, but its functions are unknown. OBJECTIVE: This study investigates the effects and regulation of CNPY2 in the apoptosis of neurons in PD. METHODS: We established a PD model in vivo by a five consecutive days-injection of 1-methyl-4- phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to mice. In vitro, the human SH-SY5Y neuroblastoma cells, after differentiation, were treated with 1-Methyl-4-phenylpyridinium iodide (MPP+) for modeling. The cells were transfected with a recombinant vector overexpressing CNPY2 followed by MPP+ treatment. Expression of CNPY2 and proteins related to apoptosis was detected by real-time PCR, western blot, or immunofluorescence staining. The ROS level and mitochondrial membrane potential were determined by flow cytometry. Cell viability and apoptosis were measured by MTT assay and TUNEL staining. RESULTS: CNPY2 level was down-regulated both in the brain and retina of PD mice and also inhibited in neurons by MPP+ in vitro. Overexpression of CNPY2 repressed the level of Bax and cleaved caspase-3, enhanced Bcl-2 level, and promoted neurite length under MPP+ treatment. CNPY2 overexpression reduced the accumulation of ROS and mitochondria dysfunction in neurons. The AKT/ GSK3ß signaling pathway was activated by overexpressed CNPY2 to inhibit MPP+-induced neuronal apoptosis, which was confirmed using an AKT inhibitor MK-2206 2HCl. CONCLUSION: CNPY2 alleviates oxidative stress, mitochondria dysfunction, and apoptosis of neurons induced by MPP+ by activating the AKT/ GSK3ß signaling pathway.

Activation of Insulin-Like Growth Factor-2 Ameliorates Retinal Cell Damage and Exerts Protection in in vitro Model of Diabetic Retinopathy.

BACKGROUND: The major event in the development of diabetes-related blindness and vision impairment is the onset of retinal cell damage. Overall awareness of insulin-like growth factor-2 (IGF2) mechanisms emphasizes its protective behavior in retinal cells that help to provide new information about the development of treatment for retinal complications. OBJECTIVES: This study analyzes the effect of in vitro changes associated with the cell survival and rescue mechanism in IGF2 inhibition and activation using chromeceptin and IGF2 peptides in ARPE-19 cells cultured in high glucose conditions. METHOD: Cell death was induced using high glucose (15 mmol/L), IGF2 inhibition was done using chromeceptin (1 µM) (Sigma Aldrich, Saint Louis, MO, USA), and IGF2 activation was done using IGF2 peptide (10 ng/mL). The cells were analyzed for changes in cell proliferation, apoptosis markers, antioxidant molecules, and alteration of cytokines. RESULTS: The study demonstrated that cells lacking IGF2 exhibited a significant increase in reactive oxygen levels with apoptosis patterns. Also, gene expression analysis by qRT-PCR demonstrated a significant increase in Yes-associated protein 1, CDK2, TNF-α, and BIRC5 genes in cells under high glucose stress and IGF inhibition compared to control. Further, the cytokine analysis also revealed that cells devoid of IGF2 activated an increase in cytokines such as IL-8, CX43, ICAM-1, IL-17, CCL3, and MCP-1 and decreased paraoxonase compared to normal control cells. On the other hand, ARPE-19 cells grown in high glucose shows that IGF2 increases the survival genes with reduced levels of inflammatory cytokines. CONCLUSION: The finding of the investigation, therefore, shows that the use of IGF2 activators may prevent the progression of ocular dysfunction in the control of diabetes-related complications.

Alcohol drinking inhibits NOTCH-PAX9 signaling in esophageal squamous epithelial cells.

Alcohol drinking has been established as a major risk factor for esophageal diseases. Our previous study showed that ethanol exposure inhibited PAX9 expression in human esophageal squamous epithelial cells in vitro and in vivo. In this study, we aimed to investigate the molecular pathways through which alcohol drinking suppresses PAX9 in esophageal squamous epithelial cells. We first demonstrated the inhibition of NOTCH by ethanol exposure in vitro. NOTCH regulated PAX9 expression in KYSE510 and KYSE410 cells in vitro and in vivo. RBPJ and NOTCH intracellular domain (NIC) D1 ChIP-PCR confirmed Pax9 as a direct downstream target of NOTCH signaling in mouse esophagus. NOTCH inhibition by alcohol drinking was further validated in mouse esophagus and human tissue samples. In conclusion, ethanol exposure inhibited NOTCH signaling and thus suppressed PAX9 expression in esophageal squamous epithelial cells in vitro and in vivo. Our data support a novel mechanism of alcohol-induced esophageal injury through the inhibition of NOTCH-PAX9 signaling. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

[Down-regulated PTTG1 expression promotes the senescence of human prostate cancer LNCaP-AI].

OBJECTIVE: To investigate the effect of the down-regulated expression of pituitary tumor-transforming gene 1 (PTTG1) on the senescence of human castration-resistant prostate cancer LNCaP-AI cells. METHODS: Human castration-resistant prostate cancer LNCaP-AI cells were induced in vitro and transfected with siRNA targeting PTTG1 (the siRNA-PTTG1 group), the reagent lip3000 only (the mock group) or siRNA negative control vector (the NC group). All the cells were cultured in fetal bovine serum (FBS) or charcoal-stripped bovine serum (CSS) and counted with the cell counting chamber. The senescence characteristics of the transfected LNCaP-AI cells were examined by senescence-associated ß-galactosidase (SA-ß-Gal) staining, and the expressions of the senescence-related ß-galactosidase-1-like proteins (Glb1), the cyclin-dependent kinase inhibitors p-21CIP1 and p-27Kip1, and the chromatin-regulating heterochromatin protein 1γ (HP1γ) were detected by Western blot. RESULTS: The expression of PTTG1 in the human prostate cancer LNCaP-AI cells was significantly reduced in the siRNA-PTTG1 group compared with those in the mock and NC groups (0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05). Culture with FBS markedly increased while that with CSS decreased the number of LNCaP-AI cells transfected with siRNA, but both FBS and CSS enhanced the proliferation of the LNCaP-AI cells in the mock and NC groups. SA-ß-Gal staining revealed that reducing the expression of PTTG1 induced a remarkably higher positive rate of the LNCaP-AI cells in the siRNA-PTTG1 than in the mock and NC groups (ï¼»63.5 ± 2.35ï¼½% vs ï¼»11.3 ± 1.24ï¼½% and ï¼»12.4 ± 1.15ï¼½%, P < 0.05). The siRNA-PTTG1 group, in comparison with the mock and NC groups, showed a significantly down-regulated expression of PTTG1 (0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05), but up-regulated expressions of p-21CIP1 (0.32 ± 0.03 vs 0.20 ± 0.02 and 0.21 ± 0.03, P < 0.05), p-27Kip1 (0.38 ± 0.02 vs 0.20 ± 0.03 and 0.22 ± 0.01, P < 0.05), Glb1 (0.24 ± 0.01 vs 0.13 ± 0.01 and 0.15 ± 0.01, P < 0.05), and HP1γ (0.41 ± 0.01 vs 0.26 ± 0.01 and 0.27 ± 0.02, P < 0.05) in the LNCaP-AI cells. CONCLUSIONS: Down-regulated expression of PTTG1 induces senescence of human castration-resistant prostate cancer LNCaP-AI cells.

Hyperactivity of the transcription factor Nrf2 causes metabolic reprogramming in mouse esophagus.

Mutations in the genes encoding nuclear factor (erythroid-derived 2)-like 2 (NRF2), Kelch-like ECH-associated protein 1 (KEAP1), and cullin 3 (CUL3) are commonly observed in human esophageal squamous cell carcinoma (ESCC) and result in activation of the NRF2 signaling pathway. Moreover, hyperactivity of the transcription factor Nrf2 has been found to cause esophageal hyperproliferation and hyperkeratosis in mice. However, the underlying mechanism is unclear. In this study, we aimed to understand the molecular mechanisms of esophageal hyperproliferation in mice due to hyperactive Nrf2. Esophageal tissues were obtained from genetically modified mice that differed in the status of the Nrf2 gene and genes in the same pathway (Nrf2-/-, Keap1-/-, K5Cre;Pkm2fl/fl;Keap1-/-, and WT) and analyzed for metabolomic profiles, Nrf2 ChIP-seq, and gene expression. We found that hyperactive Nrf2 causes metabolic reprogramming and up-regulation of metabolic genes in the mouse esophagus. One of the glycolysis genes encoding pyruvate kinase M2 (Pkm2) was not only differentially up-regulated, but also glycosylated and oligomerized, resulting in increased ATP biosynthesis. However, constitutive knockout of Pkm2 failed to inhibit this esophageal phenotype in vivo, and this failure may have been due to compensation by Pkm1 up-regulation. Transient inhibition of NRF2 or glycolysis inhibited the growth of human ESCC cells in which NRF2 is hyperactive in vitro In summary, hyperactive Nrf2 causes metabolic reprogramming in the mouse esophagus through its transcriptional regulation of metabolic genes. Blocking glycolysis transiently inhibits cell proliferation and may therefore have therapeutically beneficial effects on NRF2high ESCC in humans.

PAX9 regulates squamous cell differentiation and carcinogenesis in the oro-oesophageal epithelium.

PAX9 is a transcription factor of the PAX family characterized by a DNA-binding paired domain. Previous studies have suggested a potential role of PAX9 in squamous cell differentiation and carcinogenesis of the oro-oesophageal epithelium. However, its functional roles in differentiation and carcinogenesis remain unclear. In this study, Pax9 deficiency in mouse oesophagus promoted cell proliferation, delayed cell differentiation, and altered the global gene expression profile. Ethanol exposure downregulated PAX9 expression in human oesophageal epithelial cells in vitro and mouse forestomach and tongue in vivo. We further showed that PAX9 was downregulated in human oro-oesophageal squamous cell carcinoma (OESCC), and its downregulation was associated with alcohol drinking and promoter hypermethylation. Moreover, ad libitum feeding with a liquid diet containing ethanol for 40 weeks or Pax9 deficiency promoted N-nitrosomethylbenzylamine-induced squamous cell carcinogenesis in mouse tongue, oesophagus, and forestomach. In conclusion, PAX9 regulates squamous cell differentiation in the oro-oesophageal epithelium. Alcohol drinking and promoter hypermethylation are associated with PAX9 silencing in human OESCC. PAX9 downregulation may contribute to alcohol-associated oro-oesophageal squamous cell carcinogenesis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Esophageal cancer in Kenya.

Kenya belongs to a high incidence region known as Africa's esophageal cancer (EC) corridor. It has one of the highest incidence rates of EC worldwide, but research on EC in Kenya has gone highly unnoticed. EC in Kenya is unique in its high percentage of young cases (< 30 years of age). In this review, we show the current status of EC in the country. We mainly focus on significant risk factors such as alcohol drinking, genetic factors, malnutrition and hot food/drink. Future directions in the study and prevention of EC in Kenya are also discussed.

Chemoprevention of oxidative stress-associated oral carcinogenesis by sulforaphane depends on NRF2 and the isothiocyanate moiety.

Oxidative stress is known to play an important role in oral cancer development. In this study we aimed to examine whether a chemical activator of NRF2, sulforaphane (SFN), may have chemopreventive effects on oxidative stress-associated oral carcinogenesis. We first showed that Nrf2 activation and oxidative damage were commonly seen in human samples of oral leukoplakia. With gene microarray and immunostaining, we found 4-nitroquinoline 1-oxide (4NQO) in drink activated the Nrf2 pathway and produced oxidative damage in mouse tongue. Meanwhile whole exome sequencing of mouse tongue identified mutations consistent with 4NQO's mutagenic profile. Using cultured human oral keratinocytes and 4NQO-treated mouse tongue, we found that SFN pre-treatment activated the NRF2 pathway and inhibited oxidative damage both in vitro and in vivo. On the contrary, a structural analogue of SFN without the isothiocyanate moiety did not have such effects. In a long-term chemoprevention study using wild-type and Nrf2-/- mice, we showed that topical application of SFN activated the NRF2 pathway, inhibited oxidative damage, and prevented 4NQO-induced oral carcinogenesis in an Nrf2-dependent manner. Our data clearly demonstrate that SFN has chemopreventive effects on oxidative stress-associated oral carcinogenesis, and such effects depend on Nrf2 and the isothiocyanate moiety.

Personalized and targeted therapy of esophageal squamous cell carcinoma: an update.

Esophageal squamous cell carcinoma (ESCC) is a deadly disease that requires extensive research. In this review, we update recent progress in the research area of targeted therapy for ESCC. SOX2 and its associated proteins (e.g., ΔNP63α), which regulate lineage survival of ESCC cells, are proposed as therapeutic targets. It is believed that targeting the lineage-survival mechanism may be more effective than targeting other mechanisms. With the advent of a new era of personalized targeted therapy, there is a need to move from the tumor-centric model into an organismic model.

TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis.

Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.

Sensitive and rapid detection of viruses associated with hand foot and mouth disease using multiplexed MALDI-TOF analysis.

BACKGROUND: Human enterovirus (HEV) is the major cause of hand foot and mouth disease (HFMD). A powerful method for detecting HEVs associated with HFMD can provide results in a clinically relevant time frame. However, the limitations of the current enterovirus test make it difficult to identify multiple genotypes on the first pass. OBJECTIVE: To develop a more sensitive and easy applicable assay for detecting 18 HFMD-associated HEV serotypes in multiplex PCR products. STUDY DESIGN: : A total of 241 clinical specimens were collected from HFMD patients during the 2010 outbreak in China. These samples were tested by DNA sequencing and MassARRAY analysis, respectively. RESULTS: Analysis of a dilution series of plasmids revealed the detection limit per PCR reaction for the MassARRAY method was one copy for the tested HEVs. We compared results from 241 samples to those of the sequence analysis of the VP1 gene. The MassARRAY method detected all samples found positive by consensus PCR and sequencing method. Comparison of the results of MassARRAY and the DNA sequencing method found concordant results for 225 (93.4%) of the 241 samples. In 14 (5.8%) samples, the MassARRAY method detected multiple types, whereas the DNA sequencing method detected a single type. In another 2 (0.8%) samples, the MassARRAY method detected single types, whereas the DNA sequencing method detected no HEV. CONCLUSIONS: The MassARRAY assay is a highly sensitive and accurate method for the type-specific detection of 18 HEVs in HFMD and is a powerful complement to current detection methods.