SOX9 Modulates the Transformation of Gastric Stem Cells Through Biased Symmetric Cell Division.

BACKGROUND & AIMS: Transformation of stem/progenitor cells has been associated with tumorigenesis in multiple tissues, but stem cells in the stomach have been hard to localize. We therefore aimed to use a combination of several markers to better target oncogenes to gastric stem cells and understand their behavior in the initial stages of gastric tumorigenesis. METHODS: Mouse models of gastric metaplasia and cancer by targeting stem/progenitor cells were generated and analyzed with techniques including reanalysis of single-cell RNA sequencing and immunostaining. Gastric cancer cell organoids were genetically manipulated with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for functional studies. Cell division was determined by bromodeoxyuridine-chasing assay and the assessment of the orientation of the mitotic spindles. Gastric tissues from patients were examined by histopathology and immunostaining. RESULTS: Oncogenic insults lead to expansion of SOX9+ progenitor cells in the mouse stomach. Genetic lineage tracing and organoid culture studies show that SOX9+ gastric epithelial cells overlap with SOX2+ progenitors and include stem cells that can self-renew and differentiate to generate all gastric epithelial cells. Moreover, oncogenic targeting of SOX9+SOX2+ cells leads to invasive gastric cancer in our novel mouse model (Sox2-CreERT;Sox9-loxp(66)-rtTA-T2A-Flpo-IRES-loxp(71);Kras(Frt-STOP-Frt-G12D);P53R172H), which combines Cre-loxp and Flippase-Frt genetic recombination systems. Sox9 deletion impedes the expansion of gastric progenitor cells and blocks neoplasia after Kras activation. Although Sox9 is not required for maintaining tissue homeostasis where asymmetric division predominates, loss of Sox9 in the setting of Kras activation leads to reduced symmetric cell division and effectively attenuates the Kras-dependent expansion of stem/progenitor cells. Similarly, Sox9 deletion in gastric cancer organoids reduces symmetric cell division, organoid number, and organoid size. In patients with gastric cancer, high levels of SOX9 are associated with recurrence and poor prognosis. CONCLUSION: SOX9 marks gastric stem cells and modulates biased symmetric cell division, which appears to be required for the malignant transformation of gastric stem cells.

Helicobacter pylori pathogen inhibits cellular responses to oncogenic stress and apoptosis.

Helicobacter pylori (H. pylori) is a common gastric pathogen that infects approximately half of the world's population. Infection with H. pylori can lead to diverse pathological conditions, including chronic gastritis, peptic ulcer disease, and cancer. The latter is the most severe consequence of H. pylori infection. According to epidemiological studies, gastric infection with H. pylori is the strongest known risk factor for non-cardia gastric cancer (GC), which remains one of the leading causes of cancer-related deaths worldwide. However, it still remains to be poorly understood how host-microbe interactions result in cancer development in the human stomach. Here we focus on the H. pylori bacterial factors that affect the host ubiquitin proteasome system. We investigated E3 ubiquitin ligases SIVA1 and ULF that regulate p14ARF (p19ARF in mice) tumor suppressor. ARF plays a key role in regulation of the oncogenic stress response and is frequently inhibited during GC progression. Expression of ARF, SIVA1 and ULF proteins were investigated in gastroids, H. pylori-infected mice and human gastric tissues. The role of the H. pylori type IV secretion system was assessed using various H. pylori isogenic mutants. Our studies demonstrated that H. pylori infection results in induction of ULF, decrease in SIVA1 protein levels, and subsequent ubiquitination and degradation of p14ARF tumor suppressor. Bacterial CagA protein was found to sequentially bind to SIVA1 and ULF proteins. This process is regulated by CagA protein phosphorylation at the EPIYA motifs. Downregulation of ARF protein leads to inhibition of cellular apoptosis and oncogenic stress response that may promote gastric carcinogenesis.

Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in oesophageal adenocarcinoma.

OBJECTIVE: Oesophageal adenocarcinoma (EAC) arises in the setting of Barrett's oesophagus, an intestinal metaplastic precursor lesion that can develop in patients with chronic GERD. Here, we investigated the role of acidic bile salts, the mimicry of reflux, in activation of NOTCH signaling in EAC. DESIGN: This study used public databases, EAC cell line models, L2-IL1ß transgenic mouse model and human EAC tissue samples to identify mechanisms of NOTCH activation under reflux conditions. RESULTS: Analysis of public databases demonstrated significant upregulation of NOTCH signaling components in EAC. In vitro studies demonstrated nuclear accumulation of active NOTCH1 cleaved fragment (NOTCH intracellular domain) and upregulation of NOTCH targets in EAC cells in response to reflux conditions. Additional investigations identified DLL1 as the predominant ligand contributing to NOTCH1 activation under reflux conditions. We discovered a novel crosstalk between APE1 redox function, reflux-induced inflammation and DLL1 upregulation where NF-κB can directly bind to and induce the expression of DLL1. The APE1 redox function was crucial for activation of the APE1-NF-κB-NOTCH axis and promoting cancer cell stem-like properties in response to reflux conditions. Overexpression of APE1 and DLL1 was detected in gastro-oesophageal junctions of the L2-IL1ß transgenic mouse model and human EAC tissue microarrays. DLL1 high levels were associated with poor overall survival in patients with EAC. CONCLUSION: These findings underscore a unique mechanism that links redox balance, inflammation and embryonic development (NOTCH) into a common pro-tumorigenic pathway that is intrinsic to EAC cells.

Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma.

OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.

Multivalent tyrosine kinase inhibition promotes T cell recruitment to immune-desert gastric cancers by restricting epithelial-mesenchymal transition via tumour-intrinsic IFN-γ signalling.

OBJECTIVE: Gastric cancer (GC) ranks fifth in incidence and fourth for mortality worldwide. The response to immune checkpoint blockade (ICB) therapy in GC is heterogeneous due to tumour-intrinsic and acquired immunotherapy resistance. We developed an immunophenotype-based subtyping of human GC based on immune cells infiltration to develop a novel treatment option. DESIGN: A algorithm was developed to reclassify GC into immune inflamed, excluded and desert subtypes. Bioinformatics, human and mouse GC cell lines, syngeneic murine gastric tumour model, and CTLA4 blockade were used to investigate the immunotherapeutic effects by restricting receptor tyrosine kinase (RTK) signalling in immune desert (ICB-resistant) type GC. RESULTS: Our algorithm restratified subtypes of human GC in public databases and showed that immune desert-type and excluded-type tumours are ICB-resistant compared with immune-inflamed GC. Moreover, epithelial-mesenchymal transition (EMT) signalling was highly enriched in immune desert-type GC, and syngeneic murine tumours exhibiting mesenchymal-like, compared with epithelial-like, properties are T cell-excluded and resistant to CTLA4 blockade. Our analysis further identified a panel of RTKs as potential druggable targets in the immune desert-type GC. Dovitinib, an inhibitor of multiple RTKs, strikingly repressed EMT programming in mesenchymal-like immune desert syngeneic GC models. Dovitinib activated the tumour-intrinsic SNAI1/2-IFN-γ signalling axis and impeded the EMT programme, converting immune desert-type tumours to immune inflamed-type tumours, sensitising these mesenchymal-like 'cold' tumours to CTLA4 blockade. CONCLUSION: Our findings identified potential druggable targets relevant to patient groups, especially for refractory immune desert-type/ 'cold' GC. Dovitinib, an RTK inhibitor, sensitised desert-type immune-cold GC to CTLA4 blockade by restricting EMT and recruiting T cells.

NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1.

BACKGROUND: H. pylori infection is the main risk factor for gastric cancer. In this study, we investigated H. pylori-mediated activation of STAT3 and NF-κB in gastric cancer, using in vitro and in vivo models. METHODS: To investigate the activation of NF-κB and STAT3 by H. pylori strains we used in vitro and in vivo mouse models, western blots, immunofluorescence, ChIP Assay, luciferase and quantitative real-time PCR assays. RESULTS: Following infection with H. pylori in vitro, we found an earlier phosphorylation of NF-kB-p65 (S536), followed by STAT3 (Y705). Immunofluorescence, using in vitro and in vivo models, demonstrated nuclear localization of NF-kB and STAT3, following H. pylori infection. NF-kB and STAT3 luciferase reporter assays confirmed earlier activation of NF-kB followed by STAT3. In vitro and in vivo models demonstrated induction of mRNA expression of IL-6 (p < 0.001), VEGF-α (p < 0.05), IL-17 (p < 0.001), and IL-23 (p < 0.001). Using ChIP, we confirmed co-binding of both NF-kB-p65 and STAT3 on the IL6 promoter. The reconstitution of Trefoil Factor 1 (TFF1) suppressed activation of NF-kB with reduction in IL6 levels and STAT3 activity, in response to H. pylori infection. Using pharmacologic (BAY11-7082) and genetic (IκB super repressor (IκBSR)) inhibitors of NF-kB-p65, we confirmed the requirement of NF-kB-p65 for activation of STAT3, as measured by phosphorylation, transcription activity, and nuclear localization of STAT3 in in vitro and in vivo models. CONCLUSION: Our findings suggest the presence of an early autocrine NF-kB-dependent activation of STAT3 in response to H. pylori infection. TFF1 acts as an anti-inflammatory guard against H. pylori-mediated activation of pro-inflammatory networks.

Targeting SOX2 Protein with Peptide Aptamers for Therapeutic Gains against Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a predominant cancer type in developing countries such as China, where ESCC accounts for approximately 90% of esophageal malignancies. Lacking effective and targeted therapy contributes to the poor 5-year survival rate. Recent studies showed that about 30% of ESCC cases have high levels of SOX2. Herein, we aim to target this transcription factor with aptamer. We established a peptide aptamer library and then performed an unbiased screening to identify several peptide aptamers including P42 that can bind and inhibit SOX2 downstream target genes. We further found that P42 overexpression or incubation with a synthetic peptide 42 inhibited the proliferation, migration, and invasion of ESCC cells. Moreover, peptide 42 treatment inhibited the growth and metastasis of ESCC xenografts in mouse and zebrafish. Further analysis revealed that P42 overexpression led to alternations in the levels of proteins that are important for the proliferation and migration of ESCC cells. Taken together, our study identified the peptide 42 as a key inhibitor of SOX2 function, reducing the proliferation and migration of ESCC cells in vitro and in vivo, and thereby offering a potential therapy against ESCC.

Microbial Regulation of p53 Tumor Suppressor.

p53 tumor suppressor has been identified as a protein interacting with the large T antigen produced by simian vacuolating virus 40 (SV40). Subsequent research on p53 inhibition by SV40 and other tumor viruses has not only helped to gain a better understanding of viral biology, but also shaped our knowledge of human tumorigenesis. Recent studies have found, however, that inhibition of p53 is not strictly in the realm of viruses. Some bacterial pathogens also actively inhibit p53 protein and induce its degradation, resulting in alteration of cellular stress responses. This phenomenon was initially characterized in gastric epithelial cells infected with Helicobacter pylori, a bacterial pathogen that commonly infects the human stomach and is strongly linked to gastric cancer. Besides H. pylori, a number of other bacterial species were recently discovered to inhibit p53. These findings provide novel insights into host-bacteria interactions and tumorigenesis associated with bacterial infections.

Prevention of DNA damage in Barrett's esophageal cells exposed to acidic bile salts.

Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the USA. The major risk factor for EA is gastroesophageal reflux disease (GERD). During GERD, esophageal cells are exposed to refluxate which contains gastric acid frequently mixed with duodenal bile. This may lead to mucosal injury and Barrett's metaplasia (BE) that are important factors contributing to development of EA. In this study, we investigated DNA damage in BE cells exposed to acidic bile salts and explored for potential protective strategies. Exposure of BE cells to acidic bile salts led to significant DNA damage, which in turn, was due to generation of reactive oxygen species (ROS). We found that acidic bile salts induce a rapid increase in superoxide radicals and hydrogen peroxide, which were determined using electron paramagnetic resonance spectroscopy and Amplex Red assay. Analyzing a panel of natural antioxidants, we identified apocynin to be the most effective in protecting esophageal cells from DNA damage induced by acidic bile salts. Mechanistic analyses showed that apocynin inhibited ROS generation and increases the DNA repair capacity of BE cells. We identified BRCA1 and p73 proteins as apocynin targets. Downregulation of p73 inhibited the protective effect of apocynin. Taken together, our results suggest potential application of natural compounds such as apocynin for prevention of reflux-induced DNA damage and GERD-associated tumorigenesis.

Bacterial CagA protein induces degradation of p53 protein in a p14ARF-dependent manner.

OBJECTIVE: Infection with Helicobacter pylori is the strongest known risk factor for adenocarcinoma of the stomach. Tumorigenic transformation of gastric epithelium induced by H. pylori is a highly complex process driven by an active interplay between bacterial virulence and host factors, many aspects of which remain obscure. In this work, we investigated the degradation of p53 tumour suppressor induced by H. pylori. DESIGN: Expression of p53 protein in gastric biopsies was assessed by immunohistochemistry. Gastric cells were co-cultured with H. pylori strains isolated from high-gastric risk and low-gastric risk areas and assessed for expression of p53, p14ARF and cytotoxin-associated gene A (CagA) by immunoblotting. siRNA was used to inhibit activities of ARF-BP1 and Human Double Minute 2 (HDM2) proteins. RESULTS: Our analysis demonstrated that H. pylori strains expressing high levels of CagA virulence factor and associated with a higher gastric cancer risk more strongly suppress p53 compared with low-risk strains in vivo and in vitro. We found that degradation of p53 induced by bacterial CagA protein is mediated by host HDM2 and ARF-BP1 E3 ubiquitin ligases, while the p14ARF protein counteracts H. pylori-induced signalling. CONCLUSIONS: Our results provide novel evidence that tumorigenicity associated with H. pylori infection is linked to inhibition of p53 protein by CagA. We propose a model in which CagA-induced degradation of p53 protein is determined by a relative level of p14ARF. In cells in which p14ARF levels were decreased due to hypermethylation or deletion of the p14ARF gene, H. pylori efficiently degraded p53, whereas p53 is protected in cells expressing high levels of p14ARF.

Mitochondrial STAT3 contributes to transformation of Barrett's epithelial cells that express oncogenic Ras in a p53-independent fashion.

Metaplastic epithelial cells of Barrett's esophagus transformed by the combination of p53-knockdown and oncogenic Ras expression are known to activate signal transducer and activator of transcription 3 (STAT3). When phosphorylated at tyrosine 705 (Tyr705), STAT3 functions as a nuclear transcription factor that can contribute to oncogenesis. STAT3 phosphorylated at serine 727 (Ser727) localizes in mitochondria, but little is known about mitochondrial STAT3's contribution to carcinogenesis in Barrett's esophagus, which is the focus of this study. We introduced a constitutively active variant of human STAT3 (STAT3CA) into the following: 1) non-neoplastic Barrett's (BAR-T) cells; 2) BAR-T cells with p53 knockdown; and 3) BAR-T cells that express oncogenic H-Ras(G12V). STAT3CA transformed only the H-Ras(G12V)-expressing BAR-T cells (evidenced by loss of contact inhibition, formation of colonies in soft agar, and generation of tumors in immunodeficient mice), and did so in a p53-independent fashion. The transformed cells had elevated levels of both mitochondrial (Ser727) and nuclear (Tyr705) phospho-STAT3. Introduction of a STAT3CA construct with a mutated tyrosine phosphorylation site into H-Ras(G12V)-expressing Barrett's cells resulted in high levels of mitochondrial phospho-STAT3 (Ser727) with little or no nuclear phospho-STAT3 (Tyr705), and the cells still formed tumors in immunodeficient mice. Thus tyrosine phosphorylation of STAT3 is not required for tumor formation in Ras-expressing Barrett's cells. We conclude that mitochondrial STAT3 (Ser727) can contribute to oncogenesis in Barrett's cells that express oncogenic Ras. These findings suggest that agents targeting STAT3 might be useful for chemoprevention in patients with Barrett's esophagus.

Pathogenic bacterium Helicobacter pylori alters the expression profile of p53 protein isoforms and p53 response to cellular stresses.

The p53 protein plays a central role in the prevention of tumorigenesis. Cellular stresses, such as DNA damage and aberrant oncogene activation, trigger induction of p53 that halts cellular proliferation and allows cells to be repaired. If cellular damage is beyond the capability of the repair mechanisms, p53 induces apoptosis or cell cycle arrest, preventing damaged cells from becoming cancerous. However, emerging evidence suggests that the function of p53 needs to be considered as isoform-specific. Here, we report that the expression profile of p53 can be shifted toward inhibitory p53 isoforms by the pathogenic bacterium Helicobacter pylori, which is known for its strong association with gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. We found that interaction of H. pylori with gastric epithelial cells, mediated via the cag pathogenicity island, induces N-terminally truncated Δ133p53 and Δ160p53 isoforms in human cells. Induction of an orthologous p53 isoform, Δ153p53, was also found in H. pylori-infected Mongolian gerbils. The p53 isoforms inhibit p53 and p73 activities, induce NF-κB, and increase survival of infected cells. Expression of Δ133p53, in response to H. pylori infection, is regulated by phosphorylation of c-Jun and activation of activator protein-1-dependent transcription. Together, these results provide unique insights into the regulation of p53 protein and may contribute to the understanding of tumorigenesis associated with H. pylori.

Glutathione peroxidase 7 has potential tumour suppressor functions that are silenced by location-specific methylation in oesophageal adenocarcinoma.

OBJECTIVE: To investigate the potential tumour suppressor functions of glutathione peroxidase 7 (GPX7) and examine the interplay between epigenetic and genetic events in regulating its expression in oesophageal adenocarcinomas (OAC). DESIGN: In vitro and in vivo cell models were developed to investigate the biological and molecular functions of GPX7 in OAC. RESULTS: Reconstitution of GPX7 in OAC cell lines, OE33 and FLO-1, significantly suppressed growth as shown by the growth curve, colony formation and EdU proliferation assays. Meanwhile, GPX7-expressing cells displayed significant impairment in G1/S progression and an increase in cell senescence. Concordant with the above functions, Western blot analysis displayed higher levels of p73, p27, p21 and p16 with a decrease in phosphorylated retinoblastoma protein (RB), indicating its increased tumour suppressor activities. On the contrary, knockdown of GPX7 in HET1A cells (an immortalised normal oesophageal cell line) rendered the cells growth advantage as indicated with a higher EdU rate, lower levels of p73, p27, p21 and p16 and an increase in phosphorylated RB. We confirmed the tumour suppressor function in vivo using GPX7-expressing OE33 cells in a mouse xenograft model. Pyrosequencing of the GPX7 promoter region (-162 to +138) demonstrated location-specific hypermethylation between +13 and +64 in OAC (69%, 54/78). This was significantly associated with the downregulation of GPX7 (p<0.01). Neither mutations in the coding exons of GPX7 nor DNA copy number losses were frequently present in the OAC examined (<5%). CONCLUSIONS: Our data suggest that GPX7 possesses tumour suppressor functions in OAC and is silenced by location-specific promoter DNA methylation.

SOX9 is regulated by AURKA in response to Helicobacter pylori infection via EIF4E-mediated cap-dependent translation.

Helicobacter pylori (H. pylori) infection is the main risk factor for gastric cancer. The SRY-Box Transcription Factor 9 (SOX9) serves as a marker of stomach stem cells. We detected strong associations between AURKA and SOX9 expression levels in gastric cancers. Utilizing in vitro and in vivo mouse models, we demonstrated that H. pylori infection induced elevated levels of both AURKA and SOX9 proteins. Notably, the SOX9 protein and transcription activity levels were dependent on AURKA expression. AURKA knockdown led to a reduction in the number and size of gastric gland organoids. Conditional knockout of AURKA in mice resulted in a decrease in SOX9 baseline level in AURKA-knockout gastric glands, accompanied by diminished SOX9 induction following H. pylori infection. We found an AURKA-dependent increase in EIF4E and cap-dependent translation with an AURKA-EIF4E-dependent increase in SOX9 polysomal RNA levels. Immunoprecipitation assays demonstrated binding of AURKA to EIF4E with a decrease in EIF4E ubiquitination. Immunohistochemistry analysis on tissue arrays revealed moderate to strong immunostaining of AURKA and SOX9 with a significant correlation in gastric cancer tissues. These findings elucidate the mechanistic role of AURKA in regulating SOX9 levels via cap-dependent translation in response to H. pylori infection in gastric tumorigenesis.

Targeting SMAD3 Improves Response to Oxaliplatin in Esophageal Adenocarcinoma Models by Impeding DNA Repair.

PURPOSE: TGFß signaling is implicated in the progression of most cancers, including esophageal adenocarcinoma (EAC). Emerging evidence indicates that TGFß signaling is a key factor in the development of resistance toward cancer therapy. EXPERIMENTAL DESIGN: In this study, we developed patient-derived organoids and patient-derived xenograft models of EAC and performed bioinformatics analysis combined with functional genetics to investigate the role of SMAD family member 3 (SMAD3) in EAC resistance to oxaliplatin. RESULTS: Chemotherapy nonresponding patients showed enrichment of SMAD3 gene expression when compared with responders. In a randomized patient-derived xenograft experiment, SMAD3 inhibition in combination with oxaliplatin effectively diminished tumor burden by impeding DNA repair. SMAD3 interacted directly with protein phosphatase 2A (PP2A), a key regulator of the DNA damage repair protein ataxia telangiectasia mutated (ATM). SMAD3 inhibition diminished ATM phosphorylation by enhancing the binding of PP2A to ATM, causing excessive levels of DNA damage. CONCLUSIONS: Our results identify SMAD3 as a promising therapeutic target for future combination strategies for the treatment of patients with EAC.

Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy.

While cell fate determination and maintenance are important in establishing and preserving tissue identity and function during development, aberrant cell fate transition leads to cancer cell heterogeneity and resistance to treatment. Here, we report an unexpected role for the transcription factor p63 (Trp63/TP63) in the fate choice of the squamous versus neuroendocrine lineage in esophageal development and malignancy. Deletion of p63 results in extensive neuroendocrine differentiation in the developing mouse esophagus and esophageal progenitors derived from human embryonic stem cells. In human esophageal neuroendocrine carcinoma (eNEC) cells, p63 is transcriptionally silenced by EZH2-mediated H3K27 trimethylation (H3K27me3). Up-regulation of the major p63 isoform ΔNp63α, through either ectopic expression or EZH2 inhibition, promotes squamous transdifferentiation of eNEC cells. Together, these findings uncover p63 as a rheostat in coordinating the transition between squamous and neuroendocrine cell fates during esophageal development and tumor progression.

The combination of alisertib, an investigational Aurora kinase A inhibitor, and docetaxel promotes cell death and reduces tumor growth in preclinical cell models of upper gastrointestinal adenocarcinomas.

BACKGROUND: Upper gastrointestinal adenocarcinomas (UGCs) respond poorly to current chemotherapeutic regimes. The authors and others have previously reported frequent Aurora kinase A (AURKA) gene amplification and mRNA and protein overexpression in UGCs. The objective of the current study was to determine the therapeutic potential of alisertib (MLN8237) alone and in combination with docetaxel in UGCs. METHODS: After treatment with alisertib and/or docetaxel, clonogenic cell survival, cell cycle analyses, Western blot analyses, and tumor xenograft growth assays were carried out to measure cell survival, cell cycle progression, apoptotic protein expression, and tumor xenograft volumes, respectively. RESULTS: By using the AGS, FLO-1, and OE33 UGC cell lines, which have constitutive AURKA overexpression and variable tumor protein 53 (p53) status, significantly enhanced inhibition of cancer cell survival was observed with alisertib and docetaxel treatment in combination (P < .001), compared with single-agent treatments. Cell cycle analyses, after 48 hours of treatment with alisertib, produced a significant increase in the percentage of polyploidy in UGC cells (P < .01) that was further enhanced by docetaxel (P < .001). In addition, an increase in the percentage of cells in sub-G1-phase observed with alisertib (P < .01) was significantly enhanced with the combination treatment (P < .001). Western blot analysis demonstrated higher induction of cleaved caspase 3 protein expression with the combined treatment compared with single-agent treatments. In addition, FLO-1 and OE33 cell xenograft models demonstrated enhanced antitumor activity for the alisertib and docetaxel combination compared with single-agent treatments (P < .001). CONCLUSIONS: The current study demonstrated that alisertib combined with docetaxel can mediate a better therapeutic outcome in UGC cell lines.

Glutathione peroxidase 7 protects against oxidative DNA damage in oesophageal cells.

OBJECTIVE: Exposure of the oesophageal mucosa to gastric acid and bile acids leads to the accumulation of reactive oxygen species (ROS), a known risk factor for Barrett's oesophagus and progression to oesophageal adenocarcinoma (OAC). This study investigated the functions of glutathione peroxidase 7 (GPX7), frequently silenced in OAC, and its capacity in regulating ROS and its associated oxidative DNA damage. DESIGN: Using in-vitro cell models, experiments were performed that included glutathione peroxidase (GPX) activity, Amplex UltraRed, CM-H(2)DCFDA, Annexin V, 8-oxoguanine, phospho-H2A.X, quantitative real-time PCR and western blot assays. RESULTS: Enzymatic assays demonstrated limited GPX activity of the recombinant GPX7 protein. GPX7 exhibited a strong capacity to neutralise hydrogen peroxide (H(2)O(2)) independent of glutathione. Reconstitution of GPX7 expression in immortalised Barrett's oesophagus cells, BAR-T and CP-A led to resistance to H(2)O(2)-induced oxidative stress. Following exposure to acidic bile acids cocktail (pH4), these GPX7-expressing cells demonstrated lower levels of H(2)O(2), intracellular ROS, oxidative DNA damage and double-strand breaks, compared with controls (p<0.01). In addition, these cells demonstrated lower levels of ROS signalling, indicated by reduced phospho-JNK (Thr183/Tyr185) and phospho-p38 (Thr180/Tyr182), and demonstrated lower levels of apoptosis following the exposure to acidic bile acids or H(2)O(2)-induced oxidative stress. The knockdown of endogenous GPX7 in immortalised oesophageal squamous epithelial cells (HET1A) confirmed the protective functions of GPX7 against pH4 bile acids by showing an increase in the levels of H(2)O(2), intracellular ROS, oxidative DNA damage, double-strand breaks, apoptosis, and ROS-dependent signalling (p<0.01). CONCLUSION: The dysfunction of GPX7 in oesophageal cells increases the levels of ROS and oxidative DNA damage, which are common risk factors for Barrett's oesophagus and OAC.

Urgent need to mitigate disparities in federal funding for cancer research.

We evaluate National Cancer Institute (NCI) funding distribution to the most common cancers, considering their respective public health burdens, and explore associations between funding and racial and ethnic burden of disease. The NCI's Surveillance, Epidemiology and End Results, US Cancer Statistics database, and Funding Statistics were used to calculate funding-to-lethality (FTL) scores. Breast and prostate cancer had the first (179.65) and second (128.90) highest FTL scores, and esophagus and stomach cancer ranked 18th (2.12) and 19th (1.78). We evaluated whether there were differences between the FTL and cancer incidence and/or mortality within individual racial and ethnic groups. NCI funding correlated highly with cancers afflicting a higher proportion of non-Hispanic White individuals (Spearman correlation coefficient = 0.84; P < .001). Correlation was stronger for incidence than mortality. These data reveal that funding across cancer sites is not concordant with lethality and that cancers with high incidence among racial and ethnic minorities receive lower funding.

Multi-cancer analysis reveals universal association of oncogenic LBH expression with DNA hypomethylation and WNT-Integrin signaling pathways.

Limb-Bud and Heart (LBH) is a developmental transcription co-factor deregulated in cancer, with reported oncogenic and tumor suppressive effects. However, LBH expression in most cancer types remains unknown, impeding understanding of its mechanistic function Here, we performed systematic bioinformatic and TMA analysis for LBH in >20 different cancer types. LBH was overexpressed in most cancers compared to normal tissues (>1.5-fold; p < 0.05), including colon-rectal, pancreatic, esophageal, liver, stomach, bladder, kidney, prostate, testicular, brain, head & neck cancers, and sarcoma, correlating with poor prognosis. The cancer types showing LBH downregulation were lung, melanoma, ovarian, cervical, and uterine cancer, while both LBH over- and under-expression were observed in hematopoietic malignancies. In cancers with LBH overexpression, the LBH locus was frequently hypomethylated, identifying DNA hypomethylation as a potential mechanism for LBH dysregulation. Pathway analysis identified a universal, prognostically significant correlation between LBH overexpression and the WNT-Integrin signaling pathways. Validation of the clinical association of LBH with WNT activation in gastrointestinal cancer cell lines, and in colorectal patient samples by IHC uncovered that LBH is specifically expressed in tumor cells with nuclear beta-catenin at the invasive front. Collectively, these data reveal a high degree of LBH dysregulation in cancer and establish LBH as pan-cancer biomarker for detecting WNT hyperactivation in clinical specimens.