Nicholas J. Clemons

Hsp72 inhibits apoptosis upstream of the mitochondria and not through interactions with Apaf-1.

Hsp72 protects cells against apoptosis in response to various stresses. By simultaneously measuring cytochrome c localization and nuclear morphology in mouse embryo fibroblasts, we have shown that Hsp72 blocks cytochrome c release from mitochondria in response to cytotoxic stress and that permeabilization of the outer mitochondrial membrane is the critical point in deciding the fate of the cell. Hsp72 did not inhibit apoptosis in mouse embryo fibroblasts once cytochrome c had been released from the mitochondria. Recent reports have claimed that Hsp72 can prevent caspase activation by inhibiting the oligomerization of Apaf-1 in the presence of cytochrome c and dATP. We now show that this apparent function of recombinant Hsp72 is due to the presence of salt in the Hsp72 preparation and that the same response can be achieved by the addition of heat-denatured Hsp72 in the same high salt buffer or by the high salt buffer alone. Hsp72 expressed in a range of different cell lines had no inhibitory effect on cytochrome c-stimulated caspase activity of cytosolic extracts. We conclude that the protective effect of Hsp72 occurs upstream of the mitochondria and not through the inhibition of the apoptosome.

Aberrant Epithelial–Mesenchymal Hedgehog Signaling Characterizes Barrett's Metaplasia

BACKGROUND & AIMS The molecular mechanism underlying epithelial metaplasia in Barretts esophagus remains unknown. Recognizing that Hedgehog signaling is required for early esophageal development, we sought to determine if the Hedgehog pathway is reactivated in Barretts esophagus, and if genes downstream of the pathway could promote columnar differentiation of esophageal epithelium. METHODS Immunohistochemistry, immunofluorescence, and quantitative real-time polymerase chain reaction were used to analyze clinical specimens, human esophageal cell lines, and mouse esophagi. Human esophageal squamous epithelial (HET-1A) and adenocarcinoma (OE33) cells were subjected to acid treatment and used in transfection experiments. Swiss Webster mice were used in a surgical model of bile reflux injury. An in vivo transplant culture system was created using esophageal epithelium from Sonic hedgehog transgenic mice. RESULTS Marked up-regulation of Hedgehog ligand expression, which can be induced by acid or bile exposure, occurs frequently in Barretts epithelium and is associated with stromal expression of the Hedgehog target genes PTCH1 and BMP4. BMP4 signaling induces expression of SOX9, an intestinal crypt transcription factor, which is highly expressed in Barretts epithelium. We further show that expression of Deleted in Malignant Brain Tumors 1, the human homologue of the columnar cell factor Hensin, occurs in Barretts epithelium and is induced by SOX9. Finally, transgenic expression of Sonic hedgehog in mouse esophageal epithelium induces expression of stromal Bmp4, epithelial Sox9, and columnar cytokeratins. CONCLUSIONS Epithelial Hedgehog ligand expression may contribute to the initiation of Barretts esophagus through induction of stromal BMP4, which triggers reprogramming of esophageal epithelium in favor of a columnar phenotype.

Stromal genes discriminate preinvasive from invasive disease, predict outcome, and highlight inflammatory pathways in digestive cancers

The stromal compartment is increasingly recognized to play a role in cancer. However, its role in the transition from preinvasive to invasive disease is unknown. Most gastrointestinal tumors have clearly defined premalignant stages, and Barrett’s esophagus (BE) is an ideal research model. Supervised clustering of gene expression profiles from microdissected stroma identified a gene signature that could distinguish between BE metaplasia, dysplasia, and esophageal adenocarcinoma (EAC). EAC patients overexpressing any of the five genes (TMEPAI, JMY, TSP1, FAPα, and BCL6) identified from this stromal signature had a significantly poorer outcome. Gene ontology analysis identified a strong inflammatory component in BE disease progression, and key pathways included cytokine–cytokine receptor interactions and TGF-β. Increased protein levels of inflammatory-related genes significantly up-regulated in EAC compared with preinvasive stages were confirmed in the stroma of independent samples, and in vitro assays confirmed functional relevance of these genes. Gene set enrichment analysis of external datasets demonstrated that the stromal signature was also relevant in the preinvasive to invasive transition of the stomach, colon, and pancreas. These data implicate inflammatory pathways in the genesis of gastrointestinal tract cancers, which can affect prognosis.

Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma

Interactions between cancer cells and cancer‐associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α‐SMA‐positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7–29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro‐invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvβ3 and αvβ5, promoting activation of the PI3kinase–Akt pathway. In patient samples, periostin expression at the tumour cell–stromal interface correlates with poor overall and disease‐free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF‐secreted periostin and EAC‐expressed integrins results in PI3 kinase–Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF‐rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients.

Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett's metaplasia.

Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barretts esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barretts metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barretts pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barretts metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barretts esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barretts metaplasia.

Sox9 drives columnar differentiation of esophageal squamous epithelium: a possible role in the pathogenesis of Barrett's esophagus

The molecular mechanism underlying the development of Barretts esophagus (BE), the precursor to esophageal adenocarcinoma, remains unknown. Our previous work implicated sonic hedgehog (Shh) signaling as a possible driver of BE and suggested that bone morphogenetic protein 4 (Bmp4) and Sox9 were downstream mediators. We have utilized a novel in vivo tissue reconstitution model to investigate the relative roles of Bmp4 and Sox9 in driving metaplasia. Epithelia reconstituted from squamous epithelial cells or empty vector-transduced cells had a stratified squamous phenotype, reminiscent of normal esophagus. Expression of Bmp4 in the stromal compartment activated signaling in the epithelium but did not alter the squamous phenotype. In contrast, expression of Sox9 in squamous epithelial cells induced formation of columnar-like epithelium with expression of the columnar differentiation marker cytokeratin 8 and the intestinal-specific glycoprotein A33. In patient tissue, A33 protein was expressed specifically in BE, but not in normal esophagus. Expression of Cdx2, another putative driver of BE, alone had no effect on reconstitution of a squamous epithelium. Furthermore, epithelium coexpressing Cdx2 and Sox9 had a phenotype similar to epithelium expressing Sox9 alone. Our results demonstrate that Sox9 is sufficient to drive columnar differentiation of squamous epithelium and expression of an intestinal differentiation marker, reminiscent of BE. These data suggest that Shh-mediated expression of Sox9 may be an important early event in the development of BE and that the potential for inhibitors of the hedgehog pathway to be used in the treatment of BE and/or esophageal adenocarcinoma could be tested in the near future.

Signaling pathways in the molecular pathogenesis of adenocarcinomas of the esophagus and gastroesophageal junction

Esophageal adenocarcinoma develops in response to severe gastroesophageal reflux disease through the precursor lesion Barrett esophagus, in which the normal squamous epithelium is replaced by a columnar lining. The incidence of esophageal adenocarcinoma in the United States has increased by over 600% in the past 40 years and the overall survival rate remains less than 20% in the community. This review highlights some of the signaling pathways for which there is some evidence of a role in the development of esophageal adenocarcinoma. An increasingly detailed understanding of the biology of this cancer has emerged recently, revealing that in addition to the well-recognized alterations in single genes such as p53, p16, APC, and telomerase, there are interactions between the components of the reflux fluid, the homeobox gene Cdx2, and the Wnt, Notch, and Hedgehog signaling pathways.

Inhibiting the system xC(-)/glutathione axis selectively targets cancers with mutant-p53 accumulation.

TP53, a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC−, through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System xC− inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system xC− antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11–glutathione axis.

APR-246 potently inhibits tumour growth and overcomes chemoresistance in preclinical models of oesophageal adenocarcinoma

Objectives p53 is a critical tumour suppressor and is mutated in 70% of oesophageal adenocarcinomas (OACs), resulting in chemoresistance and poor survival. APR-246 is a first-in-class reactivator of mutant p53 and is currently in clinical trials. In this study, we characterised the activity of APR-246 and its effect on p53 signalling in a large panel of cell line xenograft (CLX) and patient-derived xenograft (PDX) models of OAC. Design In vitro response to APR-246 was assessed using clonogenic survival, cell cycle and apoptosis assays. Ectopic expression, gene knockdown and CRISPR/Cas9-mediated knockout studies of mutant p53 were performed to investigate p53-dependent drug effects. p53 signalling was examined using quantitative RT-PCR and western blot. Synergistic interactions between APR-246 and conventional chemotherapies were evaluated in vitro and in vivo using CLX and PDX models. Results APR-246 upregulated p53 target genes, inhibited clonogenic survival and induced cell cycle arrest as well as apoptosis in OAC cells harbouring p53 mutations. Sensitivity to APR-246 correlated with cellular levels of mutant p53 protein. Ectopic expression of mutant p53 sensitised p53-null cells to APR-246, while p53 gene knockdown and knockout diminished drug activity. Importantly, APR-246 synergistically enhanced the inhibitory effects of cisplatin and 5-fluorouracil through p53 accumulation. Finally, APR-246 demonstrated potent antitumour activity in CLX and PDX models, and restored chemosensitivity to a cisplatin/5-fluorouracil-resistant xenograft model. Conclusions APR-246 has significant antitumour activity in OAC. Given that APR-246 is safe at therapeutic levels our study strongly suggests that APR-246 can be translated into improving the clinical outcomes for OAC patients.

Nitric oxide-mediated invasion in Barrett's high-grade dysplasia and adenocarcinoma

Nitric oxide (NO) has been shown to induce double strand DNA breaks in Barretts oesophagus (BO) and in other cancers has a role in invasion. The specific aims of this study were to investigate whether NO can induce invasion in cells representative of different stages of Barretts progression and to determine possible underlying mechanisms. Physiological concentrations of NO that mimic luminal production of NO from dietary sources enhanced invasion in cell lines from high-grade dysplasia (GihTERT) and oesophageal adenocarcinoma (FLO) but not a non-dysplastic Barretts cell line (QhTERT). Real-time reverse transcription-polymerase chain reaction revealed that NO induced expression of matrix metalloproteinase (MMP)-1, -3, -7, -9 and -10 and tissue inhibitor of metalloproteinase (TIMP)-1, -2 and -3 in these cell lines. Furthermore, ex vivo treatment of Barretts biopsy samples with NO induced increases in MMP-1 and TIMP-1 expression, suggesting that NO enhances invasion through deregulating MMP and TIMP expression in epithelial cells. In keeping with these findings, microarray analysis and immunohistochemistry performed on biopsy samples showed enhanced expression of MMP-1, -3, -7 and -10 and TIMP-1 in the progression from non-dysplastic BO to adenocarcinoma, although this could not be directly attributed to the effect of NO. Thus, NO may play a role in Barretts carcinogenesis through deregulating MMP and TIMP expression to enhance invasive potential.