Modelling esophageal adenocarcinoma and Barrett's esophagus with patient-derived organoids.

Currently, esophageal adenocarcinoma (EAC) research is hindered by a dearth of adequate models to study this disease. Traditional cell line and genetically engineered mouse models are lacking in biological and physiological significance, whilst the inefficiency of patient-derived xenografts limit their potential applications. This review describes the landscape of EAC research using patient-derived organoids (PDOs). Here, we detail the methods of establishment and optimization of EAC PDO cultures, as well as current and prospective applications of these models. We further highlight a crucial knowledge gap in the mechanisms of EAC transformation from its precursor lesion, Barrett's esophagus (BE). As such, we also describe the culture requirements of BE PDOs and attempts to model tumorigenesis using PDO models.

Caspase-2 protects against ferroptotic cell death.

Caspase-2, one of the most evolutionarily conserved members of the caspase family, is an important regulator of the cellular response to oxidative stress. Given that ferroptosis is suppressed by antioxidant defense pathways, such as that involving selenoenzyme glutathione peroxidase 4 (GPX4), we hypothesized that caspase-2 may play a role in regulating ferroptosis. This study provides the first demonstration of an important and unprecedented function of caspase-2 in protecting cancer cells from undergoing ferroptotic cell death. Specifically, we show that depletion of caspase-2 leads to the downregulation of stress response genes including SESN2, HMOX1, SLC7A11, and sensitizes mutant-p53 cancer cells to cell death induced by various ferroptosis-inducing compounds. Importantly, the canonical catalytic activity of caspase-2 is not required for its role and suggests that caspase-2 regulates ferroptosis via non-proteolytic interaction with other proteins. Using an unbiased BioID proteomics screen, we identified novel caspase-2 interacting proteins (including heat shock proteins and co-chaperones) that regulate cellular responses to stress. Finally, we demonstrate that caspase-2 limits chaperone-mediated autophagic degradation of GPX4 to promote the survival of mutant-p53 cancer cells. In conclusion, we document a novel role for caspase-2 as a negative regulator of ferroptosis in cells with mutant p53. Our results provide evidence for a novel function of caspase-2 in cell death regulation and open potential new avenues to exploit ferroptosis in cancer therapy.

Tumor-Infiltrating Neutrophils after Neoadjuvant Therapy are Associated with Poor Prognosis in Esophageal Cancer.

BACKGROUND: In esophageal cancer (EC), there is a paucity of knowledge regarding the interplay between the tumor immune microenvironment and response to neoadjuvant treatment and, therefore, which factors may influence outcomes. Thus, our goal was to investigate the changes in the immune microenvironment with neoadjuvant treatment in EC by assessing the expression of immune related genes and their association with prognosis. METHODS: We examined the transcriptome of paired pre- and post-neoadjuvant treated EC specimens. Based on these findings, we validated the presence of tumor-infiltrating neutrophils using CD15+ immunohistochemistry in a discovery cohort of patients with residual pathologic disease. We developed a nomogram as a predictor of progression-free survival (PFS) incorporating the variables CD15+ cell count, tumor regression grade, and tumor grade. RESULTS: After neoadjuvant treatment, there was an increase in genes related to myeloid cell differentiation and a poor prognosis associated with high neutrophil (CD15+) counts. Our nomogram incorporating CD15+ cell count was predictive of PFS with a C-index of 0.80 (95% confidence interval [CI] 0.68-0.9) and a concordance probability estimate (CPE) of 0.77 (95% CI 0.69-0.86), which indicates high prognostic ability. The C-index and CPE of the validation cohort were 0.81 (95% CI 0.69-0.91) and 0.78 (95% CI 0.7-0.86), respectively. CONCLUSIONS: Our nomogram incorporating CD15+ cell count can potentially be used to identify patients at high risk of recurrent disease and thus stratify patients who will benefit most from adjuvant treatment.

Eprenetapopt triggers ferroptosis, inhibits NFS1 cysteine desulfurase, and synergizes with serine and glycine dietary restriction.

The mechanism of action of eprenetapopt (APR-246, PRIMA-1MET) as an anticancer agent remains unresolved, although the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis (SLC7A11, SHMT2, and MTHFD1L), as well as the enzymes required to synthesize glutathione (GCLC and GCLM), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limiting the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.

Loss of SMAD4 Is Sufficient to Promote Tumorigenesis in a Model of Dysplastic Barrett's Esophagus.

BACKGROUND & AIMS: Esophageal adenocarcinoma (EAC) develops from its precursor Barrett's esophagus through intermediate stages of low- and high-grade dysplasia. However, knowledge of genetic drivers and molecular mechanisms implicated in disease progression is limited. Herein, we investigated the effect of Mothers against decapentaplegic homolog 4 (SMAD4) loss on transforming growth factor ß (TGF-ß) signaling functionality and in vivo tumorigenicity in high-grade dysplastic Barrett's cells. METHODS: An in vivo xenograft model was used to test tumorigenicity of SMAD4 knockdown or knockout in CP-B high-grade dysplastic Barrett's cells. RT2 polymerase chain reaction arrays were used to analyze TGF-ß signaling functionality, and low-coverage whole-genome sequencing was performed to detect copy number alterations upon SMAD4 loss. RESULTS: We found that SMAD4 knockout significantly alters the TGF-ß pathway target gene expression profile. SMAD4 knockout positively regulates potential oncogenes such as CRYAB, ACTA2, and CDC6, whereas the CDKN2A/B tumor-suppressor locus was regulated negatively. We verified that SMAD4 in combination with CDC6-CDKN2A/B or CRYAB genetic alterations in patient tumors have significant predictive value for poor prognosis. Importantly, we investigated the effect of SMAD4 inactivation in Barrett's tumorigenesis. We found that genetic knockdown or knockout of SMAD4 was sufficient to promote tumorigenesis in dysplastic Barrett's esophagus cells in vivo. Progression to invasive EAC was accompanied by distinctive and consistent copy number alterations in SMAD4 knockdown or knockout xenografts. CONCLUSIONS: Altogether, up-regulation of oncogenes, down-regulation of tumor-suppressor genes, and chromosomal instability within the tumors after SMAD4 loss implicates SMAD4 as a protector of genome integrity in EAC development and progression. Foremost, SMAD4 loss promotes tumorigenesis from dysplastic Barrett's toward EAC.

Transketolase regulates sensitivity to APR-246 in p53-null cells independently of oxidative stress modulation.

The prevalence and dire implications of mutations in the tumour suppressor, p53, highlight its appeal as a chemotherapeutic target. We recently showed that impairing cellular antioxidant systems via inhibition of SLC7A11, a component of the system xc- cystine-glutamate antiporter, enhances sensitivity to mutant-p53 targeted therapy, APR-246. We investigated whether this synergy extends to other genes, such as those encoding enzymes of the pentose phosphate pathway (PPP). TKT, one of the major enzymes of the PPP, is allegedly regulated by NRF2, which is in turn impaired by accumulated mutant-p53 protein. Therefore, we investigated the relationship between mutant-p53, TKT and sensitivity to APR-246. We found that mutant-p53 does not alter expression of TKT, nor is TKT modulated directly by NRF2, suggesting a more complex mechanism at play. Furthermore, we found that in p53null cells, knockdown of TKT increased sensitivity to APR-246, whilst TKT overexpression conferred resistance to the drug. However, neither permutation elicited any effect on cells overexpressing mutant-p53 protein, despite mediating oxidative stress levels in a similar fashion to that in p53-null cells. In sum, this study has unveiled TKT expression as a determinant for sensitivity to APR-246 in p53-null cells.

GRB7 is an oncogenic driver and potential therapeutic target in oesophageal adenocarcinoma.

Efficacious therapeutic approaches are urgently needed to improve outcomes in patients with oesophageal adenocarcinoma (OAC). However, oncogenic drivers amenable to targeted therapy are limited and their functional characterisation is essential. Among few targeted therapies available, anti-human epidermal growth factor receptor 2 (HER2) therapy showed only modest benefit for patients with OAC. Herein, we investigated the potential oncogenic role of growth factor receptor bound protein 7 (GRB7), which is reported to be co-amplified with HER2 (ERBB2) in OAC. GRB7 was highly expressed in 15% of OAC tumours, not all of which could be explained by co-amplification with HER2, and was associated with a trend for poorer overall survival. Knockdown of GRB7 decreased proliferation and clonogenic survival, and induced apoptosis. Reverse phase protein array (RPPA) analyses revealed a role for PI3K, mammalian target of rapamycin (mTOR), MAPK, and receptor tyrosine kinase signalling in the oncogenic action of GRB7. Furthermore, the GRB7 and HER2 high-expressing OAC cell line Eso26 showed reduced cell proliferation upon GRB7 knockdown but was insensitive to HER2 inhibition by trastuzumab. Consistent with this, GRB7 knockdown in vivo with an inducible shRNA significantly inhibited tumour growth in cell line xenografts. HER2 expression did not predict sensitivity to trastuzumab, with Eso26 xenografts remaining refractory to trastuzumab treatment. Taken together, our study provides strong evidence for an oncogenic role for GRB7 in OAC and suggests that targeting GRB7 may be a potential therapeutic strategy for this cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.