Role of mTOR through Autophagy in Esophageal Cancer Stemness

Abstract

Esophageal cancer (EC) is a highly aggressive disease with a poor prognosis. Therapy resistance and early recurrences are major obstacles in reaching a better outcome. Esophageal cancer stem-like cells (CSCs) seem tightly related with chemoradiation resistance, initiating new tumors and metastases. Several oncogenic pathways seem to be involved in the regulation of esophageal CSCs and might harbor novel therapeutic targets to eliminate CSCs. Previously, we identified a subpopulation of EC cells that express high levels of CD44 and low levels of CD24 (CD44+/CD24−), show CSC characteristics and reside in hypoxic niches. Here, we aim to clarify the role of the hypoxia-responding mammalian target of the rapamycin (mTOR) pathway in esophageal CSCs. We showed that under a low-oxygen culture condition and nutrient deprivation, the CD44+/CD24− population is enriched. Since both low oxygen and nutrient deprivation may inhibit the mTOR pathway, we next chemically inhibited the mTOR pathway using Torin-1. Torin-1 upregulated SOX2 resulted in an enrichment of the CD44+/CD24− population and increased sphere formation potential. In contrast, stimulation of the mTOR pathway using MHY1485 induced the opposite effects. In addition, Torin-1 increased autophagic activity, while MHY1485 suppressed autophagy. Torin-1-mediated CSCs upregulation was significantly reduced in cells treated with autophagy inhibitor, hydroxychloroquine (HCQ). Finally, a clearly defined CD44+/CD24− CSC population was detected in EC patients-derived organoids (ec-PDOs) and here, MHY1485 also reduced this population. These data suggest that autophagy may play a crucial role in mTOR-mediated CSCs repression. Stimulation of the mTOR pathway might aid in the elimination of putative esophageal CSCs.

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

“…The PDO derivation process involves mechanically fragmenting and enzymatically digesting EAC tissue specimens (diagnostic biopsies or surgically resected tumor tissues), followed by seeding cells into or onto a 3D extracellular matrix (e.g., Matrigel) and propagating them through successive passaging rounds ( Li et al, 2018 ; Karakasheva et al, 2020 ; Karakasheva et al, 2021 ). Digestion media typically include a combination of collagenase, dispase and hyaluronidase to obtain a single cell suspension and remove non-tumor contaminants such as stroma ( Karakasheva et al, 2020 ; Du et al, 2022 ). Given the anatomical location of the tumors and association with food matter, EAC primary cultures are particularly susceptible to contamination by bacteria and fungi.…”

“…The self-organizing, self-renewal and differentiation potential of organoids renders them an ideal model for investigating cancer stem cells. A recent study discovered a hypoxia-tolerant, CD44+/CD24-subpopulation within EAC PDOs that possessed stem cell properties and was enriched after exposure to γ-radiation ( Du et al, 2022 ). Whilst it is difficult to compare stemness in organoids to that of cell lines, this finding points to a correlation between stemness and inherent resistance to radiation, consistent with that observed in EAC cell lines ( Smit et al, 2013 ; Wang et al, 2014 ).…”

“…Whilst it is difficult to compare stemness in organoids to that of cell lines, this finding points to a correlation between stemness and inherent resistance to radiation, consistent with that observed in EAC cell lines ( Smit et al, 2013 ; Wang et al, 2014 ). The organoid-forming potential of EAC PDOs was increased when treated with Torin-1, an inhibitor of the mammalian target of rapamycin (mTOR), and reduced with mTOR stimulation with MHY1485, suggesting a role for mTOR in regulating stemness of cancer cells ( Du et al, 2022 ). Together, this study presents a blueprint for using EAC PDOs to interrogate regulators of cancer cell stemness.…”