Abstract
BackgroundWith a less than 5% overall survival rate, esophageal adenocarcinoma (EAC) is one of the leading causes of death in the United States. Epithelial cell adhesion molecule (EpCAM) is a cancer stem cell (CSC) marker that is expressed in various epithelial carcinomas, including EAC. Accumulating evidence indicates that CSC subpopulations can initiate cancer development and, in addition, drive metastasis, recurrence and drug resistance. It has also been reported that EpCAM up-regulation in EAC may lead to an aggressive behavior and, thus, an adverse clinical outcome. Here, we aimed to determine whether treatment with standard chemotherapeutic agents may induce EpCAM expression and, concomitantly, increases in malignant potential and drug resistance in EAC.MethodsEpCAM expression was assessed in 20 primary human EAC/adjacent normal tissues, as well as in a human EAC-derived cell line (OE-19), in a pre-malignant Barrett’s Esophagus cell line (Bar-T) and in a benign esophageal cell line (HET 1-A), using immunohistochemistry, Western blotting and qRT-PCR, respectively. Drug-induced resistance was investigated in OE-19-derived spheres treated with (a combination of) adriamycin, cisplatin and 5-fluorouracil (ACF) using survival, adhesion and flow cytometric assays, respectively, and compared to drug resistance induced by standard chemotherapeutic agents (CTA). Finally, ACF treatment-surviving cells were evaluated for their tumor forming capacities both in vitro and in vivo using spheroid formation and xenograft assays, respectively.ResultsHigh EpCAM expression was observed in esophageal cancer tissues and esophageal cancer-derived cell lines, but not in adjacent benign esophageal epithelia and benign esophageal cell lines (HET 1-A and Bar-T). The OE-19 cell spheres were drug resistant and EpCAM expression was significantly induced in the OE-19 cell spheres compared to the non-sphere OE-19 cells. When OE-19 cell spheres were challenged with ACF, the EpCAM mRNA and protein levels were further up-regulated up to 48 h, whereas a decreased EpCAM expression was observed at 72 h. EpCAM down-regulation by RNA interference increased the ACF efficacy to kill OE-19 cells. Increased EpCAM expression coincided with the CSC marker CD90 and was associated with an aggressive growth pattern of OE-19 cell spheres in vivo.ConclusionsFrom our data we conclude that an ACF-induced increase in EpCAM expression reflects the selection of a CSC subpopulation that underlies tumor development and drug resistance in EAC.
Highlights
Esophageal carcinoma ranks among the deadliest malignancies known, with an increasing incidence rate during the past decades [1]
The expression of Epithelial cell adhesion molecule (EpCAM) was determined by Western blotting in three cell lines, HET 1-A representing benign esophageal epithelium, Barrett’s Esophagus cell line (Bar-T) representing pre-malignant Barrett’s metaplasia and OE-19 representing esophageal adenocarcinoma, respectively
We found that the OE-19 cells showed a higher EpCAM expression than the HET 1-A and Bar-T cells (Fig. 1b)
Summary
Esophageal carcinoma ranks among the deadliest malignancies known, with an increasing incidence rate during the past decades [1]. The low overall survival associated with EAC may be attributed to the fact that patients typically only present once they have developed an advanced stage of the disease. This delay in diagnosis and the lack of effective treatment options for advanced EAC have greatly contributed to the deadliness of the disease. The most commonly used therapeutic agents include cisplatin/platinum-based drugs, 5-fluorouracil (5-FU) and anthracycline derivatives such as adriamycin. Drug-induced resistance was investigated in OE-19derived spheres treated with (a combination of) adriamycin, cisplatin and 5-fluorouracil (ACF) using survival, adhesion and flow cytometric assays, respectively, and compared to drug resistance induced by standard chemotherapeutic agents (CTA). ACF treatment-surviving cells were evaluated for their tumor forming capacities both in vitro and in vivo using spheroid formation and xenograft assays, respectively
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