Modulation of Spheroid Forming Capacity and TRAIL Sensitivity by KLF4 and Nanog in Gastric Cancer Cells

Abstract

The expression of pluripotency factors, and their associations with clinicopathological parameters and drug response have been described in various cancers, including gastric cancer. This study investigated the association of pluripotency factor expression with the clinicopathological characteristics of gastric cancer patients, as well as changes in the expression of these factors upon the stem cell-enriching spheroid culture of gastric cancer cells, regulation of sphere-forming capacity, and response to cisplatin and TRAIL treatments by Nanog and KLF4. Nanog expression was significantly associated with the emergence of a new tumor and a worse prognosis in gastric cancer patients. The expression of the pluripotency factors varied among six gastric cancer cells. KLF4 and Nanog were expressed high in SNU-601, whereas SOX2 was expressed high in SNU-484. The expression of KLF4 and SOX2 was increased upon the spheroid culture of SNU-601 (KLF4/Nanog-high) and SNU-638 (KLF4/Nanog-low). The spheroid culture of them enhanced TRAIL-induced viability reduction, which was accompanied by the upregulation of death receptors, DR4 and DR5. Knockdown and overexpression of Nanog in SNU-601 and SNU-638, respectively, did not affect spheroid-forming capacity, however, its expression was inversely correlated with DR4/DR5 expression and TRAIL sensitivity. In contrast, KLF4 overexpression in SNU-638 increased spheroid formation, susceptibility to cisplatin and TRAIL treatments, and DR4/DR5 expression, while the opposite was found in KLF4-silenced SNU-601. KLF4 is supposed to play a critical role in DR4/DR5 expression and responses to TRAIL and cisplatin, whereas Nanog is only implicated in the former events only. Direct regulation of death receptor expression and TRAIL response by KLF4 and Nanog have not been well documented previously, and the regulatory mechanism behind the process remains to be elucidated.

Delta/Notch-like Epidermal Growth Factor-Related Receptor (DNER), a Potential Prognostic Marker of Gastric Cancer Regulates Cell Survival and Cell Cycle Progression

“…Tumor spheroid culture under a special non-adherent condition has been widely used to enrich tumor stem cells and stem cell-like cells, and several gastric cancer cell lines are known to form tumor spheroids [ 38 , 39 ]. In this study, five out of six gastric cancer cell lines formed spheroids or clusters, and the role of DNER in spheroid formation was examined by knocking down its expression.…”

“…The NICD insert fragment (aa 1755-2556) was obtained by single cutting with Bam HI from pCMV4-Flag-NICD and was cloned into the Bam H1 site of pBABE-zeo. The production and transduction of the retroviruses were performed as previously described in To et al [ 38 ].The retrovirus-transduced cells were selected by zeocin treatment (100 µg/mL) for seven days.…”

“…Scrambled and DNER-specific shRNA-coding sequences (Supplementary Table S1) were inserted at the Age1/EcoR1 double-digested site of the Tet-pLKO-puro (Addgene, Cambridge, MA, USA). Production and transduction of the lentiviruses were performed as previously described by To et al (2023) [38].The gastric cancer cells transduced with the lentiviruses were selected with puromycin (2 µg/mL) for seven days and were used for further experiments. The expression of p53 and DNER was silenced by the transfection of gene-specific siRNAs (Supplementary Table S1) [37,55].…”