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KCTD12 Regulates Colorectal Cancer Cell Stemness through the ERK Pathway.

ABSTRACT: Targeting cancer stem cells (CSCs) in colorectal cancer (CRC) remains a difficult problem, as the regulation of CSCs in CRC is poorly understood. Here we demonstrated that KCTD12, potassium channel tetramerization domain containing 12, is down-regulated in the CSC-like cells of CRC. The silencing of endogenous KCTD12 and the overexpression of ectopic KCTD12 dramatically enhances and represses CRC cell stemness, respectively, as assessed in vitro and in vivo using a colony formation assay, a spheroid formation assay and a xenograft tumor model. Mechanistically, KCTD12 suppresses CRC cell stemness markers, such as CD44, CD133 and CD29, by inhibiting the ERK pathway, as the ERK1/2 inhibitor U0126 abolishes the increase in expression of CRC cell stemness markers induced by the down-regulation of KCTD12. Indeed, a decreased level of KCTD12 is detected in CRC tissues compared with their adjacent normal tissues and is an independent prognostic factor for poor overall and disease free survival in patients with CRC (p?=?0.007). Taken together, this report reveals that KCTD12 is a novel regulator of CRC cell stemness and may serve as a novel prognostic marker and therapeutic target for patients with CRC.

SUBMITTER: Li L

PROVIDER: S-EPMC4742820 | biostudies-other | 2016 Feb

REPOSITORIES: biostudies-other

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KCTD12 Regulates Colorectal Cancer Cell Stemness through the ERK Pathway.

Li Liping L Duan Tingmei T Wang Xin X Zhang Ru-Hua RH Zhang Meifang M Wang Suihai S Wang Fen F Wu Yuanzhong Y Huang Haojie H Kang Tiebang T

Scientific reports 20160205

Targeting cancer stem cells (CSCs) in colorectal cancer (CRC) remains a difficult problem, as the regulation of CSCs in CRC is poorly understood. Here we demonstrated that KCTD12, potassium channel tetramerization domain containing 12, is down-regulated in the CSC-like cells of CRC. The silencing of endogenous KCTD12 and the overexpression of ectopic KCTD12 dramatically enhances and represses CRC cell stemness, respectively, as assessed in vitro and in vivo using a colony formation assay, a sphe ...[more]

PMID: 26847701

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Project description:Aims: Ribosomal protein L17 (RPL17), a 60S subunit component, is up-regulated in colorectal cancer (CRC). However, its oncogenic role in CRC progression remains unexplored. Thus, we aimed to investigate the effect of RPL17 targeting on CRC in vitro and in vivo and whether RPL17 gained an extra-ribosomal function during CRC development. Methods: RPL17-specific siRNAs complexed with cationic lipids were transfected to CRC cells to silence target gene expression and then real-time RT-PCR and western blotting were applied to observe the change of expression or activity of genes or proteins of interest. Cell proliferation assay, clonogenic assay and cell cycle analysis were used to determine the in vitro effects of RPL17siRNAs on CRC cell growth, and a subcutaneous xenograft assay was applied to test the effect of RPL17siRNAs on in vivo tumor growth. RNA sequencing and western blotting were used to investigate the underlying mechanisms. Sphere-forming assay, invasion assay and migration assay were used to evaluate the effects of RPL17siRNAs on CRC stemness. Results: siRNA-mediated inhibition of RPL17 expression suppressed CRC cell growth and long-term colony formation by inducing apoptotic cell death. Similarly, targeting RPL17 effectively suppressed tumor formation in a mouse xenograft model. RNA sequencing of RPL17-silenced CRC cells revealed the same directional regulation of 159 (93 down- and 66 up-regulated) genes. Notably, NIMA-related kinase 2 (NEK2), which functionally cooperates with extracellular-regulated protein kinase (ERK) and plays a pivotal role in mitotic progression and stemness maintenance, was down-regulated. RPL17 silencing reduced NEK2, β-catenin, and p-ERK protein levels. These molecular alterations reflected the reduction in sphere-forming capacity, expression of stem cell marker genes, migration, and invasion. Reversely, RPL17 overexpression increased the ability of long-term colony formation, migration, and invasion. Conclusion: Our findings indicate that RPL17 promotes CRC proliferation and stemness via the ERK and NEK2/β-catenin signaling axis, and targeting RPL17 could be the next molecular strategy for both primary CRC treatment and prevention of secondary tumor formation.

Project description:BackgroundAbnormal expression of ribosomal proteins has an important regulatory effect on the progression of cancer. RPL5 is involved in the progression of various malignancies, however, the role of RPL5 in colon cancer remains is still unclear.MethodsData from TCGA and GTEx databases were used to analyze the RPL5 expression in pan-cancer. The expression level of RPL5 in clinical colon cancer tissue samples and human colon cancer cell lines was detected by western blotting; siRNA targeting RPL5 was designed, and its interference efficiency was verified by western blotting and RT-qPCR; CCK8 assay, clone formation assay, cell cycle assay, and cell scratch assay were used to observe the effect of RPL5 on colon cancer cell proliferation and migration; the changes of proteins related to MAPK/ERK signaling pathway were also detected using western blotting.ResultsThe expression level of RPL5 in colon cancer tissues and cell lines was significantly higher than that in adjacent tissues and NCM460 cells, respectively, and its expression level was higher in HCT116 cells and RKO cells. Knockdown of RPL5 significantly inhibited the proliferation and migration of HCT16 and RKO cells, and arrested the cell cycle in G0/G1 phase. Mechanistic studies revealed that the expression of p-MEK1/2, p-ERK, c-Myc were down-regulated, and the expression of FOXO3 was up-regulated after down-regulation of RPL5, ERK activator (TBHQ) could partially reverse the above-mentioned effects caused by siRPL5. Moreover, TBHQ could partially reverse the inhibitory effect of siRPL5 on the proliferation and migration of colon cancer cells. Collectively, RPL5 promoted colon cell proliferation and migration, at least in part, by activating the MAPK/ERK signaling pathway.ConclusionRPL5 promoted colon cell proliferation and migration, at least in part, by activating the MAPK/ERK signaling pathway, which may serve as a novel therapeutic target for cancers in which MAPK/ERK signaling is a dominant feature.

Dataset Information

KCTD12 Regulates Colorectal Cancer Cell Stemness through the ERK Pathway.

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KCTD12 Regulates Colorectal Cancer Cell Stemness through the ERK Pathway.

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