Dataset Information

RPL17 Promotes Colorectal Cancer Proliferation and Stemness through ERK and NEK2/β-catenin Signaling Pathways.

ABSTRACT: 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.

SUBMITTER: Ko MJ

PROVIDER: S-EPMC9174872 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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RPL17 Promotes Colorectal Cancer Proliferation and Stemness through ERK and NEK2/β-catenin Signaling Pathways.

Ko Min Ji MJ Seo Yu-Ri YR Seo Daekwan D Park So-Young SY Seo Ji Hae JH Jeon Eun-Hye EH Kim Sang-Woo SW Park Keon Uk KU Koo Deog-Bon DB Kim Shin S Bae Jae-Hoon JH Song Dae-Kyu DK Cho Chi Heum CH Kim Kwang Seok KS Lee Yun-Han YH

Journal of Cancer 20220513 8

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 ...[more]

PMID: 35711835

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Project description:Distant metastasis is, unfortunately, the leading cause of death in colorectal cancer (CRC). Approximately 50% of CRC patients develop liver metastases, while 10-30% of patients develop pulmonary metastases. The occurrence of metastasis is considered to be almost exclusively driven by cancer stem cells (CSCs) formation. However, the key molecules that confer the transformation to stem cells in CRC, and subsequent metastasis, remain unclear. Far upstream element-binding protein 1 (FUBP1), a transcriptional regulator of c-Myc, was screened in CSCs of CRC by mass spectrometry and was examined by immunohistochemistry in a cohort of CRC tissues. FUBP1 was upregulated in 85% of KRAS-mutant and 25% of wild-type CRC patients. Further, whether in KRAS-mutant or wild-type patients, elevated FUBP1 was positively correlated with CRC lymph node metastasis and clinical stage, and negatively associated with overall survival. Overexpression of FUBP1 significantly enhanced CRC cell migration, invasion, tumor sphere formation, and CD133 and ALDH1 expression in vitro, and tumorigenicity in vivo. Mechanistically, FUBP1 promoted the initiation of CSCs by activating Wnt/β-catenin signaling via directly binding to the promoter of DVL1, a potent activator of β-catenin. Knockdown of DVL1 significantly inhibited the transformation to stem cells in, as well as the tumorigenicity of, CRC. Activation of Wnt/β-catenin signaling by DVL1 increased pluripotent transcription factors, including c-Myc, NANOG, and SOX2. Moreover, FUBP1 was upregulated at the post-transcriptional level. Elevated FUBP1 levels in KRAS wild-type CRC patients is due to the decrease in Smurf2, which promotes ubiquitin-mediated degradation of FUBP1. In contrast, FUBP1 was upregulated in KRAS-mutant patients through both inhibition of caspase 3-dependent cleavage and decreased Smurf2. Our results demonstrate, for the first time, that FUBP1 is an oncogene, initiating the development of CSCs, as well as a new powerful endogenous Wnt-signaling agonist that could provide an important prognostic factor and therapeutic target for metastasis in both KRAS-mutant and wild-type CRC.

Project description:BackgroundColorectal cancer (CRC) is one of the most detrimental tumors to human health. Although multimodal therapeutic approaches can improve patient survival rates, the prognosis for advanced-stage patients remains poor. It has been reported that family with sequence similarity 60, member A (FAM60A), a component of the SIN3 transcription regulator family member A (SIN3A)/histone deacetylase (HDAC) complex, plays a significant role in tumorigenesis. However, the precise function and mechanisms of action of FAM60A in CRC have not been fully elucidated. In this study, we aim to further delineate the role of FAM60A in CRC by assessing the protein expression levels of FAM60A and β-catenin in CRC tissues and to explore the potential mechanisms by which FAM60A may promote CRC cell proliferation and invasion through a suite of cellular assays.MethodsTumor tissues of 195 CRC patients and 65 adjacent non-neoplastic tissues were collected to construct tissue microarrays. The expression levels of FAM60A, c-Myc, cyclin D1, and β-catenin were detected using immunohistochemistry (IHC) staining, and the relationship between the results and the patients' clinicopathological characteristics and prognosis was analyzed. HCT116 and HT-29 cell lines with overexpression/knockdown of FAM60A were constructed. Western blot (WB) was used to detect the protein expression of FAM60A and β-catenin. Cell proliferation, apoptosis rate, cell cycle, and cell migration and invasion abilities were assessed using cell counting kit-8 (CCK-8) assay, flow cytometry, wound healing assay, and transwell assay, respectively.ResultsFAM60A demonstrated elevated expression in CRC tissues and was positively correlated with tumor infiltration depth, Ki67 proliferation index, and poor prognosis in patients. A positive correlation was observed between FAM60A and the expression of β-catenin, c-Myc, and cyclin D1, and patients with co-expression of FAM60A and β-catenin had a significantly higher rate of distant metastasis. The knockdown of FAM60A markedly reduced the proliferation, migration, and invasive capabilities of HCT116 cells, induced cell cycle arrest, and enhanced apoptosis, whereas its overexpression produced the converse effects. In HT-29 cells, FAM60A knockdown also reduced cell proliferation and impaired wound healing, with overexpression showing opposing outcomes. WB analysis revealed that modulation of FAM60A influenced β-catenin protein levels, suggesting a regulatory link between the two proteins.ConclusionsFAM60A may be a key regulator factor that modulates proliferation and invasion in CRC cells via the Wnt/β-catenin signaling pathway. Elevated FAM60A expression is associated with an adverse prognosis in CRC, underscoring its potential as a prognostic biomarker.

Dataset Information

RPL17 Promotes Colorectal Cancer Proliferation and Stemness through ERK and NEK2/β-catenin Signaling Pathways.

Publications

RPL17 Promotes Colorectal Cancer Proliferation and Stemness through ERK and NEK2/β-catenin Signaling Pathways.

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