Correlation analysis of cancer stem cell marker CD133 and human endogenous retrovirus (HERV)-K env in SKOV3 ovarian cancer cells

Kim Do-Ye,Kim Heungyeol,Ko Eun-Ji,Koh Suk Bong,Kim Hongbae,Lee Ji Young,Lee Chul Min,Eo Wan Kyu,Kim Ki Hyung,Cha Hee-Jae 한국유전학회 2024 Genes & Genomics Vol.46 No.4

Background Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. Objective To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. Method To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. Results In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. Conclusion These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells. Background Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. Objective To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. Method To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. Results In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. Conclusion These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells.

Cancer upregulated gene 2 (CUG2), a novel oncogene, promotes stemness-like properties via the NPM1-TGF-β signaling axis

Kaowinn, Sirichat,Seo, Eun Jin,Heo, Woong,Bae, Jae-Ho,Park, Eun-Jung,Lee, Soojin,Kim, Yeon Jeong,Koh, Sang Seok,Jang, Il Ho,Shin, Dong Hoon,Chung, Young-Hwa Elsevier 2019 Biochemical and biophysical research communication Vol.514 No.4

<P><B>Abstract</B></P> <P>Our previous study reported that cancer upregulated gene (CUG)2, a novel oncogene, induces both faster cell migration and anti-cancer drug resistance. We thus wonder whether CUG2 also induces stemness, a characteristic of cancer stem cells (CSCs) and further examine the molecular mechanism of this phenotype. To test that CUG2 induces stemness, we examined expression of stemness-related factors. Overexpression of CUG2 enhanced expression levels of stemness-related factors in human lung carcinoma A549 and immortalized bronchial BEAS-2B cells. Consequently, CUG2 increased cellular spherical cluster forming ability. Overexpression of CUG2 also induced tumor formation in xenotransplanted nude mice whereas transplantation of control cells failed to, implying that CUG2 possesses malignant tumorigenic potential. We paid attention to nucleophosmin (NPM1) for its known interaction with CUG2. Suppression of NPM1 hindered the CUG2-mediated stemness-like phenotypes and diminished TGF-β transcriptional activity and signaling. TGF-β increased stemness-like phenotypes in the control cells whereas TGF-β inhibitor blocked induction of the phenotypes, indicating that NPM1 is required for CUG2-mediated stemness-like phenotypes through TGF-β signaling. Furthermore, the suppression of Smad- and non-Smad-dependent TGF-β signaling pathways also prevented CUG2 from inducing stemness-like phenotypes. Altogether, we suggest that the novel CUG2 oncogene promotes cellular transformation and stemness, mediated by nuclear NPM1 protein and TGF-β signaling.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Overexpression of CUG2 promotes stemness-like properties. </LI> <LI> NPM1 is involved in CUG2-mediated stemness-like phenotypes. </LI> <LI> NPM1 utilizes TGF-β signaling for CUG2-mediated stemness-like phenotypes. </LI> <LI> TGF-β signaling plays a crucial role in CUG2-mediated stemness-like phenotypes. </LI> </UL> </P>

Machine Learning Identifies Stemness Features Associated with Oncogenic Dedifferentiation

Malta, Tathiane M.,Sokolov, Artem,Gentles, Andrew J.,Burzykowski, Tomasz,Poisson, Laila,Weinstein, John N.,Kamiń,ska, Boż,ena,Huelsken, Joerg,Omberg, Larsson,Gevaert, Olivier,Colaprico, Anto Elsevier 2018 Cell Vol.173 No.2

<P><B>Summary</B></P> <P>Cancer progression involves the gradual loss of a differentiated phenotype and acquisition of progenitor and stem-cell-like features. Here, we provide novel stemness indices for assessing the degree of oncogenic dedifferentiation. We used an innovative one-class logistic regression (OCLR) machine-learning algorithm to extract transcriptomic and epigenetic feature sets derived from non-transformed pluripotent stem cells and their differentiated progeny. Using OCLR, we were able to identify previously undiscovered biological mechanisms associated with the dedifferentiated oncogenic state. Analyses of the tumor microenvironment revealed unanticipated correlation of cancer stemness with immune checkpoint expression and infiltrating immune cells. We found that the dedifferentiated oncogenic phenotype was generally most prominent in metastatic tumors. Application of our stemness indices to single-cell data revealed patterns of intra-tumor molecular heterogeneity. Finally, the indices allowed for the identification of novel targets and possible targeted therapies aimed at tumor differentiation.</P> <P><B>Video Abstract</B></P> <P>Display Omitted</P> <P><B>Highlights</B></P> <P> <UL> <LI> Epigenetic and expression-based stemness indices measure oncogenic dedifferentiation </LI> <LI> Immune microenvironment content and PD-L1 levels associate with stemness indices </LI> <LI> Stemness index is increased in metastatic tumors and reveals intratumor heterogeneity </LI> <LI> Applying stemness indices reveals potential drug targets for anti-cancer therapies </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

microRNA-26a-5p Regulates the Cancer stemness and Enhances the Chemosensitivity of Lung Adenocarcinoma

( Minhyeok Lee ),( Ji Woong Son ),( Chang Ryul Park ),( Daeun Kang ),( Su Yel Lee ),( Se Jin Park ),( Wan Jin Hwang ),( Gwan Woo Ku ),( Seong Lan Yu ),( In Beom Jeong ),( Sun Jung Kwon ),( Jaeku Kang 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

Purpose Cancer stem cells (CSCs) identified in lung cancer exhibit resistance to chemotherapy, radiotherapy, and targeted therapy. Therefore, a technology to control of CSCs is needed to overcome such resistance to cancer therapy. Various evidences about the association between epithelial-mesenchymal transition related transcriptomic alteration and acquisition of CSC phenotype have been proposed recently. In our previous research, down-regulated miR-26a-5p is closely related to mesenchymal-like lung cancer cell lines. These findings suggest that miR-26a-5p might be involved in lung cancer stemness. Methods RNA polymerase III subunit G (POLR3G) was selected as a candidate target of miR-26a-5p related to cancer stemness. its quantitative relationship was investigated by polymerase chain reaction, western blot after transfection of miR-26a-5p. luciferase assay were done for investigating the direct regulation of miR-26a-5p on POLR3G expression. After transfection of miR-26a-5p, colony formation assay and sphere formation assay were performed to evaluate the effect on cancer stemness. By treating cancer cell by miR-26a-5p and paclitaxel, cell viability was checked by 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and Muse cell analyzer. Expression level of each gene and its impact on survival were revealed by the cancer genome atlas pancancer database. Results miR-26a-5p regulated the expression of POLR3G directly. Overexpression of miR-26a-5p induced down regulation of POLR3G and a marked reduction of colony formation and sphere formation. Co-treatment of miR-26a-5p with paclitaxel decreased cell growth, suggesting that miR-26a-5p might play a role as a chemotherapy sensitizer. In the cancer genome atlas data, downregulated miR-26a-5p and up-regulated POLR3G were shown compared to adjacent normal tissue. High miR-26a-5p and low POLR3G expression were also related to higher survival rate of patients with lung adenocarcinoma. Conclusions Overexpression of miR-26a-5p can suppress lung cancer stemness and make cancer cell become sensitive to chemotherapy. This finding provides a novel insight into a potential lung cancer treatment by regulating stemness.

Loss of Stemness by Suppression of Kruppel-Like Factor 5 in Human Limbal Stem Cells

( Woo June Hur ),( Jun Sub Choi ),( Choun Ki Joo ) 한국조직공학·재생의학회 2011 조직공학과 재생의학 Vol.8 No.2

The aim of this study is to investigate the role of endogenous Kruppel-like factor 5 (KLF5) in human limbal stem cells (hLSCs). The hLSCs were isolated from the corneoscleral rims of donors after corneal transplantation. Isolated cells were co-cultured with mitomycin-c (MMC)-treated fibroblasts. HLSCs were treated with siRNA directed against KLF5, and effects on stemness were analyzed using colony forming efficacy (CFE). ABCG2, OCT4 and P63 as markers of hLSCs, cytokeratin 3 (CK3) and E-cadherin as markers of differentiation were confirmed by RT-PCR, western blotting assay, immunocytochemistry. And corneal epithelialization of the limbal stem cells was confirmed by air-lift 3d-culture. The number of colony (larger than 1 mm in diameter) in KLF5 siRNA treated-group was observed lower than that of control groups (non-treated group and negative siRNA treated-group). And expression of PCNA, OCT4, ABCG2 and P63 were decreased by KLF5 siRNA treatment. On the other hand, expression of cyclin D1, CK3 and E-cadherin were increased in KLF5 siRNA treated-group. Additionally, the limbal stem cells with klf5 siRNA treatment had not formed multi-layers in air-lift 3d-culture. In this study, suppression of KLF5 expression in hLSCs induced the loss of their stemness. These results suggested that KLF5 is closely relative to proliferation and stemness of hLSCs in cornea.

β-hydroxybutyrate impairs nasopharyngeal carcinoma cell aggressiveness via histone deacetylase 4 inhibition

Huang Jinqiao,Chen Xian,Lin Hong,Chen Xiufen 대한독성 유전단백체 학회 2024 Molecular & cellular toxicology Vol.20 No.3

Background Cancer stem cell phenotype confers tumor aggressiveness in multiple malignancies, including nasopharyngeal carcinoma (NPC). Many studies supported that β-hydroxybutyrate (BHB), a ketone body, acts as an epigenetic factor with an anticancer effect. Nevertheless, the effects of BHB on NPC remain elusive. Objective This study explored whether BHB suppressed the aggressive phenotype of NPC cells by suppressing their stemness-like characteristics and exerting an anti-NPC effect. Results The proliferation, migration, and invasion abilities of NPC cells after BHB intervention were attenuated, the protein levels of E-cadherin were downregulated, that of N-cadherin and vimentin were upregulated, the volume and number of cell spheres were reduced, and the number of CD44 + cells (cell surface stem cell marker) was reduced. Knockdown of HDAC4 abrogated the effects of BHB on cell proliferation, invasive phenotype, and stemness. The molecular docking map of BHB-HDAC4 displayed that BHB binds the catalytic domain in HDAC4, speculating that BHB functions as an HDAC4-specific inhibitor, preventing the catalytic function of HDAC4 protein rather than inhibiting the translational synthesis of HDAC4 protein. Conclusions BHB inhibited NPC cells’ proliferation, stemness characteristics, and invasive phenotypes by specifically restraining HDAC4 expression. Background Cancer stem cell phenotype confers tumor aggressiveness in multiple malignancies, including nasopharyngeal carcinoma (NPC). Many studies supported that β-hydroxybutyrate (BHB), a ketone body, acts as an epigenetic factor with an anticancer effect. Nevertheless, the effects of BHB on NPC remain elusive. Objective This study explored whether BHB suppressed the aggressive phenotype of NPC cells by suppressing their stemness-like characteristics and exerting an anti-NPC effect. Results The proliferation, migration, and invasion abilities of NPC cells after BHB intervention were attenuated, the protein levels of E-cadherin were downregulated, that of N-cadherin and vimentin were upregulated, the volume and number of cell spheres were reduced, and the number of CD44 + cells (cell surface stem cell marker) was reduced. Knockdown of HDAC4 abrogated the effects of BHB on cell proliferation, invasive phenotype, and stemness. The molecular docking map of BHB-HDAC4 displayed that BHB binds the catalytic domain in HDAC4, speculating that BHB functions as an HDAC4-specific inhibitor, preventing the catalytic function of HDAC4 protein rather than inhibiting the translational synthesis of HDAC4 protein. Conclusions BHB inhibited NPC cells’ proliferation, stemness characteristics, and invasive phenotypes by specifically restraining HDAC4 expression.

Arctigenin protects against ultraviolet-A-induced damage to stemness through inhibition of the NF-κB/MAPK pathway

Park, See-Hyoung,Cho, Jae Youl,Oh, Sae Woong,Kang, Mingyeong,Lee, Seung Eun,Yoo, Ju Ah,Jung, Kwangseon,Lee, Jienny,Lee, Sang Yeol,Lee, Jongsung Elsevier 2018 Chemico-biological interactions Vol.282 No.-

<P><B>Abstract</B></P> <P>The stemness of stem cells is negatively affected by ultraviolet A (UVA) irradiation. This study was performed to examine the effects of arctigenin on UVA-irradiation-induced damage to the stemness of human mesenchymal stem cells (hMSCs) derived from adipose tissue. The mechanisms of action of arctigenin were also investigated.</P> <P>A BrdU-incorporation assay demonstrated that arctigenin attenuated the UVA-induced reduction of the cellular proliferative potential. Arctigenin also increased the UVA-induced reduction in stemness of hMSCs by upregulating stemness-related genes such as <I>SOX2</I>, <I>OCT4</I>, and <I>NANOG</I>. In addition, the UVA-induced reduction in the mRNA expression level of hypoxia-inducible factor (HIF)<I>-</I>1α was significantly recovered by arctigenin. The antagonizing effect of arctigenin on UVA irradiation was mediated by reduced PGE<SUB>2</SUB> production through the inhibition of MAPKs (p42/44 MAPK, p38 MAPK, and JNK) and NF-κB. Overall, these findings suggest that arctigenin can ameliorate the reduced stemness of hMSCs induced by UVA irradiation. The effects of arctigenin are mediated by PGE<SUB>2</SUB>-cAMP signaling-dependent upregulation of <I>HIF-1α</I>. Therefore, arctigenin could be used as an antagonist to attenuate the effects of UVA irradiation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Arctigenin attenuated the UVA-induced reduction of the proliferative potential. </LI> <LI> Arctigenin increased the UVA-induced reduction of the stemness of the hMSCs. </LI> <LI> Arctigenin recovered UVA-induced reduction of the expression level of HIF-1α gene. </LI> <LI> Arctigenin induces an PGE2-cAMP signaling-dependent upregulation of HIF-1α. </LI> <LI> Arctigenin could be used as an antagonist to attenuate the UVA effects. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

mTOR Plays an Important Role in the Stemness of Human Fetal Cartilage Progenitor Cells (hFCPCs)

Shin Him-Cha,Kim Jiyoung,Park So Ra,Choi Byung Hyune 한국조직공학과 재생의학회 2024 조직공학과 재생의학 Vol.21 No.2

BACKGROUND: Mammalian target of rapamycin (mTOR) is known to regulate self-renewal ability and potency of embryonic stem cells (ESCs) and adult stem cells in opposite manners. However, its effects vary even among adult stem cells and are not reported in fetal stem/progenitor cells. This study investigated the role of mTOR in the function of human fetal cartilage-derived progenitor cells (hFCPCs). METHODS: mTOR activity in hFCPCs was first examined via the level of phosphor-mTOR until passage 19, together with doubling time of cells and senescence-associated b-galactosidase (SA-bGal). Then, the effect of 100 nM rapamycin, the inhibitor of mTOR, was investigated on self-renewal ability, proliferation rate and osteogenic/adipogenic potential of hFCPCs in vitro. Expression of stemness genes (Oct-4, Sox2 and Nanog) and cell cycle regulators (CDK4 and Cyclin D1) was measured at mRNA or protein levels. RESULTS: mTOR activity was maintained constantly at high levels in hFCPCs until passage 19, while their proliferation rate was decreasing from 48 h at passage 13 to 70 h at passage 9 and senescent cells were observed at passage 18 (8.3 ± 1.2%) and 19 (15.6 ± 1.9%). Inhibition of mTOR in hFCPCs impaired their colony forming frequency (CFU-F) by 4 folds, while showing no change in their doubling time and expression of CDK4 and Cyclin D1. Upon mTOR inhibition, Oct4 expression decreased by 2 folds and 4 folds at the mRNA and protein levels, respectively, while that of Sox2 and Nanog did not change significantly. Finally, mTOR inhibition reduced osteogenic and adipogenic differentiation of hFCPCs in vitro. CONCLUSION: This study has shown that mTOR plays an important role in the self-renewal ability of hFCPCS but not in their proliferation, The effect of mTOR appears to be associated with Oct-4 expression and important in the osteogenic and adipogenic differentiation ability of hFCPCs. BACKGROUND: Mammalian target of rapamycin (mTOR) is known to regulate self-renewal ability and potency of embryonic stem cells (ESCs) and adult stem cells in opposite manners. However, its effects vary even among adult stem cells and are not reported in fetal stem/progenitor cells. This study investigated the role of mTOR in the function of human fetal cartilage-derived progenitor cells (hFCPCs). METHODS: mTOR activity in hFCPCs was first examined via the level of phosphor-mTOR until passage 19, together with doubling time of cells and senescence-associated b-galactosidase (SA-bGal). Then, the effect of 100 nM rapamycin, the inhibitor of mTOR, was investigated on self-renewal ability, proliferation rate and osteogenic/adipogenic potential of hFCPCs in vitro. Expression of stemness genes (Oct-4, Sox2 and Nanog) and cell cycle regulators (CDK4 and Cyclin D1) was measured at mRNA or protein levels. RESULTS: mTOR activity was maintained constantly at high levels in hFCPCs until passage 19, while their proliferation rate was decreasing from 48 h at passage 13 to 70 h at passage 9 and senescent cells were observed at passage 18 (8.3 ± 1.2%) and 19 (15.6 ± 1.9%). Inhibition of mTOR in hFCPCs impaired their colony forming frequency (CFU-F) by 4 folds, while showing no change in their doubling time and expression of CDK4 and Cyclin D1. Upon mTOR inhibition, Oct4 expression decreased by 2 folds and 4 folds at the mRNA and protein levels, respectively, while that of Sox2 and Nanog did not change significantly. Finally, mTOR inhibition reduced osteogenic and adipogenic differentiation of hFCPCs in vitro. CONCLUSION: This study has shown that mTOR plays an important role in the self-renewal ability of hFCPCS but not in their proliferation, The effect of mTOR appears to be associated with Oct-4 expression and important in the osteogenic and adipogenic differentiation ability of hFCPCs.

The role of hypoxia on the acquisition of epithelial-mesenchymal transition and cancer stemness: a possible link to epigenetic regulation

( Chang Dong Yeo ),( Nahyeon Kang ),( Su Yeon Choi ),( Bit Na Kim ),( Chan Kwon Park ),( Jin Woo Kim ),( Young Kyoon Kim ),( Seung Joon Kim ) 대한내과학회 2017 The Korean Journal of Internal Medicine Vol.32 No.4

A hypoxic microenvironment leads to cancer progression and increases the metastatic potential of cancer cells within tumors via epithelial-mesenchymal transition (EMT) and cancer stemness acquisition. The hypoxic response pathway can occur under oxygen tensions of < 40 mmHg through hypoxia-inducible factors (HIFs), which are considered key mediators in the adaptation to hypoxia. Previous studies have shown that cellular responses to hypoxia are required for EMT and cancer stemness maintenance through HIF-1α and HIF-2α. The principal transcription factors of EMT include Twist, Snail, Slug, Sip1 (Smad interacting protein 1), and ZEB1 (zinc finger E-box-binding homeobox 1). HIFs bind to hypoxia response elements within the promoter region of these genes and also target cancer stem cell-associated genes and mediate transcriptional responses to hypoxia during stem cell differentiation. Acquisition of stemness characteristics in epithelial cells can be induced by activation of the EMT process. The mechanism of these phenotypic changes includes epigenetic alterations, such as DNA methylation, histone modification, chromatin remodeling, and microRNAs. Increased expression of EMT and pluripotent genes also play a role through demethylation of their promoters. In this review, we summarize the role of hypoxia on the acquisition of EMT and cancer stemness and the possible association with epigenetic regulation, as well as their therapeutic applications.

Ribosomal Protein L9 Maintains Stemness of Colorectal Cancer via an ID-1 Dependent Mechanism

Eun-Hye Jeon,So-Young Park,Keon Uk Park,Yun-Han Lee 대한암예방학회 2024 Journal of cancer prevention Vol.29 No.2

The identification of therapeutic target genes that are functionally involved in stemness is crucial to effectively cure patients with metastatic carcinoma. We have previously reported that inhibition of ribosomal protein L9 (RPL9) expression suppresses the growth of colorectal cancer (CRC) cells by inactivating the inhibitor of DNA-binding 1 (ID-1) signaling axis, which is functionally associated with cancer cell survival. In addition to cell proliferation, ID-1 is also involved in the maintenance of cancer stemness. Thus, we aimed in this study to investigate whether the function of RPL9 could correlate with CRC stem cell-like properties. Here, we demonstrated that siRNA silencing of RPL9 reduced the invasiveness and migrative capabilities of HT29 and HCT116 parental cell populations and the capacity for sphere formation in the HT29 parental cell population. CD133+ cancer stem cells (CSCs) were then separated from CD133- cancer cells of the HT29 parental cell culture and treated with RPL9-specific siRNAs to verify the effects of RPL9 targeting on stemness. As a result, knockdown of RPL9 significantly suppressed the proliferative potential of CD133+ colorectal CSCs, accompanied by a reduction in CD133, ID-1, and p-IκBα levels. In line with these molecular alterations, targeting RPL9 inhibited the invasion, migration, and sphere-forming capacity of CD133+ HT29 CSCs. Taken together, these findings suggest that RPL9 promotes CRC stemness via ID-1 and that RPL9 could be a potential therapeutic target for both primary CRC treatment and the prevention of metastasis and/or recurrence.