A Novel TOX3-WDR5-ABCG2 Signaling Axis Regulates the Progression of Colorectal Cancer by Accelerating Stem-like Traits and Chemoresistance
Jiaojiao Hao, Jinsheng Huang, Chunyu Hua, Yan Zuo, Wendan Yu, Xiaojun Wu, Liren Li, Guoqing Xue, Xinyu Wan, Liyuan Ru,Ziyue Guo, Shilong Han, Wuguo Deng, Fei Lin,Wei Guo
Abstract
The eradication of cancer stem cells (CSCs) with drug resistance confers the probability of local tumor control after chemotherapy or targeted therapy. As the main drug resistance marker, ABCG2 is also critical for colorectal cancer (CRC) evolution, in particular cancer stem–like traits expansion. Hitherto, the knowledge about the expression regulation of ABCG2, in particular its upstream transcriptional regulatory mechanisms, remains limited in cancer, including CRC. Here, ABCG2 was found to be markedly up–regulated in CRC CSCs (cCSCs) expansion and chemo–resistant CRC tissues and closely associated with CRC recurrence. Mechanistically, TOX3 was identified as a specific transcriptional factor to drive ABCG2 expression and subsequent cCSCs expansion and chemoresistance by binding to −261 to −141 segments of the ABCG2 promoter region. Moreover, we found that TOX3 recruited WDR5 to promote tri–methylation of H3K4 at the ABCG2 promoter in cCSCs, which further confers stem–like traits and chemoresistance to CRC by co–regulating the transcription of ABCG2. In line with this observation, TOX3, WDR5, and ABCG2 showed abnormal activation in chemo–resistant tumor tissues of in situ CRC mouse model and clinical investigation further demonstrated the comprehensive assessment of TOX3, WDR5, and ABCG2 could be a more efficient strategy for survival prediction of CRC patients with recurrence or metastasis.
Introduction
As the most common malignancy of the digestive system, colorectal cancer (CRC) also remains one of the leading causes of cancer-related deaths in both economically developed and developing countries [1], and the pathogenic mechanisms related to CRC initiation and development have always been the hotspots in oncology. However, the enthusiasm generated by recent progress in CRC prevention, diagnosis, and intervention, has been tempered by the recurrence and drug resistance in the clinic, which greatly reduces drug efficacy and worsens patient outcome [2,3]. Cancer stem cells (CSCs) with stem-like characteristic, that is, the ability to self-renew and differentiate into multiple tumor cell types, are generally considered to be the key cells to build for the initiation, propagation, chemoresistance, metastasis, and relapse of different cancers, of course, including CRC [4–6]. Targeting CSC has been considered as a promising anticancer strategy. Therefore, there is an urgent need to further elucidate the molecular signatures affecting colorectal cancer stem cells (cCSCs) evolution, clarify what might confer these molecular signatures and consequently identify new effective intervention targets.
Materials and methods
Cell lines
CRC cell lines (RKO, LoVo, DLD1, HCT116) were obtained from American Type Culture Collection (Manassas, Virginia, United States of America). RKO were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS. LoVo, DLD1, and HCT116 were maintained in RPMI-1640 medium containing 10% FBS. All the cell lines mentioned above were cultured in a humidified atmosphere with 5% CO2 at 37°C.
Results
ABCG2 expression is preferentially up-regulated in cCSCs
In order to obtain CRC spheres with self-renewal, chemoresistance, and distinct characteristics of cCSCs, we enriched cCSCs by inducing CRC spheroid formation in the presence of chemotherapeutics (oxaliplatin 1 μg/ml). To confirm the stem-like traits of these spheres, colony formation assay, cell invasion experiments, and detection of cell surface markers were respectively performed. Compared to adherent cells, these spheres showed higher colony-forming ability, cell invasion capability, and cancer stem-like traits-related protein expressions (S1A and S1B Fig). Consistently, higher xenograft tumor formation ability of these CRC spheres than adherent cancer cells was observed in in vivo experiments (S1C Fig), and primary cells obtained from xenografts of CRC spheres displayed stronger colony formation and spherization capacity again in vitro (S1D and S1E Fig). According to the results proven above, we initially concluded that these tumor spheres cultured with oxaliplatin could be defined as cCSCs by exhibiting a series of CSC traits, including great proliferative potential and increased epithelial–mesenchymal transition.
Discussion
As a highly prevalent digestive system cancer, CRC shows a low survival rate at advanced stages of the disease. In recent years, CSCs have been found to play critical effects on tumor formation, heterogeneity, therapeutic resistance, and eventual recurrence and metastasis. However, our knowledge about the target genes involved in cCSCs expansion and therapeutic resistance and the related regulatory mechanisms for these genes remains largely limited. Other than cell sorting using specific cell surface markers, cCSCs could also be enriched according to their particular characteristics such as side population, low proteasome activity and low intracellular ROS, spheroid formation, or chemoresistance [36–38]. In the current study, cCSCs were enriched by spheroid formation upon chemotherapeutic treatment, and ABCG2 was focused among the up-regulated genes in cCSCs by comparation to the adherent cancer cells. Moreover, ABCG2 was further identified based on its elevated expression in chemo-insensitive, recurrent, and metastasized CRC tissues. In the subsequent study, our data showed that ABCG2 promoted the self-renewal and expansion of cCSCs, thereby contributing to drug resistance production and CRC progression.
Conclusions
In brief, our study uncovers that ABCG2 is overactivated transcriptionally by TOX3 via recruiting WDR5 and the subsequent histone H3K4 tri-methylation in CRC, leading to the enhanced self-renewal of cCSCs and drug resistance, particularly chemoresistance, and finally contributing to the recurrence and metastasis of CRC patients after receiving chemotherapy, highlighting the potential of targeted interventing TOX3-WDR5-H3K4me3/ABCG2 signaling axis in overcoming the challenges of drug tolerance and antagonizing CRC recurrence or metastasis, and also the potential of developing TOX3-WDR5/ABCG2 signaling axis as a group of cascade biomarkers for predicting the survival of CRC patients with recurrence or metastasis.
Acknowledgments
We would like to thank Dr. Wei Cheng (ICSC Core Facility, Dalian Medical University) for her material and technical support.
Citation: Hao J, Huang J, Hua C, Zuo Y, Yu W, Wu X, et al. (2023) A novel TOX3-WDR5-ABCG2 signaling axis regulates the progression of colorectal cancer by accelerating stem-like traits and chemoresistance. PLoS Biol 21(9): e3002256. https://doi.org/10.1371/journal.pbio.3002256
Academic Editor: Connie J. Eaves, B.C. Cancer Agency, CANADA
Received: September 23, 2022; Accepted: July 17, 2023; Published: September 14, 2023
Copyright: © 2023 Hao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files. RNA-seq raw data files are available from the GEO database (accession number GSE236764). All FACS files are available from the FlowRepository database (accession number Repository ID: FR-FCM-Z6KB).
Funding: This study was supported by the funds from the National Natural Science Foundation of China [grant number: 82072711 WG, 82172949 WD, 81972569 WD, 82100780 JH, 82172802 LL], Liaoning Revitalization Talents Program [grant number: XLYC2007172 WG], the Guangdong Basic and Applied Basic Research Foundation (2022A1515012508 WD). The funders provided research funds for the implementation of the experiments involved in the whole study, but did not participate in the design of the study, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: BSA, bovine serum albumin; cCSC, colorectal cancer stem cell; CRC, colorectal cancer; CSC, cancer stem cell; DMEM, Dulbecco’s Modified Eagle’s Medium; GEO, Gene Expression Omnibus; LDA, limited dilution assay; RT, room temperature; SD, standard deviation; SNP, single-nucleotide polymorphism; SP, side population; TNM, tumor-node-metastasis; WDR5, WD Repeat Domain 5
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002256#abstract0
