Original ArticlesNuclear EGFR-PKM2 axis induces cancer stem cell-like characteristics in irradiation-resistant cells
Introduction
Radiation therapy is one of the major tools of cancer treatment and is broadly used for a variety of malignant tumors. Within tumors, a very small subpopulation of cells is considered to possess the ability to self-renew and differentiate, leading to radioresistance, metastasis and recurrent disease. Such cells have been designated “cancer stem cells” (CSCs) [1,2] or “stem-like cancer cells” (SLCCs) [2]. Subsequently, CSCs have been discovered and isolated from many types of cancer [[3], [4], [5], [6], [7], [8], [9]]. Treatments targeting CSCs may substantially improve therapy in cancer patients. Hence, recent studies have increasingly focused on the identification of CSC-specific markers, such as CD44, CD133, ABCG2 (CD338), and ALDH1, and the embryonic stem cell transcription factors Sox2, Oct4 and Nanog [[10], [11], [12]]. However, tumor cells with CSC-like phenotypes or markers have been found to be radiation resistant, and the molecular mechanisms of the radioresistance of CSCs are poorly understood [13].
EGFR is a receptor tyrosine kinase that belongs to the ErbB family, which includes ErbB1, ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) [14]. Importantly, EGFR and its downstream signaling pathway play important roles in the control of cell proliferation, survival, epithelial-to-mesenchymal transition (EMT), migration and differentiation in a wide variety of human cancers [15]. Over-expression or elevated activity of EGFR is associated with unfavorable prognosis and resistance against chemotherapy and radiation therapy [16]. In addition, EGFR signaling has been implicated in the functions of CSCs in various cancers and is known to increase tumor invasiveness. There is a growing body of evidence that activation of the EGFR pathway mediates the regulation of multiple CSC-related genes, including Sox2 [17,18], CD44 [19], CD133 [20], Oct4 [21], and ALDH1 [22], although the underlying molecular mechanisms are not clear.
PKM2 is a rate-limiting enzyme, which catalyzes the final step in glycolysis by converting phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) to pyruvate and adenosine-triphosphate (ATP) [23]. It has been demonstrated to play a critical role in tumor metabolism and be involved in the Warburg effect, conferring cancer cells with the glycolytic phenotype to generate the energy required for cellular processes [[24], [25], [26]]. Consistently, enhanced PKM2 expression has been found in multiple human cancers, suggesting that PKM2 is a potential diagnostic biomarker and therapeutic target for tumors.
Beyond its metabolic function, PKM2 has recently received much attention for its noncanonical roles in tumorigenesis. The PKM2 dimer is also well known as a nuclear protein kinase that regulates gene transcription induced by Oct4, STAT3, β-catenin, FGFR1 or HIF-1α [24]. In addition, nuclear translocation of PKM2 may occur in response to EGFR activation. For instance, cytosolic PKM2 can directly interact with and stabilize mutant EGFR protein in lung cancer cells [27]. Activation of EGFR can also induce the translocation of PKM2 to the nucleus, where PKM2 promotes the transcriptional activity of β-catenin by binding to c-Src-phosphorylated β-catenin, resulting in transcriptional activation of cyclin D1 and Myc [28].
The aim of the present study is to investigate whether activation of the EGFR-PKM2 axis in the nucleus contributes to radioresistance and induces CSC-like properties in irradiation-resistant cells. We also examined whether the EGFR-PKM2 axis affects colony formation, tumor sphere formation, tumorigenicity and expression of stem cell-like genes in irradiation-resistant cells. Finally, we propose a new concept for targeting EGFR-PKM2 to overcome radioresistance.
Section snippets
Cell lines and culture conditions
Lung carcinoma cell lines (A549-P and A549-IR) were obtained from Xinming Deng (Emory University School of Medicine and Winship Cancer Institute of Emory University, Atlanta, USA). The cells were maintained in DMEM/F12 containing 10% fetal bovine serum. Nasopharyngeal carcinoma cell lines (HK1-P and HK1-IR) were cultured in RPMI 1640 medium with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator. All cell lines were passaged less than 10 times after their initial thaw from frozen
Isolation and characterization of CSCs from irradiation-resistant cells
To determine whether SP cells exist in irradiation-resistant cells, we stained the irradiation-resistant (IR) cells and non-irradiated parental (P) cells with Hoechst 33342 to detect and sort these side populations by flow cytometry based on their capacity to efflux the fluorescent dye. Using this procedure, we identified a very small subpopulation of total HK1-P cells (0.67 ± 0.42%) as SP cells, while the percentage of SP cells in IR cells increased to 10.05% (left panel of Fig. 1A). As
Discussion
In this study, we explored the potential function of the interacting complex of EGFR and PKM2 on stem-like gene expression in radioresistant sublines. We found that the IR cells displayed increased stem cell-like properties. Elevated nuclear localization of EGFR was found by analyzing the nuclear fraction of IR sublines, and EGFR and PKM2 were co-localized. In addition, silencing of EGFR or PKM2 significantly decreased the stem-like properties of cells and inhibited cell proliferation,
Funding
This work was supported by the National Natural Science Foundation of China [81302354 (Y. Shi), 81672787 and 81372427 (Y. Tao), 81672991 and 81271763 (S. Liu)]; the National Key Basic Research Program of China [2015CB553903 (Y. Tao)] and the Fundamental Research Funds for the Central Universities [2013ZZTS074 (B. Yan)].
Author contributions
YS designed most of the study, performed most of the experiments and wrote the manuscript. NL, WL and BY assisted with some experiments involving animals. SL, JJ, DX, and XW conducted the bioinformatics analysis. LC, XL, CM, YJ, YL, and RY contributed to the administrative, technical, or material support. YC and YT participated in discussing, revising and editing the manuscript. All authors read and approved the final manuscript.
Conflicts of interest
The authors declare that they have no competing interests.
Acknowledgments
The authors thank Prof. Xinming Deng (Emory University School of Medicine and Winship Cancer Institute of Emory University, Atlanta, USA) for his kind help in providing the lung carcinoma cell lines (A549-P and A549-IR). The authors are also grateful to the Institute of Medical Sciences flow cytometry platform for their excellent technical assistance.
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2021, Biomedicine and PharmacotherapyCitation Excerpt :Furthermore, CSCs exhibit certain characteristics such as efflux of Hoechst stain, portraying a range of cell/tumor type specific surface markers including CD133, CD44, CD24 and epithelial cell adhesion molecule (EPCAM), and aldehyde dehydrogenase-I (ALDH-I) [124–126]. In addition, expression of transcription factors such as Octamer binding transcription factor 4 (Oct4), Sex determining region Y-box 2 (Sox2), Krüppel-like factor 4 (KLF4) and Homeobox transcription factor (Nanog) among others also used as markers of CSCs [127–129]. Here in brief, we have reviewed recent updates about the role of ROS in CSCs stemness including how ROS modulate the expression and functioning of different signaling pathways associated with the maintenance of CSCs characteristics.