Inhibition of NPC1L1 disrupts adaptive responses of drug‐tolerant persister cells to chemotherapy
2022; Springer Nature; Volume: 14; Issue: 2 Linguagem: Inglês
10.15252/emmm.202114903
ISSN1757-4684
AutoresZhe Zhang, Siyuan Qin, Yan Chen, Li Zhou, Mei Yang, Yongquan Tang, Jing Zuo, Jian Zhang, Atsushi Mizokami, Edouard C. Nice, Hai‐Ning Chen, Canhua Huang, Xiawei Wei,
Tópico(s)Cancer Cells and Metastasis
ResumoArticle13 January 2022Open Access Source DataTransparent process Inhibition of NPC1L1 disrupts adaptive responses of drug-tolerant persister cells to chemotherapy Zhe Zhang Zhe Zhang orcid.org/0000-0001-7509-6965 Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Conceptualization, Data curation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Siyuan Qin Siyuan Qin State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation, Methodology Search for more papers by this author Yan Chen Yan Chen State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation, Methodology Search for more papers by this author Li Zhou Li Zhou State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation Search for more papers by this author Mei Yang Mei Yang orcid.org/0000-0002-0423-6358 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation Search for more papers by this author Yongquan Tang Yongquan Tang Department of Pediatric Surgery, West China Hospital, Sichuan University, Chengdu, China Contribution: Data curation Search for more papers by this author Jing Zuo Jing Zuo State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation Search for more papers by this author Jian Zhang Jian Zhang School of Medicine, Southern University of Science and Technology Shenzhen, Guangdong, China Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Shenzhen, China Contribution: Data curation Search for more papers by this author Atsushi Mizokami Atsushi Mizokami Department of Urology, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan Contribution: Data curation Search for more papers by this author Edouard C Nice Edouard C Nice Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic, Australia Contribution: Supervision, Writing - review & editing Search for more papers by this author Hai-Ning Chen Corresponding Author Hai-Ning Chen [email protected] orcid.org/0000-0003-0104-8498 Department of Gastrointestinal Surgery, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Conceptualization, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Canhua Huang Corresponding Author Canhua Huang [email protected] orcid.org/0000-0003-2247-7750 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Project administration Search for more papers by this author Xiawei Wei Corresponding Author Xiawei Wei [email protected] orcid.org/0000-0002-6513-6422 Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Project administration, Writing - review & editing Search for more papers by this author Zhe Zhang Zhe Zhang orcid.org/0000-0001-7509-6965 Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Conceptualization, Data curation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Siyuan Qin Siyuan Qin State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation, Methodology Search for more papers by this author Yan Chen Yan Chen State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation, Methodology Search for more papers by this author Li Zhou Li Zhou State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation Search for more papers by this author Mei Yang Mei Yang orcid.org/0000-0002-0423-6358 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation Search for more papers by this author Yongquan Tang Yongquan Tang Department of Pediatric Surgery, West China Hospital, Sichuan University, Chengdu, China Contribution: Data curation Search for more papers by this author Jing Zuo Jing Zuo State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Data curation Search for more papers by this author Jian Zhang Jian Zhang School of Medicine, Southern University of Science and Technology Shenzhen, Guangdong, China Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Shenzhen, China Contribution: Data curation Search for more papers by this author Atsushi Mizokami Atsushi Mizokami Department of Urology, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan Contribution: Data curation Search for more papers by this author Edouard C Nice Edouard C Nice Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic, Australia Contribution: Supervision, Writing - review & editing Search for more papers by this author Hai-Ning Chen Corresponding Author Hai-Ning Chen [email protected] orcid.org/0000-0003-0104-8498 Department of Gastrointestinal Surgery, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Conceptualization, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Canhua Huang Corresponding Author Canhua Huang [email protected] orcid.org/0000-0003-2247-7750 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Project administration Search for more papers by this author Xiawei Wei Corresponding Author Xiawei Wei [email protected] orcid.org/0000-0002-6513-6422 Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Project administration, Writing - review & editing Search for more papers by this author Author Information Zhe Zhang1,2,†, Siyuan Qin2,†, Yan Chen2,†, Li Zhou2, Mei Yang2, Yongquan Tang3, Jing Zuo2, Jian Zhang4,5, Atsushi Mizokami6, Edouard C Nice7, Hai-Ning Chen *,8, Canhua Huang *,2 and Xiawei Wei *,1 1Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China 2State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China 3Department of Pediatric Surgery, West China Hospital, Sichuan University, Chengdu, China 4School of Medicine, Southern University of Science and Technology Shenzhen, Guangdong, China 5Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Shenzhen, China 6Department of Urology, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan 7Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic, Australia 8Department of Gastrointestinal Surgery, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China † These authors contributed equally to this work *Corresponding author. Tel: +86 18980606468; E-mail: [email protected] *Corresponding author. Tel: +86 13258370346; E-mail: [email protected] *Corresponding author. Tel: +86 18081954096; E-mail: [email protected] EMBO Mol Med (2022)14:e14903https://doi.org/10.15252/emmm.202114903 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Entering a drug-tolerant persister (DTP) state of cancer cells is a transient self-adaptive mechanism by which a residual cell subpopulation accelerates tumor progression. Here, we identified the acquisition of a DTP phenotype in multidrug-resistant (MDR) cancer cells as a tolerance response to routine combination treatment. Characterization of MDR cancer cells with a DTP state by RNA-seq revealed that these cells partially prevented chemotherapy-triggered oxidative stress by promoting NPC1L1-regulated uptake of vitamin E. Treatment with the NPC1L1 inhibitor ezetimibe further enhanced the therapeutic effect of combinatorial therapy by inducing methuosis. Mechanistically, we demonstrated that NRF2 was involved in transcriptional regulation of NPC1L1 by binding to the −205 to −215 bp site on its promoter. Decreased DNA methylation was also related partially to this process. Furthermore, we confirmed that a triple-combination of chemotherapeutic agents, verapamil, and ezetimibe, had a significant anti-tumor effect and prevented tumor recurrence in mice. Together, our study provides a novel insight into the role of DTP state and emphasizes the importance of disrupting redox homeostasis during cancer therapy. Synopsis Drug-tolerant persister (DTP) state is a driver of therapy failure and cancer relapse. This study identified a key role for NPC1L1 in multidrug-resistant (MDR) cancer cells with the DTP state, where NPC1L1 orchestrated a redox signaling against the harsh environment caused by cancer therapy. MDR cancer cells possess a stronger capacity to enter the DTP state than the non-resistant cancer cells. NPC1L1 is a key supporter of the DTP state to counter therapy-induced oxidative stress in MDR cancer cells. NPC1L1 inhibition interrupts the uptake of vitamin E and cholesterol, triggering lipid ROS accumulation and consequent lipotoxicity during chemotherapeutic agents/verapamil treatment in MDR cancer cells. A combination of chemotherapeutic agents, verapamil, and NPC1L1 inhibitor ezetimibe exerts a significant anti-tumor effect and prevents tumor recurrence in mice. The paper explained Problem The failure of chemotherapy treatment is mostly due to the occurrence of multidrug resistance (MDR). A subset of residual cells exhibits a transient adaptive resistance mechanism by entering a drug-tolerant persister (DTP) state after treatment. This emerging concept explains the generation of resistant clones with clinically relevant MDR. Accordingly, exploring the specific role and behavior of MDR cancer cells with DTP state in response to current therapy is important for delaying recurrence or even eradicating cancer by targeting specific vulnerabilities. Results We identified the acquisition of DTP phenotype in MDR1-mediated MDR cancer cells as a tolerance response to the routine combination of chemotherapeutic agents and MDR1 inhibitor verapamil. MDR cancer cells with DTP state survived chemotherapy-induced oxidative stress primarily by scavenging lipid ROS through NRF2-NPC1L1 axis-regulated vitamin E uptake. Based on nanoparticle-related drug delivery systems to alleviate verapamil side effects in vivo, the combination of chemotherapeutic agents, verapamil, and NPC1L1 inhibitor ezetimibe demonstrated a significant anti-tumor effect and prevented tumor recurrence in mice. Impact Our study provides key bearings on the relationship between DTP state and NPC1L1-modulated oxidative stress defense by using MDR1-mediated MDR cancer cells, which establishes a novel therapeutic strategy for treating MDR cancer cells and preventing tumor recurrence. Introduction Drug resistance, either intrinsic or acquired, is omnipresent in the clinical treatment of cancer, limiting durable therapeutic benefits and even accelerating tumor recurrence or metastasis (Szakacs et al, 2006; Andrei et al, 2020; Dallavalle et al, 2020). Multidrug resistance, one of the most difficult problems occurring during chemotherapy, is commonly related to the expression of ATP-binding cassette (ABC) transporters, especially transporter—multidrug resistance protein 1 (MDR1) encoded by ABCB1 (Hall et al, 2009). Along with the emergence of appropriate analytical tools, advanced medicinal techniques, and multidisciplinary research, ample evidence not only identifies high expression of MDR1 portending a poor response to chemotherapy and adverse outcomes in patients with cancers but also confirms the necessity of applying new drug or drug delivery protocols to prevent cancer MDR in the clinic (Fan et al, 2017). Unfortunately, the growing realization that cancer cells, rather than being either sensitive or resistant, can be dynamic and transient in nature within the context of treatment is detracting from such studies (Qin et al, 2020). There is actually, a particular state lying between sensitivity and resistance, termed the "drug-tolerant persister (DTP) state," in which a cell population is endowed with a dormant, slow-cycling state and a stem-like signature (Shen et al, 2019, 2020). The concept of DTP originates from an early observation of bacterial response to antibiotics, where the existence of residual bacteria exposed to antibiotics is due to non-genetic variations and resumption of their initial characteristics upon interruption of treatment (Balaban et al, 2019). In the context of cancer, tumor cells share a similar situation in which drug resistance is, in a similar fashion to antibiotic resistance, driven by epigenetic inheritance of variant gene expression patterns. This could result in not only inhibiting the therapeutic efficacy but also providing a reservoir for further evolution (Shen et al, 2019, 2020). Given that this DTP state has been hypothesized to be part of an alternative approach toward a bona fide drug resistance mechanism, this line of research is attracting considerable attention. In addition to the recognition that DTP could serve as a target for therapy (i.e., incorporation of an epigenetic modulator), studies focusing on DTP have deepened our understanding of the mechanisms driving DTP. This encourages the development of methods aimed at disposing of these cells, including sustaining a harmless dormant state, and reactivating proliferation to enhance response to anti-proliferative drugs to eradicate them (Recasens & Munoz, 2019). However, there remains some confusion on how best to target DTP cells, indicating the requirement for a further understanding of both DTP itself and DTP-mediated drug tolerance in order to devise countermeasures against these persisters. To date, multiple studies have emphasized the impact of stress response on persister generation, and several teams have highlighted the capacity to adapt to oxidative stress as a common characteristic of cancer cells in a DTP state (Raha et al, 2014; Sahu et al, 2016; Hangauer et al, 2017; Anand et al, 2019; Dhimolea et al, 2021). Based on this characteristic, increasing numbers of drug targets involved in oxidative stress defense of DTP have been investigated. Among them the phospholipid glutathione peroxidase 4 (GPX4) is crucial for the survival of cancer cells in a therapy-resistant state. In this context, subsequent work has shown that targeting GPX4-dependent oxidative stress defense can almost completely eradicate persister cells by induction of ferroptosis, an oxidative cell death, suggesting a potential treatment strategy by disturbing the redox homeostasis (Hangauer et al, 2017). Additionally, activated NF-E2-related factor 2 (NRF2), detected in HER2-inhibited persistent breast cancer, has been found to drive the re-establishment of redox homeostasis in a glutathione metabolism-dependent manner, thereby promoting the reactivation of dormant tumor cells. This reactivation triggered by NRF2 signaling can be prevented by glutaminase inhibition. This has also been shown to impair the growth of recurrent tumors with high levels of NRF2, suggesting a novel approach to treat NRF2 high dormant and recurrent cancer (Fox et al, 2020). Such findings confirm persister population adaption to therapy-induced oxidative stress. Furthermore, MDR cancer cells might raise a robust antioxidant system for resisting oxidative stress caused by chemotherapeutic agents (Trachootham et al, 2009). We therefore questioned whether persister cancer cells arising from MDR cancer cells prefer to orchestrate the evolutionarily conserved antioxidant system against treatment. In this study, we investigated how MDR cancer cells characterized by overexpression of MDR1 underwent a combinatorial therapy (chemotherapeutic agent with MDR1 inhibitor verapamil)-induced DTP state. To gain insight into the alterations of gene expression profile in the course of non-mutationally acquired resistance, we performed RNA-seq comparing MDR persister cells to MDR cancer cells. We also investigated the function of screened genes in MDR cancer cells with a DTP status. This revealed that NPC1L1, an important regulator for redox homeostasis promoting uptake of vitamin E which can interact directly with lipid peroxyl radicals, thus preventing oxidative stress, was highly expressed in DTP. By adding the NPC1L1 inhibitor ezetimibe into the combinatorial therapy, the function of NPC1L1 on vitamin E absorption was compromised and additional cell death was observed as a consequence of macropinocytosis induction. Mechanistically, our results demonstrated a link between both NRF2 transcriptional activation and decreased DNA methylation with NPC1L1 expression. Using a nanomedicine approach to alleviate side effects of verapamil in vivo, we further confirmed the anti-tumor effect of a triple-combinatorial therapy strategy (a combination of chemotherapeutic agents, verapamil, and ezetimibe) that prevented tumor recurrence in vivo. Together, our study progresses our understanding of MDR persistence from a redox perspective, repositioning the potential of MDR therapy for treating cancer cells. Results Oncotherapy induces the transformation of MDR cancer cells into a DTP state To elucidate the molecular mechanisms underlying the established drug-resistant cancer cell models (Du145TXR resistant to taxol; MCF-7ADR resistant to adriamycin) (Appendix Fig S1A and B), we first examined the expression of MDR1 (also known as p-glycoprotein), which has been extensively studied in the context of drug-resistant phenotype (Gouaze et al, 2005; Takeda et al, 2007; Robey et al, 2018) and confirmed the marked upregulation of its protein expression (Appendix Fig S1C). We further determined the role of MDR1 played in regulating the MDR phenotype using rhodamine123 (a substrate of MDR1) and MDR1 inhibitor verapamil. Fluorescence analysis indicated that MDR1 high expressing cancer cells showed increased efflux of rhodamine123 compared to control cancer cells, and verapamil could partially reverse this phenotype (Appendix Fig S1D). To investigate which combination of chemotherapeutic agent and verapamil contributed most to the anti-cancer ability and synergistic effects, we examined cell viability using the Chou–Talalay method (combination index (CI) < 1 representing synergism) in different drug combination-treated MDR cancer cells. Our results showed that 50 μM verapamil combined with 20 nM taxol or 200 nM adriamycin exhibited beneficial anti-cancer effects in Du145TXR cells or MCF-7ADR cells (Appendix Fig S1E and F). Furthermore, the proliferation of MDR cancer cells was significantly inhibited under the combination treatment, as evidenced by reduced colony formation (Appendix Fig S2A and B). Consistently, knockdown of ABCB1 clearly decreased both cell viability and clonogenic growth capacity in chemotherapy-treated MDR cancer cells (Appendix Fig S2C–G). Together with previous reports, our data support MDR1 as the key factor driving the MDR phenotype of these cancer cells and establish that targeting MDR1, at least in part, enables MDR cancer cells to recover chemosensitivity. Notably, we observed the occurrence of residual MDR cancer cells following combination treatment. These cells were similar to those described for drug-tolerant persister (DTP) cells, which survive cytotoxic exposure via reversible and non-mutational mechanisms. To evaluate whether these residual MDR cancer cells were associated with the DTP state, we examined the cell viability and observed morphological changes in Du145TXR cells and MCF-7ADR cells following combination treatment (50 μM verapamil/20 nM taxol or 200 nM adriamycin). Surprisingly, compared with MDR cancer cells (MCs), which had not been treated with the combinatorial therapy, residual MDR cancer cells (RMCs) showed dramatic morphological alterations following combination treatment for 3 days and obvious tolerance to a second exposure of treatment applied 1, 3, 6, and 9 days after treatment withdrawal as shown by a cell viability assay (Fig 1A–D). Subsequently, a long-term "drug holiday" (30 days) allowed residual MDR cancer cells to regrow (regrown cells, RCs) and resulted in re-acquisition of sensitivity to combination treatment (Fig 1E). The reversibility of further drug resistance in residual MDR cancer cells implies that oncotherapy drives MDR cancer cells into a DTP state, termed as MDR persister cells (MPCs). To compare the differences between MPCs and persister cancer cells (PeCs), we used a similar protocol to generate DTP cells in control cells (CCs) (Fig EV1A). Our results indicated that prolonged drug exposure in control cells for > 3 days could generate a small population of DTP cells (Fig EV1B–D). In addition, DTP cells progressively re-acquired drug sensitivity following withdrawal of chemotherapy and eventually became non-differential compared with control cells by Day 6 as evidenced by cell viability (Fig EV1E and F). Accordingly, we converted the above data obtained by cell viability assays (Figs 1C–E and EV1C–F) into a drug-tolerant rate. As shown in Fig 1F and G, MDR cancer cells can further evolve upon treatment compared with control cells as evidenced by faster induction and a longer duration of the DTP state. Taken together, our results demonstrate that MDR cancer cells can transform into a DTP state resembling control cells, possibly leading to a worse outcome such as modest MDR reversal and tumor recurrence. Figure 1. Oncotherapy induces the transformation of MDR cancer cells into a DTP state A. Schematic of residual MDR cell generation and subsequent analyses. The circular arrow represents the DTP state. B. Phase contrast images of the morphological changes in MDR cells, residual MDR cells, and regrown cells for Du145TXR or MCF-7ADR. Red circles mark magnified areas (bottom). Scale bars, 50 μm (low magnification images). C, D. Cell viability of residual MDR cells (RMCs) and MDR cells (MCs) of (C) Du145TXR or (D) MCF-7ADR cells using the same combination treatment for 24 h on the indicated days. Student's t-test was used to analyze statistical differences. Mean with ± SD. E. Cell viability of regrown cells (RCs) and Du145TXR (left) or MCF-7ADR (right) cells with the same combination treatment for 24 h. Student's t-test was used to analyze statistical differences. Mean with ± SD. F, G. Drug-tolerant rate of (F) Du145/Du145TXR (20 nM taxol/20 nM taxol plus 50 μM verapamil) or (G) MCF-7/MCF-7ADR (200 nM adriamycin/200 nM adriamycin plus 50 μM verapamil) cells treated with drugs on the indicated days. Drug-tolerant rate defined by comparing cell viability followed treatment, RMCs vs. MCs or CCs vs. PeCs, > 1 represents drug tolerant. Induction time indicates the time of entering DTP state; and sustaining time indicates the time of maintaining DTP state. Blue colors represent control groups; Red colors represent MDR cancer cells. Mean with ± SD. Data information: Results are representative of three independent experiments. Source data are available online for this figure. Source Data for Figure 1 [emmm202114903-sup-0004-SDataFig1.jpg] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Control cancer cells transform into DTP states in response to chemotherapy A. Schematic of drug persister cancer cell generation and subsequent analyses. B. Phase contrast images of the morphological change in Du145 or MCF-7 cells followed by chemotherapy treatment for 72 h. Red circles mark magnified areas (bottom). Scale bar, 50 μm (low-magnification images). C, D. Cell viability of control cancer cells (CCs), 3 days treated control cancer cells (CCs-3), and persister cancer cells (PeCs) of (C) Du145 or (D) MCF-7 cells treated with the indicated concentrations of taxol (TAX) or adriamycin (ADM) for 24 h. Mean with ± SD. E, F. Cell viability of CCs, PeCs, 3 days drug withdrawn PeCs (PeCs-3), and 6 days drug withdrawn PeCs (PeCs-6) of (E) Du145 or (F) MCF-7 cells treated with the indicated concentrations of taxol (TAX) or adriamycin (ADM) for 24 h. Mean with ± SD. Data information: Results are representative of three independent experiments. Source data are available online for this figure. Download figure Download PowerPoint Upregulation of NPC1L1 supports cell survival in response to cytotoxic stress in MPCs Given the well-known characteristics of DTP cells, such as induction of cell cycle arrest, increased expression of stemness markers, as well as activation of epithelial–mesenchymal transition (EMT) (Shen et al, 2019, 2020), we investigated whether MPCs had similar phenotypes. Our data indicated that MPCs seemed to be in a state of cell cycle arrest, as evidenced by significant increases in p27 and p21 expression (Appendix Fig S3A). In addition, we further performed flow cytometry analysis which demonstrated that cell cycle arrest at G0/G1 phase was induced in MPCs, showing a non-proliferative or slowly proliferative state (Appendix Fig S3B–D). We also observed the upregulation of stemness markers ALDH1A1, Oct-4A, Sox2, KLF4, and CD44 in MPCs (Appendix Fig S3A). Consistently, sphere formation assays showed that MPCs enhanced self-renewal ability due to the increased stemness, as evidenced by a marked increase in both the number and size of MPCs spheres (Appendix Fig S3E–G). Interestingly, cell viability and colony formation assays revealed that ALDH1A1 inhibitor CM10 was most selectively lethal to MPCs cells and sensitized MCs to the combination treatment consistent with previous persister cell studies (Raha et al, 2014; Appendix Fig S3H–J). In addition to these characteristics, downregulat
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