Research progress on lung cancer stem cells in epidermal growth factor receptor-tyrosine kinase inhibitor targeted therapy resistance in lung adenocarcinoma

Hong Zhang,Yanbin Wang,Xianglin Yuan,Yanmei Zou,Hua Xiong

Abstract Lung cancer is the leading cause of cancer-related deaths globally.In recent years,with the widespread use of genetic testing,epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)-targeted drugs have been efficacious to patients with lung adenocarcinoma exhibiting EGFR mutations.However,resistance to treatment is inevitable and eventually leads to tumor progression,recurrence,and reduction in the overall treatment efficacy.Lung cancer stem cells play a crucial role in the development of resistance toward EGFR-TKItargeted therapy for lung adenocarcinoma.Lung cancer stem cells possess self-renewal,multilineage differentiation,and unlimited proliferation capabilities,which efficiently contribute to tumor formation and ultimately lead to tumor recurrence and metastasis.In this study,we evaluated the origin,markers,stemness index,relevant classic studies,resistance mechanisms,related signaling pathways,and strategies for reversing lung cancer stem cell resistance to EGFR-TKIs to provide new insights on delaying or reducing resistance and to improve the treatment efficacy of patients with EGFR-mutated lung adenocarcinoma in the future.

Keywords: Drug resistance;EGFR-TKIs;Lung cancer stem cells;Lung adenocarcinoma

1.Introduction

Lung cancer is the second most common malignant tumor globally and remains the leading cause of cancer-related deaths worldwide.China has the highest incidence and mortality rates of malignant tumors.Lung cancer has become a global challenge[1]with lung adenocarcinoma as the predominant pathological type,accounting for approximately 60% of all lung cancer cases.[2]With the advancement of precision medicine,genetic testing has been widely applied in the field of oncology,and targeted therapy has emerged as one of the important treatment modalities for advanced lung cancer patients.Lung adenocarcinoma patients have the highest rate of epidermal growth factor receptor (EGFR) mutations,accounting for approximately 50.2%of all cases.[3]Epidermal growth factor receptor–tyrosine kinase inhibitors (TKIs) such as gefitinib,erlotinib,and afatinib are commonly used targeted drugs for patients with lung adenocarcinoma containing EGFR mutations.However,the majority of EGFR mutation–positive lung adenocarcinoma patients develop resistance to targeted therapy,leading to tumor progression and recurrence and affecting overall treatment efficacy.[4]Overcoming EGFR-TKI resistance is a pressing challenge in clinical practice.

In recent years,with deepening research on cancer stem cells(CSCs),lung cancer stem cells have been found to play a crucial role in the development of resistance to EGFR-TKIs in lung adenocarcinoma.Lung cancer stem cells differ from general tumor cells as they possess strong self-renewal and differentiation abilities,playing significant roles in tumor formation,progression,metastasis,and drug resistance.[5]This review will focus on the research landscape of lung cancer stem cells in the context of resistance to EGFR-TKI–targeted therapy in lung adenocarcinoma.It will also discuss relevant mechanisms and potential therapeutic strategies,aiming to provide new insights for delaying or reducing drug resistance in the future.

2.Lung cancer stem cells

2.1.Source of lung cancer stem cells

Cancer stem cells were initially identified and characterized in acute myeloid leukemia,and subsequent studies confirmed their existence in various solid tumors.Moreover,CSCs are closely associated with tumor initiation,metastasis,drug resistance,and relapse.In 2005,Kim et al[6]used injury models to identify a regional population of lung stem cells,termed bronchioalveolar stem cells,which exhibited self-renewal and multipotency in subsequent cloning analyses,highlighting their stem cell characteristics and the progression to lung adenocarcinoma.In 2007,Ho et al[7]used stem-like lung cancer cells to efflux Hoechst33342 dye and,by flow cytometry,isolated and identified these cells in human lung cancer cells and tissues,demonstrating a higher invasive capacity and high expression of ABCG2(ATP-binding cassette transporter G2)and other ATP-binding cassette transporters resistant to various chemotherapy drugs.Currently,lung cancer stem cells are mainly sourced from the malignant transformation of normal adult stem cells or precursor cells and the acquisition of stemness through dedifferentiation of differentiated tumor cells.[8]

2.2.Biomarkers of lung cancer stem cells

The isolation and identification of lung cancer stem cells are based on their biological characteristics and specific surface markers,which serve as the cornerstone of basic research.Although there is currently no specific marker exclusively for lung cancer stem cells,several markers have been widely studied.

Cluster of differentiation 133(CD133),also known as prominin-1,is a 5-transmembrane cell surface glycoprotein.Its gene is specifically located on chromosome 4p15,which contains genes related to organ maturation,homeostasis,tumor formation,and tumor invasion.CD133 consists of 865 amino acids,including an extracellular N-terminal region composed of 85 amino acids,5 transmembrane domains,2 large extracellular loop regions,and an intracellular C-terminal tail composed of 50 amino acids.CD133 is commonly regarded as a marker of CSCs.CD133 is detected in approximately 50%of cases of non–small cell lung cancer(NSCLC),colon cancer,gastric cancer,and ovarian cancer.Bertolini et al[9]found that the population of CD133-positive cells increases in NSCLC compared with that in normal lung tissues.These CD133-positive cells have a higher tumorigenic potential in severe combined immunodeficiency mice and exhibit a higher expression of stemness genes,adhesion properties,motility,and drug efflux rates than CD133-negative cells.In NSCLC patients treated with platinum-based regimens,a high expression of CD133 is associated with shorter progression-free survival.These findings suggest that CD133+cells possess CSC-like biological characteristics,and CD133 can serve as a prognostic indicator for NSCLC patients.

CD44 is a transmembrane glycoprotein involved in cell growth,survival,differentiation,and motility.In addition,CD44,along with P-glycoprotein,contributes to multidrug resistance in cells.As a marker of CSCs,CD44 plays a significant role in regulating tumor initiation,self-renewal,metastasis,and therapeutic resistance.The expression of CD44 is associated with the characteristics of lung cancer stem cells in NSCLC.Leung et al[10]found that when NSCLC tumor cells from patients were implanted into nude mice,both the original CD44-positive cells and newly differentiated CD44-positive cells exhibited higher tumorigenicity,tumor sphere-forming ability,and migration properties compared with CD44-negative cells.Furthermore,CD44+cells expressed pluripotency/stemness genes (Oct4[Octamer-binding transcription factor 4],Nanog [Nanog homeobox],Sox2[SRY-box transcription factor 2]),whereas CD44-cells did not.The freshly sorted CD44-positive cells showed stronger resistance to cisplatin and lower levels of apoptosis compared with CD44-negative cells.Chen et al[11]discovered that the expression of Oct4 in CD133+cells was higher than in CD133-cells in lung cancer tissue.When the expression of Oct4 in CD133 cells was inhibited,their ability to form serum-free spheres decreased,and there was a tendency toward differentiation into CD133-cells.These findings suggest that Oct4 plays an important role in maintaining tumor stem cells.Therefore,CD44 can serve as a reliable marker for screening CSCs.

Aldehyde dehydrogenase (ALDH) is a class of enzymes widely present in the body that are involved in the metabolism of aldehyde compounds.Increased expression of ALDH has been confirmed to be associated with poor prognosis,cancer stemness,and drug resistance in various solid tumors.[12]ALDH1 is one of the members of the ALDH enzyme family.In recent years,ALDH1 has been recognized as an important marker for CSCs in various solid tumors,including lung cancer,colon cancer,breast cancer,and others.Jiang et al[13]found that the expression of ALDH1 in cell lines derived from NSCLC patients is associated with proliferation,self-renewal,and cellular differentiation of lung cancer cells.These cells also express the lung cancer stem cell marker CD133,indicating that CD133 and ALDH1 are involved in the proliferation,self-renewal,and cellular differentiation of lung cancer cells.The functional characteristics of tumor stem cells contribute to the increasing tumorigenicity and drug resistance of tumors.Sullivan et al[14]applied immunohistochemistry to detect the expression of ALDH1A1,ALDH3A1,and CD133 on the cell surface of NSCLC samples.The results revealed that the expression of ALDH1A1 was closely associated with poor prognosis in lung cancer patients.These findings collectively suggest that ALDH1 can serve as a functional marker for identifying lung cancer stem cells and as a prognostic indicator for lung cancer.

2.3.The stemness index of lung cancer stem cells

Because of the continuous development of high-throughput sequencing technology,many cancer gene profiles and sequencing results have provided researchers with comprehensive molecular maps of cancer.Extracting and quantifying stem cell characteristics from sequencing data will contribute to the development of tumor-targeted therapeutic drugs and diagnostic tools based on CSC and its stemness features.In 2016,Sokolov et al[15]first developed a 1-class logistic regression algorithm(OCLR)based on logistic regression.This algorithm can accurately identify specific cellular subtypes in tumor tissues and infer the association between specific functional features and tumor subtypes.

In 2018,Malta et al[16]used the OCLR algorithm to study the genetic and epigenetic expression characteristics of pluripotent stem cells,induced pluripotent stem cells,and different germ layer–like cells.They conducted a comprehensive analysis of the transcriptomes and DNA methylomes of 33 cancer types in The Cancer Genome Atlas database.As a result,they obtained 2 independent stemness indices:mDNAsi,which is based on DNA methylation–based stemness features,and mRNAsi,which is based on gene expression–based stemness features.In 2020,Zhang et al[17]published relevant work that explored biomarkers of lung adenocarcinoma stem-like tumor cells using this index.Through an integrated analysis of data from The Cancer Genome Atlas and Gene Expression Omnibus databases,Zhang et al[17]confirmed that the score of mRNA expression-based stemness index is positively correlated with clinical stage and negatively correlated with 5-year overall survival rate in lung adenocarcinoma patients.Furthermore,they identified cell cycle–related genes as important regulators of stem-like features in lung cancer cells.

Quantitative calculation of stemness can aid in understanding the regulatorymechanismsof stemness inmalignanttumors.Modelingand prediction based on stemness indices and other indicators can guide the evaluation of treatment and prognosis for cancer patients in a clinical setting.

3.Lung cancer stem cells and EGFR-TKItargeted therapy resistance

3.1.Classic studies

In the study by Hashida et al,[18]various resistant cell lines to afatinib were established,and the mechanisms of resistance were investigated.Direct sequencing did not reveal any EGFR T790M mutation in the developed resistant cells.After inducing cell resistance with afatinib,they found that this cell line displayed epithelial-mesenchymal transition(EMT)amplification.However,when adding erlotinib on top of afatinib,the cells remained sensitive.They further conducted in-depth research and established a cell line called HCC827-ACR,which exhibited resistance to both afatinib and erlotinib while overexpressing stem cell markers.In conclusion,this study established cell lines resistant to EGFR-TKIs and observed the presence of stem cell characteristics within these cell lines.

The study by Ghosh et al[19]involved continuous exposure of NSCLC cell line NCI-H1650 to erlotinib,leading to the development of an erlotinib-resistant subline(H1650-ER1).Tumor stem cell–like characteristics were evaluated in the erlotinib-resistant H1650-ER1 cells,including the expression of stem cell markers,enhanced self-renewal and differentiation capabilities,and increased in vitro tumorigenicity.The subline that was erlotinib-resistant had a group of cells that resembled tumor stem cells.Measurement of cell proliferation and tumor sphere formationinthepresenceof erlotinibrevealed reduced sensitivitytoerlotinib treatment in these cells.This study suggests that in NSCLC patients who have been treated with EGFR-TKIs to inhibit EGFR kinase activity,there may be a subpopulation of cells within the original lung cancer cells that acquire stem-like characteristics.This can lead to resistance to targeted drugs against EGFR-sensitive mutations.

The study conducted by Huang et al[20]investigated the relationship between ALDH1 expression and EGFR-TKI resistance in lung cancer stem cells.They discovered that compared with ALDH1-negative lung cancer cells,ALDH1-positive lung cancer cells displayed resistance to gefitinib.In addition,the proportion of ALDH1-positive cells was higher in PC9/gef cells(gefitinib-resistant lung cancer cells)compared with PC9 cells(gefitinib-sensitive lung cancer cells).Consistent results were observed in both clinical samples and cell culture model systems,indicating a significant increase in the proportion of ALDH1-positive cells in lung cancer cells resistant to EGFR-TKIs and chemotherapy drugs.Collectively,these findings suggest that high expression of ALDH1 in lung cancer stem cells contributes to the development of resistance to EGFR-TKIs.

3.2.Mechanismsof drug resistance in lung cancer stem cells

The development of drug resistance is the main reason for treatment failure in cancer patients,and it is also a key factor contributing to tumor recurrence and metastasis.The use of EGFR-TKI–targeted therapy has brought hope to patients with EGFR gene mutations in lung adenocarcinoma.However,the emergence of resistance is inevitable and can significantly impact treatment efficacy.Lung cancer stem cells play a crucial role in the development of resistance to EGFR-TKI–targeted therapy in lung adenocarcinoma.Researchers have been continuously exploring the mechanisms underlying this resistance,which can be summarized as follows:

Lung cancer stem cells express multiple ABC transporters on their cell surface.ABCtransportersare knownasefflux pumpsand are considered as oncogenic proteins.They can transport drugs out of the cells,reducing intracellular drug concentration and minimizing drug-induced damage to the cells.Therefore,most of these ABC transporters directly contribute to the acquisition of drug resistance,and the reversal of their efflux activity can reverse drug resistance.[21]Among them,ABCB1 and ABCG2 have been shown to be highly expressed in stem-like cells and are associated with multidrug resistance phenotypes in various stem-like tumor cells.[22]Chen et al[11]identified that high expression of ABCB1 is a major cause of docetaxel resistance in NSCLC cells HCC827,HCC4006,and H1299.

One of the characteristics of cancer is the decreased ability of tumor cells to repair DNA damage,leading to the accumulation of mutations and genomic instability.However,tumor stem cells possess robust DNA damage repair capabilities,allowing them to efficiently repair DNA damage and maintain their integrity to a maximum extent.[23]Lung cancer stem cells often exist in a quiescent state,in the G0 phase of the cell cycle,where they do not actively synthesize DNA or undergo cell division.This enables them to effectively evade the cytotoxic effects of drugs on proliferating cells,resulting in the persistence of lung cancer stem cells.Moreover,when activated by external signals,lung cancer stem cells can enter the cell cycle and undergo proliferation,leading to tumor recurrence and metastasis.[24]Furthermore,the tumor microenvironment in which lung cancer stem cells reside plays a crucial role in determining their stem-like properties.In fact,lung cancer stem cells cannot survive and function independently but are greatly influenced by the microenvironment.The tumor microenvironment provides a favorable condition for lung cancer stem cells to escape the cytotoxic effects of drugs.In addition,the continuous differentiation and self-renewal abilities of lung cancer stem cells,combined with the influence of the microenvironment,ultimately contribute to the development of drug resistance.

3.3.The signaling pathways associated with lung cancer stem cells

The Wnt pathway mainly consists of three signaling pathways,among which the Wnt/β-catenin pathway is most closely associated with tumor development.[25]The Wnt/β-catenin pathway is an evolutionarily conserved signaling pathway that primarily participates in processes such as cell proliferation,survival,migration,differentiation,and apoptosis.Studies have found that in 2 lung cancer cell lines,which have high expressions of Oct4 and Nanog and are resistant to gefitinib,β-catenin staining is evident.There is also an overexpression of Wnt pathway–related mRNA and proteins as the cells undergo EMT.[26]This indicates that activation of the Wnt/β-catenin pathway can confer drug resistance to lung cancer stem cells and promote EMT in tumor cells.

The Hedgehog(Hh)signaling pathway is physiologically activated during early human embryogenesis and organ development.Aberrant activation of this pathway can lead to the development of various tumors.[27]The Hh pathway can confer resistance to platinum-based drugs in lung tumor cells through upregulation of ABC transporters and induction of EMT.[28]However,inhibiting the Hh pathway can increase the sensitivity of cells to platinum-based drugs and decrease the expression of CSC phenotypes.

Notch is a highly conserved signaling pathway that mediates cell differentiation,proliferation,survival,and apoptosis.[29]Recent research has shown that microRNAs play a crucial role in regulating CSC properties.Jiang et al[30]found that miR-1275 can enhance the stemness of lung adenocarcinoma cells by activating the Notch pathway.On the other hand,antagonizing miR-1275 or inhibiting the Notch pathway can effectively reverse the pathway coactivation and stem-like characteristics induced by miR-1275.

3.4.Strategies for targeting lung cancer stem cells and reversing drug resistance

Lung cancer stem cells have been implicated in the development of resistance to EGFR-TKIs,playing a significant role in the emergence of drug resistance.Targeting lung cancer stem cells and reversing their resistance to EGFR-TKIs hold important clinical significance for patients with lung adenocarcinoma.

Lung cancer stem cells exist in a dedifferentiated state and possess stem-like characteristics,including self-renewal and multilineage differentiation potential.They can differentiate into various cell types within lung cancer tissue,and their invasiveness and resistance to drugs are enhanced.Inducing the differentiation of lung cancer stem cells can effectively reduce the tumor-forming ability of lung cancer cells and decrease drug resistance.[31]Through a series of in vitro experiments,Huang et al[32]have demonstrated chlorogenic acid as a potential differentiation inducer.Their studies explored the proliferation and migration/invasion capabilities of cancer cells,the production of mitochondrial ATP,and in vivo experiments on lung cancer growth in tumor-bearing mice.Inducing the differentiation of CSCs represents a novel strategy for cancer treatment.A phase I clinical trial[33]of chlorogenic acid injection in glioma patients has shown good safety and clinical benefits for patients.

Inhibiting ABC transporters can enhance the intracellular drug concentration,increase the cytotoxic effects of drugs on lung cancer stem cells,and reduce the development of drug resistance.When using elacridar[34]to inhibit ABCB1,it can suppress the stem-like characteristics of this drug-resistant cancer cell subset.

Monoclonal antibody molecules targeting the Wnt,Hh,and Notch signaling pathways have entered preclinical or clinical trial stages.

In lung cancer patients,inhibitors targeting β-catenin in the Wnt signaling pathway present a challenging therapeutic approach.β-Catenin inhibitors[35]block T Cell Fator/lef-dependent transcription by disrupting protein-protein interactions between β-catenin and other transcriptional regulatory factors,promoting β-catenin degradation,or inhibiting β-catenin kinases such as Traf2-and Nck-interacting kinase(TNIK).Small molecule β-catenin protein-protein interaction inhibitors include BC2059,[36]LF3,[37]and PRI-724.[38]MSAB[39]is a small molecule compound that binds to β-catenin,promoting its proteasomal degradation.TNIK inhibitors such as ky-05009[40]and PF-794[41]inhibit the phosphorylation of TNIK substrates,thereby suppressing β-catenin–T Cell Fator/lef-dependent transcription and various cellular processes.Because these CSC regulating pathways also play important roles in normal adult stem cells,caution is required in their clinical applications.In addition,it is important to actively explore specific markers and signaling pathways that are distinct to lung cancer stem cells compared with ordinary lung cancer cells to target and eliminate them effectively.

4.Summary and outlook

The concept of lung cancer stem cells has provided new insights for basic research in lung cancer and brought new hope for clinical treatment of lung cancer patients.With the widespread use of genetic testing,EGFR-TKI–targeted therapy has greatly benefited patients with EGFR-mutant lung adenocarcinoma.However,the inevitable emergence of drug resistance has compromised the effectiveness of targeted drugs.Lung cancer stem cells have played a subtle but significant role in the development of resistance to EGFR-TKIs in lung adenocarcinoma.Although lung cancer stem cells can be identified and sorted based on surface marker proteins,their specificity is not high.In the future,if specific markers that exclusively represent lung cancer stem cells can be accurately identified and targeted for elimination,it will have profound implications for the treatment of lung cancer patients.In recent years,many studies have been exploring the intrinsic correlation between lung cancer stem cells and resistance to EGFR-TKI–targeted therapy,as well as investigating the mechanisms of drug resistance.However,the research on lung cancer stem cells is still in its early stages,and more comprehensive,in-depth,and detailed studies are expected to be conducted in the future.

Acknowledgments

Not applicable.

Financial support and sponsorship

This project was supported by the Natural Science Foundation of Hubei Province(no.2021CFB372 to Hua Xiong).

Conflicts of interest statement

The authors declare that they have no conflict of interest with regard to the content of this report.

Author contributions

All authors were involved in the collection,writing,and revision of the manuscript.

Data availability statement

Not applicable

Ethical approval

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The study was conducted in accordance with the Declaration of Helsinki.