JMJD2C Facilitates Self-Renewal Of Stem-Like Cells And Confers Resistance To EGFR Inhibitors In Non-Small Cell Lung Cancer

BackgroundLung cancer is a leading cause of cancer death worldwide,and poses a significant risk to human health.Chemotherapeutic agents like gemcitabine,platinum compounds,and taxanes improve survival to a limited extent,but overall survival rates remain low because of recurrence of more aggressive,drug-resistant tumors.The advent of molecularly targeted therapies has shed much light on lung cancer treatment.Epidermal growth factor receptor tyrosine kinase inhibitors（EGFR-TKIs）have become the standard first-line treatment for advanced NSCLC with sensitive EGFR mutations.However,almost all patients inevitably develop resistance within6-12 months after the initiation of EGFR-TKI treatment.In all the cases,the recurrence can be local or metastatic,and commonly occur after a period of clinical dormancy.Although several mechanisms,including the T790M secondary mutation have been reported,overcoming EGFR-TKI resistance remains a challenge in clinical practice.Accumulating evidence supports the concept that the cancer stem cell（CSC）,also designated‘tumor-initiating cell’,is a rare population of undifferentiated tumorigenic cells responsible for tumor initiation,maintenance and metastasis.These cells are capable of self-renewing,differentiating,and maintaining tumor growth and heterogeneity,and play an important role in tumorigenesis and resistance to therapy.CSCs have been isolated from leukemia and several solid cancers.Resistance to conventional chemotherapeutic drugs is a common characteristic of CSCs.However,less studies have focused on the characterization of CSCs contributing to EGFR TKI resistance in lung cancer.In the current study,we explored the expression of ALDH1A1,with a view to its application as a potential marker for lung cancer stem cells.Our main aim was to determine the significance of ALDH1A1-positive CSCs in the mediation of EGFR TKI resistance in lung cancer.Histone demethylases constitute a large and diverse family of enzymes,which is regarded as an important type of histone modification during CSCs maintenance.The JMJD2 subfamily（JMJD2A-D）comprises the recently discovered Jumonji C（JmjC）domain containing（JMJD）histone demethylases,which demethylates mainly H3K9me2/3 or H3K36me3.As there is increasing evidence that JMJDs are associated with various disease states,they have emerged as attractive targets for the development of new therapeutic drugs.JMJD2C,also known as KDM4C/GASC1,is a member of the JMJD2 subgroup of JmjC domain-containing proteins which catalyzes the demethylation of tri-and dimethylated lysine 9 and lysine 36 on histone H3.Recent studies have implicated a role for JMJD2C in self-renewal and pluripotency in preimplantation embryos and embryonic stem（ES）cells.So far,the specific roles of JMJD2C involved in maintenance of CSCs or whether it can be used for CSCs-directed therapy are largely unexplored.PurposeWe aim to identify and characterize the molecular signatures and biological properties associated with JMJD2C in NSCLC ALDH^bri+CSCs and highlight JMJD2C-mediated epigenetic modifications as a means by which it is involved in orchestrating CSCs physiology.Furthermore,we highlighted the aberrantly activated JMJD2C in TKI-accumulated NSCLC ALDH1^bri+CSCs as one of the mechanisms for the acquired resistance to gefitinib.In addition,we are going to investigate whether JIB-04,the small-molecule inhibitor of JMJD2C could effectively overcome TKIs resistance both in TKI-resistant cell lines and xenograft models.We believe that these studies will shed light on novel mechanisms underlying the genesis of NSCLC and will lead to the identification of novel therapeutic modalities to combat NSCLC by overcoming resistance to EGFR inhibitors.We thus provide rationale and experimental evidence for the combined use of histone demethylase inhibitors and TKIs to overcome TKI resistance in patients with NSCLC with EGFR mutations.MethodsWe tested the expression of JMJD2C in FACS-sorted ALDH^bri+CSCs and ALDH^low-tumor cells from NSCLC specimens by qPCR and Western blotting assay;JMJD2C’s involvement in NSCLC ALDH^bri+CSCs maintenance was rigorously demonstrated by a variety of standard assays including spheres formation in vitro,tumorigenic potential in vivo,serial transplantations/passaging and lung-seeding assays,ALDH1 was identified as a marker of NSCLC CSCs in our study as assessed by Aldefluor flow cytometry assay;the regulation of JMJD2C on the pluripotency-related genes was assessed via qPCR.TKI-resistant PC9/GR was induced by stepwise selection after exposure to increasing doses of Gefitinib.Cell proliferation assay was applied to determinate IC₅₀,draw cell growth curve and calculate resistance index;The morphologic changes of PC9/GR were observed under ordinary optical microscope;flow cytometry was applied for cell cycle detection;identification of ALDH^bri+components was performed via Aldefluor flow cytometry assay;expression levels of JMJD2C in PC9/GR and PC9 cell lines were determined by qPCR and Western blotting assay;Cell viability of PC-9/GR cells treated with the indicated doses of JIB-04 alone,gefitinib alone,and combinations were assessed with the MTT method.TKI-resistant cell lines and xenograft mouse model were used to investigate whether JIB-04 could effectively overcome TKIs resistance both in vitro and in vivo.IHC was used to analyze the differences and correlation of JMJD2C and ALDH1 between EGFR-TKI-sensitive and-secondary resistant NSCLCs.ResultsFor the first part,we are going to provide insight into the functional relevance and underlying mechanisms of the JMJD2C expression in NSCLC.First and formost,we tested the expression of JMJD2C in FACS-sorted ALDH^bri+CSCs and ALDH^low-tumor cells from NSCLC specimens and PC9 cell line,JMJD2C expression was enriched in the ALDH^bri+subpopulation as compared with the ALDH^low-counterparts.Moreover,our study described increased expression of JMJD2C in the NSCLC ALDH^bri+CSCs and its involvement in CSCs maintenance was rigorously demonstrated by a variety of standard assays including spheres in vitro,tumorigenic potential in vivo,serial transplantations/passaging,lung-seeding assays and Aldefluor flow cytometry assay.Subsequent studies of the JMJD2C functional network identified a subset of pluripotency-associated genes（PAGs）to be preferentially down-regulated in JMJD2C inhibited NSCLC ALDH^bri+CSCs.Among these genes,c-Myc,a key transcription factor important for the maintenance of pluripotency and self-renewal of ES cells was strikingly down-regulated and was chosen as a target for these studies.For the second part,we objective to establish a gefitinib-resistant PC9/GR cell line of human lung adenocarcinoma,and explore its biological characteristics and drug resistance mechanisms.A gefitinib-resistant lung adenocarcinoma cell line PC9/GR were successfully established with higher resistance index and a markedly lower proliferation rate than parental cells.Loss of polarity and elongated spindle-shaped changes were observed under ordinary optical microscope.In cell cycle analysis,PC9/GR cells had a higher proportion at the G0/G1 phase and a lower proportion at the S phase when compared to parental cells.This indicates that PC9/GR cells underwent a more decreased G1-S phase transition.Aldefluor flow cytometry assay data showed that PC9/GR,gefitinib-resistant lung cancer cells,present a significantly higher ALDH^bri+cell population than PC9 cells.Moreover,JMJD2C expression was enriched in the TKIs-resistant subpopulation as compared with the sensitive counterparts.Furthermore,we show that JIB-04,the small-molecule inhibitor of JMJD2C in combination with gefitinib has a synergistic inhibitory effect on the survival and clonogenicity of cell lines resistant to TKIs.In addition,this combinational therapy reduces tumor masses much more effectively than gefitinib or JIB-04 alone in a xenograft mouse model,and this synergistic interaction is attributed to the ability of JIB-04 to decrease the activation of JMJD2C and reverse CSCs phenotype.Thus,JMJD2C may be a potential biomarker of the suitability of the combined application of a histone demethylase inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs.Last but not least,EGFR TKIs sensitive mutations-harboring NSCLCs have lower JMJD2C and ALDH1 expression compared with those with secondary resistance.Conclusions1.JMJD2 C expression was enriched in the FACS-sorted ALDHbri+ subpopulation as compared with the ALDHlow-counterparts from NSCLC specimens.In light of this commonality between sorted subpopulations,we hypothesized that JMJD2 C is involved in the maintenance of human NSCLC ALDHbri+ cells.2.JMJD2 C,a H3K9me3 histone demethylase,is involved in the maintenance and self-renewal of human NSCLC ALDHbri+ CSCs.3.JMJD2 C played roles in NSCLC CSCs physiology is proposed to,in part,through diverse pluripotency-related genes.4.A gefitinib-resistant lung adenocarcinoma cell line PC9/GR have been successfully built by stepwise selection after exposure to increasing doses of gefitinib.5.Aberrantly activated JMJD2 C in TKI-acumulated ALDH1bri+ CSCs may be one of the mechanisms for the secondary resistance to gefitinib.6.In patients with NSCLC with EGFR mutations,JIB-04,a new epigenetic anticancer drug might be used in combination with TKIs to delay or overcome TKI resistance.Therefore,reversing CSCs phenotype and decreasing JMJD2 C activation may be an effective way to improve the response to EGFR-TKI treatment.We thus provide rationale and experimental evidence for the combined use of histone demethylase inhibitors and TKIs to overcome TKIs resistance in patients with NSCLC with EGFR mutations.