STAT3 induces G9a to exacerbate HER3 expression for the survival of epidermal growth factor receptor-tyrosine kinase inhibitors in lung cancers
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HER3 mediates drug resistance against epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), resulting in tumor relapse in lung cancers. Previously, we demonstrated that EGFR induces HER3 overexpression, which facilitates the formation of cancer stem-like tumorspheres. However, the cellular mechanism through which EGFR regulates HER3 expression remains unclear. We hypothesized that EGFR downstream of STAT3 participates in HER3 expression because STAT3 contributes to cancer stemness and survival of EGFR-TKI resistant cancers.
First, RNAseq was used to uncover potential genes involved in the formation of lung cancer HCC827-derived stem-like tumorspheres. EGFR-positive lung cancer cell lines, including HCC827, A549, and H1975, were individually treated with a panel containing 172 therapeutic agents targeting stem cell-associated genes to search for potential agents that could be applied against EGFR-positive lung cancers. In addition, gene knockdown and RNAseq were used to investigate molecular mechanisms through which STAT3 regulates tumor progression and the survival in lung cancer.
BBI608, a STAT3 inhibitor, was a potential therapeutic agent that reduced the cell viability of EGFR-positive lung cancer cell lines. Notably, the inhibitory effects of BBI608 were similar with those associated with YM155, an ILF3 inhibitor. Both compounds reduced G9a-mediated HER3 expression. We also demonstrated that STAT3 upregulated G9a to silence miR-145-5p, which exacerbated HER3 expression in this study.
The present study revealed that BBI608 could eradicate EGFR-positive lung cancers and demonstrated that STAT3 enhanced the expression of HER3 through miR-145-5p repression by G9a, indicating that STAT3 is a reliable therapeutic target against EGFR-TKI-resistant lung cancers.
KeywordsBBI608 EGFR G9a HER3 Lung cancer STAT3
Epidermal growth factor receptor
Fetal bovine serum
Interleukin enhancer-binding factor 3
Transducer and activator of transcription 3
Tyrosine kinase inhibitors
Transcripts per million clean tags
The overexpression and activation of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that belongs to the ERBB family, facilitates tumor survival, proliferation, and cancer stemness in lung cancer [1, 2]. Therefore, EGFR-tyrosine kinase inhibitors (TKIs), such as gefitinib, afatinib, and osimertinib, specifically targeting EGFR wild-type (WT), EGFR and HER2 dual targets, and EGFR T790 M, respectively, are effective therapeutic agents for the eradication of lung cancers . However, resistance to EGFR-TKIs still occurs and leads to tumor recurrence [4, 5].
Various mechanisms promote EGFR-TKI resistance. In addition to KRAS and EGFR T790 mutations, the expression of oncogenes, including MET [5, 6, 7], HER2 (ERBB2) , and epidermal growth factor receptor 3 (HER3, ERBB3) [5, 9, 10], is associated with drug resistance against EGFR-TKIs and leads to tumor recurrence in lung cancers. For example, long-term treatment leads to complement activation of the MET-mediated signaling pathway in lung HCC827 cells and consequent overexpression of HER3 against gefitinib . Antitumor therapeutics by antibodies targeting HER3 triggers a response to EGFR-TKI erlotinib in refractory non–small-cell lung cancer . Constitutive overexpression of HER2 forms dimerization with HER3, leading to the downstream activation of PI3K signaling and tumor survival [12, 13]. Such results indicate that HER3 causes EGFR-TKI resistance. Therefore, it is vital to explore the molecular mechanism through which HER3 expression is regulated in EGFR-positive lung cancers.
We previously demonstrated that EGFR induces HER3 overexpression to promote the formation and survival of HCC827- and A549-derived cancer stem-like tumorspheres . Because transducer and activator of transcription 3 (STAT3) contributes to cancer stemness [15, 16] and EGFR-TKI survival , we assume that STAT3 plays a major role in the regulation of HER3 expression. In addition, we revealed that EGFR phosphorylation participates in tumorsphere formation through the upregulation of the expression of G9a histone methyltransferase (HMT) . Because YM155, an interleukin enhancer-binding factor 3 (ILF3) inhibitor , can block EGFR autophosphorylation to inhibit G9a-mediated stemness , EGFR downstream of G9a may also regulate cancer stemness and HER3 expression. Thus, the present study investigated the role of STAT3 and G9a in cancer stemness and HER3 expression.
G9a (EHMT2) has been reported to be an epigenetic regulator, which biochemically catalyzes the mono- and di-methylation of H3K9 (H3K9me1 and H3K9me2) in euchromatin , leading to gene repression . Recently, G9a has been demonstrated to interact with several transcriptional factors, including GATA3 and ZEB2 , STAT3 , and MYC , leading to the repression of gene transcription, while enhancing tumor survival. G9a is a potential mediator that silences tumor suppressors based on interacting partners. Particularly, G9a was reported to reduce the expression levels of microRNAs, such as miR-200c, under the mediation of STAT3-G9a, which causes the astrocyte leptin receptor to exacerbate tumor progression in breast cancer . In addition, G9a interacts with MYC, which drives transcriptional repression and tumorigenesis . To the best of our knowledge, STAT3 also induces MYC expression, and both transcriptional factors have been reported to participate in tumor stemness [24, 25, 26, 27]. STAT3 was demonstrated as a target against EGFR-TKI resistance . Therefore, we assumed that EGFR promotes HER3 overexpression through the STAT3-mediated activation of G9a to repress the expression of HER3-targeted microRNAs, because we identified that STAT3, an EGFR downstream phosphorylated target, participates in tumorsphere formation and survival in EGFR-positive colorectal cancer cell lines .
To validate the aforementioned assumptions, we used RNAseq to explore differential genes participating in the formation of HCC827-derived stem-like tumorspheres and in the knockdown of A549shSTAT3 and A549shG9a lung cancer cell lines compared with A549shLuc controls. The RNAseq-based gene profiling by treatment of BBI608, which selected by a screened panel targeting to stem-associated genes, and the inhibitory effects of BBI608 were compared with those of YM155, which particularly inhibited the formation of tumorspheres. BBI608, a STAT3 inhibitor, reduced not only the viability of EGFR-positive lung cancer cell lines but also the expression of G9a and HER3. These results were consistent with findings obtained following treatment with YM155, an ILF3 inhibitor. In addition, we demonstrated that STAT3 upregulated G9a expression, which significantly silenced miR-145-5p and exacerbated HER3 expression. The present study revealed the role of the transcriptional repression of miRNAs, such as miR-145-5p, by the STAT3-G9a axis in EGFR-positive lung cancers, which exacerbated HER3 expression and led to EGFR-TKI resistance.
Cell culture and tumorsphere formation
HCC827 (CRL-2868), A549 (CCL-185), H1975 (CRL-5908), and H520 (HTB-182) lung cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines were free from Mycoplasma. HCC827, H1975, and H520 cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. A549 was cultured in Dulbecco’s modified Eagle medium with the same additives. The cell lines were reauthenticated through short tandem repeat profiling (Applied Biosystems, Massachusetts, USA): HCC827 on May 8, 2015; A549 on June 4, 2014; H1975 on May 23, 2019; H520 on December 13, 2016. For tumorsphere formation, cells were cultured in low-attached six-well plates with serum-free medium containing B27 (Invitrogen, Waltham, MA), 20 ng/mL of EGF (Sigma, Missouri, TX), 20 ng/mL of fibroblast growth factor (bFGF, Sigma), 5 μg/mL of bovine insulin (Sigma), and 4 μg/mL of heparin (Sigma) for at least a 7-day incubation period. The sizes of tumorspheres were examined under an inverted microscope (Axio Observer 3, ZEISS, Oberkochen, Germany). All cells were incubated at 37 °C and 5% CO2.
Male NOD/SCID mice were purchased from BioLASCO Taiwan Co., Ltd., Taiwan. Five-week-old mice were maintained under a 12-h light/dark cycle at 22 °C. Animal studies were approved by the Institutional Ethical Review Committee at Mackay Memorial Hospital, Taiwan, and were performed according to NIH guidelines on the care and welfare of laboratory animals. Tumor xenografts were established by injecting 2 × 106 of A549shLuc (n = 4) or A549shSTAT3 (n = 4) into the subcutaneous legs of 5-week-old mice. For tumor growth inhibition, 10 mg/kg of BBI608 was injected via tail vein in A549-derived tumor xenografts (n = 3 for each group). Tumors were externally measured using a digital caliper, and tumor volumes were calculated using the following formula: 0.52 × width2 × length, where the smaller tumor diameter represented the width. Animals were sacrificed using carbon dioxide inhalation.
RNAseq, small RNAseq profiling, and bioinformatics analysis
RNAseq was performed to determine differentially expressed mRNAs in (1) HCC827-derived tumorspheres compared with parental HCC827 cells, (2) A549 cells treated with 1 μg/mL of BBI608 and 1 μg/mL of YM155, and (3) A549shSTAT3 and A549shG9a compared with A549shLuc (luciferase). A HiSeq 4000 with paired-end 150-bp sequencing was used for experiments. Genes upregulated with a > 2-fold change (log2) with a p value of < 0.05 in HCC827-derived tumorspheres were selected for bioinformatics analyses by using NetworkAnalyst (http://www.networkanalyst.ca/) , and pathway activations were selected and matched based on the KEGG database. Genes downregulated with a less than − 1-fold change (log2) with a p value of < 0.05 in (1) BBI608- and YM155-treated A549 cells compared with parental A549 cells and (2) A549shSTAT3 and A549shG9a compared with A549shLuc cells were compared using List Operations (http://www.molbiotools.com/listoperations.html) to determine common genes that were differentially expressed. In addition, differentially expressed genes were analyzed using NetworkAnalyst to determine major signaling pathways involved and key genes. Differentially expressed microRNAs were investigated using small RNA digitalization analysis through sequencing by synthesis (Illumina, San Diego, California, USA). The expression levels of known and unique miRNAs in each sample were statistically analyzed and normalized using transcripts per million clean tags (TPMs) . Common differential miRNAs in A549shILF3 and A549shG9a identified using List Operations were compared with predictable HER3-binding miRNAs selected by TargetScan (http://www.targetscan.org/vert_72/) based on conserved sites for broadly conserved miRNA families among vertebrates .
The mRNA extraction and cDNA preparation were performed as described previously . Quantitative PCR (Applied Biosystems, California, USA) was performed using the SYBR Green system (Applied Biosystems, California, USA) according to manufacturer’s instructions. Primers used for PCR were as follows: ERBB3 (HER3): forward, 5′-GCCAATGAGTTCACCAGGAT-3′ and reverse, 5′-ACGTGGCCGATTAAGTGTTC-3′. GAPDH: forward, 5′- GAGTCAACGGATTTGGTCGT-3′ and reverse, 5′- TTGATTTTGGAGGGATCTCG-3′.
Gene knockdown and overexpression
Gene knockdown was performed using a short-hairpin RNA (shRNA)-expression lentivirus system that contained the specific shRNA (target sequence of STAT3, ILF3, and EHMT2 (G9a): GCACAATCTACGAAGAATCAA, GCCATGTGATGGCAAAGCATT, and GCTCCAGGAATTTAACAAGAT for shG9a#1 / CGAGAGAGTTCATGGCTCTTT for shG9a#2, respectively) in the pLKO.1-puro vector generated in a 293 T cell line. For G9a overexpression in A549 cells, pLenti6-MK1-EHMT2-V5 based on lentiviral system (Addegene, Massachusetts, USA) was purchased and used. The procedure followed was the same as that in our previous study .
Western blot analysis
Western blot analysis was performed as described previously . Specific antibodies against ILF3, G9a, di-mH3K9, STAT3, pSTAT3, HER3, and GAPDH were purchased from Cell Signaling (Danvers, Massachusetts, USA). Image J software was used to calculate the G9a, di-mH3K9, HER3 ratio divided by GAPDH.
The Alarmar Blue assay was performed according to manufacturer’s instructions to determine cell viability. To find therapeutic agents that can be applied against EGFR-positive lung cancers, a panel containing 172 compounds targeting stemness (MCE, Monmouth Junction, NJ, USA) was added to HCC827, A549, H1975, and H520 cell lines separately at doses of 1 μM and incubated for 48 h. To examine cell viability, cells were treated using afatinib, BBI608, or YM155 for 48 h.
Transwell migration assay (8 μm) was used to detect A549 cell migration capacity. In brief, 5 × 104 cells were placed in the upper layer of a cell culture insert with 200 μL of serum free DMEM medium. To test cell migration of A549-derived tumorspheres, serum-free DEME medium containing B27 (Invitrogen, Waltham, MA), 20 ng/mL of EGF (Sigma, Missouri, TX), 20 ng/mL of fibroblast growth factor (bFGF, Sigma), 5 μg/mL of bovine insulin (Sigma), and 4 μg/mL of heparin (Sigma) was used. Then, each 750 μL of DMEM medium with 10% FBS and test agents, including 1 μg/mL BBI608, 1, 5, 10 μg/mL UNC0642, was loaded into the below 24-well culture plate. Cells were incubated at 37 °C and 5% CO2 for 16 h. The membrane inserts were fixed in 3.7% formaldehyde for 5 min and consequently incubated in 100% methanol for 20 min at room temperature. After 0.5% crystal violet in 2% ethanol to stain the membrane inserts for 15 min at room temperature, non-migrated cells on the upper membrane were scraped with cotton swabs. PBS wash for twice was necessary between operations. The cells migrated through the membrane were imaged and counted using an inverted microscope (Axio Observer 3, ZEISS, Oberkochen, Germany).
Measurement of miR-145-5p
A TaqMan advanced miRNA assay (Applied Biosystems, California, USA) was used to detect the expression of miR-145-5p in (1) lung cancer cell lines, including HCC827, A549, and H1975, (2) A549 cells treated with 1 μg/mL of BBI680 for 48 h, and (3) A549shG9a with or without 20 ng/mL of EGF treatment in comparison with A549shLuc. The detection of miR-145-5p was according to manufacturer’s instructions.
Statistical analyses were performed using GraphPad Prism v5.01 (GraphPad Software, Inc., California, USA). All analytical data with more than two groups were evaluated using analysis of variance, followed by post hoc analysis with Bonferroni’s test. Student’s t test was used to compare two groups. In addition, p < 0.05 was considered to indicate a statistically significant difference.
HER3 overexpressed in HCC827-derived stem-like tumorspheres
BBI608, a STAT3 inhibitor, significantly reduced EGFR-positive lung cancers to against EGFR-TKI-resistance
STAT3 contributes to G9a and HER3 expression and influences cell survival in HCC827-derived tumorspheres
G9a mediated STAT3-regulated HER3 expression in EGFR-positive lung cancer
G9a repressed miR-145-5p and exacerbated HER3 expression
We previously demonstrated higher EGFR phosphorylation in HCC827-derived stem-like tumorspheres even without EGF treatment ; therefore, in the present model, the activation of STAT3 was considerable. Compared with HCC827 with an EGFR E746-A750 deletion, there was higher STAT3 phosphorylation and HER3 expression in H1975 cells (Fig. 3c), indicating that EGFR autophosphorylation led to the subsequent cascades. Previous studies have revealed that the activation of STAT3 is not only achieved by EGFR  but also by IL6-gp130 [34, 35]. Particularly, the inhibition of STAT3 phosphorylation increased the inhibitory effects of afatinib, an EGFR-TKI, in T790-mutant H1975 cells . Therefore, targeting STAT3 is considered a potential strategy for overcoming EGFR-TKI resistance associated with KRAS and T790 M mutations in lung cancers. In addition, because we demonstrated that STAT3 induced HER3 expression in EGFR-positive lung cancers, the inhibition of STAT3 could prevent HER3-mediated EGFR-TKI resistance. Previous studies have indicated that the amplification of MET participates in EGFR-TKI resistance, which could be associated with MET-mediated HER3 expression  and MET-mediated AKT and MAPK signaling pathways . A combined strategy simultaneously targeting STAT3 and MET could effectively prevent HER3-derived EGFR-TKI resistance.
In clinical trials, STAT3 inhibition has been reported to have remarkable therapeutic effects against various cancer types. For example, OPB-51822, a small-molecule STAT3 phosphorylation inhibitor targeting the SH2 domain (Tyr705/Ser727), exhibited high antitumor activity in EGFR-TKI-resistant NSCLC patients on a first-in-man phase I study . In addition, STAT3-targeted RNAi therapies, such as AZD9150 (16-oligonucleotide antisense molecule targeting the 3′ untranslated part of STAT3), also exhibited single-agent antitumor activity in patients with lymphoma or NSCLC in a phase I dose escalation study . However, the STAT3-HER3 cascade is a source of EGFR-TKI resistance. The amplification of oncogenes, such as HER2, also results in EGFR-TKI resistance . In addition, previous studies have revealed that cancer stem cells that exhibit self-renewal and pluripotency are responsible for drug resistance and cancer recurrence . The present study revealed that HER2 was overexpressed in HCC827-derived tumorspheres (Fig. 1d), and HER2 expression decreased following treatment with BBI608 (Fig. 3g) and A549shSTAT3 (Fig. 4e). Because there was no change in the expression of HER2 in A549shG9a (Fig. 4e), we concluded that STAT3 was a potential therapeutic target against lung cancers with EGFR-TKI resistance that was superior to G9a.
Increasing evidence suggests that microRNAs are involved in EGFR-mediated signaling pathways in lung cancers, including miR-145, which is downregulated and associated with TKI resistance targeting ERK, AKT, Oct4, c-MYC, EGFR, and NUDT1 . The results indicate that miR-145 is a cell proliferation suppressor in lung adenocarcinoma by targeting EGFR and NUDT1 as an ERK and AKT phosphorylation inhibitor, which enhances gefitinib cytotoxicity in NSCLC [41, 42]. We further demonstrated that miR-145 was inhibited by G9a in the present study; thus, it played a major role in the regulation of HER3 expression. Because miR-145 has been reported to be a suppressor of HER3 translation in breast cancer , miR-145 downregulation by G9a could promote the overexpression of HER3 in lung cancers. In addition, we observed the downregulation of HER3 in A549shILF3 cells compared with A549shLuc cells, which validated that ILF3 enhanced HER3 expression . A previous study reported that IL3 mediates miR-145 biogenesis and enhances the development of cancer stem cells in bladder cancer . miR-145 targets EGFR , resulting in the downregulation of EGFR in A549ILF3 cells . Therefore, YM155, an ILF3 inhibitor, caused a substantial decrease in EGFR , which led to the decrease in EGF-mediated G9a levels in the present study. Similarly, we confirmed that miR-200c, which is highly expressed and associated with epithelial–mesenchymal transition, invasion, and migration in NSCLC patients , was inhibited by G9a in A459 cells measured based on small RNAseq analyses, indicating that G9a not only regulated HER3 through the repression of miR-145-5p expression but also increased other oncogenes, such as the astrocyte leptin receptor, through the repression of miR-200c expression, which enhanced tumorigenesis in lung cancers.
In conclusion, we demonstrated the STAT3-G9a-HER3 axis in lung cancer that evades EGFR-TKI therapies. The potential mechanism is through the repression of miR-145-5p expression, which specifically targets HER3 (Fig. 6). BBI608 and YM155 demonstrated similar effects, reducing the viability of lung cancer A549 cells and inhibiting STAT3-mediated G9a and HER3 expression. We propose that BBI608, which was selected from a panel kit, and YM155 are potential therapeutic agents against EGFR-positive lung cancers and may be combined with EGFR-TKIs for application in the eradication of lung cancers.
The authors thank the Radiation Biology Core Laboratory of Institute for Radiological Research, Chang Gung Memorial Hospital, for technical support. This manuscript was edited by Wallace Academic Editing.
CCC and YFC conceptualized and designed the study. CCC and ASH developed the methodology. CCC and YWC collected the data. CCC and JC analyzed and interpreted the data (e.g., statistical analysis, biostatistics, and computational analysis). CCC, KHL, ZLS and YFC contributed toward writing, reviewing, and revising the manuscript. YFC and KHL supervised the study. All authors read and approved the final manuscript.
This study was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 106-2320-B195-003), Cheng Hsin General Hospital (CHGH 106-06), and Mackay Memorial Hospital (MMH-CT-10605 and MMH-106-61). Funding bodies did not have any influence in the design of the study and data collection, analysis and interpretation of data or in writing the manuscript.
Ethics approval and consent to participate
Animal studies were approved by the Institutional Ethical Review Committee at Mackay Memorial Hospital, Taiwan, and were performed according to NIH guidelines on the care and welfare of laboratory animals.
Consent for publication
The authors declare that they have no competing interests.
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