Clinical Implications of the Autophagy Core Gene Variations in Advanced Lung Adenocarcinoma Treated with Gefitinib
SCienTifiC REPORTS |
Clinical Implications of the Autophagy Core Gene Variations in Advanced Lung Adenocarcinoma Treated with Gefitinib
Nasha Zhang 0 1
Hui Zhu 1
Li Zhang 2
Liqing Zhou 3
0 Shandong Provincial Key Laboratory of Radiation Oncology, Cancer Research Center, Shandong Cancer Hospital affiliated to Shandong University, Shandong Academy of Medical Sciences , Jinan, Shandong Province , China
1 Department of Radiation Oncology, Shandong Cancer Hospital affiliated to Shandong University, Shandong Academy of Medical Sciences , Jinan, Shandong Province , China
2 Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, Hubei Province , China
3 Department of Radiation Oncology, Huaian No. 2 Hospital , Huaian, Jiangsu Province, China. JupengYuan, Nasha Zhang and Longbin
OPEN Published: xx xx xxxx EGFR-TKIs show dramatic treatment benefits for advanced lung adenocarcinoma patients with activating EGFR mutations. Considering the essential role of autophagy in EGFR-TKIs treatments, we hypothesized that genetic variants in autophagy core genes might contribute to outcomes of advanced lung adenocarcinoma treated with gefitinib. We systematically examined 27 potentially functional genetic polymorphisms in 11 autophagy core genes among 108 gefitinib-treated advanced lung adenocarcinoma patients. We found that ATG10 rs10036653, ATG12 rs26538, ATG16L1 rs2241880 and ATG16L2 rs11235604 were significantly associated with survival of lung adenocarcinoma patients (all P < 0.05). Among EGFR-mutant patients, ATG5 rs688810, ATG5 rs510432, ATG7 rs8154, ATG10 rs10036653, ATG12 rs26538, ATG16L1 rs2241880 and ATG16L2 rs11235604 significantly contributed to disease prognosis. We also found that ATG5 rs510432, ATG5 rs688810, ATG10 rs10036653 and ATG10 rs1864182 were associated with primary or acquired resistance to gefitinib. Functional analyses of ATG10 rs10036653 polymorphism suggested that ATG10 A allele might increase transcription factor OCT4 binding affinity compared to theT allele in lung cancer cells. Our results indicate that autophagy core genetic variants show potential clinical implications in gefitinib treatment, especially among advanced lung adenocarcinoma patients, highlighting the possibility of patient-tailored decisions during EGFR-TKIs based on both germline and somatic variation detection.
degrading damaged organelles and long-lived proteins11,12. It has been reported that autophagy is involved in
multiple diseases, for example cancers, infections, neurodegeneration and aging13?16. During cancer
development, autophagy is considered as a non-apoptotic cell death pathway and suppresses tumorigenesis under certain
circumstances. However, autophagy facilitates tumorigenesis in most contexts17?19. Autophagosome is a kind of
spherical organelle with double layer membranes during autophagy. Establishment of autophagosome is
controlled by several autophagy core genes20, which might be involved in cancer initiation and progression21.
Accumulating evidences indicate that germline genetic variants may also play a part in resistance to
EGFR-TKIs. For instance, Ng et al. reported that NSCLC patients harboring EGFR mutations showed better
clinical response to TKIs if the patients carried a germline deletion polymorphism in BCL2L11 (BIM) at the same
time22. Moreover, we also found that EGFR germline polymorphisms (rs2293347 and rs4947492) might be
potential predictive markers of overall survival (OS) in advanced lung adenocarcinoma patients treated with gefitinib23.
In the current study, we hypothesized that genetic variants of autophagy core genes may contribute to differential
prognostic outcomes of advanced lung adenocarcinoma patients treated with gefitinib. To address this, we
systematically examined the clinical implications of 23 potentially functional polymorphisms in ten autophagy core
genes (ATG2B, ATG3, ATG4C, ATG5, ATG7, ATG9B, ATG10, ATG12, ATG16L2 and BECN) in advanced lung
adenocarcinoma who received gefitinib therapy.
Materials and Methods
Study subjects. There is a total of 108 patients with advanced lung adenocarcinoma treated with gefitinib in
this study (Supplementary Table?1). Patients were recruited between July 2003 and July 2012 at Tongji Hospital,
Tongji Medical College, Huazhong University of Science and Technology (Wuhan, Hubei Province, China). As
reported previously, eligible patients had at least one measurable lesion with a minimum size in at least one
diameter of ?10 mm for liver, lung, brain or lymph node metastases, WHO performance status of 0-1, and life
expectancy of ?3 months23. Each patient was treated with gefitinib orally at a daily dose of 250 mg as 2nd or 3rd
line monotherapy. The exclusion criteria included previous other EGFR-TKIs treatment, pneumonectomy or
severe cardio-pulmonary diseases23. This study was approved by the Review Boards of Tongji Hospital, Tongji
Medical College and Shandong Cancer Hospital affiliated to Shandong University. Written informed consent
from each patient for the use of his/her DNA and clinical information was obtained. The methods were carried
out in accordance with the approved guidelines.
Genetic variants selection of autophagy core genes. Single nucleotide polymorphisms (SNPs) of
autophagy core genes were selected as previously described24. In briefly, common SNPs (MAF ? 0.05 in Chinese
Han population) in eleven autophagy core genes (ATG2B, ATG3, ATG4C, ATG5, ATG7, ATG9B, ATG10, ATG12,
ATG16L1, ATG16L2 and BECN) were screened in the gene regions including a 10-kb up-stream region of each
gene based on the HapMap database. A total of 27 potentially functional SNPs were finally selected according to
linkage disequilibrium (LD) analyses with an r2 threshold of 0.80 as well as prediction with SNPinfo Web Server
Genotyping. Genomic DNA was extracted from blood sample which was collected from each patient upon
recruitment. The ATG3 rs2705507 polymorphism was excluded from the 27 SNPs since it cannot be analyzed
by the MassArray system (Sequenom Inc., San Diego, California, USA). The other 26 SNPs were finally
determined to be genotyped as described previously25?28. However, BECN rs9890617, rs9891429 and rs10512488 were
excluded because of genotyping failure. As a result, a total of 23 SNPs were successfully genotyped. A 15% blind,
random samples were genotyped in duplicates and the reproducibility was 100%.
Quantitative reverse transcription PCR (qRT-PCR). After lung cancer A549 cells were transfected with
siRNAs of OCT4, MTF1 or SOX5 (Supplementary Table?2), total RNA was isolated from cells with Trizol reagent
(Invitrogen) and treated with RNase-Free DNase to remove genomic DNA (Invitrogen). These RNA samples were
then reverse transcribed into cDNAs using Revert Ace kit (TOYOBO, Osaka, Japan). OCT4, MTF1, SOX5 ATG5,
ATG10 and ?-actin mRNAs were measured through the SYBR-Green qRT-PCR. The OCT4, MTF1, SOX5 ATG5
or ATG10 expression was calculated relative to the ?-actin expression.
Electrophoretic Mobility-Shift Assays (EMSA). Synthetic double-stranded and 3? biotin-labeled
oligonucleotides corresponding to the ATG10 rs10036653T or rs10036653A sequences (Supplementary Table?2)
and A549 cell nuclear extracts were incubated at 25 ?C for 20 min using the Light Shift Chemiluminescent EMSA
Kit (Pierce, Rockford, IL). The reaction mixture was separated on 6% PAGE, and the products were detected by
Stabilized Streptavidin-Horseradish Peroxidase Conjugate (Pierce).
Statistics. The differences in patients? characteristics were assessed by Pearson?s ?2 tests or Student?s t test.
Univariate and multivariate Cox proportional hazard regression analyses were utilized to calculate hazard ratios
(HRs) and 95% confidence intervals (CIs). Sex, age, smoking status, ECGO and stages were used as adjustment
factors during multivariate analyses. Survival differences were examined using the log-rank test. P values less than
0.05 were considered significant. All P values represent two-sided statistical tests. All statistical procedures were
conducted using the SPSS software (version 19.0) and GraphPad Prism7.
Advanced lung adenocarcinoma patients? characteristics and clinical outcomes. As shown in
Supplementary Table?1, the distribution of demographic and clinical characteristics of patients were summarized.
A total of 108 advanced lung adenocarcinoma patients were enrolled in this study. All individuals were ethnic
Han Chinese. The mean age of subjects was 56 ranging from 46 to 66. There were 53 males and 55 females. All the
patients were treated with gefitinib and 75% patients were detected with EGFR mutations. By the time of the final
analysis, the median follow-up time was 29.0 months, and the median PFS and OS was 12.4 and 24.4 months,
Effects the autophagy core gene SNPs on PFS and OS of gefitinib-treated advanced lung
adenocarcinoma patients. The detailed information of selected 26 autophagy core gene SNPs were summarized
in Supplementary Table?3. A total of 23 potentially functional SNPs from 11 autophagy core genes were
successfully genotyped. These SNPs were located in introns, exons, 3? UTR and promoters of autophagy core genes.
Associations between the 23 SNPs and PFS or OS were examined using multivariate Cox regression analyses
Impacts of autophagy core gene SNPs on gefitinib-resistance. Drug resistance to EGFR-TKIs
inevitably develops after a period of effective drug treatment. Here we investigated whether autophagy core gene
SNPs could be used as reasonable biomarkers for gefitinib-resistance in advanced lung adenocarcinoma. As
shown in Table?2, ATG5 rs510432 acts as a protective SNP significantly associated with 55% decreased risk of
primary gefitinib resistance (95% CI = 0.24-0.87, P = 0.017). ATG10 rs1864182 was significantly associated with
0.65 (0.26-1.62) 0.353
0.77 (0.29-2.05) 0.605
0.74 (0.34-1.60) 0.448
0.94 (0.45-1.99) 0.877
1.21 (0.46-3.23) 0.700
1.07 (0.54-2.10) 0.854
2.27-fold elevated risk of primary gefitinib resistance (95% CI = 1.04-4.97, P = 0.040). On the contrary, ATG10
rs1864182 might be a protective SNP for acquired gefitinib resistance (HR = 0.30, 95% CI = 0.09-0.97, P = 0.044).
Among patients with EGFR mutations, ATG5 rs510432 and rs688810 genetic variations were significantly
associated with primary gefitinib resistance (rs510432 A allele: HR = 0.39, 95% CI = 0.18-0.85, P = 0.018; rs688810
C allele: HR = 3.01, 95% CI = 1.26-7.22, P = 0.014). Additionally, ATG10 rs10036653 was significantly associated
with acquired gefitinib resistance of EGFR mutant patients (T allele: HR = 0.37, 95% CI = 0.19-0.72, P = 0.004),
which was verified in log-rank test (AT and TT vs. AA: 16 months vs. 6.5 months, P = 0.009) (Fig.?3). However,
other autophagy core gene SNPs did not significantly affect primary or acquired gefitinib-resistance (all P> 0.05)
(Supplementary Table?6 and Supplementary Table?7).
ATG5 rs510432 and ATG10 rs10036653 may influence binding of transcription factors. All
autophagy core gene SNPs investigated in the current study were selected based on their MAF in Chinese Han
population and potential function to their host genes. We found that ATG5 rs510532 and ATG10 rs10036653
contributed to not only survival but also drug resistance among gefitinib-treated advanced lung
adenocarcinoma patients. Interestingly, both SNPs are upstream gene variants (Supplementary Table?3), which leads us to
examine whether they could change transcription factor (TF) binding affinities to genomic sequences and, thus,
affect gene regulation. By using RegulomeDB, an online bioinformatics tool (http://regulome.stanford.edu/)29, we
found that ATG5 rs510432 might change the binding ability of Mtf1 to ATG5 promoter, and ATG10 rs10036653
may affect the binding affinities of Sox5 as well as Oct4 to ATG10 promoter (Table?3). We validated the
bioinformatics prediction in A549 cells. After silencing endogenous expression of OCT4, MTF1 or SOX5 with siRNAs
(siSOX5-1, siSOX5-2, siMTF1-1, siMTF1-2, siOCT4-1 and siOCT4-2), we found that only decreased expression
of OCT4 can suppress ATG10 expression (Fig.?4A). These results indicate that OCT4 might acts as important TF
impacting ATG10 expression. Because ATG10 rs10036653 SNP is located in a predicted OCT4 binding sequence,
we then conducted EMSA to distinguish the differences in binding capacity between the rs10036653T or
rs10036653A alleles. As shown in Fig.?4B, we found that OCT4-containing A549 nuclear extracts bound more to
the biotin-labeled oligonucleotide probe with the A allele sequence compared to the T allele probe. Interestingly,
although we did not find super-shift bands, we did observe attenuated OCT4 binding band with OCT4 antibody
used (Fig.?4B). These observations may explain the possible correlations between these SNPs with prognosis of
EGFR-TKIs have been proved to be promising treatment of NSCLC, especially for lung adenocarcinoma patients
harboring EGFR mutations. In addition to the EGFR mutations, we and others also found that germline
variations might be prognostic markers of gefitinib treatment22,23. In this study, we systematically evaluated 23 SNPs
from eleven autophagy core genes and treatment outcomes of advanced lung adenocarcinomas patients. Multiple
genetic variations in autophagy core genes, i.e. ATG5 rs510532 and ATG10 rs10036653, were found to be
significantly associated with clinical outcomes, especially in those with EGFR mutations. To the best of our
knowledge, our study is the first to examine clinical implications of autophagy SNPs in patients with EGFR mutant
Genetic variations of autophagy core genes are investigated in several human cancers. Qin et al. examined
14 potentially functional polymorphisms in six autophagy-related genes (ATG3, ATG5, ATG7, ATG10, ATG12
and LC3) in breast cancer susceptibility and found that ATG10 rs1864182 and rs10514231 were associated with
significantly decreased risk of breast cancer24. After genotyping 40 tagging SNPs from 7 core autophagy
pathway genes in 458 localized prostate cancer patients, Huang et al. observed the association between ATG16L1
rs78835907 and recurrence of localized disease, which was replicated in more advanced disease30. White et al.
examined five SNPs in three ATG genes (ATG5, ATG10 and ATG16L) and found that ATG SNPs might be
differentially associated with specific host and melanoma characteristics including age at diagnosis, tumor infiltrating
lymphocytes, and stage31. Berger et al. genotyped 12 SNPs in eight autophagy-related genes among patients with
mCRC treated with first-line FOLFIRI and bevacizumab in two phase III randomized trials and found that the
FIP200 rs1129660 variant showed significant associations with hypertension32. In head and neck squamous cell
carcinoma, Fern?ndez-Mateos et al. observed the associations between ATG10 rs1864183 and a higher
susceptibility to develop laryngeal cancer, ATG2B rs3759601 and pharyngeal cancer as well as ATG16L1 rs2241880 and
oral carcinoma33. However, it is still unclear if genetic variations of autophagy core genes would impact prognosis
of advanced lung adenocarcinomas patients.
Accumulated evidences demonstrated that autophagy plays an essential role in escaping from the
anti-neoplastic effects of drugs34?36. In NSCLC cells, gefitinib treatment can induce elevated ATG5 expression
and increased autophagy34. Cytotoxicity induced by gefitinib was greatly enhanced after autophagy inhibition by
ATG5 silencing34, which suggests that ATG5-regulated autophagy inhibition represents a promising approach to
improve the efficacy of EGFR-TKIs. Similarly, Sakuma et al. found that depletion of ATG5, an autophagy
inhibitor, markedly reduces gefitinib-resistant cell viability of EGFR-mutated lung adenocarcinoma cells under hypoxic
conditions36. These results elucidated that ATG5 might be a crucial gene impacting clinical outcomes of gefitinib
treatments. As a result, it is biologically plausible that the potential functional ATG5 rs510532 genetic variant may
also be a prognostic marker for gefitinib therapy.
ATG10 is an E2-like enzyme involved in E2 ubiquitin-like modifications essential for autophagosome
formation. Jo et al. found that ATG10 was increased in colorectal cancer and associated with lymphovascular invasion
and lymph node metastasis37. Qin et al. demonstrated that potentially functional polymorphisms in ATG10 were
associated with risk of breast cancer in a Chinese population24. These results indicated that ATG10 and its genetic
polymorphisms might be an important component during carcinogenesis. In line with this, we observed
significant association between the ATG10 rs1864182 SNP with prolonged survival and gefitinib-resistance of EGFR
mutant NSCLC patients.
In summary, ATG5 rs510532 and ATG10 rs10036653 genetic variations in autophagy core genes are
significantly associated with clinical outcomes of advanced lung adenocarcinoma treated with gefitinib. Genotyping
of these genetic variations with detection of EGFR mutations may improve the prediction of the treatment
outcomes. Our study also highlights the possibility of patient-tailored decisions especially during EGFR-TKIs based
on combination of germline and somatic variation detection.
This study was partially supported by National Natural Science Foundation of China (31671300, 81572934);
Taishan Scholars Program of Shandong Province (tsqn20161060); the National High-Tech Research and
Development Program of China (2015AA020950); the Science and Technology Planning Project of Huaian
(HAS2015013); Foundation of Shandong Academy of Medical Sciences (2015-44); Medicine and Health Science
Technology Foundation (2015WSA18054).
M.Y. and J.Y. conceived and designed the experiments; J.Y., N.Z. and L.Y. performed the experiments; J.Y. and
N.Z. analyzed the data; H.Z., L.Zhang. and L.Zhou. contributed materials/analysis tools; M.Y., J.Y. and N.Z. wrote
the manuscript. All authors reviewed and approved the manuscript prior to submission.
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-18165-5.
Competing Interests: The authors declare that they have no competing interests.
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