High-throughput RNAi screen identifies ILK as a target to overcome intrinsic EGFR-TKI resistance of lung squamous carcinoma

Although targeted therapy has made a significant efficacy in lung adenocarcinoma, there are still no effective targeted drugs for lung squamous cell carcinoma (SqCC). Moreover, immunotherapy only prolonged the OS of lung SqCC for less than 5 months, so till now, chemotherapy and radiotherapy are still the main treatments of advanced SqCC. Lung SqCC patients express higher epithelial growth factor receptor (EGFR) than adenocarcinoma patients, but they are intrinsic resistant to EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Therefore, if this resistance can been reversed, it would benefit to most of the SqCCs. Herein a high-throughput RNAi technology was used to screen the genes related with EGFR-TKI erlotinib resistance of lung SqCCs, and then Integrin-linked kinase (ILK) was found to be the most effective gene. We also studied the effects of ILK on erlotinib resistance in cell line and ILK expression in SqCC and adenocarcinoma patients. At last we analyzed the mechanism of ILK in EGFR-TKIs resistance using Pathway Analysis, GO Analysis and Ingenuity Pathway Analysis (IPA). ILK knockout could overcome erlotinib resistance via inhibited cell proliferation, induced apoptosis and blocked the cells at G2/M phase. ILK was expressed significantly higher in SqCC patients than in adenocarcinoma patients with sensitizing EGFR mutations. Moreover, the cell cycle pathway of G2/M DNA Damage and checkpoint regulation was identified significantly inhibited by ILK knockout in IPA, Pathways and GO analysis. Our results may provide further understanding of EGFR-TKIs resistance in lung SqCCs and thus aiding the development of potential targeted therapies for lung SqCCs.

(EGFR) than adenocarcinoma patients, but they are intrinsic resistant to EGFRtyrosine kinase inhibitors (EGFR-TKIs). Therefore, if this resistance can been reversed, it would benefit to most of the SqCCs. Herein a high-throughput RNAi technology was used to screen the genes related with EGFR-TKI erlotinib resistance of lung SqCCs, and then Integrin-linked kinase (ILK) was found to be the most effective gene. We also studied the effects of ILK on erlotinib resistance in cell line and ILK expression in SqCC and adenocarcinoma patients. At last we analyzed the mechanism of ILK in EGFR-TKIs resistance using Pathway Analysis, GO   Lung SqCC accounts for about 30% of non-small cell lung cancer (NSCLC) [1]. In the past years, therapeutic progress in lung SqCC has lagged far behind lung adenocarcinoma. Although great efforts to investigate the complex biology underlying SqCC have begun to bear fruits in a multitude of ways, there is still no effective agent. In the era of immunotherapy, Pembrolizumab was listed as the firstline treatment for NSCLC patients with PD-L1 > 50% by 2019 NCCN guideline, but the median Overall Survival (OS) is only 20 months treated with Pembrolizumab alone [2], which is very limited compared with the targeted therapy in adenocarcinomas as we known [3]. In the most important clinical trials of SqCC CheckMate-017, the median OS of Nivolumab for the second-line treatment of advanced lung SqCC was prolonged by only 3 months compared with traditional second-line chemotherapy docetaxel [4]. Pembrolizumab 10 mg/kg group and 2 mg/kg group in KEYNOTE-010 also achieved the OS benefit of 4 months and 2 months compared with docetaxel [5]. So till now, chemotherapy and radiotherapy are still the main treatments of advanced lung SqCC. The response rate was less than 30% in sensitizing EGFR-mutated lung SqCC patients, and the median progression-free survival (PFS) was only two to three months [8][9][10], which were dramatically inferior to in sensitizing EGFR-mutated adenocarcinoma patients. Therefore, lung SqCC patients are intrinsic resistance to EGFR-TKIs, which has become an urgent problem to be solved at present.
Recently, application of next generation sequencing (NGS) to investigate the genomic characterization of lung SqCC led to a further understanding of the possible targets. Filipits M et al. found that the available targeted therapies in lung SqCC include EGFR, FGFR1, MET, PI3K, DDR2, BRAF, AKT, CTLA4, and PD1 [11].
High-throughput RNAi technology is the combination of high-throughput chip and siRNAs library that makes RNA interference no longer limited to one gene silence, but simultaneously screening of large-scale genes and their functions. In this study, high-throughput RNAi technology was used to screen the most effective gene Cell proliferation analysis 6 The cells (3×10 3 /well) were seeded into 96-well plates in triplicate and were exposed to various concentrations of erlotinib. After 72 hours, 20μl of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5mg/ml) was added to each well and incubated. After 4 hours, crystalline formation was dissolved with Dimethyl sulfoxide (DMSO) and the absorbance at 530nm was read using the microplate-reader for ELISA MK-2 Labsystems Dragon. Percent survival was calculated as: (mean absorbance of the replicate wells containing drugs -mean absorbance of the replicate background wells) / (mean absorbance of the replicate drug-free wells -mean absorbance of the replicate background wells). The test was In TUNEL assay, cells were seeded in 24-well plates and exposed to 2µmol/L erlotinib for another 72 h. Apoptosis was assessed by the TUNEL assay kit (GENMED, China) following the manufacturer's protocol. Apoptotic index (AI) (%) was calculated by the formula: positive staining cells / tumor cells number × 100%.

Western blotting
Cells were washed twice with ice-cold PBS and lysed in 0.1ml of lysis buffer on ice for 30 min. Insoluble debris was removed by centrifuging at 13,000 rpm for 15 min at 4°C. Electrophoresis and blotting procedures were done according to methods described previously [15][16][17]. Intrinsic antibody against human ILK (R D, USA) was used according to the manufacturer's instructions. Blotting quantification was done with an Odyssey® Infrared Imaging system (LI-COR, USA).
Quantitative polymerase chain reaction (qPCR) 1μg cDNA was used for qPCR analysis using Lightcylcer (Roche, Switzerland) following the manufacturer's instructions. GAPDH was used as the internal control.
Amplifications were carried out in the 20µl reaction mixtures in the following conditions 95℃ for 2min and followed by 40 cycles of 95℃ for 20 s, 55℃ for 20 s and 72℃ for 35s; and then 72℃ for 3min. The copy numbers of ILK gene were http://www.ingenuity.com). The IPA software is based on computational algorithms that analyze the functional connectivity of the genes from information obtained within the IPA database. The "core analysis" function included in the software was used to interpret the differentially expressed data, which included biological processes, canonical pathways, upstream transcriptional regulators, and gene networks. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathway Knowledge Base (IPKB).

Statistical analyses
Values were expressed as mean ± SD. Statistical analyses were done by independent-samples t test. Differences were considered to be statistically significant if P < 0.05.

High-throughput RNAi screening identified that ILK knockout improved the effect of erlotinib in lung SqCC
The MTT results identified that the IC50 of SK-MES-1 cell line was 11.35umol/L and this cell line expressed EGFR ( Figure 1A, B). SK-MES-1 cells were treated with a gradient concentrations of erlotinib after transfected with lentiviral NC shRNA (Nontarget control). The cells began to slow down its proliferation significantly after administered with 2μm erlotinib, so 2μm was determined to be the screening concentration in next studies ( Figure 1C).
To confirm the role of ILK in EGFR-TKI resistance, the cell survival rates of SK-MES-1 cells transfected with negative control and the two target shRNAs were measured after 72h incubation with erlotinib of different concentrations (2μm, 4μm, 10μm).
Survival rate = (shRNA group + drug) / shRNA group. It can be seen that PSC1369 significantly decreased the cell survival rate of SK-MES-1 cells, suggesting that ILK knockout can improve the effect of erlotinib ( Figure 1E). The results of qPCR confirmed that ILK was expressed in lung cancer cell lines, including SqCC cell line SK-MES-1 and H226 ( Figure 1F).

The effects of ILK on erlotinib resistance in lung SqCC cells
The results of qPCR and western blotting confirmed that the expression of ILK was significantly inhibited in SK-MES-1 cells after RNA interference, P<0.05 (Figure 2A Figure 2C, D, E). The result of apoptosis was verified by TUNEL, in which the apoptosis rate was 83.24% in cells treated with erlotinib after ILK knockout, much more than that treated with erlotinib alone ( Figure 2F). We also found that the ability of cell clone formation decreased significantly in the group treated with erlotinib after ILK knockout, compared with the group treated with erlotinib alone, P<0.05 ( Figure 2G).

The expression of ILK in lung SqCC patients
We detected ILK expression in tumors of patients with lung squamous cell carcinoma, adenocarcinoma with wild-type EGFR and sensitizing mutated EGFR using qPCR. There were 31 patients with SqCC, 9 patients with wild-type EGFR adenocarcinoma and 10 patients with sensitizing mutated-EGFR adenocarcinoma.
The results showed that the expression of ILK in patients with sensitizing mutated EGFR adenocarcinoma was significantly lower than that in patients with wild-type with NC group, based on the threshold of fold change > 2 and p<0.05, passed the filtering process and were selected for the cluster analysis. (Figure 4A).
Moreover, functional analysis of the gene expression profiling using Pathways Analysis according to KEGG and BioCarta Database revealed that the top three enriched targets by ILK knockout were cell cycle, ECM-receptor interaction and mitosis, as shown in Figure 4B and Table S1.
The DEGs were also subjected to GO analysis. From the aspect of molecule function, the DEGs influenced kinase activity and DNA binding, which associated with the action mechanism of EGFR-TKIs. From the aspect of the cellular component, changes occurred in intracellular non-membrane bound organelle, spindle and cytoplasm.
These parts are closely correlated with cell proliferation and intracellular signal transduction, which are the important influence factors of EGFR-TKIs efficacy. From the aspect of biological process, the changes involved cell cycle, mitosis, response to stress and cell proliferation, suggesting that ILK knockout may overcome EGFR-TKIs resistance via these cell process (Table S2). Organization, DNA Replication, Recombination, and Repair ( Figure 4D).

Discussion
In this work, it was found that knockout of ILK could improve erlotinib efficacy. And after that we investigated the effect of ILK knockout on the cell biological activities of the SqCC cell line SK-MES-1. As a result, cell proliferation and cell clone formation was inhibited, cell apoptosis was up-regulated, and cells was arrested in G1 and G2/M phase when ILK was inhibited. Furthermore, we found that ILK was expressed significantly higher in the tumors of lung SqCC patients than adenocarcinoma patients with sensitizing EGFR mutations.
ILK, an important serine/threonine protein phosphatase, plays a key role in regulation of signal transduction and remodeling of the tumor extracellular matrix (ECM) [18]. High expression of ILK was closely related with the occurrence and development of lung cancer, and also correlated with anti-cancer drugs resistance.
A study conducted by Chen D et al. showed that ILK could promote the process of epithelial-to-mesenchymal transition (EMT) in lung cancer, and knockdown of ILK could significantly inhibit the invasion and metastasis of lung cancer [19,20]. In Yu's study, ILK was positively expressed in 39 (68.42%) SqCC patients and associated with high microvessel density (MVD) in lung squamous cell carcinoma (P < 0.001) [21]. Posch F et al. reported that increased serum ILK (sILK) was associated with adverse survival (p < 0.001) and the Kaplan-Meier survival analysis showed that ILK, E-cadherin, and MVD were all statistically significant prognostic factors in patients with lung SqCC [22]. Jia Z verified that ILK was involved in drug (gemcitabine) resistant of lung cancer, and this function might be mediated by EMT process and MRP1 pathway [23]. Augustin A et al. indicated that the ILK-PINCH-Parvin (IPP) complex has been shown to be involved in EMT and erlotinib sensitivity.
A retrospective analysis of the MERIT trial indicated that a low level of ILK could be linked to clinical benefit with erlotinib [24], so it was suggested that ILK may be a target of erlotinib resistance. Moreover, our results also confirmed that the expression of ILK was remarkably higher in lung SqCC patients than in sensitizing EGFR-mutated adenocarcinoma patients, suggesting that ILK may be a key marker of EGFR-TKIs resistance in lung SqCC.
In the present results, in order to gain further insights into the mechanism of ILK in EGFR-TKIs resistance of lung SqCC, Pathways Analysis, GO analysis and IPA were used to explore the connected signaling pathways and genes network. Then cell cycle, mitosis, ECM-receptor interaction and kinase activity, DNA binding were found closely correlated with the effect of ILK in EGFR-TKIs resistance. As they have been known, the cell cycle [25,26], ECM-receptor interaction [27,28], mitosis [29], were identified typically activated in cancer drug resistance, suggesting that ILK played a crucial role in EGFR-TKIs resistance of lung SqCC via these pathways. GO analysis also found that cell cycle and mitosis were the important signaling pathways involved in ILK knockout in EGFR-TKIs resistance. IPA further confirmed that G2/M DNA Damage and checkpoint regulation was the most key signaling pathway regulated by ILK knockout in EGFR-TKI resistance of lung SqCC.
Overall, ILK may represent the starting point of a new natural history of lung SqCC and may be a breakthrough in the therapy for lung SqCC that we will see over the next months to years.

Conclusions
In conclusion, high-throughput RNAi screen identified that knockout of ILK can overcome erlotinib resistance in lung SqCC cells via inhibited cell proliferation, induced apoptosis and blocked the cells at G2/M phase. Moreover, ILK was found to be expressed significantly higher in lung SqCC patients than in adenocarcinoma patients with sensitizing EGFR mutations. The top enriched targeted signaling pathway by ILK knockout was cell cycle. Our results may provide further understanding of EGFR-TKIs resistance in lung SqCCs and thus aiding the development of potential targeted therapies for lung SqCCs.

Availability of data and material
The datasets analyzed during the current study are not publicly available due to patient privacy.

Competing interests
The authors declare that they have no conflict of interest.    Figure 1 High-throughput RNAi identified that ILK knockout improved the effect of erlotinib in lung Sq 24 Figure 2 The effects of ILK on erlotinib resistance in lung SqCC cells. A and B: The results of qPCR and 25 Figure 3 The expression of ILK in lung SqCC patients was significantly higher than that in patients wit 26 Figure 4 Genome-wide transcriptional analysis of the key pathways of ILK in EGFR-TKI resistance of SK 27 Supplementary Files This is a list of supplementary files associated with the primary manuscript. Click to download. Table S1.doc Table S2.docx