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Year : 2019  |  Volume : 15  |  Issue : 4  |  Page : 876-881

Multiple MicroRNAs synergistically promote tolerance to epidermal growth factor receptor-targeted drugs in smoked lung cancer therapies

1 Department of General Surgery, CNOOC General Hospital, Tianjin, China
2 Laboratory of Cancer Cell Biology, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China

Date of Web Publication14-Aug-2019

Correspondence Address:
Guoguang Ying
Laboratory of Cancer Cell Biology, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin
Li Pan
Department of General Surgery, CNOOC General Hospital, Tianjin
Yongzi Chen
Department of General Surgery, CNOOC General Hospital, Tianjin
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_208_18

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 > Abstract 

Aims: Lung cancer is one of the leading causes of cancer-related mortality. Tobacco usage is considered as associated with the carcinogenesis, progression, and prognosis of lung cancer. Previous studies have demonstrated that the smoking inhibited medical therapy results from K-Ras gene mutation through suppressing the epidermal growth factor receptor (EGFR) pathway. However, recent clinical trials have revealed that few smoked lung cancer patients present K-Ras gene mutation; yet, the majority of smoked lung cancer patients remain K-Ras nonmutation. The chemo-resistant mechanism remains unclear. Recently, microRNA (miRNA) interaction has been found to play an important role in drug resistance process. We hypothesized that miRNA may exert medicine resistance during the processes of lung cancer therapy.
Methods: Here, we analyzed miRNA array data from the GEO database.
Results: Our results showed that the interaction network among hsa-miR-30d-3p, hsa-miR-184, hsa-miR-500a, hsa-miR-542-3p, among others, inhibited EGFR-targeted medicine therapy.
Conclusion: The research will provide evidence that promotes novel therapy of lung cancers.

Keywords: Epidermal growth factor receptor, lung cancer, microRNA, smoking

How to cite this article:
Pan L, Wang H, Jiang C, Li W, Chen Y, Ying G. Multiple MicroRNAs synergistically promote tolerance to epidermal growth factor receptor-targeted drugs in smoked lung cancer therapies. J Can Res Ther 2019;15:876-81

How to cite this URL:
Pan L, Wang H, Jiang C, Li W, Chen Y, Ying G. Multiple MicroRNAs synergistically promote tolerance to epidermal growth factor receptor-targeted drugs in smoked lung cancer therapies. J Can Res Ther [serial online] 2019 [cited 2021 May 8];15:876-81. Available from: https://www.cancerjournal.net/text.asp?2019/15/4/876/264280

Authors Li Pan and Hailong Wang contributed equally to this work

 > Introduction Top

Lung cancer is the leading cause of cancer mortality worldwide, and non-small cell lung cancer (NSCLC) represents the major histological subtype of lung cancer.[1] Almost two-thirds of patients with NSCLC have an oncogenic driver mutation, approximately half of whom have therapeutically targetable damage, which amplifies treatment options and leads to improvements in safety and survival compared with conventional chemotherapy. Various strategies involving small-molecule inhibitors have also been developed to target epidermal growth factor receptor (EGFR) mutation, including EGFR-tyrosine kinase inhibitors (TKIs) gefitinib,[2] erlotinib,[3] or afatinib.[4] However, compared with the nonsmoked NSCLC, the majority of lung cancer patients arising from tobacco usage would have preferred the EGFR nonmutation, leading to targeted EGFR medicine resistance. Determining whether targeted drugs for the EGFR mutation can be used for the EGFR nonmutation in NSCLC has become a major research interest during lung cancer treatment.

Previous studies have shown approximately 10%–20% of patients with a partial response to gefitinib do not have identifiable EGFR mutations, indicating that EGFR mutations are not the only determinants of the TKI response.[5] There are other mechanisms to regulate drug sensitivity of TKIs. The EGFR receptor downstream proteins may play important roles in regulating the TKI response. There was an observation that gefitinib and erlotinib responses in sensitive cells result in the down-regulation of extracellular signal-regulated kinases (ERK), Akt, STAT3, and STAT5, whereas a similar down-regulation is not evident in insensitive or resistant cells.[6] At the same time, preclinical studies in an NSCLC cell line xenograft model have suggested that a PI3K inhibitor, PX-866, sensitizes otherwise insensitive tumors to gefitinib 149.[7] Efforts are currently underway to develop PI3K inhibitors with greater specificity. The serine-threonine kinase mammalian target of rapamycin (mTOR) lies downstream of PI3K and is inhibited by rapamycin and rapamycin analogs.[8] Preclinical studies suggest that mTOR inhibitors might also have synergistic effects when combined with targeted EGFR inhibitors.

MicroRNA (miRNA) is a small noncoding RNA molecule (containing approximately 22 nucleotides) found in animals, which functions in RNA silencing and posttranscriptional regulation of gene expression.[9] Substantial studies have revealed that miRNA is closely related to the sensitivity of tumor cells to chemotherapeutic drugs, including EGFR receptor downstream proteins. MiR-196a can be targeted to Phosphatase and tensin homologue deleted on chromosome ten (PTEN), resulting in low expression of PTEN protein and excessive activation of the Akt pathway, thereby enhancing cell proliferation and resulting in resistance to cisplatin in ovarian cancer patients.[10] miR-143 increases drug resistance to prostate cancer by suppressing the expression of the KRAS target protein. Conversely, overexpression of miR-143 also participates in the regulation of the EGFR/RAS/MAPK pathway and improves sensitivity of prostate cancer cells to docetaxel.[11] The increase of miR-146b-3p expression can enhance the insensitivity of HCT-116 colon cancer cells to cetuximab.[12] Hence, whether miRNA can regulate the smoke-related NSCLC EGFR-targeted medicine resistance needs to be elucidated.

Here, we analyzed the miRNA array data by the GEO database, and the results showed that the interaction network among hsa-miR-30d-3p, hsa-miR-184, hsa-miR-500a, hsa-miR-542-3p, and others promoted increases in EGFR receptor downstream proteins levels that inhibit EGFR-targeted medicine therapy. Therefore, the research will provide evidence that promotes therapy of lung cancers.

 > Materials and Methods Top

Target predictions of lung cancer-related microRNAs

A web-based software Targetscan (http://www.targetscan.org) was used to generate lists of possible gene targets of each miRNA. Subsequently, the targeted genes were input into another web server miRpath v. 3 (http://snf-515788.vm.okeanos.grnet.gr), which is designed for gene cluster function, and we gained the protein class from the PANTHER analysis. After which, we clustered the top ten classes of proteins with the same functions.

Signaling pathway mapping of lung cancer-related miRNAs

The signaling pathways and processes were explored using the systems biology tool KEGG Mapper (http://www.genome.jp/kegg/tool/map_pathway2.html), which is a collection of tools for KEGG mapping: KEGG pathway mapping, BRITE mapping, and MODULE mapping. The KEGG database consists of 16 main databases (systems information, KEGG PATHWAY, KEGG BRITE, KEGG MODULE, KEGG DISEASE, KEGG DRUG, and KEGG ENVIRON; genomic information, KEGG ORTHOLOGY, KEGG GENOME, KEGG GENES, KEGG SSDB, and KEGG; chemical information, KEGG COMPOUND, KEGG GLYCAN, KEGG REACTION, KEGG RPAIR, KEGG RCLASS, and KEGG ENZYME).

 > Results Top

Tobacco promotes miRNA downregulation in lung cancer patients

Aiming to study, the effect of miRNA on targeted therapy following smoking, we analyzed three groups of microarray data in the GEO database (GSE56264). Compared between the smoking group and nonsmoking group, we analyzed miRNAs expression levels following smoking. The results showed that the nonmutation group has few differential miRNAs, and several miRNAs were downregulated. At the same time, we found miRNAs of the KRas and EGFR gene mutations were mainly downregulated after smoking. Because the function of miRNAs is to reduce expression of the target protein by inhibiting translation of the target gene, the data showed that in smoked lung cancer patients, miRNAs promoted increases in target protein levels [Figure 1].
Figure 1: The miRNAs changed in the smoked lung cancer patients. (a) The common and specific distribution of the changed miRNAs in the three groups of samples; (b) upregulation and downregulation miRNAs distribution in each group

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Functional classification of miRNAs target genes

By excluding the effect of KRas and EGFR gene mutations on the miRNAs expression, we found ten miRNAs, including seven downregulated miRNAs and three upregulated miRNAs. Using the miRpath v3 database, we analyzed the miRNAs target gene function. The results showed that the three upregulated miRNAs target genes regulated mainly extracellular matrix (ECM)-receptor interaction and focal adhesion. These target genes would affect cancer cell adhesion. The seven downregulated miRNAs target genes functioned in adhesion, RNA metabolism, protein complex formation, and cell cycle regulation. Therefore, the downregulated miRNAs had diverse functions in lung cancer cells following smoking. It also showed that the smoking-induced miRNAs target genes had many functions in lung cancers, which may be the cause of the downregulated miRNAs [Figure 2].
Figure 2: Smoking caused up and down miRNA. (a) Downregulation of miRNA corresponding to the target gene go classification; (b) upregulation of miRNA corresponding target gene of go classification because the cell function is regulated

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Pathway classification of miRNAs target genes

According to the KEGG pathway analysis, we found three upregulated miRNAs target genes that mainly regulated adhesion pathways, including ECM-receptor interaction and focal adhesion. The seven downregulated miRNAs target genes had a relationship to the PI3K-Akt signaling pathway, cell cycle, small cell lung cancer, and focal adhesion. These down-regulation miRNAs target genes affected many different functions, leading to EGFR-targeted medicine resistance. The results showed that five miRNAs affected small cell lung cancer proteins and the PI3K-Akt signaling pathway [Table 1]. These results explained that decreased miRNA expression promoted targeted medicine resistance by regulating target gene translation and promoting increased target protein expression.
Table 1: The targeted gene number of miRNAs

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We analyzed the small cell lung cancer pathway and found many target genes, including anti-apoptosis, cell cycle, and DNA repair in small cell lung cancer [Figure 3]. We found that the expression of anti-apoptosis genes, including XIAP, TRAF4, and APAF1, and cell cycle promotion genes, including MYC, SKP2, CD6, and E2F3.
Figure 3: miRNA target genes small cell lung cancer KEGG pathway. Yellow color represents a target gene regulated by miRNA, and brown yellow represents two miRNA regulated target genes

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miRNAs regulate epidermal growth factor receptor downstream proteins expression

According to the pathway analysis, we found enrichment of 52 downregulated miRNAs target genes. These miRNAs target genes participated in the PI3K-Akt signaling pathway, and the pathway had significant statistical significance (P < 0.05). We found that the down-regulated hsa-miR-184 increased expression of the key regulatory protein Akt2, promoting pathway activation and further increasing cell survival. The hsa-miR-30d-3p affected the Toll-like receptor signaling pathway by increasing PDGFC protein levels and promoting PI3K phosphorylation. The hsa-miR-542-3p and hsa-miR-500a regulated FGFR1 and GSK3b, increasing the activation of the PI3K-Akt signaling pathway and promoting cell survival [Figure 4]. These results suggested that down-regulation of miRNAs-enhanced drug resistance in lung cancer patients.
Figure 4: miRNA target genes PI3K-AKT KEGG pathway. Yellow color represents a target gene regulated by miRNA, and brown yellow represents two miRNA-regulated target genes

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miRNAs affect lung cancer patient survival following smoking

Aiming to analyze the relationship between smoking-induced miRNAs target genes and patient prognosis, we study the survival of lung cancer patients using the Kaplan–Meier Plotter database. We found that there are three survival pathways: target genes promoting survival, target genes inhibiting survival, and no relation. The first pathway included FGFR1; the second included AKT2, and the last included PDGFC [Figure 5].
Figure 5: The miRNAs target genes regulated the lung cancer survival. (a) FDFR1; (b) Akt2; (c) PDGFC

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 > Discussion Top

miRNAs exert functions in lung cance;[13],[14],[15] therefore, miRNAs have been developed as a molecular diagnostic tool for lung cancer.[15] We analyzed miRNA array data and found that smoking mainly decreased miRNA expression in NSCLC patients. In a rat injury model, miRNA expression was downregulated in the lungs of rats exposed to cigarette smoke.[16],[17] It indicates that the effects of smoking in lung cancer patients are due to decreased miRNAs expression levels, promoting an increase of its corresponding target proteins. We analyzed the function of down-regulated miRNAs target proteins by the GO database and found that there were many changes in cell functions, including RNA metabolism, cell adhesion, cell cycle, and other biological functions. Subsequently, we analyzed the miRNA corresponding target proteins signal pathways by KEGG. We found that the main signal pathway involved in down-regulation of miRNA was more than that of miRNA. The down-regulation of miRNA target proteins, such as hsa-miR-30d-3p, hsa-miR-184, hsa-miR-500a, hsa-miR-542-3p, among others, affected the small cell lung cancer pathway, and the PI3K-Akt pathway. The reduction amount may improve the therapeutic effect of early treatment of junior high school PI3k-Akt reported the expression of related proteins, prolonging the survival time of patients. Therefore, we infer that the increased protein expression in this pathway has an inhibitory function on the targeting therapy of lung cancer.

PI3K-Akt pathway has an important regulatory function in NSCLC. There are many reports on miRNAs that target EGFR, for example, miR-134, miR-145, miR-146a, and others.[18],[19],[20],[21],[22] Currently, there are mechanisms of acquired resistance to EGFR-tyrosine TKIs, and updated therapy strategies have been explained in human NSCLC.[23] The miR-134/487b/655 cluster contributed to the TGF-β1-induced epithelial-mesenchymal transition (EMT) phenomenon and affected the resistance to gefitinib by targeting directly MAGI2, in which suppression subsequently caused loss of PTEN stability in lung cancer cells. The miR-134/miR-487b/miR-655 cluster may be a new therapeutic target in patients with lung adenocarcinoma, depending on the EMT phenomenon.[24] miR-145-3p may inhibit cell growth, motility, and chemotaxis in NSCLC by targeting PDK1 through suppression of the mTOR pathway.[25] Overexpression of miR-143 decreased cell proliferation, promoted apoptosis, and suppressed phosphorylation of AKT, EGFR, and ERK1/2; thus, miR-143 may play an important role in NSCLC treatment to enhance therapeutic efficacy. miR-146a and miR-149-5p increased inhibition of cell proliferation by drugs targeting EGFR, including both TKIs (erlotinib, gefitinib, and afatinib) and a monoclonal antibody (cetuximab).[26],[27] miR-7 may have potential clinical effects in the reversal of drug resistance. From miRNA in-depth analysis of PI3K-Akt pathway regulation, we found that the expression of hsa-miR-30d-3p, hsa-miR-184, hsa-miR-500a, hsa-miR-542-3p, and others was downregulated. In addition, we found in the previous study, which can be used as NSCLC biomarkers, that these were decreased in NSCLC compared with normal lung tissue expression. It shows that it has important influences on PDGFC, JAK1, and PTEN in the regulation of PI3K-Akt. It may have a possible inhibitory effect on targeted drugs. This study has a certain effect on targeted therapy of lung cancer.

 > Conclusion Top

The miRNAs data of the GEO database was used to analysis the interaction network. The results (from this research) indicated that Multiple MicroRNAs synergistically promote tolerance to epidermal growth factor receptortargeted drugs in smoked lung cancer therapies including hsamiR30d3p, hsamiR184, hsamiR500a, hsamiR5423p et al. Therefore, the research will provide evidence that promotes therapy of lung cancers.

Financial support and sponsorship

The article was supported by the Scientific Research Fund Project of Binhai New Area, Health Bureau, Tianjin (No. 2011 BHKL004).

Conflicts of interest

There are no conflicts of interest.

 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1]


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