#Equal Contribution: Hangxing Wang, Jingyun Fang and Yujiao Wang contributed to this work equally and should be regarded as co-first authors
Lung cancer is the most common malignant tumor with the highest morbidity and mortality in the world, and non-small cell lung cancer (NSCLC) accounts for the vast majority of cases. At present, its main treatment methods are still traditional surgery, radiotherapy and chemotherapy, with disadvantages such as a high recurrence rate and limited effectiveness. Therefore, a new, better treatment method is urgently needed. Gene editing technology, as a new genetic engineering approach, has shown great potential in gene research, gene therapy and genetic improvement. It has also emerged as a promising treatment for lung cancer. This paper reviews the current research and applications of gene editing technology in NSCLC and other aspects of NSCLC, the therapeutic principle and mechanism of action, and the existing problems and prospects. This review aims to provide a basis for the prevention and treatment of NSCLC in the future and improve the survival rate of lung cancer patients.
Lung cancer is one of the most common malignant tumors and accounts for 20% of all cancer-related deaths globally [
Although new treatments such as targeted therapy, immunotherapy, and gene therapy are being researched, the current treatment of non-small cell lung cancer mainly relies on traditional surgery, radiotherapy and chemotherapy. However, different treatment regimens are urgently needed because traditional treatment methods cannot completely overcome the side effects of metastasis, recurrence and resistance of cancer cells.
Gene editing, as a newer and more accurate genetic engineering technology that can modify specific target genes in the genome of organisms, has shown great potential in gene research, gene therapy and genetic improvement because of its high efficiency of site-specific editing. Currently, it is a promising method for tumor therapy. This article reviews the principles and mechanisms of gene editing technology in the treatment of NSCLC, other applications related to NSCLC, and the existing problems and prospects to provide new ideas and references for the treatment and prevention of lung cancer.
Gene editing involves the removal of a nucleotide sequence or even a single nucleotide from a target gene in a cell’s genome and the addition or insertion of an exogenous DNA sequence [
HR, the earliest gene editing technique, involves the exchange of genetic information (recombination) between two similar (homologous) strands of DNA. By producing and isolating DNA fragments with genome sequences similar to those of the genome to be edited, followed by injecting these fragments into monocytes or by making the cells absorb them with special chemicals, these fragments, once inside the cell, can be recombined with the cell’s DNA to replace the target part of the genome. However, due to its low frequency of recombination, high error rate, time consumption, need for extensive labor [
ZFN is an artificially modified nuclease obtained by fusion of a zinc finger DNA binding domain and a DNA cutting domain from a nuclease [
TALEN is a restriction enzyme that has been genetically engineered to cut precise DNA sequences. The actual process is to fuse a TAL effector DNA binding domain with a DNA cleavage domain from a nuclease. Terence can be designed to bind to any desired DNA sequence, so the DNA can be cut at a specific location. Talen has unique advantages over traditional ZFN technologies, namely, a simpler design and higher specificity. The main disadvantages include a certain cytotoxicity and cumbersome module assembly processes.
The CRISPR/Cas system was first discovered in bacteria and archaea, where it functions as a form of adaptive immunity against viruses [
Although gene modification-mediated gene therapy still faces many problems, such as off-target effects, the development of this technology has undoubtedly brought hope for the treatment of many diseases. In recent years, researchers have used gene editing for molecular targeted intervention therapy of tumors [
At present, gene editing technology has been implemented in the clinical treatment of NSCLC. Depending on the different types of lung cancer, different editing strategies have been adopted. Currently, its participation in the treatment of NSCLC mainly includes the following three methods: gene knockout, special targets in the regulatory signaling pathways and combined effects with other therapeutic methods. Currently, gene editing technology can be used to knock out the PD-1 gene of T cells to enhance their ability to attack tumor cells or by inhibiting a specific target in a signaling pathway related to the occurrence of NSCLC to achieve a therapeutic effect against NSCLC. However, gene editing technology is currently most often used in clinical practice in combination with chemotherapy, medicinal plant therapy or other therapeutic methods to achieve therapeutic effects.
To date, in a clinical trial conducted by Sichuan University, CRISPR/Cas9 technology has been shown to be an effective intervention for lung cancer [
Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis and intracellular transcription. At present, studies have found that CRISPR/Cas9-mediated MMP3 gene knockout has an obvious antitumor effect, inhibiting the migration and invasion of tumor cells and reducing the activity of the CCN2/CTGF promoter and causing
Studies have shown that MYC gene knockout using CRISPR/Cas9 gene editing can block the silencing or overexpression of long noncoding RNA EPIC1 (LNC-EPIC1) to induce the death of human lung cancer cells [
Gene/RNA | Function | The impact of knockout/silencing | |
---|---|---|---|
PD-1 [ |
Combination with PD-L1 promotes programmed T-cell death | Increase the lifespan of T cells | |
MMP3 [ |
Protein hydrolysis and transcription play a variety of roles; high expression is not conducive to a good prognosis of lung cancer | Antitumor activity | |
LNC-EPIC 1(RNA) [ |
High expression in lung cancer cells | Induction of G1-S cell cycle arrest and apoptosis | |
MYC [ |
It interacts with LNC-EOIC 1 to induce human lung cancer progression | Promote LNC-EOIC 1 silencing |
In addition, gene editing technology can be employed to regulate specific targets in the corresponding signaling pathways to participate in the treatment of lung cancer. In the PTEN/Akt signaling pathway, the expression of miR-92a inhibited the expression of PTEN, which regulated the activity of the PI3K/Akt pathway. In the WNT/β-catenin (CTNNB1) pathway, MUC1-C silencing was achieved by genome editing to inhibit MYC and its target genes. In addition, in the GRM8/MAPK signaling pathway, suppressing the transmission of TGF-β signaling by gene editing can inhibit the occurrence and spread of non-small cell lung cancer (NSCLC) and reduce the resistance of NSCLC to targeted drugs and anticancer drugs so they can play a better role in the treatment progress.
PTEN, as a member of the protein tyrosine phosphatases (PTP) gene family, is an important antioxidant that negatively regulates the activity of the PI3K/Akt signaling pathway, thus affecting the proliferation, apoptosis, autophagy and other biological activities of tumor cells. Zhao et al. [
Wnt signaling pathways consist of three types: the Wnt/Ca2+ signaling pathway, the cell plane polarity pathway, and the classical Wnt signaling pathway. The classical Wnt signaling pathway is closely related to tumor formation, and its regulation is mediated by the phosphorylation/degradation of cytoplasmic catenin [
Dysregulation of MYC expression is a hallmark of cancer. Studies have shown that MUC1-C is a common cancer transmembrane protein and can induce the expression of MYC in KRAS mutant NSCLC. This effect can be a result of targeting MUC1-C by shRNA silencing, CRISPR editing or GO-203 pharmacological inhibition. MUC1-C activates the WNT/CTNNB1 pathway [
The TGF-β signaling pathway plays a major role in the occurrence and development of NSCLC. It promotes the invasion and metastasis of NSCLC by promoting epithelial-mesenchymal transformation and angiogenesis and is closely linked to the drug resistance of NSCLC. EMT induced by TGF-β plays a major role in targeted therapy and chemotherapy against NSCLC. Asakura et al. [
Compared with gene knockout and regulation of special targets, the current clinical use of gene editing technology is usually via combining it with other treatments. The most commonly used combination is gene editing plus chemotherapy, which can enhance the drug sensitivity of lung cancer cells and improve the antitumor effect. Compared with the former, BRM270, as a medicinal plant, in combination with gene editing can greatly reduce the toxicity and side effects of treatment. The combination of gene editing and immunotherapy is the most rapidly emerging curative method in recent years. It has concentrated targeting and can specifically enhance the immune response. In addition, gene editing could be associated with a variety of other therapies to improve survival in patients with certain types of non-small cell lung cancer (
Therapeutic method | Principle | Gene editing site | Advantage |
---|---|---|---|
Gene editing and chemotherapy [ |
Small molecule inhibitors of KRAS | Inhibition of the cascade of KRAS mutations and KRAS signaling | Precisely targeted therapy for KRAS-mutated lung cancer |
The expression of Nrf2 was up regulated in tumor cells, and the resistance to cisplatin and other drugs increased | Knockout Nrf2 gene | The sensitivity of lung cancer cells to cisplatin is enhanced, the therapeutic effect is better than that of chemotherapy alone, and the tumor volume is reduced | |
Gene editing medicinal plants [ |
BRM270: ⊕ miRNA-128 ⊖ miRNA-21 |
Silencing miRNA-21 |
BRM270 has less toxicity and fewer side effects |
Gene editing and immunotherapy [ |
CAR-T cells continue to kill tumor cells | Knock out TRAC, B2M |
The number and quality of CAR-T cells produced are good, and they have anti apoptotic properties and have a long time of action |
CRISPR/Cas9 used in antibody preparation | The coding site of the antibody | Specific antibodies can be obtained to achieve antitumor effects | |
Gene editing and multiple therapies [ |
EGFR tyrosine kinase can promote cell proliferation, angiogenesis, invasion and metastasis, reduce apoptosis and activate the Warburg effect | Knockout EGFR in EGFR mutated patients | Reduce the research and development of TKI drugs, and use them together with various therapies to treat lung cancer |
Note: ⊕: Promotion ⊖: Inhibition.
Currently, chemotherapy remains an important treatment for lung cancer. KRAS mutation is one of the most common gene mutations in NSCLC, and inhibitors have been used to inhibit mutated KRAS and oncogenic KRAS signaling in recent decades. Today, KRAS small molecule inhibitors are entering the clinic. Their tight integration with gene editing technology enhances the precise targeting of cancer cell therapy as a way to combat KRAS-driven refractory cancers [
The body’s resistance to chemotherapy drugs may increase over time. A recent study found that nuclear factor red pigment 2-related factor (Nrf2) is an important transcription factor regulating the cellular oxidative stress response and is an important regulator involved in maintaining internal REDOX homeostasis. Chemotherapeutic drugs have been shown to activate the transcriptional activity of the Nrf2 gene and upregulate Nrf2 expression. This increases the resistance of cells to chemotherapeutic drugs. CRISPR/Cas9 technology was used to knock out the Nrf2 gene in lung cancer cells, which inhibited their expression of functional proteins. At the same time, the knockout lung cancer cells were more sensitive to chemotherapy drugs such as display, carboxylates, and chlororubin [
The upregulated expression of miRNA-21 in NSCLC can promote the growth and invasion of lung cancer cells. However, the expression of miRNA-128 in lung cancer is inhibited due to its inhibitory effects on lung cancer cells [
Car-T therapy (chimeric antigen receptor-T-cell therapy) is immunotherapy currently approved for the treatment of acute lymphoblastic leukemia and non-Hodgkin’s lymphoma [
The study found that CRISPR/Cas9 plays a role in the preparation of antibodies. Some researchers have used CRISPR/Cas9 technology to achieve IgM-IgG-IgA antibody type conversion [
Under the premise of the existing technology, for individual EGFR-mutated NSCLC patients, the virus-transmitted CRISPR/Cas system can be used to repair or destroy the EGFR-mutated gene to achieve therapeutic purposes. The most common real-life cases of primary and secondary EGFR mutations demonstrate the feasibility of this approach [
In addition to clarifying the pathogenesis of non-small cell lung cancer and playing a role in its treatment, gene editing technology can also be used to establish animal models of NSCLC, diagnose and screen for early clinical NSCLC, monitor the effects after treatment, explore the mechanism of action of drugs and promote the development of new anticancer drugs.
Gene editing technology can be used to construct experimental animal NSCLC models, which provides more convenience for NSCLC-related experiments. The establishment of an experimental animal NSCLC model is conducive to further study of the etiology and pathogenesis of NSCLC to explore therapeutic methods. Meanwhile, it is also conducive to the study of the effectiveness of certain therapeutic means or therapeutic drugs to obtain the best treatment plan. At present, there are three main methods that have been used to apply gene editing technology to the construction of animal models of NSCLC.
The first one was a mouse model of the conditional expression of Cas9 constructed by Zhang Feng and his team [
The second was to fuse the EML4 and ALK genes by using gene translocation caused by chromosome 2 breakage of tumor cells in NSCLC and applied to study the mechanism of the potential antigenic effect of the fused genes [
The third was constructed by Sanchez-Rivera et al. [
Traditionally, to study the tumor suppressor or carcinogenic role of a protein in lung cancer, laboratories have had to generate new strains of mice for each protein, an extremely time-consuming and laborious task. Therefore, the introduction of CRISPR gene targeting enables the realization of the 3R principles of
Gene editing technology also plays a major role in the screening and diagnosis of lung cancer. Ubiquitin is a protein modifier and can play a role in posttranslational modification. Ubiquitin-specific peptidase 24 (USP24) was found by gene editing to be highly expressed in tumor-enhanced cell lines and advanced lung cancer tissues, especially the expression level of USP24-930T/USP24-7656C, which was significantly increased [
The use of gene editing technology to determine the prognosis of patients with lung cancer is convenient for clinicians to more accurately grasp the patient’s physical condition and provide more information for subsequent treatment and program selection [
ZAR1 is known to play a role in lung cancer through epigenetic inactivation. ZAR1 promoter methylation can occur throughout CpG islands, and its hypermethylation is closely associated with its decreased expression in cancer. Researchers used CRISPR/Cas9 to reactivate ZAR1 to study the role of ZAR1 in human cancer. It was found that ZAR1 is a novel lung cancer biomarker that can be silenced by DNA methylation in various cancers. In addition, ZAR1 exerts its tumor-suppressive function through p53 and zinc finger domains [
Adenosine to inosine (A to I) microRNA editing is well known to be associated with tumor phenotypes in various cancer types. By comparing the small RNA in-depth sequencing data of 74 publicly released lung adenocarcinoma (AD) and corresponding normal corresponding (NC) specimens with the latest analysis of the Cancer Genome Atlas (TCGA) dataset, researchers confirmed that changes in the editing level of microRNAs in lung adenocarcinoma could be used as a potential biomarker [
Glutamate receptor 8 (GRM8) is one of the G-protein-coupled receptors of the glutamate family, which can bind with various intracellular second messengers to regulate the excitability and development of neurons. Studies have found through gene editing technology that GRM8 mediates a reduction of intracellular cAMP concentration by inhibiting the activity of adenylate cyclase, and the amplification of GRM8 can promote the progression of lung squamous cell carcinoma (LUSC) [
Gene editing technology can also be employed to study the drug resistance mechanism of NSCLC. Researchers using CRISPR/Cas9 technology inserted the EGFR T790 M mutation into the drug-resistant pc9 human lung cancer cell line and found that its drug resistance was increased [
In addition, gene editing techniques can be used to explore tumor development. Compared with NSCLC, small cell lung cancer (SCLC), another highly aggressive subtype of lung cancer, is one of the deadliest solid tumors [
Gene editing technology is also commonly used to create specific cell lines for different types of NSCLC. For example, CRISPR/Cas9 gene editing technology was used to construct A549 cell lines with ubiquity C-terminal hydrolase L1 (UCHL1) gene knockout to explore the effect of UCHL1 on A549 cells in NSCLC [
In recent years, the morbidity and lethality of NSCLC have been increasing, and it has become one of the most common cancer types, posing a major threat to people’s lives and health. Currently, gene editing is one of the hottest fields in cancer research and has important application prospects in the diagnosis, screening and treatment of lung cancer. The occurrence and development of cancer are closely related to genetic changes. Compared with traditional cancer treatment, gene therapy can treat cancer more thoroughly and fundamentally. Its targeting is restricted. At the same time, it also has the advantages of fewer side effects and an obvious effect of improving patient prognosis. In addition, targeted modification of endogenous loci using gene editing technology can edit mutated genes directly or regulate signaling pathways, resulting in better therapeutic effects.
Nevertheless, there are some drawbacks of gene editing. The first is the difficulty of design and the cost. Even though the use of ZFNS and TALEN simplifies the work of gene editing, there are still difficult, time-consuming and laborious problems [
Finally, there is the issue of the ethics and the regulation of gene editing. On the one hand, gene editing technology offers a viable prospect for the treatment and prevention of human diseases. However, the International Bioethics Committee (IBC) of UNESCO points out that the application of gene editing technology should be restricted to prevention, diagnosis and treatment procedures and should not be used to alter human embryos. At the same time, scientists recognize that the use of CRISPR/Cas9 in human diseases is not entirely safe and effective [
In addition, most of the drugs used to treat non-small-cell lung cancer are administered intravenously and work in patients but are affected by poor pharmacokinetics. There are some limitations to their role. Studies are currently being reported of the development of CRISPR/Cas9 inhaled therapeutics [
With the continual development of gene editing technology, it is believed that it will play a more significant role in the prevention and treatment of non-small cell lung cancer in the future.