Targeting lncRNAs in programmed cell death as a therapeutic strategy for non-small cell lung cancer

Facts

• Lung cancer remains the leading cause of cancer-related mortality worldwide, with NSCLC being the most common histological type.

• LncRNAs are non-protein-coding RNAs with a transcript length of more than 200 nucleotides, which play a vital role in the tumorigenesis and progression of NSCLC.

• Diverse forms of PCD pathways participate in the initiation and development of NSCLC, and aberrant regulation of PCD can eventually result in cell carcinogenesis and tumour formation.

• Induction of PCD is the main mechanism leading to tumour cell death in most cancer treatments.

• LncRNAs are closely correlated with PCD including apoptosis, pyroptosis, autophagy and ferroptosis, which can regulate PCD and relevant death pathways to affect NSCLC progression and clinical therapeutic efficacy.

• Targeting lncRNAs is expected to be a promising strategy for lung cancer treatment.

Open questions

• How does programmed cell death affect the occurrence and development of NSCLC?

• What is the regulatory mechanism of lncRNAs on different types of programmed cell death in NSCLC?

• How to regulate programmed cell death by targeting lncRNA, thus inhibiting the onset and progression of NSCLC?

• How to use the relationship between lncRNA and programmed cell death to promote the sensitivity of NSCLC cells to chemotherapy and radiation therapy, thereby improving patient outcomes?

• How to seek and develop novel drugs targeting lncRNA, especially herbal medicine?

Introduction

Lung cancer, the second most common type of malignant tumours following breast cancer, remains the leading cause of cancer-related deaths worldwide. According to the data of the International Agency for Research on Cancer (IARC) of the World Health Organization, almost 2.2 million new cases of lung cancer were reported worldwide in 2020, with the mortality ranking the top in all types of cancers [1]. Identified as the major histological category of lung cancer, non-small cell lung cancer (NSCLC) constitutes approximately 85% of lung cancer cases, comprising three main subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma [2]. Because no evident clinical manifestations are observed at the early stage and diagnostic approaches are not effective, more than 75% of patients with NSCLC are diagnosed in the advanced stage, with the 5-year overall survival rate of less than 20% [3]. Despite great advances in the understanding of tumour onset and progression, the underlying mechanisms of NSCLC require further investigation to be comprehensively elucidated. Although currently used treatment approaches for NSCLC have evolved with the introduction of several novel strategies such as molecular target therapy and immunotherapy based on conventional chemotherapy and radiotherapy, the prognosis of many patients is still unfavourable owing to the limited clinical therapeutic efficacy and some unpleasant side effects [4]. Therefore, systematic and in-depth study on the molecular mechanisms of the pathogenesis of NSCLC, identification of new effective therapeutic targets and novel efficacious agents are of great significance for improving patient outcomes and enhancing the quality of life.

Long non-coding RNAs (lncRNAs) are defined as a class of non-protein-coding RNAs with a transcript length of more than 200 nucleotides [5]. They are involved in controlling and regulating various cellular biological processes such as cell proliferation, growth, differentiation, migration and apoptosis via modulation of gene expression and signalling pathways at epigenetic, transcriptional, RNA splicing and post-transcriptional levels by interacting with chromatin, proteins and RNA targets [6, 7]. Recent evidence suggests that abnormally expressed lncRNAs exert a vital role in the tumorigenesis and progression of multiple cancers, including NSCLC [8]. Furthermore, oncogenic and tumour-suppressive lncRNAs have negative or positive impacts, respectively, on tumour formation, proliferation, differentiation, invasion, epithelial–mesenchymal transition, metastasis, cancer stem cell maintenance and drug and radiotherapy resistance in NSCLC [6, 9]. Induction of programmed cell death (PCD) is the primary mechanism of most cancer treatments, which lead to tumour cell death. Studies on molecular mechanisms have demonstrated that lncRNAs are closely correlated with PCD, which include apoptosis, autophagy, ferroptosis and pyroptosis. Furthermore, lncRNAs can regulate PCD and protein expression of relevant death pathways to affect cancer progression and clinical therapeutic efficacy [10]. Therefore, in this review, we summarized the relationship between lncRNAs and four types of PCD (apoptosis, autophagy, pyroptosis and ferroptosis) in NSCLC and clarified the therapeutic role of lncRNAs in PCD for lung cancer treatment, aiming to shed new light on the pathogenic mechanisms of lung cancer and provide a novel therapeutic target for the treatment of lung carcinomas.

Relationship between lncRNAs and PCD in NSCLC

PCD refers to a genetically controlled active process that functions during cellular development and in response to stress. It is involved in self-clearance and renewal to maintain homoeostasis and normal physiological activities [11]. Several studies have indicated that PCD plays a critical role in the pathogenesis of NSCLC. Diverse forms of PCD pathways participate in the initiation and development of NSCLC, and aberrant regulation of PCD can eventually result in cell carcinogenesis and tumour formation (Fig. 1). Moreover, PCD pathways are reported to be strongly correlated with resistance in clinical cancer treatment.

Growing evidence has demonstrated that lncRNAs are strongly associated with PCD. LncRNAs regulate different types of PCD by directly influencing the expression of downstream protein molecules and multiple signalling pathways or acting as competing endogenous RNAs (ceRNAs) for binding and sponging target miRNAs to counteract their functions in the tumorigenesis and progression of NSCLC (Fig. 1). The relationship between lncRNAs and PCD provides novel insights into the pathogenic mechanisms of lung carcinomas, and targeting lncRNAs can be exploited as a promising therapeutic strategy for NSCLC treatment.

LncRNAs regulate apoptosis in NSCLC

Apoptosis is a non-inflammatory form of PCD initiated either via an extrinsic or intrinsic pathway, eventually inducing cellular demolition (Fig. 2A) [11]. A dynamic balance between pro-apoptotic and anti-apoptotic proteins is key to determining whether a cell maintains survival or undergoes apoptosis. However, dysregulation of apoptosis owing to imbalanced Bcl-2 family members, downregulation of caspases, overexpression of inhibitors of apoptosis proteins (IAPs) or impairment of death-receptor signalling is a common phenomenon exhibited in various cancers including NSCLC. These phenomena are responsible for not only tumorigenesis and cancer progression but also for resistance to multiple clinical therapies [12]. With regard to NSCLC pathogenesis, increased expression of the anti-apoptotic protein Bcl-2 and reduced levels of the pro-apoptotic protein Bax have been reported to be correlated with chemoresistance and tumour development [13, 14]. In addition, abnormalities in the activation of pivotal caspases, such as caspase 3/6/7/9, have also been observed in NSCLC progression. Moreover, studies have also suggested that caspase-8 is involved in the development of chemoresistance, which may serve as a potential target to improve chemosensitivity in NSCLC [15]. Poly (ADP-ribose) polymerase (PARP) plays a significant role in DNA damage repair and cell apoptosis, and dysregulated expression of PARP has been investigated as a possible biomarker and clinically useful target of lung cancer. In addition, several signalling pathways including STAT3, Wnt/β-catenin, NF-κB, PI3K/AKT, mTOR, MAPK/Slug, ROS, p53 and Nrf2 pathways, can regulate apoptosis through intrinsic or extrinsic mechanisms in the development of NSCLC [13, 16–20]. Explicit regulatory mechanisms of these pathways in apoptosis have been discussed in previous reviews. Accordingly, restoration of apoptosis by targeting both apoptotic pathways constitutes a feasible direction for developing promising anti-cancer drugs and enhancing the efficacy of clinical therapies.

LncRNAs regulate autophagy in NSCLC

Autophagy refers to a lysosome-mediated catabolic process dependent of multiple related proteins and signalling pathways, which plays a double-edged role in cancer (Fig. 3). On the one hand, autophagy restrains tumorigenesis by removing harmful cytotoxic agents to reduce stress injury, prevent genome damage and maintain cellular integrity [64]. In addition, defects in autophagy can promote inflammasome-mediated chronic inflammation and further induce the onset of lung cancer [65]. On the other hand, autophagy can be activated as a protective mechanism that helps to prevent intracellular metabolic and environmental stresses, such as nutrient starvation, energy shortage, hypoxia and cancer therapy, by regulating mitochondrial quality control and supply of nutrients required for cancer cell growth under nutrient-deprived conditions to facilitate cancer cell survival and tumour progression [66, 67]. Numerous studies have shown that protective autophagy is strongly correlated with resistance to anti-cancer drugs, and enhanced autophagy levels have been detected in patients with lung cancer with a poor prognosis [68]. Besides, it is reported that aberrant expression of autophagy-associated genes and proteins including unc-51-like kinase 1 (ULK1), ATG5, LC3B and p62 is involved in the onset and progression of NSCLC. In the lung tissues of NSCLC patients, LC3B expression was reduced, whereas the levels of p62, ULK1 and ATG5 were significantly increased, indicating that enhanced autophagy may be associated with the deterioration of lung cancer [69]. Furthermore, abnormal changes in the expression of genes such as epidermal growth factor receptor (EGFR), PTEN, LKB1 and AKT1 have been observed in NSCLC. These genes regulate EGFR-mediated signalling, which plays both promotive and suppressive roles in autophagic response via the RAS/RAF/MEK/ERK pathway to induce the serine phosphorylation of Beclin1 and the PI3K/AKT1 axis to inactive ULK1 by activating the negative modulator mTORC1, respectively [70, 71]. Furthermore, EGFR has emerged as a valuable target for anti-cancer therapy. Several studies have suggested that abnormal EGFR signalling participates in the induction of protective autophagy, which may be a promising approach to overcome resistance to anti-EGFR treatment in NSCLC [71]. In addition, p53 can regulate autophagic activity. Autophagy suppresses p53 by inhibiting AMPK activation and promotes tumorigenesis. Consequently, inhibition of p53 activates the transcription of autophagy-related genes, which further protects cells from apoptosis during hypoxia or nutrient starvation, resulting in tumour survival [13, 72]. Therefore, autophagy plays a pivotal role in the pathogenesis of NSCLC, and targeting autophagy is a promising approach to cancer treatment.

LncRNAs regulate pyroptosis in NSCLC

Pyroptosis is a particular lytic and inflammatory form of PCD, which can be induced via the caspase-1-dependent canonical pathway and caspase-4,5 (for human)- or caspase-11 (for mouse)-mediated non-canonical pathway (Fig. 4) [82].

Evidence shows that the induction of pyroptosis via inflammasome/caspase-1 signalling stimulates a series of inflammatory cascades by releasing inflammatory mediators, which are strongly pertinent to tumorigenesis and cancer development [83]. Furthermore, pyroptosis has been reported to play a crucial role in suppressing the proliferation of tumour cells in vitro and tumour growth in vivo, suggesting that it is a promising strategy for NSCLC therapy [84]. The well-studied NLRP3 (NLR family pyrin domain-containing 3) inflammasome plays a dominant role in inducing pyroptosis and promoting inflammation, acting as an essential regulator of immune response that can be activated by chemotherapy or radiation treatment in various cancers, including NSCLC [85]. Therefore, the NLRP3 inflammasome is considered a promising therapeutic target for lung cancer prevention and treatment. For example, simvastatin suppressed NSCLC proliferation and migration by inducing cell pyroptosis through activating NLRP3 and caspase-1 and promoting the maturation of downstream IL-1β and IL-18 (ref. [86]). Several studies have indicated that mitochondrial reactive oxygen species (ROS) play a significant role in controlling tumour growth as crucial upstream regulators to trigger NLRP3 inflammasome activation and subsequent pyroptosis, wherein the signal transduction of ROS generation requires the involvement of NF-κB pathway, which increases the expression of NLRP3 and pro-caspase-1 proteins [87]. Moreover, p53 can inhibit cancer cell proliferation, tumour growth and NSCLC development as the key mediator activating pyroptosis via upregulation of NLRP3, ASC and cleaved caspase-1 (ref. [84]).

LncRNAs regulate ferroptosis in NSCLC

As a newly discovered non-apoptotic oxidative form of PCD, ferroptosis is primarily characterized by intracellular iron-dependent excessive accumulation of lipid ROS [88]. It is initiated by the blockade of the cellular antioxidant defence depending on glutathione (GSH), which is essential for restoring the intracellular redox homoeostasis upon ROS generation (Fig. 5) [89].

Glutathione peroxidase 4 (GPX4) plays a predominant role in the prevention of ferroptosis by decreasing cellular ROS levels and effectively repairing cell damage caused by lipid oxidation, and it was demonstrated to be high in NSCLC [90]. However, inactivation of GPX4 owing to GSH depletion and direct inhibition of GPX4 via RSL3- or FIN56-mediated GPX4 degradation lead to reduced antioxidant capacity and excessive production of lipid ROS, inducing ferroptosis as a result of unchecked lipid peroxidation [91]. Erastin, an exogenic small-molecule inducer of ferroptosis, not only promotes iron accumulation in cells by improving transferrin receptor 1 (TFR1) expression but also reduces cellular GSH levels by directly suppressing the activity of system Xc-, and it has been developed as an agent to treating various cancers, including NSCLC [92]. Furthermore, p53 has been reported to inhibit system Xc- uptake of cysteine through downregulating solute carrier family 7 member 11 (SLC7A11) by binding to nuclear factor erythroid 2-related factor 2 (NRF2), thereby promoting ferroptosis in cancer cell lines [93]. It has been demonstrated that repressing SLC7A11 transcription enhances the susceptibility of lung cancer cells to an oxidant insult, enabling cells to undergo ferroptosis-like cell death [94]. A study reported that NRF2 protected malignant cells from oxidative stress and chemotherapeutic agents, thus facilitating cancer progression [95]. Moreover, an elevated level of NRF2 rendered NSCLC cells insensitive to erastin-induced ferroptosis [96]. Therefore, suppression of NRF2 may result in an enhanced ROS-related oxidative stress level conducive to accelerating ferroptosis in NSCLC cells. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is considered an essential pro-ferroptotic gene, and lower expression of ACSL4 has been associated with decreased ferroptosis in NSCLC cells [90, 97]. Several studies have shown oxidative stress resistance in NSCLC and weak sensitivity of tumour cells to ferroptosis, suggesting that ferroptosis has an important influence on the occurrence and progression of NSCLC [98]. Moreover, it was demonstrated that ferroptosis participated in modulating the sensitivity of NSCLC cells to chemotherapy and radiotherapy [99, 100]. Therefore, targeting ferroptosis is a novel approach to developing therapeutic strategies for NSCLC.

Potential chemical drugs and herbal medicine targeting lncRNAs in PCD for the treatment of NSCLC

Targeting lncRNAs displays great potential in NSCLC therapy. Recent studies have shown that some drugs can act on lncRNAs to regulate PCD in cancer cells, thus suppressing NSCLC development. To date, chemotherapeutic drugs are the most commonly used therapeutic agents for NSCLC. Paclitaxel (PTX) serves as a first-line chemotherapeutic drug for advanced NSCLC in clinical settings. A study demonstrated that PTX treatment (10 μM) inhibited A549 cell proliferation and partially induced cell apoptosis by upregulating lncRNA MEG3 and activating the p53 pathway [102]. In addition, another study indicated that nedaplatin (2 mg/mL), a functional analogue of the anti-cancer drug cisplatin, could restore the chemotherapeutic sensitivity of cisplatin-resistant A549 and H1650 cells. The underlying mechanism may involve the induction of apoptosis by regulating targets that included caspase-3, caspase-6 and cleaved PARP through downregulation of lncRNA MVIH, thus providing a rationale for nedaplatin use in the treatment of NSCLC [23].

Conventional herbal medicine also exhibits good anti-cancer effects, which is widely used as an auxiliary or alternative medication for cancer therapy in China [103]. Polyphyllin I (0, 0.4, 0.8 and 1.2 μmol/L), a steroidal saponin extracted from the rhizome of the Chinese herbal medicine Paris polyphylla with strong inhibitory effects on various cancers, was reported to induce apoptosis by downregulating Bcl-2 and upregulating Bax and cleaved caspase-3 in a dose-dependent manner through impeding the STAT3/HOTAIR/EZH2 signalling axis, thus inhibiting A549 and NCI-H358 cell growth [28]. Moreover, Yang et al. [38] suggested that treatment with polyphyllin I (0.1, 1, 2, 4, 6, 8 and 10 μg/ml in vitro; 5 mg/kg/d for 4 weeks in vivo) suppressed the viability of gefitinib-resistant PC-9-ZD cells in vitro and tumour growth in PC-9-ZD xenograft in vivo in a dose-dependent manner. This anti-tumour activity was mediated through induction of apoptosis via an increase in the level of cleaved PARP and caspase-3 by downregulating lncRNA MALAT1 and inactivating STAT3 signalling. Curcumin, a flavonoid compound widely found in various medicinal herbs, has been reported to inhibit multiple human cancer types, especially lung cancer. It was demonstrated that curcumin (50 and 100 μM in vitro; 100 mg/kg/d for 3 weeks in vivo) significantly suppressed gemcitabine-resistant NSCLC cell proliferation in a concentration-dependent manner and restrained tumour growth by promoting cell apoptosis through upregulation of lncRNA MEG3-mediated PTEN signalling [45]. Xiaoji decoction (XJD) is a Chinese medicinal formulation that has been used for the treatment of lung cancer for decades. It is comprised of Psoralea corylifolia Linn, Coriolus versicolor, Astragalus membranaceus (Fisch) Bunge, Curcumae Rhizomae, Hedyotis diffusa Willd, Buthus martensii Karsch, Scolopendra subspinipes and Rheum palmatum L. In a study by Wu et al., XJD (40 mg/mL in vitro; 13.4 g/kg/d for 25 days in vivo) enhanced the inhibitory effects of DDP not only on A549 and H1975 cell growth but also on lung tumour size by inducing a high magnitude of cell apoptosis via downregulation of SP1 through regulating the reciprocal interaction between lncRNA PVT1 and miR181a-5p [48].

Conclusion and future prospects

Recent studies have recognized that aberrant expression of lncRNAs is strongly correlated with the initiation and development of multiple cancers, including NSCLC. Abnormal regulation of PCD, including apoptosis, autophagy, pyroptosis and ferroptosis, has been demonstrated to play a critical role in the pathogenesis of NSCLC. LncRNAs can regulate PCD and relevant pathways in lung cancer cells and exert oncogenic or anti-carcinogenic effects by either directly influencing protein expression or functioning as a sponge of miRNAs (Table 1). As previously described, a considerable number of lncRNAs have been implicated in lung carcinogenesis and have been associated with resistance to anti-cancer drugs, thereby representing a potential value as predictive biomarkers for chemotherapeutic sensitivity and clinical prognosis of cancer. For example, circulating lncRNA SOX2OT and ANRIL, which were markedly overexpressed in the serum of patients with NSCLC, have been evaluated as ideal biomarkers for diagnosing NSCLC and predicting the overall survival rate of NSCLC patients, yielding superior sensitivity and specificity with remarkable potential in distinguishing patients with cancer from controls [104]. Therefore, an in-depth understanding of the relationship between lncRNAs and PCD may provide novel insights into the pathogenic molecular mechanisms involved in NSCLC, and targeting lncRNAs is expected to be a promising strategy for lung cancer treatment.

Multiple RNA interference technologies, such as small interfering RNAs (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASOs), exhibit the most intriguing approach to selectively depleting or knocking down the target oncogenic lncRNAs. For instance, MALAT1 knockdown through siRNA has been reported to block cancer cell proliferation and reduce tumour growth in NSCLC by downregulating COMMD8 [57]. He et al. [105] demonstrated that silencing CCAT2 with shRNA inhibited the proliferation, invasion and tumour formation of NSCLC cisplatin-resistant cells. In addition, Gong et al. [106] constructed MALAT1-specific ASO and nucleus-targeting TAT peptide co-functionalized Au nanoparticles, namely, ASO-Au-TAT NPs, to precisely target and degrade nuclear MALAT1, thus effectively preventing lung cancer metastasis. It is noteworthy that compared with free ASO, ASO conjugated with Au-TAT NPs represented much higher nucleus-specific delivery efficiency and more efficiently inhibited target lncRNA. Owing to low stability and poor penetration into the nucleus, free ASO exhibited limited anti-metastasis capacity in vivo even at a very high dose [107]. Therefore, this novel nanostructure has great potential for being used in cancer metastasis therapy in the future. The expression of tumour-suppressive lncRNAs was reported to be lower in lung cancer specimens than in healthy tissues. Accordingly, restoring the normal expression levels of these lncRNAs may provide a promising therapeutic approach beneficial to patients with NSCLC. For example, Li et al. [46] demonstrated that AOCP4 was downregulated in NSCLC samples, and upregulation of AOCP4 via transfection of pcDNA suppressed cell viability and invasion in NSCLC but induced cell apoptosis by inactivating the Wnt/β-catenin pathway. However, there are several issues concerning lncRNA-based therapies in clinical practice. It is important to develop a reliable delivery system to safeguard the stability of lncRNA vectors in the bloodstream and improve the efficiency of selectively attacking the tumour targets. In addition, deciding an appropriate dosage is necessary to ensure the safety and effectiveness of therapeutic lncRNAs, which requires further investigation [6, 108].

To date, a few chemical drugs and herbal medicines have been reported to target lncRNAs in PCD for NSCLC treatment, whose regulatory mechanisms mostly focus on cell apoptosis. Therefore, developing related anti-cancer medication, especially herbal medicines, which have an excellent prospect, will help to establish a more comprehensive clinical approach to improving the therapeutic outcomes of NSCLC, with the final goal to benefit the patients.

Author contributions

JL, PY and JS collected and analysed the literature data. YL contributed to the manuscript writing, figures and submission of this paper. YH modified the grammar. LX and XM performed the study design, review and proofread this manuscript. All authors read and approved the final manuscript.

Funding

The National Natural Science Foundation of China (NSFC: 81903902 and 81773974), China Postdoctoral Science Foundation (2019M663457), Sichuan Science and Technology Programme (2020YJ0172), and Xinglin Scholar Research Premotion Project of Chengdu University of TCM (BSH2019001).

Data availability

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests

The authors declare no competing interests.

Ethics statement

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.