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Long Non-Coding RNAs as Emerging Targets in Lung Cancer

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10 May 2023

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12 May 2023

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Abstract
Long non-coding RNAs (LncRNAs) are mRNA-like molecules that do not encode for proteins and that are longer than 200 nucleotides. LncRNAs play important biological roles in normal cell physiology and organism development. Therefore, deregulation of their activities is involved in disease processes such as cancer. Lung cancer is the leading cause of cancer-related deaths due to late stage at diagnosis, distant metastasis, and high rates of therapeutic failure. LncRNAs are emerging as important molecules in lung cancer for their oncogenic or tumor suppressive functions. LncRNAs are highly stable in circulation, presenting an opportunity for use as non-invasive and early-stage cancer diagnostic tools. Here, we summarize latest works providing in vivo evidence available for LncRNAs role in cancer development, therapy-induced resistance, and their potential as biomarkers for diagnosis and prognosis, with a focus on lung cancer. Additionally, we discuss current therapeutic approaches to target LncRNAs. The evidence discussed here strongly suggests that investigation of LncRNAs in lung cancer in addition to protein-coding genes will provide a holistic view of molecular mechanisms of cancer initiation, development, and progression, and could open a new avenue for cancer treatment.
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Subject: Biology and Life Sciences  -   Life Sciences

1. Introduction

Long non-coding RNAs (LncRNAs) are broadly defined as RNAs that usually do not encode for proteins and that are longer than 200 nucleotides. These are messenger RNA (mRNA)-like molecules that are transcribed by polymerase II, 5’ capped, and have a 3’ poly-A tail. Because they can form complex secondary structures, they often have functions. Many LncRNAs are preferentially found in the nucleus, where they participate in regulation of chromatin organization and transcription, as well as formation of nuclear speckles and regulation of splicing. In the cytoplasm, LncRNAs can regulate mRNA stability, bind to other non-coding RNAs, modulate protein post-translational modifications and protein function [1,2,3,4,5].
LncRNAs have been studied in mammals since the early 90’s due to their involvement in developmental processes. For example, the Xist (X-inactive specific transcript) lncRNA contributes to reshaping chromatin architecture to achieve X chromosome silencing in early embryonic development [6]. The H19 LncRNA is involved in genomic imprinting and regulation of the insulin growth factor 2 (IGF2) and other genes involved in embryonic growth [7,8]. The study of HOX genes, master regulators of embryonic development, led to the discovery of the LncRNA HOTAIR (Homeobox transcript antisense intergenic RNA). HOTAIR is transcribed from the antisense strand of the HOXC gene. HOTAIR has been reported to repress transcription of the HOXD loci via interaction with PRC2 (polycomb repressive complex 2) [9], although in vivo models developed later report conflicting results regarding HOXC or HOXD genes regulation by HOTAIR [10,11]. Because of the importance of physiological context to understand LncRNA molecular function, more recently other groups report exclusively in vivo approaches using animal models of LncRNA genetic ablation. For example, a comprehensive study developed 18 knock-out (KO) mouse models for less well-known LncRNAs with human orthologs. Certain LncRNAs expression was highly tissue-specific (such as Fendrr, Manr and linc–Cox2 expressed mainly in lung), supporting a unique physiological role, while other LncRNAs were more ubiquitously expressed. Three of the analyzed LncRNAs were required for embryonic development (Fendrr, Mdgt, Peril), speaking to their fundamental functions [12]. Thus, the involvement of LncRNAs in normal cell physiology and organism development suggests that these may also control disease-related processes such as cancer.
Lung cancer is the leading cause of cancer-related mortality in the U.S., and non-small cell lung cancer (NSCLC) is the most common subtype. Lung cancer is commonly diagnosed in late stages, where patients present distant metastasis with 9% of 5-year survival rate [13]. Although the development of targeted therapies (e.g., tyrosine kinase inhibitors, TKIs) has improved patient outcomes, their clinical efficacy is often limited by both innate and acquired resistance, permitting tumor progression and recurrence leading to poor survival rates [14]. It is imperative to better understand molecular drivers of tumorigenesis, metastasis, and therapy resistance in lung cancer to develop improved therapeutic strategies. LncRNAs are emerging as important molecules in cancer due to their oncogenic or tumor suppressive functions. Here, we summarize latest works providing in vivo evidence available for the role of lncRNAs in lung cancer, therapy resistance, and their potential as biomarkers.

2. Role of LncRNAs in lung cancer

Because of the role of LncRNAs in regulating a diverse array of cellular functions, deregulation of their activities is involved in cancer. Some mechanisms reported for LncRNAs functions in cancer are: acting as miRNA sponges to modulate activity on their targets; interacting with histone-modifier enzymes to modulate known oncogene/tumor suppressor gene expression; interacting with transcription factors to repress/activate their transcriptional programs; acting as anti-sense molecules for tumor suppressor mRNAs, among other mechanisms [15]. Importantly, regulation of LncRNA expression and function in cancer follows similar principles to that of known oncogenes and tumor suppressors, as it can be mediated by DNA methylation [16], amplification or deletion [17], and mutation or SNPs of DNA sequences [18,19]. In this section, we provide examples of well-studied LncRNAs that are reported to have oncogenic or tumor suppressive roles in lung cancer, as well as those with controversial functions.
MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) was one of the first lncRNAs described to be associated with cancer. In 2003, Ji et al analyzed the gene expression profile in human primary lung cancer tumors that subsequently metastasized or those that did not metastasize and compared their transcriptional signatures. They identified a Metastasis-Associated Lung Adenocarcinoma Transcript 1, named MALAT1, for its higher expression in primary tumors that metastasized. They also found a significant correlation between higher level of MALAT1 expression in stage I lung cancer and worse survival outcomes [20]. Several studies since then have described MALAT1 function in normal physiology and cancer [21,22,23]. Three independent groups developed an in vivo approach to describing MALAT1 physiological function. They found that Malat1 is highly abundant in several mouse tissues and highly conserved across species. Genetic perturbation of the Malat1 locus in mice (by genetic deletion [24,25] or genetic inactivation approaches [26]) did not alter animal development, did not alter nuclear speckle formation, splicing, or mRNA stability. However, they described a role for Malat1 in controlling neighboring genes expression in a tissue-specific manner that was not consistent between the three studies, especially that of Neat1, another LncRNA [24,26]. In the context of lung cancer, MALAT1 silencing did not show effects on lung cancer cell proliferation or viability in vitro [25]. To further understand the role of MALAT1 in lung cancer metastasis, Gutschner et al implanted EBC-1 lung cancer cells into nude mice and treated them with subcutaneous administration of an anti-sense oligonucleotide (ASO) targeting MALAT1. After five weeks of treatment, all primary tumors were excised. Metastasis nodules were analyzed at 12 weeks, indicating fewer and smaller metastatic nodules in the treated group, suggesting a role for MALAT1 in promoting metastasis. Through additional in vitro studies, they report MALAT1 inhibition results in aberrant expression of metastasis-associated genes in cell lines [27]. Although these studies support a role for MALAT1 in promoting metastasis and regulating certain genes expression, other evidence exists for different roles. Kim et al describe an elegant study that challenges previously reported roles for MALAT1 in metastasis. Using the Malat1 knock-out (KO) mouse model developed by Nakagawa et al (LacZ and Poly-A sequences were used as transcriptional terminators inserted 69 bp downstream of the transcription start site of Malat1 without deletion of the DNA sequence), Kim et al crossed these mice with a breast cancer model driven by MMTV-PyMT that mimics human disease. Surprisingly, they found a 7.2-fold increase in metastatic foci and 31-fold increase in percent of lung areas with metastatic lesions in Malat1-KO mice as compared to Malat1 WT mice, suggesting a role for Malat1 in suppressing breast cancer metastasis to the lung. This phenotype was rescued by transgenic expression of Malat1, suggesting that the RNA product itself diminished metastasis. Additionally, they identified Malat1 interaction with TEAD proteins in mouse-derived tumors and cell lines, which suppressed TEAD-YAP interaction and, therefore, inhibited their pro-metastatic transcriptional program [28]. This study and others highlight the importance of context (lung model vs. breast model) as well as experimental methodology to approach LncRNAs functional characterization (such as LncRNA genomic DNA loss vs. RNA loss reviewed in detail elsewhere [21]). Of note, DNA elements themselves within a LncRNA locus may be responsible for regulatory functions that are independent from transcript function [29,30,31,32]. In summary, MALAT1 promotes metastasis in lung cancer, but may show opposite functions in different types of cancer depending on cellular context.
The GAS5 (growth arrest-specific 5) gene was first described as a G0-specific gene that is inhibited by serum and growth factors [33]. In vivo, Gas5 genetic deletion (Gas5+/-) in mice decreased bone mass and impaired bone repair leading to osteoporosis. Mechanistically, GAS5 positively influenced proper cell differentiation through interaction with UPF1 (a DNA/RNA helicase) to accelerate SMAD7 mRNA decay [34]. In lung cancer, GAS5 was found downregulated in 72 NSCLC tumor samples as compared to their paired adjacent normal tissues, suggesting a tumor-suppressive role. Additionally, low GAS5 expression was correlated with larger tumor size, lower differentiation levels, and higher staging of tumor-node metastasis [35]. A xenograft model of GAS5 overexpression (OE) showed that GAS5 OE markedly decreased tumor size compared with control [35]. Although this study did not explore a mechanism for GAS5-mediated tumor suppression, other studies implicate a role for GAS5 as miRNA sponge, to negatively influence cell cycle activator genes [36] or positively influence PTEN levels [37]. Additionally, a recent study revealed GAS5 is partially localized to the mitochondria where it modulates energy homeostasis by promoting de-acetylation of malate dehydrogenase, suppressing breast cancer [38]. Taken together, a role for GAS5 in halting the cell cycle as well as promoting cell differentiation in normal cells support its tumor suppressive role reported in lung cancer. Besides GAS5, other LncRNAs have been studied for their tumor suppressive functions, such as MEG3 and TUG1, reviewed elsewhere [39].
LUCAT1 (Lung Cancer Associated Transcript 1), first identified as smoke-induced and cancer associated LncRNA1 (SCAL1) [40], has higher expression in lung cancer as compared to normal controls, and is also found overexpressed in several cancer types [41]. To our knowledge, a LUCAT1-deficient mouse model has not been reported. Additionally, patients with tumors that express high levels of LUCAT1 showed poorer overall survival as compared to those with lower LUCAT1 expression. Moreover, high LUCAT1 levels were associated with late staging in tumor-lymph node metastasis and higher tumor volume. In NSCLC cell lines A549 and SPC-A1, LUCAT1 modulates p21 and p57 expression by promoting loci methylation through PRC2 [42].
More recently, a role for LUCAT1 in regulating immune responses has been described. LUCAT1 genetic deletion in myeloid cells is found to enhance interferon-mediated gene transcription. LUCAT1 acts as an immune suppressor by interacting with STAT1 and chromatin in the nucleus. It may also act by inhibiting NF-kB functions [43]. These findings suggest a tumor-promoting role for LUCAT1 that is tumor cell-intrinsic, in addition to a potential non cell-autonomous mechanism by inhibition of immune surveillance, although this mechanism remains to be explored in the lung cancer context.
HOTAIR has been vastly studied in cancer contexts [44,45]. In NSCLC, tumor samples and cell lines expressed higher HOTAIR levels as compared to normal counterparts [46]. Additionally, high HOTAIR levels correlated with higher tumor grade and presence of lymph node metastases [46,47]. In vitro, HOTAIR has been reported as a direct target of the Hypoxia-inducible-factor-1α (HIF-1α), therefore enhancing A549 NSCLC cells proliferation, migration, and invasion [48]. In vivo, tail vein injections of SPC-A1 cells with or without siRNA targeting HOTAIR showed that the knock-down condition reduced number of metastasis nodules found in the lungs of immunocompromised mice [46]. HOTAIR silencing resulted in a decrease in matrix metalloproteinases (MMPs, which promote invasion and migration) expression and an increase in HOXA5 levels (a tumor suppressor) in cell lines, suggesting HOTAIR acts through regulation of expression of cancer-related genes [46]. Although this xenograft assay does not account for all steps required for a tumor cell to achieve metastatic colonization (extravasation, survival in blood, seeding of new site, proliferation in new site), and they measured colonization of lungs by a lung cancer cell line (same tissue), it raises the possibility that HOTAIR may be involved in seeding and survival of cancer cells. Additionally, the absence of a competent immune system challenges interpretation of these results. Development of a HOTAIR transgenic mouse model to understand in vivo implications in lung cancer initiation and progression is necessary, similar to a HOTAIR inducible system recently reported for breast cancer [49]. This model showed that sustained HOTAIR overexpression promotes breast cancer metastasis to lungs. Overall, with the data available, HOTAIR seems to play an oncogenic role in lung cancer; however, robust mechanisms through which this lncRNA function remain to be uncovered.
Most studies focus on the contribution of a single lncRNA to cancer phenotypes. However, whether the lncRNAs described above are expressed simultaneously in tumors with unique or redundant functions remains to be explored in depth. For example, Esposito et al showed that at least 80 oncogenic lncRNAs are active in NSCLC through a lncRNA-focused CRISPR screen. By further dissecting the role of two candidate lncRNAs, CHiLL1 and GCAWKR, they showed these have distinct cellular localization and non-overlapping targets. Importantly, ASOs targeting both these lncRNAs yielded additive effects, suggesting that they have cooperating functions in NSCLC progression [50]. LncRNAs are generally expressed at lower levels than protein-coding genes [51]. Because of this, we speculate that lncRNAs with redundant functions may be expressed simultaneously to compensate for a higher expression of their targets in disease conditions. By examining available TCGA lung adenocarcinoma datasets containing mRNA expression data, we did not find significant correlations (negative or positive) among the expression of lncRNAs described here. However, such an analysis in combination with functional studies could shed light on mutual exclusivity relationships between certain lncRNAs. Additionally, whether certain lncRNAs are predominantly expressed at different stages of tumor progression remains to be explored. A new online resource, lncRNAfunc https://ccsm.uth.edu/lncRNAfunc, provides insights on differentially expressed lncRNAs across different cancer types and stages available in TCGA, as well as functional predictions [52]. Although this analysis did not detect any correlations between the lncRNAs mentioned here and lung cancer stages, possibly due to lack of sufficient sample sizes, these lncRNAs did show correlation with stage in other cancers; for example, LUCAT1 was correlated with cancer stage in kidney cancer.
In summary, LncRNAs have oncogenic and tumor suppressive roles in lung cancer, illustrated in Figure 1. LncRNAs interact with protein-coding molecules resulting in activation or inactivation of specific signaling pathways in cancer cells. We speculate more lung cancer specific lncRNAs will be identified with genome-wide transcriptomic studies.

3. LncRNAs in lung cancer therapy resistance

The role of non-coding RNAs in resistance to cancer therapies has been documented [45,53,54]. Mechanistically, LncRNAs can contribute to therapy resistance by promoting cell survival pathways (including autophagy, DNA repair), inhibiting apoptosis and cell cycle checkpoints, increasing self-renewal capacity and epithelial to mesenchymal transition (EMT), modulating the tumor microenvironment as well as the cellular xenobiotic stress response (drug efflux mechanisms), among others [45,55]. Here, we provide evidence for the roles of the LncRNAs discussed above in resistance to chemotherapy, radiotherapy, and targeted therapy in lung cancer. Additionally, we discuss new advances in understanding the role of LncRNAs in immune checkpoint therapy in lung cancer.

3.1. Role of LncRNAs in resistance to chemotherapy, radiotherapy, and targeted therapy in lung cancer

In a tumor xenograft model, silencing of MALAT1 in cisplatin-resistant A549 cells led to decreased growth in nude mice (subsequently treated with cisplatin), as compared to non-targeting control. Similarly, overexpression of MALAT1 in cisplatin-sensitive A549 lung cancer cells increased tumor volume as compared to empty vector control [56]. These data suggest a role for MALAT1 in promoting cisplatin resistance in lung cancer. The authors suggest that modulation of STAT-3 function by MALAT1 drives this phenotype although direct interaction was not confirmed [56]. While additional lung cancer-focused studies are lacking, MALAT1 role in cisplatin resistance was also found in a xenograft model of oral squamous cell carcinoma [57]. In radiotherapy, MALAT1 also promotes resistance, although this function has not been explored in lung cancer. In a xenograft model of esophageal squamous cell carcinoma, MALAT1 levels were found reduced upon radiation in tumors that respond to treatment. Additionally, overexpressing MALAT1 in xenografts did not decrease in size upon radiation exposure while controls showed regression [58]. Furthermore, in colorectal carcinoma cell lines, MALAT1 knockdown enhanced radiosensitivity [59]. Therefore, MALAT1 can impact sensitivity to radiation therapy in cancer. To our knowledge, there are no robust in vivo studies addressing the role of MALAT1 in lung cancer targeted therapy resistance (TKIs), only those in cell lines. Cheng et al characterized differentially expressed LncRNAs in gefitinib (EGFR TKI)-sensitive PC9 cells and gefitinib-resistant PC9 cells. They found H19 and BC200 LncRNAs as upregulated in resistant cells vs. sensitive ones, while MALAT1 and HOTAIR were downregulated in the resistant setting. These data suggest that MALAT1 may promote sensitivity to targeted therapy in lung cancer cells [60]. However, opposite roles for MALAT1 in targeted therapy resistance in other cancers have been described. For example, MALAT1 is overexpressed in Sutinib-resistant renal cell carcinoma tumors vs. sensitive tumors [61]. These findings suggest MALAT1 plays a role in therapy resistance that can be highly context-specific in regard to type of therapy (cisplatin vs. targeted therapy) or cancer primary site (lung vs. kidney) and support the need to study these functions and mechanisms in physiologically-relevant settings.
GAS5, a tumor suppressive LncRNA, plays a role in sensitization of lung cancer cells to therapy. In cisplatin-resistant A549 and H1299 cells, GAS5 overexpression reduced IC50 (half-maximal inhibitory concentration) values to cisplatin. In vivo, cisplatin-resistant A549 cells stably overexpressing GAS5 yielded lower tumor volumes when injected into nude mice as compared to vector controls [62]. Additionally, a role for GAS5 has been reported in sensitivity to targeted therapy (gefitinib, EGFR TKI). A xenograft mouse model of GAS5 overexpression (OE), GAS5 OE plus gefitinib, gefitinib alone, or vehicle, showed that GAS5 OE plus gefitinib yielded the best tumor size reduction outcomes. This suggests that GAS5 can synergize with targeted therapy to achieve better clinical outcomes [35]. Lastly, roles for GAS5 in sensitizing lung cancer cells to radiotherapy have also been reported [63]. This evidence suggests GAS5 as a promising target to sensitize lung tumors to cancer therapy.
LUCAT1 can contribute to cisplatin resistance in NSCLC. Shen et al describe a role of LUCAT1 as sponge of miR-514-3p, whose target is ULK1, a protein involved in autophagy. Therefore, LUCAT1 promotes cisplatin resistance by modulating autophagy [64]. Although further investigation in the context of lung cancer is limited, LUCAT1 is known to promote resistance to DNA-damaging agents in colorectal carcinomas [65]. Because LUCAT1 has been recently reported to play a role in immune cell regulation [43,66], its role in immune checkpoint blockade therapies should be explored in detail as a possible target for combination therapy.
The role of HOTAIR in therapy resistance, similar to findings described for MALAT1, can be complex. For example, HOTAIR was found in higher levels in cisplatin-resistant NSCLC tumors as compared to sensitive ones [47]. In radiotherapy, HOTAIR can promote resistance to radiation therapy through inhibition of p21 in cervical cancer [67], it can modulate β -catenin signaling in Lewis lung cancer tumors [68], and it modulates Akt signaling in breast cancer cell lines [69]. On the contrary, in targeted therapy HOTAIR was reported downregulated in tumors derived from acquired and primary resistant states to EGFR-TKIs as compared to treatment naïve tumors. Here, higher levels of HOTAIR expression were correlated with better survival outcomes [70]. Consistent with these findings, HOTAIR was downregulated in gefinitib-resistant PC9 cells vs. sensitive ones [60]. However, another study reports higher HOTAIR expression in gefitinib-resistant PC9 cells as compared to gefitinib-sensitive cells [71]. Lastly, in vitro assays suggest that HOTAIR may mediate Crizotinib (ALK/ROS1 inhibitor) resistance through upregulation of autophagy [72]. Taken together, HOTAIR promotes resistance to cisplatin and radiotherapy in lung cancer. In targeted therapy, the type of drug and differences in experimental methodologies employed may account for the confounding role of HOTAIR.

3.2. Role of LncRNAs in immunotherapy responses in lung cancer

The role of LncRNAs in resistance to immune checkpoint inhibitors (ICI) is emerging as a field of study in many cancers [73]. ICIs target immune inhibitory molecules such as PD-1, PD-L1, and CTLA-4 with the goal of re-activating immune surveillance and tumor-cell killing [74]. Even though these therapies have favorable outcomes in certain tumor types, an effective and durable response in lung cancer is achieved only in ~25% of cases [75]. Therefore, understanding the underlying molecular mechanisms of response or resistance to ICIs is critical to improve lung cancer outcomes.
MALAT1 may be involved in regulating responses to immunotherapy. In the study of 113 NSCLC tumor samples, MALAT1 expression was positively correlated with PD-L1 mRNA as well as PD-L1 protein levels [76]. Here, the authors propose MALAT1 acts as a sponge of miR-200a-3p, whose target is PD-L1 [76]. Similarly, another study proposed LINC01140 directly represses two miRNAs (miR-377 and miR-155-5p) whose target is PD-L1. Therefore, LINC01140 expression promotes PD-L1 expression and a potential pro-tumorigenic microenvironment [77]. In a co-culture assay, LINC01140 silencing in lung cancer cells promoted higher IFN-γ secretion from cytokine-induced killer cells, as compared to non-targeting control. In a xenograft model, lung cancer cells with knock-down of LINC01140 were injected into immunocompromised mice and received peritumoral administration of cytokine-induced killer cells upon tumor establishment. Further tumor growth was inhibited in the knock-down condition as compared to non-targeting controls. Importantly, higher levels of pro-inflammatory cytokines were found in circulation of mice injected with LINC01140 knock-down tumor cells compared to controls [77]. Moreover, a recent study reports an unbiased approach to understanding LncRNAs relationship to the tumor immune microenvironment and prediction of response to immune checkpoint therapy in NSCLC [78]. Based on LncRNAs that were correlated with immune-checkpoint expression, and taking into account overall survival data, Zhang et al identified a signature of ten LncRNAs that they used to separate patients into “low” and “high” risk groups. They analyzed immune infiltrates in tumor samples and found a significantly higher density of T-cells (CD4+ and CD8+) and dendritic cells in the low-risk group (suggesting responsiveness to immunotherapy), while macrophages were higher in the high-risk group tumors (suggesting unresponsiveness to immunotherapy) [78]. Although the gold-standards for prediction of immunotherapy response are still levels of immune-checkpoint molecules, the evidence discussed here suggests there is potential for LncRNAs to function as biomarkers to predict immunotherapy response in lung cancer, as well as to be therapeutic targets in combination with immune checkpoint inhibitors.
In summary, the LncRNAs discussed above have all been reported to modulate resistance to cancer therapies, although evidence for some therapy types is limited to other cancer settings. A caveat in the data presented is the focus on association studies, in vitro and xenograft assays (immunocompromised mice), without strong mechanistic insights.

4. LncRNAs as biomarkers in NSCLC

LncRNAs are highly stable molecules that can be found in the systemic circulation. They are resistant to degradation due in part to their secondary structures, transport by exosomes, and stabilizing post-translational modifications [79,80]. Therefore, the study of LncRNAs in circulation is a plausible non-invasive method of detecting and following cancer progression. Currently, the most commonly used biomarkers for NSCLC diagnosis from circulation are carcinoembryonic antigen (CEA), cytokeratin-19 fragment (CYFRA21-1), squamous cell carcinoma antigen (SCCA), prolactin (PRL), and carbohydrate antigen 125 (CA125), which can be used individually or combined as a signature [81,82]. Additionally, LncRNAs can also be stably found in urine [83] and even in nasal mucosa [84], although the latter has not been explored in the context of cancer. In this section, we review evidence for the potential of LncRNAs as disease biomarkers in lung cancer.
Tang et al analyzed LncRNAs expression in the blood from 232 patients diagnosed with NSCLC as compared to healthy controls. Expression levels of three LncRNAs (RP11-397D12.4, AC007403.1, ERICH1-AS1) was higher in disease versus health states. Importantly, this expression pattern was stable even after freeze-thaw cycles [85]. In a similar study, the LncRNAs SPRY4-IT1, ANRIL, and NEAT1 were found overexpressed in NSCLC versus healthy controls (N=50/group), and were detected stably after several freeze-thaw cycles and after samples exposure to room temperature for up to 24h [86]. A more recent study analyzed exosomal LncRNAs from blood of NSCLC patients vs. Tuberculosis patients or healthy controls. They report higher levels of RP5-977B1 in NSCLC compared to the two non-cancer groups. The diagnostic power of this LncRNA was greater than that of conventional biomarkers such as CEA and CYFRA21-1, and additionally worked for early-stage NSCLC, speaking to the promise of LncRNAs to detect early disease [87]. HOTAIR has also been evaluated for its diagnostic value for pathological staging of NSCLC. It was determined to have similar power as that of biomarkers CEA and CYFRA21-1 [88]. ESCCAL-1 is an oncogenic lncRNA, initially identified in esophageal cancer [89]. Results from a large cohort lung cancer screen show that ESCCAL-1 has increased expression in serum samples from lung cancer patients as compared to serum from patients with benign nodules or healthy individuals (unpublished data). These studies support the use of elevated lncRNAs expression individually or as signatures as biomarkers to predict disease or as staging tools in NSCLC. Clinical trials that evaluate the use of LncRNAs as biomarkers are currently ongoing for other cancers, according to the ClinicalTrials.gov database.
Comparable to tumor-promoting lncRNAs, tumor-suppressive lncRNAs such as GAS5 also show potential as biomarkers in NSCLC. Liang et al compared GAS5 levels in the plasma of 90 NSCLC patients vs. 33 healthy controls. They detected significantly lower levels of GAS5 in plasma derived from cancer patients. Additionally, they measured the dynamics of GAS5 before and after surgery, detecting an increase in GAS5 levels seven days after patients had surgery [90]. As discussed above, GAS5 has a clear tumor suppressive role in lung cancer, therefore, these studies are consistent and support the use of GAS5 as biomarker for diagnosis and for responses to clinical intervention.

5. LncRNAs as therapeutic targets

The main approaches to targeting LncRNAs are similar to protein-coding genes: inhibiting oncogenic LncRNAs or restoring function of tumor-suppressive LncRNAs. Here, we briefly describe advances in LncRNA therapeutics as well as some challenges.
Therapeutic approaches to target LncRNAs with oncogenic functions mainly use ASOs (anti-sense oligonucleotides). The core mechanisms of action of ASOs involve promoting cleavage of their RNA targets, impeding their translation, or modulating splicing [91]. There are several types of ASOs depending on their chemical modifications, such as locked nucleic acids and morpholinos, that may make them more resistant to degradation, provide better cellular availability, and lower off-target events [91]. As mentioned in sections above, ASOs are a strategy widely used in cell line and animal-based assays to elucidate LncRNAs role in cancer [27]. Although ASOs that target miRNAs have entered clinical trials [4,92], to our knowledge there are no therapies in clinical trials targeting LncRNAs directly. Small molecules are another strategy to interfere with oncogenic LncRNAs function [93]. For example, quercetin, a recently developed small molecule, binds to a MALAT1 triplex and modulates its transcript levels and functions in vitro [94]. Another small molecule recently identified, AC1NOD4Q, blocks the interaction between HOTAIR and EZH2 (a PRC2 subunit), impeding methylation of downstream targets [95]. Moreover, emerging strategies to silence LncRNAs in pre-clinical models use CRISPR-based approaches although these have not reached the clinical setting [96].
Restoring function of tumor suppressive LncRNAs can be achieved by gene therapy or administration of synthetic RNA molecules approaches. Although gene therapy has not reached the clinic for LncRNAs as targets, there are pre-clinical models exploring it in non-cancer contexts. For example, the LncRNA LeXis has been explored for exogenous administration using an adeno-associated virus vector in a murine model of Familial Hypercholesterolemia [97].
There are several challenges for RNA-based therapies reported from the field of miRNAs. For example, in the phase-I clinical trial for MRX34, a double-stranded miR-34a mimic encapsulated in liposomal nanoparticles, serious adverse events were immune-mediated toxicities, such as cytokine release syndrome, that resulted in 4 patient deaths and caused the trial to terminate early [98]. The delivery of these therapies is another challenge, as they may accumulate in detoxifying organs such as kidneys and liver, causing associated toxicities [99,100]. Additionally, off-target events and on-target events in non-tumor tissues can account for toxicities. Therefore, there is a need to detect immune-related toxicities in pre-clinical studies and design strategies to reduce their prevalence, to engineer delivery strategies that target organs of interest with lower off-site effects, as well as to increase target molecule specificity.

6. Conclusions/Perspectives

In summary, LncRNAs regulate key biological pathways of lung cancer such as tumor development, metastasis, and resistance to current therapies, summarized in Figure 1 and Table 1. Although the LncRNAs described in this review have been studied for at least 10 years, several aspects (such as roles in immunotherapy responses) remain to be explored in the context of lung cancer (Table 1). Because of the ability of LncRNAs to modulate function of other biomolecules, a comprehensive approach to studying their role in signaling pathways is necessary and should take into account the interactome between coding and non-coding molecules. Such approach has been recently reported for neural cell differentiation processes using CRISPRi and single-cell RNA-seq approaches [101]. Additionally, with the increasing access to single-cell sequencing technologies, it would be important to question cell of origin of LncRNAs expression within a tumor, such as for LUCAT1, reported to modulate immune responses.
LncRNAs have highly context-dependent roles. Therefore, robust in vivo studies to dissect mechanisms that account for physiological functions of LncRNAs are essential. Additionally, studies should be conducted in the presence of a competent immune system. Assays relying on xenografts (using immunocompromised mice) have two important caveats: 1) they do not account for potential physical interactions between tumor cells and microenvironment that may alter outcomes, and 2) they fail to predict potential side effects of LncRNA-targeting therapies that are immune system-dependent. This gap in knowledge presents an opportunity for the field to develop such approaches.
Lastly, even though the role of LncRNAs in therapy resistance is documented, a role in the persister cell state (minimal residual disease) has not been explored. Such studies could shed light on important survival mechanisms that drive therapeutic failure and disease recurrence. LncRNAs show promise as disease biomarkers based on their highly stable nature in circulation. An important aspect to explore deeper is their potential to detect disease in early stages and temporal circular detection of lncRNAs to monitor the therapeutic response. Such approach could be implemented for routine surveillance in advanced cancer patient groups.

Acknowledgments

T.G.B. is supported by funding from NIH/NCI and the Chan-Zuckerberg Biohub. J.G-A. is a recipient of a University of California President’s Post-Doctoral Fellowship.

Conflicts of Interest

T.G.B. receives funding support from Revolution Medicines, Kinnate, and Verastem and is an advisor to Revolution Medicines, Relay, Rain, Scorpion, Engine, Deciphera.

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Figure 1. Illustration of roles of LncRNAs in lung cancer.GAS5 acts as a miRNA sponge to activate their mRNA targets and inhibit cancer cell proliferation, therefore acting as a tumor suppressor. LUCAT1 can promote cancer cell proliferation by epigenetic silencing of p21 and p57 loci. Additionally, LUCAT1 may create a pro-tumorigenic microenvironment by inhibiting interferon-mediated responses through sequestration of STAT1. HOTAIR can promote cancer cell proliferation and metastasis by recruitment of PRC2 to methylate loci and repress gene expression, such as HOXA5 which is a tumor suppressor. MALAT1 stimulates lung cancer metastasis by potential recruitment of transcription factors (TF) to promoters of metastasis-associated genes. Green arrows represent positive influence. Red arrows represent inhibition.
Figure 1. Illustration of roles of LncRNAs in lung cancer.GAS5 acts as a miRNA sponge to activate their mRNA targets and inhibit cancer cell proliferation, therefore acting as a tumor suppressor. LUCAT1 can promote cancer cell proliferation by epigenetic silencing of p21 and p57 loci. Additionally, LUCAT1 may create a pro-tumorigenic microenvironment by inhibiting interferon-mediated responses through sequestration of STAT1. HOTAIR can promote cancer cell proliferation and metastasis by recruitment of PRC2 to methylate loci and repress gene expression, such as HOXA5 which is a tumor suppressor. MALAT1 stimulates lung cancer metastasis by potential recruitment of transcription factors (TF) to promoters of metastasis-associated genes. Green arrows represent positive influence. Red arrows represent inhibition.
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Table 1. Summary of roles of LncRNAs MALAT1, GAS5, LUCAT1, and HOTAIR in normal physiology and in lung cancer.
Table 1. Summary of roles of LncRNAs MALAT1, GAS5, LUCAT1, and HOTAIR in normal physiology and in lung cancer.
LncRNA Normal function Role in lung cancer Role in chemotherapy Role in radiotherapy Role in
targeted therapy		 Role in immunotherapy Potential as biomarker
in lung cancer
MALAT1 Regulation of neighboring genes expression [24,26] Oncogene
[20,27]		 Promotes resistance to cisplatin[56] Not explored May promote sensitivity: Down-regulated in EGFR-TKI resistant PC9 cells [60] May be associated with therapeutic failure: correlated with PD-L1 expression [76] Yes – higher levels in NSCLC [102]
GAS5 Cell cycle inhibition[33], cell differentiation[34] Tumor suppressor
[35]		 Promotes sensitivity to cisplatin[62] Promotes sensitivity [63] Promotes sensitivity to EGFR TKI [35] Not explored Yes – lower levels in NSCLC [90]
LUCAT1 Inhibition of immune responses [43,66] Oncogene
[40,42]		 Promotes resistance to cisplatin [64] Not explored Not explored Not explored Yes – higher levels in LUAD [103]
HOTAIR Regulation of HOX genes expression [9,10,11] by recruitment of histone-modifier enzymes [104] Oncogene [46,47,69] Promotes resistance to cisplatin [47] Promotes resistance [68] Controversial roles:
• Downregulated in EGFR-TKI resistant tumors [70]
• Downregulated in EGFR-TKI resistant PC9 cells [60]
• Upregulated in EGFR-TKI resistant PC9 cells [71]
• Promotes resistance to Crizotinib (ALK/ROS1 inhibitor) [72]		 Not explored Yes – higher levels in NSCLC [88]
TKI=tyrosine-kinase inhibitor, NSCLC=Non-Small Cell Lung Cancer, LUAD= Lung Adenocarcinoma.
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