RNA-Binding Proteins as a Molecular Link between COPD and Lung Cancer

Abstract Chronic obstructive pulmonary disease (COPD) represents an independent risk factor for lung cancer development. Accelerated cell senescence, induced by oxidative stress and inflammation, is a common pathogenic determinant of both COPD and lung cancer. The post transcriptional regulation of genes involved in these processes is finely regulated by RNA-binding proteins (RBPs), which regulate mRNA turnover, subcellular localization, splicing and translation. Multiple pro-inflammatory mediators (including cytokines, chemokines, proteins, growth factors and others), responsible of lung microenvironment alteration, are regulated by RBPs. Several mouse models have shown the implication of RBPs in multiple mechanisms that sustain chronic inflammation and neoplastic transformation. However, further studies are required to clarify the role of RBPs in the pathogenic mechanisms shared by lung cancer and COPD, in order to identify novel biomarkers and therapeutic targets. This review will therefore focus on the studies collectively indicating the role of RBPs in oxidative stress and chronic inflammation as common pathogenic mechanisms shared by lung cancer and COPD.


Introduction
Chronic obstructive pulmonary disease (COPD) and lung cancer are two major non-communicable diseases that share many important features -from genetic background and environmental triggers, to pathophysiological mechanisms and societal impact, as they dramatically affect public health worldwide.
The epidemiological and clinical association of lung cancer in COPD patients led to the search and identification of a complex network of shared pathogenic mechanisms of genetic and environmental nature.In particular, oxidative stress-driven inflammation and accelerated cellular senescence featured in COPD are key drivers of alteration of lung microenvironment -driven by aberrant signaling pathways, transcriptional regulation and epigenetic mechanisms -also conductive for lung cancer [1][2][3][4].
In the last two decades, the contribution of posttranscriptional gene regulation (PTGR) has been increasingly described in human cancer pathophysiology, while much less has been characterized regarding these mechanisms in chronic inflammatory diseases, in particular those conductive of increased cancer risk as COPD [5,6].Posttranscriptional regulatory mechanisms are fully integrated with transcriptional control through coordinated signaling and are dedicated to the regulation of RNA turnover and translation rates in all basic homeostatic process like cell cycle, proliferation, stress responses as well as in disease process [7].
PTGR is mediated mainly by two molecular species, RNA-binding proteins (RBPs) and noncoding RNAs (ncRNA), the latter being mainly represented by microRNAs (miRNAs).Both factors associate with mRNA molecules forming ribonucleoprotein complexes (mRNPs) through sequence-specific or conformational interactions and regulate every step of mRNA lifespan: from pre-mRNA splicing to cytoplasmic translocation, compartmentalization and translation of mature mRNAs [8].The role of miRNA in homeostatic and pathogenic process such as lung cancer and COPD has been subjected to many studies [9,10].A common miRNA signature, including miR-106a, miR-17, miR-15b, miR-107, and miR-103, has been recently identified in both COPD and lung cancer [10].RBPs have been studied in this field more recently, based on strong preclinical evidence implicating them in oxidative stress responses and inflammation [11].This review will therefore focus on the studies collectively indicating the role of RBPs in oxidative stress and chronic inflammation as common pathogenic mechanisms shared by lung cancer and COPD, in order to highlight their potential role as disease determinant/biomarker and therefore for prospective therapeutic targeting [12][13][14].

Molecular mechanisms linking COPD and lung cancer: current view
A complex interaction between multiple environmental especially cigarette smoke exposure and genetic factors underlies the development of both COPD and lung cancer [15].The existence of a genetic predisposition for those diseases has been pointed out by many genome-wide association studies (GWAS).Genetic-mapping studies have identified several single nucleotide polymorphisms (SNPs) in candidate genes associated with both conditions.
It is well established that COPD and lung cancer are associated with the same risk locus on chromosome 15q25 containing nicotinic acetylcholine receptor alpha subunits (CHRNA)-3 and CHRNA5 genes, which encode nicotinic acetylcholine receptors [16,17].Moreover, the rs1422795 SNP on the 5q33 locus, containing a disintegrin and metalloproteinase 19 (ADAM19) gene, confers susceptibility to both COPD and lung cancer, while the rs7671167 SNP on the 4q22 locus, containing family with sequence similarity 13 member A (FAM13A), appears to be protective for both COPD and lung cancer [18].Acquired somatic mutations, as result of cigarette smoke-induced mutagenesis, in genes involved in cell cycle regulation can lead to malignant transformation [15].Polymorphisms in p21 WAP/CIP1 gene, a cyclin-dependent kinase inhibitor known as a promoter of stimulus-induced cell cycle [19], have been associated to lung cancer risk [20] and increased expression of p21 WAP/ CIP1 is described in immune and structural cells of COPD patients [21].Along with increased rate of cell division, increased DNA damage and repression of the DNA repair mechanisms strongly support carcinogenesis.To this end, significant increase of 8-hydroxy-2-deoxyguanosine (8-OH-dG), a marker of oxidant-induced DNA damage, was detected in peripheral lung of smokers with and without COPD [4,22].The same research group demonstrated a selective decrease of Ku86 in the bronchiolar epithelium of patients with COPD and in a smoke-exposed mouse model susceptible to lung cancer [22].Ku86, together with Ku70, is part of DNA-binding regulatory subunits of the DNA-dependent protein kinase (DNA PK), initiating the repair of DNA double-strand breaks [23].They also showed that oxidative stress, reproduced in primary human bronchial epithelial cells upon stimulation with hydrogen peroxide, is able to reduce Ku86 protein as observed in COPD patients, suggesting a contribution of DNA damage/repair imbalance to increased risk of lung carcinoma in COPD [22].In general, oxidative DNA modifications, including 8-OH-dG mentioned above and 8-oxo-7-8-dihydroguanine (8-oxoG), localized in promoter or enhancer regions could act as epigenetic regulator of gene expression [24].Particularly, the signal-induced formation of localized oxidized bases in the promoter regions in many oncogene promoters is responsible of malignant transformation [24].Of note, differential expression of glutathione peroxidase (GPX) isoforms, a class of enzymes that protect cells from oxidative damages, was reported in lung cancer.Indeed, GPX2 was found significantly overexpressed in lung adenocarcinoma patients, while GPX3 expression was reduced in lung squamous cell carcinoma patients, both associated with poor prognosis [25].
Besides genetic factors, bioinformatics analysis building on large transcriptomic datasets are unbiasedly identifying novel gene expression patterns and pathways more closely related to disease phenotypes.Weighted gene co-expression network analysis (WGCNA), a bioinformatic method to analyze the relationship between different gene sets or between gene sets and clinical phenotype [26], was conducted on 13865 genes from 62 lung tissues of COPD patients with or without lung adenocarcinoma [27].20 co-expressed clusters were identified and one of them was specifically related to cell cycle, DNA transcription/replication and cancer pathways.In vitro, validation of selected genes identified increased gene levels of fizzy-related protein homolog 1 (FZR1), a gene involved in cell cycle as a specific activator of anaphase-promoting complex or cyclosome ubiquitin ligase [28], whose role in tumorigenesis is still controversial [29][30][31], in both airways and alveoli of COPD patients with lung adenocarcinoma and lung carcinoma epithelial cells exposed to cigarette smoke extract (CSE) [27].Along the same lines, a gene expression profile study identified 133 and 145 differentially expressed genes (DEGs) in patients with COPD and lung cancer, respectively, compared with healthy controls [32].The 1544 DEGs (889 up-regulated and 655 down-regulated genes) identified in patients with lung cancer coexisting with COPD, compared to COPD group, were associated with oxidation-reduction process, positive regulation of transcription from RNA polymerase II promoter and apoptosis.The secreted phosphoprotein 1 (SPP1), also known as osteopontin, a secreted extracellular matrix protein, was the only overlapping DEG that was up-regulated in COPD patients, lung cancer patients and patients with both of them.Increased expression of SPP1 in patients with lung cancer was associated with shorter survival time.Interestingly, SSP1 upregulation was previously correlated with non-small cell lung cancer (NSCLC) growth and progression [33] and its increase was also detected in epithelial cells of the small airways of patients with COPD [34].Boelens et al compared 35 squamous cell carcinoma (SCC) patients with and without COPD with whole-genome gene expression profile [35].They identified 309 genes differentially expressed in SCC from non-COPD and COPD patients.In particular, 248 genes had a decreased expression in SCC without COPD and 61 genes had a decreased expression in SCC with COPD.Most of the genes belonging to the latter group were involved in mitochondrial localization and function.Moreover, 33 of 309 genes were located on chromosomal arm 5q.SCC patients without COPD showed decreased expression of these genes and a more frequent copy number loss of 5q.
Lamontagne and his group mapped COPD candidate causal genes by integrating GWAS and lung expression quantitative trait loci (eQTL) data [36] Chronic pro-inflammatory status characterizing COPD is a potent driver for lung cancer development [37,38].The inflammatory network that characterizes COPD involves several immune cells such as neutrophils, alveolar macrophages, CD8 + T cytotoxic and CD4 + T lymphocytes that are activated and recruited though chemotactic mediators released upon cigarette smoke [39].In addition, the increase of innate lymphoid cells (ILCs) suggests that these cells contribute to induce and maintain pro-inflammatory status even after smoking cessation [40].The initial mechanistic studies indicated the key role of overexpression of proinflammatory transcription factors, like nuclear factor kappaB (NF-κB), in mediating the increased production of pro-inflammatory mediators such as cytokines and chemokines including interleukin (IL)-6, IL-8, IL-1β, tumor necrosis factor (TNF)-α, C-C motif ligand (CCL)-2, etc.These mediators further sustain their release by tumor cells, generating a complex inflammatory microenvironment [41].It is also now fully established that the inflammatory microenvironment in COPD lungs may influence lung cancer development through epigenetic alterations including DNA methylation, miRNA expression and histone acetylation [3] and new molecular species contributing to these mechanisms are being discovered.In an epigenome-wide association study, increased methylation of CCDC37 and MAP1B promoters and the consequent repression of those genes were prevalent in patients with both lung cancer and COPD [42].CCDC37 belongs to the gene family coding for coiled-coil domain containing proteins [43], and its hypermethylation was previously detected also in SCC [44].MAP1B, encoding for microtubule-associated protein 1B, one of the major cytoskeletal proteins, was described as positive modulator of transforming growth factor (TGF)-β1-induced epithelialmesenchymal transition (EMT) in NSCLC [45].Importantly, DNA methylation profiling of NSCLC revealed a COPD-driven immune-related signature, characterized by differentially methylated status of genes involved in immune responses, reinforcing the immune-based link between lung cancer and COPD [46].Recently, DNA hypermethylation of prostaglandin E receptor 4 gene (PTGER4), one of four receptors identified for the proinflammatory prostaglandin E2 (PGE2), was detected in the serum of patients with lung cancer compared to patients with benign lesions and patients with COPD [47].
Inflammation is a major endogenous contributor of oxidative stress burden, being a source of reactive nitrogen and oxygen species (RNOS), whose levels are persistently elevated in COPD patients [3,4].Chronic, unresolved inflammation is associated with an increased risk of malignant disease and RNOS, along with many inflammatory mediators influence cancer microenvironment, altering cell growth and differentiation, apoptosis and immune responses [48][49][50][51].Oxidative stress induces inflammation and accelerated senescence by many mechanisms, chiefly through activation of the cyclin-dependent kinase inhibitors p16 INK4   and p21 WAP/CIP1 , inducing cell cycle arrest.This condition, known as stress-induced senescence phenotype (SIPS) is one of the hallmarks of COPD [52].Senescent cells acquire a senescence-associated secretory phenotype (SASP) characterized by the activation of pro-inflammatory pathways resulting in the secretion and release of several pro-inflammatory mediators such as cytokines, chemokines, proteins, growth factors, prostanoids and proteases.This chronically expressed inflammatory secretome alters the local microenvironment leading to structural cell activation and reinforcing a skewed Th1/Th17-driven inflammation.The end damage of such tissue dysfunction manifests as fibrosis, parenchymal loss and may lead to malignant transformation [52][53][54].
The SASP is also fundamental in the context of tumor biology, can act as both pro-tumorigenic, but also tumor-suppressive factors.For example, the release of SASP-associated proteases induces the remodeling of extracellular matrix and tissue structure, promoting tumor cell invasion and metastasis [55].Senescent fibroblasts acquiring SASP are able to induce EMT in adjacent cancerous epithelial cells [56].As counterpart, SASP can recruit immune cells, including CD4 + T cells, macrophages and natural killer (NK) cells, that suppress tumorigenesis [55].
Chronic inflammation also enhances EMT and it is associated to invasive and metastatic lung cancer.Finally, active EMT was detected in airway epithelial basal cells of stable COPD patients, characterized by an irregular and fragmented epithelial reticular basement membrane and loss of epithelial markers [57].Increased levels of absent in melanoma 2 (AIM2), involved in innate immune response by recognizing cytosolic double-stranded DNA and inducing caspase-1-activating inflammasome formation [58], have been shown in cancerous tissues of both non-COPD and stable COPD patients with adenocarcinoma.Interestingly, non-cancerous tissues of smoking COPD patients had increased expression of AIM2 than smokers with normal lung function and it was correlated to an increased hazard ratio of poor survival rate [59].
Additional mechanisms and molecules are increasingly identified as potential link between COPD to lung cancer, although causative relations are yet to be fully established.For example, the release of circulating cell-free mitochondrial DNA (cf-MtDNA), as a consequence of lung tissue injury, further sustains inflammation and increased copy number of cf-MtDNA has been demonstrated both in serum sampled from COPD and NSCLC patients [60].
Interestingly, patients with both SCC and COPD show a significant overexpression of mitochondrial localization-related genes and reduced expression of mitochondrial transcription factor A (mtTFA), which regulates mtDNA replication [61].
Additionally, the increased expression of the immune checkpoint protein programmed death ligand 1 (PD-L1) in alveoli, bronchioles, and vessels of COPD patients with milder stages, along with similar increased expression of PD-L1 in alveolar macrophages in both COPD and NSCLC patients [62], suggests a potential link between the two diseases also on immune side.
The growing knowledge on how lung microenvironment shapes homeostatic and pathological tissue responses is increasingly uncovering the role of extracellular vesicles (EVs) as mediators of intercellular communication and regulator of many fundamental biological processes, including inflammation and neoplastic transformation [63].EVs are a group of membraned vesicles characterized by different size and origin.Microvesicles are the larger size class of EVs with a diameter of 50-500 nm and they are generated by budding of the plasma membrane.Exosomes are smaller EVs of 50-150 nm size originated in the lumen of multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell surface [64].EV intravescicular and membrane-bound contents appears to be tightly regulated.In EVs isolated from bronchoalveolar lavage (BAL) of stable COPD patients, increased expression of miR-451a and miR-663a was found compared to EV from healthy subjects [65].Similarly, overexpression of miR-27a-3p and miR-106b-3p was detected in EVs obtained from lung cancer cohort compared to stable COPD patients [66].Specific miRNA enrichment and, more in general, a specific signature of EVs cargo can be predictive of the physiological or pathological state of the origin cell and indicate how they could alter biological processes in recipient cells.

RNA-binding proteins: general outlook of structure and pathophysiological functions
As regulatory factors, RBPs constitute a heterogeneous group of modulators of PTGR that control maturation, stability, transport and degradation of cellular RNAs [67].RBPs mainly act as part of ribonucleoprotein (RNP) complexes, by binding conserved sequences mostly located in the untranslated regions (UTRs) of targeted mRNAs [8].The modular structure of RBPs, composed by the repetition of multiple domains, defines their functional activity [68].The majority of RBPs contain canonical RNA-binding domains (RBDs), such as RNA recognition motif (RRM), K homology (KH) domain, DEAD motif, double-stranded RNA-binding motif (DSRM) or zinc-finger domain [8,69].Moreover, the specificity of RBPs-RNA association is enhanced by the co-existence of multiple binding domains, along with onset of chemical interactions (hydrogen bonds, stacking interactions, weaker interactions) [70].Recent proteome-wide approaches further uncovered a number of highly conserved "unconventional" RBPs lacking the canonical RBDs.They mediate both highly specific and nonspecific RNA binding through unfolded and flexible regions of the protein (containing repeated motifs rich in glycine, lysine and arginine), so called intrinsically disordered regions (IDRs) [71].Interactions mediated by unconventional RBPs promote protein-RNA co-folding, interaction by shape complementary, scaffolding protein-RNA complexes or altering the activity of the bound protein.Unconventional RBPs include metabolic enzymes, such as 3-hydroxyacyl-CoA dehydrogenase type 2 (HSD17B10), regulators of alternative splicing, the E3 ubiquitin and ISG15 ligase TRIM25 and others.
RBP-RBP dynamic interplay further give complexity to the regulatory mechanisms of common target mRNAs.Particularly, the synergistic interaction between two RBPs with the same regulatory aim is known as cooperative model, in opposition to the competitive one, in which the antagonistic interaction leads to different regulatory outcome [72].
Particular sequences present in the 3'UTRs and 5'UTRs and, less often, in the coding sequence of the transcripts act as binding sites for RBPs, whose interaction determines their fate, controlling the stability and/or translation of targeted mRNAs.Computational studies identified more than 400 motifs for a collection of over 200 RBPs, conserved across 5 species [73].Highly relevant to PTGR regulation in lung disease context, genes involved in immune responses, inflammation and carcinogenesis are highly enriched for some of most conserved and well-characterized RBP binding sequences, the adenosine/uridine-rich elements, called AU-rich elements (AREs) [74][75][76].Several RBPs associating with ARE sequences of gene transcripts involved in these processes mediate mRNA destabilization, such as tristetraprolin (TTP), AU-rich element binding factor 1 (AUF-1) and KH-type splicing regulatory protein (KSRP), while others, like human antigen R (HuR) have a stabilizing effect [77][78][79].A group of ARE-binding proteins, including T-Cell-Restricted Intracellular Antigen-1 (TIA-1) and T-cell internal antigen-1 related protein (TIAR), induce translational silencing of targeted transcripts [80] [ Table 1].By binding to ARE-bearing transcripts induced by a specific response, RBPs coordinately regulate PTGR for multiple transcripts that are also functionally related.The regulatory activity of RBPs on gene expression is dynamic and adapts to cell conditions continuously.Several evidence of cross-talk between ARE-RBPs reveal that multiple factors contribute to determining the prevalence of one RBP over the others when recognizing the same mRNA target, by modulating RBP-RNA transient interactions and ultimately determining the functional outcome [81].Among these, the differential expression or subcellular localization of RBP isoforms, their conformational state or their post translational modifications can strongly determine the fate of the protein-RNA complexes.On the mRNA side, presence of specific sequences, secondary structure and epitranscriptomic modifications are also key determinants to take into account.
Alternative splicing of RBP-coding genes allows the synthesis of proteins with slight differences in amino acids sequence, yet conferring isoform-specific functions: for instance, the gene encoding for RBP AUF-1, also known as heterogeneous nuclear ribonucleoprotein (HNRNP)-D, originates four isoforms (p37, p40, p42, p45) deriving from the same pre-mRNA by differential splicing of exons 2 and 7, whose inclusion affects the isoforms' cellular localization and binding affinity [82].
Post translational modifications (e.g.phosphorylation, methylation, acetylation, ubiquitination and others) control several proprieties of RBPs, influencing their activity and localization, as well as the interaction with both target mRNAs and other proteins [83].Relevant to lung biology is the example of the RBP TTP, which presents several phosphorylation sites and is highly phosphorylated in vivo [84].Coelho et al. demonstrated how the phosphorylation status of TTP regulates the expression of PD-L1.In non-cancerous cells the functionally active form of TTP, associated to a low phosphorylation status, binds to the 3'UTR of PD-L1 transcript, accelerating its mRNA degradation and thus decreasing its expression.The activation of oncogenic RAS pathway in tumor cells, including lung cancer cells, makes TTP highly phosphorylated which inhibits its function, leading to higher PD-L1 expression and occurrence of tumor immune resistance [85].
A further level of regulation is given by modifications of mRNAs that modulate all aspects of RNA metabolism and related physiological and pathological processes.Reversible biochemical modifications of RNA -the epitranscriptome -trigger changes in RNA structure which modulate the accessibility of RBPs to RNA: as these changes can be induced also aberrantly by pathogenic changes, they can become a targetable event for therapeutic interference.In particular, the biological functions of N 6 -adenosine methylation (m6A) are mediated by special RNPs (i.e.methyltransferases, demethyltransferases) called writers, erasers and readers as they can respectively install, remove or recognize this modification.Through recognition and binding of reader RBPs, this modification can influence cellular processes, such as mRNA stability and translation, splicing, miRNA biogenesis, X-chromosome inactivation, in homeostatic and disease conditions [86,87].In fact, alterations in m6A patterns are present in pathologies such as cancer, obesity [88,89].

Role of RBPs in oxidative stress, inflammaging and SASP: a link between COPD e lung cancer
RBPs are recognized mediators of post-transcriptional regulation of genes involved in both oxidative stress and chronic inflammation [11], that have a key role in sustaining the accelerated, stress-induced premature cell senescence (SIPS) also defined as inflammaging [90,91], correlated with COPD and lung cancer pathogenesis.SASP mediator transcripts, including TNF-α, IL-1β, interferon (IFN)-γ, TGF-β, vascular endothelial growth factor (VEGF), C-X-C motif ligand (CXCL)-1, CXCL5, CXCL8, CCL2, CCL1 are largely regulated by PTGR.In particular, both preclinical in vitro and in vivo studies showed that they are targeted by the RBPs HuR, TTP, and AUF-1 [92,93].As general function, HuR acts as a positive regulator of mRNA stability and translation, while TTP and AUF-1 have been described as promoting mRNA decay of their targets, although AUF-1 function is indeed more complex, involving translational and transcriptional regulatory roles [82,94] [Table 2, Figure 1].Endogenous and exogenous triggers lead to aberrant activation of mechanistic target of rapamycin (mTOR) as a consequence of phosphatidylinositol 3-kinase (PI3K) activation due to loss of phosphatase and tensin homolog (PTEN) activity [52].Increased in mTOR activity is also triggered by decreased activation of AMP kinase (AMPK), leading to accelerated senescence and established of SASP through miR-34a-mediated inhibition of sirtuin-1 and sirtuin-6, key controllers of cellular senescence through multiple mechanisms [52].However, it is still now poor known that SASP factors are regulated by mTOR pathway also in RBPs-dependent manner [95].Indeed, inhibition of mTOR inhibits SASP by specifically downregulating MAPKAPK2 (also known as MK2) translation.MAPKAPK2 specifically phosphorylates at Ser 54, Ser 92 and Ser 203 the RBP ZFP36L1, also known as Tis11b or butyrate response factor (BRF-1), member of TTP zinc finger RBP family, inhibiting its binding to target mRNAs [96].Among the anti-inflammaging effects mediated by mTOR inhibition, downregulation of MAPKAPK2 decreases ZFP36L1 phosphorylation, a modification that blocks ZFP36L1 ability to degrade targeted SASP transcripts including IL-8, IL-1β, matrix metalloproteinase (MMP)-3, MMP10 and cyclin dependent kinase inhibitor (CDKN)-1A mRNA [95].
Moreover, mouse models TTP -/-and AUF-1 -/-, RBPs that enhances mRNA decay of inflammatory mediators, clearly indicate their relevance as endogenous 'brakes' for inflammatory responses.A particular relevance for pathogenic mechanisms shared by COPD and lung cancer -chronic inflammation and accelerated aging -is suggested by the phenotype of the mouse model knock out for the RBP AUF-1.This model shows, in different experimental settings, a complex phenotype with both overexpressed inflammatory process and accelerated aging [82].Decreased AUF-1 levels and activity induced accelerated aging as a result of influence both on transcriptional and posttranscriptional mechanisms: in fact, AUF-1 can act as transcription factor, upregulating transcription of telomerase enzyme (TERT) mRNA and as RBP, promoting the decay of the cyclin-dependent kinase inhibitors p21 WAP/CIP1 , contrasting senescence-induced cell cycle arrest [82].Moreover, mice AUF-1 -/-model are highly susceptible to endotoxin-induced septic shock with increased mortality due to an overexpressed inflammatory response, mediated by the lack of AUF-1-mediated degradation of TNF-α and IL-1β mRNA [5].
Besides demonstration of pathogenic potential for RBPs in mechanisms bridging inflammation and cancer in preclinical in vitro and in vivo, alterations of RBPs expression and intracellular localization have been increasingly identified also in human neoplastic diseases.In fact, AUF-1 is also implicated in proliferation and invasiveness of breast cancer cells through aberrant regulation of several targets: suppression of the cell cycle inhibitor CDKN2A, increased secretion of stromal cell-derived factor 1 (SDF1) and MMP-2, and facilitation of the EMT [97].Moreover, the RBP HNRNPC1/C2, part of the same protein family of AUF-1, was found significantly increased in COPD patients and in cigarette smoke-induced mouse model of COPD.As Figure 1.involvement of rbPs in senescence-associated secretory phenotype (sasP).exogenous (cigarette smoke) and endogenous (rOs/rns, inflammation, genetic predisposition) factors trigger changes in cellular phenotypes regulated by transcriptional and post-transcriptional events, coordinated by stress-induced signaling pathways.exemplary of post-transcriptional events, mtOr activation via Pi3K signaling leads to the inhibition of sirtuin-1 and sirtuin-6 through mir-34a upregulation, along with the downregulation of mrna degrading activity of the rbP ZfP36l1 via maPKaPK2-mediated phosphorylation.Pi3K signaling also acts as inhibitor of the rbP ttP.Dna damage induced by stress conditions results in cell cycle arrest as a consequence of the activation of cyclin-dependent kinase inhibitors p16 inK and p21 CiP1 , negatively regulated by the rbPs auf-1 and ttP.upon these conditions, targeted cells acquire sasP resulting in the secretion of multiple inflammatory cytokines, chemokines, proteins, growth factors vastly regulated by rbPs (short-listed in the figure), though complex changes in mrna stability and translation.among the rbPs characterized in these processes, hur has been reported to act globally as a positive regulator while auf-1, ttP and ZfP36l1 generally decrease mrna stability.auf-1 has also regulatory roles in transcription.Dysregulation of sasP factors leads to the development of COPD and lung cancer.see discussion [52,53].Created with biorender.com.abbreviations: amPK: amP-activated protein kinase; auf-1: au-rich element binding factor 1; CCl: C-C motif ligand; COPD: chronic obstructive pulmonary disease; CXCl: C-X-C motif ligand; hur: human antigen r; il: interleukin; maPKaPK2: mitogen-activated protein kinase-activated protein kinase 2; mir: microrna; mmP: matrix metalloproteinase; mtOr: mechanistic target of rapamycin; Pi3K: phosphatidylinositol 3-kinase; Pten: phosphatase and tensin homolog; rbP: rna binding protein; rOs: reactive oxygen species; rns: reactive nitrogen species; sasP: senescence-associated secretory phenotype; tGf: transforming growth factor; tnf: tumor necrosis factor; ttP: tristetraprolin; veGf: vascular endothelial growth factor a; ZfP36 l1 Zinc finger protein 36 -like 1.
regulator of telomerase biogenesis and telomere length regulation [98], upregulation of HNRNPC may promote accelerated aging of lung tissue in COPD [99].
The RBP HuR, mainly characterized as functional antagonist of mRNA-decay promoting RBPs, is also widely described in the pathogenesis of cancers in colon, pancreas, brain, lung and others [78].HuR localization in the cytoplasm of cancer cells is associated to the increased stabilization of important mediators of neoplastic transformation, such as the cationic amino acid transporter 1 (CAT1) and cyclooxygenase (COX)-2, acting as a repressor for miRNA binding [100,101].The RBP TTP is an immediate-early response gene and it regulates mitotic signaling pathways in many cancer types [102] and shares regulation of key inflammatory and stress response genes with other ARE-binding RBPs including HuR [103].The interplay between these two RBPs can regulate the expression of common targets in an agonistic or antagonistic manner [104].Loss of TTP has been identified as pathogenic due to the ensuing stabilization, with increased translation, of multiple cancer hallmark genes: in particular, what is chiefly relevant for cancer pathogenesis is the loss of balance between levels of TTP and its functional antagonist HuR: loss of TTP is paralleled by increase of expression and activation of HuR [105,106].HuR was found to be one of the most upregulated RBPs in small cell lung cancer (SCLC) [107] and cytoplasmic levels of HuR has been shown associated with high tumor grade and poor survival rate in NSCLC [108].In lung adenocarcinoma, TTP overexpression inhibits cell proliferation by inducing cell cycle arrest in the S phase and decreases the expression of autophagy-related transcripts, including Beclin1 and LC3II/I [109].
Sandri and colleagues conducted an unbiased multi-omic analysis to clarify the role of lung stroma in the malignant transformation that occurs in patients with COPD [110].The concurrent application of quantitative mass spectrometry, RNA sequencing on total cytoplasmic mRNA and polysomal-associated mRNA allowed to compare tumor tissue, tumor-adjacent stroma and stroma from stable COPD patients without and with lung cancer in terms of proteomics, transcriptomics and translatomics.Strikingly, this multi-omic approach identified that the regulation of gene expression in COPD lung tissue adjacent to the lung cancer -compared to that occurring in tissue not associated with lung cancer -occurs primarily through changes in translational efficiency, a chief posttranscriptional mechanism in which RBPs hold a master regulatory role [111][112][113][114]. Interestingly, pathway analysis showed activation of extracellular matrix differentiation pathway and PI3K-Akt signaling in the tumor adjacent tissue, suggesting that those intracellular signals could have a role in pre-malignant transformation in COPD.A significant part of downstream targets of this signaling pathways is regulated at the level of mRNA stability [115].In particular, PI3K signaling act as important functional inhibitor of the mRNA destabilizing activity of TTP, whose loss and functional inhibition are both well-described in cancer pathogenesis [6,116].
The same research group has applied a similar multi-omic approach in tissues derived from patients with and without lung cancer matched on the basis of lung function, ranging from normal to severe airflow obstruction [117].They showed that the translational changes resulting in the activation of mTOR pathway in tumor cells are associated to subjects with normal lung function or mild airflow obstruction while translational modifications leading to extracellular matrix pathway activation are observed in subjects with severe airflow obstruction.As PI3k-dependend and independent signaling pathway, mTOR impacts RBPs (reviewed in [111]).In particular, phosphorylation at Ser202 of HuR mediated by mTOR appears necessary for its regulatory function of cell proliferation [118,119].
There are emerging evidences regarding the role of RBPs in the pathogenesis of chronic inflammatory diseases, in particular of COPD, on the strong preclinical evidence of HuR, TTP, and AUF-1 as the three RBPs mainly regulating genes involved in proliferation/apoptosis, oxidative stress responsiveness, angiogenesis, immune response skewing in immune cells, and their targets [78,106].Earlier data indicated increased level of cold-inducible RNA-binding protein (CIRP) in patients with COPD and in rats with chronic airway inflammation [120] and decreased levels of AUF-1 mRNA were found in BAL cells and PBMC of patients with sarcoidosis, another chronic inflammatory lung disease [121].However, there are conflicting evidences regarding airway epithelial HuR expression in COPD [122,123].Recently, an immunohistochemical expression profile of RBPs has been described in lower airway samples of stable COPD patients.In particular, decreased AUF-1 levels, but not of HuR and TTP, were found in bronchiolar epithelium compared to smokers with normal lung function.The same pattern was reproduced in human bronchial epithelial cells upon inflammatory stimulation and, additionally, increased levels of inflammatory mediators regulated by AUF-1 were detected in condition of AUF-1 loss [123].Furthermore, in silico analysis of bronchiolar epithelial cell transcriptome from COPD patients and matched smoker and nonsmoker control subjects showed a global downregulation of RBPs expression; interestingly, several clusters of co-regulated RBPs were identified, revealing the potential for RBPs interplay and suggesting shared post-transcriptional regulation of biological pathways involved in COPD pathogenesis [124].
RBPs can also act as carriers of cellular RNAs into EVs released by immune and structural cells [125].The release of EVs containing RBPs could be a mechanism for spreading inflammation and accelerated aging to nearby cells, by transferring RNAs critically involved in these biological processes or by acting on transcripts present in the recipient cell.

Targeting RBPs: new tools and indications arising from cancer treatments
An important role of RBP-mediated control of pathogenic mechanisms shared by COPD and lung cancer can be inferred by pharmacological data.Aspirin is a widely used non-steroidal anti-inflammatory drug (NSAID) that acts not only by covalent modification of the COX2 enzyme but also through transcriptional regulation of immune-related genes [126].Its use in COPD patients was associated decreased risk of developing lung carcinoma and decreased lung carcinoma-related mortality [127].It is not known whether aspirin is able to interfere with RBP-mediated functions; to this end, HuR is a major positive regulator of COX2 mRNA stability [128] and this function has been experimentally targeted in colon cancer, where COX2 is a key pathogenic factor [129].Along the same lines, the anti-inflammatory effect of aged citrus peel (chenpi), commonly used in China as a dietary supplement for respiratory diseases, seems to prevent COPD progression to lung cancer through acting on PI3K-Akt-MAPK signaling pathway [130], opening to the hypothesis that this effect could affect downstream RBPs, regulated post translationally through these pathways [111].The risk of developing lung cancer in COPD patients treated with inhaled glucocorticoids (ICS) is still controversial.Systemic glucocorticoids in vivo induce the expression of TTP [131] and in agreement with the role of this RBP in cancer so far described the use of ICS seems to be protective against lung cancer, nevertheless other evidence suggest that ICS can correlate to lung cancer development as a consequence of increased respiratory infections [132].
Natural or synthetic modulators of NF-κB/ROS signaling show anti-cancer activity, controlling the levels of ROS, oxidative stress, cell cycle and apoptosis [133].It has been shown that quercetin, member of phenolic ROS/NF-κB signaling modulators, inhibits cell growth and proliferation of NSCLC cells [134].In the same manner, the treatment of dioscin-60 -O-acetate, a natural steroidal saponin, suppresses the growth of NSCLC and SCLC cells, through NF-κB signaling pathway [135].NF-κB-mediated inflammatory responses are strongly regulated by TTP [136] and this class of compounds could have an affect also on TTP and, in general, on RBPs.
Besides these indirect leads, RBPs targeting has been tackled as therapeutic approach in order to inhibit their expression or function, and several inhibition strategies have been developed to target RBPs especially involved in cancer.Small-molecules are able to inhibit RBPs function in multiple ways, from preventing the interaction with target RNAs to altering their enzymatic activity or inducing their degradation [137].MS-444 was the first small molecular inhibitor developed to target HuR, interfering with its dimerization and, consequently, inhibiting its binding to target mRNAs [138].RK-33 was found to specifically bind to RNA helicase DDX3 and block its helicase activity [139].The RBP Musashi protein is strongly inhibited by oleic acid, that prevent its interaction to target mRNA by binding to RRM1 protein domain and inducing conformational changes [140].
Nanoparticle delivery of small interfering RNA (siRNA) targeting RBPs is another promising approach.Inhibition of lung cancer cell proliferation was observed after HuR reduced expression achieved by delivering HuR-targeted siRNA [141].Interestingly, this approach is able to reduce the tumor burden in mouse models of lung cancer [142].
Natural or recombinant circular RNAs (circRNAs) may be another therapeutic strategy, since their ability to sequestrate single or multiple RBPs and regulate their activity [143].For example, HuR is bound by circPABPN1 preventing its binding to PABPN1 mRNA, resulting in decreased PABPN1 translation [144].
Attractive is the application of CRISPR/Cas9 system to directly target an RBP with multiple functional effect.For example, it could be possible to knockout an oncogenic RBP in cancer cells or modify the binding site in the target mRNA [137].

Conclusions
COPD represents a well-established risk factor for lung cancer development, although the full spectrum of molecular mechanisms underlying their associated incidence is not fully characterized.The complex biology of RBPs, critically shaped by their post translational and epitranscriptomic modifications, makes them able to rapidly adapt to homeostatic and pathologic cell conditions and exert a large functional impact, coordinately regulating multiple sets of genes and adjusting their protein output to the microenvironmental demands.In particular, RBPs roles in oxidative stress-sustained inflammation and neoplastic transformation, described so far in COPD and lung cancer preclinical and clinical models, could be particularly valuable as therapeutic targets not only, as presently pursued, when cancer is established but also to curb the neoplastic progression from COPD status.Further preclinical studies and specific mouse models are clearly needed to better understand RBPs role in responses shared by COPD and lung cancer and its therapeutic targetability.Specifically, mounting evidence indicate a key connection between chronic inflammation and progression to cancerous transformation through SASP.Acquisition of SASP by lung senescent cells, as a result of oxidative stress triggered by both endogenous and exogenous sources, leads to the onset of an inflammatory microenvironment that drives small airways remodeling and lung parenchyma destruction in COPD while, in lung cancer, it regulates tumor cells growth, invasion and metastasis.The data presented herein indicate that as SASP factors transcripts are widely regulated through RBPs, which in turn are targeted by major signaling pathways involved in COPD to cancer transition, their regulatory role could be a specific target in therapeutic strategies addressing accelerated, stress-induced cellular senescence.Furthermore, RBPs localized into EVs could be harnessed as new therapeutic strategies (e.g.drug delivery) or biomarkers for disease states, since their cargo reflects the physiology and microenvironment of the cells of origin.