Noncoding RNAs in Lung Cancer Angiogenesis

Lung cancer is the major death-related cancer in both men and women, due to late diagnostic and limited treatment efficacy. The angiogenic process that is responsible for the support of tumor progression and metastasis represents one of the main hallmarks of cancer. The role of VEGF signaling in angiogenesis is well‐established, and we summarize the role of semaphorins and their related receptors or hypoxia‐related factors role as prone of tumor microenvironment in angiogenic mechanisms. Newly, noncoding RNA transcripts (ncRNA) were identified to have vital functions in miscellaneous biological processes, including lung cancer angiogenesis. Therefore, due to their capacity to regulate almost all molecular pathways related with altered key genes, including those involved in angiogenesis and its microenvironment, ncRNAs can serve as diagnosis and prognosis markers or therapeutic targets. We intend to summarize the latest progress in the field of ncRNAs in lung cancer and their relation with hypoxia‐related factors and angiogenic genes, with a particular focus on ncRNAs relation to semaphorins.


Noncoding RNAs (ncRNAs)-definition, biogenesis and classification
The noncoding RNAs evolved in the last few years as important regulators of numerous physiological and pathological processes with increased attention regarding cancer diagnosis, prognosis, and therapeutics [1].The concept known as "dark matter" defined by the lack of function and lack of genetic information is now long gone, being replaced by the regulatory ncRNAs involved in cancer development and progression [1].The transcription of the noncoding regions produces RNA sequences that can vary in size, short, mid-size, and long noncoding RNAs, and are able to influence the expression of tumor suppressor or tumor promoting coding genes, activity that further classifies this class of RNAs into oncogenic or tumor suppressor sequences [2].
The noncoding niche is rapidly expanding as new sequences are discovered and characterized.The ncRNAs, as their name underline, are RNAs that do not codify for proteins but new molecular concepts are revealed regarding the interplay between these types of RNA sequences and protein coding genes [3].ncRNAs are also known as regulatory RNAs.
One of the most studied ncRNAs class is represented by microRNAs (miRNAs) that are small single-stranded nucleotide sequences (18-22 nucleotide length) capable of gene regulation through sequence complementarity [2], being involved in all hallmarks of cancer [4].The biogenesis mechanism is presented in Figure 1.The discovery of miRNAs has enabled new Figure 1.miRNA biogenesis mechanism.microRNAs are situated in the genome of the host as individual transcriptional units but also as clusters of a number of distinct microRNAs.For the first step, RNA polymerase II transcribes the target sequence resulting in a primary transcript named pri-miRNAs.This unprocessed sequence is then subjected to the activity of RNase III-type enzyme Drosha that transforms the pri-miRNA sequence into a transcript of approximatively 70 nt, pre-miRNA.This precursor is then transferred in the cytoplasm via Exportin-5, followed by another miRNA manipulation step governed by the RNase III protein Dicer, resulting in a double stranded RNA called miRNA-miRNA duplex.The less stable strand is further captured by the RISC complex, association that facilitates specific gene regulation through complementary interactions.
Physiologic and Pathologic Angiogenesis -Signaling Mechanisms and Targeted Therapy noninvasive diagnosis methods and also has conducted towards the development of more targeted therapeutics alternatives in a large number of cancers and other pathological states [4,5].Despite numerous discoveries in the ncRNA field, the two main noncoding fronts in cancer are still represented by microRNAs and the more recent characterized long noncoding RNAs (lncRNAs) [6].As the technology advances, these last sequences are increasingly mentioned in pathological contexts, where differential expression levels are associated with malignant states and other diseases [6].Despite the associations between lncRNAs expression patterns and different types of cancers, there are still many unknowns regarding the complex mechanism of action.
MiRNAs revolution has stimulated the investigation of other types of small ncRNAs such as small interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs) [3,7,8].These last two types of molecules are similar to miRNAs in length and function, where siRNAs mediate posttranscriptional inhibitory processes and piRNAs act particularly on transposable elements and are capable of forming complexes with Piwi proteins [7,8].piRNAs transcribed from kiwi clusters together with Piwi proteins are capable of transposon modulation through interruption of the specific transcript that will be no longer able to exercise their specific activity.Other types of ncRNAs, circularRNA (ciRNA) are formed through base pairing of intronic repeats that ends up with a complete circular fragment that is able to act as a miRNA sponge through complementary interactions [3,9].
Supplementing the complex regulatory networks of miRNAs, ciRNAs have recently emerged as new cancer modeling tools through miRNA targeting, escaping from the initial characterization as transcriptional "noise" [9,10].These types of transcripts are ubiquitous present in eukaryotic cells and competitively bind microRNAs sequences, functioning like an inhibitory sponge; process that could attribute a significant therapeutic potential to these circular fragments [9,10,11,12].In this sense, specific microRNAs are eliminated from the regulatory networks, influencing the expression scheme of target genes.Competitive endogenous RNA (ceRNA) describes a new mechanism of gene regulation, being involved in physiological and pathological processes [13].
The traditional concept that RNA molecules are just intermediary sequences between DNA and proteins is now replaced with more advanced molecular data, where shortand long-noncoding sequences play a key role in normal development and disease progression [14].SiRNAs and miRNAs are similar in length, approximatively 22 nucleotides, and are both processed by Dicer through cleavage.SiRNAs are derived from complementary dsRNA duplexes, where miRNAs originate from imperfect RNA hairpins from short introns or long transcripts [15][16][17][18].Both small noncoding types of sequences associate with Argonaute proteins in order to manipulate gene expression (generally through 3′UTRs) [19], although siRNAs are also involved in viral defense and transposon regulation.piR-NAs are the longest fragments from the small RNAs group, having approximatively 26-30 in length.This class associates with PIWI-clade Argonaute proteins in order to guide transposon activity and chromatin status [15,17].Long noncoding RNA group consist in all RNA sequences that are not responsible for protein generation and their length exceed 200 nucleotides, being further grouped in concordance with their genomic localization: intronic, intergenic, sense, and antisense ncRNAs to host gene locus [6,20].Biogenesis of lncRNAs is very similar with the processing activity of mRNAs molecule, being transcribed by RNA Pol II and also being subjected to the same epigenetic modifications and splicing signals.The functional roles of lncRNAs are more extended than in the case of small ncRNAs, a significant part being still incompletely understood.Briefly, this type of sequences is not so well conserved as miRNAs and also can control gene activity at different levels in a more complex scheme [2,6,16].

Lung cancer-molecular classification and survival rates
Lung cancer occupies the first place regarding the mortality rates from the oncological field, being characterized by an aggressive profile that ends with numerous deadly metastatic sites.One of the main reasons for the high mortality rates consists in the late diagnosis [21].According to the characteristics of the cancer cells, this malignancy presents itself in two major forms, one being small-cell lung cancer (SCLC), and the other being named non-small-cell lung cancer (NSCLC) according to the histological classification and another rare subtype, lung carcinoid tumor (LCT) [22,23].NSCLC ranks as the number one diagnosed type of lung cancer in the oncological field, being further divided into three histologic types: squamous cell carcinoma, large-cell carcinoma, and adenocarcinoma.Adenocarcinomas represent the most common subtype of NSCLC, with an incidence of 35-40% from all lung cancer cases, being the most lethal type of cancer in male population, and the second in women.This type of pulmonary malignancy frequently presents distant metastases and pleural effusions.Between a quarter and 30% of all lung cancer cases belong to the squamous cell carcinoma category.These particular tumors are mostly located in the central areas of the lungs, and were shown to be connected to tobacco smoking [24].Lung carcinoid tumors are very rare and represent about 5% of the lung cancers which grows very slowly and are rarely associated with metastasis [25].Despite the frequency drop, pulmonary tumors remain the major cause of death and morbidity around the world, being very aggressive and refractory to standard oncologic therapy [26], due to the late diagnostic [27].
Environmental and occupational exposure to different agents and an individual's susceptibility for these agents were associated with a risk of lung cancer in approximately 9-15% of cases.The cigarette smoke is the primary risk factor for the development of lung cancer and is estimated to be responsible for approximately 90% of all lung cancers [24], followed by asbestos [28], and radon [27].More than 300 harmful substances with 40 known potent carcinogens were discovered in tobacco smoke.
The classical therapeutic strategies like surgery and chemotherapy or radiation fail to accomplish their purpose in advanced pathological states.In the case of patients diagnosed early in the disease, the chances of survival are more promising, being observed a partial response to drugs based on platinum.However, even in this case, the final outcome is not necessary a positive one due to acquisition of treatment resistance.According to National Cancer Institute, survival rates for early stages of NSCLC are extremely low compared to other types of cancer, where the rate for the late stages of the same malignancy can reach even 1%: the 5 years survival rate for stage IA is approximately 49%, 45% for stage IB, 30% for stage IIA and 31% for IIB.The next stages, IIIA and IIIB, are associated with even more dramatically numbers, 14% and 5% respectively (Figure 2).For the case of metastatic lung cancer, where the tumor has spread within different body sites, the survival rates are extremely low (1%) [29,30].Therefore, a critical part of lung cancer management is represented by the discovery of specific molecular carcinogenic pathways in order to precisely target key molecules that are responsible for tumor development and avoid treatment resistance.ncRNAs study represents an important research direction for achieving these goals.

Angiogenesis-beyond hallmarks of lung cancer
Nowadays, the cancer hallmarks are at the center of carcinogenesis: prolonged proliferation signals, escaping of growth inhibitors, apoptosis inhibition, indefinite replicative potential, vascular network development (angiogenesis) and activation of cell invasion, and thus metastasis (Figure 3) [31].Although all of these hallmarks represent key elements without which tumorigenesis could not more or less advance, angiogenesis surpasses this listing of malignant processes: without the ability to receive oxygen and nutrients and evacuate waste products, the spreading of the tumor is naturally restricted.Moreover, the vessel network is one of the invasion routes used by transformed mesenchymal cell in order to evade from the original carcinogenic site and invade other tissues [31].All these features stand at the base of the therapeutic concept, where angiogenesis is one of the main signaling pathway targeted in the treatment of cancer patients, including individuals with lung cancer.Inhibition of this malignant progression pathway through exogenous administration of targeted agents in the form of ncRNAs/anti-ncRNAs will enable the proper management of tumor spreading and will serve as a feasible therapeutic strategy for lung cancer [33].
The most promising proangiogenic target in lung cancer is VEGF (vascular endothelial growth factor), more precisely the interaction of VEGF with the transmembrane receptors or receptors downstream the signaling pathways.However, prolonged exposure to VEGF/VEGFR inhibitors may force tumor cells to find alternative pathways for vascular Noncoding RNAs in Lung Cancer Angiogenesis http://dx.doi.org/10.5772/66529development [34].Additionally, some other angiogenic pathways have been explored with the same purpose, where FGFRs (fibroblast growth factor receptors), angiopoietin, PDGFRs (platelet-derived growth factor receptors), and, in the last few years, semaphorins and the related receptors captured the attention [32,34].The metastatic cascade, a multievent process that leads to the spreading of the tumor cells to numerous sites in the organism and causes death, represents the main challenge in cancer treatment and angiogenesis plays a major role in this progression [35].

Implication of ncRNAs in regulation of lung cancer angiogenesis
As a result of the limited success of the classical antiangiogenic therapies targeting VEGF and its related receptors [35,36], researchers have deepened their knowledge by analyzing the expression of ncRNAs sequences in this pathology (Figure 4) [37,38].The mechanism of lung cancer angiogenesis is far from being completely deciphered and implicit the process of therapeutic inhibition via ncRNAs remains to be further investigated.Targeting ncRNAs will enable a more precise treatment and will avoid compensatory mechanisms retrieved in lung cancer [2,37,38].Physiologic and Pathologic Angiogenesis -Signaling Mechanisms and Targeted Therapy

miRNAs related to lung cancer angiogenesis
Among all types of ncRNAs, miRNAs molecules are the most intensive studied in what regards novel cancer therapies.Although the majority of the studies are concentrated on oncogenic miRNA inhibition via exogenous delivery of complementary (antisense) sequences through different vectors, it seems that another therapeutic alternative consists in miRNA replacement.This last type of targeted treatment may be even more effective due to the fact that the predominant pathological model consists more in dowregulated tumor suppressor sequences than overexpressed oncogenic genes [5,21,39].
Until this moment, several miRNA patterns involved in different lung cancer processes such as cell proliferation, resistance to therapy, invasion, metastasis, and angiogenesis have been identified.We will focus on some important miRNAs that presented the most aberrant expression related to lung cancer angiogenesis (Tables 1 and 2).Malignant cells lacking nutrients and oxygen enter in hypoxic stress, state that promotes the signaling pathways related to angiogenesis in order to sustain cell proliferation.The same process is present at the metastatic sites, where mesenchymal cells that went through epithelial to mesenchymal transition are establishing new malignant formations.The complex malignant scheme is strictly regulated by noncoding RNAs (miRNAs and lncRNAs).Red -overexpressed ncRNAs; Green-downregulated ncRNAs. Noncoding

LncRNAs related to lung cancer angiogenesis
The number of lncRNAs has significantly increased due to the progresses offered by sequencing methods in genomic research.Long noncoding transcripts act as gene regulators via a wide range of mechanism [70], those related to lung cancer being summarized in Table 3.
The first long noncoding sequence associated with lung cancer was MALAT1 that through increased expression and gene targeting (caspase-8, caspase-3, BCL-XL, BCL-2, and BAX) promotes the proliferation and invasiveness of cancer cells.Recently it was emphasized to target SLUG gene via a competitively "sponging" miR-204 [71].Following this initial lncRNA, a significant list of lncRNA was associated with lung cancer progression or inhibition through modulation of key mechanisms involved in the hallmarks of lung pathology.The regulatory process is complex, lncRNAs being able to escort chromatin modifying enzymes to target loci within the genome, to bind the promoter of genes and modify the transcription process, to be processed into miRNAs and further act as short noncoding transcripts, and finally to modify the stability of specific mRNAs through direct binding [70].
Recent evidences suggested the role of PANDAR in lung cancer cell proliferation through p53/ PANDAR/NF-YA/Bcl-2 axis [72].Another lncRNA positively regulated by p53 is TUG1, whose downregulation is associated with increased cell proliferation and poor survival rate in lung cancer patients [73].Also, considering the antiangiogenetic role of the p53 gene and the positive correlation between the two sequences, there is a possible role for TUG1 in angiogenesis suppression, however further investigations are necessary.HOTTIP is a long noncoding transcript that is associated with tumor growth [74], process that involves the formation of new blood vessel network, fact that could transform HOTTIP into a new target for antiangiogenetic therapies.MVIH is associated with microvascular invasion in HCC, being upregulated in this type of cancer with an increased oncogenic potential.Further studies have investigated the possible role of the same lncRNA in lung cancer and the results were increasingly similar with the previous pathology, MVIH representing a biomarker for poor prognosis and associated tumor cell proliferation [75].There are also other lncRNAs with tumor suppressor or tumor promoting roles in lung cancer malignancies, like MEG3 (tumor suppressor), ANRIL, and AK001796 (oncogenic role) that are involved in cell proliferation and cell viability, processes that go hand in hand with the angiogenetic transformation [76][77][78].lnRNA BC087858 is overexpressed in NSCLC and was demonstrated to be connected with drug resistance via EGFR-TKIs axis [80].MEG3 was proved to be downregulated in tumoral tissue, and directly related with high tumoral stage.Preclinical studies demonstrated a reduced proliferation rate in the case of MEG3 overexpression, by targeting MDM2 and p53 proteins.MEG3 is presented not only as prognostic marker but also as important therapeutic target [76].ANRIL is overexpressed in lung cancer tissue, being correlated with tumor-node-metastasis stages and tumor size, but until now there are not presented data with a direct connection with angiogenesis [78].

Ultraconserved regions (UCRs)
Ultraconserved regions (UCRs) are genome sequences longer than 200 bp and, as the name suggests, are conserved within humans, rats, and mouse, preserving their nucleotide LincRNA-p21 p53 and HIF1A Regulation of TP53-dependent apoptosis and Warburg effect and angiogenesis [84] Table 3.The main lncRNAs involved in lung cancer angiogenesis and possible therapeutic targets for inhibiting lung cancers.
succession during the evolution [85,86].Until this moment there are a number of 481 conserved sequences, a part of them being situated at sensitive sites regarding cancer susceptibility and are further transcribed (T-UCR) into pathological expression patterns.Considering this recent discovery, it has been postulated that the differential expression pattern could serve as stratification tool in the oncology domain, being able to differentiate between human cancers and possible between molecular subtypes of carcinomas [85,86].
The exact mechanism that leads to aberrant expression of T-UCR is not fully deciphered, although it is thought that the primary regulation models are represented by miRNAs interactions and epigenetic modifications in CpG islands hypermethylation [85].
Calin et.al. were the first to discover the T-UCR spectrum in malignant cells compared with healthy ones and found significant differences between the two states [85].So far, molecular analysis have revealed different T-UCR signatures in a number of carcinomas, including prostate, hepatocellular, and colorectal cancer, as well as in chronic lymphocytic leukemia and neuroblastoma.Presently was observed upregulation of several T-UCRs and demonstrated by multiple investigations to be related with increased risks for tumour occurrence and a high metastatic rate.Therefore, the main investigation area is focused on integration of synthetic antisense oligonucleotides (ASOs) to inhibit T-UCR functions [85].In lung cancer, an important number of T-UCRs need to be characterized and then used for developing novel therapies.In spite of the interest on the T-UCR, there are only few investigations on T-UCR therapy.

ncRNAs related to hypoxia in lung cancer
Hypoxia is a preangiogenetic process driven by specific gene modifications, alterations that are able to induce the installation of the mesenchymal phenotype through epithelial to mesenchymal transition (EMT), acquisition of drug and radiation resistance, and propagation of lung cancer stem cells [87,88].Compared to other cancers, lung malignancy is severely sustained by the installation of hypoxia through complex interactions between specific molecules (HIF1α and miRNAs or other ncRNAs, as displayed in Tables 4 and 5) and establishment of noncoding regulatory networks related to connection with the cell cycle regulation, apoptosis or autophagy [88].
In terms of lung cancer hypoxia, miRNAs play a pivotal role through the ability to orchestrate extensive signaling networks involved in this carcinogenic step.MiR-200 family has been extensively characterized in numerous malignant scenarios and miR-200b member seems to have a role that could be exploited in the context of the clinical area regarding hypoxia induced EMT where cells acquire motility characteristic and are able to invade secondary sites within the organism promoting lung cancer metastasis [89].Reinforced expression of the tumor suppressor miRNAs inhibited EMT through regulation of key genes involved in this pathway [88].Another possible therapeutic target is represented by miR-21, that is elevated in NSCLC-derived cells grown under hypoxic conditions [90].Hypoxic conditions also triggered miR-155 overexpression and downregulation of FOXO3A target gene, and protects lung cancer cells to irradiation, elucidating a possible course of treatment through inhibition of miR-155 combined with radiotherapy [65].Hypoxia management has led to reduced angiogenesis and thus obtuse the malignant cell proliferation and survival due to deprivation of nutrients and oxygen via various molecules including the noncoding transcripts represented by miRNAs and lncRNAs.Multiple targeting through ncRNAs that are able to influence the fate of the hypoxic microenvironment will bring new insights into the pathogenesis of lung cancer, permitting the development of new clinical tools for cancer management, improving the concerning survival rate of this pathology.The list of miRNAs and lncRNAs implicated in the vascular invasion of the pulmonary malignancy is presented in Tables 4 and 5. LncRNAs have recently emerged as important prognosis and therapeutic tolls in different malignancies and even for specific carcinogenic processes as lung cancer hypoxia.One of the main studied lncRNAs is HOTAIR, pathological expressed in numerous malignant scenarios, being associated with tumor promoting roles and a negative outcome in oncological patients.It was demonstrated that this lncRNA is a direct target for HIF-1α that act as an enhancer of expression and contribute together to the securitization of hypoxia followed by cell proliferation, migration, and metastasis.This information could transform HOTAIR in a possible therapeutic target under hypoxic conditions for NSCLC, that is limited in what regards the therapeutic options [99,100].Another newly discovered lncRNAs in lung cancer hypoxia that is lncRNA-LET targeted by HIHDR.The interaction between these two molecules ends with reduction of histone acetylation at the promoter region of the noncoding transcripts and thus decreased expression.Moreover, the downregulation process secures the expression of nuclear factor 90 proteins, a key element for cell migration induced by hypoxia.This data suggest that lncRNA-LET can be used as a clinical tool against cancer promotion [97].
It was proved to target the apoptosis pathway via regulation by p53 and the Warburg effect.LincRNA-p21 is downregulated in tumor tissue, and has effect on the lung cancer patients via angiogenesis regulation [84].
Other important ncRNA structures with a significant role in the development of novel molecular therapies are represented by PIWI-interacting RNAs (piRNAs).piRNAs are recognized to be involved in transposon silencing and gene expression during development and the complete role on the somatic cells remains to be deciphered [8].In a recent paper were emphases a different piRNASs expression profiles between normal bronchial epithelial cells and lung cancer cells.The most downregulated piRNAs in lung cancer cells was piRNA-like-163 (piR-L-163) having as direct target the phosphorylated ERM (p-ERM) [102].S100A4-small interfering RNA (S100A4-siRNA) was proved to activate the apoptosis and increase the radiosensitivity of A549 lung cells.S100A4 may promote A549 cell proliferation but also invasion, and metastasis by regulating the expression of E-cadherin and p53 protein [103].

ncRNAs targeting semaphorines and its related receptors in lung cancer
Semaphorins are guidance molecules which were characterized initially as directing elements for axon outgrowth; however advances in genomic and translational medicine revealed a more complex role for these proteins, being involved in cell migration, vascular network, and tissue development [32,104].Considering their vital role in physiological processes is not surprising that these guidance proteins are also involved in similar pathological processes especially from the oncologic area, where they exercise the same functions, but in a negative manner [32].Therefore, semaphorins are implicated in carcinogenic establishment, metastasis, and especially angiogenesis in numerous cancers, including lung cancer.Regarding their role in angiogenesis, the family of semaphorins is divided into two main pathological classes: tumor suppressors inhibiting the angiogenic process and oncogenes through promotion of vascular invasion.Therefore, loss of expression in the case of antiangiogenic semaphorins and/or increased expression pattern for the procarcinogenic ones translates into sustaining of the malignant cells [106].Immediately after the establishment of their newly discovered role, in vitro and in vivo studies confirmed the ability of semaphorins to serve as therapeutic targets in the form of suppression or enhancement [32].Despite the fact that their role in pulmonary malignant processes is quite extensively studied, little is known about the ncRNAs regulatory action on the expression pattern of semaphorins.Deciphering the regulatory noncoding sequence panel for these proteins will enable a more advanced and specific molecular management of lung cancer, especially in angiogenesis that has a vital role regarding the maintenance of tumor cells integrity and proliferation.
The process of angiogenesis, can also occur through semaphorin receptors, neuropilins, and plexins (Figure 5).In the case of neuropilins, we encounter a multiple ligation system, this membrane proteins being able to bind both class-3 semaphorins, VEGF and growth factors.Also, this type of receptors that are essential to proper vascular development during organism development are generally mutated in lung cancer.On the other hand, in vivo suppression of neuropilins led to improper vascular network.
Among the first studies that elucidated the role of neuropilins in vascular development is the research where the authors observed that overexpressing of Nrp1 was lethal for embryos due to extensive vascular defects like overdevelopment of blood vessel network and deformed hearts [107].This discovery paved the way for further research in the area of cancer management with focus on targeted therapy.Therefore, it has been proven that a combined form of therapy represented by neuropilins inhibitors (semaphorin, anti-NRP, soluble NRP -B domain, and VEGF mimetics) administrated concomitant with anti-VEGF signaling molecules (kinase inhibitors, anti-VEGF, anti-VEGFR-2, and soluble VEGFR for VEGF) is more efficient than the classical antiangiogenic therapeutic strategy targeted towards VEGF alone [104].Research studies demonstrated a role for NRP1 and NRP2 in lung cancer progression and angiogenesis where these two molecules were observed as normally expressed in bronchial basal cells, and as it progressed in the severity of the cell lesions, the level of neuropilins increased significantly, concomitant with VEGF expression [104].NRP1 has been previously associated with cancer angiogenesis: overexpression of NRP1 in AT2.1 cells (in vitro model of prostate cancer) resulted in advanced vascular density, cell proliferation, and also inhibited apoptosis [108]; rat estrogen-induced pituitary tumors presented increased levels of NRP1, level that was also correlated in a positive manner with the aggressiveness of angiogenesis development [109].
The competitive binding of class-3 semaphorins and VEGF that in physiological conditions leads to the proper development of the vascular platform is changed during malignant scenarios where proangiogenic VEGF takes the lead due to mutations in the structure of the binding domain that decreases the complementarity with semaphorins or enhances the expression of receptors.Therefore, an alternative therapeutic pathway could be represented by the Physiologic and Pathologic Angiogenesis -Signaling Mechanisms and Targeted Therapy modulation of neuropilins (NRPs) expression.Furthermore, the specific malignant expression is most likely influenced by other molecules such as miRNAs and lncRNAs (Table 6).
Lung cancer therapies focused on semaphorins and their receptors are still an insufficiently explored domain that could hold great promises regarding the inhibition of cancer spreading.Considering the competitive binding between class-3 semaphorins and VEGF in vascular development, antiangiogenic strategies as antibodies for VEGF or NRP inhibition, soluble NRP or NRP blocking peptides have been tested with effective results [104,106].A more recent treatment compromising both VEGF and SEMA3A inhibitors have been applied in vitro and in vivo for colon cancer [105].Another type of action could be represented by the induced internalization of the neuropilins through administration of dextran sulfate and fucoidan that significantly decreased the number of endothelial surface receptors, including VEGFR [131].Although anti-VEGF molecules are well-known as efficient angiogenesis inhibitors, combining the modulation of VEGF/VEGFR with SEMA/NRP may hold significant clinical usage.Moreover, extension of the molecular insight regarding noncoding RNAs regulation of semaphorins and their receptor could improve even more the inhibition of angiogenesis if we take in consideration the ability of noncoding RNAs to regulate waste singling networks that involve more than one target gene.Physiologic and Pathologic Angiogenesis -Signaling Mechanisms and Targeted Therapy

ncRNAs therapies targeting lung cancer angiogenesis
Once considered the "trash" of the genome, the noncoding RNA sequences are now emerging as important therapeutic targets (Table 7).Due to the complex regulatory network involving ncRNAs and also because of the personalized pathological expression pattern among cancer types, subtypes and malignant stages, ncRNAs are subjected to numerous preclinical studies regarding their silencing or induced expression [2].A lipid-based delivery vehicle for tumor suppressor miR-34 was developed in order to enhance the expression of the specific molecule in a mouse model of non-small-cell lung cancer [132].This approach has demonstrated to be efficient in both locally and systemically administration, being observed a reinforced miR expression concomitant with downregulation of the specific targets.Moreover, the intravenous delivery of miR-34 mimic did not produce an immune reaction in mice, but unfortunately this was not the case in humans.Very recently, MRX34, the miR-34 mimic, was stopped to be administrated in a cancer clinical trial due to major immune reactions [133].
Another therapeutic alternative that is currently on the scientific spotlight consist in the manipulation of the ciRNAs that can function as microRNA sponges, modulating their oncogenic or tumor suppressor activity [136].Despite the fact that there is a number of research studies focused on this type of noncoding RNAs, relatively little is known about the regulatory mechanism of circRNAs in cancer development.Future perspectives imply ciRNAs-based therapy that can stand as "super-sponges" and modulate the activity of extended regulatory miRNA networks, influencing at a superior level the carcinoma progression [136].The discovery of lncRNAs as regulators of cancer development has naturally conducted towards potential therapeutic alternatives using these long fragments as direct targets.The expression pattern of these sequences has been also investigated in lung cancer and the list of oncogenic and tumor suppressor pathological expressed lncRNAs is continuously growing.Administration of siRNA, shRNA, and miRNAs or antisense oligonucleotides in order to inhibit oncogenic lncRNAs is currently under investigation [137].HOTAIR has been on spotlight of artificial knockdown via siRNA delivery with great rates of success in lung cancer and also breast and pancreatic malignancies [100].
Moreover, the same approaches have been shown to be effective for the reverse of cisplatin resistance through reduced expression of p21 [138].Downregulation of MALAT1 through shRNA delivery is also a potent therapeutic approach for lung cancer as it was shown reduced cell viability after this type of treatment [71].MALAT1 has been also inhibited by exogenous antisense oligonucleotides, approach that induced reduced cancer progression through cell cycle arrest [151].Considering that in lung cancer there are also downregulated tumor suppressor lncRNAs, the replacement therapy could also stand as an effective therapeutic approach for this type of carcinoma, but nonetheless the scientific information are quite limited regarding this area.
The discovery that miRNA sequences can act as key regulators in cancer pathways through aberrant expression has led to the idea that these fragments could serve as potent therapeutic targets [139].In this sense, several strategies have been implemented until now: inhibition strategies-inhibitory antisense oligonucleotides and delivery vectors (miRNA sponges) and enhancement strategies-miRNA replacement therapy (Figure 6) [139,140].
For the case of therapeutics that follow an antagonistic pattern, the activity of tumor promoting miRNAs that are hazardous expressed is inhibited via administration of single stranded oligonucleotides complementary with the specific molecule or with the target binding site on the mRNA molecule; in either situation, the interaction between miRNAs and mRNA molecules is blocked and the downstream pathological pathway is strongly affected [140].
Delivery of anti-miRNA oligonucleotides (AMOs) in the context of preclinical studies is still a problematic area considering the necessity of target administration, prolonged stability, and increased pharmacokinetic properties [140].In this means, there is an urgent need for efficient delivery vectors/vehicles that are able to fulfill the reminded request in order to accomplish the treatment purpose.The majority of the freely administrated oligonucleotides is retrieved in the liver and kidneys and then eliminated through the urine.Also, the necessary dose of synthetic sequences is usually very high and the chance for off-target delivery is also increasing.Establishment of an effective delivery system will break the grounds of miRNAs therapeutics and also other noncoding treatment strategies [139].The current strategy for in vivo administration implies conjugation-based methods, where miRNA sequences are conjugated with different molecules like cholesterol [141] and α-tocopherol [142].Although these studies have demonstrated promising results, the efficiency of miRNA targeting is still limited.Another type of delivery method consists in liposome-mediated delivery of siRNAs, where the first attempt [143] was to inhibit the replication of hepatitis B virus (HBV) in an animal model through administration of siRNAs integrated in as polyethylene glycol (PEG)-lipid conjugate (SNALP).Since then, different liposome-based vehicles have been tested and the results are encouraging considering that the administration dose is significantly decreased Physiologic and Pathologic Angiogenesis -Signaling Mechanisms and Targeted Therapy comparing to the naked oligonucleotides [144][145][146][147]. Progresses in the area of material science produced a promising in vivo delivery system in the form of polymer-based nanoparticles that are more flexible than liposomes and also can be produced in a more homogenous manner regarding their size and form [139].In respect of siRNAs and anti-miRs delivery, the size of the vehicle is very important in order to permit the passing through cellular compartments, where nanoparticles can fulfill this request having a size between 10 and 100 nm [139].Moreover, in order to avoid the stimulation of the immune system, cyclodextrin-PEG conjugated nanoparticles have been developed and tested for the inhibition of EWS-FLI1 in an in vivo model of Ewing's sarcoma [148].Attracting strategies for targeted therapies consist in the conjugation of siRNAs or anti-miR sequences with specific antibodies able to conduct the small fragments towards distinctive cells expressing the desired antigen [147,149,150].MicroRNAs have emerged as important regulators of lung cancer angiogenesis and also as key therapeutic targets regarding inhibition or enhancement strategies.MiRNAs sequences that are marked with green composed the tumor suppressor group that have been tested in the context of replacement therapies; the genes marked with the same color represent the target genes that have been downregulated after therapeutic modulation of miRNAs.Inversely, miRNAs sequences marked with red are oncogenic ones proposed for inhibition in what regards antiangiogenic programs; the genes marked with the same color also represent the target genes, but in this case their expression has been augmented.
Noncoding RNAs in Lung Cancer Angiogenesis http://dx.doi.org/10.5772/66529 MiRNA replacement therapy is more limited regarding the current attempts and results, although it seems to emerge as a more efficient form of treatment considering that the majority of pathological expressed miRNAs consist in downregulated or inhibited tumor suppressor sequences [140].Even if the success of this therapeutic strategy could be greater than miRNA inhibition workflows, the requirements for the structure and composition of the replacement fragment are much more stringent considering the necessity of RISC uptake.Furthermore, the impediments regarding the delivery system for these oligonucleotides are the same as in the case of inhibitory antisense attempts.

Conclusions and future perspectives
Lung cancer remains the most deadly disease from the oncological field, being an aggressive form of cancer that is usually diagnosed in late stages with minimal therapeutic alternatives.Even in the case of early discovery, the classical treatments have failed numerous times due to compensatory mechanisms developed within the tumor environment leading to the same negative outcome.Therefore, we face a crisis situation where we need to develop new therapeutic tools for lung cancer management able to target key elements/pathways, but avoid in the same time the possibility of alternative carcinogenic pathways activation.One of the hallmarks of cancer is represented by angiogenesis, process that is in the sight of researchers for some time, but the classical inhibition of central molecules like VEGF has failed to deliver long-lasting results.Therefore, ncRNAs have emerged as potential lifesaving agents due to the capacity of extensive modulation, where the same ncRNA is able to target multiple genes and regulate their function.Also the same microRNA, or more recently discovered, the lncRNA can be encounter in different consecutive processes in pulmonary carcinogenesis, as in the case of hypoxia and angiogenesis.Development of novel therapeutic tools able to transform the pathological expression of ncRNAs, mainly through silencing of upregulated patterns, will enable a more extensive, and in the same time, specific approach that will probably excludes the installation of compensatory mechanism and significantly contribute to a better outcome in lung cancer patients.The concept of noncoding RNAs as therapeutic targets in the clinical context is now more feasible than ever, being supported by numerous preclinical studies.One of the main approaches should involve manipulation of miRNAs that are actively implicated in the regulation of VEGF genes expression, genes that hold a key role in the vascular development process.Even more, a heterogeneous approach that implies the administration of different miRNA sequences able to target multiple genes and naturally multiple pathological pathways within the angiogenic process will represent a more extended form of therapy that could modify extensive regulatory networks.This type of targeting will also minimize the compensatory mechanisms that are usually encountered after the implementation of classical therapeutic strategies due to concomitant regulation of multiple signaling pathways.Additionally, some other approaches may be used for the inhibition of angiogenesis.For example, semaphorins are now emerging as important regulators of vascular density in malignancies, with possible roles as prognostic tools or even therapeutic targets.Inhibition of procarcinogenic semaphorins would represent a novel course of action regarding cancer treatment considering their central role in vascular density.Moreover, the Physiologic and Pathologic Angiogenesis -Signaling Mechanisms and Targeted Therapy receptors associated with semaphorins contain binding sites for both semaphorins and VEGF molecules, engaging the competitive binding between these types of molecules.Managing the expression of VEGF via miRNA therapy concomitant with the levels of neuropilins (semaphorins receptors) will enable a more dramatic approach that could have more drastic results for cancer development.
Current therapeutic programs are promoting the effectiveness of specific sequence inhibition or enhancement through administration of antisense oligonucleotides or supplementation of the same sequence through exogenous enhancement.Development of chemically modified oligonucleotides under the form of medication for individuals diagnosed with cancer is now at the close horizon.Administration of synthetic oligonucleotides for noncoding RNAs inhibition or upregulation will enhance the effect of the current therapeutic strategies by modulation of specific gene expression able to influence the carcinogenic process or even reverse the malignant installation.In this sense, it is now clearly understood that the major strategy towards cancer treatment is focused on taking advantage of the key roles of noncoding sequences regarding the modulation of entire aberrant regulatory networks through manipulation of central molecules.

Figure 2 .
Figure 2. The overall survival rates associated with different lung cancer subtypes (NSCLC, SCLC) and the 5-year survival rate based on lung cancer stages.

Figure 3 .
Figure 3. Lung cancer hallmarks with focus on angiogenesis.

Figure 4 .
Figure 4. Evolution of vascular network within lung cancer.Malignant cells lacking nutrients and oxygen enter in hypoxic stress, state that promotes the signaling pathways related to angiogenesis in order to sustain cell proliferation.The same process is present at the metastatic sites, where mesenchymal cells that went through epithelial to mesenchymal transition are establishing new malignant formations.The complex malignant scheme is strictly regulated by noncoding RNAs (miRNAs and lncRNAs).Red -overexpressed ncRNAs; Green-downregulated ncRNAs.

Figure 6 .
Figure 6.MicroRNAs have emerged as important regulators of lung cancer angiogenesis and also as key therapeutic targets regarding inhibition or enhancement strategies.MiRNAs sequences that are marked with green composed the tumor suppressor group that have been tested in the context of replacement therapies; the genes marked with the same color represent the target genes that have been downregulated after therapeutic modulation of miRNAs.Inversely, miRNAs sequences marked with red are oncogenic ones proposed for inhibition in what regards antiangiogenic programs; the genes marked with the same color also represent the target genes, but in this case their expression has been augmented.

66529 No. Name Location Length (nucleotides) Expression level Target gene Activity Possible role in lung cancer angiogenesis Clinical potential References
RNAs in Lung Cancer Angiogenesis http://dx.doi.org/10.5772/

Table 1 .
The main tumor suppressor altered miRNAs implicated in lung cancer angiogenesis.

Type of miRNA Expression level Name Target gene miRNA role in lung cancer hypoxia Possible clinical role of miRNAs in lung cancer References
Physiologic and Pathologic Angiogenesis -Signaling Mechanisms and Targeted Therapy

Table 4 .
Tumor suppressor and oncogenic miRNAs involved in lung cancer hypoxia with possible roles in diagnosis, prognosis, and therapy.

Table 5 .
LncRNAs involved in lung cancer hypoxia with possible roles in diagnosis, prognosis, and therapy.

Table 6 .
Semaphorin and the targeting miRNAs with implication in lung cancer.

Table 7 .
Some relevant miRNAs tested on cell culture and animal models as potential therapeutic agents in lung cancer.