**6. 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].



**miRNAs**

**Lung cancer** 

**Experimental model**

**Therapeutic** 

**Delivery** 

**Target gene**

**Obtained results**

Inhibition of VEGF‐C expression concomitant

[53]

with angiogenesis restriction

Low expression of the target genes that are

critical factors for angiogenesis

Inhibition of lymphangiogenesis

**References**

**system**

**approach**

Ectopic miR‐128

VEGF‐C VEGF‐A,

VEGFR‐2 and

VEGFR‐3

overexpression

**subtype**

miR‐128

NSCLC

NSCLC cells HUVECs and

NSCLC cells

Nude mice (A549

*In vivo*

replacement

therapy

cells)

miR‐497

NSCLC

NSCLC cells

Over expression

VEGF‐A

Decreases in the levels of VEGF‐A protein

[123]

274 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

with no significant changes for the VEGF‐A

mRNA; inhibition of cell invasion

Increased levels of VEGF‐A protein with no

significant changes for the VEGF‐A mRNA;

increased cell invasion

Restoration of the miR‐497 levels reduced

[54]

tumor development and angiogenesis in both

*in vitro* and *in vivo* experimental models

mir‐378 over expression models presented

[48]

tumors with decreased vascularisation

compared to the models with HMOX1

induced over expression; increased oxygen

partial pressure; increased MUC5AC,

Ang‐1,MMP12 levels and decreased TNF‐α

and IL‐1β levels ‐ all essential genes for

angiogenesis

Dowregulation of VEGF‐A gene correlated

[42]

with inhibited cell growth

of the miRNA

miRNA inhibition

NSCLC cells SCID mouse

xenograft model

miR‐378

NSCLC

Swiss nude

Overexpression

Lentiviral

HMOX1

vectors

particles

(pEZX‐MR03

backbone)

of miR‐378

immunodeficient

murine model

(NCI‐H292‐Luc

cells overexpressing

miR‐378—

subcutaneous

xenografts)

A549, Y‐90 and SPC‐

Overexpression

mir-126

VEGF‐A

expression

vector

(LV‐miR‐126)

of miR‐126

A1 cells

Tumor xenograft

mir-126

expression vector

(LV‐miR‐126)

model (A549

infected with

LV‐miR‐126)

miR‐126

Ectopic

HDGF

expression of the

miRNA sequence

Noncoding RNAs in Lung Cancer Angiogenesis http://dx.doi.org/10.5772/66529 275 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 the 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

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 the spotlight of artificial knockdown via siRNA delivery with great rates of success in lung cancer

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

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

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

enhancement strategies—miRNA replacement therapy (**Figure 6**) [139, 140].

and also breast and pancreatic malignancies [100].

276 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

limited regarding this area.

**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.

comparing to the naked oligonucleotides [144–147]. 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].

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.

## **7. 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 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.
