Emerging roles of RAC1 in treating lung cancer patients

Ting Zou1,2, Xiaoyuan Mao1,2, Jiye Yin1,2, Xi Li1,2, Juan Chen1,2, Tao Zhu1,2, Qiuqi
Accepted Article
Li1,2, Honghao Zhou1,2, Zhaoqian Liu1,2*

1
Department of Clinical Pharmacology, Xiangya Hospital, Central South University,
Changsha 410008; P. R. China;
2
Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory
of Pharmacogenetics, Changsha 410078; P. R. China.

*To whom correspondence should be addressed:

Professor Zhao-Qian Liu, Department of Clinical Pharmacology, Xiangya Hospital,
Central South University, Changsha 410008; P. R. China; Institute of Clinical
Pharmacology, Central South University; Hunan Key Laboratory of
Pharmacogenetics, Changsha 410078; P. R. China. Tel: +86 731 84805380, Fax: +86
731 82354476, E-mail: liuzhaoqian63@126.com.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgements
This work was supported by the National High-Tech R&D Program of China (863
Program) (2012AA02A517), the National Natural Science Foundation of China
(81373490, 81573508), and the Hunan Provincial Science and Technology Plan of
China (2015TP1043).

This article has been accepted for publication and undergone full peer review but has not gehprt
been through the copyediting, typesetting, pagination and proofreading process, which a
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1111/cge.12908

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 Graphical Abstract

In this review, we summarized the association between RAC1 and lung cancer. We

overview the classical binding cycle of RAC1 and GTP/GDP. We also summarized
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the upstream regulators and downstream effectors of RAC1. And we highlight the

feasibility of RAC1 in treating lung cancer patients as a novel drug target, which may

provide an important clue for future lung cancer treatments.

This article is protected by copyright. All rights reserved.

 Abstract

The Ras-related C3 botulinum toxin substrate 1 (RAC1), a member of the Rho

family of small guanosine triphosphatases (GTPases), is critical for many cellular

Accepted Article
activities, such as phagocytosis, adhesion, migration, motility, cell proliferation, and

axonal growth. Additionally, RAC1 plays an important role in cancer angiogenesis,

invasion, and migration, and it has been reported to be related to most cancers, such as

breast cancer, gastric cancer, testicular germ cell cancer, and lung cancer. Recently,

the therapeutic target of RAC1 in cancer has been investigated. In addition, some

investigations have shown that inhibition of RAC1 can reverse drug resistance in

non-small-cell lung cancer (NSCLC). In this review, we summarize the recent

advances in understanding the role of RAC1 in lung cancer and the underlying

mechanisms and discuss its value in clinical therapy.

Key words: Cell proliferation and apoptosis; Invasion; Lung cancer; Migration;

RAC1.

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 Introduction

The Ras-related C3 botulinum toxin substrate 1 (RAC1) is a member of the

Rho family of small GTPases and belongs to the Ras superfamily (1). Rho GTPases,
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including RHO, RAC1, and CDC42, are molecular switches that control a wide

variety of signal transduction pathways in all eukaryotic cells (2). RAC1 is localized

to chromosome 7p22 and comprises 7 exons over a length of 29 kb (3). It is critical

for many cellular activities, such as phagocytosis, adhesion and motility, cell

proliferation, axonal and dendrite growth, and angiogenesis. It is also a key regulator

of platelet functions and vascular pathology (4). RAC1 can mediate intracellular

transport and cellular transformation by interacting with its effectors, such as PI3

kinase, synaptojanin 2, IQGAPE, and phospholipase D1. The dysregulation of RAC1

can lead to numerous disorders and malignant transformation (5). Overexpression of

RAC1 was associated with lymph node metastasis, high TNM stage, and poor

differentiation in NSCLC patients (6). Downregulation of RAC1 can reduce cell

activation, such as migration, invasion, and the formation of lamellipodia (7).

Moreover, the inhibition of RAC1 can also sensitize gifitinib-resistant NSCLC cells to

gifitinib (8). Finally, we speculate that the dysregulation of RAC1 can lead to massive

cellular disorders, including platelet function, cell migration, adhesion, intracellular

transport, and cellular transformation.

Like other small GTPases, RAC1 can change its form by binding GTP or GDP

(9). RAC1 is activated when bound to GTP and thereby exerts its function to regulate

its downstream effectors. Otherwise, it cannot function in an inactive GDP-bound

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 form (10). There are three main upstream regulators of RAC1, including guanine

nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and guanine

nucleotide dissociation stimulators (GDSs) (11). GEFs can shift RAC1 from the
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GDP-bound form to the GTP-bound form, and the latter promotes active regulation.

GAPs stimulate GTP hydrolysis, which can lead to accumulation of the inactive

GDP-bound form. GDSs are involved in RAC1 inactivation by binding to C-terminal

prenyl groups on GTPases as cytosolic chaperones (12). Through this binding cycle,

RAC1 can regulate a system of cellular activity in an orderly manner (Figure 1).

Lung cancer is one of the leading causes of cancer-related deaths worldwide

(13). Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of lung

cancer (14). Moreover, the majority of patients are diagnosed in advanced stages,

which means that these patients have already missed their best treatment opportunity,

making this disease incurable (15). Platinum-based chemotherapy is the first-line

treatment for advanced lung cancer. In addition, surgical treatment, radiation

treatment, and molecular targeted therapy are also therapeutic options for lung cancer

patients in clinical settings (16) (Table 1). However, the efficiency and adverse effects

induced by platinum-based chemotherapy differ between individuals, and the

five-year survival rate of the majority of lung cancer patients remains very low (17).

In recent years, certain mutations in important genes, such as eIF3a and WISP1, have

been found to be associated with chemotherapy responses in NSCLC, and

corresponding drugs targeting these genes have been applied to clinical treatment (18,

19). RAC1 shows increased expression in lung cancer tissues compared with normal

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 tissues (20). Combined, these findings suggest that RAC1 may be an attractive

potential therapeutic target for lung cancer treatment.

In this review, we summarize the recent advances of RAC1 in lung cancer and
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its underlying mechanisms and discuss its applicable value in clinical therapy. First,

we overview the classical signalling pathway mediating the interaction between

RAC1 and GTP or GDP. We also classify the biological functions of RAC1, including

platelet function, cell adhesion and migration, and intracellular transport. Then, we

explore the relationship between RAC1 and lung cancer systematically from cellular

and clinical investigations. Moreover, we summarize the probable signalling pathways

mediating the relationship between RAC1 and lung cancer. Finally, we evaluate the

possibility of using RAC1 inhibitors for treating lung cancer patients. We highlight

the feasibility of RAC1 in treating lung cancer patients as a novel drug target, which

may provide an important clue for future lung cancer treatments.

Biological functions of RAC1

RAC1 in platelet function

Rho GTPases achieve their biological function by controlling downstream

effectors (21). Phosphatidylinositol 3 kinase (PI3K) is one of the crucial effectors of

Rho GTPases and can regulate the complex (22). RAS proteins can activate type I

PI3Ks, such as p110, p110, and p110, by interacting with them directly via an

amino-terminal RAS-binding domain (RBD) (23). Interestingly, p110, as a

ubiquitous member of PI3Ks, cannot bind RAS proteins. However, RAC1 can

activate p110 by binding to its RBD (24). These type I PI3Ks are also critical

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 proteins in the regulation of cellular transport and growth, including cell survival, cell

apoptosis, cell motility, adhesion, migration, cell proliferation, and metabolism (25).

p110, p110, p110, and p110 are ubiquitously expressed, and among them, p110
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also plays an important role in breast and prostate carcinogenesis, and particularly in

PTEN loss (26). Moreover, p110 is a crucial factor in integrin-dependent adhesion

and clot formation in platelets (27). RAC1 can activate these three type I PI3Ks,

including p110 (28). The molecular events of platelet activation are essential in

sepsis, atherothrombosis, and cancer in terms of both physiology and pathology (29).

RAC1 is highly expressed in platelets and has emerged as a crucial regulator in the

dynamics of the platelet actin cytoskeleton, playing a central role in platelet secretion,

aggregation, spreading, and thrombus formation (30). RAC1 may also have potential

effects in anti-angiogenesis therapy of human diseases that are associated with blood

vessel overgrowth and aberrant neovascularization (31).

The on-off relationship of RAC1 during cell adhesion and migration

Rho family GTPases are key regulators of cell adhesion and migration (32).

RAC1 activation regulates cell motility during cell migration and actin-driven

protrusions (33). E-cadherin is a classical cell-to-cell adhesion receptor. RAC1 can be

activated by E-cadherin contacts among different pathways (34). RAC1 also plays a

crucial role in the differentiation of epithelial organs by controlling polarization.

Some special activators or GEFs of RAC1 have been reported to play an essential role

in regulating adhesion junctions in epithelial cells (35). The adhesion of cells to the

extracellular matrix (ECM) plays an essential role in regulating important cellular

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 functions, such as migration, adhesion, and proliferation, and it is also important in

mammalian physiology. RAC1 plays an important role in mediating cellular responses

downstream of ECM factors (36).

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RAC1 in intracellular transport

The RAC subfamily, including RAC1, RAC2, RAC3, and RHOG, is involved

in cellular endocytic and exocytosis transport (37). As the best tagged member of the

RAC subfamily, RAC1 has been found to regulate endocytic and exocytosis

trafficking pathways (38). However, RAC1 does not regulate these pathways through

the common ER/Golgi interface or participate in the early secretory pathway, as do

other Rho GTPases, such as CDC42. RAC1 performs its function as a transporter in

aclathrin-dependent endocytosis by modulating synaptojanin 2 (PIP2) and PI3K (39).

PIP2 is a poly phosphoinositide phosphatase, which can induce the activation of

RAC1 GEFs to regulate vesicular trafficking and then mediate rearrangement in

diverse cell types by inducing PI3K-mediated PIP3 phosphorylation. PI3K is an

upstream regulator of RAC1 and a member of the phosphatidylinositol 4-phosphate

5-kinase family (40). RAC1 can regulate the subunit of PI3K in return and then

mediate the endothelial permeability to influence cellular transport (41). The

dysregulation of RAC1 may lead to malignant transformation in cells and result in

cancer development.

Association between RAC1 and lung cancer

In recent years, several studies have been conducted to investigate the

association between RAC1 and lung cancer in cellular and clinical studies. During

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 lung cancer, RAC1 has been shown to be overexpressed, and mutational or splicing

events have been found (42). RAC1 inhibitors can enhance chemotherapy sensitivity

in lung cancer patients and at least partially overcome drug-resistance in NSCLC.

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RAC1 and lung cancer in cellular studies

Overexpression of RAC1 has been reported in lung cancer. However, the

underlying mechanisms are not entirely clear despite many investigations.

Gastonguay A et al. found that RAC1 can regulate cell migration and cell proliferation

in non-small cell lung carcinoma, likely through its ability to promote NF-kappa B

(NF-B) activation (43). Knockdown of RAC1 by siRNA was correlated with the

activity of NF-B transcription and resulted in decreased cell migration and

proliferation. Moreover, the RAC1 inhibitor NSC23766 could also strongly inhibit

cell cycle progression, cell proliferation, and NF-B activity in lung cancer cells. The

RAC1 inhibitor NSC23766 could inhibit RAC1 to an even greater extent than siRNA.

This research highlighted the possibility of using RAC1 pathways as therapeutic

targets for the treatment of lung carcinoma. Shailaja Akunuru et al. showed that

blocking RAC1 could suppress NSCLC stem cell proliferation and metastasis activity

(20). Downregulation of RAC1 by shRNA suppressed the malignant phenotypes of

primary cells from NSCLC patients with respect to cell growth, proliferation, invasion,

sphere formation, and lung colonization assays. Downregulation of RAC1 could also

inhibit the activity of human NSCLC cell lines; the researchers observed that isolated

side population (SP) cells from human NSCLC cells contained elevated levels of

RAC1-GTP, and these were identified as putative cancer stem cells (CSCs). They

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 showed enhanced in vitro invasion, migration, and increased in vivo tumour initiating

and lung colonizing activities in xenografted mice. This evidence suggested that high

expression of RAC1 in CSCs was associated with increased activity of malignant

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cellular behaviour (44). shRNA targeting of RAC1 could inhibit the tumourigenic

activities of CSCs, which implies the potential of RAC1 as a drug target in the clinical

therapy of NSCLC. RAC1 has also been reported to play an important role in the

regulation of DNA damage responses (45). RAC1 might integrate DNA

damage-dependent and independent cellular stress responses upon genotoxin

treatment, which is in accordance with the mechanisms of DNA damage responses

(DDRs) associated with cell death, survival, and DNA repair (46). RAC1 can mediate

lung cancer development and progress through the regulation of cellular proliferation,

migration, and adhesion. It may also play important roles in DNA damage, which can

affect drug sensitivity in the clinical therapy of lung cancer.

RAC1 and lung cancer in clinical studies

As the importance of RAC1 in lung cancer treatment has been deeply studied,

many investigations have aimed at unravelling the role of RAC1 in lung

carcinogenesis based on clinical studies. Kai Yuan et al. performed a trial in early

operable non-small cell lung cancer patients and found that the RAC1 expression was

associated with prognosis (47). In their immunohistochemistry study of 111 tissue

samples from patients with early operable NSCLC, they found that RAC1 showed a

cytoplasmic pattern of expression, and its expression was higher than in normal lung

tissues that showed negative or weak cytoplasmic staining. The statistical analysis

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 showed that RAC1 expression significantly increased with the advancement of the

TNM stage (P<0.05) and T stage (P<0.01). RAC1 expression was related to poor

outcome according to overall survival analysis (P=0.012), even in stage I patients

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(P=0.023). Multivariate analysis showed that RAC1 overexpression was an

independent marker of overall survival after adjusting for other prognostic factors

(P=0.023) (47). Recently, we discovered that RAC1 mutations can affect

platinum-based chemotherapy toxicity in lung cancer patients. RAC1 rs836554,

rs4720672, and rs12536544 were associated with haematologic and gastrointestinal

toxicity. These polymorphisms of RAC1 may be novel and crucial genetic markers to

predict platinum-based chemotherapy toxicity in lung cancer patients (48) (Table 2).

The association between RAC1b and lung cancer

RAC1b, an alternatively spliced isoform of RAC1, is also preferentially

upregulated in lung cancer, especially stage I and II human lung adenocarcinoma, and

it is a key effector of lung cancer progression (49). RAC1b has also been found to

accelerate K-ras-induced lung tumourigenesis and act as an oncogene in lung cancer

(42). Research based on cultured cells has discovered that RAC1b may exhibit

oncogenic activities through the promotion of the epithelial-mesenchymal transition

and induction of mitochondrial reactive oxygen species (ROS) (50). The expression of

RAC1b was significantly correlated with sensitivity to the MAP2K (MEK) inhibitor

PD-0325901 in a cellular study (51). Clinical research based on genome and

transcriptome sequencing in lung cancer revealed that RAC1b is significantly

upregulated in lung cancer (52). In conclusion, as a spliced mutation of RAC1,

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 RAC1b plays an important role in the tumourigenesis and progression of lung cancer.

It is worthwhile to focus on the consequences of RAC1b in lung cancer therapy.

RAC1 signalling pathway in lung cancer

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RAC1 signalling pathways play important roles in tumour evolution and

progression. Given that RAC1 participates in many types of cellular signalling

pathways upon stimulation of cell surface receptors, deregulation of these signalling

pathways can give rise to a wide range of diseases (53). RAC1 has been reported to be

overexpressed in lung cancer, and RAC1 signalling pathways can contribute to

tumourigenesis, leading to lung cancer (54). RAC1 has many effectors related to lung

cancer, such as p21-activated kinases (PAKs), COX-2, Armus, E-cadherin, N-cadherin,

fibronectin, MMP2, and others (55). There are also many upstream regulators in the

RAC1 signalling pathways that can control cell tumourigenesis, angiogenesis,

haematogenous metastasis, cell-to-cell contacts, cellular invasion, adhesion, and

migration (Figure 2-3). The classic regulators of RAC1 are GEFs, GAPs, and GDS.

There are also other regulators belonging to the RAC1 signalling pathway.

An important branch of EGFR signalling can be activated through the

activation of RAC1, which promotes cell proliferation, survival, and cancer metastasis

through T-cell lymphoma invasion and metastasis 1 (Tiam1) in non-small-cell lung

cancer cells. There is an EGFR/PI3K/AKT axis that stimulates RAC1-GEF Tiam1

(56). Vascular endothelial growth factor (VEGF) can activate RAC1 through VEGF

receptors/PI3K and result in haematogenous metastasis to the lung (57). Armusasa

TBC/RABGAP protein can regulate the signalling pathway between ARF6, RAC1,

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 and RAB7 during junction disassembly and regulate the stability of cell-cell contact in

lung cancer cells (58).

RAC1 is essential in the regulation of cell invasion, adhesion, and migration.

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The deregulation of its signalling pathway is a clear path to malignant transformation

(59). Numerous upstream regulators participate in RAC1 signalling to modulate these

cellular activities. Vimentin is an intermediate filament protein, and its depletion can

lead to dephosphorylation of VAV2 (a GEF), a novel regulator of lung cancer cell

motility through the regulation of focal adhesion kinase (FAK) activity (60). PARD3,

a cell polarity regulator, can promote malignant invasion and affect tumour

aggressiveness and metastasis by stimulating TIAM to activate RAC1 and STAT3 in

lung squamous cell carcinomas (LSCC) (61). Loss of the small GTPase RhoB can

lead to the PP2A inhibition, E-cadherin repression, and Akt1 activation, which in turn

can activate RAC1 through the GEF Trio in lung cancer cells (62). Curcumin is a

crystalline and natural compound isolated from the plant Curcuma longa and has low

toxicity in normal cells. The inhibition of curcumin can inhibit RAC1/PAK1

signalling. Inhibition of MMP-2 and MMP-9 expression can result in the inhibition of

cell migration and invasion in lung cancer cells (63). RAC1 can also promote NF-B

activity to regulate cell proliferation and migration in non-small cell lung carcinoma

(43). The protein kinase Ciota (PKCiota), or Par6, can regulate the activation of Ect2

and then RAC1 to influence cell proliferation and invasion in NSCLC cells (64).

KAI1/CD82 can inhibit the PI3K/Akt/mTOR pathway to inhibit the expression of

RAC1, leading to decreased cell proliferation in H1299 lung cancer cells (65).

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 Phosphoprotein enriched in diabetes (PED) is upregulated in human lung cancer; it

can interact with RAC1-ser phosphorylation by regulating AKT and then modulate

ERK 1/2 nuclear localization to regulate cell migration and invasion processes in lung
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cancer cells (66). GPC receptors, integrins, ion channels, and GF receptors are

upstream regulators of RAC1 in the actin reorganization signalling pathway. Citron,

PKN, mDia, ROCK1/2, Rhotekin, and PIP5-K are the receptors for RAC1 to regulate

actin reorganization in cancer progression (67). Inhibition of RAC1 and IKK can lead

to TNF-mediated NF-B apoptosis of A549 lung cancer cells (68).

As a potential drug target, there are many regulators and effectors of RAC1

that modulate cellular activities. Any disorder of the RAC1 signalling pathway may

result in malignancy. This mechanistic evidence from lung cancer investigations

shows that RAC1 is of great importance and could be applied to clinical therapy as an

antitumour drug.

RAC1 and lung cancer in antitumour drug studies

Drug resistance and abundant unpredictable dose-limiting adverse drug

reactions (ADR) are the largest challenges preventing clinical therapy benefits in lung

cancer (69). Many endeavours have been undertaken to develop novel therapeutic

strategies. PAK1 is a major common downstream effector for RAC1 and can also play

an important role in RAC1-mediated invasion and metastasis (70). Naoki Kaneto et al.

also found that inhibiting RAC1 expression could suppress migration and growth of

epidermal growth factor receptor (EGFR) mutants in NSCLC cell lines. EGFR is

overexpressed by nearly 50% in NSCLC patients and is also associated with poor

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 prognosis and tumour growth. Although surgical resection is always the first line

treatment for early stage or operable lung cancer, with or without EGFR mutations,

EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy could follow first-line

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chemotherapy or replace first-line chemotherapy when an EGFR mutation is

discovered prior to first-line chemotherapy (71). Targeting the RAC1 pathway can

overcome resistance to EGFR-TKI in NSCLC patients, which may open a novel

avenue to avoid drug resistance in lung cancer patients with EGFR mutations (8). An

investigation showed that inhibition of RAC1-GEF DOCK3 by miR-512-3p could

also contribute to suppression of metastasis in non-small cell lung cancer (72).

miR-512-3p may play a critical role in tumour development as the expression of

miR-512-3p was low in most NSCLC samples compared with paired normal controls.

miR-512-3p may be an upstream regulatory factor of RAC1 and can also be applied to

clinical therapy for lung cancer as a potential target.

There are many cellular studies on RAC1 and its potential as a target for

antitumour drugs in lung cancer therapy. Drug resistance is a significant challenge in

clinical cancer treatment. There is a pressing need to identify new targets against

cellular transformation and drug resistance.

RAC1 inhibitors for antitumour therapy in lung cancer

RAC1 is an intracellular signal conductor, and it can regulate various cellular

functions, including cell migration, invasion, and cytoskeletal organization. Given

that RAC1 is upregulated in lung cancer, its inhibitor has been investigated to

determine its potential in clinical therapy. The most popular inhibitors of RAC1 are

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 NSC23766 and dominant negative RAC1 (RAC1N17) (73). NSC23766 can inhibit

the activity of RAC1 and also inhibit cell migration, invasion, and induce

rearrangements of the actin cytoskeleton in lung cancer cells (7). NSC23766 can
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reduce tumour cell and endothelial cell adhesion (74). In addition, it can sensitize

cells to antitumour drugs, which may be applied to drug resistant lung cancer patients.

NSC23766 can suppress cell migration and growth in EGFR-mutant NSCLC cells,

even in gefitinib-resistant cells. It can inhibit cell growth in vivo through MEK- or

PI3K-independent mechanisms (8). NSC23766 and RAC1N17 incompletely inhibited

tumour metastasis and led to a more efficient and specific attenuation of cancer cell

migration by simultaneous inactivation of RAC1 and RAP1 (75). Since these

NSC23766 and RAC1N17 studies were all performed in vitro, research on NSC23766

and RAC1N17 in clinical trials should be designed. These reports indicate that

inhibition of RAC1 can selectively inhibit tumour metastasis and be applied to clinical

therapy in lung cancer (Figure 4).

Many RAC1 inhibitors have already been invented, and their functions to

overcome drug resistance or as independent targets to reduce tumour progression in

lung cancer cells have also been identified. It is of great potential value to investigate

their possibility in clinical therapy and to apply them to lung cancer treatment as a

valid antitumour drug.

Association between RAC1 and other cancers

As an oncogene, RAC1 is associated with various cancers besides lung cancer,

such as melanoma, colorectal cancer, breast cancer, and glioma. P29S, a classical

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 mutation of RAC1, can regulate cell migration and invadopodia-mediated matrix

degradation in melanoma cells (76). Decreasing the expression of RAC1 can inhibit

cell migration and invasion in colorectal cancer cells, which indicates that RAC1 has
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potential value as a novel anticancer drug target in colorectal cancer (77). RAC1 is

overexpressed and correlated with poor prognosis in breast cancer, which may be due

to its regulation of the anti-apoptotic proteins Bcl-xL and Mcl-1, and it is also

associated with chemotherapy- or radiation-induced DNA damage (78). RAC1 is also

upregulated in glioma, and it can regulate cell migration and invasion in glioma cells.

RAC1 plays an important role in the treatment resistance and disease progression of

glioma (79).

Conclusions

RAC1 has long been recognized as an oncogene in antitumour drug discovery.

In this review, we summarized the biological function of RAC1 and the relationship

between RAC1 and lung cancer. We summarized the RAC1 signalling pathways that

are involved in lung cancer development and progression. There are many upstream

regulators and downstream effectors of RAC1 involved in this signalling pathway,

and any element with aberrant activity may lead to malignant tumour development. In

our previous study, RAC1 mutations were associated with platinum-based

chemotherapy toxicity in lung cancer patients. We also analysed the possibility of

applying RAC1 inhibitors to clinical therapy in lung cancer. In conclusion, this review

draws further attention to the relationship between RAC1 and lung cancer. Studies

focusing on the potential and significant value of RAC1, RAC1 polymorphisms, and

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 RAC1 inhibitors in lung cancer are meaningful.

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unrestricted use, distribution, and reproduction in any medium, provided appropriate

credit is given to the original author(s) and the source, a link is provided to the

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Accepted Article

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 Figu
ure legendss

Figu
ure 1 RAC1 regulatess many celllular functiions in two classical co
onformatioons.

RAC
C1 can exch
hange its acctive-inactiv
ve status to regulate
r thee activity off its
Accepted Article
dow
wnstream eff
ffectors. Whhen it is bouund to GDP, RAC1 is in
n an inactive state, and it

cannnot control the relevantt moleculess. GEFs (Guuanine Nuclleotide Exchhange Factoors)

can convert RA
AC1 to a GT
TP-binding activated foorm that can
n regulate th
he specific

effeectors dominnated by RA
AC1. GAPs (GTPase Activating
A Proteins) can
n change RA
AC1

from
m the activee GTP-bindiing form to the inactivee GDP-bind
ding form. GDSs
G (Guannine

nucleotide Disssociation Sttimulators) can

c situate RAC1
R in an
n inactive GDP-binding
G g

statee through binding to itss C-terminaal prenyl grooup as cytossolic chaperrones. These

threee regulatorss (GEFs, GA

APs, and GDSs) can modulate
m thee activity off RAC1 in a

metthodical man
nner, whichh contributes to the orderly regulattion of multtiple cellularr

and biological functions.

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 Figu
ure 2 RAC1 signallingg pathwayss associated
d with lungg cancer celll migration
n

d invasion. RAC1 is knnown to affeect tumour-rrelated celluular functioons. The

and

folloowing are several

s popuular regulato
ors and effeectors that participate
p inn RAC1
Accepted Article
signnalling-indu
uced regulattion of cell adhesion
a annd migration
n in lung caancer. PKC iota
i

can activate RA
AC1 throughh interaction with Ect22 to promotee cell migraation and

adhesion. PAR3 can stimuulate TIAM to active RA

AC1 and reegulate STA
AT3 expressiion

to ddrive malignnant invasioon. The loss of RhoB caan inhibit PP

P2A and E--cadherin,

whiich may indduce Akt1 acctivation, leeading to celll invasion through

t stim
mulating Triio (a

GEF
F) to regulaate RAC1. Curcumin
C caan inhibit thhe RAC1/PA
AK1 signallling pathwaay

and then inhibiit MMP-2 annd MMP-9 expression, which resuults in the innhibition off cell

miggration and invasion.

i PE
ED can inteeract with RAC1-ser
R phhosphorylatiion throughh the

conttrol of AKT
T and then activate
a the ERK1/2 patthway to modulate
m celll migration and

invaasion.

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 Figu
ure 3 RAC1 signallingg pathwayss associated
d with cellu
ular biologiical functioons

ung cancerr. As a moleecular switchh, RAC1 caan regulate various

in lu v celllular

phyysiopatholog
gical events, such as ceell adhesion, cell prolifeeration, tum
mourigenesiss,
Accepted Article
and apoptosis. There are several upstrream and doownstream molecules
m involved
i in

C1 signallin
RAC ng pathways. Vimentinn can stimulate VAV2 (aa GEF) andd then regulaate

RAC
C1 activity to affect ceell adhesion through FA
AK. KAI1/C
CD82 can innhibit the

PI3K
K/AKT/mT
TOR pathwaay to regulatte cell proliiferation by modulatingg RAC1.

EGF
FR/PI3K/AKT can conntrol the actiivity of TIA
AM1 to affect cell proliiferation,

survvival, and innvasion throough the reggulation of RAC1.

R RAC
C1 and IKK
K can affect

TNF
F-mediated NF-B to regulate
r cell apoptosis. RAC1b, a splice variaant of RAC11,

can interact witth K-RAS to

t affect tum
mourigenesiis.

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 ure 4 NSC 23766 is a selective in
Figu nhibitor of the RAC1--GEF interaaction.

Thee chemical name

n of NSC 23766 is N6-[2-[[4-(D
Diethylaminno)-1-methyylbutyl]
Accepted Article
amiino]-6-meth
hyl-4-pyrimiidinyl]-2-m
methyl-4, 6-qquinolinediaamine trihyddrochloridee.

NSC
C 23766 can
n prevent RAC1
R activaation by the RAC-speciific guaninee nucleotidee

exchhange factoors (GEFs) TrioN

T and Tiam1
T withoout affectingg CDC42 orr RhoA

activvation. NSC
C 23766 inhhibits RAC1
1-dependentt cellular fu
unctions and
d is reportedd to

reveerse tumourr cell phenottypes in lunng cancer ceells.

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 Table 1 Alternative treatments against lung cancer in clinical therapy

Alternative treatments and internal

Applicable patients Reference
classifications
Early and advanced lung
Surgery cancer excluding extensive (80)
Accepted Article
metastasis
Radiotherapy Most lung cancer (81)
Platinum/gemcitabine
Platinum/paclitaxel
Platinum-based
Platinum/navelbine Advanced stage lung cancer (82-86)
chemotherapy
Platinum/etoposide
Platinum/irinotecan
Patients with VEGF
receptor mutation and
VEGF inhibitors (87)
mostly in combination with
chemotherapy
Lung adenocarcinoma
EGFR inhibitors patients with EGFR (88)
Molecular mutations
targeted therapy Patients with ALK
ALK inhibitors mutations and EML4 as a (89)
fusion gene
Considering the severe
Other targets without
toxicity of the present
inhibitors, such as K-RAS, (90-92)
inhibitors, they are not
IGFR1, DDR and so on
effective therapy
Activate the immune system
Immunotherapy against the tumour as an (93)
assistant treatment
VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor;
ALK, anaplastic lymphoma kinase; K-RAS, Kirsten rat sarcoma 2 viral oncogene
homolog (a member of GTPase molecules); IGFR1, insulin-like growth factor 1
receptor (a mediator of cellular proliferation); DDR, discoidin domain receptors
(receptor tyrosine kinases that belong to the EGFR family).

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 Table 2 RAC1 mutations associated with the outcomes of lung cancer treatment
Polymorphisms Outcomes References
Accepted Article
RAC1b (a spliced It is highly expressed in lung cancer, and its
variant of RAC1, expression is significantly associated with
(52)
includes one sensitivity to a MAP2K (MEK) inhibitor
additional exon) PD-0325901 in clinical therapy.

RAC1 rs836554, These polymorphisms are significantly

rs4720672, and associated with platinum-based chemotherapy (48)
rs12536544 toxicity in lung cancer patients.

Several polymorphisms have been associated with lung cancer, and the increased
expression of RAC1 has long been recognized. The RAC1 polymorphisms rs836554,
rs4720672, and rs12536544 were significantly associated with platinum-based
chemotherapy toxicity (such as haematologic toxicity and gastrointestinal toxicity)
(P=0.018, P=0.044, and P=0.021, respectively). MAP2K, mitogen-activated protein
kinase.

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