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Long interspersed nucleotide element-1 (LINE-1) methylation in colorectal
cancer
Article in Clinica Chimica Acta · November 2018
DOI: 10.1016/j.cca.2018.11.018

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Contents lists available at ScienceDirect

Clinica Chimica Acta
journal homepage: www.elsevier.com/locate/cca

Review

Long interspersed nucleotide element-1 (LINE-1) methylation in colorectal
cancerT⁎

Mohammad Amin Kerachiana,b,c, , Matin Kerachiand,eaMedical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iranc
Cancer Genetics Research Unit, Reza Radiotherapy and Oncology Center, Mashhad, IrandFaculty of Medicine, McGill University, Montreal, CanadaeResearch Institute at McGill University Health Center, Montreal, Canadab

A R T I C LE I N FO

A B S T R A C T

Keywords:
Long interspersed nucleotide element-1
LINE
SINE
SVA
Methylation
Colon
CancerColorectal cancer (CRC) represents a group of molecularly heterogeneous diseases characterized by genetic andepigenetic alterations. Long interspersed nuclear elements (LINEs) are a form of retrotransposable element foundin many eukaryotic genomes. These LINEs, when active, can mobilize in the cell and steadily cause genomic
rearrangement. Active LINE reorganization is a source of endogenous mutagenesis and polymorphism in the cellthat brings about individual genomic variation. In normal somatic cells, these elements are heavily methylated
and thus mostly suppressed, in turn, preventing their potential for bringing about genomic instability. When
LINEs are inadequately controlled, they can play a role in the pathogenesis of several genetic diseases, such as
cancer. In tumor cells, LINE hypomethylation can reactivate the mobilization of these elements and is associatedwith both an advanced stage and a poor prognosis. In this article, we summarize the current knowledge surrounding LINE methylation, its correlation to CRC and its application as a diagnostic, prognostic and predictive
biomarker in colon cancer.

1. Introduction
Colorectal cancer (CRC) constitutes a signiﬁcant global healthburden, having led to over 700,000 deaths globally in 2012. It currently
stands as the third most common cancer in men and the second mostcommon in women [3], with a yearly incidence of over one million
cases worldwide [1,2]. In the United States and Europe, it is considered
the third and second leading cause of cancer mortality, respectively [1].
The primary cause of death is due to liver metastasis [4] and the mediansurvival rate with metastases is approaching 30 months [5,6]. Approximately 50% of patients with CRC develop tumor recurrences[7–9]. Fortunately, due to the implementation and growth of wide
spread cancer screening assays, such as colonoscopy and image-based
detection, as well as increasingly eﬀective therapies, the mortality of
CRC is subsiding in many countries [1].
CRC represents a group of molecularly heterogeneous diseases
characterized by genetic and epigenetic alterations along a tumorigenesis sequence [10,11]. In addition to genetic mutations, it is known
that epigenetic changes such as DNA methylation, the loss of genome

imprinting, and histone modiﬁcation are important factors for tumor
initiation, development and progression [12–14].
DNA methylation is a fundamental epigenetic process that, in mostcases, modulates genetic expression levels [15]. In tumor cells, abnormal DNA methylation may be classiﬁed into two categories: sitespeciﬁc CpG island promoter hypermethylation and global DNA hypomethylation [16]. Several studies in cancer cells have reported sitespeciﬁc CpG island promoter hypermethylation in tumor suppressor
genes and global DNA hypomethylation in repetitive sequences[17–20]. This is contrasted in normal somatic cells, where long interspersed nuclear elements (LINEs) are heavily methylated, limiting theactivities of retrotransposal elements and thus preventing genomic instability [21,22].
There exists a group of LINEs named, LINE-1 retrotransposon, whichconstitute a notable portion (approximately 17%–18%) of the humangenome. Accordingly, the methylation status of LINE-1 retrotransposons could reﬂect the global DNA methylation level of the
genome [16,23–28]. A low degree of methylation correlates to chromosomal instability and CRC dysplasia progression [10,29,30]. The⁎
Corresponding author at: Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Azadi Square, 917794-8564 Mashhad,Iran.
E-mail addresses: amin.kerachian@mail.mcgill.ca, kerachianma@mums.ac.ir (M.A. Kerachian).

https://doi.org/10.1016/j.cca.2018.11.018
Received 16 September 2018; Received in revised form 11 November 2018; Accepted 12 November 2018
Available online 13 November 2018
0009-8981/ © 2018 Elsevier B.V. All rights reserved.

Clinica Chimica Acta 488 (2019) 209–214

M.A. Kerachian, M. Kerachianand stomach) and wide in other tissues (such as the thyroid and esophagus) [46]. The LINE-1 methylation status has been also proposed tovary at individual genomic locations. One study of human cancer celllines demonstrated that the methylation levels diﬀer at nine LINE-1 loci[47]. Another study that evaluated the methylation patterns of 17 LINE1 loci in several cell types has shown that the methylation levels atthese loci are inﬂuenced diﬀerentially, depending on the location of theparticular sequences in the genome [48]. In can be said that thechanging methylation status observed in diﬀerent sets of LINE-1 loci
may lead to diﬀerent cellular phenotypes [16].

structure, retrotransposition process and correlation to CRC of LINE-1s
are discussed in detail in this literature review.
2. The structure of LINE-1
Repetitive sequences compose half of the human genome and are
subdivided into two main types. The ﬁrst type is referred to as satellite
repeats, also called tandem repeats. These refer to a repeating sequenceof one or more nucleotide located adjacent to one another. The second
type consists of interspersed repeats, or repeated sequences, that, unlike
satellite repeats, are scattered throughout the genome [16]. The humangenome has numerous interspersed repetitive sequences originatingfrom DNA transposons and retrotransposons [31]. The former of these
two elements moves through a “cut-and-paste mechanism” mediated byan element-encoded protein called transposase [16]. Three categories
of retrotransposons are known: LINE-1, SINE or Alu, and SVA elements.
The ﬁrst of these categories are autonomous element, capable of selfpropagation through RNA intermediates, and the latter two are nonautonomous, which depend on LINE-1 for activation. LINE-1retrotrasposons are present on both homologous chromosomes [16]. Nearly
500,000 of these LINE-1 s are truncated [32] such that only 3000 to5000 can be considered full length LINE-1 sequences throughout thehuman genome [25]. In general, the majority of LINE-1 s have lost theirretrotransposition competency due to 5′ truncations, inverted rearrangements, point mutations occurring during reverse transcription
or subsequent chromosomal replication of the inserted element.
The average human genome contains approximately 50–120 active
LINE-1 s, with highly active groups (about 5–10% of active elements)[33]. These active LINE-1 s utilize a “copy-and-paste” mechanism toinsert themselves throughout the genome, with potentially disruptive
eﬀects on neighboring genes or regulatory sequences. A full-length
LINE-1 has an internal promoter in its 5- untranslated region (UTR),
which ranges from +1 to 909 base pairs (bp) [34]. The initial 460 bp
region of the 5′- UTR includes 29 CpG sites, whose methylation status
has been extensively investigated and shown to be heavily methylated
in normal somatic cells [35–37]. Most methylated CpGs are located in
the 5′ region and can behave as internal promoters [22]. A full-length
LINE-1 element contains two promoters within its 5′-UTR region thatare aligned in opposing directions. One of these promoters acts as asense-promoter and regulates the transcription of retrotransposon-related genes such as an RNA-binding protein, an endonuclease, and areverse transcriptase. The second promoter which is aligned in the
opposite orientation, functions as an antisense promoter, and generates
an alternative transcription start site that results in induction of transcription of downstream genes [26,38]. In addition, LINE-1 retrotransposons consist of two open reading frames (ORF1 and ORF2)
ﬂanked by a 5′- and 3′- UTR. These two ORFs encode an RNA-binding
protein, and proteins with endonuclease and reverse-transcriptase activities, respectively [39].

3.2. Activity in tumor cells
LINE-1 mobilization can, over time, rearrange the genome, and thus
aﬀect genetic expression in many ways. Active LINE-1 s can continuallyreorganize the human genome, becoming a source of in-house mutagenesis and polymorphism, which results in individual genome variation via recombination and rearrangement. These events can participate
in the pathogenesis of many genetic diseases, such as cancer[16,49–53]. For instance, in cancer cells, LINE-1 retrotransposons caninactivate surrounding gene function through insertional mutagenesis,
aberrant splicing or DNA breaks, leading to genomic instability [41,54](Fig. 2). It should be noted that somatic LINE-1 insertions occur more
often in intergenic or heterochromatic regions [45], in cancer-speciﬁc
hypomethylation regions [43], and in genes commonly mutated incancer, suggesting an oncogenic eﬀect of LINE-1 insertions [38,55–57].
As a protective response to the LINE-1-induced mutagenesis eﬀect intumorogenesis, eukaryotic cells have developed several mechanisms toneutralize LINE-1 mobilization including siRNAs or piRNA-mediatedmechanisms. The bidirectional promoters within the LINE-1 5′-UTR cangive rise to sense and antisense RNAs, which, when bound to eachother, form double-stranded RNAs (dsRNAs). These dsRNAs are subsequently sliced by the enzyme Dicer into endogenous (endo)-siRNAs.
The resultant endo-siRNAs can then degrade LINE-1 mRNAs and silence
LINE-1 retrotransposition by triggering the RNA interference mechanism, thereby creating a negative regulatory loop. Like siRNA,
piRNA can also exert a negative regulation eﬀect on LINE-1 s. In addition to siRNA and piRNA, other defensive strategies against LINE-1
mobilization include the RNA helicase MOV10 (which degrades LINE-1
mRNAs and suppresses their translation) and APOBEC3 family members (which inhibits LINE-1 retrotransposition by varying mechanisms)[31].
4. LINE-1 methylation levels and molecular alterations in CRC
Some molecules such as folate play a crucial role in cellular methylation, and their deﬁciency can contribute to DNA hypomethylation[2]. Figueiredo et al. reported that folic acid supplementation does notinﬂuence LINE-1 methylation levels in the normal colonic mucosa [58].
In contrast, Liu et al. elucidated an association between the relative
distribution of folate species and global DNA hypomethylation in thenormal human colorectal mucosa [59].
The relationship between LINE-1 methylation levels and endogenous molecular alterations in CRC is relevant to our general understanding of the disease. The hypomethylation of LINE-1 was shownto inversely correlate with microsatellite instability (MSI) and the CpG
island methylator phenotype (CIMP) in CRC according to a study performed by Hur et al. [26]. On the contrary, Kuan et al. were not able toelucidate an association between tumor LINE-1 hypomethylation andthe molecular alterations including speciﬁc gene mutations, MSI andelevated microsatellite alterations at selected tetranucleotides [60].
Similarly to the Hur study, Iacopetta and colleagues showed that theLINE-1 methylation levels were inversely associated with the CpG island methylation of speciﬁc genes including MLH1, P16 (INK4A), APC,
TIMP3, ER, MYOD in normal colonic mucosa [61]. Ogino et al. indicated that LINE-1 methylation level was higher in MSI-positive

3. LINE-1 mobilization
3.1. Activity in normal cells
LINE-1 hypomethylation is normally active in the germline andduring embryogenesis, while being epigenetically suppressed in somatic cells. For the most part, LINE-1 s are highly methylated and inactivated in these somatic cells, however some continue to retain theircapacity to retrotranspose into new genomic locations [40] (Fig. 1). In
tumor cells, LINE-1 s can be reactivated through hypomethylation andparticipate in the pathological processes of cancer initiation and progression [25,31,40,41]. LINE-1 activity diﬀers in cellular function,varies within and among cancer types and ﬂuctuates during cancer
evolution [42–45]. The LINE-1 methylation levels in normal tissues arestrongly based on the tissue type; the range of LINE-1 methylation levels is narrow in some tissues (such as the liver, kidneys, breast, lungs
210

Clinica Chimica Acta 488 (2019) 209–214

M.A. Kerachian, M. KerachianFig. 1. Mobilization of a LINE-1 element. LINE-1 is a retrotransposon, meaning that its replication cycle involves an RNA intermediate. It is also an autonomous
element, which means that it encodes its own protein machinery for reverse transcription and integration of its sequence into the genome. Full-length LINE1 elements
comprise a 5′ UTR sequence that contains an internal bidirectional promoter (sense promotor, SP and antisense promotor, ASP), followed by open reading frame −1(ORF1) and − 2 ORF2, a 3′ UTR and a poly (A) tail. ORF1 encodes an RNA-binding protein (L1ORF1p) and the protein encoded by ORF2 (L1ORF2p) has anendonuclease (EN) domain and reverse transcriptase (RT) domain. It is assumed that nuclear import is required for ORF2p to access the genome. ORF2p hasendonuclease activity that nicks the DNA at an insertion site and leaves a 3′OH group. ORF1p is a major component of the LINE-1 RNP complexes formation. The
order of events during the mobilization of a LINE-1 element, which results in a 5′ truncated insertion is: Step 1: LINE1 transcription in the nucleus; Step 2: LINE-1
mRNA exportation to the cytoplasm; Step 3: ORF1, ORF2 translation and ribonucleoprotein (RNP) complex formation; Step 4: reverse transcription of the RNA by
ORF2p-mediated target primed reverse transcription (TPRT); and Step 5: de novo double-stranded insertion.

with a CRC family history include Lynch syndrome cases [66].
In CRC, LINE-1 hypomethylation may also signiﬁcantly correlatewith various clinical and pathological variables. The relationship between LINE-1 hypomethylation and early-onset CRC has recently been
the focus of much scientiﬁc attention. Early-onset CRC presents aclinically distinct CRC phenotype and is often associated with an unfavorable prognosis. Baba et al. showed that severe LINE-1 hypomethylation (methylation < 40%) appear signiﬁcantly more in younger
patients than in older individuals [63]. Antelo et al. identiﬁed a similar
relationship between early-onset CRC (≤50 years of age) and LINE-1
hypomethylation [67]. Thus, LINE-1 hypomethylation may be a potentially important marker for early-onset CRC [16]. Furthermore, arecent study has shown that LINE-1 hypomethylation is a prognostic
biomarker in early-stage rectal cancer, although the study did not examine the entire lower bowel for possible cancer [68]. An additional

tumors than in MSI-negative tumors, as well as in BRAF mutation tumors than in BRAF wild-type tumors in primary CRCs [62]. Baba et al.
manifested no signiﬁcant relationship between KRAS and PIK3CA mutations and LINE-1 methylation level in primary CRCs [63]. In a study
by Murata et al., it was demonstrated that in metastatic CRC tumors,the LINE-1 methylation level was not associated with MSI status or
KRAS, BRAF and PIK3CA mutations. However, their study was limited
by a rather small sample size [24]. A few other studies have shown theassociation of LINE-1 hypomethylation in CRCs with inferior survival,but, once again, their relatively small sample sizes might also aﬀect
their results [64,65]. Moreover, the association of LINE-1 hypomethylation with inferior survival appeared to be stronger in MSI-high caseswith a family history of CRC rather than in MSI-high cases without asimilar family history [10]. This conclusion may be overinterpreted
since the MSI-high/BRAF wild-type subtype and the MSI-high subtype

Fig. 2. The potential pathogenic functions of LINE-1 in cancer.
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M.A. Kerachian, M. Kerachiancolonic mucosa have been shown to be independent of age, body massindex, sex, smoking status, alcohol consumption and race [58]. In addition, LINE-1 methylation has minimal variability throughout thecolon in contrast to other loci [79], which is an important characteristic
for a diagnostic biomarker. Hur et al. reported that in CRCs with liver
metastasis, LINE-1 methylation levels were lower in the metastasis
versus the primary CRC tissue [26]. These examples indicate that LINE
methylation can change with tumor development and progression but
not all studies have conﬁrmed this ﬁnding. In one particular study of869 population-based CRC tumors, Ogino et al. demonstrated that
LINE-1 methylation levels were not associated with tumor stage [62].
Murata et al. also found identical LINE-1 methylation levels between
primary tumors and liver metastases. In addition, they reported theabsence of heterogeneity of the LINE-1 methylation level in superﬁcial
regions with that of the invasive front regions in primary tumors. It was
assumed that cancer cells acquired the tumoral characteristic of global
DNA hypomethylation during the early stage of tumor development andthe cells maintained this characteristic throughout the process of metastasis [24]. These studies were in accord with an additional study by
Matsunoki et al. [80], which reported that LINE methylation was relatively stable during CRC progression. More studies with larger sample
sizes are needed to explain the current discrepancies.

study by Rhee et al. showed that LINE-1 hypomethylation is associatedwith adverse prognoses in MSI-high CRCs [69]. The association of LINE1 hypomethylation with lower bowel cancer survival rates was found to
be stronger in the MSI-high/BRAF wild-type subtype, which is linked tothe most favorable prognosis among subtypes [70]. The potential mechanisms of interaction between LINE-1 methylation levels and MSI
status have been reported in several studies. Compared to microsatellite
stable (MSS) tumors, MSI-high variants are characterized by numerous
somatic mutations [71] that may be more readily inﬂuenced by LINE-1
hypomethylation leading to chromosomal instability. Other possible
mechanisms may involve inﬂammatory mediators, variation in locus
speciﬁc methylation patterns, and non-coding RNAs describing the interaction of LINE-1 methylation level and MSI status [10].
5. Role of LINE-1 hypomethylation in CRC
In 1992, cancer-associated with LINE-1 mutagenesis was reported
when a somatic LINE-1 insertion into the APC gene was found to havecaused a gene disruption in a colon cancer sample [50]. In a study by
Lee et al., somatic LINE-1 insertions were more frequently found in CRC
than other cancers such as prostate and ovarian cancers, and interestingly, no somatic LINE-1 insertions were detected in glioblastoma andmultiple myeloma [43]. In cancer tissues obtained from CRC patients,
the numbers of somatic LINE-1 insertions ranged from 2 to 106, indicating the possibility of the LINE-1 retrotransposition proﬁle as asignature for diﬀerent subtypes of cancer [31]. In 2014, Barchitta et al.
reported a meta-analysis explaining the role of LINE-1 hypomethylationin human cancer. They demonstrated that LINE-1 methylation levels
were signiﬁcantly lower in cancer patients than in control samples,
especially in certain cancer types. The results were conﬁned to tissue
samples, both fresh/frozen and FFPE specimens [72]. Another metaanalysis supported the correlation between LINE hypomethylation andpoor prognosis in cancer patients [73]. More speciﬁcally, LINE-1 hypomethylated in CRC had been associated with advanced stage andpoor prognosis [10,74].

5.2. LINE-1 as a prognostic biomarker in CRC
Most research on the LINE-1 methylation levels in gastrointestinal
cancers has originated from CRC tumors, in which the levels of LINE-1
methylation are variably diminished. In in vitro studies, the levels ofLINE-1 methylation in colon cancer cell lines (COLO205, HCC-2998,
HCT116, HCT15, HT29, RKO, SW48, KM12, LOVO, SW620) are highlyvariable, ranging from 30 to 70% [81]. In a study of 643 CRCs in two
independent prospective cohorts, Ogino et al. demonstrated that theextent of LINE-1 hypomethylation is associated linearly with tumor
invasiveness. They observed an approximately ﬁve-fold increase incancer-speciﬁc mortality as the LINE-1 methylation levels of the tumor
ranged from high to low [64]. Ahn et al. showed that a lower LINE-1
methylation level in CRC is more commonly associated with unfavorable prognoses in patients with resected stage III proximal, but notdistal cancers [65]. If a potential informative high-risk biomarker exists
in adenomas associated with concomitant or synchronous CRCs, a potential biomarker with the ability to predict the risk of metachronous
neoplasia and provide revealing information on potentially missed
synchronous lesions could very well be considered [11]. In a metaanalysis performed by Ye et al., LINE-1 methylation was signiﬁcantly
associated with the survival of CRC patients, which could be a predictive factor for CRC prognosis [82]. Hypomethylation in LINE-1 andMSI-high in CRC have been associated with inferior and superior survival, respectively; however, it remains uncertain whether the prognostic association of LINE-1 hypomethylation diﬀers by MSI status.
Inamura et al. elucidated that the association of LINE-1 hypomethylation with inferior survival is stronger in MSI-high CRCs than in MSS
CRCs. Hence, the low LINE-1 methylation level in CRC could also berecognized as a prognostic biomarker. It could be a useful prognostic
biomarker to identify aggressive carcinomas among MSI-high CRCs
[10] and LINE-1 methylation assay may also be a useful prognostic
marker in both sporadic and hereditary CRCs for the early detection ofcancer [9,70].

5.1. LINE-1 as a diagnostic biomarker in CRC
Several studies have investigated the associations between LINE-1
methylation levels and cancer risk, progression, and prognosis, with amajority of these studies supporting the correlation between tissue
LINE-1 hypomethylation and increased cancer risk [42,44,75]. For instance, in patients with a family history of CRC, colonic LINE-1 hypomethylation was linked to a higher CRC risk indicating an association
between genetic predisposition and somatic epigenetic changes [75].
LINE-1 methylation levels in normal colon tissues have also been explored in multiple studies. Kamiyama et al. evaluated the LINE-1 methylation levels in cancer tissues obtained from CRC patients andmatched them with non-cancerous colonic mucosa, The study foundelevated LINE-1 relative demethylation levels in the non-cancerous
colonic mucosa compared to tumor lines [76]. In African Americans
with CRC, LINE-1 was found to be progressively hypomethylated in thenormal adenoma cancer sequence [42]. Bariol et al. also demonstrated
that the global DNA hypomethylation level was greater in CRC neoplastic lesions (including hyperplastic polyps and adenoma) than in thenormal colonic mucosa [77]. Similarly, Sunami et al. showed a linear
correlation between LINE-1 demethylation progression and TNM stage
progression, suggesting that the onset of LINE-1 demethylation occurs
early during the normal colorectal mucosa's progression to adenoma.
Moreover, genomic methylation levels have been shown to continuously decrease during CRC development and progression, whichsuggests that aberrant DNA methylation analysis allows for CRC subtype identiﬁcation [74]. This phenomenon strengthens the potential
utility of methylation tests for detection of CRC and suggests that LINE1 hypomethylation may be a useful diagnostic marker in both sporadic
and inherited CRCs [78]. The LINE-1 methylation level in normal

5.3. LINE-1 as a preventive biomarker in CRC
LINE-1 hypomethylated CRC has been associated with a young age
at onset [71] and CRC familial clustering [83]. Tumor LINE-1 methylation levels are an increasingly attractive potential biomarker forpredicting survival beneﬁts from adjuvant chemotherapy. Surgery
supplemented with adjuvant chemotherapy using oral ﬂuoropyrimidines extends the lifespan of patients with low LINE-1 methylation
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M.A. Kerachian, M. Kerachianlevels, while apparently conferring no survival beneﬁts in patients withhigh LINE-1 methylation levels [84]. LINE-1 hypomethylation, a surrogate biomarker for global DNA methylation, is associated with worse
progressive-free survival and overall survival following FOLFOX (5ﬂorouracil, folinic acid and oxaliplatin)-based chemotherapy. In otherwords, LINE-1 hypomethylationis have resistance to FOLFOX treatment[85].

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6. Conclusion and future directions
As mentioned above, not all studies have conﬁrmed that LINE-1
methylation levels vary with tumor development and progression.
These discrepancies may be due to diﬀerences in molecular methods
used to assess the LINE-1 methylation or simply due to chance variation
between independent studies. Factors such as the side of tissue samples
could inﬂuence the experimental results since LINE-1 methylation levels in normal mucosa obtained from the right side of the colon are
noticeably lower than those obtained from the left side of the colonwhen observed [58]. In addition, an important problem in the assessment of LINE-1 methylation status in CRC is that this disease is highly
heterogeneous and contains diﬀerent inﬁltrating cells including stromalcells, lymphocytes, and endothelial cells. This heterogeneity may confound molecular analysis of LINE methylation, since LINE-1 retrotransposon are highly methylated in normal cells. It is necessary to
precisely isolate tumor cells from normal tissue specimens, in order toobtain accurate interpretations. In this regard, Sunami et al. used integrated lasers to capture microdissection in their LINE-1 methylation
analyses and showed that a mean methylation level of 80% was foundin normal subjects, indicating a potential threshold for pathologic activity [74]. These issues should be further examined with both increasingly standard and precise techniques and in independent studies
with greater sample sizes.
Determining the histopathology associated with the onset of LINE-1
hypomethylation and studying its trend along its progression sequence
can illuminate the role of LINE-1 hypomethylation in CRC. In contrast
to irreversible genetic changes, epigenetic changes of LINE hypomethylation may provide potentially reversible molecular targets forboth cancer cell therapies and chemoprevention. Further investigationsin this ﬁeld would provide new insights into the pathogenesis of CRC
and could help to develop new therapeutic strategies.
Disclosure
The authors have declared no conﬂict of interest.
Acknowledgements
Our sincere thanks go to Ms. Afsaneh Mojtabanezhad Shariatpanahi,
for providing valuable feedback, Ms. Mona Nematy for making theﬁgures and Mr. Ebrahim Pouladin for his support in cancer research
programs.
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