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Role of microRNA in the Diagnosis and Therapy of Hepatocellular Carcinoma: a New Frontier.
Author: Georgios Tsoulfas1
1
Department of Surgery, Aristotle University of Thessaloniki, Thessaloniki 54622, Greece
Running title: MicroRNA and hepatocellular carcinoma
Author contributions: Tsoulfas G solely contributed to this paper.
Correspondence to: Georgios Tsoulfas, MD, PhD, FICS, FACS, Assistant Professor of Surgery, Aristotle
University of Thessaloniki, 66 Tsimiski Street, Thessaloniki 54622, Greece. [email protected]
Telephone: +30-6971895190
Fax: +1-30-2310332022
Keywords: Biomarkers; hepatocellular carcinoma; microRNA; orthotopic liver transplantation; surgical resection;
therapy
1
Abstract
The incidence of hepatocellular carcinoma continues to increase worldwide, representing one of the premier causes of
cancer-related deaths.
It is closely related to liver fibrosis and cirrhosis (and their underlying etiologies), thus
making its treatment a challenge, as one has to simultaneously manage both the liver disease, as well as the
malignancy.
Although there are a variety of different therapies proposed, the two main ones that can actually lead to
a therapeutic result, are surgical resection and orthotopic liver transplantation.
However, the challenge remains in
terms of early diagnosis of the disease, identifying the steps that lead to its progression and choosing the best
treatment for each patient.
Regarding this latter comment, it has been determined that not all cancers are the same,
in the sense that their molecular make-up and alterations are what dictate their specific behavior and aggressiveness;
which would be helpful to know if the goal is to choose a patient- and tumor-oriented individualized approach, that
will yield better results.
Critical in this effort has been and continues to be the role of microRNA, which appears to
play a central role in the progression, diagnosis and management of hepatocellular carcinoma.
This paper will
attempt to clarify the role of microRNA in these areas and provide a window to the future.
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INTRODUCTION
Hepatocellular carcinoma (HCC) can occur in an otherwise normal liver, although frequently it is at the end of the
line of progressive liver disease, which leads to hepatitis, fibrosis and subsequently to cirrhosis.
No matter what the
avenue, its incidence is increasing worldwide, making it one of the leading causes of cancer-related deaths [1].
Despite its global reach and a significant effort to identify methods of prevention or increase the cure rate, these goals
remain elusive, mainly because of the multifactorial nature of the disease.
Specifically, although the original liver
disease can vary (with etiologies such as alcoholic liver disease, viral hepatitis, hemochromatosis, autoimmune
hepatitis, primary biliary cirrhosis, just to name a few), the different initiating factors converge to a final pathway,
which through changes in the liver architecture and physiology can lead to the appearance of HCC [2-4].
The major
challenge in the case of an existing underlying liver disease is that any treatment should be able to address both the
hepatic disease, as well as the malignancy.
Despite the fact that there is a multitude of treatments, ranging from more invasive to less so (such as
radiofrequency ablation, microwave ablation, chemoembolization, radiotherapy), combined with the fact that there is
essentially no effective chemotherapy against HCC, have left surgical resection and orthotopic liver transplantation
(OLT) as the two main therapeutic options.
A multidisciplinary approach and a careful patient selection can lead to
5-year survivals of 40% after hepatic resection and 60-70% after OLT, with the advantage of the latter being that it
addresses both the primary hepatic disease, as well as the malignancy [5-6].
Even so and despite the numerous
technical improvements in surgical technique and methods, the limiting factors remain those of the ability to detect
disease at an early stage, as well as that of recurrence.
This has led to renewed effort into investigating the
molecular nature of the disease, with the hope that by identifying pathways involved in the pathogenesis and
progression of HCC, it will be possible to achieve earlier diagnosis, and more patient-oriented and tumor-targeted
approaches. The multitude of oncogenes identified (such as c-FOS, c-JUN, RHO, IGF-II for example) serve to
remind us of the complexity of most processes in nature, and explain the fact that how tumors behave in each patient
is the result of both the tumor’s molecular basis, in addition to the patient’s and the tumor’s microenvironment [7].
One of the newest and more interesting players in this evolving tale are microRNAs (miRNAs), which are
post-transcriptional regulators of genes involved in different cellular processes, ranging from apoptosis to metabolism
and carcinogenesis.
The goal of this paper is to look into the role of miRNAs in the liver, with special emphasis on the diagnosis and
treatment of HCC.
Gaining an understanding of their strengths and limitations in the fight against HCC, will create
a bridge between the laboratory and clinical practice, a process already underway.
3
MiRNA AND THE LIVER: MORE THAN MEETS THE EYE!
Discovery and action of miRNA
MiRNAs were first described in 1993, and they were found to be endogenous, small, noncoding RNAs, which control
the expression of protein-coding genes on a post-transcriptional level through imperfect base pairing [8].
There
have been approximately 1733 mature miRNAs identified as part of the human genome, with a registry, called
miRBase, established and maintained by the University of Manchester and which can be found at the web address
http://www.sanger.ac.uk/Software/Rfam/mirna/ or http://www.mirbase.org/ [9].
Their biogenesis is the result of a
highly conserved chain of events starting with the primary miRNA transcript in the nucleus, to the finished product in
the cytosol, a process that has been well described (Figure 1) [10-12].
Figure 1 Schematic of miRNA biogenesis. MiRNA genes are transcribed into long, primary miRNAs (pri-miRNAs),
which generate mature, functional miRNAs.
These pri-miRNAs are processed in the nucleus by the RNAse III
endonuclease Drosha and DGCR8 to become pre-miRNAs. The pre-miRNAs are transported into the cytoplasm by
exportin 5 and are processed into miRNA duplexes by the RNAse III enzyme Dicer.
stem-loop, leading to two short, complementary RNA molecules.
RNA-induced silencing complex (RISC).
Dicer cleaves the pre-miRNA
Only one of these is incorporated into the
The RISC complex contains members from the Argonaute family of
proteins, which have endonuclease activity directed against mRNA strands that are complementary against the
miRNA fragment bound to them.
Additionally, Argonaute is partly responsible for the selection of the guide strand
and destruction of the passenger strand.
The guide strand is chosen by the Argonaute and is incorporated into the
RISC complex, whereas the passenger strand is degraded by the RISC. These processed miRNAs are loaded onto the
RISC and led to their mRNA targets, where the formation of the double-stranded RNA (result of the miRNA binding)
can lead to translational repression.
In the figure the miRNA guide strand is shown with the thinner line, whereas
the passenger strand is represented by the thicker line.
4
The interaction between the finished miRNA product and the mRNA, and the miRNA-mediated gene activation or
suppression that follows, is the mode of miRNA action on their targets. The critical factor explaining the importance
of miRNA is that a single miRNA can affect several target mRNAs, thus enabling miRNAs to essentially modulate up
to a third of the human genome [13].
This versatile action is responsible both for the role of miRNA in organ
development, and the possibility of leading to carcinogenesis by affecting the maturation process.
MiRNA and the liver
The liver is rich with pluripotent cells, such as hepatic stem cells and progenitor cells, in addition to hepatocytes,
hepatic stellate cells and immune cells, all of which play a central role in the ability of the liver to regenerate and
actively control the immune system.
If we add to these the various chronic insults, such as viral hepatitis, alcohol
consumption, non-alcoholic steatohepatitis and the metabolic syndrome, we have the theater of action for miRNAs,
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where by affecting the development and behavior of these cells, they can lead to processes such as hepatitis, fibrosis,
cirrhosis and carcinogenesis, with HCC being the end result.
As far as fibrosis is concerned, hepatic stellate cells and Kupffer cells are key players, which are activated by
inflammatory cytokines, such as interleukin -6 (IL-6), IL-1, tumor necrosis factor-a (TNF-a) and transforming growth
factor-a (TGF-a) [14].
Some of these actions are regulated by miRNAs, such as miR-29, which is part of the
signaling pathway between TGF-b and Nuclear factor kappa B (NFkB), and whose down-regulation can lead to
hepatic fibrosis [15-16].
The other side of this coin is that enabling miR-29 to suppress collagen synthesis and
deposition, can lead to a therapeutic target to prevent fibrosis and the resultant cirrhosis.
Another miRNA, that is
perhaps the most famous one in the liver, is miR-122, as it is liver-specific, with target genes involved in the acute
stress response, viral infection and hepatic carcinogenesis (Figure 2) [17-19].
The ability of miRNAs to act as
oncomirs, that is miRNAs with oncogenic potential, should come as no surprise and can explain the role of miRNAs
in cancer pathogenesis through the regulation of tumor suppressor genes or oncogenes [20].
This makes them prime
targets in the quest for early diagnosis, as well as for more targeted therapeutic approaches.
Figure 2 Effect of miR-122 loss on hepatocyte function. Normal functions of miR-122 in the hepatocytes include
carbohydrate and lipid metabolism, bilirubin excretion and detoxification of endogenous compounds.
Loss of
miR-122 results in increased lipid synthesis and decreased lipid export; however, the more critical loss of the
miR-122 tumor suppressive role, is the resultant increased fibrosis and inflammation, which can lead to HCC.
The
loss of miR-122 normal function essentially strengthens the axis of steatosis, inflammation and cancer, which is an
active process.
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THE ROLE OF MiRNAs IN HCC DIAGNOSIS
MiRNAs as HCC biomarkers
Identifying the relevant miRNAs involved in the carcinogenic process is the first step.
Oncomirs, such as miR-21
by being upregulated can suppress phosphatases and tensin homolog deleted on chromosome ten (PTEN), leading to
HCC progression [21].
Although not unique to HCC (as it is involved in cancers such as breast, colon and pancreas),
miR-21 has the potential to become a marker for HCC, and potentially a therapeutic target down the road [22]. The
authors of another study used microarray analysis to compare the miRNA expression profiles of 25 pairs of HCC and
the adjacent noncancerous tissue [23]. There was up-regulation of 30 miRNAs vs. down-regulation of 5 other ones,
with this differential expression of miRNA in tissue involved with HCC vs. that which has no tumor, being
encountered in several reports [24-25].
In another paper, Li et al. in looking for a biomarker for HCC, used five
different miRNAs (miR-96, miR-18a, miR-10b, miR-125a, and miR-378), which had previously been reported as
being dysregulated in HCC and tested for a serum biomarker in a population with HBV-related HCC [26]. The
authors identified miR-18a as a candidate for a blood-based biomarker for the early diagnosis of HCC, thus providing
a potentially simple and cost-effective method of early diagnosis.
Signaling networks have also been targets of research, as miRNAs can affect positive and negative feedback
loops involved in various cell processes, with an example being miR-181, which regulates the Wnt/Beta-catenin
7
pathway in stem cells, and whose members of its family are found in HCC cells [27].
The ability of miR-181 to
create a positive feedback loop by targeting mRNA of hepatic transcriptional regulators can lead to uncontrolled HCC
growth.
An example of a negative feedback loop is that of the let-7 miRNA, which binds to Lin28 (a marker of
stem cells), and which in turn inhibits let-7 and the expression of the oncogene c-Myc [28].
negatively affect the progression of HCC.
This last pathway can
The first step in deciphering the role that miRNAs can play as potential
diagnostic markers, is to clarify what constitutes a clinically useful biomarker [29].
Complexities of identifying the proper miRNA biomarker
What is apparent from the various studies is the complexity of the role of miRNA in hepatic carcinogenesis, as some
miRNAs are up-regulated (such miR-181 and miR-221), whereas others are down-regulated (such as miR-26, let-7,
miR-122).
To make things even more interesting, there are miRNAs, such miR-195, whose expression is
up-regulated in some studies and down-regulated in others [23, 25]. There are several possible explanations for this
apparent inconsistency among the different reports.
They include the molecular heterogeneity of HCC (which
explains the different rates of clinical progression and aggressiveness observed), the ability of miRNA to affect
different transcriptional pathways which in turn may act in different directions, and, last but not least, technical issues,
as the techniques used in the different studies may not be the same.
Specifically, in some studies microarrays are
used, whereas in others reverse transcription polymerase chain reaction assays or high-throughput sequencing [30].
The question is not necessarily one of reliability, rather a possible explanation for the discordance seen in the results.
Additionally, apart from the differences of the HCC tumor microenvironment itself, there are also differences in the
microenvironment of the surrounding healthy tissue used to compare, as that in turn can be affected to various
degrees by the existence of primary hepatic disease with its own intricacies.
A critical element of the importance and usefulness of miRNAs as HCC biomarkers is the fact that they are
present in different mediums, such as tissue, blood, plasma and urine, with each one having advantages and
disadvantages (Table 1).
The use of miRNA from different samples – advantages and disadvantages. Table 1
Advantages
Disadvantages
Noninvasive
Allows
Plasma
Validation of normal ranges
monitoring
progression
and
of
disease
response
Standardization of assays
to
treatment
Enables diagnosis
Unclear mechanism
8
Urine
Noninvasive
Tissue specificity of the markers
Enables diagnosis
Timing
between
collection
and
processing
Enables monitoring of disease
Peripheral Blood
Noninvasive
Depends on cell counts
Diagnosis possible
Variability
based
on
time
of
collection
Allows monitoring of disease
Heterogeneous sample
The accuracy of tissue miRNAs as biomarkers is counterbalanced by the need for invasive biopsies or surgery to
obtain them, which makes their use as biomarkers in the case of early HCC less than ideal.
interest in serum miRNA.
This has led to increased
However, there are several questions, such as how are miRNAs delivered into the
extracellular space, how can they reach the circulation, how do they avoid degradation by endogenous ribonuclease,
and whether there is an interaction between cancer and normal cells regarding the release of circulating miRNAs.
Some theories referring to the release of the circulating miRNAs describe it as a passive process from injured cells
from tissue inflammation, apoptosis or necrosis, whereas others describe a more active process, possibly with the aid
of microvesicles [31-33].
Additionally, in order to explain the stability and protection of circulating miRNA, it is
believed that apart from the microvesicles, lipoprotein complexes (such as high density lipoprotein, HDL) or
apoptotic bodies are involved in their transport [34]. This raises the question of how the changing serum levels of
these lipoprotein complexes, affects the miRNA quantification.
These circulating miRNAs represent a mirror of several of the processes going on in the organism, thus
providing a readily accessible biomarker.
However, in order to become clinically applicable, the results from the
different studies looking at various miRNAs as biomarkers need to be validated in cross-sectional trials following the
currently accepted guidelines for the assessment of diagnostic biomarkers [35-36].
It is vital to maintain high
analytical standards for study design and statistics, despite the early stage of miRNA research.
Essentially, a trial
would be needed, where randomization occurs based on the specific biomarker signature.
Furthermore, large
sample, population-based studies would be needed, with an ethnic mix, while at the same time taking into
consideration that the different miRNA expression profiles in the case of HCC may differ based on the underlying
etiology of HCC, as well as the immune status of the HCC patient.
THE ROLE OF MiRNA IN HCC TREATMENT
9
The ability of miRNA to regulate a variety of cell processes through post-translational modifications places them in
an ideal position as therapeutic targets.
The two main strategies for therapeutic intervention have been miRNA
replacement or overexpression and miRNA inhibition.
MiRNA replacement/overexpression
In the diseased HCC tissue there are miRNAs which are underexpressed there, as opposed to the healthy tissue,
where they are found in normal amounts.
This means that replacing these miRNAs in the diseased cells would not
affect the healthy ones, something which can be done with the use of short RNA duplexes mimicking these miRNAs.
Examples of this strategy include the delivery of miR-124 systemically to induce apoptosis and the delivery of
miR-26 to suppress Myc-induced tumorigenesis [37-38].
Pursuing this strategy has led to the development of
potential therapeutic interventions at the preclinical stage, such as those with the use of Let-7 and miR-34, which
supplement an underexpressed or missing miRNA) [39].
The risk remains that this method would affect healthy
cells that normally do not express these miRNAs.
MiRNA inhibition
Modified, single-stranded oligonucleotides serve as miRNA inhibitors or antagonists, called antagomirs or antimirs,
based on whether they work by degradation or sequestration of the mature miRNA.
Antimirs are complementary to
only part of the mature miRNA (although they bind with high affinity), whereas antagomirs show complementarily to
the entire mature miRNA.
An excellent example of an antagomir is miR-221, which has been use to block HCC
progression in mice and increase survival when given systemically [40-41].
In another study the authors treated
HCV-infected chimpanzees with an miR-122 specific oligonucleotide and observed suppression of HCV viremia,
thus showing that the liver-specific miR-122 is necessary for HCV RNA collection in the hepatocytes [42]. This
would be of immense value given the world-wide prevalence of HCV as an etiology for cirrhosis and HCC.
Antimirs because of their strong affinity and despite their partial complementarity, have the advantage that they can
be administered in small doses and be able to have a significant effect by binding to a variety of tissues, something
which makes them an appealing therapeutic target.
The difficulty lies in the fact that human miRNAs have
significant familial redundancy, thus making it harder to achieve a targeted effect [43].
MiRNAs and HCC: response to chemotherapy and molecular therapies
HCC is known as a type of tumor without significant response to chemotherapy.
However, there are medications,
10
such as adriamycin and vincristine, which have been shown to have an effect.
Increasing susceptibility of HCC to
different chemotherapeutic regimens or different types of molecular is one of the areas where miRNAs can prove
useful.
One example is miR-122 which makes HCC cells more responsive to the agents previously mentioned,
through the down-regulation of the Multidrug Resistance Related (MDR) genes [44].
Another example is the
altered expression of drug-transporters, who are up-regulated by the down-regulation of a group of miRNAs [45].
One of the more recent medications used as chemotherapy in cases of advanced HCC is sorafenib, which is a tyrosine
and Raf kinase inhibitor, and which has been found to affect 14 different miRNAs, with miR-122 as the prime suspect,
by being able to make cells more responsive to sorafenib [46-47].
Along the same lines is the action of small
interfering RNA (siRNA), which has been investigated in advanced stages, in cancers refractory to most standard
treatments [48].
Their mode of action differs from miRNAs in that they are made to target and degrade a single
gene, whereas miRNAs can bind to the target with a completely or not completely complementary sequence, thus
affecting a greater number of cells, which may, unfortunately, include cells that do not have anything to do with the
cancer [49-50].
Clinical application of miRNAs against HCC
Although we do not have a complete understanding of the complex nature of miRNA and are only beginning to
understand its action, its versatility combined with the current sequencing technologies, have allowed the possibility
of moving ahead with therapeutic interventions, some of which are reaching the clinical stage.
Such an example is
Miravirsen, which is an antimiR targeting miR-122, and which led to the inhibition of miR-122 and increased mRNA
targets, when it was given intravenously in mice and African monkeys [51-52].
Most importantly, it led to a
reduction in HCV RNA serum levels, which lasted for 3 months, without any evidence of resistance or adverse affects
[53]
.
Based on these results, Miravirsen was the first miRNA inhibitor to enter clinical trials, with two phase 1 safety
studies revealing no safety issues [53].
Even more exciting are preliminary results showing a decrease in the HCV
RNA of patients, which can lead to possible therapies for a great number of patients [54].
In a multicenter phase 2a
study the safety and efficacy of Miravirsen in 36 patients with HCV genotype 1 infection was investigated, with
results showing prolonged dose-dependent reduction in HCV RNA levels without evidence of viral resistance [55].
This has direct implications for HCC, given the linear relation between HCV, liver disease and HCC.
Challenges in the use of miRNA against HCC
11
There is a multitude of miRNAs, who are regulated in alternate ways in HCC (Table 2).
It is obvious from the
discussion above that with continued effort miRNA, whether in the form of miRNA inhibitors or siRNAs, has the
potential to play a significant role in the clinical stage in the fight against HCC.
point, there are still certain issues that need to be examined in more detail.
However, before we get to that
Those include the lack of an ideal
delivery system, the problem of tissue specificity, undependable cellular uptake and possible systemic effects and
toxicities.
Table 2. MicroRNAs as biomarkers for HCC diagnosis and prognosis
miRNA
Molecular alteration
Clinical Significance
Reference
miR-10b
Upregulated in HCC
Diagnostic
56
miR-18a
Upregulated
with HBV
Diagnosis
26
miR-21
Upregulated in HCC,
resistance to combined
therapy with IFN-a and
5-FU
Diagnosis, prognosis
56
miR-26
Overexpression predicts
good
response
to
Interferon-a
Therapy and prognosis
for HCV
57
miR-29
Downregulated in HCC Diagnosis
with HBV, chronic liver
injury
58
miR-34
Upregulated in HCC
Diagnosis
23
miR-96
Upregulated
with HBV
Diagnosis
59
miR-122
Differentiate HCC from
chronic HBV/cirrhosis
and
response
to
chemotherapy
Therapy
60
miR-124
Downregulated in HCC
Diagnosis
61
miR-125a
Downregulated in HCC
Diagnosis
23
miR-181
Upregulated in HCC
Diagnosis
27
miR-195
Downregulated in HCC
with HBV or HCV
Diagnosis
58
in
in
HCC
HCC
miR-
62
miR-378
Downregulated in HCC
with HBV
Diagnosis
58
Let-7
Upregulated
with HBV
Diagnosis
63
in
HCC
12
Regarding the issue of delivery to the target cells, the ideal vector is one that is aimed at the target cancer cells,
while avoiding destruction by ribonucleases in the circulation or significant uptake by the healthy cells.
There are
multiple candidates, including atecollagen, a type of collagen solubilized by protease, and which has physical
properties similar to natural collagen [64].
candidate [65].
Its low antigenicity and its resistance to nucleases make it an excellent
Another possibility has been the use of nanoparticle technology, and specifically
liposome-nanoparticles, which have the ability to encapsulate miRNAs [65-66].
The evolution of gene therapy has
raised the possibility of viral vectors for the miRNA, with the main advantage being sustained expression after single
dosing, the ability to achieve a tumor-specific effect, avoid hepatotoxicity and unwanted immune responses.
Using
an appropriate vector it can be possible to up-regulate tumor suppressor genes in the tumor, while increasing gene
silencing in the normal tissue [67-68].
It is interesting that although there have been virus-delivered gene therapies
for HCC going through the clinical testing phase, the same cannot be said for miRNA [69].
Another issue is that of targeting the tumor cells.
Therein lies one of the main advantages of miRNA, which is
none other than its ability to modulate a significant number of different genes and pathways, thus increasing its
effectiveness. Additionally, using combinations of different miRNAs may decrease the chances of resistance to a
certain therapy.
The other side of this, is that the more pathways that are activated, the greater the chance of
encountering side-effects and unwanted actions.
CONCLUSION
In this review we have seen the intimate relation shared by miRNA and the liver, which makes it a prime candidate
for the diagnosis and management of hepatocellular carcinoma.
The greatest appeal for miRNA comes from the fact
that despite its size, it can affect a great number of significant genes and pathways, something which increases
tremendously its biological efficacy.
an excellent biomarker.
Additionally, its “omni-presence” in the human body, raises the possibility of
However, there are still challenges that remain. We do not know enough regarding the role
of miRNA in the early stages of carcinogenesis, something which would allow us to prevent the development of HCC,
rather than fighting an uphill battle; although it should be said that the advent of Miravirsen can change this, if indeed
it proves to be as effective in stopping HCV.
Another challenge is that of the variability seen in the different results
and the lack of reproducibility, as we see miR behaving in different ways in different models.
This could be due to
inadequate technical means or simply a matter of the complex nature of HCC and the versatile “personality” of
miRNA.
Finally, identifying the proper delivery mode for the miR is just as important, so that their actions are
13
specific and targeted.
All in all, miRNA may be a “David”, ready to fight the “Goliath” that HCC represents.
Conflict of interest: The author has no conflict of interest to report.
14
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