siRNA PREPARATIONS IN GENE THERAPY OF MELANOMA

siRNA PREPARATIONS IN GENE THERAPY OF MELANOMA

Developmental Period Medicine, 2013, XVII, 3
196
© IMiD, Wydawnictwo Aluna
Joanna Bogusławska1, Maciej Małecki1,2
siRNA PREPARATIONS IN GENE THERAPY
OF MELANOMA
PREPARATY siRNA W TERAPII GENOWEJ CZERNIAKA
1
Department of Applied Pharmacy and Bioengineering,
Medical University of Warsaw, Poland
2
Institute of Mother and Child, Warsaw, Poland
Abstract
Melanoma is a type of malignant skin cancer, characterized by a steadily increasing rate of morbidity,
making it a continuous challenge for modern oncology. In recent years there has been a significant
increase in developing gene therapy approaches for the treatment of cancer. The phenomenon of RNA
interference initiated studies on the inhibition of the expression of selected genes by small interfering RNA
(siRNA) in cells. The use of siRNA preparations for the treatment of patients depends on the transfection
efficiency and the level of silencing the targeted genes. Currently, preclinical and clinical studies are being
conducted in order to test the effectiveness and safety of siRNA preparations.
Key words: RNA interference, siRNA preparations, gene therapy, skin cancer
Streszczenie
Czerniak należy do złośliwych nowotworów skóry, charakteryzuje się wzrastającym współczynnikiem
zachorowalności i jest nadal wyzwaniem dla współczesnej onkologii. W ostatnich latach obserwuje się
znaczący rozwój terapii genowej, która ma zastosowanie w leczeniu nowotworów. Odkrycie zjawiska
interferencji RNA (RNAi) zapoczątkowało badania dotyczące hamowania ekspresji wybranych genów
przez małe interferujące RNA (siRNA) w komórkach nowotworowych. Przeciwnowotworowa aktywność
preparatów siRNA zależy od wydajności transfekcji oraz stopnia wyciszenia docelowych genów. Obecnie
prowadzone są badania przedkliniczne i kliniczne, dzięki którym ocenia się skuteczność i bezpieczeństwo
preparatów siRNA.
Słowa kluczowe: interferencja RNA, preparaty siRNA, terapia genowa, czerniak
DEV. PERIOD MED., 2013, XVII, 3, 196201
INTRODUCTION
Melanoma is a malignant skin cancer, which is difficult
to treat. Compared with other malignant neoplasms, it
is characterized by high growth dynamics in both the
incidence rate and – to a lesser extent – mortality rate
(1). Each year there are approximately 160 thousand
new cases of melanoma in the world (2). The highest
incidence rate is observed in Australia (40 to 60 cases
per 100 thousand inhabitants per year) and the lowest
in Japan (0.2 cases per 100 thousand inhabitants) (3).
In Poland, melanoma is a relatively rare cancer but
the incidence rate is increasing. The number of cases
is doubling every 10 years and at present amounts to
about 2.2 thousand new cases per year (one thousand
males and 1.2 thousand females) (1, 4).
Currently, next to chemotherapy, radiotherapy and
surgery, patients with melanoma are recommended
immunotherapy, biochemotherapy or targeted therapy
(5). A special type of targeted therapy is gene therapy,
which allows for the treatment of genomic diseases at the
molecular level. Over recent years a significant development
of gene therapy strategies has been observed. One of
them consists in blocking the biosynthesis of defective
proteins by altering gene expression. The discovery of
siRNA preparations in gene therapy of melanoma
RNA interference (RNAi) phenomena initiated studies
on the inhibition of the expression of selected genes by
small interfering RNA (siRNA) in the cells (6). The natural
occurrence of RNAi phenomena in eukaryotic organisms
makes the siRNA molecule much more effective and
special in comparison with other methods of silencing,
such as antisense oligonucleotides and ribozymes (7, 8).
Despite the difficulties associated with the specificity,
synthesis and administration of siRNA, the molecule
provides great therapeutic possibilities in the treatment
of oncological patients.
The purpose of this article is to characterize siRNA
preparations primarily regarding their use in planning
gene therapy protocols for patients with melanoma.
INHIBITION OF GENE EXPRESSION
BY RNA INTERFERENCE
The term ,,RNA interference’’ (RNAi) was used by Craig
C. Mello and Andrew Z. Fire) to describe gene silencing
after Caernohabditis elegans nematode (C. elegans): long,
double-stranded RNA molecules were introduced to the
organism (9). Fire and Mello were awarded the 2006
Nobel Prize in physiology and medicine since they were
the first to indicate the double-stranded RNA molecule
as an inducer of homologous gene silencing (10). Three
years after the publication of this groundbreaking work
a group of researchers led by Thomas Tuschl identified
the so-called short interfering RNA (siRNA) (11, 12)
in extracts of insect effector molecules (the cells which
had been cut from the long RNA). This discovery paved
the way to inhibiting gene expression in mammalian
cells by synthetic siRNA (13). The research led to the
description of several types of short regulatory RNA
(srRNA). Besides the aforementioned siRNA, an important
group are also miRNA molecules (called microRNA),
which are endogenous. In the case of siRNA molecules,
gene silencing takes place at the transcriptional stage
(TGS) and the post-transcriptional stage (PTGS), whilst
messenger RNA (miRNA) at the post-transcriptional stage
(14, 15). Regulation at the post-transcriptional level may
take place through the degradation of messenger RNA
(mRNA) or the inhibition of translation. However, RNA
may control the expression of genes at the transcriptional
level by epigenetic modification of genetic material.
Interfering RNA interacting with the RNA-dependent
RNA polymerase and histone methyltransferase promotes
the process of histone methylation. This leads to the
silencing of centromeric DNA and/or the formation of
heterochromatin, which is not subject to transcription.
This mechanism has been observed in yeast, plants and
fruit flies, but so far there have been no conclusive reports
on its role in mammals (16).
The molecules of long, double-stranded RNA (dsRNA)
induce RNAi in lower organisms such as C. elegans or
in plants. In mammalian cells, RNAi can be induced by
synthetic siRNA duplexes or hydrolyzed by the nuclease of
Dicer RNA molecules of hairpin structure (siRNA, short
hairpin RNA) (17). siRNA molecules are 21-23 nucleotides
(18). Dicer nuclease, which takes place in human cells,
is the type III ribonuclease. It has two domains of the
197
RNase III activity and additional functional domains: a
binding dsRNA, helicase and PAZ domain (19). Further
research into the molecular mechanism of the process
of RNA interference showed colocalization of short
interfering RNA with a protein complex of complicated
enzyme activity called RISC (RNA Induced Silencing
Complex). The RISC complex binds preferentially to
the antisense siRNA strand whilst the sense strand is
degraded (20). Single-stranded antisense siRNA located
within RISC recognizes complementary mRNA sequences
(21). Endonuclease Ago2 (part of the Argonaute protein
family) intersects the formed mRNA/siRNA duplexes
and further degradation occurs due to the action of
exonucleases (22). The RISC complex includes for example
the Argonaute protein family, dsRNA-binding proteins,
as well as a number of proteins of helicase and nuclease
activity. Argonuate proteins contain two characteristic
PAZ and PIWI domains. The PAZ domain turned out to
be crucial for binding to siRNA, by interacting with the
Dicer PAZ domain. However, the catalytic center of all
the RISC complex is located within the PIWI domain.
The PAZ domain of RISC complex binds the 3 ‘end of
an siRNA molecule whilst the 5’ end is bound to the
PIWI domain. After the degradation of the target RNA
molecule, RISC is released and can be reused in the
RNAi process (23).
METHODS OF INTRODUCING siRNA
TO CELLS
The introduction of biomolecules to cells may be
conducted by physical, physicochemical and biological
methods. Currently, viral vectors are the main carriers
used in gene therapy (24). The following systems deserve
special attention in the case of RNAi: retroviruses, including
lentiviruses, adenoviruses, adeno-associated viruses (AAV)
and the herpes virus type one. At the same time other
non-viral systems (25) are sought, which include both
methods based on the direct transfer of the so-called
,,naked siRNA” and the introduction of genetic material
combined with the carrier. Despite quite effective and
reliable methods of delivering siRNA in vitro preparations,
only a few have found their application in vivo. Some
of the reasons for this are due to the rapid enzymatic
degradation of siRNA, renal clearance and non-specific
absorption of the reticuloendothelial system (26, 27).
There are various non-viral methods of introducing
duplexes into an organism, including electroporation,
microinjection, direct injection into the place of use (such as
brain tumors), inhalation, or using an eye drop solution in
the treatment of age-related macular degeneration (AMD)
(28). Work on increasing the efficiency of introducing
siRNA into in vivo cells (26) is being continued.
siRNA PREPARATIONS IN GENE THERAPY
In the course of melanoma the occurrence of changes in
the functioning of proteins involved in intracellular signal
transduction pathways (Table I) is recorded most often. The
pathways listed in table I (29) are linked and are regulated
in reply to changing the environmental conditions and
Joanna Bogusławska, Maciej Małecki
198
Table I. Molecular characteris!cs of melanoma, molecular subtypes [29, changed].
Tabela I. Charakterystyka molekularna czerniaka, podtypy molekularne [29, zmienione].
Subtypes
Podtypy
Pathways
Szlak
Gene
Gen
BRAF
1.1
BRAF/PTEN
1.2
MAPK
1.3
BRAF/AKT
1.4
BRAF/CDK4
2.1
c-KIT
c-KIT
3.1
GNAQ
GNAQ
3.2
GNA11
GNA11
4.1
NRAS
NRAS
5.1
MITF
MITF
PTEN
6.1
6.2
AKT/PI3K
PI3K
6.3
7.1
AKT
CDK
ARF/INK4a
7.2
CDK4
7.3
CCND1
Therapeu!c strategies
Strategie terapeutyczne
BRAF, MEK, Hsp90 inhibitors
Inhibitory BRAF, MEK, Hsp90
Combina!on treatment: BRAF and PI3K or AKT or mTOR inhibitors
Terapia skojarzona: inhibitory BRAF i PI3K lub AKT lub mTOR
Combina!on treatment: BRAF and AKT or mTOR inhibitors
Terapia skojarzona: inhibitory BRAF i AKT lub mTOR
Combina!on treatment: BRAF and CDK4 inhibitors
Terapia skojarzona: inhibitory BRAF i CDK4
Gleevec and c-KIT inhibitors
Gleevec i inhibitory c-KIT
MEK inhibitors
Inhibitory MEK
MEK inhibitors
Inhibitory MEK
MAPK and PI3K/AKT pathway inhibitors; Farnesyl transferase inhibitors
Inhibitory szlaków MAPK i PI3K/AKT; inhibitory transferazy farnezylowej
Histone acylotransferase inhibitors
Inhibitory acylotransferazy histonów
PI3K, AKT, mTOR inhibitors
Inhibitory PI3K, AKT, mTOR
AKT, mTOR inhibitors
Inhibitory AKT, mTOR
PI3K, AKT, mTOR inhibitors
Inhibitory PI3K, AKT, mTOR
CDK inhibitors
Inhibitory CDK
CDK inhibitors
Inhibitory CDK
CDK inhibitors
Inhibitory CDK
the situation of metabolic cells. Regulatory dysfunction
within signaling pathways affects the functioning of the
cells and the cancer growth potential. The mechanism
of antitumor activity of the preparation using siRNA
molecules may be associated with the MAPK pathway,
namely the inhibition of the growth and invasive capacity
of melanoma by inactivation of the mutant BRAF (V600E).
The kinase signaling pathway (RAS/RAF/MEK/ ERK)
activated by mitogens (MAPK) is an intracellular pathway
transmitting mitogenic signals to the nucleus through a
series of phosphorylation of the molecules that make up
the path. The consequence of MAPK activation is its effect
on biological processes - proliferation, differentiation,
stress response or the action of cytokines, apoptosis
(30). The mutation of the BRAF gene, encoding kinase,
is found in 40-70% of melanomas. The most common is
the V600E substitution - the replacement of valine for
glutamic acid at position 600, representing nearly 90%
of all the above-mentioned mutations (31). In 2004,
The Oncogene journal published articles on the inactivation
of the mutant BRAF (V600E) by siRNA molecules both
in vitro and in vivo in mouse cells. In one of the studies
the molecules were introduced into cells by lentiviral
vectors (32). It is worth noting that these vectors allow
for the stable expression of RNA interference in target
cells, since unlike retroviruses they tend to integrate into
the host genome at a site distant from the promoter, in
the intron area, potentially limiting their oncogenicity
(33). The results of these studies have shown that the
BRAF molecules (V600E) siRNA inhibit most of 10
melanoma cell lines with this mutation in vitro and in
vivo, thus contributing to reducing both mutant BRAF
protein and excessive ERK phosphorylation. Scientists
believe that the future of clinical trials lies in gene therapy
using BRAF (V600E) siRNA molecules (32).
The aim of molecular therapies are also genes involved
in the functioning of the metabolic MAPK and PI3K/
AKT pathways. NRAS “hyperactivity” occurs in 15-30% of
melanomas. PI3K/AKT pathway is an important regulator
of survival, proliferation, adhesion, migration, invasion
and cell metabolism (34). Together with the MAPK
pathway it is a pillar in the process of carcinogenesis
in the case of malignant melanoma (35, 46). In 2005,
researchers from Sweden conducted a study on human
melanoma cells cultured in vitro with NRAS (Q61R)
mutation using siRNA molecules and plasmid vectors
siRNA preparations in gene therapy of melanoma
as non-viral carriers (37). The suppression of oncogenic
NRAS in two cell lines (A375 and 397) reduced their
proliferation, apoptosis and the phosphorylation of ERK
and AKT, and reduced the expression of NF-kappaB and
cyclin D1. Further studies also confirmed the reduction
of the sensitivity of cells to EphA2 (receptor tyrosine
kinase), uPAR (urokinase receptor), cyclin E2 and some
cytoskeletal proteins. The use of siRNA against NRAS
(Q61R) may contribute to the development of melanoma
treatment in the subgroup of patients with this mutation
(38).
The inhibition of gene expression by means of
siRNA also found its application found in the oncogene
c-myc. C-myc proto-oncogene plays a significant role
in the regulation of the cell cycle. It is involved in cell
proliferation, differentiation and apoptosis. Extracellular
signal-regulated kinase (ERK), which is a mitogen-activated
protein kinase (MAP), is responsible for the activation
of a series of transcription factors, such as Myc (c-myc).
The proto-oncogene c-myc expression is frequently
disturbed in many cancers (including melanoma) and
amplification is one of the mechanisms of its activation.
C-myc siRNA is the preparation silencing the gene, the
efficiency of which was tested in B16F10 melanoma cells
in a syngeneic mouse model both in vitro and in vivo.
siRNA cationic liposomes were the carrier of c-myc.
The studies have shown that the preparation increases
the inhibition of c-myc expression, induces apoptosis
inhibiting the anti-apoptotic protein Bcl-2 and decreases
melanoma tumor growth. It also causes the sensitivity
of melanoma cells to paclitaxel. An adverse reaction
observed in mice was low immunotoxicity (39). Similar
results were also obtained in studies on the cells of the
human melanoma cell line MDA-MB-435. The results
proved that the siRNA preparation inhibits the growth
of the MDA-MB-435 tumour (40).
MITF is a transcription factor that plays a significant
role in the differentiation, growth and control of the
survival signals of melanocytes (41). MITF is responsible
for the induction of the expression of genes involved in
melanogenesis (e.g. tyrosinase) and the regulation of
the cell cycle (e.g. CDKN1A and CDKN2A) (41). MITF
amplification occurs in 10-20% melanomas (according to
Garraway and coworkers it concerns only 10% of primary
tumors and 21% of metastatic tumours) (42). Japanese
scientists have conducted research on the effectiveness
of MITF-specific siRNA based on B16 melanoma cells
in a syngeneic mouse model in vitro and in vivo. The
transfection method used in the study was electroporation.
The results confirmed that the MITF-specific siRNA
inhibits the expression of MITF in B16 melanoma cells
and reduces their viability by inducing apoptosis (43).
In 2006 research was conducted in Germany on the
induction of apoptosis in melanoma cells by inhibiting the
uPAR receptor and thereby activating the p53 protein. The
protein stops the cell cycle at the stage of G1/S transition
and runs mechanisms to repair minor damage to DNA,
induces apoptosis in cells with significant DNA damage
and interacts with all the proteins involved in the above
processes, and ,,monitors” their course (44). The loss
of the Tp53 function is detected in approximately 80%
199
of all cancers (45). The mutations associated with the
loss of the Tp53 function is present in about 10% of all
the melanomas (46). The increased expression of the
urokinase uPAR receptor (the plasminogen activator) is
associated with metastatic cancers including melanoma.
The above-mentioned studies were based on the use of
uPAR siRNA molecules that had been introduced into
three human melanoma cell lines (WM239A, WM9,
1205Lu) by plasmid vectors, both in vitro and in vivo.
The studies have shown the inhibition of uPAR expression
contributing to p53 induction and the change in the
expression of proteins from the Bcl-2 family to proapoptotic proteins (47).
Currently, for example, the siRNA preparation
marked with the CALAA-01 symbol is being tested
in phase I of clinical trials in patients with melanoma.
The siRNA molecule is directed to the M2 subunit of
ribonucleotide reductase (RR) (86). Ribonucleotide
reductase is an enzyme involved in the synthesis of
deoxyribonucleotides, consisting of two subunits of
RRM1 and RRM2, which participate in the differentiated
cell cycle. Whilst the RRM1 expression remains constant
throughout the cell cycle, the RRM2 subunit is expressed
only at the stage of the interphase of the G1/S cell
cycle. The study used cyclodextrin and transferrin
as a marker (48).
The study of the siRNA (siR2B 5) RRM2
preparation both in vitro and in vivo showed the
reduced activity of ribonucleotide reductase inhibiting
the proliferation of human melanoma cell lines
alone or synergistically with temozolomide (49).
Other studies have shown that such a combination of Rad51
siRNA preparation and dacarbazine may synergistically
inhibit melanoma. Dacarbazine (DTIC) induces apoptosis
in melanoma cells by double-stranded DNA breakage
(DSBs). When defending themselves, the cancer cells
increase the expression of DNA repair molecules they are
responsible for. Rad51 siRNA preparation was introduced
by the viral (HVJ-E) method to B16F10 cells contributing
to the increase in the amount of DSBs and therefore the
apoptotic death of melanoma cells (50).
SUMMARY
Currently extensive studies are being conducted on
the use of siRNAs molecules specific for the genes which
are a well-defined target in the treatment of diseases
with known origins. This area of research involves many
diseases, not only neoplastic ones but also for example eye
diseases, neurodegenerative diseases and metabolic diseases.
Reports on the successful use of siRNA for gene silencing
in melanoma cells are promising and allow to hope for
the wider use of siRNA preparations in clinical trials.
It remains to develop effective solutions for the specific
transport of siRNAs into cells and extend the duration
of the effect of silencing.
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Author’s contributions/Wkład Autorów
According to the order of the Authorship/Według kolejności
Conflicts of interest/Konflikt interesu
The Authors declare no conflict of interest.
Autorzy pracy nie zgłaszają konfliktu interesów.
Received/Nadesłano: 02.07.2013 r.
Accepted/Zaakceptowano: 09.07.2013 r.
Published online/Dostępne online
Address for correspondence:
Maciej Małecki
Department of Applied Pharmacy and Bioengineering,
Medical University of Warsaw
Banacha 1, 02-097 Warsaw, Poland
tel. 0048 572-09-65
e-mail: [email protected]