Cyclodextrin mediated delivery of NF -κB and SRF siRNA reduces the [619113]

Cyclodextrin mediated delivery of NF -κB and SRF siRNA reduces the
invasion potential of prostate cancer cells in vitro

Prostate cancer is the most prevalent cancer in men worldwide.
Nearly 85% of diagnosed cases of prostate cancer will be localised to
the prostate gland itself.
When the prostate cancer is localised, several forms of treatment
can be administered, including active surveillance, surgery
(prostatectomy) and radiation therapy.
When the disease becomes invasive and spreads beyond the
capsu le of the prostate, alternative treatment options are required such
as androgen -deprivation therapy or chemotherapy (such as Docetaxel).
Although initially effective, androgen -deprivation therapy will
eventually fail leading to castration -resistant prosta te cancer, which is
far more difficult to treat, with established (Docetaxel) and newly
developed drugs (Sipuleucel -T, Abiraterone Acetate, Denosumab,
Cabazitaxel and MDV -3100) offering only a modest survival time
(~2.4 –4.8 months depending on the treatmen t used).
Resistance to current therapies has been associated with a number
of mechanisms including the expression of transcription factors NF -κB
and SRF.

Since its discovery by Fire and Mello in 1998, RNA interference
(RNAi) has shown potential as an effe ctive treatment for a wide range
of disease states.7 One of the major challenges to translating this
discovery into an effective RNAi therapeutic is the design of a delivery
system that is able to encapsulate and protect the small interfering RNA
(siRNA) w hile simultaneously facilitating delivery to the intended
target site .
A CD modified with a hepta -guanidino group on one side and a
polyethylene glycol -anisic acid moiety targeting the sigma receptor on
the other has been synthesised for use in prostate c ancer. These targeted
CD molecules were shown to be capable of knocking down the
expression of target genes in the PC -3 prostate cancer cell line and
resulted in reduced tumour growth in a TRAMP -C1 induced mouse
model of prostate cancer.
A recent study id entified 11 potential transcription factors associated
with castration -resistant prostate cancer, among these, was the central
transcription factor SRF. The serum response factor (SRF) is a key
transcription factor that regulates the expression of genes as sociated

with cellular growth and differentiation. SRF has been implicated in
prostate cancer progression via its role in the activity of the androgen
receptor through FHL2. FHL2 is frequently over -expressed in prostate
cancer and is associated with a poor patient outcome.
Increased levels of SRF have also been implicated in a
biochemical recurrence of prostate cancer following prostatectomy. NF-κB is a transcription factor with a role in the establishment of
an effective immune system as well as being integral to other
developmental processes.
NF-κB has been defined as having a critical role in the initiation
and progression of many different cancer types including prostate,
breast and colorectal.
The degree of nuclear localisation of NF -κB in primary p rostate
cancer has been shown to be an effective prognostic marker in
determining the probability of the cancer becoming metastatic.
Cancer metastasis is a complicated process with many events, the
underlying mechanisms of which are not completely understo od. In
order for a prostate cancer cell to become metastatic it has to acquire
the ability to disrupt cell –cell interactions, become motile and gain the
ability to enzymatically degrade the extracellular matrix.
A proposed initiator of prostate cancer metastasis is epithelial -to-
mesenchymal transition (EMT). In EMT, epithelial cells lose their
inherent characteristics and become more mesenchymal -like. This
transition results in cells losing cell –cell adhesion and becoming more
motile. This facilitates t he escape of the cell from the primary tumour
and allows it to become metastatic. A critical EMT event is the
downregulation of the transmembrane protein E -cadherin, which
functions in binding intracellular catenin and binding cadherins on
adjacent cells.
Another critical event in prostate cancer is the breakdown of cell –
matrix binding. This has been shown to be facilitated in malignant
tumours by the production of matrix metalloprotei -nases (MMPs),
which function in cancer metastasis through the enzymatic degradation
of extracellular matrix.
The main aim of this study was to investigate the effect that silencing
two transcription factors (SRF and the RelA subunit of NF -κB) would
have on the ability of prostate cancer cells to invade. This was tested as
either a single therapy (silencing one gene), or alternatively as a
combination therapy approach (silencing both genes).
The genes were silenced using the commercially available
Lipofectamine2000 (LF2000, Invitrogen, Carlsbad, CA, USA) vector
and a well -established modified CD delivery vector.

The mechan -ism behind the invasion inhibition was also
investigated by quantifying the expression of E -cadherin and MMP9 in
the treated PC -3 cells.
An effective treatment for metastatic prostate cancer is still an
unmet clinical need.
RNAi offers potential as an effective therapy in many disease
states, with the main barrier to development being the lack of a suitable
delivery vector. Since 2004, there has been a surge in the number of
RNAi therapeutics for cancer en tering clinical trials.
In the early stages, these were mainly modified free siRNA, with
the first actively targeted CD -containing nanoparticle incorporating
siRNA developed by Calando Pharmaceuticals (Pasadena, CA, USA).
This demonstrated the in vivo pote ntial of treating cancer in
humans with CDs.
In this study, the CD vector was shown to be capable of delivering
siRNA and inducing RNAi in PC -3 cells. The level of knockdown of
RelA and SRF (at both mRNA and protein level) was shown to be
significant and s imilar to levels of knockdown previously observed in
other cell lines.
These studies indicate that targeting of RelA and SRF may offer
a beneficial inhibition of prostate cancer invasion.
The SC12 click -propyl amine CD used in this study has
previously exh ibited efficacy as an siRNA delivery vector in in vivo
models of inflammatory bowel disease10 and Huntington’s disease.
The focus on this study was metastatic prostate cancer.
The results from the in vitro invasion assay have indicated the
potential of us ing a CD -based, non -viral gene delivery vector
complexed with siRNA to combat metastasis of prostate cancer. In
terms of designing a successful RNAi therapeutic agent, the choice of
target gene is vital, and this study shows there is no significant benefit
(in this case) in using a combination gene therapy approach as a means
of reducing metastasis.
The results demonstrate that knockdown of two central
transcription factors (RelA and SRF) using the modified CD resulted in
a significant reduction in a cytok ine well known to be involved in
cancer metastasis and we hypothesise that this reduction in part,
explains the mechanism of invasion inhibition.

To our knowledge this is the first report of a CD -based
combination siRNA approach for the treatment of metastatic prostate
cancer and the results presented indicate the potential for future
development of an RNAi -based therapeutic.