Alexis M Ball

Gregory Ziomek

The treatment of prostate cancer (PCa) has seen significant progress in recent years.  Unfortunately, these treatments are not perfect.  Different treatment options are available depending on the progress of the disease, but all come with varying side effects that significantly reduce the overall quality of life of the patient (Debes et al 2004, Korfage et al 2006).  The possibility of metastasis or a cancer coming out of remission after treatment also remains an issue with any form of cancer.  In this paper, we propose a treatment method using small interfering RNA (siRNA) that targets the expression of the Androgen Receptor (AR) and the anti-apoptotic Bcl-2 genes combined with a prostate specific membrane antigen (PSMA) specific aptamer attached to nanoparticles that may exclusively and effectively eliminate only PCa cells and prevent metastasis.

There are several reasons to target Bcl-2 and AR over other possible genes involved in the development, growth, and metastasis of PCa.  AR is a very important component to PCa.  It is so important that many current treatments revolve around this receptor as a target to treat this disease, with up to 80% of patients responding to therapies that block or remove androgens (Heinlein et al 2004).  A big problem with this approach is that within 12-18 months, a metastatic case will begin to progress again (Heinlein et al 2004).  Over time, AR expression increases and loses specificity to ligands.  This way, the cancer cells are hypersensitive to androgens and will divide even more rapidly.  The progression of cancer also occurs through deregulation of AR activity through signal transduction cascades, alteration in expression of AR coregulators, and mutations of the AR that enable it to become transcriptionally active in response to tumor suppressor gene products (Heinlein et al). Other pathways are known to interact with AR activity that increase cell proliferation- such as those of insulin-like grow factor, epidermal growth factor, and keratinocyte growth factor that increase AR and AR associated gene expression (Feldman and Feldman 2001).  It is clear that just inhibiting or blocking the usual substrates for AR doesn’t have a long therapuetic effect, but preventing it from getting being fully expressed is a better approach.   

Bcl-2 has been studied in various cancers since its gene product inhibits apoptosis, programmed cell death, and is involved in metastasis (Hockenberry et al 1990, Hotchkiss et al 2009).  With androgen sensitive tumors in the prostate, Bcl-2 expression has been observed to increase when the androgen pathway is blocked through androgen suppression therapy (Fuzio et al 2011).  As a way to adapt to the treatment that reduces AR activity, Bcl-2 increases cancer growth.  Bcl-2 has also been observed to mutate in early stages of cancer, so the sooner its function is blocked the chance for cancer metastasis or the return of cancer after initial treatment are both reduced.  One cannot simply knock out the expression of AR and that Bcl-2 is a logical candidate target for treatment. 

We propose to silence the genetic sequences of AR and Bcl-2 by using RNAi. siRNAs are used to block the translation of mRNA into a functional protein in a process known as RNA interference, or RNAi (Reynolds et al 2004).  RNAi has recently been used as a method of discovering the molecular mechanisms of cancer and is being explored as a possible method of cancer treatment (Abdelrahim et al 2006).  We can use the process of RNAi as a way to knock out genes such as AR and Bcl-2 that are important to the growth and survival of PCa cells by delivering siRNA specific to each protein in cancerous cells.  As a vector to get the siRNA into cells, they can be attached to nanoparticles.

Nanotechnology is a growing field that has a variety of uses across disciplines.  Using nanoparticles to carry the therapuetic nucleotides, instead of other delivery methods, allows for high specificty and thus is speculated to have minimal side effects.   In addition to the nanoparticle, an aptamer (a short piece of DNA or RNA) designed to bind to PSMA would be included.  In prostate cancer the most well established prostate restricted cell surface antigen is PSMA.  PSMA is an ideal target since it is expressed by all prostate cancers and expression levels increase progressively in more poorly differentiated, metastatic and hormone refractory cancers (Bander et. al 2005). Utilizing an aptamer binding specifically to PSMA will ensure that the siRNA be endocytosed only by the target cells of interest, greatly reducing the chance of damaging normal tissue.  Support from previous studies on this work by McNamara et al showed that using a PSMA specific aptamer was an effective way to target the correct cell type and ensured that the therapeutic molecules get through the plasma membrane.  

To confirm the potential use and efficacy of the nanoparticles loaded with our siRNA-aptamer chimera,  we would evaluate in vivo mouse models.  Nanoparticles will be administered intravenously to murine models with PCA.  To ensure that our nanoparticles are able to diffuse through the blood stream to our tumors, we will attach of polyethylene glycol (PEG) polymer to our vector.  Attaching PEG to the complex will prevent the nanoparticles from binding proteins in the blood (Zhou et al 2010).   It also allows the nanoparticles to go without detection and destruction by the host’s immune system (Zhou et al 2010).  Avoiding an immune response is a great reason to use nanoparticles instead of a viral vector for the siRNAs.  

In addition to PEG, a poly (DL-lactide) (PLA) polymer will be fused with our vector. PLA is a biodegradable carrier and will be used to reduce toxicity and avoid accumulation of the polymer in the cell (Zhou et al 2010). Moreover, the degradation of the polymer can be used as a tool to release the plasmid DNA into the cytosol and the degradation products are removed through the citric acid cycle (Zhou et al 2010).  The nanoparticles will be fused with synthetically constructed siRNA sequence 5’ CAAGGGAGGT TACACCAAAdTdT-3’ for  AR (Dehm, 2011) and 5′-AAUGGAUGUACUUCAUCACdTdT-3′for Bcl-2 (Satoru et al 2008). We will also trial the siRNA sequences, 5’GAAATCAGCGCAGGTAATCATdTdT 3’ for the AR and 5’GCAAATCACGGTCTTATTATdTdT 3’ for Bcl-2. These scrambled sequences were generated via InVivoGen online siRNA scrambler. They will not have the same nucleotide composition as our siRNA sequences and do not code for any miRNA.  They serve as a negative control to demonstrate that the transfection does not induce nonspecific effects on gene expression. Murine models will allow us to test various dosing amounts of our vector and the frequency of admission for optimal treatment response.  These models will also allow ensure that RNAi-mediated inhibition of AR and Bcl-2 does not influence growth rate or viability of nonmalignant cells as well as the efficacy of our treatment. These steps are important in validation of our treatment prior to moving to clinical trials. 

There are potential issues with this treatment method.  It is unknown what the bombardment with siRNA will do to normal RNAi function in cells.  There is a potential for the exogenus siRNA to outcompete for the normal cellular machinery (Castanotto et al 2009).  Gene expression that regularly involves RNAi could be changed in a harmful manner as well.  Careful dosing to maximize therapeutic effect while minimizing exposure to novel siRNAs will be a challenge in testing protocols.  Since Bcl-2 and AR are expressed at varying rates depending on the progession of the cancer, some doses may be more effective or toxic than others.  Choosing the correct shape for nanoparticle is also very important since the aptamer and siRNAs are negatively charged and need a positively charged carrier.  If attached to a linear-shaped nanoparticle, the aptamer and siRNAs will collapse onto it, but a more three dimensionally shaped particle should help prevent this by aiming the charges away from the nanoparticle (Bagalkot and Gao 2011).

If  our treatment is successful, the future of prostate cancer will be very different.  It is our hope that this treatment is effective and as painless as possible, eliminating the chance for the return of prostate cancer afterward.  However, we understand that cancer is not derived from a single mutation and there has been great difficulty identifying which genes initiate the progression of cancer rather from those that are merely markers of the neoplastic state (Tomlins et al 2006). Most human cancers contain multiple genetic abnormalities not present in the normal tissues from which the tumors arise, which is in fact, a heterogeneous population of several mutant clones (Bapat 2007). All this variety means that the population of tumor cells could easily contain a mutant resistant to our treatments. This mutant then gains selective advantage over other cells. Thus, over time these resistant cells will proliferate and further evolve with new adaptations. Therefore in the event that the cancer comes back, it developed different molecular pathways to gain oncogenicity, allowing it to grow even more aggressively.

Recently developed technologies are allowing scientists to develop more innovative methods for treating difficult diseases. Combining the versatility of nanoparticles with RNAi and aptamers is one potentially effective way to treat an aggressive and deadly cancer like PCa.    Within a large population of cancer cells, the odds are relatively high that at least one cell carries a mutation conferring resistance to a particular drug. However, the odds of a single cell being resistant to several different drugs are much lower (Merlo et al 2006). We expect the dual approach of targeting Bcl-2 and AR should greatly reduce the chances of metastasis of cancerous cells.   Lastly, due to the specificity of this approach, the side effects that patients suffer would be reduced, increasing their quality of life. 

References

Abdelrahim, M., Safe, S., Baker, C., Abudayyeh A. (2006). RNAi and cancer: Implications and applications. Journal of RNAi and Gene Silencing. 2(1): 136-145

Bagalkot, V., Gao, X. (2011). siRNA-Aptamer chimeras on nanoparticles: Preserving targeting functionality for effective gene silencing.  ACS Nano. Published online

Bander NH, Milowsky MI, Nanus DM, Kostakoglu L, Vallabhajosula S, Goldsmith SJ.(2005) Phase I trial of 177 lutetium-labeled J591, a monoclonal antibody to prostate-specific membrane antigen, in patients with androgen-independent prostate cancer. J Clin Oncol 23:4591-4601.

Bapat S. (2007). Evolution of cancer stem cells. Seminars in Cancer Biology. 17: 204-213.

Castanotto, D., Rossi, J.J. (2009). The promises and pitfalls of RNA interference-based therapeutics. Nature.  457: 426-433.

Debes J.D., Tindall, D.J. (2004). Mechanisms of androgen-refractory prostate cancer.  New England Journal of Medicine.  351:1488-1490

Dehm S. (2011) Androgen Receptor Isoforms and methods

Feldman, B., Feldman, D.  (2001) The development of Androgen-independent Prostate Cancer.  Nature Reviews:Cancer. 1: 34-46

Fuzio, P., Ditonno, P., Lucarelli, G., Battaglia, M., Bettocchi, C., Senia, T., Perlino, E.  (2011). Androgen deprivation therapy affects Bcl-2 expression in human prostate cancer. International Journal of Oncology. 39(5):1233-1242

Heinlin, C.A., Chang., C. (2004) Androgen receptor and prostate cancer.  Endocrine Reviews. 25(2): 276-308

Hockenberry, D., Nunez, G., Milliman, C., Schreiber, R.D., Korsemyer, S.J. (1990). Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 22;348(6299):334-336 

Hotchkiss, R.S., Strasser, A., McDunn, J.E., Swanson, P.E. (2009) Cell Death. New England Journal of Medicine. 361:1570-1583

Korfage, I.J., Hak, T., de Koning, H.J., Essink-Bot, M.L. (2006). Patient’s perceptions of the side- effects of prostate cancer treatment— A qualitative interview study. Social Science and Medicine. 63(4):911-919

Merlo, L. M. F., Pepper, J. W., Reid, B. J., and Maley, C. C. (2006). Cancer as an evolutionary and ecological process. Nature Reviews Cancer 6:924-935.

Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S., Khvorova, A. (2004) Rational siRNA design for RNA interference. Nature Biotechnology 22(3):326-330

Satoru S., Toshihro U., Kae F., Yohei S., Kazuchika T., Kazuko H., Tadaaki O., Junichi Y. (2008) 

Tumor Regression in mice by delivery of Bcl-2 Small interfering RNA with Pegylated Cationic Liposomes. Cancer Research. 68;8843

Tomlins S., Rubin M., Chinnaiyan A. (2006). Integrative Biology of Prostate Cancer. Annual Review of Pathology: Mechanisms of Disease. 1:243-271

Zhou J., Rossi J. (2010) Aptamer-targeted cell specific RNA interference. Silence. 1-10