User:331pten/sandbox

Question of Interest
In a mouse model of prostate cancer featuring reduced levels of PTEN expression, will the combined use of mesenchymal stem cells with adenovirus-mediated gene delivery of exogenous PTEN yield a higher rate of cancer regression compared to adenovirus gene delivery of PTEN alone?

This question aims to target cancer, a common condition and a serious health issue, with annual cases of cancer projected to rise from 14 million in 2012 to 22 million within the next two decades. The relevance of the question lies in its potential to elucidate a major component of tumour cell proliferation using PTEN and the Akt signalling pathway.

PTEN
PTEN encodes for the PTEN protein and acts as a tumor suppressor gene through the action of its phosphatase protein product. The phosphatase protein performs a regulatory role within the cell cycle, preventing uncontrolled growth and division of cells. Mutation of the gene consequently results in tumor formation and is observed in large number of cancers at high frequency, especially in prostate cancer (the focus of my experiment).

PTEN acts as a phosphatase to dephosphorylate phosphatidylinositol-3,4,5-triphosphate (PIP3). It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-triphosphate in cells and functions as tumor suppressor by negatively regulating Akt/PKB signalling pathway. PTEN is a phosphoinositide 3-phosphatase that is capable of inhibiting proliferation of cells, the survival and growth of cells, by inactivating the PI 3-kinase-dependent signalling pathway. It is one of the most commonly lost tumor suppressors in human cancer. PTEN specifically catalyses dephosphorylation of 3’ phosphate of inositol ring in PIP3, resulting in biphosphate product PIP2. This dephosphorylation is important because it results in inhibition of Akt signaling pathway. Inhibition of the Akt pathway helps prevent uncontrolled cell growth, leading to tumor formation.





The PTEN/Akt Pathway: Upstream regulation of Akt
The activation of receptor tyrosine kinases initiate the signalling of a kinase called phosphoinositol 3-kinase to trigger the Akt pathway. 3-kinase works to phosphorylate PIP2, in order to generate a second messenger, PIP3. PIP3 then directly activates the kinase Akt.

The PTEN/Akt Pathway: Downstream regulation of Akt
Once activated by PIP3, Akt becomes a proto-oncoprotein that has many different corresponding substrates and outcomes. The most well-known role that Akt plays is the phosphorylation of proapoptotic proteins such as Bax and Bad to inhibit their ability to form holes in the outer mitochondrial membrane. Consequently, without Akt the cell would undergo apoptosis through the mobilization of a caspase cascade, a series of reactions that lead to apoptosis.

Background: Limitations of Current Cancer Treatments
Current cancer treaments include: chemotherapy, radiation, hormone therapy, and surgery. Essentially, the main flaw with all these treatments is that they lack specificity in targeting only the cancerous cells in the body, as they also destroy normal cells in the process.

Background: What is the alternative to current cancer treatments? Gene therapy
Gene therapy is the use of functional DNA introduced to recipient cells via vectors to replace mutated genes that cause diseases. The functional DNA containing the therapeutic gene is then expressed within the recipient cell to produce the desired function.

Adenovirus-mediated gene therapy
The adenoviruses are 90-100nm viral envelope-lacking nucleocapsids containing double-stranded linear DNA. The adenovirus is commonly used as a gene therapy vector because its genome is relatively simple to alter using recombination techniques and its presence in numerous species. Also, the inserted therapeutic gene is often replicated without mutation and the adenovirus affects the host without severe side effects.

Limitations of adenovirus-mediated gene delivery
One limitation is the trigger of the host immune response to adenovirus vectors, which in vivo is triggered by chemical signals such as cytokines along with the activation of leukocytes. Furthermore, it is often detrimental and sometimes even fatal to the patient being treated.

What is a possible solution?
In order to overcome this, researchers have begun to experiment with utilizing mesenchymal stem cells (MSCs) as a protective storage system for recombinant adenovirus vectors. This is largely due to reports that studies showing that proteins needed for the complete activation of T cells, such as CD40, CD86, CD80 are all not expressed in MSCs. Interestingly enough, there is evidence that shows adenoviral modification having no significant influence on the immunological properties of MSCs, thus revealing the therapeutic potential of an adenovirus-MSC coupled approach to the treatment of cancer.

More about mesenchymal stem cells


In addition to its other properties, MSCs have also been shown to migrate specifically to tumor sites. The basis for this migration seems to lie in the evidence that tumors release cytokines, chemokines and other chemical signals that recruit MSCs to home in to their respective locations. This unique tumor-tropic ability of MSCs is called homing capacity. Unfortunately, the molecular mechanisms underlying homing capacity is still unclear.

Hypothesis
Based on the evidence mentioned above, I hypothesize that an adenovirus-MSC-PTEN coupled approach will yield a far more robust effect on combating tumourigenesis than adenovirus gene delivery of exogenous PTEN alone.

Prediction
Given the immunoprivileged nature of MSCs and their homing capacity as well as PTEN's action on the Akt signalling pathway, I predict that the adenovirus-MSC-PTEN coupled approach will allow for a more direct influx of PTEN protein into the target tumor cells which will lead to reduced levels of activated Akt (via reduced levels of PIP3). Consequently, this will yield reduced tumorigenesis and potentially regression of the cancer.

Animal Model
The model organism for my experiment is a mouse model of prostate cancer. There are several reasons why I choose mice to be the model organism for research regarding cancer: First, mice are just as likely as humans to develop cancer. Also, the human and mouse genomes are about 95% identical, and similar genes as well as changes in genomic integrity have been found in cancer when comparing the two genomes. Moreover, mice are relatively easy to genetically manipulate, relatively simple to store and can be bred quite easily to produce large populations. Since prostate cancer affects males, only male mice will be used in this experiment.

The specific type of mice I will be using in this experiment is a heterozygous conditional deletion of the PTEN gene in the mouse prostate. The mouse must be heterozygous for the PTEN deletion because it has been found that homozygous deletions of the PTEN gene leads to inviable organisms.

Transduction of PTEN into MSCs via adenovirus-mediated gene delivery
First, a recombinant adenovirus vector with the PTEN gene inserted will be created. A recombinant adenovirus vector with junk DNA of the same length as the PTEN gene inserted will also be created. Next, each recombinant adenovirus vector (PTEN and junk DNA) will be transduced into separate plates of mouse mesenchymal stem cells isolated from the bone marrow of C57BL/6 mouse (available from Gibco) using a commercially available transfection reagent.

The advantage of using MSCs as a storage for PTEN expression is the fact that as mentioned before, MSCs have the unique property of migrating specifically to tumour sites and being able to bypass the immune response.

Western Blot Analysis
To allow for stable expression of the exogenous PTEN gene, the mice will be sacrificed (only the prostate will be extracted) 12 hours after intravenous injection of MSCs (containing either PTEN or junk DNA). Afterwards, a SDS-PAGE and western blot analysis will be run. Rabbit anti-PTEN and anti-PIP3 will be used as the primary antibodies for the Western blot analysis on the prostate samples. Fluorescently labelled goat anti-rabbit will act as the secondary antibody.

The advantage of using western blot analysis is that it shows a direct representation of protein levels in a sample in a relatively short amount of time. One round of western blotting takes around one day in the lab. Moreover, anti-PTEN and anti-PIP3 primary antibodies are very simple to find since there is so much cancer research happening.

Lastly, beta-actin will act as the loading control to ensure that all the samples have been loaded in relatively equal volumes.

Live/Dead Cell Viability Assay
For this assay, I will use the "LIVE/DEAD Viability/Cytotoxicity Kit for Animal Cells" (available via 'Molecular Probes') to determine the proportion of dead and living prostate tumour cells. This will be applied to the prostate samples, with red cells (via action of ethidium homodimer-1) signifying dead cells and green cells (via action of calcein AM) signifying live cells.

Treatment groups, result predictions
The table below outlines the different steps of the experiment from transduction to the viability assay. It also summarizes the predicted results assuming that my hypothesis is supported. The subject of all the treatment groups is the mice model with the heterozygous conditional deletion of the PTEN in the mouse prostate. Predicted results from treatment group 2-4 are all relative to baseline levels observed in treatment group 1 (control group).



Treatment group 1: This group is the negative control with just MSCs injected intranveously (no adenovirus vector). All the other results are relative to the baseline data from this group.

Treatment group 2: This group is treated with MSCs transduced with an adenovirus vector containing junk DNA. Since the vector contains junk DNA, the results should be very similar to that of treatment group 1. However, it is possible that there will be more cell death due to the introduction of foreign genetic material. This phenomenon is also seen in techniques such as DNA transfection. Essentially, this is a control meant to ensure that any differences in results are not due to the process of transducing the MSCs with the adenovirus vector.

Treatment group 3: This group is treated with MSCs transduced with an adenovirus vector containing the PTEN gene. Based on our hypothesis, results should show that from the addition of exogenous PTEN, levels of PIP3 will decrease due to more PTEN protein dephosphorylating PIP3, converting it to PIP2. In turn, this leads to reduced activation of the Akt signalling pathway which leads to programmed cell death of the tumour cells. In relation to my prediction, cell death of the tumour cells could potentially mean cancer regression.

Treatment group 4: This group is treated with the adenovirus vector containing the PTEN gene. However, no MSCs are transduced for this group. Based on the migration towards tumor sites that mesenchymal stem cells have displayed, a treatment of the adenovirus vector with the PTEN gene should be less specific than one with MSCs transduced with that same vector. Therefore, we would expect similar results to treatment group 3, but to a lesser extent.

Some necessary precautions
Certain things to look out for throughout the experiment are:

1) Sample size. Different sources recommend different sample sizes. The sample size will need to be adjusted accordingly to ensure that it is neither too small or too large. If it is too small, you risk the experimental results being unreliable. If it is too large, this will lead to inefficient use of time and resources in the lab.

2) The short-lived nature of gene therapy. The problem with introducing therapeutic DNA into the genome is that usually, this effect is not permanent and is short-lived. Therefore, the subjects may have to undergo multiple sessions of gene therapy for there to be an appreciable effect on cancer regression.

3) Immune response associated with adenoviruses. When adenoviruses are injected into the body, the immune system is naturally triggered to combat this "invasion". However, I feel that the use of MSCs in this experiment properly accounts for this problem as MSCs do not activate any responses from the body's immune system due to its unique properties mentioned in the "background" section.

4) It may turn out that the use of MSCs do not yield an enhanced effect on tumour cells when compared to injection of adenoviruses containing PTEN alone. Rather than immediately refuting my hypothesis, this result could just be a case of the PTEN protein in MSCs being unable to bypass the MSC membrane and make its way into the tumour cells. To address this potential issue, an interesting follow-up experiment to perform would be the create a new recombinant adenovirus vector with the PTEN gene fused to a HIV gene called "TAT". The protein product of This HIV gene has the unique ability of being able to translocate across membranes, as it is a cell-penetrating peptide. The idea is that the PTEN gene fused to TAT will be able to translocate across multiple membranes as well. This would address the aforementioned issue.

Overall, I believe the results should agree with our hypothesis and this experiment would provide clues to new and more efficient methods of cancer treatment.