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In-vitro Validation of Prostate Cancer Genes

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Yu Bin Tan

on 21 May 2014

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Transcript of In-vitro Validation of Prostate Cancer Genes

Backgrounder!
Methodology
Aims
1) To identify and select a couple of candidate prostate cancer genes for in vitro testing.

2) To evaluate expression of candidate prostate cancer genes in human prostate cancer cell lines.

PTEN Overexpression
Overexpression could be attributed to the tumour suppressing function of the gene not properly carried out due to
mutation
deletion
malfunctioning
To verify the cause, the gene can be sequenced to check for mutations that may have occurred.

RPTOR Overexpression
Overexpression observed is in line with current literature.

An increase in RPTOR gene expression would result in more mTOR/RPTOR protein complex and enhance protein synthesis while increasing the rate of cell growth.

In this way, overexpression of RPTOR could encourage tumour progression.

In-vitro Validation of Prostate Cancer Genes
Backgrounder!
Our Hypothesis
Dys-regulation of PTEN and RPTOR expression would result in prostate cancer development.
RPTOR and PTEN were chosen from a prioritised list of potential genes

Experiments were conducted on 48 cDNA samples
rtPCR was used for amplification and quantification
Cq value obtained were analysed
Relationship between RPTOR and PTEN is visually represented
Conclusion
No significant correlations observed between the expression levels and any of the 3 factors - age of patient, Gleason score and cancer stage.
Obtaining Results
Functionality primers were tested
Expression of the 2 genes were tested in 48 tissue array samples using q-PCR.
Cq values from rtPCR analysis were used to derive ΔΔCq values
ΔΔCq values plotted it against:

1. Stage of prostate cancer (stage 2-4)
2. Gleason score (6-9)
3. Age groups (45-70 and above)

Results
Age group
Gleason score
Cancer stage
Figure 1a. RPTOR
Figure 1b. PTEN
Figure 2a. RPTOR
Figure 2b. PTEN
Figure 3a. RPTOR
Figure 3b. PTEN
Overexpression of RPTOR was observed in all stages of prostate cancer
PTEN overexpression in all stages of prostate cancer
This is unexpected as loss of PTEN expression is usually observed in prostate cancer

Expression of RPTOR was much lower in grade 6 tumours compared to other grades of prostate tumours
Expression of PTEN is lower in grade 6 tumours compared to other grades of prostate tumours
Expression of RPTOR is
Highest in prostate tumours from ages 60-64
Lowest for tumours from ages 70 and above
The expression of PTEN is
Highest in tumours from ages 50-54
Lowest for tumours from ages 70 and above
Limitations
1. Small prostate sample size of 48 (Samples were hard to come by!)

2. Unbalanced sample representation of each group --> affect quality of analysis --> inconclusive results despite the quality controls carried out before the actual experiment.
Future work

Fluidgm Access ArrayTM system: Use of microfluidics to battle small sample volume
Test for the function of the genes in a tumourous sample via functional assays.

Interesting findings!
Heat map observation
Majority of samples (20 out of 39) showed
overexpression of both genes
What could this mean?
RPTOR and PTEN may be co-expressed.
Using the GeneMANIA analysis, the greatest factor that relates both genes (37.73%) is also co-expression.

Heat map
Legend
Purple: ΔΔCq values more than 2.000
Yellow: ΔΔCq values less than 0.500
Bibliography
Reflections
1. Lee SC, Ban K. manuscript in preparation. 2011.
2. RPTOR regulatory associated protein of MTOR, complex 1 [ Homo sapiens (human) ]. National Center for Biotechnology Information.
3. Sun C, Southard C, Witonsky DB, Kittler R, Di Rienzo A. Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations. PLoS Genetics. 2010;6(10).
4. Wazir U, Newbold RF, Jiang WG, Sharma AK, Mokbel K. Prognostic and therapeutic implications of mTORC1 and Rictor expression in human breast cancer. Oncol Rep 2013;29(5):1969-74.
5. Okumura K, Mendoza M, Bachoo RM, DePinho|| RA, Cavenee WK, Furnari FB. PCAF Modulates PTEN Activity. J Biol Chem. 2006;281(36).
6. Weng L-P, Gimm O, Kum JB, Smith WM, Zhou X-P, Wynford-Thomas D, et al. Transient ectopic expression of PTEN in thyroid cancer cell lines induces cell cycle arrest and cell type-dependent cell death. Oxford Journals, Human Molecular Genetics 2000;10(3):251-8.
7. Chen L, Loh WY, See YLS, Kenneth B, Choon LS. Validating the Role of Potential Genes and Pathways in the Development of Cancer. 2012.
8. GeneMANIA 2007. Available from: http://www.genemania.org.
9. Takei Y, Saga Y, Mizukami H, Takayama T, Ohwada M, Ozawa K, et al. Overexpression of PTEN in ovarian cancer cells suppresses i.p. dissemination and extends survival in mice. Mol Cancer Ther. 2008;7(3):704-11.
10. Kappes H, Goemann C, Bamberger AM, Löning T, Milde-Langosch K. PTEN expression in breast and endometrial cancer: correlations with steroid hormone receptor status. Pathobiology. 2001;69(3):136-42.
11. Asnaghi L, Bruno P, Priulla M, Nicolin A. mTOR: a protein kinase switching between life and death. Pharmacol Res 2004;50(6):545-9.
12. Ukraintseva S, Yashin A. Individual Aging and Cancer Risk: How are They Related? Demographic Research. 2003;9(8):163-96.
13. Fluidgm, inventorAccess Array™ System.
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