PTEN Information
PTEN (Phosphatase Tensin Homolog) is an oncogene whose mutation is associated with many clinical situations, such as cancer, Alzheimer's Disease, and autism [1] [2] [3]. Biochemically, the PTEN gene encodes an enzyme which functions in modifying different families of macromolecules via a process known as dephosphorylation. In human cells, the PTEN locus is located on chromosome 10. Individuals with cells that possess only one copy of the PTEN gene, usually due to the inheritance of a loss-of-function mutation in their parent's germ cell line, are often found to have developed some form of cancer in later life.
Signalling Pathway
PTEN acts as a tumor suppressor gene [4] by negatively regulating the P13K/Akt signalling pathway in somatic cells. In this pathway, external cell growth and survival signals are detected and relayed to the P13K molecule via receptor tyrosine kinases (RTKs), which activates it. Upon activation, P13K proceeds to convert phosphatidylinositol (4,5)-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3) via a process known as phosphorylation. The role of PTEN in this pathway is to dephosphorylate PIP3 back into PIP2. This makes PTEN an antagonist of P13K [5].
As PIP3 activates a family of molecules known as protein kinase B (abbreviated as Akt), conversion of PIP3 into PIP2 by PTEN results in the depletion of PIP3 and the inactivation of Akt. When active, Akt can further activate or inhibit downstream molecules of the P13K/Akt pathway via phosphorylation of these molecules. This series of reactions is responsible for the activation of mammalian target of rapamycin (mTOR), a signalling molecule which inhibits cell death and promotes cell growth and proliferation by allowing the cell to proceed past the G1 phase of the cell cycle. PTEN, by the dephosphorylation of PIP3, thus serves to negatively inhibit expression of mTOR, resulting in the arrest of cell growth at G1 [1].
Significance in the Clinical Field
Cancer
As a tumor-suppressor gene, PTEN is one of the most common genes found to be mutated in cancerous cell lines. The most noticeable example is that of endometrial carcinomas, where PTEN is mutated in 50% of the tumours. High rates of PTEN mutation exist in other types of cancer as well, mainly in glioblastoma, melanoma, and prostate cancer [4].
When a mutation in the PTEN gene eliminates the enzymatic activity of its protein product, PIP3 builds up, resulting in constitutive activation of Akt. This ultimately leads to increased cell growth and proliferation, along with decreased rates of cell death.
Alzheimer's disease
PTEN positively regulates neuronal insulin signaling and glucose uptake. Alzheimer's Disease, which can be thought of as a brain-specific form of diabetes [6], has been found to be present when mutated forms of PTEN are also present. PTEN, acting through FAK (a kinase that is involved in neurodegeneration) prevents ERK activation (another kinase that is involved in neurodegeneration). When PTEN is mutated, ERK is constitutively expressed and can lead to neurodegeneration.
Autism Spectrum Disorder
Via the PIP3/Akt pathway, mutations in PTEN typically lead to an increase in the activation of Akt. This in turn affects multiple genes further downstream in the pathway. One such gene is GSK3, which has been shown to control neuronal polarity, and is downregulated in cells with mutant PTEN. Other genes that are affected include those of the TSC/mTOR pathway, a pathway which regulates protein synthesis, and which is upregulated in cells with mutant PTEN, resulting in increased levels of protein synthesis.
Clearly, PTEN controls neural growth via the regulation of genes important for protein synthesis, cell survival/proliferation, axon growth, and neuronal polarity [7]. The importance of PTEN in such processes allows for mutations in PTEN to lead to numerous neurological illnesses such as macrocephaly, seizures and epilepsy as well as symptoms reminiscent of autism spectrum disorder. Although more research is needed in this area, social behaviour deficits as well as increased anxiety has been observed in mice whose PTEN was ablated in the cortex and hippocampus [7].
Medical Application
Chemotherapy is a common form of treatment for many types of cancers. However, this method of treatment affects the entire body and causes many undesirable side effects, including hair loss, nausea, vomiting, diarrhea, loss of appetite, and mouth sores [8]. In addition to these side effects, it is difficult to effectively kill the cancer stem cells that maintain the cancer cell population.
In the prostate gland, castrate-resistant Nkx3-1-expressing cells (CARNs) are multipotent stem cells that line the lumen of the gland. [9]. PTEN mutations in CARNs have been associated with prostate cancer in humans because the stem cell properties of CARNs allow them to become cancer repopulating cells when PTEN is mutated. Its is therefore more effective to directly target the PTEN mutations within these CARNs in patients with prostate cancer
Method of Treatment
Targeting these PTEN mutations is possible using adenovirus vectors. The pathogenic adenoviral genes are removed, and replaced with a large enough portion of the wild-type PTEN to restore proper function, usually around 1.2kb. [10] The patient is then infected with the vector. To target prostate cancer, efficiency may be increased by injecting the virus directly into the prostate.
Purpose of Experiment and Overview
The efficacy and potential issues of the vector therapy should be estimated before therapeutic use in humans. Therefore, it is pertinent to model this treatment in a mouse. mTOR increases tumor growth and is inhibited by wild-type PTEN. Theoretically, if PTEN function is restored in cancerous CARNs, mTOR levels should be decreased in mouse prostates [11]. In our experiment, we attempt to visualize and quantify the effects of reversing the PTEN mutation both in vivo and through biochemical assay.
All mice are transgenic, with GFP attached to the Nestin gene. Nestin is a gene expressed mainly in development as an intermediate filament protein. Nestin is also expressed in some neuronal precursor cells in the adult [12], so it won’t be expressed in any cell populations we are interested in to interfere with the results. Since nestin is also expressed in endothelial cells during angiogenesis of the cancerous tumour, we can attach GFP to the nestin regulatory element to visualize the tumour in vivo [13] before taking some of the cells for assaying to determine mTOR expression. This allows us to quantitatively determine how much angiogenesis the tumour is undergoing via measurement of GFP fluorescence intensity, and subsequently estimate the rate of tumor growth.
Experimental Protocol
Controls and Experimental Groups
Group 1: Positive control (Healthy mice without PTEN mutations in CARNs)
Group 2: Negative control (mice with cancer induced by PTEN mutation in prostate CARNs, no treatment)
Group 3: Control for injection (mice with cancer induced by PTEN mutation in prostate CARNs, saline injection)
Group 4: Experimental (mice with cancer induced by PTEN mutation in prostate CARNs, viral injection)
Procedure
1.) Infect Animals with adenovirus vector via injection into lumen of prostate gland
2.) View the cells of the tumor in vivo
2.) Isolate prostate gland from mouse. Homogenize prostate tissue
3.) Purify mTOR protein
4.) Dilute pure mTOR extraction from each group into an equal amount of Bovine Serum Albumin
4.) Perform Bradford protein assay on purified mTOR extracts to determine mTOR concentration
5.) Determine amount of mTOR using concentration and the initial volume of extract
References
- ^ a b Isabelle Sansal and William R. Sellers (2004). "The Biology and Clinical Relevance of the PTEN Tumour Suppressor Pathway". Journal of Clinical Oncology 22 (14):2954-2963. doi: 10.1200/JCO.2004.02.141
- ^ Griffin et al (2005). "Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer’s disease pathology". Journal of Nerochemistry 93 105-117. doi:10.1111/j.1471-4159.2004.02949.x
- ^ Buxbaum et al (2012). "Mutation Screening of the PTEN Gene in Patients with Autism Spectrum Disorders and Macrocephaly". National Centre for Biotechnology Information 144b (4): 484-491. doi: 10.1002/ajmg.b.30493
- ^ a b Laura Simpson and Ramon Parsons (2001). "PTEN: Life as a Tumor Suppressor". Experimental Cell Research 264 (1):29-41. doi: http://dx.doi.org/10.1006/excr.2000.5130
- ^ Stambolic et al. (1998). "Negative Regulation of PKB/Akt-Dependent Cell Survival by the Tumor Suppressor PTEN". Cell 95 (1): 29-39. doi:10.1016/S0092-8674(00)81780-8
- ^ Gupta, A. & Dey, C.S. (2012). PTEN, a widely known negative regulator of insulin/PI3K signaling, positively regulates neuronal insulin resistance. Mol Biol Cell. 23 (19): 3882–98. doi:10.1091/mbc.E12-05-0337
- ^ a b Zhou, J. & Parada, L.F. (2012). "PTEN signaling in autism spectrum disorders". Current Opinion in Neurobiology. 22 (5):873–79.doi: http://dx.doi.org/10.1016/j.conb.2012.05.004
- ^ Jay Zatzkin (2011). "Prostate Cancer: Chemotherapy". WebMD.com. Retrieved 15 Nov 2012
- ^ Renea A Taylor, Roxanne Toivanen and Gail P Risbridger (2010). "Stem cells in prostate cancer: treating the root of the problem". Endocrinology Related Cancer". 17 273-285. doi:10.1677/ERC-10-0145
- ^ Chen et al (2006) "Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector" Journal of Huazhong University of Science and Technology. 26 (3):325-328
- ^ David A. Guertin and David M. Sabatini (2007). "Defining the Role of mTOR in Cancer". Cancer Cell Review. 12 (1):9-22. doi:10.1016/j.ccr.2007.05.008
- ^ Dahlstrand et al (1992). "Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments" Journal of Cell Science. 103 589-597.
- ^ Robert M. Hoffman (2005). "The Multiple Uses of Fluorescent to Visualize Cancer in vivo". Nature Reviews Cancer. 5 796-806. doi:10.1038/nrc1717
PTEN Information
PTEN (Phosphatase Tensin Homolog) is an oncogene whose mutation is associated with many clinical situations, such as cancer, Alzheimer's Disease, and autism [1] [2] [3]. Biochemically, the PTEN gene encodes an enzyme which functions in modifying different families of macromolecules via a process known as dephosphorylation. In human cells, the PTEN locus is located on chromosome 10. Individuals with cells that possess only one copy of the PTEN gene, usually due to the inheritance of a loss-of-function mutation in their parent's germ cell line, are often found to have developed some form of cancer in later life.
Signalling Pathway
PTEN acts as a tumor suppressor gene [4] by negatively regulating the P13K/Akt signalling pathway in somatic cells. In this pathway, external cell growth and survival signals are detected and relayed to the P13K molecule via receptor tyrosine kinases (RTKs), which activates it. Upon activation, P13K proceeds to convert phosphatidylinositol (4,5)-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3) via a process known as phosphorylation. The role of PTEN in this pathway is to dephosphorylate PIP3 back into PIP2. This makes PTEN an antagonist of P13K [5].
As PIP3 activates a family of molecules known as protein kinase B (abbreviated as Akt), conversion of PIP3 into PIP2 by PTEN results in the depletion of PIP3 and the inactivation of Akt. When active, Akt can further activate or inhibit downstream molecules of the P13K/Akt pathway via phosphorylation of these molecules. This series of reactions is responsible for the activation of mammalian target of rapamycin (mTOR), a signalling molecule which inhibits cell death and promotes cell growth and proliferation by allowing the cell to proceed past the G1 phase of the cell cycle. PTEN, by the dephosphorylation of PIP3, thus serves to negatively inhibit expression of mTOR, resulting in the arrest of cell growth at G1 [1].
Significance in the Clinical Field
Cancer
As a tumor-suppressor gene, PTEN is one of the most common genes found to be mutated in cancerous cell lines. The most noticeable example is that of endometrial carcinomas, where PTEN is mutated in 50% of the tumours. High rates of PTEN mutation exist in other types of cancer as well, mainly in glioblastoma, melanoma, and prostate cancer [4].
When a mutation in the PTEN gene eliminates the enzymatic activity of its protein product, PIP3 builds up, resulting in constitutive activation of Akt. This ultimately leads to increased cell growth and proliferation, along with decreased rates of cell death.
Alzheimer's disease
PTEN positively regulates neuronal insulin signaling and glucose uptake. Alzheimer's Disease, which can be thought of as a brain-specific form of diabetes [6], has been found to be present when mutated forms of PTEN are also present. PTEN, acting through FAK (a kinase that is involved in neurodegeneration) prevents ERK activation (another kinase that is involved in neurodegeneration). When PTEN is mutated, ERK is constitutively expressed and can lead to neurodegeneration.
Autism Spectrum Disorder
Via the PIP3/Akt pathway, mutations in PTEN typically lead to an increase in the activation of Akt. This in turn affects multiple genes further downstream in the pathway. One such gene is GSK3, which has been shown to control neuronal polarity, and is downregulated in cells with mutant PTEN. Other genes that are affected include those of the TSC/mTOR pathway, a pathway which regulates protein synthesis, and which is upregulated in cells with mutant PTEN, resulting in increased levels of protein synthesis.
Clearly, PTEN controls neural growth via the regulation of genes important for protein synthesis, cell survival/proliferation, axon growth, and neuronal polarity [7]. The importance of PTEN in such processes allows for mutations in PTEN to lead to numerous neurological illnesses such as macrocephaly, seizures and epilepsy as well as symptoms reminiscent of autism spectrum disorder. Although more research is needed in this area, social behaviour deficits as well as increased anxiety has been observed in mice whose PTEN was ablated in the cortex and hippocampus [7].
Medical Application
Chemotherapy is a common form of treatment for many types of cancers. However, this method of treatment affects the entire body and causes many undesirable side effects, including hair loss, nausea, vomiting, diarrhea, loss of appetite, and mouth sores [8]. In addition to these side effects, it is difficult to effectively kill the cancer stem cells that maintain the cancer cell population.
In the prostate gland, castrate-resistant Nkx3-1-expressing cells (CARNs) are multipotent stem cells that line the lumen of the gland. [9]. PTEN mutations in CARNs have been associated with prostate cancer in humans because the stem cell properties of CARNs allow them to become cancer repopulating cells when PTEN is mutated. Its is therefore more effective to directly target the PTEN mutations within these CARNs in patients with prostate cancer
Method of Treatment
Targeting these PTEN mutations is possible using adenovirus vectors. The pathogenic adenoviral genes are removed, and replaced with a large enough portion of the wild-type PTEN to restore proper function, usually around 1.2kb. [10] The patient is then infected with the vector. To target prostate cancer, efficiency may be increased by injecting the virus directly into the prostate.
Purpose of Experiment and Overview
The efficacy and potential issues of the vector therapy should be estimated before therapeutic use in humans. Therefore, it is pertinent to model this treatment in a mouse. mTOR increases tumor growth and is inhibited by wild-type PTEN. Theoretically, if PTEN function is restored in cancerous CARNs, mTOR levels should be decreased in mouse prostates [11]. In our experiment, we attempt to visualize and quantify the effects of reversing the PTEN mutation both in vivo and through biochemical assay.
All mice are transgenic, with GFP attached to the Nestin gene. Nestin is a gene expressed mainly in development as an intermediate filament protein. Nestin is also expressed in some neuronal precursor cells in the adult [12], so it won’t be expressed in any cell populations we are interested in to interfere with the results. Since nestin is also expressed in endothelial cells during angiogenesis of the cancerous tumour, we can attach GFP to the nestin regulatory element to visualize the tumour in vivo [13] before taking some of the cells for assaying to determine mTOR expression. This allows us to quantitatively determine how much angiogenesis the tumour is undergoing via measurement of GFP fluorescence intensity, and subsequently estimate the rate of tumor growth.
Experimental Protocol
Controls and Experimental Groups
Group 1: Positive control (Healthy mice without PTEN mutations in CARNs)
Group 2: Negative control (mice with cancer induced by PTEN mutation in prostate CARNs, no treatment)
Group 3: Control for injection (mice with cancer induced by PTEN mutation in prostate CARNs, saline injection)
Group 4: Experimental (mice with cancer induced by PTEN mutation in prostate CARNs, viral injection)
Procedure
1.) Infect Animals with adenovirus vector via injection into lumen of prostate gland
2.) View the cells of the tumor in vivo
2.) Isolate prostate gland from mouse. Homogenize prostate tissue
3.) Purify mTOR protein
4.) Dilute pure mTOR extraction from each group into an equal amount of Bovine Serum Albumin
4.) Perform Bradford protein assay on purified mTOR extracts to determine mTOR concentration
5.) Determine amount of mTOR using concentration and the initial volume of extract
References
- ^ a b Isabelle Sansal and William R. Sellers (2004). "The Biology and Clinical Relevance of the PTEN Tumour Suppressor Pathway". Journal of Clinical Oncology 22 (14):2954-2963. doi: 10.1200/JCO.2004.02.141
- ^ Griffin et al (2005). "Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer’s disease pathology". Journal of Nerochemistry 93 105-117. doi:10.1111/j.1471-4159.2004.02949.x
- ^ Buxbaum et al (2012). "Mutation Screening of the PTEN Gene in Patients with Autism Spectrum Disorders and Macrocephaly". National Centre for Biotechnology Information 144b (4): 484-491. doi: 10.1002/ajmg.b.30493
- ^ a b Laura Simpson and Ramon Parsons (2001). "PTEN: Life as a Tumor Suppressor". Experimental Cell Research 264 (1):29-41. doi: http://dx.doi.org/10.1006/excr.2000.5130
- ^ Stambolic et al. (1998). "Negative Regulation of PKB/Akt-Dependent Cell Survival by the Tumor Suppressor PTEN". Cell 95 (1): 29-39. doi:10.1016/S0092-8674(00)81780-8
- ^ Gupta, A. & Dey, C.S. (2012). PTEN, a widely known negative regulator of insulin/PI3K signaling, positively regulates neuronal insulin resistance. Mol Biol Cell. 23 (19): 3882–98. doi:10.1091/mbc.E12-05-0337
- ^ a b Zhou, J. & Parada, L.F. (2012). "PTEN signaling in autism spectrum disorders". Current Opinion in Neurobiology. 22 (5):873–79.doi: http://dx.doi.org/10.1016/j.conb.2012.05.004
- ^ Jay Zatzkin (2011). "Prostate Cancer: Chemotherapy". WebMD.com. Retrieved 15 Nov 2012
- ^ Renea A Taylor, Roxanne Toivanen and Gail P Risbridger (2010). "Stem cells in prostate cancer: treating the root of the problem". Endocrinology Related Cancer". 17 273-285. doi:10.1677/ERC-10-0145
- ^ Chen et al (2006) "Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector" Journal of Huazhong University of Science and Technology. 26 (3):325-328
- ^ David A. Guertin and David M. Sabatini (2007). "Defining the Role of mTOR in Cancer". Cancer Cell Review. 12 (1):9-22. doi:10.1016/j.ccr.2007.05.008
- ^ Dahlstrand et al (1992). "Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments" Journal of Cell Science. 103 589-597.
- ^ Robert M. Hoffman (2005). "The Multiple Uses of Fluorescent to Visualize Cancer in vivo". Nature Reviews Cancer. 5 796-806. doi:10.1038/nrc1717