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Structure and Function
PTEN, phosphatase and tensin homologue, is a protein coded by Pten gene at the 10q23 locus. Its principal catalytic function is to dephosphorylate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3), which is an activator of PDK (3-phosphoinositide-dependent kinase) and AKT. Loss of PTEN allele function increases cellular PtdIns(3,4,5)P3 level and thus deregulates and increase the potency of phosphoinositide 3-kinase (PI3K) – AKT pathway, a pathway that stimulates cell growth and survival .. Downregulation of PTEN level increases cancer susceptibility and favours tumour progression. PTEN is a haploinsufficient tumour suppressor and its function is dependent on the level of expression or activity of PTEN protein rather than the number of copies of the Pten gene. PTEN is 403 amino acids long and contains a phosphatase domain and C2 domain. The phosphatase catalytic motif is HCXXGXXR (where X can be any amino acids), or P loop in residues 123 to 130, is encompassed in the amino-terminal region’s actin-binding protein tensin 1 homologue. C2 domain can bind to cell membranes. Tumour and cancer associated mutations targets all domains of PTEN.

PTEN is part of PTEN–PI3K–AKT–mTOR pathway and the pathway has an important role in regulating cellular metabolisms (insulin, amino acids, and growth factors), PTEN reduces the potency of the pathway. Loss of function of PTEN will result in overexpression of the pathway and thus leads to uncontrolled cell growth. PTEN proteins are also localized to the nucleus and seemingly to reduce the progression of cancers, this implies there’s more function to PTEN than its usual phosphatase role. PTEN when localization also plays a role in cell polarity and cell fate, as seen in epithelial-mesenchymal transition. Even though reduced activity of PTEN leads to proliferation of cancers, completely loss of PTEN induces cellular senescence and depletes cancer stem cells, because acute PTEN depletion triggers cellular checkpoints that irreversibly leads to senescence and apoptosis.

Regulation
PTEN regulation is a critical component of proper cell cycle maintenance, ensuring division and apoptosis when signalled. Disruption in the integrity of PTEN protein regulators can cause cancers and other diseases. Perturbation in components needed in essential regulatory mechanisms such as microRNA regulation, epigenetic silencing, transcriptional regulation, protein-protein interactions, and post-translational modifications, can result in the compromising of PTEN function, thus affecting the PI3K-Akt pathway. Understanding components that regulate PTEN is important, as mutations that affect PTEN are very common causes of many different types of cancers.

Genetic Alterations of PTEN
PTEN mutations can happen in both germline and somatic cells. Somatic mutation such as insertions, deletions and substitutions can occur throughout the whole PTEN gene but certain amino acids in the PTEN gene has higher rate of mutations. Allelic or complete deletion of PTEN often coincides with prostate and breast cancer, also in melanoma and glioma. Germline mutations of PTEN is often seen with patients of PHTS (PTEN hamartoma tumour syndromes, where a patient has a PTEN mutation and develops non-malignant tumours also patient’s cellular elements have a chaotic architecture). Mutated PTEN gene can, however, still give rise to partial or completely functional protein; suggesting other mechanisms might be at play for silencing PTEN. Germline mutations can occur in exon 5,7 and 8, promoter, in splice donor and acceptor sites of PTEN (as seen in Cowden disease).

Epigenetic Silencing and Transcriptional Regulation
In normal tissues, PTEN is constitutively expressed, but the expression of the gene can be altered epigenetically and make the expression pathological. Aberrant methylation of the PTEN promoter is linked to many types of cancer. PTEN locus can be repressed by epigenetic repressor complex containing a chromatin-remodelling ATPase and histone deacetylase. Alternative splicing that cause the retention of intron 3 and 5 regions is also associated with breast cancer. In addition, many tumour suppressors and oncoproteins can influence the transcriptional control of PTEN by affecting key junctures in the transcriptional network, but the detail mechanisms are not well understood .

Post-transcriptional regulation of PTEN by non-coding RNAs
The expression of PTEN is negatively regulated by numerous microRNAs (miRNAs) at the post‑transcriptional level. miRNA is a class of 20-25 nucleotide non-coding RNAs that anneals to complementary sites on either coding sequence or 3' UTR of target mRNAs. miRNAs promote impairment in translation and decrease in mRNA stability, down-regulating PTEN expression. This is found to be linked to human cancers and diseases, such as breast cancer and Cowden disease. On the other hand, PTEN pseudo gene 1 (PTENP1), which consist of homologous sequence of PTEN mRNA, also regulates PTEN expression by controlling the expression of PTEN-targeting miRNA. PTENP1 genes directly compete for the binding of miRNAs as competitive endogenous RNAs (ceRNAs) and inhibit the negative regulation of miRNA on PTEN expression.

Phosphorylation
When phosphorylated at its C-terminus region, PTEN is active and “open”, and so will act as an antagonizer for the PI3K/Akt pathway. The unphosphorylated PTEN gets recruited into a PTEN-associated complex (PAC) which interacts with PDZ-containing proteins that work together to block Akt activation, such as MAGI-2. The PDZ domain in the PTEN is a 80-100 residue region that allows for other proteins to assembly onto it and for the PAC by acting as a scaffold. However, phosphorylation at its C-terminal tail causes PTEN to become “closed” and more stable, through masking of the PDZ-binding domain in that region. In this “closed” conformation, PTEN has a low affinity for PDZ-domain-containing proteins and so will not participate in the PAC. This reduces the overall phosphoinositide-3,4,5-triphosphatase activity of the PTEN. Thus, phosphorylation of the C-terminal tail in PTEN prevents the recruitment of PTEN into the PAC, which prevents it from functioning and acting as an antagonist of the PI3K/Akt pathway. One such protein that phosphorylates PTEN is casein kinase 2 (CK2), which phosphorylates the Ser370, Ser380, Ser385, Thr382, and Thr383 residues located in the C-terminal tail to cause a reduction in PTEN activity. Interestingly, phosophorylation by glycogen synthase kinase 3 beta (GSK3β) at Thr366 destabilizes PTEN, suggesting that the steady state of the phosphotase is regulated by phosphorylation at different sites by different PTEN kinases.

Acetylation
Acetylation enhances the activity between PDZ domains and PTEN by increasing the affinity of PTEN to PDZ-domain containing proteins, causing stronger bonding in the PAC. One such site of acetylation of PTEN is at the Lys402 that is located in the PDZ domain-binding motif of PTEN. Acetylation at this site has shown greater interaction between the PTEN and MAGI-2 (MAGI-2 as mentioned above in phosphorylation section). Alternatively, acetylation on PTEN may also negatively regulate it. In specific, the histone acetyltransferase p300/CBP-associated factor (PCAF) will acetylate Lys125 and Lys128 in the presence of certain growth factors. As these residues are found in the catalytic region of PTEN and so are essential for PtdIns (3,4,5)P3 specificity acetylation of these residues have been found to inhibit PTEN activity.

Oxidation
In the presence of reactive oxygen species (ROS), PTEN activity is downregulated. ROS causes a disulfide to form between the Cys124 residue that is located in the PTEN active site and the Cys71 residue. The creation of the disulfide changes the conformation of PTEN and results in its deactivation.

Ubiquitylation
Ubiquitylation can increase PTEN degradation but can also increase nuclear import of PTEN. An important component of ubiqtuin-mediation proteasomal degradation is the ubiquitin ligase (E3). One such ligase is NEDD4-1, whose activity is normally supressed. However, NEDD4-1 function can be stimulated by specific signals, causing polyubiquitination of PTEN, and increasing PTEN degradation in the cytosol. This negatively regulates the effect of PTEN on the PI3K/Atk pathway. However, NEDD4-1 is also capable of catalyzing monoubiquitination of PTEN at Lys13and Lys289; monoubquitylation at these sites have been shown to increase the import of PTEN from the cytoplasm to the nucleus, increasing PTEN activity. Thus, ubiquitylation can have both a positive and negative effect on PTEN function.

PTEN regulation by protein–protein interactions
PTEN stability and activity can be regulated through interactions with different proteins. Most of these proteins interact with the C terminus of PTEN and also the PDZ domains. Stability is increased with phosphorylation within the C terminus of PTEN. Examples would include PICT1, DLG-1, and ROCK, which bind to the C terminus and phosphorylates PTEN. On the other hand, stability is decreased if there is no phosphorylation with the C terminus of PTEN or the terminus is mutated in a way that phosphorylation cannot occur. It has been noted that in the absence of the PICT1, a protein that enhances stability of PTEN by phosphorylation, rapid degradation can be observed.

Most of the proteins that increase phosphatase (PPase) activity of PTEN enhance the signaling or recruitment of PTEN to the appropriate location to restrict activation of PI3K. Examples of these proteins would include NHERF, MAGI2, β-Arrestins, MyosinV, and DLG-1 . In contrast, the proteins that decrease PPase activity inhibits PTEN by binding to it directly, which prevents it from being able to use the PIP3 phosphatase. These proteins would include PREX2a, SIPL1, and MSP58, and they bind to the carboxy terminus tail of PTEN, inhibiting its activity .

Clinical Significance and Applications
Breast cancer is one of the most common diseases amongst women. It has been noted that around 30 - 50% of breast cancer patients have low expression of PTEN in the breast cancer tissues. Based on our understanding of PTEN and its regulatory effects as a tumor suppressor gene, a downregulation of PTEN can cause the cancer cells to continue growing and proliferating. Comparisons of human miRNA sample have shown that miR-21 is found to be greatly overexpressed in breast tumours compared to normal breast tissues. This miRNA inhibits apoptosis in breast cancer and various cancer by downregulating PTEN expression as described earlier.

Based on our understanding of negative regulation of miRNA on PTEN expression, we can try to up-regulate PTEN expression by inhibiting the expression of this highly overexpressed miRNA, miR-21. This can be done by delivering competitive endogenous RNA (ceRNA), which can be synthesized to have homologous sequences to PTENP1, to a breast cancer patient by viral vector to compete for the miR-21 against the PTEN gene. PTENP1 is a pseudogene of PTEN and share similar 5' region to PTEN gene; it has been found to be selectively lost in people with cancer. Incorporation of the ceRNA would allow for the cellular PTEN to have a higher and more likely chance of being expressed since the ceRNA would lower the odds of the miR-21 binding to the PTEN mRNA and lowering its translational rate.



To promote expression of the ceRNA in the cell, we could use viral vectors. In specific, we could use adenoviral vectors, which are replicated as episomal elements in the nucleus and thus minimizing any possible adverse effects from disruption of the host cell DNA. We can generate replication-deficient adenovirus vectors through the replacement of the El gene (required for viral replication) in the adenovirus gene with our gene of interest – in this case, the ceRNA. This will ensure that the viruses will not replicate and spread, minimizing the risk of adding an exogenous element to cells, since the genes for the replication of this virus would be deleted. We would also viral envelope proteins with those from other viruses or by chimeric proteins, so that the sequence of ceRNA is able to interact with specific target host cell proteins in the patient. By injecting the ceRNA-containing virus intravenously to the patient, the virus can transport ceRNA to the cell, where the PTEN-targeting miRNA is localized, via viral infecting mechanisms. Ongoing booster sessions would be required depending on patient response, as the ceRNA-contained virus cannot replicate itself. ceRNA directly compete for the binding of miRNAs and inhibit negative regulation of miRNA on PTEN expression.

To assess whether the treatment was successful, PET (Positron Emission Tomography) or CT (Computed Tomography) can used to scan a whole-body of the patient. This result of this scan can be compared to a previous scan prior to treatment to determine the effect of our treatment. Of course, introduction of external elements into cells will have its risks. The biggest possible side effect is severe triggering of inflammatory response with the introduction of foreign material into the cell. For example, in one case of adenovirus administration, the patient suffered severe side effects caused by the immune system reaction to the viral proteins when the vector spread to other regions unintentionally. However, studies have shown that inflammatory responses are highly dependent on dosage in that no cellular toxicity is observed until the dosage exceeds a ‘threshold effect’, so observing a patient’s responses will be critical to prevent the possibility of exceeding a safe dosage limit. There is also the possibility of the viral vector leaking and disseminating into undesired areas of the body. To combat this, tissue-specific regulatory promoters can be added in the vector to limit the translation of the transgene.

It is not just breast cancer that has shown an overexpression of miR-21; elevated levels of this specific miRNA has been observed in glioblastoma tumours in the brain and spine, which suggests that such a technique can be applied to other similar situations. Other types of cancer which have been shown to have an aberrant number miRNA present in the cells, such as prostate cancer, lung cancer, colon cancer, and pancreatic tumours, and so application of this gene therapy should be considered for future studies regarding other cancers. Vectors coding products that can bind to the miRNA to down-regulate its inhibition of desired genes can be created and tailored for each therapeutic application.

Viral transduction for other diseases
Apoptosis driven by regulators, such as PTEN, is needed to avoid excessive cell division and growth. In an experiment done by Tanaka and Grossman in 2002 regarding bladder cancer, PTEn adenoviral vectors were created and injected in vivo into cell lines with tumours created as a result of upregulated Akt and a wild-type PTEN gene as well as in cell lines with upregulated Akt and a deleted PTEN gene. Results from both groups showed that there was a downregulation in phosphorylated (active) Akt and an increase in apoptosis, meaning tumour growth was suppressed. Insertions of such vectors should be considered and could prove to be an effective treatment for other cancers. Similar results were observed in adeno-viral transductions of PTEN into gastric cancer cell lines. Other viral vectors such as retroviruses, lentiviruses, adeno-associated viruses, could also be considered, depending on various factors, such as the target tissue and desired packaging capacity.

Using inhibitors
Low levels of PTEN have been observed in some diseases, similar to breast cancer. To increase PTEN activity, inhibiting enzymes that causes PTEN to become less activity could be beneficial. Recently, small CKII inhibitors were isolated and tested in a colorectal carcinoma cell line (HCT116). The results showed that addition of the inhibitors reduced phosphorylation of PTEN and inhibited HCT116 cell growth[1H]. Addition of compounds that can increase PTEN activity in tumour cell lines can prove beneficial in treating tumours

Inhibiting the mammalian target of rapapmycin (mTOR) also can be used to treat problems associated with loss of PTEN. mTOR mediates the phosphorylation of PTEN and the activity of kinase, such as Akt activity that activates p70S6 kinase (p70S6K), which is associated with increased proliferation of tumor. The study that investigated the effect of pharmaceutical drugs on cell growth in eight human breast cell lines found that CCI-7799 reduces proliferation and tumor size by inhibition of p70S6K activity and mTOK. Some chemotherapeutic drugs, such as cisplatin, paclitaxel, Adriamycin, can influence PTEN activity in similar manners. Inhibitors that mimic PTEN function are used as therapeutic agents in breast cancer and other types of cancer.

Diagnosis
Tissues from some tumour cell lines are expected to have a higher quantity of phosphorylated PTEN compared to wild-type tissues, since phosphorylated PTEN reduces the activity of the phosphatase and allows for the PI3K/Akt pathway to progress more abundantly. As PTEN phosphorylation is correlated with its activity, quantifying the amount of phosphorylated PTEN can be used as a prognostic tool for tumour development. Levels of active PTEN can be detected with phosphospecific antibodies to diagnose tumour activity.