Identification Of The Deubiquitinase For The Tumor Suppressor PTEN And The Study Of PTEN Ubiquitination Regulated Breast Cell Apoptosis

Background and objectivePhosphatase and tension homologue deleted from chromosome10(pten/mmac1/tep1) belongs to the protein tyrosine phosphatases family (PTP),which located at chromosome10q23.3. It is found to be the only one tumor suppressor that has the activity of both lipid phosphatase and protein phosphatase. PTEN antagonizes the actions of phosphatidylinositol3-kinase by dephosphorylating the second messenger phosphatidylinositol3,4,5-trisphosphate(PIP3), regulating activation of the phosphatidyl inositol3-kinase (PI3K) and kinase Akt as well as the downstream cellular growth, proliferation,migration and other cellular responses.PTEN is considered to be the most powerful tumor suppressor found after p53, which is frequently disrupted or mutated in human cancers and cancer predisposition syndromes. Tight regulation of PTEN stability is of great importance because even a slight reduction in PTEN protein levels can have tremendous consequences in tumor development.PTEN has been a hot spot both in the fields of life science and medicine since it was first identified. The importance of PTEN’s physiological function has been generally acknowledged by the academic circle.As PTEN is such an important molecule, its activity and stability is controlled strictly and accurately. In the tumors, the pten promoter methylation can be found to lead to the gene silence; in the post-transcriptional step, PTEN can be inhibited by RNA miR-21and miR-19; In the post-translational step, PTEN can be ubiquitinated, phosphated and acetylated to change its subcellular location, protein structure, protein stability and protein activity.Besides transcription and protein synthesis modulation, the protein dose in the cell is also accurately modulated by the balance of ubiquitination and deubiquitination. The ubiquitin ligase (E3) and deubiquitinase (DUB) are a couple of crucial molecules accurately controlling the protein level. Several E3ubiquitin ligases including Nedd4-1, WWP2, XIAP, CHIP (C terminus of Hsc70-interacting protein) and TRIM27/RFP have been shown either to promote PTEN polyubiquitination for proteasomal degradation or monoubiquitination for nuclear import, while the ubiquitin-specific proteases of PTEN are only HAUSP and USP13. HAUSP has been shown to remove the monoubiquitination of PTEN and induces its nuclear export. However, HAUSP has no effect on PTEN protein stability and it thus is a DUB specific for PTEN de-monoubiquitination. To date, only USP13has been identified to have the function of de-ubiquitinating the polyubiquitination of PTEN and preventing PTEN degradation. The DUB for de-polyubiquitination and stabilization of PTEN remains requiring more excavation and research.Forkhead box O (FoxO) transcription factors are crucial regulators of diverse cellular activities. These factors are also associated with multiple human cancers. Akt acts as an important upstream regulator. Activated Akt directly phosphorylates FoxO family proteins and promotes their degradation. Consequently, less FoxO protein accumulates in the nucleus to execute protranscriptional actions towards target genes involved in cell-cycle arrest and apoptosis. PI3K/AKT signaling has been shown to be downregulated by PTEN, so the protein level of PTEN is a key factor in regulating FoxO family members’ function.In this research, we identified a new DUB of PTEN, researched its functions on remaining PTEN stability and explained the molecular mechanism, and confirmed how the E3CHIP involved PTEN and its downstream apoptosis pathway.MethodsHuman DUB family can be divided into five sub-families, including UCH, USP, OTU, Josephin and JAMM/MPN+. To identify the DUB that regulates PTEN protein stability, we overexpressed each of the human DUBs in HCT116cells. PTEN levels were tested by Western blot. We found that overexpression of a member of the OTU sub-family OTU domain-containing protein3(OTUD3) can upregulate PTEN level. Coimmunoprecipitation tested the interaction of OTUD3and PTEN, and confirmed the related regions.OTUD3was overexpressed to observe its affect of Akt pathway. Endogenous OTUD3was knocked down to test the influence on cell proliferation, apoptosis and invasiveness.OTUD3and PTEN expressions in colorectal cancers and breast cancers were tested by immunohistochemistry or Western blot. The mutations of OTUD3and PTEN in breast cancer tissues were screened and the plasmids carrying the same mutation sites were used to study the influences on PTEN halflife. E3CHIP was overexpressed in the breast cancer cell MCF7and breast epithelial cell MCF10A, and the activity of Akt, FoxO proteins, protein level of Bim and cell apoptosis were examined. Immunohistochemistry was used to test CHIP and Bim expressions in breast cancer tissues。Results1. Overexpression of OTU sub-family member OTUD3can specially upregulate PTEN level, but other OTU sub-family members can’t. It showed that the regulation of PTEN by OTUD3had specificity. Knockdown of OTUD3can lead to PTEN decrease significantly, and this effect can be repeated in multiple cell lines. However, the enzyme-inactive mutant OTUD3C76A lost the ability to upregulate PTEN. Downregulation of PTEN was found when OTUD3C76A was overexpressed. It showed that OTUD3was a key modulator of PTEN protein level, and this function of OTUD3dependented on its DUB activity.2. OTUD3can bind with PTEN directly. The OTU domain of OTUD3and the C2domain of PTEN intermediated the interaction, which is independent the DUB activity of OTUD3. The mutations in PTEN C2domain reported by literatures are PTEN△M199, PTENP246L, PTEN△T319, PTENF341V, PTENV343E, PTENL345Q, PTENT3491, PTEN1-319and PTEN1-342. In the mutant PTEN proteins, PTEN△M199, PTENF341V and PTEN1-319can not bind to OTUD3, which leads to less stability, shorter halflife and lower protein level of PTEN.3. Knockdown of OTUD3can overly activate the Akt pathway, leading to faster proliferation, more invasive behavior and less sensitivity to cisplatin induced apoptosis, which is similar to the effects of PTEN knockdown. It is quite notable that the recuction of OTUD3is associated with increasing invasiveness ability of tumor cells. When OTUD3knockdown cells were subcutaneous injected into nude mice, the metastasis occurs in four weeks, which is just like the ones injected with PTEN knockdown cells. It shows how important OTUD3is in the inhibition of tumor cell migration. The decrease or deletion of OTUD3can promote the tumor matastasis.4. In human colorectal cancer tissues,OTUD3and PTEN are positively related and their tissue location is consequent.5. Because of the tumor suppressor role of PTEN, we screened the mutations of PTEN and OTUD3in breast cancer tissues, and researched if the mutations involved the function of OTUD3and stability of PTEN. The results are evidences to prove that OTUD3is a new tumor suppressor. In the screening, two OTUD3mutations were identified, OTUD3R79T and OTUD3E86K.OTUD3(wild type), OTUD3R79T and OTUD3E86K were overexpressed separately in MCF7cells. The PTEN protein level in cells expressing OTUD3R79T got higher, just like in cells expressing OTUD3. Overexpression of OTUD3E86K decreased PTEN protein level significantly. The phenomenon suggests that OTUD3R79T can stabilize PTEN, and the mutation does not reduce its DUB activity, while OTUD3E86K may have lower or lost its DUB activity, just like the enzyme-inactive mutant OTUD3C76A, which losts the ability to upregulate PTEN. Overexpression of OTUD3R79T prolonged the halflife of PTEN, but OTUD3E86K can shorten the halflife of PTEN. It proves that OTUD3R79T has the ability to stabilize PTEN, but OTUD3E86K losts and becomes a negative regulator of PTEN. Both OTUD3R79T and OTUD3E86K can combine with endogenous PTEN, and the ability has no difference from wild type OTUD3. It suggests that the difference of function between OTUD3R79T and OTUD3E86K is not related to the interaction with PTEN, but is associated with the decreasing or lost of DUB activity.6. Intriguingly, through sequencing of PTEN C2domain, we detected PTEN mutations in4out of50breast cancers, i.e. PTENF341V+I224M, PTENE307K and PTENF341V. The cancer tissues bearing PTENF341V failed to bind OTUD3and expressed PTEN with short halflife. Unlike PTENF341V, the PTENI224M and PTENE307K mutations had no significant effects on the PTEN binding of and stabilization by OTUD3. Overexpression of OTUD3can still prolong the halflife of PTENI224M and PTENE307K. These findings suggest that the amimo acid site1224and E307does not intermediate PTEN stability maintaince.7. PTEN is such a crucial tumor suppressor, and OTUD3acts as a tumor suppressor through maintaining PTEN stability, but what the role of PTEN ubiquitination played in breast cancer cells? To answer this question, how C terminus of Hsc70-interacting protein (CHIP) influences PTEN and downstream pathways in breast cancer cell MCF7and breast epithelial cell MCF10A is tested. It proves how important PTEN ubiquitination is in breast cancer tumorigenesis, development and therapy. Mumbers of studies have shown that apoptosis resistance can be observed in multiple human tumors, but the molecular mechanism is still unknown. Our research showed that CHIP downregulate PTEN level through promoting the ubiquitin-proteasome degradation, which led to the activation of PI3K-Akt. The excessively activated Akt phosphated downstream FoxO family members. The function and the protein dose of FoxO familiy members were inhibited by phosphorylation. FoxO3is a master transcriptional regulator which can modulate multiple genes’transcription. Excessive phosphorylation of FoxO3decreases its ability to binding to bim and pten promoters, which leads to transcriptional inhibition and protein synthesis reduction of Bim and PTEN. The reduction of Bim can lead to cells appearing apoptosis resistance. Decreasing of PTEN level upregulates Akt phosphorylation, which will lead to a higher level of phosphated FoxO3, severer transcriptional inhibition of pten gene. All of above make a positive feedback loop to continuously decrease PTEN protein level. In other words, PTEN is controlled in both transcriptional and post-translational steps to make PTEN dose reduce continuously. The positive feedback loop leads to MCF7and MCF10A cells obtaining apoptosis resistance. CHIP expresses in breast cancer tissues at high levels, which is negatively associated with PTEN and Bim.Conclusions1. OTUD3is a DUB of PTEN, and maintains PTEN protein stability.2. OTUD3acts as a tumor suppressor through modulating PTEN stability.3. Gene mutations affected OTUD3function can be found in breast cancer tissues.4. E3CHIP induces apoptosis resistance through forming a positive-feedback loop to reduce PTEN level.SignificanceOur study found a new DUB of PTEN, and identified the first substrate of OTUD3PTEN. Our research revealed the mechanism of PTEN stability maintaince and identified that CHIP regulated PTEN in transcriptional and posttranslational steps in breast cancer cells and breast epithelial cells. These discoveries supported the "Continuum Model" hypothesis and provided new targets for tumor therapy.