| Background and aimsProstate cancer is the second most common cancer, behind only lung caner, and it is the fifth leading causes of cancer death among men. In the recent decade, the incidence rate of prostate cancer increased dramatically. Even with the remarkable progress on prostate cancer treatment, androgen deprivation therapy is only initially effective to control prostate cancer growth, however, in most patients, these therapies eventually fail and the cancer progresses to be castration resistant. The treatment of castration resistant prostate cancer has always been the biggest challenge.Eleven-nineteen lysine-rich leukemia 2(ELL2) belongs to ELL family. As one of the important members of super elongation complex(SEC), Ell2 regulates the catalytic rate of RNA polymerase II transcription. Abnormal regulation of gene transcription in eukaryotic cells leads to various diseases, which has been a popular topic for recent years. In previous research, it has been shown that ELL is regulated by androgen and promotes prostate cell proliferation, which indicates ELL may play an important role in the generation and progression of prostate diseases. As the member of ELL family, ELL2 also binds to RNA Polymerase II and increases elongation rate during transcription, however, it has not been known if ELL2 abnormal expression or dysfunction is involved in prostate cancer.Cancer is a complex group of diseases with a wide rang of possible causes. Abnormal regulation of cell cycle results in uncontrolled cell proliferation, and these cancer cells are out of immunosurveillance and evade apoptosis. The decreased rate of cell apoptosis is one of the important markers for carcinogenesis and cancer progression. There are two signaling pathways has been shown to regulate cell apoptosis: extrinsic Fas signaling pathway which is activated by extracellular ligand binding, and intracellular programmed apoptotic signaling in response to a stress. Both of the pathways can finally activate the proteolytic enzymes, such as caspases. In previous researches, in the benign prostatic hyperplasia(BPH) tissues, ELL2 is an androgen regulated protein, however, if Ell2 regulates cell proliferation and apoptosis is not clear.Another leading cause of prostate cancer poor prognosis is its metastasis and progression. Matrix metalloproteinases(MMPs) play a crucial role during the progress of cancer cell migration and invasion. MMPs, especially MMP1, MMP2, MMP3 and MMP9, are capable of degrading extracellular matrix(ECM) and increasing the tumor cell ability to invade. In addition, cell-adhesion molecules(CAM), such as intercellular adhesion molecule-1(ICAM-1), epithelial cadherin(E-cadherin) and N-cadherin, also play important roles in cancer metastasis. The regualtion of prostate cancer cell ability of migrationa and invasion by ELL2 should also be investegated.In eukaryotes, most proteins are degraded by ubiquitin proteasome system. Ubiquitination is an enzymatic post-translational modification which plays an essential role on protein activation, concentration regulation and degradation. Inhibition of ubiquitination and degradation is an effective way to increase target protein level. The ubiquitination bonds are always formed with one of the seven lysine residues from the ubiquitin molecule, however, this binding can be blocked by the competition binding of other proteins or small molecules to this lysine site, which blocks the target protein degradation. Ubiquitin is a highly conserved small protein, and it has seven lysine residues and an N-terminus that may serve as points of ubiquitination; the addition of a single ubiquitin molecule(monoubiquitination) or different types of ubiquitin chains(polyubiquitination) to those sites leads to the variety of ubiquitin modifications. Proteins marked for degradation are covalently linked to ubiquitin and are targeted to the proteasome. The ubiquitin is released and reused, while the targeted protein is degraded. Ubiquitin molecule has seven lysine residues which can form different polyubiquitination chain, and Lys48 is the most well characterized to play an important role in ubiquitination proteasome system. But the functions of the remaining five ubiquitin chain types which are linked via Lys6, Lys11, Lys27, Lys29, Lys33 are less well defined, and those five residues are call atypical ubiquitin lys-residues. It has been found that the five atypical ubiquitin chain types are related with multiple cell function, which has been a hot research topic. In previous reports, it has been shown that deduction of E3 ubiquitin-protein ligase SIAH1 stabilizes ELL2 expression level and its activity in regulation of RNA polymerase II function. However, the molecular mechanism of ELL2 ubiquitination and degradation need to be further investigated.In this project, we explored the ELL2 expression level in human prostate specimens by comparing ELL2 expression in prostate cancer samples to normal prostate samples by immnuohistochmistry. ELL2 expression level decreased in high Gleason score prostate cancer specimens. To investigate the effect of ELL2 on proliferation, apoptosis, invasion and migration in prostate cancer cells, prostate cancer C4-2 cells were transfected with ELL2 si RNA to knockdown ELL2 expression or transfected with Flag-tagged ELL2 plasmid to overexpress ELL2, and MTT assay, flow cytometry, and transwell assay has been performed. We also used Western Blot to detect the protein expression levels of PCNA involved in cell proliferation, caspase-3 and PARP involved in apoptosis, and MMPs, N-cadherin, E-cadherin and ICAM-1 involved in cell invasion and migration to investigate the possible molecular mechanisms of ELL2 function in prostate cancer. In previous research, the degradation of ELL2 has been found to be dependent on ubiquitination-proteasome pathway by MG132 treatment and immunoprecipitation in C4-2 cells. We further identified the ELL2 ubiquitination sites by plasmid construction and site direct mutagenesis. Additionally, we further investigated the effects of EAF1 and EAF2 on ELL2 degradation by co-transfection of ELL2, EAF1 and EAF2 in 293 cells. At last, we identified the specific lys-residues on ubiquitin to be involved in ELL2 ubiquitination by co-transfection of ELL2 with wild type or mutant ubiquitins followed with immune-precipitation. Our researches focus on investigation the function of ELL2 in prostate cancer, and these findings may provide foundations on regulation of ELL2 expression levels, and development of novel target and pharmaceutical drug to retreat prostate cancer. Methods1. ELL2 expression and its effects on proliferation, apoptosis, invasion and migration in prostate cancer1.1 ELL2 expression in prostate cancer29 prostate cancer samples and 22 normal prostate samples were collected separately, and ELL2 expression was examined by immunohistochemistry.1.2 Androgen regulation of ELL2 in prostate cancer cellsProstate cancer cells LNCa P and C4-2 were treated with the synthetic androgen methyltrienolone(R1881) for various doses and time points, ELL2 was detected by Western blot and real-time PCR.1.3 ELL2 effects on proliferation, apoptosis, invasion and migration in prostate cancer cellsC4-2 cells were transfected with ELL2 si RNA or ELL2 expression vector followed with MTT assay to examine cell proliferation, flow cytometry to examine apoptosis, Western blot to examine PCNA and caspase-3 and PARP, transwell assay to check cell migration and invasion, and Western blot to check MMP1, MMp2, MMp3, MMP9, ICAM-1, N-cadherin and E-cadherin.2. Determine the specific domain and amino acid sites of ELL2 for ubiquitination, the effects of EAF1/EAF2 on ELL2 degradation, and the lysine residues of ubiquitin involved in ELL2 degradation2.1 Examination of ELL2 protein stabilityC4-2 cells were treated with R1881 to increase ELL2 expression, followed with the treatment of cycloheximide(CHX) to inhibit protein synthesis. Western blot were performed to examine ELL2 protein level hourly for five hours. Additionally, C4-2 cells and 293 cells with exogenous ELL2 expression were treated with CHX and MG132 to check stability of endogenous and exogenous ELL2.2.2 Determination of the domains and sites of ELL2 for its ubiquitination293 cells were cotransfected with Flag-tagged ELL2 plasmid and HA-tagged ubquitin plasmid, and treated with MG132. Immunoprecipitation with Flag antibody conjugated beads and Western blot were performed to check the ubiquitination of Flag-tagged ELL2. Furthermore, C4-2 cells were transfected with various vectors coding truncated ELL2 fragments(fragment 1: aa1-aa292, fragment 2: aa293-aa531, fragment 3: aa532-aa640) and treated with CHX. Expression levels of various truncated ELL2 were examined at 4hr, 8hr, 12 hr, 24 hr and 1hr, 2hr, 3hr, 4hr; for fragment 3, the expression level has been examined at 15 min, 30 min, 45 min, 60 min, and 75 min. Finally, we generated nine site directed mutant vectors of fragment 3 with K to R mutations. C4-2 cells were transfected with these nine vectors individually and treated with CHX, and the expression levels of fragments 3 were examined at 0hr, 6hr, 12 hr and 24 hr.2.3 The effects of ELL2 binding partners EAF1 and EAF2 on ELL2 degradation293 cells were cotransfected with Flag-tagged ELL2 together with Myc control vector, Myc-tagged EAF1 and Myc-tagged EAF2 individually, and treated with CHX. ELL2 expression was examined at 0hr, 6hr, 12 hr and 24 hr by Western blot. Furthermore, 293 cells transfected with Flag-tagged ELL2 and HA-tagged Ub were transfected with Myc control vector, Myc-tagged EAF1 and Myc-tagged EAF2 individually. Cells were treated and MG132, and ELL2 ubiquitination levels were examined by immunoprecipitaion with Flag antibody conjugated beads and Western blot.2.4 Determination of lysine residues on ubiquitin for ELL2 ubiquitination and degradation.293 cells were cotransfected with Flag-tagged ELL2 and various ubiquitin expression vectors with site direct mutation at K6, K11, K27, K29, K33, K48 or K63, then treated with MG132. ELL2 ubiquitination levels were examined by immunoprecipitaion with Flag antibody conjugated beads and Western blot. Results1. ELL2 expression and its effects on proliferation, apoptosis, invasion and migration in prostate cancer1.1 ELL2 expression decreased in prostate cancerAs shown by immunohistochemistry, ELL2 expression decreased in human prostate cancer specimens by comparing to normal prostate specimens, and down-regulation of ELL2 correlated with increased Gleason score(P<0.05).1.2 ELL2 is androgen responsive proteinIn C4-2 and LNCa P cells, ELL2 protein level and m RNA level were significantly induced by R1881 by a dose-/time- dependent manner in Western blot and real-time PCR(P<0.05).1.3 ELL2 effects on proliferation, apoptosis, invasion and migration in prostate cancer cellsKnockdown of ELL2 in C4-2 cells significantly induced cell proliferation(P<0.001) in MTT assay and up-regulated PCNA expression in Western-blot, while overexpression of exogenous ELL2 inhibited cell proliferation(P<0.001). Knockdown of ELL2 in C4-2 cells significantly blocked apoptosis(P<0.05) as shown in flow cytometry and down-regulated caspase-3 and PARP expression(P<0.05, P<0.01), while overexpression of ELL2 induced apoptosis(P<0.01) and up-regulated caspase-3 and PARP expressions(P<0.001, P<0.001). Knockdown of ELL2 in C4-2 cells significantly induced cell invasion and migration(P<0.01, P<0.05) in transwell assay, induced protein expressions of MMP2(P<0.001), MMP3(P<0.001), MMP9(P<0.001), N-cadherin(P<0.05) and ICAM-1(P<0.05), and inhibited E-cadherin(P<0.05), while overexpression of exogenous ELL2 inhibited cell invasion and migration(P<0.01, P<0.01), inhibited protein expressions of MMP2(P<0.05), MMP3(P<0.01), MMP9(P<0.01), N-cadherin(P<0.01) and ICAM-1(P<0.05), and upregulated E-cadherin(P<0.001).2. Determination of the specific domain and amino acid sites of ELL2 for ubiquitination, the effects of EAF1/EAF2 on ELL2 degradation, and the lysine residues of ubiquitin involved in ELL2 degradation.2.1 ELL2 is unstable proteinAs shown in Western blot, with the treatment of CHX, ELL2 expression level dramatically decreased, and decreased 80% by comparing to control sample. Additionally, both endogenous and exogenous ELL2 levels were significantly up-regulated by MG132 treatment(P<0.05, P<0.05).2.2 ELL2 is ubiquitylated and degradesFragment 2 containing aa293-531 is the most stable fragment of ELL2, and mutations at K571, K584 and K599 are beneficial for their protein stability. As shown in Western blot, the sample treated with MG132 has obvious ubiquitination bands, but not in control sample. By detecting the expression levels of various truncated ELL2, it has been found that fragments 3 containing aa 532-640 degraded faster that other two fragments, and it degraded for more than half at 75 min. Fragment 2 containing aa293-531 is the most stable fragment, and its expression level did not change for 12 hours, while the degradation rate of fragment 2 containing is in the middle. By transfection mutant ELL2 expression vectors with various K to R mutations, we found that mutant expression vectors only with K/R571, K/R584 and K/R599 degraded slower than wild type ELL2.2.3 ELL2 binding partners EAF1/EAF2 stabilize ELL2 expressionCo-transfection of Myc-tagged EAF1 or Myc-tagged EAF2 slow down ELL2 degradation by comparing to the control sample which was co-transfected with empty Myc vector. Especially, the effect of EAF2 on ELL2 stability is more significant. Furthermore, as shown in Western blots, co-transfection of HA-tagged ubiquitin and Flag-ELL2 with Myc-tagged EAF1 or Myc-tagged EAF2 individually significantly decreased the intensity of ubiquitylated ELL2 bands and increased ELL2 expression, by comparing with control samples with transfection with Myc empty vector.2.4 Determination of lysine residues on ubiquitin for ELL2 ubiquitination and degradation.As shown in Western blot, ubiquitination bands of K6, K11, K33, K48 and K63 are visible, and bands of K63, K6 and K48 are stronger. Conclusion1. ELL2 is down-regulated in prostate cancer and its expression level reversely correlated with Gleason score.2. ELL2 is responsive to androgen, and its abnormal expression may be related with castration resistance in prostate cancer.3. Up-regulation of ELL2 protein level inhibits prostate cancer cell proliferation, induces apoptosis, and inhibits cancer cell ability to migrate and invade, which indicates that ELL2 is involve in the tumorigenesis and progression of prostate cancer.4. ELL2 degrades through ubiquitination and proteasome system, and its C-terminal domain(aa532-aa640) plays the crucial on its stability. K571, K584 and K599 are the active sites to be ubiquitylated. EAF1 and EAF2 can stabilize ELL2 by block ubiquitination. The investigation of ELL2 molecular structure will provide insights on understanding the regulation of ELL2 intracellular level and further provide evidences for development of novel treatments at the molecular level.5. Except for K63 and K48, there may be multiple atypical lysine residues involved in ELL2 ubiquitination, which indicates the involvement of ELL2 in various cell functions. |
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