Cyclin-dependent Kinase (CDK11) Is Crucial In The Growth Of Liposarcoma Cells

Liposarcoma is the second (second only to malignant fibrous histiocytoma in incidence) most common soft tissue malignant tumors, account for 15% of adult soft tissue sarcoma. Liposarcoma usually arises in lipoblast cells in deep soft tissue, such as that inside the thigh or in the retroperitoneum. Patient survival is related to the degree of malignancy of the cell types, localization, size of the primary tumor and treatment protocols. For the treatment of liposarcoma, although surgical resection remains the main modality for curative therapy, extremity liposarcomas situated adjacent to vital organs and large retroperitoneal liposarcomas are difficult to manage and are associated with local recurrence and a high mortality rate. Local recurrence rates have been reported as between 15% and 75%. Incorporation of neo-adjuvant approaches such as chemotherapy or radiotherapy could reduce complications and surgical impact, doxorubicin alone or in combination with ifosfamide is often used as a first-line regimen in patients with high risk of recurrence or metastatic liposarcoma. There has been no significant progress in improving the survival rate of recurrent or metastatic liposarcoma for almost three decades. Thus, identification of new targeted therapies is critical to improving the survival rate of liposarcoma patients. New discoveries in molecular biology may one day open a door for the treatment of liposarcoma.The discovery of oncogenic kinases and target-specific small-molecule inhibitors has revolutionized the treatment of a select group of cancers, such as chronic myeloid leukaemia (CML) and gastrointestinal stromal tumors (GIST). Protein kinases play important roles in regulating tumor cellular functions-proliferation/cell cycle, cell metabolism, survival/apoptosis, DNA damage repair, cell motility, and drug resistance--so it is not surprising that they are often themselves cancer causing oncogenes. Kinases such as c-Src, c-ABL, PI3K/AKT, EGFR, MAP, IGF-1R, and JAK are commonly activated and highly expressed in cancer cells and are known to contribute to cancer progression. Kinases are now firmly established as a major class of drug targets. Significant progress has been made in understanding kinases and their functions. There has been an explosion in the number of kinase inhibitors entering the clinic, and many more are in preclinical development. The best known kinase inhibitor is Gleevec used in the treatment of patients with CML and GIST. Gleevec inhibits the kinase activity of BCR-ABL, the fusion protein that causes CML and c-Kit in GIST. However, the question remains as to whether other highly expressed and activated kinases in tumors including liposarcoma can be targeted in a similar manner.Recently, the Cyclin-Dependent Kinases (CDKs), a family of protein kinases, have been established as master regulators of cell growth/proliferation and apoptosis, and thus inhibitors of CDKs have been explored as a novel therapeutic strategy in malignancy. Although to this date no cell cycle protein kinase inhibitors have been approved as an anticancer drug, several CDKs inhibitors (flavopiridol and dinaciclib) have entered clinical trials in chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, renal, prostate, colon and gastric carcinomas patients. These ongoing clinical trials appeared promising and will facilitate the development of more potent CDK-based therapeutic compounds.To identify the potential therapeutic kinase targets in sarcoma cells, we performed a comprehensive kinome-wide screening of lentiviral shRNA kinase library in sarcoma cell line KHOS. We hypothesized that kinases are critical for sarcoma cell growth will be our top hits. Among the targeted kinase genes, we found that knocking down the expression of CDK11, PLK1, DYRK1B and ROCK1 led to inhibitory growth effects. Of those kinases that we found critical to sarcoma cell growth, the elevated expression of PLK1, Mirk/DYRKIB and ROCK1 have been intensely investigated in various human tumors, and PLK1 and ROCK1 inhibitors are already in different phases of clinical trials for the treatment of these diseases. While the role of CDK11, a member of cyclin-dependent kinase (CDK) family is less known, especially in sarcoma. CDKs are critical regulators of cell cycle progression and RNA transcription. A variety of genetic and epigenetic events cause universal overexpression and activation of CDK in human cancer, and their inhibition can lead to both cell cycle arrest and apoptosis.CDK11 (formerly known as PITSLRE or CDC2L1) is a member of the extended family of p34cdc2-related kinases. At least 10 CDK11 isoforms have been cloned in eukaryotic cells. Although no clear mechanistic functions for CDK11 are known in tumor cells, the CDK11 has been reported to have effects on the regulation of transcription and RNA splicing. CDK 11 is ubiquitously expressed in tumor cell lines and constantly through the cell cycle. Although we have observed that knockdown of CDK11 decreased cell viability and increased apoptosis in osteosarcomas, the significance of CDK11 signaling in liposarcomas is unknown.In this study, we first comparatively examined the expression of CDK11 in liposarcoma and lipoma (benign tumor) tissue microarray by Immunohistochemical analysis. Furthermore, we evaluated the function role of CDK11 in the growth and proliferation of liposarcoma.Part 1 CDK11 expression in liposarcoma and benign lipomaIntroduction and AimsProtein kinases play important roles in regulating tumor cellular functions, so it is not surprising that they are often themselves cancer causing oncogenes. Kinases are commonly activated and highly expressed in cancer cells and are known to contribute to cancer progression. Kinases are now firmly established as a major class of drug targets. Significant progress has been made in understanding kinases and their functions. There has been an explosion in the number of kinase inhibitors entering the clinic, and many more are in preclinical development. However, the question remains as to whether other highly expressed and activated kinases in tumors including liposarcoma can be targeted in a similar manner.In this study, to clarify whether CDK11 controls liposarcoma cell growth similar to other protein kinases, we first comparatively examined the expression of CDK11 in liposarcoma and lipoma (benign tumor) tissue microarray by Immunohistochemical analysis.Methods1. To compare the expression of CDK11 between benign lipoma and liposarcoma tissues, CDK11 protein levels were analyzed by immunohistochemistry using lipoma and liposarcoma tissue microarray.2. Kaplan-Meier survival analysis was used to analyze the correlation between the level of CDK11 expression and prognosis. A two-sided Student’s t-test was used to compare the differences of CDK11 expression between survivor and nonsurvior in liposarcomas.Results1. The results showed that protein expression level of CDK11 was significantly higher in liposarcoma samples (2.6±0.20) than in lipoma samples (0.56±0.18). High expression of CDK11 (CDK11 staining≥3) was identified in 58.5% of liposarcoma, whereas it was not observed in lipoma tissues, suggesting that CDK11 expression may play an important role in the growth of liposarcoma. Moreover, in the liposarcoma tissues,38 (92.7%) of 41 samples were positive when tested for CDK11 staining, yet 5 (55.6%) of lipoma tissues were positive. These results also showed that the CDK11 protein was mainly localized in the nucleus of liposarcoma cells.2. Kaplan-Meier survival analysis showed that the outcome for patients in the liposarcoma group was not significantly worse than for those in the lipoma group. By comparing the clinical characteristics of low-staining (≤2) and high-staining (≥3) liposarcomas, no correlation existed between CDK11 expression and survival time. Liposarcoma patients were grouped into survivors and nonsurvivors, comparison of CDK11 staining intensity between 2 group patients revealed that CDK11 staining for samples from nonsurvivors were not significantly higher than that of survivors.Conclusions1. Our study shows little to no expression of CDK11 in lipoma, whereas CDK11 expression is significantly higher in liposarcoma. This suggests that CDK11 may play an important role in the proliferation of liposarcoma.2. Because of the small samples, CDK11 expression failed to produce any significant relationship with the overall survival rate. In conclusion, CDK11 expression is not associated with the clinical prognosis in liposarcoma.Part 2 CDKll is crucial in the growth of liposarcoma cells and its mechanism of cell regulationIntroduction and AimsRecently, the Cyclin-Dependent Kinases (CDKs), a family of protein kinases, have been established as master regulators of cell growth/proliferation and apoptosis, and thus inhibitors of CDKs have been explored as a novel therapeutic strategy in malignancy. To identify the potential role on liposarcoma cells, effects of CDK11 siRNA on cellular growth and proliferation were assessed in vitro via RNA interfering.Methods1. To characterize the functional role of CDK11 in liposarcoma, The human liposarcoma cell lines, SW872 and SW982, were cultured and synthetic human CDK11 siRNA was transfected into two liposarcoma cell lines. Effects of CDK11 siRNA on cellular growth and proliferation were assessed in vitro using the MTT assay. Western blot analysis was performed to assess the efficiency of the transfection.2. To further analyze the long-term effects of CDK11 expression knockdown on liposarcoma cell lines, five lentiviruses carrying different shRNA sequences targeting human CDKll kinase genes were transfected into SW982 cells. After puromycin selection, lentivirus-transfected cells were collected and total cell numbers were counted.3. The effect of CDKll expression on liposarcoma cell growth and the subcellular localization of CDK11 were further evaluated by immunofluorescence assay.4. To investigate how CDK11 inhibited tumor cell growth and survival, Western blotting was performed on several apoptotic related proteins (MCL-1, Bcl-XL, Bax, Survivin, Cytochrome C, and Cyclin D1) after CDK11 knockdown in liposarcoma cell lines SW872 and SW982.Results1. CDKll siRNA inhibits liposarcoma cell growth and proliferation. Western blot analysis suggested that down-regulated expression of CDK11 protein by CDK11 siRNA is associated with the inhibition of cell growth. SW872 and SW982 cells were also transfected with CDK11 siRNA in a dose-dependent manner. The dose-dependent inhibition of liposarcoma cell growth and survival was observed, accompanied by down-regulated expression of CDK11 protein.2. Compared to SW982 cells without puromycin and negative shRNA lentiviral controls, transfection with CDK11 shRNA lentiviruses significantly reduced cell growth and survival in SW982 cells. Puromycin had little or no effect on negative shRNA lentiviral controls. The results show that CDK11 shRNA (TRCN0000006208) inhibited liposarcoma cell growth by 66.7%, whereas the other four CDK11 shRNA lentiviruses significantly decreased cell growth by 90-100%.3. The immunofluorescence assay also showed that the CDK11 protein was mainly localized in the nucleus of liposarcoma cells. The data of CDK11 immunofluo rescence analysis further confirmed that compared with cells transfected with nonspecific siRNA, SW872 and SW982 cells expressed much lower levels of CDK11 after being transfected with CDK11 siRNA.4. Two anti-apoptotic proteins (Cyclin D1 and Survivin) were highly expressed in liposarcoma, and transfection of CDK11 siRNA significantly down-regulated the expression of these anti-apoptotic proteins in both SW872 and SW982 cells.In addition, decreased expression of another anti-apoptotic protein Cytochrome C was associated with transfection of CDK11 siRNA in SW872. Thus, these results indicate that knockdown of CDK11 by siRNA could induce cell apoptosis via down-regulation of anti-apoptotic proteins Cyclin D1 and Survivin in liposarcoma cells.Conclusions1. CDK11 knockdown by synthetic siRNA and lentivirus shRNA inhibited liposarcoma cell growth and survival, which implies again that CDK11 plays an important role in liposarcoma cell proliferation and growth.2. These result of mechanism indicate that knockdown of CDK11 by siRNA could induce cell apoptosis via down-regulation of anti-apoptotic proteins Cyclin D1 and Survivin in liposarcoma cells.3. CDK11 may be a promising therapeutic target for liposarcoma patients, and CDK11 inhibitors may be a potential new cancer drugs.Part 3 The effect of CDK11 knockdown on chemotherapy drugsIntroduction and AimsDoxorubicin, a kind of antitumor antibiotics, has an effect on a wide variety of tumor, and it mainly embed DNA and inhibit the synthesis of nucleic acid. Doxorubicin alone or in combination with ifosfamide is often used as a first-line regimen in patients with high risk of recurrence or metastatic liposarcoma. inhibitors of CDKs have been explored as a novel therapeutic strategy in malignancy. Although to this date no cell cycle protein kinase inhibitors have been approved as an anticancer drug, several CDKs inhibitors (flavopiridol and dinaciclib) have entered clinical trials. Combination effect of CDKs inhibitors and chemotherapy drug are discussed at many researches, combination of flavopiridol and doxorubicin or other chemotherapy drugs have got good clinical effect at a variety of cancer clinical trials. CDK11 inhibitor can be a new potential anti-cancer drug, but there is no specific CDK11 inhibitors, so in this study we focus on whether CDK11 down-regulation influenced chemotherapeutic agents’effects on liposarcoma cells.MethodsTo determine whether CDK11 down-regulation influenced chemotherapeutic agents’effects on liposarcoma cells, SW872 and SW982 cells were transfected with CDK11 siRNA and then incubated with doxorubicin. Western blotting confirmed decreased expression of CDKll in liposarcoma cells at these concentrations of CDK11 siRNA.ResultsThe combination of CDK11 siRNA and doxorubicin significantly inhibited cell growth and survival compared to those from each treatment alone in SW872 and SW982 cell lines. Thus, the results show that CDK11 knockdown enhances the cytotoxic effect of doxorubicin to inhibit cell growth in liposarcoma cell lines.ConclusionsCDK11 knockdown also enhances the cytotoxic effect of chemotherapeutic agent doxorubicin acts synergistically with the chemotherapeutic agent doxorubicin in liposarcoma cell lines, suggesting that combination therapies could be explored in liposarcoma clinical trials.Conclusions of this paper1. CDK11 may play an important role in the proliferation of liposarcoma. CDK11 knockdown by synthetic siRNA and lentivirus shRNA inhibited liposarcoma cell growth and survival, which implies again that CDK11 plays an important role in liposarcoma cell proliferation and growth.2. These result of mechanism indicate that knockdown of CDKll by siRNA could induce cell apoptosis via down-regulation of anti-apoptotic proteins Cyclin D1 and Survivin in liposarcoma cells.3. CDK11 knockdown also enhances the cytotoxic effect of chemotherapeutic agent doxorubicin in liposarcoma cell lines. Combination therapies of CDK11 inhibition and chemotherapeutic agent could be explored in liposarcoma clinical trials.4. CDK11 may be a promising therapeutic target for liposarcoma patients, and CDK11 inhibitors may be a potential new cancer drugs.