Systematic Kinome shRNA Screening Identifies CDK11 (PITSLRE) Kinase Expression Is Critical for Osteosarcoma Cell Growth and Proliferation

Abstract

Purpose: Identification of new targeted therapies is critical to improving the survival rate of patients with osteosarcoma. The goal of this study is to identify kinase based potential therapeutic target in osteosarcomas.Experimental Design: We used a lentiviral-based shRNA kinase library to screen for kinases which play a role in osteosarcoma cell survival. The cell proliferation assay was used to evaluate cell growth and survival. siRNA assays were applied to confirm the observed phenotypic changes resulting from the loss of kinase gene expression. CDK11 (PITSLRE) was identified as essential for the survival of osteosarcoma cells, and its expression was confirmed by Western blot analysis and immunohistochemistry. Overall patient survival was correlated with the CDK11 expression and its prognosis. The role of CDK11 expression in sustaining osteosarcoma growth was further evaluated in an osteosarcoma xenograft model in vivo.Results: Osteosarcoma cells display high levels of CDK11 expression. CDK11 expression knocked down by either lentiviral shRNA or siRNA inhibit cell growth and induce apoptosis in osteosarcoma cells. Immunohistochemical analysis showed that patients with osteosarcoma with high CDK11 tumor expression levels were associated with significantly shorter survival than patients with osteosarcoma with low level of tumor CDK11 expression. Systemic in vivo administration of in vivo ready siRNA of CDK11 reduced the tumor growth in an osteosarcoma subcutaneous xenograft model.Conclusions: We show that CDK11 signaling is essential in osteosarcoma cell growth and survival, further elucidating the regulatory mechanisms controlling the expression of CDK11 and ultimately develop a CDK11 inhibitor that may provide therapeutic benefit against osteosarcoma.

Targeting Cdk11 in osteosarcoma cells using the CRISPR‐cas9 system

“…3 In this study, the optimization electroporation parameter applied was: pulse voltage, 1230; pulse width, 10; pulse number, 4, which achieved 70–80% transfection (data not showed). KHOS and U-2OS cells were successfully transfected with CDK11-Cas9-GFP or pEGFP-N3.…”

“…In an effort to identify new therapeutic targets in osteosarcoma, we found that knockdown of CDK11 (cyclin-dependent kinase 11, also known as CDC2L for cell division cycle 2-like or PITSLRE) by short hairpin RNA (shRNA) or short interfering RNA (siRNA) inhibited tumor cell growth and induced apoptosis. 3 Importantly, nuclear CDK11 expression levels correlated with clinical prognosis in osteosarcoma patients. Systemic in vivo administration of in vivo ready CDK11 siRNA reduced tumor growth in an osteosarcoma xenograft model.…”

TFAP2B overexpression contributes to tumor growth and a poor prognosis of human lung adenocarcinoma through modulation of ERK and VEGF/PEDF signaling

“…The sequence of the human TFAP2B-specific siRNA was 5′-GGA CCA GUC UGU CAU UAA ATT-3′ and 5′-CCC GAA AGA AUA UGC UGU UTT-3′, and the scramble siRNA was 5′-UUC UCC GAA CGU GUC ACG UTT-3′. The siRNA and plasmid were incorporated with additional chemical modifications for superior serum stability in the in vivo applications, and the knockdown efficiency was validated in vitro [28,29]. For the in vivo delivery of the siRNA and plasmid into tumors, the sequences were first encapsulated into DOTAP-cholesterol (DC) (Avanti Polar, Birmingham, AL, USA) nanoparticles that had validated by many analyses by dissolving in sterilized de-ionized water and then mixing with the nanosomes.…”

Cyclin-dependent kinase 11 (CDK11) is crucial in the growth of liposarcoma cells

“…In previously work we screened osteosarcoma cell lines with the lentiviral shRNA kinase library, consistently identifying that knockdown of CDK11 expression led to inhibited growth and induction of apoptosis in both in vitro and in vivo models [17]. CDK11 (formerly known as PITSLRE or CDC2L1) is a member of the extended family of p34cdc2-related kinases [18].…”