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  • br Materials and methods br Results br


    Materials and methods
    Discussion CDK4 has been identified recently as a potential therapeutic target in human breast cancer, liposarcoma, melanoma, and glioblastoma [[37], [38], [39]]. Due to the importance of CDK4 activity in cancer cells, CDK4 inhibitors have emerged as promising candidates for the treatment of many cancer types [11]. In the current study, we aimed to explore the expression and the therapeutic potentials of CDK4 in osteosarcoma. We first determined CDK4 expression in both osteosarcoma tissues and cell lines and found CDK4 was highly expressed in most of the tested osteosarcoma tissue samples and in all the human osteosarcoma cell lines. These results suggest that CDK4 may be a critical player in osteosarcoma growth and proliferation. We further explored the relationship between CDK4 expression and the clinicopathological characteristics of osteosarcoma by using TMA containing 72 tissue samples from 54 osteosarcoma patients with complete clinical information, as well as up to 252 months of follow-up data. Our results showed that CDK4 expression significantly correlated with the metastasis status and clinical prognosis of osteosarcoma patients. Previously, CDK4 amplification has been found associated with a poor prognosis in liposarcoma patients [40]. Ewing sarcoma cells also require CDK4 for survival and anchorage-independent growth. Knockdown of CDK4 abrogated proliferation and transformation of fusion-gene positive rhabdomyosarcoma cells via G1 phase Brassinolide arrest [41]. More recently, overexpression and activation of the CDK4/6 and Rb pathways have been found in chordoma, another rare type of sarcoma [42]. These findings together support that CDK4 plays important role in the development and progression in different sarcomas including osteosarcoma. Subsequently, we explored the functional roles of CDK4 in osteosarcoma cell proliferation and Brassinolide growth in vitro. We first inhibited CDK4 in osteosarcoma cells by the clinically approved CDK4 inhibitor palbociclib and investigated the cellular phenotypic alternations. Our findings demonstrated that CDK4 inhibition by palbociclib decreased osteosarcoma cell proliferation and growth in a dose-dependently manner, but exhibited only mild inhibition on CDK4-lower expressed NHOst cells. We further specifically knocked down CDK4 expression using CDK4 specific siRNA and determined osteosarcoma cell viability. Consistently, CDK4 inhibition by siRNA reduced osteosarcoma cell growth dose-dependently. These studies collectively suggest that CDK4 plays a crucial role in osteosarcoma proliferation and growth. As an important cell division modulator, CDK4 exerts its functional role mainly through phosphorylation of Rb and promotion of the transition from G1 to S phase of the cell cycle [43]. In response to mitogenic signals, CDK4/6 combines with cyclin D and transfers into the nucleus. Then, the cyclin D-CDK4/6 complex phosphorylates Rb into pRb and releases the E2F transcription factor, which activates downstream target genes and promotes cell cycle progression, and cell proliferation and growth [30]. In human cancer, the cyclin D-CDK4/6-Rb pathway is universally disrupted [44,45]. Thus, we determined the alternations of respective protein expression after CDK4 inhibition by palbociclib. We found that both palbociclib and CDK4 siRNA dose-dependently inhibited the downstream Rb phosphorylation and decreased cell survival protein expression. This finding implies that pRb and survivin act as positive regulators of cell proliferation and growth via the same signaling pathway, which has been observed in other studies [[46], [47], [48]]. To be noted, due to palbociclib mainly inhibiting CDK4 activity, but not its expression, the Western blot showed that palbociclib had no influence on CDK4 amount, but effectively inhibited CDK4 downstream Rb phosphorylation and downregulated survivin expression. Regarding survivin repression by palbociclib, it might be mediated by E2F. Several previous studies have shown E2F downregulating survivin via direct binding to the survivin promoter and inducing survivin transcription [49,50].