Studies On The Function And Mechanism Of CDK16 Promoting The Progression Of Triple-Negative Breast Cancer By Regulating Spindle Formation

Breast cancer is the most diagnosed malignancy and the second leading cause of cancer-related deaths among women.Triple-negative breast cancer(TNBC)is a subtype of breast cancer with the highest risk of recurrence,metastasis and death due to its highly heterogeneous genetic background and the lack of effective targeted therapies.Exploring oncogenic drivers of TNBC and identifying new therapeutic targets are of clinical importance to improve the prognosis of TNBC patients.Targeting specific CDK has been shown to be an effective anti-tumor strategy.PFTAIRE kinases(PFTK1-2,also known as CDK14-15)and PCTAIRE kinases(PCTK1-3,also known as CDK16-18)are newly proposed CDK subfamilies,and are divided into atypical CDKs.These atypical CDKs share cyclin Y(CCNY)and cyclin Y-like 1(CCNYL1)as cyclin partners to perform their enzymatic activity.Previous studies have found that Ccny and Ccnyl1 play an important role in regulating mammary stem cell activity as well as mammary gland development,and the oncogenic role of CCNY has been reported in various cancers including breast cancer.These evidences suggest that atypical CDKs which depend on CCNY may have similar functions.In this study,we used breast cancer models to screen potential targets for breast cancer therapy among CCNY-dependent CDKs by clinical correlation analysis and functional study.The expression pattern and prognostic relevance of these candidates were analyzed using publicly available breast cancer datasets.It is showed that CDK16 m RNA is highly expressed in breast cancer,particularly in TNBC,and the elevated CDK16 expression is correlated with poor outcomes of breast cancer patients.The upregulation of CDK16 protein expression in TNBC was further validated by IHC and WB analyses of clinical samples.It is suggested that CDK16 may have oncogenic functions in TNBC and is promising to be a potential target for the treatment of TNBC.In vitro experiments indicated that CDK16 knockdown significantly impaired the proliferative ability and three-dimensional(3D)colony formation of TNBC cells,and induced G2/M arrest and cell apoptosis,while overexpression of CDK16 dramatically promoted the proliferation,migration and invasion of TNBC cells.The results of in vivo studies showed that CDK16 silencing significantly delayed tumorigenesis and inhibited tumor growth in multiple TNBC tumor models,including cell line-derived xenografts(CDX)model,patient-derived organoids(PDO)model and patient-derived xenografts(PDX)model.In mouse models of lung metastasis and systemic metastasis,CDK16 knockdown markedly suppressed TNBC metastasis,while CDK16 hyperexpression significantly accelerated lung metastasis and multi-organ distant metastasis of TNBC.These data revealed that CDK16 plays an oncogenic role in TNBC,and CDK16 inhibition induces potent anti-tumor effects.The results of pharmacological studies indicated that pharmacological inhibition of CDK16 using its inhibitor Rebastinib,was able to reproduce the anti-tumor effects elicited by CDK16 knockdown.TNBC cells were sensitive to Rebastinib treatment in vitro,and Rebastinib administration effectively inhibited the growth and metastasis of TNBC in vivo.Together,these results demonstrated that both genetic knockdown and pharmacological inhibition of CDK16 can exert anti-tumor effects.Mechanistically,CDK16 promotes cell proliferation and tumor progression by phosphorylating Protein Regulator of Cytokinesis(PRC1)at the T481 site and regulating spindle formation during mitosis.Inhibition of CDK16 induces spindle formation defects,subsequently leading to G2/M arrest and cell apoptosis.In addition,our study revealed the similar role of CDK16 to CDK4/6 in phosphorylating Rb and regulating the Rb-E2 F signaling pathway.Other cancer-related signaling pathways,such as MAPK and PI3K-Akt signals may also contribute to the function of CDK16 in regulating TNBC progression.Collectively,our data demonstrated that CDK16 exerts its function through multiple mechanisms.In conclusion,our study systematically investigated the oncogenic role of CDK16 in TNBC,assessed the therapeutic potential of targeting CDK16 in multiple TNBC models,and elucidated the molecular mechanisms of CDK16 involved in regulating TNBC progression.Our study provides new insights into the roles of atypical CDKs in cancer,highlights the significance of CDK16 in TNBC,and reveals a promising target for TNBC therapy.