Combined Androgen Receptor Blockade Overcomes The Resistance Of Breast Cancer Cells To Palbociclib

Background and objectiveBreast cancer is the most common malignancy,and is the second cause of cancer death among females worldwide,accounting for an estimate 266120 new cases and40,920 deaths in 2018.For hormone receptor(HR)-positive breast cancer patients,which accounts for approximately 60%-70%of all cases,hormonal monotherapy including selective estrogen receptor(ER)modulator(SERM)or selective ER down-regulator(SERD),aromatase inhibitor(AI),or even their combination as doublets have contributed to the decline in breast cancer mortality.Unfortunately,disease progression occurs inevitably after endocrine treatment in patients with metastatic breast cancer.It is crucial to find new therapeutic strategies to improve the outcome of these patients.The dysregulation of cyclin D-cyclin-dependent kinase 4/6(CDK 4/6)-retinoblastoma tumor suppressor protein(Rb)axis has been implicated in breast cancer progression.Furthermore,cyclin D1 is overexpressed in 50%-70%of breast cancer with cyclin D1-encoding gene(CCND1)amplified in 15%and the high expression of cyclin D1 is common in ER-positive breast cancer.Cyclin D1,as a vital cell cycle modulator,can form a complex with CDK4/6,then phosphorylate Rb,and release E2F from the Rb-E2F complex,thereby driving cells through the checkpoint to start DNA replication and initiate cell division.Therefore,cyclin D-CDK4/6-Rb axis is considered as a potent therapeutic target in ER-positive breast cancer.Multiple clinical trials have demonstrated that the addition of CDK4/6 inhibitor is associated with disease response in females with hormone refractory ER-positive and human epidermal growth factor receptor2(HER2)-positive metastatic breast cancer.However,resistance to the small molecular inhibitor has become an inevitable issiue after the initial use.Here,we investigated the potential mechanism of resistance by establishing a CDK4/6inhibitor palbociclib-resistant breast cancer cell line(MCF-7pR)and provide evidence that androgen receptor(AR)activation promotes cell cycle progression and cell proliferation in CDK4/6 inhibitor resistance and identify AR inhibition as a putative novel therapeutic strategy to treat CDK4/6 inhibitor resistance in breast cancer.Materials and Methods1.The human palbociclib-resistant cell line MCF-7pR was established previously.Combined with CCK8 assay and western blot were performed to confirm the successful generation of palbociclib-resistant cells.Western blot,cell cycle analysis and colony formation assay were employed to investigate if there are any differences in cell cycle distribution and colony formation between the parental MCF-7 and the resistant cells.2.The expression of hormone receptors was identified with real-time PCR,western blot and immunofluorescence in order to reflect the changes of hormone receptors in resistant cells(MCF-7pR).3.Based on the literature reports and previous study results of our research group,we treated with AR agonist DHT and AR short hair RNA plasmid(shAR)in parental MCF-7 cells and resistant MCF-7pR cells.The function of AR and its related possible signaling axis in resistant cells in vitro was measured by CCK8 assay,western blot,cell cycle analysis and co-immunoprecipitation assay in order to investigate the potential mechanism of resistance.4.ShAR was transfected into resistant cells MCF-7pR.The effect of AR blockade by short hair plasmid was measured by CCK8 assay,western blot,cell cycle analysis and colony formation assay in order to verify the sensitivity to palbociclib,cell proliferation ability and cell cycle distribution.5.The resistant cells MCF-7pR were treated with palbociclib,or enzalutamide or in combination.The changes of cell cycle distribution and G1-S phase related proteins were detected by cell cycle analysis and western blot respectively.Cell viability was measured by CCK8 assay and the combination index(CI)values were analyzed by CompuSyn software.6.The xenograft model was injected subcutaneously by resistant cells MCF-7pR in order to evaluate the synergistic effect of enzalutamide and palbocicclib on palbociclib-resistant cells in vivo.When tumors reached an average of 100 mm~3,four groups were randomly divided.Mice were treated with vehicle,palbociclib,enzalutamide,or a combination.Tumors were measured once every three days.Hematoxylin and eosin(H&E)staining and immunohistochemistry staining were performed to analyze the expression of androgen receptor and Ki67 and verify the proliferation ability of the tumors.Results1.CDK4/6 inhibitor palbociclib inhibited proliferation,impaired the colony formation capacity,and mediated G1/S phase arrest in parental estrogen receptor-positive breast cancer cells MCF-7.Conversely,palbociclib had no effect on the cell proliferation,colony formation and G1/S phase transition of resistant cells MCF-7pR.Furthermore,increased multidrug resistance protein 1(MDR1)expression was detected in MCF-7pR cells compared to the parental MCF-7 cells,confirming the successful generation of palbociclib-resistant cells.2.Compared with the parental MCF-7 cells,ER and progesterone receptor(PR)expression were reduced obviously but the expression of AR was elevated in the mRNA and protein level in resistant cells.Furthermore,the immunostaining showed more localizations of AR in the cell nucleus of resistant cells compared to that in the parental MCF-7 cells.3.Contrary to the inhibitory effect of the AR agonist DHT in parental cells,the DHT treatment enhanced the cell proliferation of MCF-7pR cells.MCF-7 cells exhibited a decrease in the level of cyclin D1 following the DHT treatment while androgen activated by the DHT increased the cyclin D1 expression in MCF-7pR cells.Data from western blot experiments of resistant cells demonstrated that AR activation due to DHT treatment elevated the expression of the expression of CDK2 and CCNE1 which are highly associated with palbociclib resistance.Besides,silencing of AR inhibited the expression of these two genes.4.The expression of AR was obviously decreased in MCF-7pR cells after the transfection with shAR.The IC50 of palbociclib in MCF-7pR cells transfected with shAR compared with the control group.ShAR decreased the clonogenicity in MCF-7pR cells.The combination of AR blockade and palbociclib resulted in substantially increased G1/S phase transition arrest.Palbociclib treatment led to decreased expression of several G1-restriction point markers including RB,pRB,E2F1,cyclin A2 and cyclin E2 in cells transfected with shAR relative to control.5.In resistant MCF-7pR cells,AR antagonist could resensitize MCF-7pR cells to CDK4/6 inhibitor.The combination induced the suppression of S phase entry significantly in MCF-7pR cells.In line with the cell-cycle analysis,the combination therapy also suppressed the expression of the pRb and related G1/S phase cyclins.A fixed radio model(1:1)of combination therapy was used in MCF-7pR cells and most of the CI values were below 1 indicating a significant synergistic effect.6.The combination of enzalutamide and palbociclib reduced significantly tumor growth compared to single drug treatment in vivo.Additionally,the single drug or the combination treatment did not cause body weight loss apparently.The dual treatment was superior to any of the single treatments and resulted in significant shrinkage as evidenced by decreased levels of Ki67.ConclusionIn conclusion,AR could function as a potential biomarker and play a vital role in palbociclib resistance of breast cancer both in vitro and in vivo.AR activation in resistant cells may participate in promoting ongoing cell-cycle entry.AR could bind to and activate cyclin D1 and improve the expression of CDK2 and CCNE1 which showed that AR participated in the cells cycle progression of resistant cells to palbociclib.Inhibition of AR could resensitize resistant cells to palbociclib and yielded the synergistic growth inhibitory effect on resistant cells when given with palbociclib.Considering the synergistic growth inhibitory effect of enzalutamide and palbociclib on the palbociclib-resistant cells in vitro and in vivo,the clinical application of enzalutamide could be considered in the context of palbociclib resistance.