Enzalutamide, approved by the United States Food and Drug Administration in 2018 for the management of metastatic castration-resistant prostate cancer (CRPC), is an androgen receptor (AR) inhibitor. It blocks androgen binding to the AR, AR nuclear translocation, and AR-mediated DNA binding. Unfortunately, a considerable proportion of tumors eventually develop resistance during the treatment. The molecular mechanisms underlying enzalutamide resistance are not completely understood. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressor complex 2, has been proposed as a prognostic marker for prostate cancer (PCa). With the goal to test whether EZH2 also plays a critical role in acquisition of enzalutamide resistance in CRPC, here we examined whether EZH2 inhibition/depletion enhances the efficacy of enzalutamide in enzalutamide-resistant PCa cells. We show that combining the EZH2 inhibitor GSK126 with enzalutamide synergistically inhibits cell proliferation and colony formation and promotes apoptosis in enzalutamide-resistant PCa cells. EZH2 depletion also overcomes enzalutamide resistance in both cultured cells and xenograft tumors. Mechanistically, we found that EZH2 directly binds to the promoter of prostate-specific antigen and inhibits its expression in enzalutamide-resistant PCa cells. In agreement, bioinformatics analysis of clinical RNA sequencing data involving GSEA indicated a strong correlation between AR and EZH2 gene expression during PCa progression. Our study provides critical insights into the mechanisms underlying enzalutamide resistance, which may offer new approaches to enhance the efficacy of enzalutamide in CRPC.

Polo-like kinase 1 (Plk1) plays an important role in cell-cycle regulation. Recent work has suggested that Plk1 could be a biomarker of gemcitabine response in pancreatic ductal adenocarcinoma (PDAC). Although targeting Plk1 to treat PDAC has been attempted in clinical trials, the results were not promising, and the mechanisms of resistance to Plk1 inhibition is poorly understood. In addition, the role of Plk1 in PDAC progression requires further elucidation. Here, we showed that Plk1 was associated with poor outcomes in patients with PDAC. In an inducible transgenic mouse line with specific expression of Plk1 in the pancreas, Plk1 overexpression significantly inhibited caerulein-induced acute pancreatitis and delayed development of acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Bioinformatics analyses identified the regulatory networks in which Plk1 is involved in PDAC disease progression, including multiple inflammation-related pathways. Unexpectedly, inhibition or depletion of Plk1 resulted in upregulation of PD-L1 via activation of the NF-κB pathway. Mechanistically, Plk1-mediated phosphorylation of RB at S758 inhibited the translocation of NF-κB to nucleus, inactivating the pathway. Inhibition of Plk1 sensitized PDAC to immune checkpoint blockade therapy through activation of an antitumor immune response. Together, Plk1 suppresses PDAC progression and inhibits NF-κB activity, and targeting Plk1 can potentiate the efficacy of immunotherapy in PDAC. Significance: Inhibition of Plk1 induces upregulation of PD-L1 expression in pancreatic ductal adenocarcinoma, stimulating antitumor immunity and sensitizing tumors to immunotherapy.

Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is a Food and Drug Administration–approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment. After a short positive response period, tumors will develop drug resistance. In this study using RNA-Seq and bioinformatics analyses, we observed that NOTCH signaling is a deregulated pathway in enzalutamide-resistant cells. NOTCH2 and c-MYC gene expression positively correlated with AR expression in samples from patient with hormone refractory disease in which AR expression levels correspond to those typically observed in enzalutamide resistance. Cleaved NOTCH1, HES1 (Hes family BHLH transcription factor 1), and c-MYC protein expression levels are elevated in two enzalutamide-resistant cell lines, MR49F and C4-2R, indicating NOTCH signaling activation. Moreover, inhibition of the overexpressed ADAM metallopeptidase domain 10 (ADAM10) in the resistant cells induces an exclusive reduction in cleaved NOTCH1 expression. Furthermore, exposure of enzalutamide-resistant cells to both PF-03084014 and enzalutamide increased cell death, decreased colony formation ability, and resensitized cells to enzalutamide. Knockdown of NOTCH1 in C4-2R increased enzalutamide sensitivity by decreasing cell proliferation and increasing cleaved PARP expression. In a 22RV1 xenograft model, PF-03084014 and enzalutamide decreased tumor growth through reducing cell proliferation and increasing apoptosis. These results indicate that NOTCH1 signaling may contribute to enzalutamide resistance in prostate cancer, and inhibition of NOTCH signaling can resensitize resistant cells to enzalutamide.

Increased abundance of polo-like kinase 1 (PLK1) is observed in various tumor types, particularly in lung adenocarcinoma (LUAD). Here, we found that PLK1 accelerated the progression of LUAD through a mechanism that was independent of its role in mediating mitotic cell division. Analysis of human tumor databases revealed that increased PLK1 abundance in LUAD correlated with mutations in KRAS and p53, with tumor stage, and with reduced survival in patients. In a mouse model of KRAS ^G12D -driven, p53-deficient LUAD, PLK1 overexpression increased tumor burden, decreased tumor cell differentiation, and reduced animal survival. PLK1 overexpression in cultured cells and mice indirectly increased the expression of the gene encoding the receptor tyrosine kinase RET by phosphorylating the transcription factor TTF-1. Signaling by RET and mutant KRAS in these tumors converged to activate the mitogen-activated protein kinase (MAPK) pathway. Pharmacological inhibition of the MAPK pathway kinase MEK combined with inhibition of either RET or PLK1 markedly suppressed tumor growth. Our findings show that PLK1 can amplify MAPK signaling and reveal a potential target for stemming progression in lung cancers with high PLK1 abundance.