One of the properties of human breast cancer cells is cancer stemness, which is characterized by self-renewal capability and drug resistance. Protein kinase D1 (PRKD1) functions as a key regulator of many cellular processes and is downregulated in invasive breast cancer cells. In this study, we found that PRKD1 was upregulated in MCF-7-ADR human breast cancer cells characterized by drug resistance. Additionally, we discovered that PRKD1 expression was negatively regulated by miR-34a binding to the PRKD1 3'-UTR. PRKD1 expression increased following performance of a tumorsphere formation assay in MCF-7-ADR cells. We also found that reduction of PRKD1 by ectopic miR-34a expression or PRKD1 siRNA treatment resulted in suppressed self-renewal ability in breast cancer stem cells. Furthermore, we confirmed that the PRKD1 inhibitor CRT0066101 reduced phosphorylated PKD/PKCμ, leading to suppression of breast cancer stemness through GSK3/β-catenin signaling. PRKD1 inhibition also influenced apoptosis initiation in MCF-7-ADR cells. Tumors from nude mice treated with miR-34a or CRT0066101 showed suppressed tumor growth, proliferation, and induced apoptosis. These results provide evidence that regulation of PRKD1, a novel miR-34a target, contributes to overcoming cancer stemness and drug resistance in human breast cancer.

The role of adjuvant therapy following resection of biliary tract cancer (BTC) remains unclear. We therefore evaluated the feasibility and toxicity of adjuvant gemcitabine in patients with BTC. This clinical phase II trial was an open-label, single center, single-arm study. Within 8 weeks after gross complete resection of BTC, patients were started on intravenous infusions of gemcitabine 1000 mg/m2 over 30 min on days 1, 8, and 15 of every 28-day cycle. Intratumoral expression of cytidine deaminase (CDA), human equilibrative transporter-1 (hENT1), deoxycytidine kinase (dCK) and ribonucleotide reductase subunit 1 (RRM1) was measured by immunohistochemistry. This study enrolled 72 patients with BTC (26 with gallbladder cancer, 33 with extrahepatic cholangiocarcinoma, and 13 with intrahepatic cholangiocarcinoma). The 2-year recurrence-free survival (RFS) rate was 43% (95% CI, 33-57%). Multivariable analysis showed that DCK expression, vascular invasion, and lymph node metastasis were significantly associated with RFS. Twenty-one (31.8%) were positive for DCK immunoreactivity. The median RFS was 34.95 months for DCK-positive patients, compared with 11.41 months for DCK-negative patients. Although the primary hypothesis of this study, defined as a 2-year RFS of 60%, was not met, intratumoral DCK expression was significantly associated with RFS in patients with resected BTC treated with postoperative gemcitabine chemotherapy. Future randomized controlled trials are warranted.

The role of lymphocyte antigen 6 complex, locus K (LY6K) in breast cancer has been studied, whereas the epigenetic control of LY6K transcription is not fully understood. Here, we report that breast cancer patients with increased LY6K expression had shorter disease-free and overall survival than the patients with low levels of LY6K by multivariate analysis. LY6K also was upregulated in breast cancer patients with distant metastases than those without distant metastases, downregulating E-cadherin expression. Furthermore, xenograft tumor volumes from LY6K knockdown nude mice were reduced than those of mice treated with control lentivirus. Interestingly, LY6K has a CpG island (CGI) around the transcription start site and non-CGI in its promoter, called a CGI shore. LY6K expression was inversely correlated with methylation in not only CGI but CGI shore, which are associated with histone modifications. Additionally, LY6K methylation was increased by the PAX3 transcription factor due to the SNP242 mutation in LY6K CGI shore. Taken together, breast cancer risk and metastasis were significantly associated with not only LY6K expression, but also methylation of CGI shore which induced by SNP242 mutation. Our results suggest that an understanding epigenetic mechanism of the LY6K gene may be useful to diagnose carcinogenic risk and predict outcomes of patients with metastatic breast cancer.