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.