PKD1 is a potential biomarker and therapeutic target in triple-negative breast cancer

Abstract

Protein Kinase D1 (PKD1) is a serine/threonine kinase encoded by the PRKD1 gene. PKD1 has been previously shown to be a prognostic factor in ERα+ tamoxifen-resistant breast tumors and PKD1 overexpression confers estrogen independence to ERα+ MCF7 cells. In the present study, our goal was to determine whether PKD1 is a prognostic factor and/or a relevant therapeutic target in breast cancer. We analyzed PRKD1 mRNA levels in 527 primary breast tumors. We found that high PRKD1 mRNA levels were significantly and independently associated with a low metastasis-free survival in the whole breast cancer population and in the triple-negative breast cancer (TNBC) subtype specifically. High PRKD1 mRNA levels were also associated with a low overall survival in TNBC. We identified novel PKD1 inhibitors and assessed their antitumor activity in vitro in TNBC cell lines and in vivo in a TNBC patient-derived xenograft (PDX) model. Pharmacological inhibition and siRNA-mediated depletion of PKD1 reduced colony formation in MDA-MB-436 TNBC cells. PKD1 inhibition also reduced tumor growth in vivo in a TNBC PDX model. Together, these results establish PKD1 as a poor prognostic factor and a potential therapeutic target in TNBC.

Keywords: PKC; PKD; protein kinase D1; triple-negative breast cancer.

Figure 1. PRKD1 expression is a poor prognostic factor in the entire breast cancer cohort and in the TNBC subgroup

(A) PRKD1 mRNA levels in 527 primary breast tumors. PRKD1 mRNA expression was analyzed by RT-qPCR and normalized to that of the TBP control gene. Normal breast tissues were used as a reference (expression level =10). (B) Immunohistochemical analysis of PKD1 protein expression in primary breast tumors showing high or low PRKD1 mRNA levels (relative PRKD1 mRNA levels of 10.2 and 1.0, respectively). Original magnification x200. (C) Kaplan-Meier analysis of metastasis-free survival according to PRKD1 mRNA expression in the entire breast cancer cohort (n=527). (D) Kaplan-Meier analysis of metastasis-free survival according to PRKD1 mRNA expression in TNBC (n=102).

Figure 2. Effect of PKD1 inhibition in TNBC cells

(A) PRKD1 mRNA levels in 21 TNBC cell lines. PRKD1 mRNA expression was analyzed by RT-qPCR and normalized to that of the TBP control gene. (B) Western-blot analysis of PKD1 protein expression in six TNBC cell lines. β-actin was used as a loading control. (C) Effect of PKD1 pharmacological inhibitors on clonogenicity of MDA-MB-436 cells. CFU, colony forming unit. Untreated cells were used as a reference (100%). Mean values ± SEM from two independent experiments are shown. (D) Effect of siRNA-mediated PKD1 knockdown on MDA-MB-436 clonogenicity. MDA-MB-436 cells were transfected with 50 nM non-targeting (siCNTRL) or PKD1-targeting (siPRKD1) siRNAS during 48 hours. Left: western-blot showing the efficiency of PKD1 silencing. β-actin was used as a loading control. Right: colony formation was evaluated after two weeks. Cells transfected with non-targeting siRNAs were used as a reference (100%). Mean values ± SEM from two independent experiments are shown.

Figure 3. In vivo antitumor activity of the AB9275 PKD1 inhibitor against a TNBC PDX

(A) PRKD1 mRNA levels in 41 TNBC PDXs. PRKD1 mRNA expression was analyzed by RT-qPCR and normalized to that of the TBP control gene. (B) Western-blot analysis of PKD1 protein expression in four PDXs expressing high or intermediate PRKD1 mRNA levels (HBCx-4A, HBCx-60, BC385) and low PRKD1 mRNA levels (HBCx-12A). β-actin was used as a loading control. (C) Immunohistochemical analysis of PKD1 protein expression in the HBCx-4A, HBCx-60, BC385, and HBCx-12A tumors. Original magnification x200. (D) Effect of AB9275 on tumor growth in the HBCx-60 PDX model. Mice bearing HBCx-60 tumors were treated per os once daily during 22 days with 30 mg/kg AB9275 (n=7) or water (n=8). Mean RTV±SEM are shown. ^*, P < 0.05; Wilcoxon-Mann-Whitney test.