Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor

Abstract

Btk and Etk/BMX are Tec-family non-receptor tyrosine kinases. Btk has previously been reported to be expressed primarily in B cells and has an important role in immune responses and B-cell malignancies. Etk has been shown previously to provide a strong survival and metastasis signal in human prostate cancer cells, and to confer androgen independence and drug resistance. While the role of Etk in prostate carcinogenesis is well established, the functions of Btk in prostate cancer have never been investigated, likely due to the perception that Btk is a hematopoietic, but not epithelial, kinase. Herein, we found that Btk is overexpressed in prostate cancer tissues and prostate cancer cells. The level of Btk in prostate cancer tissues correlates with cancer grades. Knockdown of Btk expression selectively inhibits the growth of prostate cancer cells, but not that of the normal prostate epithelial cells, which express very little Btk. Dual inhibition of Btk and Etk has an additive inhibitory effect on prostate cancer cell growth. To explore Btk and Etk as targets for prostate cancer, we developed a small molecule dual inhibitor of Btk and Etk, CTN06. Treatment of PC3 and other prostate cancer cells, but not immortalized prostate epithelial cells with CTN06 resulted in effective cell killing, accompanied by the attenuation of Btk/Etk signals. The killing effect of CTN06 is more potent than that of commonly used inhibitors against Src, Raf/VEGFR and EGFR. CTN06 induces apoptosis as well as autophagy in human prostate cancer cells, and is a chemo-sensitizer for docetaxel (DTX), a standard of care for metastatic prostate cancer patients. CTN06 also impeded the migration of human prostate cancer cells based on a 'wound healing' assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model.

Figure 1

BTK is expressed in prostate cancers and cell lines. (a) The expression level of Btk in prostate cancer tissue was examined using tissue microarray. (b) The array contains 28 benign, 48 gleason 6 (CaP 6) and 46 gleason 8 (CaP 8) prostate cancer samples. (c) The antibody used was confirmed using cellblocks with PC3, PC3 with depletion of Btk by SiBtk and RAMOS cells. (d) The expression level of Etk and Btk in prostate cancer cells was examined using western blot assay. (e) Btk levels in prostate cancer cells were also measured using ELISA. (f) The antibody for tissue microarray was also confirmed using western blot with RAMOS cells and siBtk for PC3 cells, and with Daudi cells as a positive control. (g) Btk levels were further confirmed using RT-PCR. Columns, mean; bars, standard deviation, n=3

Figure 2

Both Btk/Etk (BMX) contribute to proliferation of prostate cancer cells. (a and b) The expression of Etk and Btk in PC3 and RWPE1 cells was knocked down using the corresponding siRNA. The growth of the knockdown cells was measured using MTT assay. (c) The effect of Btk on prostate cancer cell growth was further confirmed using siBtk and LFM-A13 (50 μM) in PC3 and Du145 cells. Columns, mean; bars, standard deviation, n=3

Figure 3

CTN06 is a potent Etk and Btk dual inhibitor. (a) Chemical structure of CTN06. (b) The potency of CTN06 to Btk, Etk, Src and Mer was measured using TLC to identify ³³P-phosphorylated peptide substrate. Purified TKs (20 nM), CTN06 (0–10 μM) and the peptide substrate (YIYGSFK) were incubated with ³³P-ATP in a kinase reaction. The resulting product was analyzed on a TLC plate. (c) The intensity of the radioactive spot was measured using densitometer. (d) Summarization of the kinase inhibition profile of CTN06. Columns, mean; bars, standard deviation, n=3. Molecular docking of CTN06 to Btk. (e) Surface view of Btk docked to CTN06 after 20 ns of MD relaxation. Brown: helix C (439–452); green: activation loop helix 1 (AH1) (541–546) and activation loop helix 2 (AH2) (548–553); yellow: DFG motif (538–540); blue: Thr474; red: Cys480. (f) Cartoon representation showing predicted interactions of Btk with CTN06. Thr474, Leu408, Cys480 and Asp538 side chains are shown as sticks and colored based on residue type (red: acidic, green: polar, yellow: non-polar). DFG motif is shown in red. Figures were generated using PyMol

Figure 4

CTN06 induces autophagy as well as apoptosis in prostate cancer cells. (a) Growth inhibition of CTN06 to LNCaP, Du145, PC3 prostate cancer and normal prostate (RWPE1) cells. Cells were seeded at 5000 cells/well in 96-well plate overnight and treated with CTN06 at the indicated concentrations. The cell viability was measured using MTT assay after 72 h. Columns, mean; bars, standard deviation, n=3. Inhibition of Btk by CTN06 (0.5 μM) in those cells was measured using western blot. (b) Induction of autophagy in PC3 cells by CTN06. PC3 cells were stably transfected with GFP-LC3 and were grown in 6-well plate to 50% confluence and treated with CTN06. Autophagy was visualized by GFP-LC3 ‘puncta' at 2 and 24 h and immunoblot of endogenous LC3 isoforms 24 h after treatment. (c) Induction of apoptosis of PC3 cells following treatment with CTN06. PC3 cells were seeded at 10⁶ cells/ml (2 ml) in a 6-well plate overnight and then treated with CTN06 at the indicated concentrations for 24 h. Apoptosis was analyzed using PI staining as well as Annexin V-FITC apoptosis detection kit. Columns, mean; bars, standard deviation, n=3. In all, 0.5 and 2 μM are significantly different from 0 μM (*P<0.05, one-way ANOVA with Tukey test for pairwise comparison)

Figure 5

Inhibition of cell signaling in PC3 cells following treatment with CTN06. Cells were grown in 100-mm plate to 50% confluence and treated with CTN06 at the indicated concentrations. Cells were harvested after 12 h. pEtk, Etk, pBtk, Btk, pSrc, Src, pPLCγ2, PLCγ2, pStat3, Stat3, pAkt, Akt, pERK and ERK levels were measured using the corresponding antibodies by western blot. One of three similar experiments depicted

Figure 6

CTN06 as a chemo-sensitizer. (a) Comparison of CTN06 with other kinase inhibitors. PC3 cells were seeded at 5000 cells/well in 96-well plates overnight and treated with CTN06 or the other kinase inhibitors including AZD0530, sorafenib, AG1478, 3ATA and LMF-A13 at 2 μM. The cell viability was measured using MTT assay after 72 h. (b) Growth inhibition of CTN06 and in combination with 10 μM chloroquine (CQ) or 2 ng/ml docetaxel (DTX) to PC3 human prostate cancer cells. Cells were seeded at 5000 cells/well in 96-well plate overnight and pretreated with the corresponding co-treatments for 1 h, then treated with 0.25 μM CTN06. The cell viability was measured using MTT assay after 72 h. (c) The apoptotic effect of CTN06 in combination with CQ to PC3 cells was further measured using Annexin-V and PI staining and flow cytometry. Columns, mean; bars, standard deviation, n=3

Figure 7

Inhibition of PC3 xenograft tumor growth by CTN06. 2 × 10⁶ PC3 cells were injected subcutaneously to nude mice. The tumors were grown to the indicated size and the mice were randomly divided into two groups (8 mice/group). The control group was treated with vehicle. The treatment group was treated with CTN06 at 10 mg/kg daily via IP injection. (a and b) The tumor size and body weight were measured once a week. Marks, mean; bars, mean; n=8 (*P<0.05, one-way ANOVA with Tukey test for pairwise comparison). (c and d) The tumor samples were further analyzed for Ki67 and cleaved caspase 3 using immunohistochemistry and LC3I to LC3II conversion using western blot