Inhibition of SYK kinase does not confer a pro-proliferative or pro-invasive phenotype in breast epithelium or breast cancer cells

Abstract

SYK has been reported to possess both tumour promotor and repressor activities and deletion has been linked to a pro-proliferative / pro-invasive phenotype in breast tumours. It is unclear whether this is a consequence of protein deletion or loss of kinase activity. The SYK inhibitor, BI 1002494, caused no increase in proliferation in breast cancer cells or primary mammary epithelial cells in 2D or 3D cultures, nor changes in proliferation (CD1/2, CDK4, PCNA, Ki67) or invadopodia markers (MMP14, PARP, phospho-vimentin Ser56). BI 1002494 did not alter SYK protein expression. There was no change in phenotype observed in 3D cultures after addition of BI 1002494. Thirteen weeks of treatment with BI 1002494 resulted in no ductal branching or cellular proliferation in the mammary glands of mice. An in silico genetic analysis in breast tumour samples revealed no evidence that SYK has a typical tumour suppressor gene profile such as focal deletion, inactivating mutations or lower expression levels. Furthermore, SYK mutations were not associated with reduction in survival and disease-free period in breast cancer patients. In conclusion, small molecule inhibition of the kinase function of SYK does not contribute to a typical tumour suppressor profile.

Keywords: 3D culture; SYK inhibitor; breast carcinoma; murine mammary gland; proliferation.

Figure 1. Western blot: effect of 16-hour incubation of BI 1002494 (0, 0.032, 0.16, 0.8, 4 and 20 μM) on selected proliferative (cyclin D1/2, CDK4, PCNA, p21 Waf1/Cip1) and EMT/invadopodia (MMP14, PARP, phospho-vimentin Ser56) marker proteins in a non-tumorigenic, spontaneously immortalized human breast epithelial cell line MCF-10A and in two breast cancer cell lines MDA-MB-468 and DU4475.

+ anti-IgM F (ab)₂ indicates the presence of 10 μg/mL, -anti-IgM F (ab)₂ its absence.

Figure 2

Effect of 16-day incubation of BI 1002494 (0, 1 or 10 μM) on cell number in MCF-10A (A), DU4475 (B), MDA-MB-468 (C) and T-47D (D) cell lines. Cell numbers and viability were analysed using a Vi-CellXR cell counter (Beckman Coulter) following the manufacturer’s instructions.

Figure 3

Effect of 16-day incubation of BI 1002494 (0, 1 or 10 μM) on selected proliferative (cyclin D1/2, PCNA) and EMT/invadopodia (MMP14, PARP) markers in DU4475 and MDA-MB-468 breast cancer cell lines (A) and effect of 13-day incubation of BI 1002494 at the indicated concentrations (0 to 3200 nM) on Cyclin D1/2, CDK4, PCNA, PARP, p21 (Waf1/Cip1) and SYK protein expression in the MDA-MB-468 breast cancer cell line (B).

Figure 4

Box plot analysis of a time course experiment (0 h–192 h) with tumour spheroids of MCF-10A (A) and T-47D (B). 32 spheroids at each time point and for each concentration (0, 0.125, 0.25, 0.5, 1, 2, 4 and 8 μM) were non-invasively imaged on the Genetix CloneSelect Imager (CSI) which detected and recorded the area as the mean loci area [mm²] of spheroids. The line in the box shows the median, the asterisk indicates the mean value, the box shows the inter-quartile range (IQR), and the whiskers show the data within 1.5 × IQR of the upper and lower quartiles. Dots represent outliers. The mean loci area [mm²] of each spheroid at each time point and concentration (red box plots) was compared with the corresponding untreated sample (green box plots). Statistical analyses were performed using unpaired Student’s t-test. Significance levels are indicated as follows: ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001; ns.: not significant.

Figure 5. Effect of 15-day incubation of BI 1002494 on T-47D breast cancer cells cultivated in alginate capsules in a bioreactor.

(A) Viability staining (FDA, fluorescein diacetate) and (B) Caspase (green) and annexin (red) live cell staining of 3D tumor cultures at different time points. (C) Cryosections of T-47D alginate capsules were stained for cell death (In Situ Cell Death Detection Kit, TMR red, Roche) and proliferation (Ki-67). Values are percent of stained positive cells compared to DAPI positive cells and are mean ± standard error of the mean (SEM) of three separate images. Statistical analysis was performed for each condition using Student’s t test and was non-significant (P > 0.5). (D) Cell death (In Situ Cell Death Detection Kit, TMR red, Roche) and Ki-67 (green) staining of 3D tumor cell cultures at day 15 after treatment.

Figure 6

Effect of 12-day incubation of BI 1002494 (0, 1, 3, 10 μM) on primary human mammary epithelial cell proliferation (A) and 4-day incubation of BI 1002494 on PARP, MMP14, PCNA and p21 protein expression in primary human mammary epithelial cells (B).

Figure 7. Ki-67 immunohistochemistry.

Mice were treated daily for 13 weeks with either vehicle (A and B) or 30 mg/kg q. d. (C), 100 mg/kg q. d. (D) and 100 mg/kg b. i. d. (E) BI 1002494. Breast tissue was excised and stained for the proliferation marker Ki-67. Representative photomicrographs are illustrated.

Figure 8. In silico analysis of SYK gene characteristics compared with the well-known tumour suppressors TP53 and PTEN in the Metabric and TCGA invasive breast cancer studies.

(A) Lollipop plot of the number of mutations observed in SYK, TP53 and PTEN genes (TCGA and Metabric), (B) Oncoprint of the somatic DNA alterations rate in the SYK and TP53 genes (TCGA and Metabric), (C) Oncoprint of DNA mutations or mRNA expression alterations in SYK and PTEN genes (Metabric only) and (D) survival in patients with or without DNA alterations of known significance in TP53 and PTEN (TCGA and Metabric), as well as SYK (Metabric; mutations, copy number alterations and mRNA expression alterations).