CHEK2 represses breast stromal fibroblasts and their paracrine tumor-promoting effects through suppressing SDF-1 and IL-6

Abstract

Background: Active fibroblasts, the predominant and the most active cells of breast cancer stroma, are responsible for tumor growth and spread. However, the molecular mediators and pathways responsible for stromal fibroblast activation, and their paracrine pro-carcinogenic effects are still not well defined. The CHEK2 tumor suppressor gene codes for a protein kinase, which plays important roles in the cellular response to various genotoxic stresses.

Methods: Immunoblotting, quantitative RT-PCR and Immunofluorescence were used to assess the expression of CHEK2 in different primary breast fibroblasts and in tissues. The effect of CHEK2 on the expression and secretion of SDF-1 and IL-6 was evaluated by immunoblotting and ELISA. The WST-1 colorimetric assay was used to assess cell proliferation, while the BD BioCoat Matrigel invasion chambers were utilized to determine the effects of CHEK2 on the migratory and the invasiveness capacities of breast stromal fibroblasts as well as breast cancer cells.

Results: We have shown that CHEK2 is down-regulated in most cancer-associated fibroblasts (CAFs) as compared to their corresponding tumor counterpart fibroblasts (TCFs) at both the mRNA and protein levels. Interestingly, CHEK2 down-regulation using specific siRNA increased the expression/secretion of both cancer-promoting cytokines SDF-1 and IL-6, and transdifferentiated stromal fibroblasts to myofibroblasts. These cells were able to enhance the proliferation of non-cancerous epithelial cells, and also boosted the migration/invasion abilities of breast cancer cells in a paracrine manner. The later effect was SDF-1/IL-6-dependent. Importantly, ectopic expression of CHEK2 in active CAFs converted these cells to a normal state, with lower migration/invasion capacities and reduced paracrine pro-carcinogenic effects.

Conclusion: These results indicate that CHEK2 possesses non-cell-autonomous tumor suppressor functions, and present the Chk2 protein as an important mediator in the functional interplay between breast carcinomas and their stromal fibroblasts.

Keywords: Breast cancer; CHEK2; Cancer-associated fibroblasts; IL-6; SDF-1.

Fig. 1

CHEK2 expression is down-regulated in cancer-associated fibroblasts. a Whole cell lysates were prepared from the indicated cells and 50 μg of proteins were used for immunoblotting analysis using antibodies against the indicated proteins. b Total RNA was extracted from the indicated cells and the amount of the CHEK2 mRNA was assessed by qRT-PCR. Error bars represent means ± S.D

Fig. 2

CHEK2 represses the expression/secretion of SDF-1 and IL-6. a Whole cell lysates were prepared from the indicated cells and were used for immunoblotting analysis. The numbers below the bands indicate the corresponding expression levels after loading correction against GAPDH. b Total RNA was extracted from the indicated cells and used to assess the mRNA levels of SDF-1 and IL-1 using qRT-PCR and specific primers. c Conditioned media from the indicated cells were collected after 24 h and the levels of the indicated proteins were determined by ELISA, and were presented in the respective histograms. Error bars represent means ± S.D, *, p value < 0.0001

Fig. 3

CHEK2 represses the migration/invasion abilities of breast stromal fibroblasts. TCF-169-CHEK2-siRNA cells and their controls (a), NBF-1-CHEK2-siRNA cells and their controls (b) as well as CAF-169 and TCF-169 cells (c) were seeded with SFM in the inserts of BD bio-coat™ chambers (BD biosciences) (without matrigel, for migration) or coated with matrigel layer (for invasion), while complete media was added as a chemo-attractant to the lower wells of the chambers. After 24 h of incubation, cells were stained with Diff-Quick stain then counted. Upper panels: images of the invaded and migrated cells. Histograms: Average numbers of invaded and migrated cells. Error bars represent means ± S.D, *, p value < 0.0001

Fig. 4

CHEK2 deficient fibroblasts enhance the proliferation of epithelial cells in a paracrine manner. SFCM were collected from TCF-169-CHEK2-siRNA cells and their controls, and then were added to MCF10A (a) or MDA-MB-231 (b) cells that were seeded on 96-well plates. The proliferation was measured at the indicated times using the WTS-1 assay. Error bars represent ± SD, p value < 0.0001

Fig. 5

CHEK2-deficient fibroblast secretions stimulate invasion/migration in an SDF1- and IL-6-dependent manner and enhance mesenchymal features in breast cancer cells. a SFCM were collected after 24 h of incubation from TCF-169-CHEK2-siRNA and their control cells, and were added to MDA-MB-231 cells (10⁵) that were seeded onto the upper compartment of the plates and incubated for 24 h. The numbers of invaded and migrated cells were presented in histograms. Error bars represent means ± S.D. *: p value <0.001, as compared to the control lane 1. b MDA-MB-231 cells were treated as indicated, and then the migration and invasion abilities were assessed as described in A. c MDA-MB-231 cells were treated with SFCM from the indicated cells for 24 h, and then cell lysates were prepared and used for immunoblotting analysis using the indicated antibodies

Fig. 6

CHEK2 upregulation represses breast stromal fibroblasts and their paracrine procarcinogenic effects. CAF-169 cells transfected with CHEK2-ORF (CAF-169-CHEK2-ORF) or control plasmid (CAF-169-control). a Whole cell lysates were prepared from the indicated cells and were used for immunoblotting analysis. The numbers below the bands indicate the corresponding expression levels after loading correction against GAPDH. b Assessment of the migration/invasion abilities of CAF-169 cells expressing either CHEK2 ORF or control plasmid as described in Fig. 3. c SFCM were collected after 24 h of incubation from CAF-169-CHEK2-ORF cells (SFCM-CHEK2-ORF) and control cells (SFCM-control), and then were added to MDA-MB-231 cells. The migration and invasion abilities were assessed as described in Fig. 5a. Error bars represent means ± S.D. *: p value <0.001