Calreticulin mediates an invasive breast cancer phenotype through the transcriptional dysregulation of p53 and MAPK pathways

Abstract

Background: The introduction of effective novel biomarkers of invasion and metastasis is integral for the advancement of breast cancer management. The present study focused on the identification and evaluation of calreticulin (CRT) as a potential biomarker for breast cancer invasion.

Methods: Two-dimensional gel protein electrophoresis and MALDI-TOF were utilized in the analysis of fresh-frozen invasive intra-ductal carcinoma specimens. Calreticulin-associated expression was analyzed using immunohistochemistry of FFPE non-malignant/malignant breast specimens. A CRT-knockdown model of MCF7 cell line was developed using siRNA and the CRT genotype/phenotype correlations based on migration and trans-well invasion assays were determined. Finally, microarray-based global gene expression profiling was conducted to elucidate the possible calreticulin pro-invasive regulatory pathways.

Results: Two-dimensional gel protein electrophoresis and MALDI-TOF analysis showed upregulation of calreticulin expression in tumor tissues as compared to the normal adjacent tissues. Meta-analysis of the immunohistochemical results confirmed significantly higher expression of calreticulin (p < 0.05) in the stromal compartments of malignant tissues as compared to non-malignant tissues. Migration and transwell invasion assays showed significant loss in the migratory and invasive potential of CRT-knockdown cells (p < 0.05). Global gene expression profiling successfully identified various putative gene networks such as p53 and MAPK pathways that are involved in calreticulin breast cancer signaling.

Conclusion: Besides confirming calreticulin overexpression in invasive breast cancer tissues, this study reveals a calreticulin-dependent pro-invasive potential and suggests possible contributing pathways. Defining the mechanistic role of invasion and characterizing the possible calreticulin-dependent molecular targets will be the focus of future work.

Keywords: Breast cancer; Calreticulin; Invasion; MAPK; p53.

Fig. 1

Two-dimensional gel electrophoresis and MALDI-TOF mass spectra analysis reveal calreticulin as over expressed in breast cancer tissues. a A representative 2D gel electrophoresis map for the tumor tissue from a stage III sample after running on 17 cm pH 4–7 IPG strips. Spots 1 through 9 were excised and the protein identification was determined by MALDI-TOF analysis, in which spots 1–5 and spot 9 were over-expressed in the tumor tissue, while spots 6–8 were down-regulated. b MALDI-TOF mass spectrum of the tryptic peptides corresponding to calreticulin. c Expression of calreticulin in different intra ductal carcinoma (IDC) samples (T tumoral tissue, N normal adjacent tissue). d Graph represents the CRT expression fold change across different stages of infiltrating ductal breast carcinoma specimens

Fig. 2

Immunohistochemistry results and the comparison of the expression levels of calreticulin in various invasive states of breast cancer. a Immunohistochemical staining of calrecticulin expression in non-malignant lesions of human breast. (I) Negative control. (II) Positive staining in the glandular (white arrow) and stromal compartments (black arrow), ×10 magnification. (III) Positive staining in glandular (white arrow) and stromal compartments (black arrow), ×40 magnification. b Immunohistochemical staining of calreticulin expression in malignant lesions of human breast. (I) Negative control. (II) Positive staining in malignant glandular epithelium (black arrows) and stromal parts (white arrows) of the tumor, ×10 magnification. (III) Positive staining in glandular parts of breast tumor. (IV) Breast tumor with invasive pattern, ×10 magnification. (V) Breast tumor with invasive pattern, ×40 magnification. (VI) Calreticulin expression in stromal cells with spindle shapes (arrows). c (I) Differential expression of calreticulin in different stages of breast cancer. Calreticulin expression is presented based on the localization in three different tissue compartments: glandular structures, stroma and invasive areas. The expression of calreticulin is stratified according to the stage of the tumor (2, 3, 4) and the intensity of expression (low, moderate, high). (II) Differential expression of calreticulin in different grades of breast cancer. Calreticulin expression is presented based on the localization in three different tissue compartments: glandular structures, stroma and invasive areas. The expression of calreticulin is stratified according to the grade of the tumor (1, 2, 3) and the intensity of expression (low, moderate, high)

Fig. 3

The knockdown of CRT expression results in the inhibition of the migratory and invasive potential of MCF7 breast cancer cells. a Relative CRT expression analysis shows that more than 87 % decrease in expression was achieved in CRT-knockdown cells. b Western blot results confirmed the down regulation of calreticulin expression in CRT-knockdown MCF7 cells as compared to control groups. Beta actin was used as the house-keeping control. c MTT assay showed 90.7 % viability in CRT-siRNA transfected MCF7 cells as compared to the untreated group. d Migration assay results revealed a significant association between CRT expression and the migratory potential of MCF7 cells. The effect on migration was not proliferation-dependent. e Statistical analysis of the invasion assay results showed a significant decrease in the number of invading cells following CRT-knockdown of MCF7 cells

Fig. 4

Migration assay: comparing the speed in gap closure in a monolayer of cells. The images were captured at 0, 10 and 20 h post scratching. The CRT-siRNA knockdown cells displayed an obvious decrease in gap closure rate as compared to the control group in the absence and presence of mitomycin-C

Fig. 5

Matrigel invasion assay: micrograph of transwell invaded cells. Control and CRT-knockdown MCF7 cells (black arrow) are shown to have invaded through the pores (white arrow)

Fig. 6

Validation of microarray results by qPCR. The relative expression of selected genes are shown. IFI6 and PARP14 were upregulated in CRT-knockdown MCF7 cells while MNT and TACC2 were downregulated

Fig. 7

CRT-knockdown correlates with the modulation of p53 and MAPK pathways. a Possible role of calreticulin in the p53 pathway. Activated p53 suppresses the processes of tumor formation and progression through the activation of SERPINB5, TP53I11 and TP53TG5. Calreticulin possibly blocks p53 stimulatory effects of these processes. b Possible contribution of calreticulin in the MAPK pathway. MEKs are activated following the attachment of growth factors and other ligands to the surface receptors. This leads to the activation of sequential phosporylation processes, enhancement of transcriptional activities and finally regulation of gene expression. MAP2K6 and MAPK8 are two significantly modulated MAPKs following CRT knockdown (p < 0.05) while MKK1 and MKK4 have also shown some changes in their expression levels. Calreticulin possibly has a stimulatory effect on the production of MKKs