Identification of sequence-specific interactions of the CD44-intracellular domain with RUNX2 in the transcription of matrix metalloprotease-9 in human prostate cancer cells

Abstract

Aim: The Cluster of differentiation 44 (CD44) transmembrane protein is cleaved by γ-secretase, the inhibition of which blocks CD44 cleavage. This study aimed to determine the biological consequence of CD44 cleavage and its potential interaction with Runt-related transcription factor (RUNX2) in a sequence-specific manner in PC3 prostate cancer cells.

Methods: Using full-length and C-terminal deletion constructs of CD44-ICD (D1-D5) expressed as stable green fluorescent protein-fusion proteins in PC3 cells, we located possible RUNX2-binding sequences.

Results: Chromatin immunoprecipitation assays demonstrated that the C-terminal amino acid residues between amino acids 671 and 706 in D1 to D3 constructs were indispensable for sequence-specific binding of RUNX2. This binding was minimal for sequences in the D4 and D5 constructs. Correspondingly, an increase in matrix metalloprotease-9 (MMP-9) expression was observed at the mRNA and protein levels in PC3 cells stably expressing D1-D3 constructs.

Conclusion: These results provide biochemical evidence for the possible sequence-specific CD44-ICD/RUNX2 interaction and its functional relationship to MMP-9 transcription in the promoter region.

Keywords: CD44; CD44-ICD; MMP-9; Prostate cancer; RUNX2; metastasis; tumorigenesis.

Figure 1

Immunoblotting and confocal microscopy analysis of the expression and distribution of CD44, CD44-ICD and RUNX2 in PCa cell lines. A-C: an equal amount of protein lysates (40 µg) made from LNCaP (lane 1), PC3 (lane 2) and PCa2b (lane 3) cells were immunoblotted with CD44 (A), CD44-ICD (B), and RUNX2 (C) antibodies to detect total cellular levels of the respective proteins. (*) and (**) represent the ~ 20-kDa and ~25-kDa fragments of CD44 extracellular truncation fragment (CD44-EXT). CD44-ICD is ~16.5-kDa fragment of CD44. Immunoblotting with a GAPDH antibody was used as a loading control; D: immunostaining analysis of the distribution of RUNX2 (red), CD44-ICD (green), and DAPI (blue). Arrows point to the regions of colocalization (yellow) in RUNX2/CD44-ICD panel. Scale bar: 100 µm. The results represent one of the three separate experiments performed with the same results. CD44: Cluster of differentiation 44; ICD: intracellular domain

Figure 2

Immunohistochemical analysis of TMA in adjacent normal prostate tissue and adenocarcinoma (stage IV). Immunohistochemical staining was performed with an antibody to CD44-ICD in prostate cancer tissue array with adjacent normal prostate tissue. Sections were then scanned using an Aperio Scanscope^® CS instrument (Aperio Scanscope CS system, Vista, CA, USA). A, B: represent normal prostatic and adenocarcinoma (stage IV) tissue sections, respectively. These sections are magnified in A’, A’’, B’ and B’’. Staining was repeated two times. Scale bar represents 500 µm (A and B), 100 µm (A’ and B’), and 25 µm (A” and B”); C, D: the protein expression pattern is expressed as percent cells stained per core for CD44-ICD and CD44s proteins and presented as a graph. Data are given as a scatterplot for the indicated number of cores analyzed in Table 2. The number of cores that were analyzed by two investigators are provided in the parentheses of the first column denoted as “Grade” in Table 2. CD44: Cluster of differentiation 44; ICD: intracellular domain

Figure 3

Analysis of CD44-ICD overexpression and its interaction with RUNX2. A: an equal amount of protein lysates (40 μg) prepared from PC3 cells transfected with CD44-ICD or control PC3 cells were used for immunoblotting analysis with a CD44-ICD antibody. Immunoblotting with a GAPDH antibody was used as a loading control; B: equal amounts of PC3 lysates (200 µg) were immunoprecipitated with a RUNX2 antibody (lane 2-3) or a species-specific non-immune serum (NI, lane 1). Immunoprecipitates were subjected to immunoblotting with an antibody to CD44-ICD. (*) and (**) represent the ~20-kDa and ~25-kDa fragments of CD44 extracellular truncation fragment (CD44-EXT). CD44-ICD is ~16.5-kDa fragment of CD44. An equal amount of lysate (Input) used for immunoprecipitation was assessed by direct immunoblotting of lysates with an antibody to nucleoporin. CD44: Cluster of differentiation 44; ICD: intracellular domain

Figure 4

Analysis of the effect of CD44-ICD overexpression on the expression of metastasis-related genes and migration in PC3 cells. Real-time PCR analysis of SOX2 (A), MMP-9 (B) and OPN (C) expression in PC3 and PC3-CD44-ICD cells. GAPDH was used as a loading control for real-time PCR analysis. PC3 (left panel) and PC3 cells transfected with CD44-ICD (right panel) were subjected to wound-closure assay. Phase contrast micrographs show migration at 0 and 24 h (D). Scale bar: 200 µm. The results shown are representative of three independent experiments. *P < 0.05 or **P < 0.01. CD44: Cluster of differentiation 44; ICD: intracellular domain

Figure 5

Generation of CD44-ICD-EGFP plasmid constructs and transfecting into PC3 cells. A: a schematic diagram of CD44 domains is provided; B: a schematic diagram of constructs generated is shown. Full-length CD44-ICD (FL) and deletion constructs of CD44-ICD (D1-D5) were generated in EGFP vector. Numbers in the figure indicate the amino acid (aa) sequence for each construct; C: immunoblotting analysis using anti-GFP antibody is shown. Immunoblotting with a GAPDH antibody was used as a loading control. GFP/ICD fusion proteins of different sizes and GFP (~27-29 kDa) are indicated in Figure C; D: Phase contrast micrograph shows the morphology of PC3 cells transfected with indicated CD44-ICD-constructs and EGFP vector as well as untransfected PC3 cells. Magnification 100X. CD44: Cluster of differentiation 44; ICD: intracellular domain

Figure 6

Analysis of the specificity of interaction of RUNX2/CD44-ICD deletion constructs. A: immunoblotting analysis with a RUNX2 antibody (top panel): Nuclear lysates prepared from PC3 cells transfected with indicated constructs were immunoprecipitated with a GFP antibody (lane 1-6) and immunoblotted (IB) with an antibody to RUNX2 (lanes 1-6). An equal amount of nuclear lysate used for immunoprecipitation (IP) was assessed by direct immunoblotting of lysates with an antibody to nucleoporin (Input for IP). A decrease in loading was observed in D2 samples, which corresponded to the possible decrease in the coprecipitation of RUNX2; B: confocal microscopy analyses of cells transfected with CD44-ICD deletion construct and stained with an antibody to GFP (green) and RUNX2 (red). Colocalization is seen in yellow (indicated by arrows in FL, D1, D2, D3, and D4). Scale bar: 25 µm. CD44: Cluster of differentiation 44; ICD: intracellular domain

Figure 7

Analysis of mRNA expression of MMP-9 and the effect of sequence-specific interaction of CD44-ICD/RUNX2 on the promoter region of the MMP-9 gene. A: real-time PCR analysis of MMP-9 expression was done in PC3 cells expressing indicated CD44-ICD deletion constructs. GAPDH was used as a loading control for real-time PCR analysis; B: ChIP assay (top): ChIP assay was performed in cells expressing indicated constructs for MMP-9 promoter. ChIP assay showed an increase in signal in PC3 cells expressing FL, D1, D2, and D3. Signal is considerably reduced in D4 and D5. Immunoblotting analysis with an antibody to MMP9 is shown (middle panel in B). The expression levels of MMP-9 protein corresponded to the observations shown in ChIP assay. Immunoblotting with a sCD44 antibody was used as loading control (B; bottom panel). *P < 0.05 vs. D4 and D5. CD44: Cluster of differentiation 44; ICD: intracellular domain

Figure 8

Schematic diagram illustrating the proposed mechanism of CD44-ICD-DelConstruct-RUNX2 interaction in PC3 cells. CD44 is sequentially cleaved to generate CD44-intracellular domain (ICD) fragment. CD44-ICD-FL, D1, D2 and D3 fragments interact with RUNX2 in the nucleus of PC3 cells to activate transcription of MMP-9 to promote tumor progression via migration and metastasis. CD44: Cluster of differentiation 44