CRP2, a new invadopodia actin bundling factor critically promotes breast cancer cell invasion and metastasis

Abstract

A critical process underlying cancer metastasis is the acquisition by tumor cells of an invasive phenotype. At the subcellular level, invasion is facilitated by actin-rich protrusions termed invadopodia, which direct extracellular matrix (ECM) degradation. Here, we report the identification of a new cytoskeletal component of breast cancer cell invadopodia, namely cysteine-rich protein 2 (CRP2). We found that CRP2 was not or only weakly expressed in epithelial breast cancer cells whereas it was up-regulated in mesenchymal/invasive breast cancer cells. In addition, high expression of the CRP2 encoding gene CSRP2 was associated with significantly increased risk of metastasis in basal-like breast cancer patients. CRP2 knockdown significantly reduced the invasive potential of aggressive breast cancer cells, whereas it did not impair 2D cell migration. In keeping with this, CRP2-depleted breast cancer cells exhibited a reduced capacity to promote ECM degradation, and to secrete and express MMP-9, a matrix metalloproteinase repeatedly associated with cancer progression and metastasis. In turn, ectopic expression of CRP2 in weakly invasive cells was sufficient to stimulate cell invasion. Both GFP-fused and endogenous CRP2 localized to the extended actin core of invadopodia, a structure primarily made of actin bundles. Purified recombinant CRP2 autonomously crosslinked actin filaments into thick bundles, suggesting that CRP2 contributes to the formation/maintenance of the actin core. Finally, CRP2 depletion significantly reduced the incidence of lung metastatic lesions in two xenograft mouse models of breast cancer. Collectively, our data identify CRP2 as a new cytoskeletal component of invadopodia that critically promotes breast cancer cell invasion and metastasis.

Keywords: LIM domain; MMP-9; actin cytoskeleton; breast cancer; invadopodia; invasion.

Figure 1. CRP2 up-regulation is associated with a significantly higher risk of metastasis in basal-like breast cancer patients, and correlates with the mesenchymal phenotype in human breast cancer cell lines

A. Kaplan-Meier survival analyses in relation to CSRP2 expression (affy ID 207030_s_at) in breast carcinoma from the basal subtype using distant metastasis free survival as an endpoint. The patient samples, hazard ratio with 95% confidence interval, and p value (Logrank test) are displayed on the chart. B. and C. CRP2 protein level in human breast cancer cell lines (B) and in MCF7-derived cells which underwent EMT through the expression of a constitutively active version of Snail (SNAIL S6A) or prolonged TNF treatment (1001; C). Relative CRP2 expression (lower panels) are calculated from at least three independent experiments and expressed as fold of CRP2 protein level in SKBR3 (B) or MCF7 cells (C). D. and E. Immunohistochemical staining of CRP2 in two cases of invasive ductal carcinoma showing strong staining in tumor cells (D), and faint staining in residual normal breast tissue (arrows; strongly stained tumor cells are indicated by asterisks; (E), respectively (magnification: 200x). Error bars denote standard error. Significant levels: *: p < 0.05 and **: p < 0.001 (unpaired, two-tailed t-test).

Figure 2. CRP2 localizes to active invadopodia

A.-D. CRP2-GFP localizes to actively degrading invadopodia. Cells were grown on Cy3-conjugated gelatin-coated coverslips (D) for 6 h and CRP2-GFP A. was co-localized with actin (B) and cortactin (C). E. Merge of (A-D) F.-I. CRP2-GFP (F) and (H) and actin (G) and (I) co-localize in elongated invadopodia. F. and G. correspond to a focal plane located at 1.4 μm underneath the ventral cell surface, whereas (F) and I show projections along the z axis (35 confocal slices) of the entire invadopodium labeled by an asterisk in (F) and (G) J.- L. CRP2-GFP (J), cortactin (K) and actin (L) in MDA-MB-231-luc cells invading a 3D EHS matrix. The image corresponds to a projection of 17 confocal slices. Bars = 5 μm (A-G), 1 μm (H) and (I), 10 μm (J-L).

Figure 3. CRP2 promotes actin bundling in in vitro reconstituted assays and in breast cancer cells

A. and B. Actin filaments (1 μM) polymerized alone (A) or in the presence of recombinant CRP2 (3 μM; B). C. Typical examples of ROI (13 × 13 μm) used for skewness measurements in Acti-stain 555 phalloidin-stained MDA-MB-231-luc cells expressing GFP alone or CRP2-GFP. D. Skewness average calculated from three independent experiments, including 200 optical sections as in (C). Error bars denote standard error. Significant level: *: p < 0.001. E. and F. Selection of images from Supplementary Movies 2 and 3 (real-time time TIRF microscopy) showing CRP2-induced crosslinking of actin filaments (E) and actin filaments elongation inside a bundle (E) and (F). In both cases 1 μM actin was copolymerized with 3 μM CRP2. Green and red arrows point to fast growing barbed ends of filaments elongating toward the left and right, respectively. For better readability some actin filaments were highlighted in color. G. Kymograph corresponding to (F) and Supplementary Movie 3, showing that CRP2 assembles bundles of mixed polarity. Bars = 20 μm (A and B), 10 μm C., 2 μm (E and F).

Figure 4. CRP2 contributes to breast cancer cell invasion

A. CRP2 protein level in MDA-MB-231-luc cell lines stably expressing two independent shRNAs targeting CRP2 transcripts (shCRP2a and shCRP2b) or a control, non-targeting, shRNA (sh-). Average expression values (lower panel) in shCRP2a and b cell lines were calculated from three independent western blot analyses and expressed as fold of CRP2 protein in sh- cells. B. Proliferation rate of sh-, and shCRP2a and b cell lines as assessed by MTT assay. C. Proliferation rate as assessed by [³H]Thymidine incorporation. The data are expressed as fold of [³H]Thymidine incorporation in control sh- cells. Unpaired two-tailed, t-test revealed no statistical difference in the proliferation rate of the three cell lines. D. 3D cell spheroid proliferation assay. The right chart indicate the average spheroid volume at 6 days of culture (n = 25). E. 2D scratch wound assay (on 2D collagen matrix surface). Gap closure was analyzed using the automated image acquisition and processing system IncuCyte (Essen BioScience). The right image panel show typical gap closure at 6h for each cell line. F. Velocity of 2D migrating cells. For each cell line, at least 150 cells migrating on collagen-coated μ-Slide Chemotaxis^2D chambers (Ibidi) were tracked over 2 days, and an average velocity was calculated. G. 3D scratch wound assay. After wounding, cells were embedded in a 3D collagen matrix. Results were normalized to sh- and are expressed as percentage of gap closure after 72h. The right image panel show typical gap closure at 72 h for each cell line. H. Velocity of invading cells. For each cell line, at least 150 cells embedded in a 3D collagen matrix were tracked over 2 days and an average velocity was calculated. I. Transwell assay performed with MDA-MB-231-luc cells transfected with control (siCtr) or CRP2 specific (siCRP2) siRNA. Invading cells at 24 h were quantified by MTT assay, and the results were normalized to siCtr cells (set at 100%). All the data originate from at least three independent experiments. Error bars denote standard error. Significant levels: *: p < 0.001, **: p < 0.05. Bars = 300 μm (D) and 150 μm (E and G).

Figure 5. CRP2 overexpression promotes MCF-7 cell invasion

A. CRP2 protein level in MCF-7 cell lines stably expressing CRP2-HA (CRP2OE) or a control empty vector (Ctr). Average total CRP2 expression (lower panel) are calculated from three independent western blot analyses and are expressed as fold of endogenous CRP2 protein in Ctr cells. B. Transwell invasion assay. Invading cells at 48 h were quantified by MTT assay, and the results were normalized to Ctr cells (set at 100%). The data originate from 5 independent experiments. Error bars denote standard error. Significant levels: *: p < 0.005.

Figure 6. CRP2 is required for ECM degradation

A. Gelatin degradation assay. Control sh- and shCRP2a and shCRP2b cells were plated on Cy3-conjugated gelatin for 18 h, fixed and stained with DAPI and Acti-stain^TM 670 phalloidin (left panels). Gelatin degraded areas appear as dark punctuate in the fluorescent background (right panels). B. Same as A for an shCRP2a-derived cell line in which CRP2 expression was restored by expressing an shRNA-resistant CRP2 coding sequence (shCRP2a/rescue), and related controls, i.e. sh- and shCRP2a cells lines transduced with an empty vector (sh-/empty and shCRP2a/empty, respectively; see also Supplementary Figure 4). C. and D. Quantitative analyses corresponding to the experiments shown in (A) and (B) Actively ECM degrading cells were scored and expressed as percentage of the total cell population (C). A degradation index corresponding to the ratio of degraded gelatin surface per cell was calculated (D). The data originate from at least three independent experiments (n ≥70 cells). Error bars denote standard error. Significant levels: *: p < 0.0001. Bars = 20 μm.

Figure 7. CRP2 regulates MMP-9 expression in MDA-MB-231-luc cells

A. CRP2 protein level in MDA-MB-231-luc cells transfected with a control siRNA (siCtr) or a CRP2 targeting siRNA (siCRP2). Forty hours after transfection, cells weretreated with or without PMA (100 ng/ml) in serum-free DMEM for 24 h. Average expression values (lower panel) were expressed as fold of CRP2 protein in untreated (−PMA) control siCtr cells which was set to 1. B. Gelatin zymography assays. The medium of the cell cultures described in A. was collected and assessed for its content in secreted pro-MMP-9. The results were expressed as fold of pro-MMP-9 secretion in PMA-treated control siCtr cells which was set to1. C. and D. Expression levels of cytosolic MMP-9 protein (C) and MMP-9 mRNA (D) in the cell cultures described in A. as assessed by western blot and real-time qPCR analyses, respectively. The results were expressed as fold of pro-MMP-9 expression in PMA-treated control siCtr cells which was set to 1. The data originate from at least 3 independent experiments. Error bars denote standard error. Significant levels: *: p < 0.01.

Figure 8. CRP2 promotes breast cancer metastasis

A. Implantation of control (sh-) and CRP2-depleted (shCRP2a) MDA-MB-231-Luc cells (1×10⁶) to the lungs of athymic nu/nu female mice was immediately controlled by bioluminescence imaging after tail vein injection. B. Typical lung metastatic lesions observed after 5 weeks with white light (four left panels) and fluorescence microscopy to detect GFP (cell transduction marker; four right panels). Lower panels are magnifications corresponding to the above white rectangles. Asterisks indicate some extended metastatic lesions. C. Number of lung metastatic lesions in sh- and shCRP2a xenograft mice (n = 8 and 9, respectively). Data are presented as standard box plots with whiskers from minimum to maximum values. D. Extensively necrotized lung of one sh- mouse. E.-G. Metastases at other sites than lungs observed in sh- mice, including a hind leg (E; the image corresponds to the excised tumor), the brain (F) and the liver (G). The lower panels show the corresponding GFP (cell transduction marker) signals. H.-K. Orthotopic tumor model. Control (sh-) and CRP2-depleted (shCRP2a) MDA-MB-231-Luc cells (5×10⁵) were implanted in a mammary fat pad of NOD scid gamma mice (n = 5 and 6, respectively). H. Bioluminescence imaging showing typical and similar signals at the primary tumor site in sh- and shCRP2a xenograft mice (19 days after tumor cell implantation). I. Primary tumor volume. J. and K. Typical lung metastases (J) and number of lung metastatic lesions (K) 20 days after tumor cell implantation. Error bars denote standard error. Significant levels: *: p < 0.05. Bars = 5 mm.