Control of MT1-MMP transport by atypical PKC during breast-cancer progression - PubMed (original) (raw)

. 2014 May 6;111(18):E1872-9.

doi: 10.1073/pnas.1400749111. Epub 2014 Apr 21.

Catalina Lodillinsky, Laetitia Fuhrmann, Maya Nourieh, Pedro Monteiro, Marie Irondelle, Emilie Lagoutte, Sophie Vacher, François Waharte, Perrine Paul-Gilloteaux, Maryse Romao, Lucie Sengmanivong, Mark Linch, Johan van Lint, Graça Raposo, Anne Vincent-Salomon, Ivan Bièche, Peter J Parker, Philippe Chavrier

Affiliations

Control of MT1-MMP transport by atypical PKC during breast-cancer progression

Carine Rossé et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Dissemination of carcinoma cells requires the pericellular degradation of the extracellular matrix, which is mediated by membrane type 1-matrix metalloproteinase (MT1-MMP). In this article, we report a co-up-regulation and colocalization of MT1-MMP and atypical protein kinase C iota (aPKCι) in hormone receptor-negative breast tumors in association with a higher risk of metastasis. Silencing of aPKC in invasive breast-tumor cell lines impaired the delivery of MT1-MMP from late endocytic storage compartments to the surface and inhibited matrix degradation and invasion. We provide evidence that aPKCι, in association with MT1-MMP-containing endosomes, phosphorylates cortactin, which is present in F-actin-rich puncta on MT1-MMP-positive endosomes and regulates cortactin association with the membrane scission protein dynamin-2. Thus, cell line-based observations and clinical data reveal the concerted activity of aPKC, cortactin, and dynamin-2, which control the trafficking of MT1-MMP from late endosome to the plasma membrane and play an important role in the invasive potential of breast-cancer cells.

Keywords: MMP14; actin cytoskeleton; membrane traffic; multi-vesicular body.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Co–up-regulation and colocalization of MT1-MMP and aPKCι in hormone receptor-negative breast tumors correlate with poor prognosis. (A) Metastasis-free survival (MFS) curves for breast-tumor patients with normal (Norm) or overexpressed (Overexp, >3) aPKCι and MT1-MMP mRNA levels (Spearman rank). (B and C) aPKCι (B) and MT1-MMP (C) immunohistochemistry staining on consecutive sections of human breast tumor TMAs showing adjacent nonneoplastic tissue and representative ER− PR− HER2−/triple-negative tumors. Note that adjacent nonneoplastic tissue shown in Top panels correspond to tumor sample shown in Middle panels. Right panels show higher magnification. [Scale bars: 20 μm (Left) and 5 μm (Right).] (D) Intensity scoring of aPKCι (black bars) and MT1-MMP (red bars) immunohistochemistry staining (on a 0-to-3 scale as documented in

Fig. S1 A and B

) of a tissue microarray of human breast tumors representative of the different molecular subtypes and adjacent nonneoplastic areas (Normal breast tissue). Breast molecular subtypes were defined as follows: luminal A+B according to ref. [luminal A, estrogen receptor (ER) ≥ 10%, progesterone receptor (PR) ≥ 20%, Ki67 < 14%; luminal B, ER ≥ 10%, PR < 20%, Ki67 ≥ 14%]; ER− PR− HER2+, ER < 10%, PR < 10%, HER2 2+ amplified or 3+ according to ref. ; ER− PR− HER2− (triple-negative), ER < 10%, PR < 10%, HER2 0/1+ or 2+ nonamplified according to ASCO guidelines (53). n, the number of tumors analyzed. *P < 0.05; ***P < 0.001; ns, non significant compared with normal adjacent tissues (χ2 test). Note that intensity scores of MT1-MMP expression in luminal A+B tumors are inferior to normal tissues.

Fig. 2.

Fig. 2.

Atypical PKCζ/ι regulate MT1-MMP–dependent matrix degradation and invasion. (A and B) Quantification of FITC–gelatin degradation by MDA-MB-231 (A) and BT-549 cells (B) treated with indicated siRNAs. Values are means ± SEM of the normalized degradation area from at least three independent experiments. (Insets) Immunoblotting with antibodies against aPKCζ/ι and MT1-MMP of cells treated with the indicated siRNAs. Immunoblotting with antibodies against GAPDH served as a control for loading. (C) Quantification of FITC–gelatin degradation by MDA-MB-231 stably expressing MT1-MMP–mCherry treated with the indicated siRNAs (red bars) compared with untransfected MDA-MB-231 cells (black bars). Note the logarithmic scale. (D) MDA-MB-231 cells treated with indicated siRNAs were tested for their ability to invade through Matrigel. Values are means ± SEM normalized to the mean for siLuc-treated cells. *P < 0.05; **P < 0.01. (E) Multicellular spheroids of MDA-MB-231 cells treated with siRNAs against luciferase or aPKCζ/ι (siaPKCζ/ι-2) were embedded in 3D acid-extracted type I collagen (T0) and further incubated for 2 d (T2). Images show phalloidin-labeled spheroids collected at T2 (Insets correspond to spheroids at T0). (Scale bars: 200 μm.) (F) Data are mean invasion area in type I collagen at T2 normalized to the mean invasion area at T0 ± SEM (n = 3 independent experiments; 15–20 spheroids were analyzed for each cell population). ***P < 0.001.

Fig. 3.

Fig. 3.

aPKCζ/ι regulate MT1-MMP trafficking to plasma membrane invadopodia. (A) Ultrathin cryosection of MDA-MB-231 cells expressing MT1-MMP–mCherry labeled with MT1-MMP antibody followed by protein A-gold. Red arrows, MT1-MMP in the limiting membrane; black arrows, MT1-MMP associated with intraluminal vesicles of late endosomes/multivesicular bodies (LE/MVB). (Scale bars: 500 nm.) (B) Confocal spinning-disk microscopy image of an MDA-MB-231 cell expressing GFP–aPKCι and MT1-MMP–mCherry. [Scale bars: 5 μm (Left) and 1 μm (Right, boxed region at higher magnification). (C) MDA-MB-231 expressing MT1-MMP–mCherry alone or together with constitutively active aPKCι were imaged by TIRFM. (Insets) Corresponding wide-field images showing equal MT1-MMP expression. (Scale bar: 5 μm.) (D and E) Integrated intensity of MT1-MMP–mCherry signal per unit membrane area measured from TIRFM images. Values represent mean percentage of membrane MT1-MMP normalized to cells expressing only MT1-MMP–mCherry (D) or siLuc-treated cells (E) ± SEM. *P < 0.05; ***P < 0.001. (F) MDA-MB-231 cells expressing MT1-MMP–pHluorin and DsRed-cortactin plated on cross-linked gelatin and analyzed by dual color TIRFM. (Insets) Split signals from the boxed region. (Scale bar: 5 μm.) (G) Plots show the percentage of cells with MT1-MMP–pHluorin–positive invadopodia. Efficiency of cortactin knockdown is shown in Fig. 4_G_. Values are means ± SEM from three independent experiments scoring a total of 150–200 cells for each cell population. **P < 0.01; ***P < 0.001 (compared with cells treated with siLuc siRNA).

Fig. 4.

Fig. 4.

aPKCζ/ι regulates cortactin and dynamin-2 recruitment on MT1-MMP–positive endosomes. (A) MDA-MB-231 cells stably expressing MT1-MMP–mCherry (red) were treated with indicated siRNAs, plated on gelatin, and immunolabeled with an antibody against cortactin (in green). [Scale bars: 5 μm (entire cell) and 1 μm (boxed region at higher magnification, Right panel of each row).] (B) Quantification of cortactin on MT1-MMP–mCherry–containing vesicles in deconvoluted image stacks of MDA-MD-231 cells as in A. The x axis indicates mean cortactin intensity associated with MT1-MMP–mCherry–containing endosomes normalized to the value in control siLuc-treated cells (in percentage) ± SEM (from 50 cells from each cell population). ***P < 0.001. (C) MDA-MB-231 cells expressing MT1-MMP–mCherry (red) were immunolabeled for cortactin (blue) and dyn-2 (green). (Left) The distribution of dyn-2 on the ventral plane of the cell in clathrin-coated pits with partial association with cortactin. (Center) Documents dyn-2’s association with cortactin-positive puncta on MT1-MMP–mCherry vesicles (arrows). (Right) A higher magnification of the boxed region. [Scale bars: 5 μm (Left and Center) and 1 μm (Right)]. (D) Confocal spinning-disk microscopy image of MDA-MB-231 cells expressing GFP–dyn2 and DsRed–cortactin (

Movie S2

). [Scale bars: 5 μm (Upper) and 1 μm (Lower). (E) Dual-color confocal spinning-disk microscopy of MDA-MB-231 cells expressing DsRed–cortactin and GFP–dyn-2 and treated with indicated siRNAs. (Scale bars: 5 μm.) (F) The ratio of signal intensities from GFP–dyn-2 and DsRed–cortactin was measured for 200 and 290 endosomal cortactin patches in cells treated with siRNAs against luciferase or aPKC, respectively. ***P < 0.001. (G) Quantification of gelatin degradation by MDA-MB-231 cells treated with indicated siRNAs. Values are means ± SEM of the normalized degradation area from three independent experiments. (Inset) Immunoblotting with indicated antibodies using tubulin as a control for loading.

Fig. 5.

Fig. 5.

Phosphorylation of cortactin by aPKCι controls dyn-2 association with cytoplasmic cortactin-positive puncta. (A) Purified GST-tagged human cortactin (2 μg) was incubated in the presence (0.5 μg) or absence of recombinant human aPKCι for 20 min at 37 °C. Cortactin phosphorylation was analyzed by immunoblotting with antibodies against cortactin pSer298 phosphopeptide (anti-P298). (B) Lysates of MDA-MB-231 cells expressing DsRed–cortactin alone or together with GFP–aPKCι were analyzed by immunoblotting with antibodies against pSer298 (anti-P298), total cortactin, or GFP. (C) Quantification of dyn-2 on cortactin patches from confocal dual-color spinning-disk images of MDA-MD-231 cells overexpressing GFP–dyn-2 together with DsRed-tagged wild-type cortactin or variants as indicated. The ratios of the signal intensities from dyn-2 and cortactin were measured from n endosome patches. ***P < 0.001; NS, not significant. (D) Whole-cell extracts from breast tumors underexpressing (low) or overexpressing (high) aPKCι mRNA were analyzed by immunoblotting with the indicated antibodies. Tubulin was used as a control for equal loading.

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