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Liprin-α1 regulates breast cancer cell invasion by affecting cell motility, invadopodia and extracellular matrix degradation

A Corrigendum to this article was published on 14 April 2011

Abstract

Migration of cells and degradation of the extracellular matrix (ECM) are required for efficient tumor cell invasion, but the underlying molecular mechanisms are only partially known. The PPFIA1 gene for liprin-α1 is frequently amplified in human breast cancers. We recently demonstrated that liprin-α1 is an important regulator of cell edge dynamics during motility. We show, herein, that the liprin-α1 protein is highly expressed in human breast tumors. Functional analysis shows that liprin-α1 is specifically required for the migration and invasion of highly invasive human breast cancer MDA-MB-231 cells. We used time-lapse analysis to demonstrate defects in the motility of liprin-α1-depleted cells that include a striking instability of the lamellipodia. Liprin-α1 levels altered by either RNA interference or overexpression affected also cell spreading and the number of invadopodia per cell, but not the density of invadopodia per unit of surface area. On the other hand, silencing of liprin-α1 inhibited the degradation of the ECM, whereas its overexpression enhanced degradation, resulting in significant negative or positive effects, respectively, on the area of degradation per invadopodium. Transfection of fluorescent-labeled cortactin revealed that depletion of liprin-α1 also affected the assembly and disassembly of invadopodia, with decrease of their lifetime. Our results strongly support a novel important role of liprin-α1 in the regulation of human tumor cell invasion.

Introduction

The liprin family includes the α- and β-subfamilies of dimeric adaptor proteins represented by four and two mammalian genes, respectively (Serra-Pagès et al., 1995, 1998). As all other members of the family, the widely expressed liprin-α1/PPFIA1 protein is made by an amino-terminal coiled-coil region and a carboxy-terminal region including three predicted steryl alpha motifs, which are present in several proteins to mediate interactions with either proteins, RNA or lipid membranes (Qiao and Bowie, 2005). Liprins show some degree of homology with kazrinE, a widely expressed cytoplasmic protein with three carboxy-terminal steryl alpha motifs that is involved in epidermal differentiation (Nachat et al., 2009). Liprin-α proteins may interact with different ligands, including liprin-β, the tyrosine phosphatase receptor LAR, kinesin motor proteins, the ArfGAP GIT1 and the adaptor proteins ERC and CASK (see de Curtis, 2011 for a review). Liprin-α1 is required for the assembly of neuronal synapses (Spangler and Hoogenraad, 2007) and is implicated in the regulation of non-neuronal cell migration (Shen et al., 2007). We have recently shown that liprin-α1 is an essential regulator of cell spreading on extracellular matrix (ECM) (Asperti et al., 2009, 2010). Interestingly, the gene for liprin-α1 is included in the human chromosomal region 11q13 (Katoh and Katoh, 2005; Järvinen et al., 2006) that is frequently amplified in various malignant tumors, including 20% of breast cancers (Al-Kuraya et al., 2004). Based on this information, we have addressed the role of liprin-α1 in the regulation of the different cellular processes required for the invasion of the MDA-MB-231 breast cancer cells.

Results and discussion

Liprin-α1 is required for tumor cell invasion

Amplification of the PPFIA1/liprin-α1 gene has been reported in various cancers including 20% of breast cancers. Whether this amplification reflects an increase in the liprin-α1 protein is not known. By using affinity-purified antibodies, we found that liprin-α1 is highly expressed in breast cancer (Figure 1a). In support of a possible role of liprin-α1 in breast cancer, we found that 57 of 116 human breast cancer samples examined by immunohistochemistry show a clear increase in the expression of the endogenous protein in tumor cells (Supplementary Figure S1).

Figure 1
figure1

Liprin-α1 is required for human breast cancer cell invasion. (a) Expression of liprin-α in human tumors, detected with affinity-purified anti-liprin-α1 antibodies. Sections from paraffin-embedded tissues were incubated for 1 h with 0.25 μg/ml affinity-purified anti-liprin-α1 immunoglobulin G and peroxidase-conjugated secondary antibodies, and counterstained with hematoxylin. Bars=100 μm. (b) Downregulation of endogenous proteins in MDA-MB-231 cells transfected with small interfering RNAs (siRNAs). Bars are percentages±s.e.m. of protein after silencing (n=2−4 experiments). MDA-MB-231 cells from ATCC (American type culture collection, Manassas, VA, USA) were used up to the tenth passage. Antibodies for liprin-α1 and βPIX have been characterized (Asperti et al., 2009). We utilized monoclonal antibodies against Flag and tubulin (Sigma-Aldrich, St Louis, MO, USA), GIT1/2 (BD Biosciences, San Jose, CA, USA) and cortactin (Millipore, Billerica, MA, USA); polyclonal antibodies against Flag (Sigma-Aldrich), GIT1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GFP (green fluorescent protein) (Molecular Probes, Eugene, OR, USA). siRNAs (from Invitrogen, Paisley, Scotland, UK or Qiagen, Hilden, Germany) for human liprin-α1 (liprins1a and b), human GIT2, human βPIX and control (luciferase) were as described (Frank et al., 2006; Asperti et al., 2009). The siRNA for human GIT1 targeted the sequence 5′-GCCTGGATGGAGACCTAGA-3′. Cells transfected with Lipofectamine 2000 (Invitrogen) and 50–100 nM of siRNAs were used after 2 days for immunoblotting (Asperti et al., 2009). (c, d) MDA-MB-231 siRNA-transfected cells were tested for migration (c) and invasion (d). For migration, siRNA-transfected serum-starved cells (30 000/well) were seeded in 8 μm 24 Transwells (Corning Inc. Life Sciences, Lowell, MA, USA) with lower chamber coated with 20 μg/ml fibronectin and analyzed after 8 h. For invasion, cells (100 000/well) were seeded on Matrigel (BD Transduction, San Jose, CA, USA)-coated Transwells with lower chambers filled with NIH 3T3-conditioned medium (Shaw, 2005). After 5 h, cells invading to the lower side were counted (n4 experiments, in duplicate). All quantitative analyses presented in this and the following figures were blinded. Bars are normalized means±s.e.m. (migration assays: 4–6 experiments; 2 experiments with βPix siRNAs. Invasion assays: 4–5 experiments; 2 experiments with liprin1b siRNA). Significance was set at P<0.05 by the Student's t-test (*<0.05; **<0.01).

Given the published effects of liprin-α1 on cell motility, we used RNA interference to study the function of this protein in the motility of human MDA-MB-231 breast cancer cells, an established system to analyze invasion in vitro. Liprin-α1 was efficiently depleted (89–98% silencing) in MDA-MB-231 cells by either of two different small interfering RNAs (Figure 1b). Liprin-α1 depletion had no effect on cell adhesion to fibronectin (Supplementary Figure S2), but it was required for migration (Figure 1c). MDA-MB-231 cells are highly invasive in response to specific stimuli (Supplementary Figure S3), and liprin-α1 depletion strongly inhibited also invasion of these cells through Matrigel (BD Transduction) (Figure 1d). Conversely, depletion of components of the PIX/GIT complexes that may interact with liprin-α1 (Totaro et al., 2007) did affect neither migration nor invasion (Figures 1c and d). These data demonstrate a unique role of liprin-α1 in breast cancer cell invasion.

The LAR tyrosine phosphate receptor is another binding partner of liprin-α1 (Serra-Pagès et al., 1995). LAR is expressed in MDA-MB-231 cells (Figure 2a). We found that LAR depletion by small interfering RNA inhibited MDA-MB-231 cell spreading on fibronectin (Figures 2b and c). Moreover, the steryl alpha motif2 domain required for the interaction of liprin-α1 with LAR was required for the positive effects of liprin-α1 on cell spreading (Figures 2e and f) and on the redistribution of active β1-integrins (Figures 2g–i), thus confirming the effects of these proteins described in non-tumorigenic COS7 cells (Asperti et al., 2009). On the other hand, LAR depletion had no detectable effects on invasion by MDA-MB-231 cells (Figure 2d). Therefore, the effects of liprin-α1 on tumor cell invasion appear to be independent from LAR.

Figure 2
figure2

LAR is required for spreading, but not for invasion. (a) Immunoblotting with the indicated antibodies on equal amounts of lysates from MDA-MB-231 cells transfected with control or specific small interfering RNAs (siRNAs). LAR-E indicates the 150 kDa form representing the larger mature cleaved LAR polypeptide. (b) Immunofluorescence on MDA-MB-231 cells transfected with control and LAR-specific siRNA, plated on fibronectin and stained for the indicated proteins. Bar=20 μm. (c) Quantification of cell spreading on fibronectin: bars are normalized means±s.e.m. (**P<0.01). For spreading, cells trypsinized 2 days after transfection were plated on coverslips (50 000 cells) coated with 10 μg/ml fibronectin (BD-Transduction) and fixed after 18 h at 37 °C for immunofluorescence. Fixed cells incubated with the indicated antibodies and fluorescent phalloidin to reveal filamentous actin were observed with Zeiss Axiophot or UltraviewERS (PerkinElmer, Waltham, MA, USA) microscopes. Projected cell areas were evaluated from 40 cells per condition from two independent experiments. (d) LAR depletion does not affect invasion: MDA-MB-231 cells were transfected with control or specific siRNAs, and invasion was quantified as described in Figure 1. Bars are normalized mean values±s.e.m. from three experiments (**P<0.01). (e) Immunofluorescence on MDA-MB-231 cells transfected with plasmids for β−galactosidase, liprin-α1 or liprin-ΔSAM2 (steryl alpha motif 2), and plated on fibronectin for 18 h before staining for the indicated proteins. Bar=20 μm. (f) Quantification of cell spreading on fibronectin: bars are normalized mean values±s.e.m (*P<0.05); n=40 cells per condition from two experiments. (g) Transfected cells were used for immunofluorescence with the 9EG7 mAb recognizing active β1-integrins (Lenter et al., 1993). Bar=20 μm. (h, i) Bars represent mean values±s.e.m. of the fraction of projected cell area occupied by active β1-integrins (h), and of the percentage of projected cell area occupied by active β1-integrins that localize at the cell periphery (i); n=23–25 cells per condition from two experiments (**P<0.01). The 9EG7 mAb and the anti-LAR mAb recognizing the 150 kDa E subunit were from BD Biosciences. The siRNA for LAR and the plasmid for FLAG-liprin-ΔSAM2 have been described previously (Asperti et al., 2009).

Liprin-α1 regulates cell edge dynamics and the motility of tumor cells

We investigated the possible causes of the observed inhibition of MDA-MB-231 cell migration and invasion by time-lapse analysis during random migration (Pankov et al., 2005). Liprin-α1 silencing had a striking effect on the behavior of cells freely moving on fibronectin (Figure 3a), with a significant decrease of both the path (−17%) and Euclidean distance (−38%) covered by the liprin-α1-depleted cells, and a non-significant decrease in spontaneous directionality (−24%), suggesting a defect in the persistence of directed movement (Figures 3b and c). Interestingly, time-lapse analysis highlighted a dramatic effect of liprin-α1 silencing on lamellipodia, the lamellar protrusions driving locomotion of cells on ECM (Supplementary movies S1 and S2). Quantification revealed in liprin-α1-depleted cells a twofold increase in the number of lamellipodia per cell and a 60% decrease in the average life of lamellipodia (Figure 3d). These data show that liprin-α1 is important for the stabilization of lamellipodial protrusions, and suggest that the defect in lamellipodial persistence may underlie the defect in migration and invasion observed in MDA-MB-231 tumor cells depleted of liprin-α1.

Figure 3
figure3

Liprin-α1 is required for the persistence of lamellipodia and for the efficient migration of human breast cancer cells. (a) Frames from time lapse of cells transfected with small interfering RNAs (siRNAs) and plasmid for GFP (not shown), and plated on fibronectin. (b) Cell tracking for 5 h of control and liprin-α1-depleted cells. Bars=20 μm. (c) For random migration, 50 000 transfected cells were seeded for 3 h on 2.5 μg/ml fibronectin-coated dishes before time lapse with an Axiovert microscope (Zeiss). Path, mean velocity, directionality and lamellipodia dynamics were evaluated during 5 h with ImageJ (Bethesda, MD, USA). Cells undergoing division and non-moving cells were ignored. Analysis of random migration over 5 h: bars represent normalized mean values±s.e.m. of cell tracks (path), Euclidean distance (displ.), path rate (Vp), Euclidean rate (Vd) and persistence of migration (persist=path/displ). (d) Left: frames from time lapse of siRNA-transfected cells: arrows indicate lamellipodia. Center: average number of lamellipodia per cell formed in 5 h. Right: average lamellipodia persistence (90 and 188 lamellipodia from 10 control or liprin-α1-depleted cells, respectively). *P<0.05, **P<0.01.

We also tested the effects of liprin-α1 in other human tumor cell lines that include cervical cancer HeLa cells, poorly invasive breast cancer MCF7 cells, fibrosarcoma HT1080 cells and epidermoid carcinoma A431 cells. All these cells express liprin-α1, but only invasion by HeLa cells was inhibited by the depletion of endogenous liprin-α1 (Supplementary Figures S4a–b), whereas for MCF7, invasion was very poor under the experimental setting utilized (data not shown). We then compared the effects of liprin-α1 depletion on the motile behavior of HeLa and HT1080 cells on fibronectin (Supplementary Figure S4c). As for MDA-MB-231 cells, liprin-α1 depletion inhibited both the path and the path rate (Vp) of HeLa cells, whereas it had an opposite effect in HT1080 cells, where liprin-α1 depletion resulted in an increase of both parameters (Supplementary Figure S4d). On the other hand, overexpression of liprin-α1 did not significantly affect the behavior of either cell types (Supplementary Figure S4e). As observed in MDA-MB-231 cells, liprin-α1 depletion in HeLa cells caused also an increase in the number of lamellipodia per cell and a decrease in the average life of the lamellipodia (Supplementary Figure S4f). Interestingly, depletion of liprin-α1 caused similar effects on the number and persistence of lamellipodia even in the poorly invasive MCF7 breast cancer cells, although these cells were almost non-motile on fibronectin (Supplementary Figure S4f). Altogether, these results indicate that different types of human tumor cells respond differently to liprin-α1 depletion and suggest that liprin-α1 is important for the invasion of different types of tumor cells.

Liprin-α1 is required for efficient ECM degradation

Invadopodia are dynamic filamentous actin-rich protrusions effecting ECM degradation and tumor cell invasion (Weaver, 2008; Albiges-Rizo et al., 2009). We investigated whether liprin-α1 influences the formation and/or function of invadopodia. Endogenous liprin-α1 did not colocalize with cortactin-positive, filamentous actin-positive invadopodia (Supplementary Figure S5). We quantified the number of both centrally located (perinuclear) and total invadopodia per cell. Liprin-α1 silencing decreased the number of invadopodia per cell, whereas liprin-α1 overexpression increased the number of invadopodia per cell (Figures 4a and b). On the other hand, no significant differences in the density of invadopodia were observed in cells with altered liprin-α1 levels when compared with controls (Figure 4c). These results suggest that the effects of liprin-α1 on the number of invadopodia per cell may simply reflect the inhibitory or promoting effect on spreading induced by liprin-α1 silencing or overexpression, respectively (Figure 4d).

Figure 4
figure4

Liprin-α1 affects cell spreading, but not the density of invadopodia. (a) Confocal images of cells transfected for 1 day with Flag-βgalactosidase or Flag-liprin-α1, or cotransfected for 2 days with GFP and small interfering RNAs (siRNAs), and cultured 18 h on fibronectin. Bar=20 μm. Insets: threefold enlargements of perinuclear areas indicated by arrows. (b–d) Liprin-α1 depletion and overexpression affect the number of invadopodia per cell (b) and cell spreading (d), but not the density of perinuclear invadopodia (c). Bars: means±s.e.m. (n=40 cells per condition, two experiments). For spreading, cells were treated as described in the legend of Figure 2. Projected cell areas and numbers of invadopodia were evaluated from at least 40 cells per condition from 2 experiments. *P<0.05, **P<0.01.

We then tested whether liprin-α1 influences the function of invadopodia by assessing ECM degradation. Active invadopodia can be found both in the perinuclear region and at the periphery of cells (Supplementary Figure S6). Liprin-α1 silencing evidently inhibited ECM degradation, both as percentage of cells showing matrix degradation (48% after liprin-α1 silencing versus 90% in control cells) and as the fraction of cell area overlapping with matrix degradation, which was reduced by 7.7-fold (Figure 5a). After normalization for the reduction in spreading induced by liprin-α1 silencing (Figure 5a), the fraction of basal cell area associated with ECM degradation was still reduced by fourfold, and the area of ECM degradation per invadopodium was reduced by 4.5-fold. On the other hand, liprin-α1-overexpressing cells showed a 2.6-fold increase of ECM degradation (Figure 5b). Normalization for the increase in projected cell area induced by liprin-α1 overexpression revealed a twofold increase in the average fraction of cell area associated with ECM degradation in liprin-α1-transfected cells, whereas the area of ECM degradation per invadopodium was increased by 1.6-fold. The effects of liprin-α1 levels on ECM degradation suggest a role of this protein in the regulation of invadopodia function.

Figure 5
figure5

Liprin-α1 affects ECM degradation and invadopodia dynamics. MDA-MB-231 cells transfected for 2 days with small interfering RNAs (siRNAs) (a) or 1 day with plasmids for Flag-liprin-α1 (b) were replated for 5 h (50 000 cells per coverslip) on Oregon-green 488-conjugated gelatin (Invitrogen) and 10 μg/ml fibronectin (Artym et al., 2006). An Olympus IX70 microscope (Olympus, Tokyo, Japan) was used to measure degradation, projected cell areas and number of cortactin-, filamentous actin-positive invadopodia (60 cells per condition, three experiments). (a) Depletion of liprin-α1 inhibits ECM degradation. (b) Liprin-α1 overexpression stimulates ECM degradation. Arrowhead: area enlarged in the insets (3 × ), showing the colocalization of cortactin-positive invadopodia with ECM degradation (arrows). Bars=20 μm. Graphs on the right show normalized means±s.e.m. of the area of degradation per cell (left bars), cell area (central bars) and number of invadopodia per cell (right bars) (n=60 cells per condition, three experiments). (c) Time lapse showing the distribution of DS-Red-Cortactin (Cao et al., 2003) in cells cotransfected with siRNAs and plated on fibronectin. Bar=20 μm. (d) Left: distribution of invadopodial lifespan. Center: lifespan of invadopodia±s.e.m. (n=100 invadopodia per condition). Right: liprin-α1 depletion increases the rates of invadopodial assembly and disassembly. Bars: rates±s.e.m., determined as the number of cortactin-positive dots appearing (assembly) or disappearing (disassembly) per min; n=10; 287 (control) and 398 (liprin-α1 siRNA) invadopodia were considered. For invadopodia dynamics, cells cotransfected for 18 h with DS-Red-Cortactin, siRNA and pEGFP-N1 (not shown) were replated for 1 day on fibronectin-coated coverslips (10 μg/ml) and recorded at an UltraviewERS microscope. Z-stack projections of images captured every min for 60 min were analyzed with ImageJ after background subtraction. Lifespan of invadopodia was measured as the time elapsed between the first and last frames in which they were detected. A total of 100 invadopodia from five different cells were analyzed per condition. Rates of assembly and disassembly were measured as number of invadopodia forming or disappearing per min, respectively, in 50 × 50 pixel perinuclear regions (five cells per condition, two intervals of 10 min per cell). *P<0.05, **P<0.01.

Liprin-α1 influences the dynamics of invadopodia

Time-lapse imaging on cells cotransfected with DS-Red-Cortactin and liprin-α1 small interfering RNA (Supplementary movies S3 and S4) revealed a significant decrease (30%) of the average lifespan of invadopodia compared with controls (Figures 5c and d). Both the rates of assembly and disassembly of invadopodia were markedly higher in liprin-α1-depleted cells (Figure 5d). Altogether, these findings indicate a higher turnover of invadopodia upon liprin-α1 depletion and suggest a role of this protein in the stabilization of invadopodia. The higher turnover of invadopodia may underlie the inhibitory effects of liprin-α1 silencing on matrix degradation (Figure 5) and invasion (Figure 1).

Conclusions

Our data show that liprin-α1 is required for breast and cervical cancer cell invasion, and suggest that this protein is highly expressed in human breast cancer. We have shown that the inhibitory effects of the depletion of endogenous liprin-α1 on the motility of highly invasive breast cancer cells correlates with alterations in the turnover of both lamellipodia and invadopodia, two cellular structures important for invasion. Similar effects were also observed on the motile properties of cervical cancer HeLa cells. The effects of liprin-α1 depletion on the stability of lamellipodia and invadopodia support the hypothesis of a role of liprin-α1 in the regulation of dynamic events associated with invasion.

In apparent contrast with our conclusion, liprin-α1 depletion increases invasion of head and neck squamous cell carcinoma cells (Tan et al., 2008). Therefore, a correlation between liprin-α1 expression and invasiveness cannot be general for tumors. On the other hand, it is reasonable to postulate that the positive effects of liprin-α1 in the invasion of MDA-MB-231 breast cancer cells may depend on specific liprin-related signaling networks available in these cells. We therefore would like to propose the hypothesis that the combination of liprin-α1 expression with specific sets of signaling molecules may result in the promotion of invasion by liprin-α1 in certain tumor cells, whereas different combinations may explain the observed lack of positive effects or even opposite effects of liprin-α1 in other tumor cell types. In this direction, the tumor suppressor ING4 may interact with liprin-α1, thus suppressing migration of colon carcinoma cells (Kim et al., 2004). Conversely, whereas ING4 gene deletion has been associated with the invasiveness of 10–20% of human breast cancer cell lines and tumors, no such deletion was detected in invasive MDA-MB-231 cells (Kim et al., 2004). Moreover, we have shown, herein, that other liprin-α1-interacting partners, those of the GIT/PIX complexes and LAR phosphatase receptors do not appear to be involved in the invasion of MDA-MB-231 cells. Future work is needed to identify the partner(s) mediating the effects of liprin-α1 on breast cancer cell invasion.

Our study strongly supports a role for liprin-α1 in tumor progression through its ability of regulating invasion, and provides the basis for further functional validation that may lead to the identification of novel candidates for prognosis and targeted therapies.

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Acknowledgements

Support to I de Curtis by the AIRC (Italian Association for Cancer Research, grant no. 5060) and by the Italian Telethon Foundation (grant no. GGP09078) is gratefully acknowledged. The plasmid for DsRed-Cortactin was generously provided by Mark A McNiven (Mayo Clinic, Rochester, MN, USA). We thank Cesare Covino of the Alembic facility at our Institute for his support in the morphological analysis, Rosanna Latino and Maurizio Ferrari for the cytogenetic analysis, and Jacopo Meldolesi for critical reading of the manuscript.

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Astro, V., Asperti, C., Cangi, G. et al. Liprin-α1 regulates breast cancer cell invasion by affecting cell motility, invadopodia and extracellular matrix degradation. Oncogene 30, 1841–1849 (2011). https://doi.org/10.1038/onc.2010.562

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Keywords

  • cell migration
  • invadopodia
  • invasion
  • lamellipodia
  • liprins

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