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.
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).
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.
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.
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).
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.
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).
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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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.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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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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