- Research article
- Open Access
- Open Peer Review
Hakai reduces cell-substratum adhesion and increases epithelial cell invasion
© Rodríguez-Rigueiro et al; licensee BioMed Central Ltd. 2011
- Received: 12 August 2011
- Accepted: 3 November 2011
- Published: 3 November 2011
The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue formation, differentiation and motility. Adherens junctions are important components of the junctional complex between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in cell-substratum adhesions and invasion in epithelial cells.
Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible manner was employed.
Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-overexpressing MDCK cells. The expression of Paxillin was found to be regulated by a proteasome-independent mechanism, possibly due to the decreased abundance of E-cadherin.
Taken together, these results suggest that Hakai may be involved in two hallmark aspects of tumour progression, the lowering cell-substratum adhesion and the enhancement of cell invasion.
- Focal Adhesion
- MDCK Cell
- Focal Contact
The most frequent types of tumours are carcinomas, which develop through the transformation of epithelial cells. Epithelial cells are polarized and are often connected with each other via cell-cell adhesions to form structured cells sheets. The formation of these tight and compact cell-cell adhesions restricts cell movement within the epithelium. Epithelial cells are also attached to an extracellular matrix substratum which is essential for their differentiation and polarization . During transformation, epithelial cells start to proliferate, acquire the ability to migrate, and lose both the intercellular adhesion, mediated by cadherins at adherens junctions, and the interactions with the extracellular matrix. All of these characteristics facilitate the invasion and metastasis of epithelial cells .
Hakai was firstly described as a RING finger-type E3 ubiquitin-ligase for the E-cadherin complex . E-cadherin is the prototypical and best characterized member of the cadherins at adherens junctions and its loss is in carcinoma associated with poor prognosis . Hakai binds to the cytoplasmic domain of E-cadherin and mediates its ubiquitination, endocytosis and degradation [3, 5]. In consequence, it leads to the disruption of epithelial cell-cell adhesions, inducing epithelial-mesenchymal transition (EMT), a key event in epithelial transformation [6, 7]. Apart from this functional role, we recently reported that Hakai is not only implicated in lowering cell-cell contacts, but can also promote proliferation in an E-cadherin-independent manner. Moreover, Hakai overexpression induces anchorage-independent cell growth, produces protrusions that are dynamically extended and retracted, and increases the ability of the RNA-binding protein PSF to bind to mRNAs that encode cancer-related proteins [8, 9].
Given the role of Hakai in tumorigenesis, we are interested to examine the possible implication of Hakai in the regulation of adhesions to the extracellular matrix (ECM) and invasion in epithelial cells, two hallmark processes in cancer and metastasis [10, 11]. To investigate this possibility, we studied Hakai's influence on cell attachment to the substrate and invasion capacity of Madin-Darby canine kidney (MDCK) cells. We report that in this system, Hakai overexpression leads to a reduction in cell adhesion to the substrate with impact on the key focal adhesion-associated protein Paxillin, and increases cell invasion. Our data suggest that Hakai may have an important functional role during oncogenesis through its implication on cell adhesion to the substrate and cell invasion.
Antibodies and materials
The rabbit polyclonal anti-Hakai antibody (Hakai-2498) was kindly provided by Yasuyuki Fujita . Anti-FAK antibody was from Upstate (Charlottesville, VA), anti-Paxillin antibody was from Transduction Laboratories, anti-Vinculin was from Chemicon International (Hampshire, UK), antibody to the extracellular portion of E-cadherin (ECCD-2) was from Zymed (South San Francisco, CA), anti-α-Tubulin antibody was from Immunologicals Direct (Oxford Biotechnology Ltd., UK), HRP-rabbit and mouse polyclonal antibodies was from GE Healthcare (UK) and Alexa-Fluor 488 mouse antibody and Alexa-mouse 568 were from Molecular Probes (Paisley, UK). All primary antibodies were used at dilutions of 1:1000 for Western blotting and 1:100 for immunofluorescence, except for anti-FAK antibody that was used at a dilution of 1:5000 for Western blotting and 1:500 for immunofluorescence. Secondary antibodies were used at dilutions 1:10000 for HRP-rabbit, 1:5000 for HRP-mouse antibody, and 1:200 for Alexa-Fluor antibodies. pcDNA-HA-Hakai was kindly provided by Walter Birchmeier . MG132 and lactacystin were obtained from Calbiochem (Darmstadt, Germany).
MDCK cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing penicillin/streptomycin and 10% FCS at 37°C and ambient air supplemented with 5% CO2. MDCK cells stably expressing Hakai were kindly provided by Yasuyuki Fujita . MDCK cells were transfected with pcDNA-HA-Hakai using Lipofectamine-Plus™ reagent (Invitrogen) and were selected in a medium containing 800 μg ml-1 of G418 (Calbiochem). More than ten stable clones were obtained from two independent transfections. All clones, including 4, 6, 7 and 8 clones, represented comparable phenotypes and the results shown were obtained using clone 4, unless otherwise indicated. MDCK cells stably expressing E-cadherin shRNA in a tetracycline-inducible manner were kindly provided by Yasuyuki Fujita .
For the adhesion assay, 1 × 105 cells were plated in a 6-well plate using human fibronectin-coated coverslips (BD Bioscience, Belgium). After 24 hours, the cells were vigorously washed five times with phosphate-buffered saline (PBS), and the remaining cells attached to the plate were analyzed by phase contrast images using a Leica DMIRB microscope with a Hamamatsu C4742-95 Orca camera.
Cells cultured in 6-well plate dishes were lysed for 30 min in 0.3 ml of 1% Triton X-100 lysis buffer (20 mM Tris-HCL [pH 7.5], 150 mM NaCl, and 1% Triton X-100) containing 5 μg ml-1 lupeptin, 50 mM phenylmethylsulfonyl fluoride, and 7.2 trypsin inhibitor units for aprotinin. After centrifugation at 14, 000 × g for 10 min, thirty micrograms of the supernatants were loaded in 10% polyacrilamide SDS-PAGE. Western blotting was performed as previously described .
For immunofluorescence, human fibronectin-coated coverslips (BD Bioscience, Belgium) were used. Cells on coverslips were washed twice in PBS and incubated in 3% parafolmaldehyde-PBS for 15 min. After washing twice in PBS, cells were permeabilized in 0.5% Triton X-100-PBS for 15 min, followed by blocking in DMEM containing 10% fetal bovine serum for 1 h. They were further incubated in primary antibodies for 1 h, washed in PBS, incubated in Alexa-Fluor 488 or Alexa-Fluor 568-conjugated secondary antibody solution for 1 h, and washed four times in PBS. The coverslip was the mounted onto Mowiol on a glass slide. Immunofluorescent images were analyzed by epifluorescence microscopy. To obtain epifluorescence images, we used a Zeiss Axioskop 1 with a Roper Scientific Coolsnap camera.
For invasion assays, 5 × 104 cells were plated in DMEM/1% FCS in a cell invasion chamber (Cell invasion assay kit, Chemicon International) in a 24-well plate, which contains an 8-μm pore size polycarbonate membrane covered with a thin layer of collagen matrix. Invasive cells migrate through a membrane according to the gradient of FCS to the lower chamber that contains DMEM/10% FCS. The invaded cells were stained with crystal violet, and the absorbance of stained cells was measured in a 565-nm wavelength.
Student t test assuming unequal variance was performed for statistical analysis.
Hakai overexpression reduces cell-substratum adhesion
Paxillin down-regulation is controlled by a proteasome-independent mechanism and results from the decreased expression of E-cadherin
However, several lines of evidence suggest that Hakai can also affect cellular phenotypes in an E-cadherin-independent manner. First, MDCK cells expressing E-cadherin shRNA do not extend spiky protrusions that are seen in Hakai-overexpressing cells . Second, in Hakai-overexpressing MDCK cells, Hakai is localized at the end of the protrusion where FAK is also enriched , suggesting that Hakai may be involved in regulating the dynamic extension and retraction of these structures and in influencing cell motility and cell attachment to the ECM. Finally, Hakai is also located in the nucleus, where it can interact with nuclear proteins such as PSF and modulate cancer-related gene expression  and it may act as a correpresor of the estrogen receptor in breast cancer cells . These findings suggest that Hakai can have ubiquitin-independent functions that may influence the cell phenotype, in addition to its influence on known substrates (like E-cadherin) and as-yet unreported substrates in the nucleus or the cytoplasm, as previously reported for other E3-ubiquitin ligases [17, 18].
Hakai overexpression enhances invasiveness
In summary, we present evidences that indicate that Hakai is implicated in the regulation of cell-substratum adhesions and in epithelial cell invasion. We have shown that the overexpression of Hakai in epithelial cells reduces Paxillin expression level and it disappears from focal adhesions and focal contacts. We demonstrated that this Paxillin reduction can not be recovered by using two important proteasome inhibitors, such as lactacystin and MG132, further supporting that Paxillin down-regulation is controlled by a proteasome-independent mechanism. Furthermore, we have shown that paxillin reduction may, at least partially, result from decreased expression level of E-cadherin. All these data suggest the implication of Hakai in controlling several important cellular processes that are crucial during tumour progression.
We thank Myriam Gorospe for critical reading the manuscript. We thank Yasuyuki Fujita for helpful discussions and for providing reagents. T.R.R and A.F. are recipients of a grant from the Secretaria Xeral I+D+I, Xunta de Galicia, Spain (10CSA916023PR and IPP.08-07) and M.H.C is supported by the Instituto de Salud Carlos III, Spain (CA09/00116). This work is supported by a grant from the Conselleria de Sanidade (PS09/24) and from the Secretaria Xeral I+D+I, (10CSA916023PR), both from Xunta de Galicia, Spain.
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