Carneiro BR et al. (2014), Acquisition of anoikis resistance up-regulates ...

ECB-ART-43783

PLoS One 2014 Dec 01;912:e116001. doi: 10.1371/journal.pone.0116001.

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Acquisition of anoikis resistance up-regulates syndecan-4 expression in endothelial cells.

Carneiro BR , Pernambuco Filho PC , Mesquita AP , da Silva DS , Pinhal MA , Nader HB , Lopes CC .

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Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding colonization of distant organs. Cell adhesion plays an important role in neoplastic transformation. Tumors produce several molecules that facilitate their proliferation, invasion and maintenance, especially proteoglycans. The syndecan-4, a heparan sulfate proteoglycan, can act as a co-receptor of growth factors and proteins of the extracellular matrix by increasing the affinity of adhesion molecules to their specific receptors. It participates together with integrins in cell adhesion at focal contacts connecting the extracellular matrix to the cytoskeleton. Changes in the expression of syndecan-4 have been observed in tumor cells, indicating its involvement in cancer. This study investigates the role of syndecan-4 in the process of anoikis and cell transformation. Endothelial cells were submitted to sequential cycles of forced anchorage impediment and distinct lineages were obtained. Anoikis-resistant endothelial cells display morphological alterations, high rate of proliferation, poor adhesion to fibronectin, laminin and collagen IV and deregulation of the cell cycle, becoming less serum-dependent. Furthermore, anoikis-resistant cell lines display a high invasive potential and a low rate of apoptosis. This is accompanied by an increase in the levels of heparan sulfate and chondroitin sulfate as well as by changes in the expression of syndecan-4 and heparanase. These results indicate that syndecan-4 plays a important role in acquisition of anoikis resistance and that the conferral of anoikis resistance may suffice to transform endothelial cells.

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Genes referenced: hpse2 LOC100889101 LOC100893907 LOC115919910 LOC590297 Sdc

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	Figure 1. Morphologic characteristics and integrin β5 expression of EC-derived cell lines.(A) Parental (EC), EJ-ras transfected endothelial cells (EJ-ras EC), and anoikis-resistant endothelial cells (Adh1−EC and Adh2−EC) were maintained in culture for 7 days and photographed under a Nikon phase contrast microscope. Bar = 100 µm. (B) Immunofluorescent analysis of F-actin and integrin β5 in EC, EJ-ras EC, Adh1−EC and Adh2−EC cells. Actin filaments were stained with Alexa Fluor-488 phalloidin (green) and integrin β5 was stained with anti-integrin β5 antibody followed by secondary Hilyte Fluor-594-labelled antibody (red). Nucleus stained with DAPI (blue). Scale bar: 50 µm. (C) The protein expression levels of integrin β5 were assessed by western blot analysis. GAPDH is shown as a protein loading control. Histogram depicting integrin β5 protein levels normalized to GAPDH. (D) Flow cytometry analysis for cell surface presentation of integrin β5 in EC, EJ-ras EC, Adh1−EC and Adh2−EC cells. Filled histogram, cells treated with anti-integrin β5 and secondary antibody; opened histogram, cells treated only with secondary antibody. In B, C and D, two independent experiments were performed in duplicates. The bars represent the standard error.
	Figure 2. Proliferation and cell cycle of EC-derived cell lines.(A) Growth curves of EC, EJ-ras transfected cells (EJ-ras EC), and EC anoikis resistant (Adh1−EC and Adh2−EC). (B) BrdU incorporation for 20 h. Serum-starved EC, EJ-ras-transfected cells (EJ-ras EC) and EC anoikis resistant (Adh1−EC and Adh2−EC) were serum-starved, as described in Methods, and stimulated to proliferate by the addition of 10% FCS. (C) Cell cycle distribution of EC, EJ-ras transfected cells (EJ-ras EC), and EC anoikis resistant (Adh1−EC and Adh2−EC). Histograms show the proportion of cells at different stages in the cell cycle (DNA content of propidium iodide–stained nuclei) analyzed by flow cytometry. ABS: absorbance. All experiments were repeated three times. The bars represent the standard error. * P≤0.05.
	Figure 3. Adhesion rate of EC-derived cell lines.(A), (B) and (C) Assay adhesion of parental (EC), EJ-ras transfected endothelial cells (EJ-ras EC), and anoikis-resistant endothelial cells (Adh1−EC and Adh2−EC) on fibronectin, laminin, and collagen IV, respectively. ABS: absorbance. The experiments were performed in triplicates and repeated three times. The bars represent the standard error. * P≤0.05.
	Figure 4. Invasion capacity and percentage of apoptosis in EC-derived cell lines.(A) Values of relative invasiveness of EJ-ras EC, Adh1−EC and Adh2−EC cells normalized with those of EC cells (100%). Invasion activity of EC-derived cell lines was analyzed by using a Transwell coated with ECL cell attachment matrix as described in Methods. (B) Percentage of apoptotic cells detected by Annexin V-FITIC/PI double staining method in EC, EJ-ras transfected cells (EJ-ras EC), and EC anoikis resistant (Adh1vEC and Adh2−EC) cells. All experiments were repeated three times. The bars represent the standard error. * P≤0.05.
	Figure 5. [35S] sulfated glycosaminoglycans synthesized by EC, EJ-ras EC, Adh1−EC and Adh2−EC cells.(A) Endothelial cells were exposed to [35S]sulfate for 18 h. The radioactive glycosaminoglycan-free chains were prepared, from both cells and conditioned medium, by incubation with proteolytic enzyme. Aliquots were subjected to electrophoresis for 60 min in 0.6% agarose (0.05 M 1,3-diaminopropane-acetate buffer pH 9.0) [38], [39]. The radioactive compounds were located in the gels, as described in Methods. CS: chondroitin sulfate; DS: dermatan sulfate; HS: heparan sulfate; OR: origin. (B) Quantification of the experiment shown in A. For further details, see Methods. The experiments were performed in duplicates and repeated three times. EC: parental endothelial cells; EJ-ras EC: EJ-ras transfected endothelial cells; Adh1−EC and Adh2−EC: anoikis-resistant endothelial cells. The bars represent the standard error. * P≤0.05.
	Figure 6. Expression of syndecan-4 and heparanase in EC, EJ-ras EC, Adh1-EC and Adh2-EC cells.(A) Representative RT-PCR products fractionated in 1% agarose gel electrophoresis and stained with ethidium bromide, showing bands of the expected sizes. (B) Expression of syndecan-4 detected by RT-qPCR. GAPDH was used as a loading control. (C) The protein expression levels of syndecan-4 were assessed by western blot analysis. GAPDH is shown as a protein loading control. Histogram depicting syndecan-4 protein levels normalized to GAPDH. (D) Flow cytometry analysis for cell surface presentation of syndecan-4 in EC, EJ-ras EC, Adh1-EC and Adh2-EC cells. Filled histogram, cells treated with anti-syndecan-4 and secondary antibody; opened histogram, cells treated only with secondary antibody. (E) Expression of heparanase detected by RT-qPCR. GAPDH was used as a loading control. In A, B and E, the experiments were repeated four times. In C and D two independent experiments were performed in duplicates. EC: parental endothelial cells; EJ-ras EC: EJ-ras transfected endothelial cells; Adh1-EC and Adh2-EC: anoikis-resistant endothelial cells. The bars represent the standard error. * P≤0.05.

References [+] :

Baciu, Protein kinase C regulates the recruitment of syndecan-4 into focal contacts. 1995, Pubmed