The Pro-Tumoral Activity of Heparan Sulfate 3-O-Sulfotransferase 3B (HS3ST3B) in Breast Cancer MDA-MB-231 Cells Is Dependent on the Expression of Neuropilin-1

Heparan sulfate 3-O-sulfotransferases (HS3STs) catalyze the maturation step of heparan sulfate (HS) 3-O-sulfation. This modification is relatively rare. Moreover, only a few biological processes have been described to be influenced by 3-O-sulfated HS, and few ligands have been identified so far. Among them, neuropilin-1 (Nrp1) was reported to exhibit tumor-promoting properties by enhancing the action of various growth factors. We recently demonstrated that transient overexpression of HS3ST2, 3B or 4 enhanced the proliferation of breast cancer MDA-MB-231 cells and promote efficient protection against pro-apoptotic stimuli. Hence, we hypothesized that the pro-tumoral activity of these HS3STs could depend on the expression of Nrp1. To test this, MDA-MB-231 cells were stably transfected with a construct encoding HS3ST3B and the expression of Nrp1 was down-regulated by RNA interference. First, we confirmed that stable expression of HS3ST3B effectively increased cell proliferation and viability. Silencing the expression of Nrp1 markedly attenuated the promoting effects of HS3ST3B, while the same treatment had only a moderate effect on the behavior of the parental cells. Altogether, our findings support the idea that the tumor-promoting effects of HS3ST3B could be dependent on the expression of Nrp1 in cancer cells

Heparan sulfate 3-O-sulfotransferases (HS3STs) involvement in cellular processes associated with cancer

Les héparanes sulfates (HS) sont des polysaccharides linéaires dont l’état de sulfatation détermine les propriétés biologiques. La dernière étape de leur maturation est catalysée par les HS 3-O-sulfotransférases (HS3STs), qui sont représentées par sept isoenzymes. La fonction de ces enzymes dans la progression tumorale reste controversée. C’est dans ce contexte que nous avons étudié leur rôle en utilisant la lignée de cancer du sein MDA-MB-231.Nous avons montré que l’expression transitoire des HS3ST2, 3B et 4 augmente la prolifération et la survie des cellules, ce qui est associé à une suractivation de Src et Akt. En complément, nous avons observé une augmentation de l’activation de la voie NF-κB et de l’expression de protéines anti-apoptotiques. Ces réponses ont été corrélées à une résistance accrue des cellules transfectées à la mort induite par des stimuli pro-apoptotiques ou par les cellules NK. Ces premiers résultats suggèrent que les HS 3-O-sulfatés pourraient avoir un rôle protecteur contre le système immunitaire. La neuropiline-1 a récemment été décrite comme un ligand des HS 3-O-sulfatés. Nous avons donc poursuivi notre étude en analysant le rôle de ce co-récepteur dans des cellules exprimant stablement la HS3ST3B. L’invalidation d’expression de la neuropiline-1 réduit la prolifération et la survie des cellules transfectées, ainsi que l’activation de Src et Akt. Ces derniers résultats suggèrent que les propriétés pro-tumorales des HS modifiés par la HS3ST3B sont dépendantes de leur interaction avec la neuropiline-1. Dans leur ensemble, nos travaux suggèrent que l’expression de certaines HS3STs dans les cellules cancéreuses pourrait être de mauvais pronostic.Heparan sulfate (HS) is a linear polysaccharide in which the sulfation pattern determines the biological properties. The last step of HS maturation is catalyzed by the HS 3-O-sulfotransferases (HS3ST), which are represented by seven isozymes. The functions of HS3STs in cancer progression are still controversial. In this context, we focused our investigations on the roles of these enzymes in MDA-MB-231 breast cancer cells. First, we found that transient expression of HS3ST2, 3B and 4 enhanced their proliferation and survival. It turns out that these effects are related to an increase in the activation of Src and Akt. Complementary to this, we observed an increase in the activation of the NF-κB pathway and the expression of anti-apoptotic proteins. In line with these findings, we showed that HS3ST-transfected cells were more resistant to cell death induced by pro-apoptotic stimuli or NK cells. These results suggest that, in addition to increasing cellular growth, 3-O-sulfated HS can also protect cancer cells against the immune system. Neuropilin-1 was recently described as a preferential ligand of 3-O-sulfated HS. Thus, we investigated the role of this co-receptor in MDA-MB-231 cells that carry a stable expression of HS3ST3B. We demonstrated that silencing neuropilin-1 resulted in reduced proliferation and survival, which was associated with a strong decrease in Src and Akt activation. These last results support a model in which HS3ST-modified HS may display tumor-promoting functions through the interaction with neuropilin-1. Overall, our findings suggest that the expression of certain HS3STs in cancer cells could be associated with a bad prognosis

The Pro-Tumoral Activity of Heparan Sulfate 3-O-Sulfotransferase 3B (HS3ST3B) in Breast Cancer MDA-MB-231 Cells Is Dependent on the Expression of Neuropilin-1

Heparan sulfate 3-O-sulfotransferases (HS3STs) catalyze the maturation step of heparan sulfate (HS) 3-O-sulfation. This modification is relatively rare. Moreover, only a few biological processes have been described to be influenced by 3-O-sulfated HS, and few ligands have been identified so far. Among them, neuropilin-1 (Nrp1) was reported to exhibit tumor-promoting properties by enhancing the action of various growth factors. We recently demonstrated that transient overexpression of HS3ST2, 3B or 4 enhanced the proliferation of breast cancer MDA-MB-231 cells and promote efficient protection against pro-apoptotic stimuli. Hence, we hypothesized that the pro-tumoral activity of these HS3STs could depend on the expression of Nrp1. To test this, MDA-MB-231 cells were stably transfected with a construct encoding HS3ST3B and the expression of Nrp1 was down-regulated by RNA interference. First, we confirmed that stable expression of HS3ST3B effectively increased cell proliferation and viability. Silencing the expression of Nrp1 markedly attenuated the promoting effects of HS3ST3B, while the same treatment had only a moderate effect on the behavior of the parental cells. Altogether, our findings support the idea that the tumor-promoting effects of HS3ST3B could be dependent on the expression of Nrp1 in cancer cells

Protective effects of the expression of HS3ST2, HS3ST3B and HS3ST4 against apoptosis.

<p>Transfected MDA-MB-231 cells were treated with staurosporin (1 μM) for 5 h or with a mixture of anti-Fas antibody (100 ng/mL) and TNF-α (100 ng/mL) for 16 h. (<b>A</b>) The percentage of apoptotic cell population was evaluated by analyzing the binding of fluorescein-conjugated annexin-V. Each bar of histogram represents mean ± S.D. of the rate of apoptotic cells by comparison with untreated control cells. Data were obtained from three distinct experiments. (<b>B</b>) In parallel experiments, the activation of caspase-3 was analysed in HS3ST-overexpressing cells and in control cells after exposure to either staurosporin or to the mixture of anti-Fas antibody plus TNF-α. Results are expressed as fold increase in caspase-3 activity by comparison with untreated control cells and correspond to means ± S.D. from three independent experiments (*<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, significantly different when compared to control cells; <i>n</i>.<i>s</i>., not significantly different).</p

The heparan sulfate 3-<i>O</i>-sulfotransferases (HS3ST) 2, 3B and 4 enhance proliferation and survival in breast cancer MDA-MB-231 cells

<div><p>Heparan sulfate 3-<i>O</i>-sulfotransferases (HS3STs) catalyze the final maturation step of heparan sulfates. Although seven HS3ST isozymes have been described in human, 3-<i>O</i>-sulfation is a relatively rare modification, and only a few biological processes have been described to be influenced by 3-<i>O</i>-sulfated motifs. A conflicting literature has recently reported that HS3ST2, 3A, 3B and 4 may exhibit either tumor-promoting or anti-oncogenic properties, depending on the model used and cancer cell phenotype. Hence, we decided to compare the consequences of the overexpression of each of these HS3STs in the same cellular model. We demonstrated that, unlike HS3ST3A, the other three isozymes enhanced the proliferation of breast cancer MDA-MB-231 and BT-20 cells. Moreover, the colony forming capacity of MDA-MB-231 cells was markedly increased by the expression of HS3ST2, 3B and 4. No notable difference was observed between the three isozymes, meaning that the modifications catalyzed by each HS3ST had the same functional impact on cell behavior. We then demonstrated that overexpression of HS3ST2, 3B and 4 was accompanied by increased activation of c-Src, Akt and NF-κB and up-regulation of the anti-apoptotic proteins survivin and XIAP. In line with these findings, we showed that HS3ST-transfected cells are more resistant to cell death induction by pro-apoptotic stimuli or NK cells. Altogether, our findings demonstrate that HS3ST2, 3B and 4 share the same pro-tumoral activity and support the idea that these HS3STs could compensate each other for loss of their expression depending on the molecular signature of cancer cells and/or changes in the tumor environment.</p></div

Effects of the expression of HS3ST2, HS3ST3B and HS3ST4 on colony formation in MDA-MB-231 cells.

<p>(<b>A</b>) Equal numbers of control and HS3ST-overexpressing cells (2000 per well) were seeded in six well plates and maintained for nine days in DMEM complemented with 1% FCS to form colonies. Fresh complete growth medium was then added for three days, after which the colonies were stained with crystal violet. The right panel (<b>B</b>) represents the quantification of the colonies per well. Results are expressed as fold changes by comparison with control cells transfected with empty vector. Data are means ± S.D. from five separate experiments performed independently (***<i>P</i> < 0.001, significantly different when compared to control cells).</p

Effects of the expression of HS3ST2, HS3ST3A, HS3ST3B and HS3ST4 on breast cancer cell growth.

<p>MDA-MB-231 and BT-20 cells were transfected with overexpression vectors and cultured in medium containing 1% FCS for 24 h and 48 h. At each time, the effects of HS3STs on the growth of MDA-MB-231 and BT-20 cells was estimated by cell counting (<b>A</b>) and MTS assay (<b>B</b>). Results are expressed as fold changes by comparison with control cells transfected with empty vector. Data are means ± S.D. from three separate experiments performed independently (*<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, significantly different when compared to control cells).</p

Overexpression of gD-type HS3STs in MDA-MB-231 cells.

<p>Cells were transiently transfected with empty vector (control) or vectors encoding HS3ST2, HS3ST3A, HS3ST3B or HS3ST4, and then cultured for 24 h and 48 h prior analysis of the expression and activity of each isozyme. (<b>A</b>) Following RNA extraction, the mRNA levels of HS3STs were quantified by real-time RT-PCR at 24 h post-transfection. Relative abundance of the transcripts was normalized to endogenous HPRT mRNA. Data are means ± SD from at least three distinct experiments. (<b>B</b>) In parallel experiments, proteins from cell lysates were separated by SDS-PAGE and subjected to Western blotting with appropriate specific antibodies. Parallel immunoblotting with antibodies to GAPDH confirmed equal loading of the samples. (<b>C</b>) Efficiency in the reaction of heparan 3-<i>O</i>-sulfation was confirmed 48 h post-transfection by using HS4C3 antibody. Cell surface fluorescent staining was detected by flow cytometry. Filled histograms represent the staining obtained with HS4C3 antibody, while open histograms correspond to the binding of the irrelevant antibody MBP49. Each panel represents the binding of the antibodies to the surface of HS3ST-expressing cells (black histograms) compared to the binding of the same antibodies to control cells (grey histograms). Data are representative of three separate experiments. (<b>D</b>) Compositional disaccharide analysis of HS from transfected MDA-MB-231 cells. Following depolymerization of HS with a mixture of heparinases, disaccharides were labelled with AMAC and resolved by RP-HPLC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194676#pone.0194676.s001" target="_blank">S1 Fig</a>). Data are expressed as frequency of each disaccharide and correspond to means ± S.D. from two sets of triplicate injections performed with distinct cell preparations (**<i>P</i> < 0.01, ***<i>P</i> < 0.001, significantly different when compared to control cells).</p

Effects of the expression of HS3STs on NK-mediated cell death.

<p>Control or HS3ST-overexpressing MDA-MB-231 cells were cultured for 32 h and then exposed to activated NK cells for 5 h at the different ratios target/effector 1:1, 1:5 and 1:10. NK-mediated cytotoxicity was estimated by using a LDH assay, as described in “Materials and Methods”. Data are means ± S.D. from three separate experiments performed with NK cells from distinct donors (*<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, significantly different when compared to control cells; <i>n</i>.<i>s</i>., not significantly different).</p

Effect of the expression of HS3ST2, HS3ST3B and HS3ST4 on cell signaling.

<p>Twenty hours post transfection, MDA-MB-231 cells were serum-starved for 3 hours, collected and lysed. Proteins were then separated by SDS-PAGE and subjected to Western blotting with antibodies to I-κB, survivin, XIAP and to the phosphorylated forms of c-Src, Akt, ERK1/2, STAT3 and NF-κB p65 subunit. Parallel immunoblotting with antibodies to GAPDH and to c-Src, Akt, ERK1/2, STAT3 and NF-κB p65 regardless of their phosphorylation status confirmed equal loading of samples. Histograms represent the quantification of the phosphorylation status of c-Src, Akt, ERK1/2, STAT3, NF-κB p65 and of the expression of I-κB, survivin and XIAP related to GAPDH. Data were normalized to control cells. Representative results from three independent experiments are shown.</p