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Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis.

Lemjabbar-Alaoui H, van Zante A, Singer MS, Xue Q, Wang YQ, Tsay D, He B, Jablons DM, Rosen SD - Oncogene (2009)

Bottom Line: We found induction of SULF2 transcripts and Sulf-2 protein in human lung adenocarcinoma and squamous cell carcinoma, the two major classes of non-small-cell lung carcinomas (NSCLCs).We confirmed widespread Sulf-2 protein expression in tumor cells of 10/10 surgical specimens of human lung squamous carcinomas.Our findings support an essential role for Sulf-2 in lung cancer, the leading cancer killer.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of California, San Francisco, CA 94143, USA.

ABSTRACT
Heparan sulfate (HS) proteoglycans (HSPGs) bind to multiple growth factors/morphogens and regulate their signaling. 6-O-sulfation (6S) of glucosamine within HS chains is critical for many of these ligand interactions. Sulf-1 and Sulf-2, which are extracellular neutral-pH sulfatases, provide a novel post-synthetic mechanism for regulation of HSPG function by removing 6S from intact HS chains. The Sulfs can thereby modulate several signaling pathways, including the promotion of Wnt signaling. We found induction of SULF2 transcripts and Sulf-2 protein in human lung adenocarcinoma and squamous cell carcinoma, the two major classes of non-small-cell lung carcinomas (NSCLCs). We confirmed widespread Sulf-2 protein expression in tumor cells of 10/10 surgical specimens of human lung squamous carcinomas. We studied five Sulf-2(+) NSCLC cell lines, including two, which were derived by cigarette-smoke transformation of bronchial epithelial cells. shRNA-mediated Sulf-2 knockdown in these lines caused an increase in 6S on their cell surface and in parallel reversed their transformed phenotype in vitro, eliminated autocrine Wnt signaling and strongly blunted xenograft tumor formation in nude mice. Conversely, forced Sulf-2 expression in non-malignant bronchial epithelial cells produced a partially transformed phenotype. Our findings support an essential role for Sulf-2 in lung cancer, the leading cancer killer.

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Sulf-2 knockdown effect on autocrine Wnt signaling (a) H292, Calu6, and P-ST cells were transfected with TOP/FOP flash reporter system, then cultured in presence of 3 μg/ml sFRP1 or WIF-1 or medium alone. Means ± SD’s for 3 determinations of TOP/FOP activity are shown. (b) Growth of H292 and P-ST cells in the presence of sFRP1 or WIF-1 or medium alone (Control). (c) Effect of Sulf-2 knockdown on TOP/FOP flash activity. (d) Effect of Sulf-2 knockdown on localization of b-catenin in nuclear (n) and plasma membrane (m) fractions, as determined by immunoblotting. (e) Growth of Sulf-2 knockdown or control-treated cells in the presence of sFRP1 (3 μg/ml), WIF-1 (3 μg/ml) or medium alone (Control). (f) TOP/FOP flash was measured in the indicated lines after transfection with empty vector pcDNA (Control), Sulf-2 cDNA or S2ΔCC cDNA. (g) TOP/FOP flash was measured in Sulf-2 or S2ΔCC-expressing BEAS2B and H140 cells and their non transduced counterparts, as well as 3 clones (2, 7 and 9, see Fig. 5). Data shown are means ± SD’s. In all panels, * denotes p<0.05.
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Figure 6: Sulf-2 knockdown effect on autocrine Wnt signaling (a) H292, Calu6, and P-ST cells were transfected with TOP/FOP flash reporter system, then cultured in presence of 3 μg/ml sFRP1 or WIF-1 or medium alone. Means ± SD’s for 3 determinations of TOP/FOP activity are shown. (b) Growth of H292 and P-ST cells in the presence of sFRP1 or WIF-1 or medium alone (Control). (c) Effect of Sulf-2 knockdown on TOP/FOP flash activity. (d) Effect of Sulf-2 knockdown on localization of b-catenin in nuclear (n) and plasma membrane (m) fractions, as determined by immunoblotting. (e) Growth of Sulf-2 knockdown or control-treated cells in the presence of sFRP1 (3 μg/ml), WIF-1 (3 μg/ml) or medium alone (Control). (f) TOP/FOP flash was measured in the indicated lines after transfection with empty vector pcDNA (Control), Sulf-2 cDNA or S2ΔCC cDNA. (g) TOP/FOP flash was measured in Sulf-2 or S2ΔCC-expressing BEAS2B and H140 cells and their non transduced counterparts, as well as 3 clones (2, 7 and 9, see Fig. 5). Data shown are means ± SD’s. In all panels, * denotes p<0.05.

Mentions: Previous work has implicated the Sulfs as positive regulators of Wnt signaling in development (Dhoot et al., 2001; Freeman et al., 2008) and in pancreatic cancer cell lines (Nawroth et al., 2007). These enhancing effects were demonstrated for canonical Wnt signaling, which involves the translocation of β-catenin to the nucleus and its participation in a transcriptional complex (Clevers, 2006; Logan and Nusse, 2004). The analysis of Emerson and colleagues (Ai et al., 2003) strongly supports a model whereby HSPGs sequester Wnt ligands and the Sulf acts to reduce the affinity of HSPG for Wnt, freeing or altering Wnt association with HSPGs, and thus allowing Wnt access to its signal-transduction receptors on the cell surface. To determine whether the five Sulf-2+ lines exhibited canonical Wnt signaling, we used the TOP/FOP flash assay (Clevers, 2006), which quantifies β-catenin-dependent transcriptional activity. All five cell lines exhibited TOP/FOP flash activity, with a range from 4 to 13 (Supplementary Fig. 9a). To check whether the signaling was autocrine in nature (i.e., triggered by extracellular Wnts), we employed sFRP and WIF-1, which are soluble extracellular inhibitors of Wnt signaling (Kawano and Kypta, 2003). When the five cell lines were transfected with sFRP or WIF cDNA, TOP/FOP flash activity was inhibited by 50–60% (Supplementary Fig. 9b). Consistent with these results, the addition of either sFRP1 or WIF protein to the culture medium of the cells reduced TOP/FOP flash activity to a comparable extent (Fig. 6a). Furthermore, the inhibitors markedly reduced cell growth by 30–50% (Fig. 6b). We next asked whether Wnt signaling was regulated by Sulf-2. Knockdown of Sulf-2 in the five cell lines resulted in a 45–50% inhibition of TOP/FOP flash activity (Fig. 6c). In addition, Sulf-2 knockdown cells demonstrated a favored plasma membrane localization for β-catenin, whereas a nuclear localization was favored in the control cells (Fig. 6d, Supplementary Fig. 9c). Also, Sulf-2 knockdown (H292 cells) led to a 3–4 fold reduction in the expression of several Wnt target genes (Cyclin D1, Cyclin D2, Cyclin D3, JUN, and MYC), which are involved in cell growth (http://www.stanford.edu/~rnusse/wntwindow.html) (Supplementary Table 3). Importantly, the addition sFRP1 or WIF-1 to the culture medium of knockdown cells produced no further inhibition of Wnt signaling (Supplementary Fig. 9d) or cell growth (Fig. 6e), consistent with the model in which Sulf-2 directly regulates the mobilization of Wnt ligands from HSPG sequestration (Ai et al., 2003). It is conceivable that Sulf-2 knockdown could also have indirect effects on Wnt signaling as there was a ≈2-fold reduction in the expression of two WNT genes among the six WNTs expressed in H292 cells (Supplementary Table 3).


Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis.

Lemjabbar-Alaoui H, van Zante A, Singer MS, Xue Q, Wang YQ, Tsay D, He B, Jablons DM, Rosen SD - Oncogene (2009)

Sulf-2 knockdown effect on autocrine Wnt signaling (a) H292, Calu6, and P-ST cells were transfected with TOP/FOP flash reporter system, then cultured in presence of 3 μg/ml sFRP1 or WIF-1 or medium alone. Means ± SD’s for 3 determinations of TOP/FOP activity are shown. (b) Growth of H292 and P-ST cells in the presence of sFRP1 or WIF-1 or medium alone (Control). (c) Effect of Sulf-2 knockdown on TOP/FOP flash activity. (d) Effect of Sulf-2 knockdown on localization of b-catenin in nuclear (n) and plasma membrane (m) fractions, as determined by immunoblotting. (e) Growth of Sulf-2 knockdown or control-treated cells in the presence of sFRP1 (3 μg/ml), WIF-1 (3 μg/ml) or medium alone (Control). (f) TOP/FOP flash was measured in the indicated lines after transfection with empty vector pcDNA (Control), Sulf-2 cDNA or S2ΔCC cDNA. (g) TOP/FOP flash was measured in Sulf-2 or S2ΔCC-expressing BEAS2B and H140 cells and their non transduced counterparts, as well as 3 clones (2, 7 and 9, see Fig. 5). Data shown are means ± SD’s. In all panels, * denotes p<0.05.
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Related In: Results  -  Collection

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Figure 6: Sulf-2 knockdown effect on autocrine Wnt signaling (a) H292, Calu6, and P-ST cells were transfected with TOP/FOP flash reporter system, then cultured in presence of 3 μg/ml sFRP1 or WIF-1 or medium alone. Means ± SD’s for 3 determinations of TOP/FOP activity are shown. (b) Growth of H292 and P-ST cells in the presence of sFRP1 or WIF-1 or medium alone (Control). (c) Effect of Sulf-2 knockdown on TOP/FOP flash activity. (d) Effect of Sulf-2 knockdown on localization of b-catenin in nuclear (n) and plasma membrane (m) fractions, as determined by immunoblotting. (e) Growth of Sulf-2 knockdown or control-treated cells in the presence of sFRP1 (3 μg/ml), WIF-1 (3 μg/ml) or medium alone (Control). (f) TOP/FOP flash was measured in the indicated lines after transfection with empty vector pcDNA (Control), Sulf-2 cDNA or S2ΔCC cDNA. (g) TOP/FOP flash was measured in Sulf-2 or S2ΔCC-expressing BEAS2B and H140 cells and their non transduced counterparts, as well as 3 clones (2, 7 and 9, see Fig. 5). Data shown are means ± SD’s. In all panels, * denotes p<0.05.
Mentions: Previous work has implicated the Sulfs as positive regulators of Wnt signaling in development (Dhoot et al., 2001; Freeman et al., 2008) and in pancreatic cancer cell lines (Nawroth et al., 2007). These enhancing effects were demonstrated for canonical Wnt signaling, which involves the translocation of β-catenin to the nucleus and its participation in a transcriptional complex (Clevers, 2006; Logan and Nusse, 2004). The analysis of Emerson and colleagues (Ai et al., 2003) strongly supports a model whereby HSPGs sequester Wnt ligands and the Sulf acts to reduce the affinity of HSPG for Wnt, freeing or altering Wnt association with HSPGs, and thus allowing Wnt access to its signal-transduction receptors on the cell surface. To determine whether the five Sulf-2+ lines exhibited canonical Wnt signaling, we used the TOP/FOP flash assay (Clevers, 2006), which quantifies β-catenin-dependent transcriptional activity. All five cell lines exhibited TOP/FOP flash activity, with a range from 4 to 13 (Supplementary Fig. 9a). To check whether the signaling was autocrine in nature (i.e., triggered by extracellular Wnts), we employed sFRP and WIF-1, which are soluble extracellular inhibitors of Wnt signaling (Kawano and Kypta, 2003). When the five cell lines were transfected with sFRP or WIF cDNA, TOP/FOP flash activity was inhibited by 50–60% (Supplementary Fig. 9b). Consistent with these results, the addition of either sFRP1 or WIF protein to the culture medium of the cells reduced TOP/FOP flash activity to a comparable extent (Fig. 6a). Furthermore, the inhibitors markedly reduced cell growth by 30–50% (Fig. 6b). We next asked whether Wnt signaling was regulated by Sulf-2. Knockdown of Sulf-2 in the five cell lines resulted in a 45–50% inhibition of TOP/FOP flash activity (Fig. 6c). In addition, Sulf-2 knockdown cells demonstrated a favored plasma membrane localization for β-catenin, whereas a nuclear localization was favored in the control cells (Fig. 6d, Supplementary Fig. 9c). Also, Sulf-2 knockdown (H292 cells) led to a 3–4 fold reduction in the expression of several Wnt target genes (Cyclin D1, Cyclin D2, Cyclin D3, JUN, and MYC), which are involved in cell growth (http://www.stanford.edu/~rnusse/wntwindow.html) (Supplementary Table 3). Importantly, the addition sFRP1 or WIF-1 to the culture medium of knockdown cells produced no further inhibition of Wnt signaling (Supplementary Fig. 9d) or cell growth (Fig. 6e), consistent with the model in which Sulf-2 directly regulates the mobilization of Wnt ligands from HSPG sequestration (Ai et al., 2003). It is conceivable that Sulf-2 knockdown could also have indirect effects on Wnt signaling as there was a ≈2-fold reduction in the expression of two WNT genes among the six WNTs expressed in H292 cells (Supplementary Table 3).

Bottom Line: We found induction of SULF2 transcripts and Sulf-2 protein in human lung adenocarcinoma and squamous cell carcinoma, the two major classes of non-small-cell lung carcinomas (NSCLCs).We confirmed widespread Sulf-2 protein expression in tumor cells of 10/10 surgical specimens of human lung squamous carcinomas.Our findings support an essential role for Sulf-2 in lung cancer, the leading cancer killer.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of California, San Francisco, CA 94143, USA.

ABSTRACT
Heparan sulfate (HS) proteoglycans (HSPGs) bind to multiple growth factors/morphogens and regulate their signaling. 6-O-sulfation (6S) of glucosamine within HS chains is critical for many of these ligand interactions. Sulf-1 and Sulf-2, which are extracellular neutral-pH sulfatases, provide a novel post-synthetic mechanism for regulation of HSPG function by removing 6S from intact HS chains. The Sulfs can thereby modulate several signaling pathways, including the promotion of Wnt signaling. We found induction of SULF2 transcripts and Sulf-2 protein in human lung adenocarcinoma and squamous cell carcinoma, the two major classes of non-small-cell lung carcinomas (NSCLCs). We confirmed widespread Sulf-2 protein expression in tumor cells of 10/10 surgical specimens of human lung squamous carcinomas. We studied five Sulf-2(+) NSCLC cell lines, including two, which were derived by cigarette-smoke transformation of bronchial epithelial cells. shRNA-mediated Sulf-2 knockdown in these lines caused an increase in 6S on their cell surface and in parallel reversed their transformed phenotype in vitro, eliminated autocrine Wnt signaling and strongly blunted xenograft tumor formation in nude mice. Conversely, forced Sulf-2 expression in non-malignant bronchial epithelial cells produced a partially transformed phenotype. Our findings support an essential role for Sulf-2 in lung cancer, the leading cancer killer.

Show MeSH
Related in: MedlinePlus