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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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Effects of Sulf-2 knockdown in lung cancer cell lines: (a) Cells were transduced with Sulf-2-specific (PLV-1413, PLV-1143) or control (PLV-Ctrl) shRNAs. Immunoblotting for Sulf-2 in CM (Upper) and cell lysates (Middle) with a loading control of b-actin (Lower). (b) Effects of Sulf-2 knockdown on the cell surface expression of the RB4CD12 epitope in 6 cell lines, one of which is negative for Sulf-2 (H1975). Mock-knockdown (PLV-Ctrl) (2) or Sulf-2-knockdown (PLV-1413) (3) cells were stained with RB4CD12 or with an irrelevant antibody as control (1). (c). Cell growth was monitored for 6 days using CellTiter-Blue. (d) Cell proliferation in mock- or Sulf-2 knockdown H292 and P-ST cells, as measured by BrdU incorporation. (e) Effect of Sulf-2 knockdown on cell apoptosis as determined by staining with APC conjugated-Annexin V. (f) Wound-healing migration assay for mock-knockdown, Sulf-2 knockdown or non-transduced cells. Healing of scratch wounds was measured at the indicated times. Scale bar = 120μm. (g) The formation of colonies in soft agar by Sulf-2 knockdown cells and control cells after 21 days of culture. The values shown in graphs are means ± SDs. (* indicates p< 0.05 relative to controls).
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Figure 3: Effects of Sulf-2 knockdown in lung cancer cell lines: (a) Cells were transduced with Sulf-2-specific (PLV-1413, PLV-1143) or control (PLV-Ctrl) shRNAs. Immunoblotting for Sulf-2 in CM (Upper) and cell lysates (Middle) with a loading control of b-actin (Lower). (b) Effects of Sulf-2 knockdown on the cell surface expression of the RB4CD12 epitope in 6 cell lines, one of which is negative for Sulf-2 (H1975). Mock-knockdown (PLV-Ctrl) (2) or Sulf-2-knockdown (PLV-1413) (3) cells were stained with RB4CD12 or with an irrelevant antibody as control (1). (c). Cell growth was monitored for 6 days using CellTiter-Blue. (d) Cell proliferation in mock- or Sulf-2 knockdown H292 and P-ST cells, as measured by BrdU incorporation. (e) Effect of Sulf-2 knockdown on cell apoptosis as determined by staining with APC conjugated-Annexin V. (f) Wound-healing migration assay for mock-knockdown, Sulf-2 knockdown or non-transduced cells. Healing of scratch wounds was measured at the indicated times. Scale bar = 120μm. (g) The formation of colonies in soft agar by Sulf-2 knockdown cells and control cells after 21 days of culture. The values shown in graphs are means ± SDs. (* indicates p< 0.05 relative to controls).

Mentions: We employed a previously developed lentiviral shRNA strategy (Nawroth et al., 2007) to achieve knockdown of Sulf-2 in NSCLC lines. We transduced five of the Sulf-2+ lines (H460, H292, Calu-6, P-ST and B-ST cells) with either a specific shRNA (PLV-1413 or PLV-1143) (Nawroth et al., 2007) or a control shRNA (PLV-Ctrl) containing an irrelevant sequence. Sulf-2 protein expression was strongly reduced (>90%) in all of the lines after transduction with PLV-1413 or PLV-1143, as compared to the control cells (Fig. 3a). To determine the effects of Sulf-2 knockdown on the sulfation status of HSPGs, we used a phage-display antibody (RB4CD12), whose binding to HSPGs depends on 6S (Dennissen et al., 2002). We have verified that this antibody can be used as a cell surface-staining reagent to report the presence of the Sulfs within cells (M. Hossain, T. Hosono, R. Tang, T. H. van Kuppevelt, G. J. Jenniskens, S. D. Rosen, and K. Uchimura, manuscript submitted). Transduction of Sulf-2+ lines with PLV-1413 increased the RB4CD12 epitope on the cell surface relative to that of control-treated cells by 30–37% (Fig. 3b). The same treatment had no effect on the epitope in a Sulf-2− line (H1975).


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)

Effects of Sulf-2 knockdown in lung cancer cell lines: (a) Cells were transduced with Sulf-2-specific (PLV-1413, PLV-1143) or control (PLV-Ctrl) shRNAs. Immunoblotting for Sulf-2 in CM (Upper) and cell lysates (Middle) with a loading control of b-actin (Lower). (b) Effects of Sulf-2 knockdown on the cell surface expression of the RB4CD12 epitope in 6 cell lines, one of which is negative for Sulf-2 (H1975). Mock-knockdown (PLV-Ctrl) (2) or Sulf-2-knockdown (PLV-1413) (3) cells were stained with RB4CD12 or with an irrelevant antibody as control (1). (c). Cell growth was monitored for 6 days using CellTiter-Blue. (d) Cell proliferation in mock- or Sulf-2 knockdown H292 and P-ST cells, as measured by BrdU incorporation. (e) Effect of Sulf-2 knockdown on cell apoptosis as determined by staining with APC conjugated-Annexin V. (f) Wound-healing migration assay for mock-knockdown, Sulf-2 knockdown or non-transduced cells. Healing of scratch wounds was measured at the indicated times. Scale bar = 120μm. (g) The formation of colonies in soft agar by Sulf-2 knockdown cells and control cells after 21 days of culture. The values shown in graphs are means ± SDs. (* indicates p< 0.05 relative to controls).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Effects of Sulf-2 knockdown in lung cancer cell lines: (a) Cells were transduced with Sulf-2-specific (PLV-1413, PLV-1143) or control (PLV-Ctrl) shRNAs. Immunoblotting for Sulf-2 in CM (Upper) and cell lysates (Middle) with a loading control of b-actin (Lower). (b) Effects of Sulf-2 knockdown on the cell surface expression of the RB4CD12 epitope in 6 cell lines, one of which is negative for Sulf-2 (H1975). Mock-knockdown (PLV-Ctrl) (2) or Sulf-2-knockdown (PLV-1413) (3) cells were stained with RB4CD12 or with an irrelevant antibody as control (1). (c). Cell growth was monitored for 6 days using CellTiter-Blue. (d) Cell proliferation in mock- or Sulf-2 knockdown H292 and P-ST cells, as measured by BrdU incorporation. (e) Effect of Sulf-2 knockdown on cell apoptosis as determined by staining with APC conjugated-Annexin V. (f) Wound-healing migration assay for mock-knockdown, Sulf-2 knockdown or non-transduced cells. Healing of scratch wounds was measured at the indicated times. Scale bar = 120μm. (g) The formation of colonies in soft agar by Sulf-2 knockdown cells and control cells after 21 days of culture. The values shown in graphs are means ± SDs. (* indicates p< 0.05 relative to controls).
Mentions: We employed a previously developed lentiviral shRNA strategy (Nawroth et al., 2007) to achieve knockdown of Sulf-2 in NSCLC lines. We transduced five of the Sulf-2+ lines (H460, H292, Calu-6, P-ST and B-ST cells) with either a specific shRNA (PLV-1413 or PLV-1143) (Nawroth et al., 2007) or a control shRNA (PLV-Ctrl) containing an irrelevant sequence. Sulf-2 protein expression was strongly reduced (>90%) in all of the lines after transduction with PLV-1413 or PLV-1143, as compared to the control cells (Fig. 3a). To determine the effects of Sulf-2 knockdown on the sulfation status of HSPGs, we used a phage-display antibody (RB4CD12), whose binding to HSPGs depends on 6S (Dennissen et al., 2002). We have verified that this antibody can be used as a cell surface-staining reagent to report the presence of the Sulfs within cells (M. Hossain, T. Hosono, R. Tang, T. H. van Kuppevelt, G. J. Jenniskens, S. D. Rosen, and K. Uchimura, manuscript submitted). Transduction of Sulf-2+ lines with PLV-1413 increased the RB4CD12 epitope on the cell surface relative to that of control-treated cells by 30–37% (Fig. 3b). The same treatment had no effect on the epitope in a Sulf-2− line (H1975).

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