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

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.

Figure 1

SULF transcript and protein expression in NSCLC tumors and lung cancer cell lines: (a) DNA microarray analysis of SULF1 and SULF2 expression in squamous cell lung carcinomas and adjacent normal lung. Results were mined from (www.functionalglycomics.org, core E, #887). SULF1 (Left) and SULF2 (Right) transcripts in normal lung vs. lung squamous cell carcinomas (10 cases, lines connect individual patient values). Mean values (horizontal black bars) increased 18-fold for SULF1 (p=0.0005) and 3-fold for SULF2 (p=0.003). qPCR determinations of SULFs (normalized to % b̃actin) in (b) lung squamous cell carcinomas (12 cases) and (c) lung adenocarcinomas (12 cases) vs. adjacent normal lung samples. Relative to normals, SULF1 and SULF2 increased 12-fold (p=0.008) and 4-fold (p=0.05) in squamous carcinomas and 3-fold for both (p=0.003 and p=0.002, respectively) in adenocarcinomas. (d) RT-PCR analysis of SULFs in NSCLC cell lines (H358; H522; H266; H1299; H1703; H1975) and smoke transformed cells (B-ST; P-ST) and their respective parentals (B–C; P-C) with b actin as control. (e) Immunoblotting of Sulf-2 in conditioned medium (Upper) and lysates (Middle) of indicated cells with b-actin as loading control (Lower).

Figure 2

Sulf-2 protein expression in NSCLC tumors: representative sections of benign lung and squamous cell carcinoma were stained with hematoxylin and eosin (H&E) and adjacent serial sections were stained with anti-Sulf-2 antibody (2B4). (a) Normal lung, H&E. (b) Normal lung stained with 2B4. (c) Squamous cell carcinoma, H&E. (d) Squamous cell carcinoma stained with 2B4 antibody demonstrates islands of tumor cells strongly positive for Sulf-2 surrounded by weakly staining desmoplastic stroma. Panels a, b, c and d are low-power micrographs (100X, scale bar = 500μm). (e and f) High-power micrographs of squamous cell carcinoma stained with 2B4 antibody. Panel f shows staining of tumor-associated stromal cells with 2B4 antibody (400X, scale bar = 100μm).

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).

Figure 4

Effects of Sulf-2 overexpression in non-malignant bronchial epithelial cells: (a) BEAS2B and H140 were transduced with either Sulf2 or inactive Sulf-2 (S2ΔCC). Immunoblotting of Sulf-2 protein in CM (Upper) and cell lysates (Middle) of transduced cells as compared to Sulf-2+ cells (H292, Calu-6 and P-ST) with b-actin as loading control (Lower). (b) Staining of parental and transduced cells with RB4CD12 or control antibody, BEAS2B (left), H140 (right). (c) Healing of scratch wounds by parental and transduced cells. Scale bar = 76μm. (d) Growth of parental and transduced cells by Cell Titer Blue assay. (e) Soft agar colony formation by parental and transduced cells after 21 days of culture. (f) 9 clones of Sulf-2 expressing BEAS2B cells were generated from colonies in soft agar. Apoptosis was determined for the clones, parentals, and transduced populations by measuring staining with APC conjugated-Annexin V. The values shown in graphs are means ± SDs for 3 independent determinations (* indicates p< 0.05).

Figure 5

Effects of Sulf-2 knockdown on the tumorigenicity of lung cancer cell lines: The indicated lines with mock knockdown (PLV-Ctrl, black lines) or Sulf-2 knockdown (PLV-1413, dashed lines) were injected subcutaneously into nude mice and tumor volume was monitored over time. The values shown are means (+SEM) of 4–5 mice. * indicates p< 0.05.

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.