Ganglioside GM3 Promotes Carcinoma Cell Proliferation via Urokinase Plasminogen Activator-Induced Extracellular Signal-Regulated Kinase-Independent p70S6 Kinase Signaling  Xiao-Qi Wang, Ping Sun, Linda Go, Viola Koti, Margarita Fliman, Amy S. Paller  Journal of Investigative Dermatology  Volume 126, Issue 12, Pages 2687-2696 (December 2006) DOI: 10.1038/sj.jid.5700469 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 GM3 overexpression stimulates uPA-induced PCNA expression and BrdU incorporation. GM3 expression in SCC12 cells was increased by treatment with either antisense oligomers to block the synthesis of downstream gangliosides of GM3 (Wang et al., 2002a) or pharmacological addition of purified GM3 (Wang et al., 2001a). (a) After starvation of serum and growth factors, cells were stimulated with 10nm uPA (top row), 100ng/ml EGF (middle row), or 10μg/ml fibronectin (bottom row) for 24hours. Eight microgram of total protein from the whole-cell lysate was applied to a 12% SDS-PAGE mini-gel and probed with anti-PCNA antibody; lanes 1 and 4, parental SCC12 cells; lane 2, cells treated with sense oligomers to serve as a control (sense); lane 3, cells treated with antisense oligomers to block the GM3 metabolism (antisense); lane 5, cells treated with vehicle, DMSO, as a control for GM3-treated cells (vehicle); and lane 6, pharmacological addition of GM3 (GM3). (b) BrdU incorporation was measured in cells prepared as indicated. The figure represents the means±SD of studies from three different experiments. The changes in GM3 expression are shown in parentheses; bar 1, parental SCC12 cells; bar 2, cells treated with sense oligomers to serve as a control (sense); bar 3, cells treated with antisense oligomers to block the GM3 metabolism (antisense); bar 4, cells treated with vehicle, DMSO, as a control for GM3-treated cells (vehicle); and bar 5, pharmacological addition of GM3 (GM3). The expression of GM3 after genetic manipulation (increased 2.35-fold, bar 3) or pharmacological addition (increased 1.98-fold, bar 5) was detected by (c) ganglioside ELISA (Wang et al., 2002b) and (d) TLC immunostaining (Wang et al., 2002a); Sta=ganglioside standard (*P<0.05; **P<0.01). Journal of Investigative Dermatology 2006 126, 2687-2696DOI: (10.1038/sj.jid.5700469) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Overexpression of GM3 in the presence of uPA inhibits ERK activation, but stimulates ERK-independent p70S6 kinase activation. Cells were prepared as indicated in Figure 1a. (a) ERK and (b, c) p70S6 kinase were immunoprecipitated from the whole-cell lysate as described in Materials and Methods. Equal loading of the immunoprecipitated kinases was confirmed by immunoblotting with either (a, top row) anti-ERK antibody or (c, top row) anti-p70S6 kinase antibody. (a) Using immunoprecipitated ERK, the effect of GM3 on the phosphorylation of ERK was detected with anti-phospho-ERK antibody (middle row). The activity of this kinase was measured using immunoprecipitated ERK to incubate with its substrate in the presence of [γ-32P]ATP as described in Materials and Methods (bottom row). Lane 1, parental SCC12 cells; lane 2, cells treated with sense oligomers to serve as a control (sense); lane 3, cells treated with antisense oligomers to block the GM3 metabolism (antisense); and lanes 4–6, cells are same as lane 1–3, but without uPA stimulation. (b) Using immunoprecipitated p70S6 kinase, the effect of GM3 overexpression on the activity of p70S6 kinase was detected in the presence or absence of PD98059, an ERK inhibitor; bars 1–2, parental SCC12 cells treated without or with PD98059; bar 3, sense oligomer-treated cells (sense); bars 4–5, antisense oligomer-treated cells in the absence or presence of PD98059 (antisense); bar 6, vehicle DMSO-treated cells (DMSO); bars 7–8, cells with pharmacological addition of GM3 in the absence or presence of PD98059 (GM3); and bars 9–16, cells are same as bar 1–8 but without uPA stimulation. (c) The phosphorylation of p70S6 kinase was detected using anti-phosphoserine (middle row) or anti-phosphothreonine antibodies (bottom row); lane 1, parental SCC12 cells; lane 2, cells treated with sense oligomers to serve as a control (sense); lane 3, cells treated with antisense oligomers to block the GM3 metabolism (antisense); and lanes 4–6, cells are same as lane 1–3, but without uPA stimulation. Data from the p70S6 kinase assay are presented as fold increase from parental SCC12 cell controls in the absence of inhibitors (n=3, means±SD). The figures are representative of all cell lines studied (**P<0.01; ***P<0.001). P-values reflect the comparison of cells with increased GM3 with their respective control cells; #P<0.05 of SCC12 cells with ERK inhibition (solid bar) versus no inhibition (open bar). Journal of Investigative Dermatology 2006 126, 2687-2696DOI: (10.1038/sj.jid.5700469) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Rapamycin, but not PD98059, blocks the uPA stimulation of PCNA expression in cells with elevated GM3. (a) GM3 was depleted either by stable transfection of cells with a human plasma ganglioside-specific sialidase (SSIA) to render GM3 non-functional or by treatment of cells with 2μm PPPP for 5 days to block the synthesis of GM3 precursor, lactosylceramide. (b) Treatment with PPPP did not affect ceramide expression. Gangliosides and ceramide were detected by TLC immunostaining (Wang et al., 2002a). Cells with or without GM3 modulation were treated with 100μm of PD98059, a specific inhibitor of ERK, or 100ng/ml of rapamycin (Chung et al., 1992), an inhibitor to mTOR and p70S6 kinase (a downstream signaling molecule of mTOR), for 12hours and then stimulated with uPA for 10minutes. (c) Eight microgram of total protein from the whole-cell lysates was applied to a 12% SDS-PAGE mini-gel and probed with anti-PCNA antibody; lanes 1–3, parental SCC12 cells treated with or without PD98059 or rapamycin; lane 4, sense oligomer-treated cells (sense); lanes 5–7, antisense oligomer-treated cells in the absence of presence of either PD98059 or rapamycin (antisense); lane 8, pcDNA-transfected mock control for SSIA cells (pcDNA); lanes 9–11, SSIA cells treated without or with PD98059 or rapamycin; lane 12, vehicle DMSO-treated cells (vehicle); and lanes 13–15, cells treated with PPPP in the absence or presence of PD98059 or rapamycin. p70S6 kinase was immunoprecipitated from the whole-cell lysates and incubated with S6 kinase peptide in the presence of [γ-32P]ATP as indicated in Materials and Methods. (d) The activity of p70S6 kinase (n=3, mean±SD) is presented as fold increase above parental SCC12 cells treated without inhibitors; bars 1–3, parental SCC12 cells treated with or without PD98059 or rapamycin; bar 4, sense oligomer-treated cells (sense); bars 5–7, antisense oligomer-treated cells in the absence of presence of either PD98059 or rapamycin (antisense); bar 8, pcDNA-transfected mock control for SSIA cells; bars 9–11, SSIA cells treated without or with PD98059 or rapamycin; bar 12, vehicle DMSO-treated cells (vehicle); and bars 13–15, cells treated with PPPP in the absence or presence of PD98059 or rapamycin. The figures are representative of all cell lines studied (*P< 0.05; **P<0.01; ***P<0.001). P-values compare the hatched or solid bars with the open bars. Sta=ganglioside standard; pcDNA=vector control for SSIA; and SSIA=cells stably transfected with human ganglioside-specific plasma membrane sialidase. Journal of Investigative Dermatology 2006 126, 2687-2696DOI: (10.1038/sj.jid.5700469) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The stimulation of p70S6 kinase activity by GM3 overexpression requires uPAR signaling. GM3 expression in SCC12 cells was endogenously modulated as described in Materials and Methods. The expression of GM3 was detected by ganglioside ELISA or TLC immunostaining as shown in Figures 1 and 3. (a) Starved cells were incubated in DMEM/F12 medium containing 0.6% BSA with or without 1μg/ml of anti-uPAR neutralization antibody in the presence or absence of PD98059 for 24hours before the stimulation with uPA. After stimulation with 10nm uPA, cells were lysed, and (a) PCNA expression or (b) p70S6 kinase activity was detected as described; lane or bar 1, parental control cells (SCC12); lane or bar 2, PD98059-treated parental control cells; lane or bar 3, PD98059- and anti-uPAR antibody-treated parental control cells; lane or bar 4, negative control cells (sense); lane or bar 5, PD98059-treated negative control cells; lane or bar 6, PD98059- and anti-uPAR antibody-treated negative control cells; lane or bar 7, cells with increased GM3 (antisense); lane or bar 8, PD98059-treated GM3-overexpressing cells; and lane or bar 9, PD98059- and anti-uPAR antibody-treated GM3-overexpressing cells. (c) Starved cells were stimulated with 10nm uPA, 100ng/ml EGF or 10μg/ml fibronectin (Wang et al., 2003a) for 10minutes. p70S6 kinase was immunoprecipitated from cells and the activity of p70S6 kinase was measured as described above (***P<0.001). Journal of Investigative Dermatology 2006 126, 2687-2696DOI: (10.1038/sj.jid.5700469) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 uPA stimulates ERK-independent p70S6 kinase phosphorylation in cells with elevated GM3, but not in cells without GM3. Cells prepared as described in Figure 3. To study ERK-independent p70S6 kinase activation, cells were treated with 100μm of PD98059, a specific inhibitor of ERK, for 12hours, and then stimulated with uPA for 10minutes. p70S6 kinase was immunoprecipitated from cells as described in Materials and Methods. An aliquot of the immunoprecipitated p70S6 kinase was applied onto a 10% SDS-PAGE mini-gel and probed with antibody directed against the p70S6 kinase (top row), p70S6 kinase phosphothreonine 421/serine 424 (second row), p70S6 kinase phosphothreonine 389 (third row), or p70S6 kinase phosphoserine 411 site (bottom row). Lanes 1, 4, and 8, parental control cells (SCC12); lane 2, sense oligomer-treated control cells (sense); lane 3, GM3-overexpressing cells (antisense); lane 5, pcDNA-transfected mock control cells; lanes 6–7, sialidase-overexpressing cells with depleted GM3 (SSIA3 and SSIA 6 clones); lane 9, vehicle DMSO-treated cells (vehicle); and lane 10, PPPP-treated cells with depleted GM3. Journal of Investigative Dermatology 2006 126, 2687-2696DOI: (10.1038/sj.jid.5700469) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 PKC-ζ and PI3K signaling pathways mediate uPA-induced ERK-independent p70S6 kinase activation and phosphorylation at specific sites in the presence of elevated GM3. (a) Cells with elevated GM3 prepared as described in Figure 1 were treated with 100μm of PD98059, a specific inhibitor of ERK, in the presence or absence of 20μm of LY294002 or 100nm of wortmannin (both inhibitors of PI3K), 50μm of the pseudopeptide inhibitor of PKC-ζ, or both LY294002 (20μm) and pseudopeptide inhibitor of PKC-ζ (50μm). The vehicle of the PI3K inhibitors or PKC-ζ pseudopeptide was applied as a control. The phosphorylation of p70S6 kinase at different sites was detected as described in Figure 5; lane 1, GM3-overexpressing cells; lane 2, cells treated with DMSO to serve as a vehicle control for PI3K inhibitor-treated cells; lane 3, cells treated with PI3K inhibitor, LY294002; lane 4, cells treated with PI3K inhibitor, wortmannin; lane 5, cells treated with PBS as a vehicle control for cells treated with PKCζ pseudopeptide inhibitor; and lane 6, cells treated with PKCζ pseudopeptide inhibitor. (b) Cells were prepared as indicated in Figure 6a. The effect of PI3K and PKC-ζ on p70S6 kinase activity was measured as described in Materials and Methods. The activity of p70S6 kinase is presented as fold increase above parental SCC12 cells treated without inhibitors (n=3, mean±SD) The effect of pseudopeptide inhibitor on uPA-stimulated PKC-ζ activity in normal SCC12 cells was detected by in vitro kinase activity assay as described in Materials and Methods and shown as the inset in 6(b) (*P<0.05; **P<0.01; ***P<0.001); bar 1, parental control cells (SCC12); bar 2, sense oligomer-treated control cells (sense); and bars 3–6, GM3-overexpressing cells (antisense) treated without (bar 3) or with PI3K inhibitor, LY294002 (bar 4), PKCζ pseudopeptide inhibitor (bar 5), or both inhibitors (bar 6). Journal of Investigative Dermatology 2006 126, 2687-2696DOI: (10.1038/sj.jid.5700469) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 uPA activates PI3K and PKC-ζ in the presence of ERK inhibitors. Cells prepared as described in Figure 1a were treated with the PD98059, an ERK inhibitor, and 10nm uPA was added for 10minutes. (a) The PI3K activity was measured using the immunoprecipitated p85 subunit of PI3K. After incubation of the immunoprecipitated PI3K with its substrate, phosphatidylinositol, in the presence of [γ-32P]ATP, PI3K activity was detected as described (Wang et al., 2002b); lane 1, parental control (SCC12); lane 2, DMSO-treated vehicle control (vehicle); lane 3, pharmacological addition of GM3 (GM3); lane 4, sense oligomer-treated control (sense); and lane 5, antisense oligomer-treated cells with increased GM3 (antisense). (b) The activity of PKC-ζ was analyzed using immunoprecipitated PKC-ζ in the presence of both MBP and [γ-32P]ATP as described in Materials and Methods (***P<0.001); bar 1, parental control (SCC12); bar 2, DMSO-treated vehicle control (vehicle); bar 3, pharmacological addition of GM3 (GM3); bar 4, sense oligomer-treated control (sense); and bar 5, antisense oligomer-treated cells with increased GM3 (antisense). Journal of Investigative Dermatology 2006 126, 2687-2696DOI: (10.1038/sj.jid.5700469) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Proposed mechanism of GM3-triggered uPAR activation and cell proliferation. uPAR associates with integrin α5β1 (Wang et al., 2005) to cross-activate EGFR signaling (Wang et al., 2003a) in the presence of uPA. uPA is able to stimulate cell proliferation via either ERK-dependent or ERK-independent pathways. Cells show increased proliferation in the presence of uPA whether GM3 is increased or decreased. However, the underlying mechanism of increasing cell proliferation is different in the presence of increased GM3 versus the absence of GM3. In contrast to (a) quiescent control cells, cells that (b) overexpress GM3 show activation of PI3K and PKC-ζ, increased p70S6 kinase activity, and cell proliferation, despite suppression of ERK signaling. Cells in which GM3 is (c) depleted show activation of ERK signaling, leading to increased cell proliferation that is independent of p70S6 kinase activation. Journal of Investigative Dermatology 2006 126, 2687-2696DOI: (10.1038/sj.jid.5700469) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions