Research ArticleCancer

The p85 isoform of the kinase S6K1 functions as a secreted oncoprotein to facilitate cell migration and tumor growth

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Science Signaling  27 Mar 2018:
Vol. 11, Issue 523, eaao1052
DOI: 10.1126/scisignal.aao1052
  • Fig. 1 p85S6K1 and p70S6K1 share similar biochemical and biological properties.

    (A) Alignment of domains between S6K1 and S6K2. As indicated, p85S6K1 contains an extra 6R residues and nuclear localization sequence (NLS) in its extreme N terminus. KD, kinase domain. (B) In vitro kinase assays were performed with S6K1 purified from mammalian cells and with the bacterially purified His-S6 as the substrate. n = 2 biological replicates. (C) Immunoblot (IB) analysis of whole-cell lysates (WCLs) derived from human embryonic kidney (HEK) 293 cells transfected with the indicated constructs encoding various forms of S6K1. n = 2 biological replicates. P, phosphorylated. (D to K) IB analysis of WCL derived from S6k1/2−/− MEFs (D) or S6K1-depleted MDA-MB-231 cells (H) stably expressing the indicated constructs. The cells were subjected to cell size detection (E and I), colony formation (F and J, left), and invasion (G and K, left) assays. Relative colony numbers or numbers of invasive cells were calculated in the right panels of (F), (G), (J), and (K). EV, empty vector. Data are means ± SD; n = 3 biological replicates for (D) to (K). *P < 0.05, **P < 0.01 (t test).

  • Fig. 2 p85S6K1 is secreted from cells and can enter cells in a manner dependent on the N-terminal 6R motif.

    (A) Schematic workflow for the identification of secreted proteins. (B) The supernatant medium harvested from HEK293T cells transfected with indicated constructs was concentrated by column or hemagglutinin (HA) immunoprecipitation (IP) and then subjected to IB analysis. n = 2 biological replicates. (C) HEK293T cells were transfected with p85S6K1 and treated with or without brefeldin A (1 μg/ml) for 20 hours. The supernatant medium was harvested and subjected to HA IP and analyzed by IB. n = 2 biological replicates. (D) Different S6K1 proteins were eluted and purified from HEK293T cells transfected with the indicated constructs by HA IP, and the purified proteins were applied to S6K1-depleted MDA-MB-231 cells for 20 hours before harvesting for HA IP and IB analysis. n = 2 biological replicates. (E) S6K1 proteins were collected by HA IP from HEK293T cells expressing p85-GFP-HA constructs, eluted, and applied to S6K1-depleted HeLa cells, which were immunostained to show cell penetration of p85. n = 2 biological replicates. Scale bar, 50 μm. (F) Workflow for the analysis of secreted p85 protein entering the surrounding fibroblasts in vivo. (G) Tumors dissected from mice harboring HA-p85– or p70-expressing MDA-MB-231 cells treated with or without brefeldin A were stained with HA antibody. Scale bars, 25 μm. (H) Bacterially produced, purified recombinant S6K1 protein was applied to S6K1-depleted MDA-MB-231 cells for 20 hours before harvesting for HA IP and IB analysis. n = 2 biological replicates. IHC, immunohistochemistry; GFP, green fluorescent protein.

  • Fig. 3 Purified recombinant p85S6K1 enters cells and activates downstream signaling to promote behaviors typical of malignant cells.

    (A to D) IB analysis of WCL derived from S6K1-depleted MDA-MB-231 cells treated with different doses of bacterially purified S6K1 proteins (A). The treated cells were assessed for cell size (B), colony formation (C, left), and invasion (D, left), and colony numbers and the number of invasive cells were quantified in the right panels of (C) and (D). Data are means ± SD; n = 3 biological replicates from (A) to (D). *P < 0.05, **P < 0.01, ***P < 0.001 (t test). (E to H) IB analysis of WCL derived from S6k1/2−/− MEFs treated with different doses of bacterially purified S6K1 proteins (E). The treated MEFs were assessed for cell size (F), colony formation (G, left), and invasion (H, left), and colony numbers and the number of invasive cells were quantified in the right panels of (G) and (H). Data are means ± SD; n = 3 biological replicates. **P < 0.01, ***P < 0.001 (t test).

  • Fig. 4 Media conditioned by p85S6K1-expressing MDA-MB-231 cells promote malignant behaviors in MCF10A cells.

    (A to E) Application of media conditioned by cancer cells onto normal epithelial cells or fibroblasts (A). Where indicated, the conditioned media from S6K1-depleted MDA-MB-231 cells transfected with p85S6K1, p70S6K1, or Δ6Rp85S6K1 were applied to nontransformed MCF10A cells. The treated cells were subjected to colony formation assays (B) and wound-healing assays (C), and colony numbers and relative open area were quantified in (C) and (E). Data are means ± SD; n = 3 biological replicates for (B) and (D). *P < 0.05, **P < 0.01 (t test). (F to H) Immunodepletion of p85S6K1 from media conditioned by MDA-MB-231 cells before application of conditioned media to MCF10A cells (F). The treated cells were subjected to colony formation (G) and wound-healing (H) assays. The relative colony numbers and open area were quantified in the right panels of (G) and (H). Data are means ± SD; n = 3 biological replicates for (G) and (H). **P < 0.01 (t test).

  • Fig. 5 Purified recombinant p85S6K1 enhances in vivo tumorigenesis and lung metastasis in xenograft mouse model.

    (A to C) S6K1-depleted MDA-MB-231 and control cells were subjected to mouse xenograft assays, in which the tumor-grafted mice were treated or not with bacterially purified His-S6K1 proteins. Tumor sizes were monitored (A), and tumor mass was weighed (B) and quantified (C). Data are means ± SD; n = 6 mice per condition. **P < 0.01 (t test). (D to F) The lung metastases from mice treated or not with recombinant S6K1 proteins were presented in (D) and (E), and the metastatic nodules of the lung were quantified in (F). Data are means ± SD; n = 12 mice per condition. **P < 0.01 (t test). Scale bar, 100 μm.

  • Fig. 6 A proposed model for the re-education of surrounding cells by p85S6K1 secreted by cancer cells.

    A proposed model to illustrate the roles of secreted p85S6K1 in mediating the cross-talk between cancer cells and surrounding stromal cells. In brief, cancer cells harboring amplified S6K1, especially in breast tumors, may secrete p85S6K1, which may enter surrounding cells and have a paracrine effect on surrounding cells including fibroblasts, adjacent normal breast epithelial cells, and other cells (such as immune cells or endothelial cells) to induce cancer-associated phenotypes in these cells. Subsequently, these tumor-associated converted cells could re-educate the microenvironment to facilitate proliferation, invasion, and migration of cancer cells.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/523/eaao1052/DC1

    Fig. S1. Whole-proteome screen of arginine-enriched proteins.

    Fig. S2. S6K1 and S6K2 are amplified in breast cancers.

    Fig. S3. The N-terminal 6R motif facilitates PTEN secretion and cell penetration.

    Fig. S4. Purified p85, but not p70 or Δ6R-p85, enters HeLa cells.

    Fig. S5. Purified p85 enters cells to promote cell proliferation and migration.

    Fig. S6. Bacterially purified recombinant p85 enters cells to facilitate proliferation and migration of HeLa cells.

    Fig. S7. Media conditioned by p85-expressing MDA-MB-231 cells promote malignant behaviors in MEFs and WI-38 cells.

    Fig. S8. Immunodepletion of p85 suppresses malignant behaviors in MEFs induced by conditioned media.

  • Supplementary Materials for:

    The p85 isoform of the kinase S6K1 functions as a secreted oncoprotein to facilitate cell migration and tumor growth

    Jianjun Zhang, Jianping Guo, Xing Qin, Bin Wang, Linli Zhang, Yingnan Wang, Wenjian Gan, Pier Paolo Pandolfi, Wantao Chen,* Wenyi Wei*

    *Corresponding author. Email: chenwantao196323{at}sjtu.edu.cn (W.C.); wwei2{at}bidmc.harvard.edu (W.W.)

    This PDF file includes:

    • Fig. S1. Whole-proteome screen of arginine-enriched proteins.
    • Fig. S2. S6K1 and S6K2 are amplified in breast cancers.
    • Fig. S3. The N-terminal 6R motif facilitates PTEN secretion and cell penetration.
    • Fig. S4. Purified p85, but not p70 or Δ6R-p85, enters HeLa cells.
    • Fig. S5. Purified p85 enters cells to promote cell proliferation and migration.
    • Fig. S6. Bacterially purified recombinant p85 enters cells to facilitate proliferation and migration of HeLa cells.
    • Fig. S7. Media conditioned by p85-expressing MDA-MB-231 cells promote malignant behaviors in MEFs and WI-38 cells.
    • Fig. S8. Immunodepletion of p85 suppresses malignant behaviors in MEFs induced by conditioned media.

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    © 2018 American Association for the Advancement of Science

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