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. 2016 Mar 9;27(9):1409-1419.
doi: 10.1091/mbc.E16-02-0101. Online ahead of print.

SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases

Nawal Bendris  1 , Karla C Williams  2 , Carlos R Reis  3 , Erik S Welf  3 , Ping-Hung Chen  3 , Bénédicte Lemmers  4 , Michael Hahne  4 , H S Leong  2 , Sandra L Schmid  1
Affiliations

SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases

Nawal Bendris et al. Mol Biol Cell. .

Abstract

Despite current advances in cancer research, metastasis remains the leading factor in cancer-related deaths. Here, we identify sorting nexin 9 (SNX9) as a new regulator of breast cancer metastasis. We detected an increase in SNX9 expression in human breast cancer metastases compared with primary tumors and demonstrated that SNX9 expression in MDA-MB-231 breast cancer cells is necessary to maintain their ability to metastasize in a chick embryo model. Reciprocally, SNX9 knockdown impairs the process. In vitro studies using several cancer cell lines derived from a variety of human tumors revealed a role for SNX9 in cell invasion and identified mechanisms responsible for this novel function. We showed that SNX9 controls the activation of RhoA and Cdc42 GTPases and also regulates cell motility via the modulation of well-known molecules involved in metastasis, namely RhoA-ROCK and N-WASP. In addition, we have discovered that SNX9 is required for RhoGTPase-dependent, clathrin-independent endocytosis, and in this capacity, can functionally substitute to the bona fide Rho GAP, GRAF1 (GTPase Regulator Associated with Focal Adhesion Kinase). Together, our data establish novel roles for SNX9 as a multifunctional protein scaffold that regulates, and potentially coordinates, several cellular processes that together can enhance cancer cell metastasis.

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Figures

FIGURE 1:
FIGURE 1:
SNX9 regulates clathrin independent endocytosis of CD44. (A, B) Kinetics of clathrin-independent endocytosis of CD44 measured by internalization of an anti-CD44 antibody (see Materials and Methods) in siRNA-treated parental MDA-MB-231 (A) or siRNA-treated 231-oxSNX9 (B). GRAF1 depletion was used as a positive control for CIE perturbation. n = 3, *p < 0.05, ***p < 0.001. (C, D) Kinetics of clathrin-mediated endocytosis of the TfnR measured by internalization of an anti-TfnR antibody (see Materials and Methods) in 231-siSNX9 (C) or 231-oxSNX9 (D) vs. 231-si dynamin 2 (siDyn2) or controls. Dynamin 2 depletion was used as a positive control for CME perturbation. n = 6 and 3, respectively. *p < 0.05, **p < 0.001, ***p < 0.0001.
FIGURE 2:
FIGURE 2:
Differential expression of SNX9 affects the activation of RhoGTPases. (A, B) Active forms of the indicated Rho-family GTPases were pulled down using beads coupled to respective effector domains that can only bind the GTP-bound (i.e., active) form of the GTPases (see Supplemental Figure S2, A and B, and Materials and Methods). Bar chart shows quantification of active forms of RhoA, RhoC, Cdc42, and Rac1 in 231-siSNX9 (A) or 231-oxSNX9 (B) after normalization to respective control cells. n = 3–6; *p = 0.02. (C) Myosin light chain (MLC2) and cofilin are phosphorylated downstream of RhoA-ROCK activation. The bar chart compares the phosphorylation of MLC2 and cofilin in control and SNX9-depleted cells. n = 3; *p = 0.05. (D) Representative Western blot of His-SNX9 interaction with GST-Cdc42 or GST-RhoA in vitro. Before transferring to nitrocellulose membranes, protein loading was measured on Stain-Free gels (see Materials and Methods) to ensure that comparable amounts of GST- vs. GST-Cdc42 or GST-RhoA beads were used in each condition. Blot is representative of three independent experiments. (E, F) Pi production after GTP hydrolysis by RhoA (E) or Cdc42 (F) either alone or incubated with SNX9 and/or p50GAP. p50GAP alone was used as a positive control for Pi production by the GTPases. n = 4; ****p < 0.0001.
FIGURE 3:
FIGURE 3:
SNX9 regulates the ability of MDA-MB-231 cells to invade through collagen matrix. (A) 231-CTR, -siSNX9, or -oxSNX9 cells were subjected to an inverted three-dimensional cell invasion assay through a bovine collagen I matrix (see Materials and Methods). Representative images of the positions of the nuclei of invading cells detected by Hoechst staining under the indicated conditions. (B, C) Quantification of nuclei distribution in inverted invasion assay of control cells vs. 231-siSNX9 (B) or 231-oxSNX9 (C). n = 10 and 6, respectively; *p = 0.02. (D) Quantification of cell invasion after specific depletion of exogenous SNX9, using an siGFP treatment of 231-oxSNX9. n = 4; *p = 0.04; ns, nonsignificant. (E) Western blot analysis of SNX9 expression in cell lines used in D. GAPDH was used as loading control. Blot is representative of three independent experiments. (F, G) Quantification of cell invasion of siRNA-treated parental MDA-MB-231 (F) or in siSNX9- or siGRAF1-treated 231-oxSNX9 (G). n = 3; ****p < 0.0001.
FIGURE 4:
FIGURE 4:
Membrane binding is not important for SNX9 function in cell invasion. (A) Schematic representation of SNX9 protein domains, indicating the mutations of BARmut and PXmut (from Yarar et al., 2008). (B) Western blot analysis of SNX9 protein expression in cell lines stably expressing WT- or mutant-mCherry-SNX9. Asterisk indicates mCherry-SNX9 band. GAPDH was used as loading control. Blot is representative of three independent experiments. (C) Fractionation experiment using the cell lines in B, showing WT- or mutant-mCherry-SNX9 in cytoplasm (S, supernatant) vs. membranes (P, pellet). The absence of actin was used as purity readout of pellet fractions. Blot is representative of three independent experiments. (D) Quantification of SNX9 distribution in conditions used in C. (E) Quantification of cell invasion abilities of the stable cell lines in B. n = 3; *p = 0.025, ***p = 0.0001; ns, nonsignificant.
FIGURE 5:
FIGURE 5:
SNX9 protein expression enhances metastatic activity and is increased in human breast cancer metastases. (A) Representative image of metastases detected after injection of zsGreen-231-siSNX9 or zsGreen-231-oxSNX9, compared with their respective controls, into the chorioallantoic membrane of chicken embryos. Stromal cells were visualized with Lectin-Dylight-649 and metastases detected throughout the embryo by zsGreen fluorescence. Bar, 20 μm. (B) Quantification of metastatic efficiency of cells and conditions described in A. n = 14–16 for each condition. Results represent three independent experiments. ****p < 0.0001, **p = 0.0012. (C) Representative image of immunohistochemical staining of SNX9 in primary human breast tumors vs. their corresponding metastases. (D) Quantification of SNX9 expression levels corresponding to C. n = 7 for matched samples. Bar, 100 μm. **p = 0.0097.
FIGURE 6:
FIGURE 6:
SNX9 controls cell invasion through the RhoA-ROCK pathway and N-WASP. (A) Bar chart representing quantification of cell invasiveness of 231-siCTR and 231-siSNX9 either mock treated or treated with ROCK inhibitor, Y27632. n = 4; ***p = 0.002. (B) Western blot analysis of N-WASP protein expression in cell lines used in C. Asterisks indicate exogenous N-WASP. GAPDH was used as loading control. Blot is representative of three independent experiments. (C) Quantification of cell invasion after SNX9 knockdown in MDA-MB-231 parental cells or N-WASP–overexpressing cells. n = 4, ****p < 0.0001. ns, nonsignificant. (D) Similar experiment as in C, except using cells that were either mock treated or treated with ROCK inhibitor.
FIGURE 7:
FIGURE 7:
SNX9 is a multifunctional scaffold regulating CIE, cell invasion, and metastasis. Model placing the findings described in this article (solid arrows) in the context of existing knowledge (dashed arrows). (A) SNX9 overexpression decreases active RhoA and increases active Cdc42, consequently enhancing in vitro cancer cell invasion and leading to increased metastasis. (B) SNX9 depletion impairs CD44 internalization and Cdc42 and N-WASP activation and enhances RhoA activation. In this condition, in vitro cell invasion and metastasis are also decreased. The question marks indicate SNX9 functions whose exact mechanisms remain unknown. Our results summarized here are in accordance with the higher expression of SNX9 in human metastases than in matched primary breast tumors.
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