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. 2017 Oct 2;127(10):3755-3769.
doi: 10.1172/JCI93172. Epub 2017 Sep 11.

Syntaphilin Controls a Mitochondrial Rheostat for Proliferation-Motility Decisions in Cancer

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Free PMC article

Syntaphilin Controls a Mitochondrial Rheostat for Proliferation-Motility Decisions in Cancer

M Cecilia Caino et al. J Clin Invest. .
Free PMC article

Abstract

Tumors adapt to an unfavorable microenvironment by controlling the balance between cell proliferation and cell motility, but the regulators of this process are largely unknown. Here, we show that an alternatively spliced isoform of syntaphilin (SNPH), a cytoskeletal regulator of mitochondrial movements in neurons, is directed to mitochondria of tumor cells. Mitochondrial SNPH buffers oxidative stress and maintains complex II-dependent bioenergetics, sustaining local tumor growth while restricting mitochondrial redistribution to the cortical cytoskeleton and tumor cell motility. Conversely, introduction of stress stimuli to the microenvironment, including hypoxia, acutely lowered SNPH levels, resulting in bioenergetics defects and increased superoxide production. In turn, this suppressed tumor cell proliferation but increased tumor cell invasion via greater mitochondrial trafficking to the cortical cytoskeleton. Loss of SNPH or expression of an SNPH mutant lacking the mitochondrial localization sequence resulted in increased metastatic dissemination in xenograft or syngeneic tumor models in vivo. Accordingly, tumor cells that acquired the ability to metastasize in vivo constitutively downregulated SNPH and exhibited higher oxidative stress, reduced cell proliferation, and increased cell motility. Therefore, SNPH is a stress-regulated mitochondrial switch of the cell proliferation-motility balance in cancer, and its pathway may represent a therapeutic target.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. SNPH isoforms.
(A) Schematic diagram of the human SNPH locus (based on the Vertebrate Genome Annotation [Vega] repository; http://vega.archive.ensembl.org/index.html). The position and sequences of intron-exon boundaries, long (L) or short (S) SNPH transcripts, and TaqMan gene expression assays utilized for mRNA amplification of the two SNPH isoforms are indicated. (B) Schematic diagram of L-SNPH or S-SNPH protein isoforms. Pro, proline. (C and D) The indicated normal human tissues (C), normal diploid (MRC5) cells, or tumor cell types (D) were analyzed for L-SNPH or S-SNPH mRNA copy number, and normalized to 1,000 molecules of β-actin. Mean ± SEM (n = 3 per tissue or cell line examined). (E) PC3 cells were fractionated in cytosol (Cyto) or mitochondrial (Mito) extracts and analyzed by Western blotting. TCE, total cell extracts. (F) MCF-7 cells devoid of endogenous SNPH as in D were transfected with SNPH cDNA and analyzed by fluorescence microscopy. Merge image includes the F-actin channel (cyan). Scale bar: 5 μm. (G) PC3 cells were fractionated in sub-mitochondrial extracts containing outer membrane (OM), inter-membrane space (IMS), inner membrane (IM), or matrix (M) and analyzed by Western blotting. The expression of SDHB, cytochrome c (Cyto c), or ClpP was used as a markers for each fraction. MTE, unfractionated mitochondrial extracts.
Figure 2
Figure 2. SNPH regulation of mitochondrial bioenergetics.
(A and B) PC3 cells stably transduced with control pLKO or shRNA-SNPH (clone 0 or 5) were analyzed for OCR (A) or ATP production (B). Data are expressed as mean ± SD of replicates of a representative experiment (n = 3). ***P < 0.0001, by 2-tailed Student’s t test (A). ***P < 0.001 by ANOVA and Bonferroni’s post-test (B). (C and D) PC3 cells transfected with control siRNA (Ctrl) or siRNA-SNPH were transduced with control adenovirus (Ad-LacZ) or SNPH-directed adenovirus (Ad-SNPH), and analyzed for OCR (C) or ATP production (D). Data are expressed as mean ± SD of replicates of a representative experiment (n = 3). ***P < 0.001 by ANOVA and Bonferroni’s post-test. (E and F) PC3 cells transduced with pLKO or shRNA-SNPH were analyzed for oxidative phosphorylation complex II (C.II) activity (E) and normalized to citrate synthase activity (F). Gray tracing, blank reaction. Data are expressed as mean ± SD of replicates of a representative experiment (n = 3). **P < 0.01 by ANOVA and Bonferroni’s post-test. (G and H) The experimental conditions were as in E and F, except that transduced PC3 cells were analyzed for oxidative phosphorylation complex I (C.I) activity (G) and normalized to citrate synthase activity (H). Gray tracing, blank reaction. Data are expressed as mean ± SD of replicates of a representative experiment (n = 3). NS, not significant (P > 0.05) by ANOVA and Bonferroni’s post-test. (I) PC3 cells transduced with pLKO or shRNA-SNPH were treated with CHX, and aliquots of cell extracts harvested at the indicated time intervals after release (h) were analyzed by Western blotting. (J and K) Protein bands from the experiment in I were quantified by densitometric scanning after CHX release. Changes in SDHA (J) or SDHB (K) protein bands in pLKO or shRNA-SNPH are shown. Data are expressed as mean ± SD (n = 4). The statistical analyses are as follows: SDHA (J), 2 hours, P = 0.05; 4 hours, P < 0.0001; 6 hours, P = 0.0001; 8 hours, P = 0.0001; 10 hours, P < 0.0001; SDHB (K), 2 hours, P = 0.14; 4 hours, P = 0.0007; 6 hours, P < 0.0001; 8 hours, P < 0.0001; 10 hours, P < 0.0001, by 2-tailed Student’s t test.
Figure 3
Figure 3. Effect of SNPH on mitochondrial oxidative stress.
(A) LN229 cells expressing control siRNA (Ctrl) or siRNA-SNPH were transfected with cDNA encoding antioxidant SOD2 or Prx3 and analyzed for total ROS production by fluorescence microscopy. Data are expressed as mean ± SD of single-cell determinations (Ctrl, n = 109; SNPH, n = 75; SOD2, n = 74; and Prx3, n = 55). **P < 0.01 by ANOVA and Bonferroni’s post-test. CellROX, ROS sensor. (B) PC3 cells transduced with pLKO or shRNA-SNPH (clones 0 and 5) were analyzed for mitochondrial superoxide production (mitoSOX) by fluorescence microscopy. Data are expressed as mean ± SD of single cell determinations (pLKO, n = 202; SNPH #0, n = 296; SNPH #5, n = 297). ***P < 0.001 by ANOVA and Bonferroni’s post-test. FU, fluorescence units. (C) PC3 shRNA-SNPH #0 cells were analyzed for NAD+/NADH ratio. Data are expressed as mean ± SD of replicates of a representative experiment (n = 3). **P = 0.001, by 2-tailed Student’s t test. (DF) PC3 cells were treated with the indicated increasing concentrations of the oxidative stimuli DMNQ (D), H2O2 (E), and paraquat (PQ, F) and analyzed by Western blotting. Bottom panels (E and F): Densitometric quantification of SDHA, SDHB, or Prx-SO3 protein bands. Data are expressed as mean ± SD (n = 3). *P < 0.05; **P < 0.01; by ANOVA and Bonferroni’s post-test. (G) PC3 shRNA-SNPH #0 cells were treated with the mitochondrial superoxide scavenger MT and incubated with CHX, and cell extracts harvested at the indicated time intervals after release (h) were analyzed by Western blotting. (H) Densitometric quantification of SDHA, SDHB, or COX-IV proteins bands from the experiment in G. Data are expressed as mean ± SD (n = 4). The statistical analyses are as follows: SDHA, 2 hours, NS; 4 hours, P = 0.001; 6 hours, NS; 8 hours, NS; 10 hours, P = 0.033; SDHB, NS for all time points; COX-IV, NS for all time points by 2-tailed Student’s t test.
Figure 4
Figure 4. SNPH regulation of mitochondrial oxidative stress controls tumor cell motility.
(A) LN229 cells were transfected with vector or Flag-SNPH cDNA and analyzed for subcellular mitochondrial localization by confocal fluorescence microscopy. Three-dimensional isosurface renderings of representative cells are shown. (B) LN229 cells transfected with control siRNA or siRNA-SNPH were treated with vehicle or MT (200 μM) and analyzed by fluorescence microscopy. Masks are superimposed to the mitochondrial fluorescence channel (MTC02) to indicate the cell border (cyan lines, based on the actin channel) and the cortical region (area between the cyan and magenta lines). Arrowheads, cortical mitochondria. Scale bar: 10 μm. (CE) LN229 cells transfected with siRNA-SNPH were transfected with vector, SOD2, or Prx3 cDNA and analyzed for cell motility in a 2D chemotaxis chamber (C), with quantification of speed of cell migration (D) and distance traveled by individual cells (E). Each tracing in C and symbol in E corresponds to an individual cell. Data are expressed as mean ± SEM (n = 91–112). ***P < 0.0001, by ANOVA and Bonferroni’s post-test. The cutoff velocities for each condition in 2D chemotaxis experiments (C) are indicated.
Figure 5
Figure 5. Mitochondrial SNPH supports tumor cell proliferation.
(A) The indicated tumor cells transfected with control siRNA (Ctrl) or siRNA-SNPH were analyzed by direct cell counting (top) or cell viability by trypan blue exclusion (bottom) after 72 hours. The same number of cells were seeded at time 0. Data are expressed as mean ± SEM (n = 4). Red boxes indicate two cell types (C4-2B and MCF-7) with low to undetectable levels of endogenous SNPH. The statistical analyses per each cell type are as follows: BPH1, P = 0.03; A549, P = 0.004; H1299, NS; C4-2B, NS; DU145, P = 0.03; PC3, P = 0.002; LN229, P = 0.002; U251, P < 0.0001; U87, P = 0.03; MCF-7, NS; MDA-231, P = 0.01; Hs578T, P = 0.02 by 2-tailed Student’s t test. (B and C) PC3 cells transduced with pLKO or shRNA-SNPH (clones 0 and 5) were analyzed in a colony formation assay, and crystal violet–stained colonies (B) were counted after 10 days (C). Data are expressed as mean ± SEM (n = 3). ***P < 0.001 by ANOVA and Bonferroni’s post-test. (D) The indicated tumor cell types were transfected with vector or SNPH cDNA and analyzed by direct cell counting after 72 hours. Data are expressed as mean ± SD (n = 3). ***P < 0.0001, by 2-tailed Student’s t test. (E) PC3 cells transduced with pLKO or shRNA-SNPH were transfected with SOD2 cDNA and analyzed by direct cell counting. Data are expressed as mean ± SD (n = 3). *P < 0.01, by ANOVA and Bonferroni’s post test. (F) PC3 cells transduced with shRNA-SNPH were reconstituted with vector or SOD2 cDNA and analyzed by propidium iodide staining and flow cytometry. The cellular fractions in the indicated cell cycle phases are indicated. Data are expressed as mean ± SD (n = 3). **P = 0.01 by 2-tailed Student’s t test.
Figure 6
Figure 6. Mitochondrial SNPH regulation of metastasis.
(A and B) siRNA-SNPH PC3 cells were reconstituted with vector (pCMV), FL SNPH, or an SNPH mutant deleted in the MLS (Δ-MLS), and analyzed for OCR (A) or ATP production (B). Data are mean ± SD (n = 3). **P < 0.01; ***P < 0.001 by ANOVA and Bonferroni’s post-test. (C and D) C4-2B cells were transfected with pCMV6, FL SNPH, or Δ-MLS SNPH and analyzed for mitochondrial trafficking to the cortical cytoskeleton (C) or direct cell counting (D). For C, data are mean ± SEM (vector, n = 89; FL SNPH, n = 96; Δ-MLS SNPH, n = 87). **P < 0.01; ***P < 0.001 by ANOVA and Bonferroni’s post-test. For D, data are mean ± SD (n = 6). (E) shRNA-SNPH PC3 cells were reconstituted with pCMV6, FL SNPH, or Δ-MLS SNPH and analyzed for Matrigel invasion. Data are mean ± SEM (n = 3). ***P < 0.001, by ANOVA and Bonferroni’s post-test. (F) Yumm1.7 cells expressing mCherry and stably transfected with pCMV6, FL SNPH, or Δ-MLS SNPH were injected subcutaneously into syngeneic C57BL/6 mice, and mCherry-positive cells (insets) disseminated to the lungs were detected by immunocytochemistry. Scale bar: 50 μm. (G) Quantification of mCherry-positive Yumm1.7 cells transfected as in F in lungs of reconstituted animals. Each symbol corresponds to the mean number of disseminated cells per lung of an individual animal. DTC, disseminated tumor cells. *P < 0.05; ***P < 0.001, by ANOVA and Bonferroni’s post-test.
Figure 7
Figure 7. SNPH regulation of cell proliferation-motility in vivo.
(A) PC3 cells transduced with pLKO or shRNA-SNPH (clones 0 and 5) were injected subcutaneously in immunocompromised mice (5 mice per group; 2 tumors/mouse), and tumor volume was quantified with a caliper at the indicated time intervals. Each symbol corresponds to an individual tumor. On day 20, pLKO vs. SNPH #0, P < 0.001; pLKO vs. SNPH #5, P < 0.05, by ANOVA and Bonferroni’s post-test. (B) pLKO-transduced PC3 cells or pLKO-transduced PC3 cells isolated from a liver or lung metastatic site (Met) from the experiment in A were analyzed for SNPH mRNA levels by qPCR. Data are expressed as mean ± SD (n = 3). ***P < 0.001 by ANOVA and Bonferroni’s post-test. (C and D) The metastatic cell lines in B were analyzed for mitochondrial superoxide production by mitoSOX reactivity and fluorescence microscopy (C) or Western blotting (D). Data in C are expressed as mean ± SEM of single-cell determinations (n = 76–200). ***P < 0.001 by ANOVA and Bonferroni’s post-test. (E) The indicated metastatic cell lines were analyzed for mitochondrial oxidative phosphorylation complex II (C.II) activity at the indicated time intervals. (F and G) The indicated metastatic cell lines were analyzed in a colony formation assay (F), and crystal violet–stained colonies were quantified after 10 days (G). Data are expressed as mean ± SD (n = 3). **P < 0.01; ***P < 0.001 by ANOVA and Bonferroni’s post-test. (H and I) The indicated metastatic cell lines were analyzed for cell motility in a 2D chemotaxis chamber (H) with quantification of speed of cell migration (I). Data are expressed as mean ± SEM (n = 104–106). ***P < 0.0001, by ANOVA and Bonferroni’s post-test. (J) The indicated metastatic cell lines were analyzed by Western blotting.
Figure 8
Figure 8. Hypoxic and oxidative stress regulation of SNPH.
(A) PC3 cells were exposed to normoxia (N) or hypoxia (H; 1% O2 for 24 hours) and analyzed by Western blotting. (B) Cases of clear cell renal cell carcinoma in the TCGA database were stratified for SNPH mRNA expression and VHL mutational status. Mut, mutations; Trunc, truncated; del, deletions. Each symbol corresponds to an individual tumor. **P < 0.01; ***P < 0.001 by ANOVA and Bonferroni’s post-test. (C and D) LN229 cells exposed to normoxia or hypoxia as in A were analyzed for mitochondrial trafficking to the cortical cytoskeleton by fluorescence microscopy (C), and cortical mitochondria (mito) were quantified (D). Each symbol corresponds to an individual cell. ***P < 0.0001, by 2-tailed Student’s t test. The representative images displayed in C are brightness- and contrast-enhanced to highlight cortical mitochondria. Quantification was done in unsaturated images. (E and F) PC3 cells were treated with the indicated concentrations of DMNQ and analyzed by Western blotting (E) or qPCR amplification of SNPH mRNA (F). Data are expressed as mean ± SD (n = 3). ***P < 0.001, by ANOVA and Bonferroni’s post-test. (G and H) Normal diploid HFFs or normal human prostate epithelial cells (RWPE1) were transfected with control siRNA or siRNA-SNPH and analyzed for OCR (G) or ATP production (H). Data are mean ± SD (HFFs, n = 3; RWPE1, n = 12). *P = 0.001 to P < 0.0001; NS, not significant, by 2-tailed Student’s t test. (I) HFFs (top) or RWPE1 cells (bottom) transfected as in G were analyzed for cell motility in a 2D chemotaxis chamber. Each trace corresponds to the movements of an individual cell. (J and K) HFFs or RWPE1 cells transfected as in G were analyzed by 2D chemotaxis, and speed of cell migration (J) and distance traveled by individual cells (G) were quantified. Data are expressed as mean ± SD (HFFs, n = 35–42; RWPE1 cells, n = 50–52). NS, 2-tailed Student’s t test.
Figure 9
Figure 9. A mitochondrial SNPH “rheostat” for cell proliferation-motility decisions in cancer.
An adequate supply of oxygen and nutrients in the tumor microenvironment maintains high levels of SNPH in mitochondria for efficient, “non-leaky” oxidative bioenergetics and low ROS generation. These conditions support continued tumor cell proliferation while halting mitochondrial trafficking to the cortical cytoskeleton as a “regional” energy source to fuel membrane dynamics of cell motility and invasion. Conversely, the emergence of an oxidative and hypoxic microenvironment, typical of advanced tumors, acutely lowers SNPH levels, compromising oxidative phosphorylation complex II integrity and mitochondrial bioenergetics. The resulting increase in ROS inhibits tumor cell proliferation, while promoting increased mitochondrial trafficking to the cortical cytoskeleton and focal adhesion kinase–dependent (FAK-dependent) tumor cell migration and invasion. KIF5, kinesin family member 5; TRAK, trafficking kinesin-binding protein 1.

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