Myc Regulation of a Mitochondrial Trafficking Network Mediates Tumor Cell Invasion and Metastasis

Abstract

The Myc gene is a universal oncogene that promotes aggressive cancer, but its role in metastasis has remained elusive. Here, we show that Myc transcriptionally controls a gene network of subcellular mitochondrial trafficking that includes the atypical mitochondrial GTPases RHOT1 and RHOT2, the adapter protein TRAK2, the anterograde motor Kif5B, and an effector of mitochondrial fission, Drp1. Interference with this pathway deregulates mitochondrial dynamics, shuts off subcellular organelle movements, and prevents the recruitment of mitochondria to the cortical cytoskeleton of tumor cells. In turn, this inhibits tumor chemotaxis, blocks cell invasion, and prevents metastatic spreading in preclinical models. Therefore, Myc regulation of mitochondrial trafficking enables tumor cell motility and metastasis.

Keywords: Myc; cell invasion; metastasis; mitochondria.

FIG 1

Oncogenic Myc regulation of mitochondrial dynamics. (A) UniProt classification categories of RNA-Seq genes most significantly affected by conditional Myc knockdown in Burkitt lymphoma P493 cells. Enr, enrichment (fold number of genes from each individual category over values expected by chance); FDR, false-discovery rate (Bonferroni-corrected P value); IM, inner membrane. (B) Ingenuity Pathway Analysis of 24 mitochondrial functions with at least 10% of genes significantly modulated by conditional Myc knockdown in P493 cells. The majority of identified functions shared at least 50% of the affected genes with other functions. Unique genes in selected mitochondrial functions are indicated. KO, knockout. (C and D) PC3 cells were transfected with control nontargeting siRNA (siCtrl) or Myc-directed siRNA (siMyc), and mitochondrial accumulation at the cortical cytoskeleton was imaged by confocal microscopy (C) and quantified (D). 3D isosurface renderings of representative cells are shown. Symbols correspond to an individual determination (n = 45 to 48). Scale bars, 5 μm. ***, P < 0.0001. (E) PC3 cells were transfected as described above for panel C, and the speed of individual MitoTracker-labeled mitochondria was quantified by time-lapse videomicroscopy. Representative tracings from two independent experiments (Exp.) are shown. (F) Experiments were performed under the conditions described above for panel C, and transfected PC3 cells expressing RFP-labeled mitochondria were imaged for mitochondrial motility by time-lapse videomicroscopy, expressed in 2D plots. Each tracing corresponds to the movement of an individual mitochondrion. The cutoff velocities for slow-moving (blue) or fast-moving (magenta) mitochondria are indicated. Data are from a representative experiment (n = 3). (G) Experiments were performed under the conditions described above for panel C, and the speed of mitochondrial movements (top) (n = 52 to 55) and total distance traveled by individual mitochondria (bottom) (n = 56) were quantified. Each symbol corresponds to an individual determination. ***, P < 0.0001.

FIG 2

Myc regulation of Drp1. (A) Chromatin immunoprecipitation sequencing (ChIP-Seq) tracks of Myc occupancy of the Drp1 promoter in Burkitt lymphoma P493 cells at three time points (t = 0, 1, and 24 h) after Dox removal and in neuroblastoma BE2C, Kelly, or NGP cells. (B) PC3 cells transfected with siCtrl or siMyc were analyzed for Myc accumulation at the Drp1 promoter by ChIP. IgG, nonbinding IgG. Means ± SD are shown (n = 3). (C and D) Experiments were performed under the conditions described above for panel B, and transfected PC3 cells were analyzed for changes in Drp1 mRNA by quantitative PCR (qPCR) (C) or protein expression by Western blotting (D). Means ± SD are shown (n = 3). ***, P < 0.0001. MTE, mitochondrial extracts; p, phosphorylated. (E and F) PC3 cells transfected as described above for panel B were labeled as indicated and analyzed for colocalization of Ser616-phosphorylated Drp1 (pDrp1) and mitochondria (TOM20) by confocal fluorescence microscopy (E), with quantification of the Pearson correlation coefficient (PCC) of Drp1-mitochondrion colocalization (F). Representative images and higher-magnification insets of selected areas are shown. Each symbol corresponds to an individual determination (n = 2). ***, P = 0.0003. (G) PC3 cells were transfected as described above for panel B, and isolated mitochondrial extracts were analyzed by Western blotting. (H) PC3 cells transfected as described above for panel B were analyzed by Western blotting. (I) PC3 cells transfected with siCtrl or siMyc were reconstituted with Drp1 cDNA and analyzed by Western blotting. (J and K) Experiments were performed under the reconstitution conditions described above for panel I, and transfected PC3 cells were imaged for individual mitochondrial motility by time-lapse videomicroscopy expressed in 2D plots (J), and the speed of mitochondrial movements (K, top) (n = 97 to 99) or total distance traveled by individual mitochondria (K, bottom) (n = 63 to 67) was quantified. The cutoff velocities for slow-moving (blue) or fast-moving (magenta) mitochondria are indicated. Each symbol corresponds to an individual determination. Data from a representative experiment are shown (n = 3). ***, P < 0.0001; ns, not significant. (L) DU145 cells were transfected as described above for panel B and analyzed for mitochondrial ROS generation by MitoSox red staining and flow cytometry. Representative tracings are shown.

FIG 3

Myc transcriptional control of a mitochondrial trafficking network. (A) ChIP-Seq tracks of Myc accumulation at the RHOT1, RHOT2, TRAK2, or Kif5B promoter in P493 cells at three time points (t = 0 h, 1 h, and 24 h) after Dox removal or in neuroblastoma BE2C, Kelly, and NGP cells. (B) PC3 cells were transfected with siCtrl or siMyc and analyzed for Myc accumulation at the RHOT1, RHOT2, TRAK2, or Kif5B promoter by ChIP. IgG, nonbinding IgG. Means ± SD are shown (n = 3). (C and D) PC3 cells transfected as described above for panel B were analyzed for changes in RHOT1, RHOT2, TRAK2, or Kif5B mRNA levels by quantitative PCR (C) or protein expression by Western blotting (D). Means ± SD are shown (n = 3). ***, P < 0.0001.

FIG 4

Myc regulation of mitochondrial trafficking. (A) PC3 cells transfected with siCtrl or individual siRNA sequences targeting RHOT1, RHOT2, TRAK2, or Kif5B were analyzed by Western blotting. (B and C) PC3 cells transfected with siCtrl or individual siRNAs targeting RHOT1, RHOT2, or Kif5B were labeled as indicated, and mitochondrial accumulation at the cortical cytoskeleton was imaged by confocal fluorescence microscopy (B) and quantified (C). 3D isosurface renderings of representative images are shown. Scale bars, 20 μm. Each symbol corresponds to an individual determination. (siCtrl, n = 43; siRHOT1, n = 43; siRHOT2, n = 39; siKif5B, n = 32). ***, P < 0.0001. (D) PC3 cells transfected with siCtrl or siMyc were reconstituted with individual cDNAs encoding RHOT1, RHOT2, or Kif5B and analyzed by quantitative PCR. Means ± SD are shown (n = 3). (E) Experiments were performed under the reconstitution conditions described above for panel D, and transfected PC3 cells were quantified for mitochondrial accumulation at the cortical cytoskeleton by confocal fluorescence microscopy. Each symbol corresponds to an individual determination (siCtrl, n = 27; siMyc, n = 30; RHOT1, n = 32; RHOT2, n = 33; Kif5B, n = 23). ***, P < 0.0001; *, P = 0.02; ns, not significant. (F and G) Experiments were performed under the reconstitution conditions described above for panel E, and transfected PC3 cells were analyzed for speed of mitochondrial movements (F) and total distance traveled by individual mitochondria (G). Each symbol corresponds to an individual determination (Ctrl, n = 148; Myc, n = 132; RHOT1, n = 32; RHOT2, n = 35; Kif5B, n = 35; Drp1, n = 97). ***, P < 0.0001.

FIG 5

Myc control of tumor chemotaxis. (A) PC3 cells transfected with siCtrl or siMyc were labeled with talin-RFP and analyzed for focal adhesion (FA) complex dynamics by time-lapse videomicroscopy. Representative merged frames at 0 h and 2 h are shown (n = 2). (B) PC3 cells transfected as described above for panel A were analyzed for FA complex dynamics, and the percentage of new, stable, or decayed FA complexes was quantified. Means ± SD are shown (n = 10 to 15). **, P = 0.006; ***, P = 0.0005 to <0.0001. (C) Experiments were performed under the conditions described above for panel A, and the speed (top) and length (bottom) of FA complexes in siRNA-transfected PC3 cells was quantified (siCtrl, n = 891; siMyc, n = 380). ***, P < 0.0001. (D) PC3 cells transfected with siCtrl or siTRAK2 (top) or siKif5B (bottom) were labeled with talin-RFP and analyzed for FA complex dynamics by time-lapse videomicroscopy as described above for panel A. Means ± SD are shown (siTRAK2, n = 5 to 9; siKif5B, n = 9 to 19). *, P = 0.02; ***, P < 0.0001. (E) PC3 cells were transfected with siCtrl or two independent siRNA sequences targeting Myc (siMyc #1 and siMyc #2) and analyzed by Western blotting. (F and G) PC3 cells were transfected as described above for panel A and analyzed for 2D cell motility by time-lapse videomicroscopy (F), with quantification of the speed of cell movements and total distance traveled by individual cells (G). Each tracing corresponds to the movements of an individual cell (n = 19 or 20). The cutoff velocities for slow-moving (blue) or fast-moving (magenta) cells are indicated. Data are from a representative experiment (n = 3). ***, P < 0.0001. (H) PC3 cells were transfected with siCtrl or siMyc and analyzed for chemotactic cell motility with quantification of the forward migration index. Each symbol corresponds to an individual cell. Data are from a representative experiment. Arrows, direction of the chemotactic gradient. (I) PC3 cells transfected as described above for panel A were analyzed by Western blotting. p, phosphorylated.

FIG 6

Myc regulation of tumor cell invasion. (A to C) PC3 cells transfected with siCtrl or siMyc were analyzed for directional cell migration in the presence or absence of mitomycin C in a wound closure assay (A), and the area covered by cell migration in the absence (−Mito C) (B) or presence (+Mito C) (C) of mitomycin C was quantified. Representative images at the indicated time point are shown. BAF, binary area fraction (n = 3). (D and E) PC3 cells transfected with siCtrl or two independent Myc-directed siRNA sequences (siMyc #1 or siMyc #2) were analyzed for invasion across Matrigel-coated Transwell inserts (D) and quantified (E). Representative images of DAPI-stained nuclei of invaded cells are shown (n = 3). Each symbol corresponds to an individual determination (siCtrl, n = 18; siMyc #1, n = 16; siMyc #2, n = 14). ***, P < 0.0001. (F and G) PC3 cells transfected with siCtrl or siMyc were embedded in 3D organotypic spheroids in a collagen matrix (F), and cell invasion was quantified after 1 to 3 days by phase-contrast microscopy (G). Representative images are shown. Red contour, mask-inverted images used to quantify the length and area between the core and the invasive edge; AU, arbitrary units. ***, P < 0.0001. (H) Neuroblastoma Shep21 cells containing a doxycycline (Dox)-regulated Myc transgene (Dox-off) were analyzed for Matrigel invasion in the presence (+) or absence (−) of Dox. Each symbol corresponds to an individual determination (n = 5). *, P = 0.02. (I) Neuroblastoma SHEP-NMycER cells expressing a 4OHT-inducible Myc transgene were analyzed for Matrigel invasion in the presence (+) or absence (−) of 4OHT. Each symbol corresponds to an individual determination (n = 11). **, P = 0.008.

FIG 7

Myc-mitochondrial trafficking regulation of tumor cell invasion. (A and B) PC3 or DU145 cells transfected with siCtrl or siMyc or neuroblastoma Shep21 cells containing a Dox-regulated Myc transgene (Dox-off) were reconstituted with Drp1 cDNA and analyzed for invasion across Matrigel-coated inserts (A) and quantified (B). Representative images of DAPI-stained nuclei of invaded cells are shown (n = 3). Each symbol corresponds to an individual determination (PC3, n = 30 [siCtrl], n = 19 [siMyc], and n = 16 [siMyc plus Drp1]; DU145, n = 13 [siCtrl], n = 16 [siMyc], and n = 18 [siMyc plus Drp1]; Shep21, n = 6 [siCtrl], n = 7 [siMyc], and n = 7 [siMyc plus Drp1]). ***, P < 0.0001; ns, not significant. (C and D) PC3 cells transfected with siCtrl or siMyc were reconstituted with cDNA encoding RHOT1, RHOT2, or Kif5B and analyzed for 2D cell motility by time-lapse videomicroscopy (C), with quantification of the speed of cell movements and total distance traveled by individual cells (D) (siCtrl, n = 21; siMyc, n = 21; RHOT1, n = 19; RHOT2, n = 22; Kif5B, n = 22). ***, P < 0.0001. (E) PC3 cells transfected with siMyc were reconstituted with the vector (top), RHOT1 (middle), or Kif5B (bottom) cDNA and analyzed for chemotactic cell motility with quantification of the forward migration index in rose plots. Arrows, direction of the chemotactic gradient. (F) Experiments were performed under the reconstitution conditions described above for panel C, and transfected PC3 cells were analyzed for Matrigel invasion. Each symbol corresponds to an individual determination (Ctrl, n = 21; Myc, n = 31; RHOT1, n = 11; RHOT2, n = 31; Kif5B, n = 19). ***, P = 0.0004 to <0.0001. (G) PC3 cells were transfected with siCtrl or siRNA targeting RHOT1, RHOT2, TRAK2, or Kif5B and analyzed for Matrigel invasion. Each symbol corresponds to an individual determination (siCtrl, n = 84; siRHOT1, n = 25; siRHOT2, n = 21; siTRAK2, n = 28; siKif5B, n = 23). ***, P = 0.0001 to 0.0002. (H) PC3 cells transfected with the vector or cDNA encoding RHOT2 or Kif5B were analyzed for Matrigel invasion. Each symbol corresponds to an individual determination (vector, n = 8; RHOT2, n = 12; Kif5B, n = 12). ***, P = 0.0001 to 0.0004.

FIG 8

Myc-mitochondrial trafficking regulation of metastasis. (A) Yumm 1.7 or PC3 cells stably transfected with control nontargeting shRNA (shCtrl) or five independent shRNA sequences targeting Kif5B were analyzed by Western blotting. (B and C) Yumm 1.7 shRNA transfectants as described above for panel A were analyzed for Matrigel invasion (B) and quantified (C). Representative images of DAPI-stained nuclei of invaded cells are shown (n = 3). Each symbol corresponds to an individual determination (Ctrl, n = 27; shKif5B 1, n = 17; shKif5B 2, n = 15; shKif5B 3, n = 24). ***, P = 0.0004 to <0.0001. (D and E) Yumm 1.7 cells expressing mCherry and stably transfected as described above for panel A were engrafted subcutaneously (s.c.) on the flanks of syngeneic C57BL/6NCr mice, and lungs harvested after 1 to 3 weeks were analyzed by immunohistochemistry (D) with quantification of mCherry⁺ disseminated tumor cells (DTC) (E). Representative images are shown (n = 23 to 26). ***, P < 0.0001. (F) PC3 cells expressing shCtrl or Kif5B-directed shRNA (shKif5B) were engrafted s.c. in athymic nude mice, and superficial tumor growth was measured at the indicated time intervals with a caliper. Each line corresponds to data for an individual tumor. (G) PC3 cells as described above for panel F were analyzed for cell cycle transitions by propidium iodide staining and flow cytometry. The percentage of cells in each cell cycle phase is indicated. Means ± SD are shown. Data are from a representative experiment. (H) PC3 cells expressing shCtrl or shKif5B were injected into the spleen of immunocompromised mice, and livers were analyzed by hematoxylin and eosin staining and light microscopy after 11 days. White circles, metastatic foci. Representative images are shown. (I and J) Experiments were performed under the conditions described above for panel H, and the surface area (n = 49 to 60) (I) and number (n = 8 to 11) (J) of liver metastatic foci per microscopy field were quantified. **, P = 0.001; ns, not significant. (K) Schematic model for Myc transcriptional regulation of a mitochondrial trafficking network in tumor cell motility and invasion.