Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis

Abstract

Rho GTPases control cytoskeletal dynamics through cytoplasmic effectors and regulate transcriptional activation through myocardin-related transcription factors (MRTFs), which are co-activators for serum response factor (SRF). We used RNA interference to investigate the contribution of the MRTF-SRF pathway to cytoskeletal dynamics in MDA-MB-231 breast carcinoma and B16F2 melanoma cells, in which basal MRTF-SRF activity is Rho-dependent. Depletion of MRTFs or SRF reduced cell adhesion, spreading, invasion and motility in culture, without affecting proliferation or inducing apoptosis. MRTF-depleted tumour cell xenografts showed reduced cell motility but proliferated normally. Tumour cells depleted of MRTF or SRF failed to colonize the lung from the bloodstream, being unable to persist after their arrival in the lung. Only a few genes show MRTF-dependent expression in both cell lines. Two of these, MYH9 (NMHCIIa) and MYL9 (MLC2), are also required for invasion and lung colonization. Conversely, expression of activated MAL/MRTF-A increases lung colonization by poorly metastatic B16F0 cells. Actin-based cell behaviour and experimental metastasis thus require Rho-dependent nuclear signalling through the MRTF-SRF network.

Figure 1. MRTFs and SRF activity is dependent on Rho-actin signalling in MDA-MB-231 and B16F2 cells.

(A) MRTF localisation is Rho- and actin-dependent. Cells expressing Flag-MRTF-A with or without C3 transferase where indicated, were stimulated by serum and/or treated with Latrunculin B. Scale bar, 20µm. (B-E) SRF reporter gene activity is MRTF- and Rho-actin signal-dependent. Cells were transfected with an SRF reporter gene together with MRTF-A, C3 transferase, and activated RhoA or RhoC expression plasmids as indicated. MRTF and SRF were depleted from MDA-MB-231 cells or B16F2 cells by short term siRNA oligonucleotide transfection (siRNA oligonucleotides: mrtfa1 targets MAL/MRTF-A; mrtfb1 targets MRTF-B; mrtfa1+mrtfb1 and mrtfa/b, simultaneously target both MRTFs, in two different ways; srf targets SRF). Data are mean ± half-range of two independent experiments. (B) Basal MRTF activity requires Rho-actin signalling; (C) MRTF activation by constitutively active RhoA and RhoC; (D) CD stimulation activates the SRF reporter gene even when Rho is inactivated by C3 transferase expression. (E) CD-stimulated reporter activity is mediated by MRTF-A, MRTF-B, and SRF. (F) Reporter activity in stable cell derivatives expressing tetracycline-inducible MRTF shRNAs. C15-4 cells, MDA-MB-231 parental Tet repressor cell line; 15-17 and 15-20, MDA-MB-231 cell lines expressing the mrtfa/b shRNA; C6 cells, B16F2 parental Tet repressor cell line; Cl10 and Cl16, B16F2 cells expressing the mrtfa/b shRNA. Tetracycline treatment (72h) was as indicated. For cell lines stably expressing inducible SRF shRNAs, see Figure S1A and S1B.

Figure 2. MRTF activity is required for cell adhesion and spreading

(A) Adhesion assay. MDA-MB-231 cells depleted of MRTFs or SRF by siRNA oligonucleotide transfection (left panels) or expression of stable MRTF shRNAs (right panels) were plated on fibronectin (top panels) or collagen I (bottom panels), and adhesion quantified by dye retention 30 minutes after plating. Data show mean ± SEM (n=3; **p<0.001, *p<0.05, Student’s t test). (B-D) Impaired cell spreading in SRF or MRTF depleted MDA-MB-231 cell lines. MDA-MB-231 parental control (C15-4), derivatives expressing MRTF shRNA (15-17, 15-20) or SRF shRNA (SRF54) were plated on fibronectin or collagen I matrices for 3h, then fixed and stained with anti-paxillin antibody (red) and phalloidin (green) to visualize focal contacts and F-actin respectively. Scale bars, 20µm. (B), MRTF depletion; (C) high-magnification of representative cells from experiment in (B); (D), SRF depletion. (E) Impaired B16F2 cell spreading. Parental control C6 or MRTF shRNA C16 cells were analysed as in panel (B). For time course, see Figure S2.

Figure 3. MRTFs and SRF are required for cell motility in culture

(A-E) Migration-speed in the scratch-wound assay is MRTF- and SRF-dependent. MRTF and SRF were depleted from MDA-MB-231 cells (A, B, D) or B16F2 cells (C) either by short term siRNA oligonucleotide transfection or in stable cell derivatives expressing tetracycline-inducible shRNAs, as described in Figure 1 legend. A scratch was made across the confluent cell monolayer and migration of individual cells monitored using time lapse microscopy. Median speed was calculated by tracking individual cell movement (~50-150 cells per condition, 3 independent experiments; for hierarchical ANOVA, comparisons were made between control and depleted cells cultured under comparable conditions, ie either with or without tetracycline. ***, p<0.001; **, p<0.01; *, p<0.05). (E) Wound-closure images before and after wounding. MRTF- and SRF-depleted MDA-MB-231, and MRTF-depleted B16F2 cells are shown in left, centre and right images. Scale bars, 200µm. (F) Directionality of migration in the scratch-wound assay is MRTF- and SRF-dependent. Median persistence in migrating MDA-MB-231 cells was calculated by tracking individual cells, 3 independent experiments; significance by ANOVA, ***, p<0.001; ** p<0.01. Bottom, overlays of representative trajectories described by parental control (C15-4) and MRTF-depleted cells. (G) MTOC reorientation 6h following scratch-wounding. Cells were fixed and stained for gamma-tubulin to reveal the MTOC as a red dot; nuclei were counterstained with DAPI (blue). Dashed white line, wound edge; arrow, direction of movement. MTOC in the first row of cells migrating into the wound scored as reoriented when located between the nucleus and the leading edge of the cells into the wound. Data are mean ± half-range of two independent experiments. Scale bar, 10µm.

Figure 4. MRTF-SRF signalling is required for invasion and tumour cell motility in vivo.

(A) MRTF- and SRF-depletion impairs invasion through collagen-coated membrane towards serum-containing medium in the Boyden chamber assay. MRTF and SRF were depleted from MDA-MB-231 cells either by short-term siRNA oligonucleotide transfection in mass culture (left; mean ± half-range, two independent experiments) or in stable cell derivatives expressing tetracycline-inducible MRTF shRNAs (right; mean ± SEM, 3 indepedent experiments; *, P<0.05; Student’s t test). (B) Fibroblast-led organotypic invasion assay. MDA-MB-231 and B16F2 derivatives were seeded on dense fibrillar collagen I-matrigel matrix containing or lacking embedded carcinoma associated fibroblasts. Invasion was measured after 7 days. Representative H-and-E stained sections; arrows, invading cells; scale bar, 100µm. Data, mean ± SEM, three independent experiments (***, p<0.001; **, p<0.01, Student’s t test), except for SRF54 (mean ± half-range, n = 2). (C) MRTF or SRF depletion does not affect proliferation of MDA-MB-231 or B16F2 cells cultured with tetracycline (n = 4; 2-way ANOVA: C15-4/15-20, p=0.54; C15-4/SRF54, p=0.91; C6/Cl10, p=0.46; C6/SRF73, p=0.36). (D) MRTF does not affect proliferation of MDA-MB-231 or B16F2 cell xenografts. MDA-MB-231 derivatives (control: C15-4; MRTF-depleted: 15-20) and B16F2 derivatives (control: C6; MRTF-depleted: Cl10, Cl16) were injected subcutaneously into SCID and C57BL/6 mice respectively (12 animals/group); tumour volume was determined weekly (MDA-MB-231) or every other day (B16F2). (E) Proliferation status of MDA-MB-231 tumours at 9 wk, revealed by Ki67-staining; scale bar, 250µm. (F) Tumour motility in vivo monitored by multi-photon confocal imaging. Image shows a mixed tumour: MDA-MB-231 C15-4-GFP parental control cells, green; 15-20-CFP MRTF-depleted cells, cyan; reflectance image, red. Middle panels, 90s time-lapse images of the boxed area, with moving control cells outlined and numbered; arrow, direction of movement. Scale bar, 25μm. (G) Quantification of intravital imaging data. Data points, proportion of motile tumour cells in a given confocal section, normalised to the total area of tumour cells in the section (typically 4-6 tumors/animal, 4 areas/tumor, 2-3 sections/area), (*, p<0.05, Mann Whitney test; **p<0.01 Wilcoxon paired test).

Figure 5. MRTF-SRF signalling is required for an early step in experimental metastasis.

(A) MDA-MB-231 control (C15-4, C3) or MRTF-depleted cells (C15-4 derivatives 15-17, 15-20; C3 derivative C3-9) cells were injected into the tail vein of 5-8 week nude mice (1x10⁶ cells each) and maintained with doxycycline. Lungs were isolated after 20 weeks, fixed, and tumour cell nodules in lung sections counted microscopically (6 mice per group; significance by Mann-Wittney test: ***, p<0.001, **, p<0.01). Right, representative H&E-stained lung sections, with tumour nodules outlined by dashes. Scale bar, 250µm. (B) Control B16F2 control cells (C6, C1) or MRTF-depleted derivatives of C6 cells (Cl10, Cl16, Cl101), were injected into the tail vein of 5-8 week C57BL/6 mice (0.3x10⁶ cells each) and maintained with doxycycline. Lungs were isolated after 14 days and the number of surface tumour nodules quantified as in (A). Right, representative lungs from animals bearing control and MRTF-depleted B16F2 cells. MRTF depletion using a different siRNA combination is shown in Figure S4C. (C) Mixtures of GFP-expressing control-transfected and CFP-expressing MRTF-depleted MDA-MB-231 cells (2x10⁶ each), or GFP-expressing control-transfected and Cherry-expressing MRTF-depleted MTLn3 cells (0.75x10⁶ each) were co-injected into the mouse tail vein, and those present at the lung counted 2h or 24h later. Left, relative proportions of control and MRTF-depleted cells present at 2h or 24h; total cells detected are indicated below (error bars, SEM; n = 5). Right, representative images; scale bar, 50µm. (D) Control or SRF-depleted MDA-MB-231 or B16F2 cells were injected into the mouse tail vein of host mice (2x10⁶ MDA-MB-231 cells, 6-11 animals; 0.3x10⁶ B16F2 cells, 5-6 animals). Lung tumour development was assessed as in panels A and B.

Figure 6. MRTF-dependent gene expression in MDA-MB-231 and B16F2 cells.

(A) MRTF depletion affects both basal-level and cytochalasin D-inducible transcript levels. The transcriptomes of normal and MRTF-depleted B16F2 and MDA-MB-231 cells were analysed by microarray. The table shows genes whose basal and/or CD-induced expression levels change upon MRTF depletion in both lines by more than 1.5-fold (yellow highlight). For CD-induction, the threshold for CD-inducibility was taken as 1.3-fold change over basal (green highlight). Red highlight, SRF target genes validated by previous functional analysis (promoter analysis, ChIP, genetics; for references see13, 42, 43, 50); brackets indicate presumptive SRF targets identified only through conservation of SRF binding sites within 5kb of the transcription start. Microarray data are summarised, and notes on the analysis are given with Supplementary Tables 1A-1D; RT-PCR validation of selected genes is shown in Figure S5A. (B,C) MYH9 and MYL9 depletion impairs MDA-MB-231 invasive growth. Cells were depleted of the proteins by transfection of siRNA oligonucleotides, and assayed as in Figure 4A, 4B. (B) Boyden chamber assay, performed as in Figure 4A; data are mean ± half-range, 2 independent experiments. (C) organotypic invasion assay, performed as in Figure 4B; data are mean ± half-range, two independent experiments. (D) MDA-MB-231-GFP cells were depleted of MYH9 and/or MYL9 by siRNA transfection and injected into the mouse tail vein (0.75x10⁶ cells per mouse), and labelled cells in the lung counted 24h later (data are mean ± SEM, n = 4; significance by Mann-Wittney test: *, p<0.05). (E) B16F2 cells were depleted of MYH9 and/or MYL9 by siRNA transfection, injected into the mouse tail vein (0.70x10⁶ cells per mouse) and lung tumours counted 11 days later (4-5 mice per group; significance by Mann-Wittney test: *, p<0.05).

Figure 7. MRTF-dependent gene expression is sufficient to promote experimental metastasis

(A) Activation of the SRF reporter gene in B16F0 cells by co-expression of the activated MRTF-A mutant MAL-xxx,. A representative experiment is shown; data, mean of duplicate determinations. (B) RT-PCR analysis of the endogenous Myl9, Myh9, Cyr61, and Tpm1 transcripts in B16F0 cells expressing MAL-xxx. Representative of 3 independent experiments; data, mean of duplicate determinations. (C) MAL-xxx expression induces a spreading phenotype in B16F0 cells. Cells were plated on collagen and stained for DNA, MRTF-A, and paxillin. Scale bar, 20µm. (D) B16F0 cells transfected with MAL-xxx were injected into the mouse tail vein (0.9x10⁶ cells per mouse) and lung tumours counted 12 days later (6 mice per group; significance by Mann-Wittney test: **, p<0.01). (E) Schematic representation of the role of Rho GTPase signaling in cytoskeletal rearrangments. Rho GTPases are activated in response to extracellular signals, external (mechanical) cytoskeletal stress and internal homeostatic controls. In the cytoplasm they act directly with effector proteins to control F-actin and G-actin levels through regulation of actin treadmilling, and the assembly and reorganisation of actin-based cell structures– (black arrows). They also signal indirectly to the nucleus to control gene expression through the actin-MRTF-SRF pathway Activity of MRTF proteins is controlled a direct repressive interaction with G-actin (red repressive arrow). Changes in G-actin concentration can be brought about by changes in tonic levels of Rho GTPase activity, or by acute activation in response to signalling, leading to alterations in MRTF activity and consequent changes in SRF-dependent gene expression (blue arrows). This defines a G-actin-MRTF-SRF autoregulatory loop (thick arrows). Our data identify Myh9 and Myl9 as among the critical MRTF-SRF targets involved in these processes. Both cytoplasmic and nuclear pathways are required for optimal control of effective cytoskeletal dynamics and their downstream biological consequences.