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. 2016 May 15;129(10):1989-2002.
doi: 10.1242/jcs.180539. Epub 2016 Apr 12.

"VSports app下载" Mechanical signals regulate and activate SNAIL1 protein to control the fibrogenic response of cancer-associated fibroblasts

Kun Zhang  1 , Whitney R Grither  2 , Samantha Van Hove  3 , Hirak Biswas  3 , Suzanne M Ponik  4 , Kevin W Eliceiri  5 , Patricia J Keely  4 , Gregory D Longmore  6
Affiliations

V体育平台登录 - Mechanical signals regulate and activate SNAIL1 protein to control the fibrogenic response of cancer-associated fibroblasts

VSports手机版 - Kun Zhang et al. J Cell Sci. .

Erratum in

Abstract

Increased deposition of collagen in extracellular matrix (ECM) leads to increased tissue stiffness and occurs in breast tumors. When present, this increases tumor invasion and metastasis. Precisely how this deposition is regulated and maintained in tumors is unclear VSports手机版. Much has been learnt about mechanical signal transduction in cells, but transcriptional responses and the pathophysiological consequences are just becoming appreciated. Here, we show that the SNAIL1 (also known as SNAI1) protein level increases and accumulates in nuclei of breast tumor cells and cancer-associated fibroblasts (CAFs) following exposure to stiff ECM in culture and in vivo SNAIL1 is required for the fibrogenic response of CAFs when exposed to a stiff matrix. ECM stiffness induces ROCK activity, which stabilizes SNAIL1 protein indirectly by increasing intracellular tension, integrin clustering and integrin signaling to ERK2 (also known as MAPK1). Increased ERK2 activity leads to nuclear accumulation of SNAIL1, and, thus, avoidance of cytosolic proteasome degradation. SNAIL1 also influences the level and activity of YAP1 in CAFs exposed to a stiff matrix. This work describes a mechanism whereby increased tumor fibrosis can perpetuate activation of CAFs to sustain tumor fibrosis and promote tumor metastasis through regulation of SNAIL1 protein level and activity. .

Keywords: Extracellular matrix; Fibrosis; Mechanotransduction; SNAIL1. V体育安卓版.

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
ROCK kinases increase the SNAIL1 protein level post-transcriptionally. Human breast CAFs were infected with lentiviruses expressing two different shRNAs targeting ROCK1 or ROCK2 (shROCK) or control scrambled shRNA (CTL). Western blotting of cell extracts was performed with the indicated antibodies (A). (B) Quantification of the relative protein level of SNAIL1, SNAIL2 and YAP in the experiment described in A. Level of protein in control cells was arbitrarily set at 1. (C) Q-PCR determination for relative mRNA levels for the indicated mRNAs from samples as in experiment A. (D) CAFs were treated with Fasudil at increasing concentrations, as indicated, for 8 h. Western blotting of cell extracts was performed with the indicated antibodies. (E) Quantification of the relative protein level of SNAIL1, SNAIL2 and YAP in the experiment described in D. The level of protein in cells at t=0 were arbitrarily set at 1. (F) Q-PCR of the relative level of mRNA of genes from the CAF cells treated with DMSO (CTL) or 10 µM Fasudil for 8 h, as in experiment D. All experiments were performed two or three times and a representative example shown. Quantifications are plotted as the mean±s.d.
Fig. 2.
Fig. 2.
Mechanical signals increase the SNAIL1 protein level post-transcriptionally. (A) Human breast tumor CAFs were cultured on soft (80–120 Pa) or stiff (120 kPa) polyacrylamide hydrogels coated with fibronectin for 12 h. Western blotting of cell extracts was performed with the indicated antibodies. (B) Quantification of the relative protein level of SNAIL1, SNAIL2, YAP and TAZ for the experiment described in A. The level of protein in cells on soft substrate was arbitrarily set at 1. (C) Q-PCR determination of relative SNAIL1, SNAIL2, YAP and TAZ mRNA level in CAFs cultured on fibronectin-coated soft or stiff hydrogels, as described in A. (D) CAFs were plated on fibronectin-coated soft (80–120 Pa) or stiff (120 kPa) hydrogels for 8 h, then treated with the proteasome inhibitor MG132 (10 µM) for 4 h or left untreated (CTL). Western blotting of cell lysates was performed with the indicated antibodies. (E) CAFs were plated on fibronectin-coated soft (80–120 Pa) or stiff (120 kPa) hydrogels for 8 h, then treated with blebbistatin (20 µM) or cytochalasin D (10 µM) for 4 h or untreated (CTL). Western blotting of cell lysates was performed with the indicated antibodies. (F) Quantification of the relative protein level of SNAIL1 and YAP in the experiment described in E. The level of protein in control cells on soft substrate was arbitrarily set at 1 (n=2). (G) WT CAFs (CTL) or CAFs expressing constitutively activated RhoA (CA-Rho) were plated on fibronectin-coated soft or stiff hydrogels for 12 h. Western blotting was performed on cell lysates with the indicated antibodies. (H) Quantification of the relative protein level of SNAIL1 and YAP in the experiment described in G (n=2). The level of protein in control cells on soft substrate was arbitrarily set at 1. All experiments were performed two or three times. Quantifications are plotted as the mean±s.d.
Fig. 3.
Fig. 3.
SNAIL1 protein level increases in breast tumors from mice with increased collagen deposition in the breast. (A–D) Picosirius Red staining of mammary gland from 13-week-old mice (A,C) and implanted PyMT breast tumor from WT (B) and Col1α1tmJae mice (D). (E) Western blot analysis of whole-tumor tissue lysates harvested from mammary tumors of FVB WT or FVB Col1α1tmJae mice transplanted with primary PyMT tumor cells. (F) Graph quantifying SNAIL1 protein level determined by densitometry and normalized to β-tubulin control. Results are presented as mean±s.d.; n=4. (G–L). Tumor epithelial K8 and SNAIL1 immunofluorescence of breast tumors from Col1α1tmJae mice (G–I) and WT mice (J–L). (M) A quantification of the percentage of K8+ tumor cells that were also SNAIL1+ in breast tumors from Col1α1tmJae mice and WT mice. Results are presented as mean±s.d. of five fields that were scored. *P<0.05 (unpaired, two-tailed Student's t-tests). Scale bars: 50 μm.
Fig. 4.
Fig. 4.
In response to mechanical signals SNAIL1 accumulates in the nucleus. (A–C) CAFs were cultured on fibronectin-coated soft (80–120 Pa) or stiff (120 kPa) hydrogels for 12 h. Immunofluorescent staining for YAP (A) or SNAIL1 (B) (both green), F-actin (Rhodamine–phalloidin, red) and nuclei (DAPI staining, blue) was performed. The nuclear-to-cytosolic ratio of the YAP or SNAIL1 fluorescent intensity was quantified using ImageJ software. At least 50 cells, in multiple fields, were quantified. (C) CAFs were treated as in A and B, but after 12 h cells were lysed and the nuclear (N) and cytosolic (C) fractions isolated. Western blotting was performed on subcellular fractions with the indicated antibodies. Lamin A/C served as a nuclear marker whereas β-tubulin served as a cytosolic marker. (D) Confocal immunofluorescence images of CAFs plated on fibronectin-coated micropatterns of indicated area sizes (μm2) for 6 h. Scale bars: 20 μm. Histograms to the right show a quantification of the nuclear-to-cytosolic fluorescent intensity ratio of YAP or SNAIL1 in cells as determined with ImageJ software. At least 20 cells were quantified per condition. Results are presented as mean±s.d. **P<0.01 (unpaired, two-tailed Student's t-tests).
Fig. 5.
Fig. 5.
ROCK increases SNAIL1 protein level and nuclear accumulation through indirect activation of ERK2. (A) CAFs treated with Fasudil (10 µM) or untreated (CTL) were added to soft (80–120 Pa) or stiff (120 kPa) fibronectin-coated hydrogels for 12 h, then the cells were lysed and western blotting performed on cell extracts with the indicated antibodies. (B) CAFs were infected with scrambled shRNA (SCR) or ROCK2-shRNA-expressing lentivirus (RK2) and then transfected with YFP-tagged constitutively activated MEK (CA-MEK) or empty plasmid DNA (CTL). Western blotting was performed on cell extracts with the indicated antibodies. (C) Quantification of the relative SNAIL1 protein level in the experiment described in B. The level of protein in control cells exposed to scrambled shRNA was arbitrarily set at 1. (D) CAFs were cultured on fibronectin-coated soft (80–120 Pa) or stiff (120 kPa) hydrogel for 12 h in the presence of dilute DMSO, MEK1 inhibitor PD98059 (50 µM), p38 MAPK inhibitor SB203580 (20 µM) or TGFβ pathway inhibitor SB431542 (10 µM). Western blotting was performed on cell extracts with the indicated antibodies. (E) Quantification of the relative protein level of SNAIL1 and YAP in the experiment described in D. The level of protein in control cells on soft substrate was arbitrarily set at 1. (F) CAFs (CTL) or CAFs depleted of ERK2 with shRNA (–ERK2) were plated on soft (80–120 Pa) or stiff (120 kPa) matrices and western blotting was performed on cell extracts with the indicated antibodies. (G) Quantification of the relative protein level of SNAIL1 and YAP in the experiment described in F. The level of protein in control cells on soft substrate was arbitrarily set at 1. All experiments were performed two or three times and a representative example is shown. Quantifications are plotted as the mean±s.d.
Fig. 6.
Fig. 6.
ROCK increases SNAIL1 nuclear accumulation through activation of ERK2. (A,B) Immunofluorescent staining for SNAIL1 (A) or YAP (B) (green), F-actin (Rhodamine–phalloidin, red), and nuclei (DAPI, blue) in CAFs cultured on fibronectin-coated soft (80–120 Pa) or stiff (120 kPa) hydrogels for 12 h that were treated with scrambled shRNA (CTL) or depleted of ERK2 with shRNA (–ERK2). Histograms on right quantify the nuclear-to-cytosolic fluorescent intensity ratio of SNAIL1 (A) or YAP (B) using ImageJ software. Bars represent the mean±s.d.; more than 50 cells, in multiple fields were scored. *P<0.05 (unpaired, two-tailed Student's t-tests). Scale bars: 20 μm. (C) Human breast tumor CAFs were depleted of SNAIL1 with shRNA (–SNAIL1) or scrambled shRNA (CTL). SNAIL1-depleted cells were concurrently rescued with FLAG-tagged shRNA-resistant WT SNAIL1, or an S82A,S104A (S82/104A) SNAIL1 mutant. Cells were then cultured on fibronectin-coated soft (80–120 Pa) or stiff (120 kPa) hydrogels for 12 h. Western blotting was performed on cell extracts with the indicated antibodies. (D) Quantification of the relative amount of SNAIL1 and YAP protein level in the experiment described in C. The protein level in CTL cells on soft substrate was arbitrarily set at 1. (E) Human breast CAFs were depleted of SNAIL1 with shRNA (–SNAIL1) or scrambled shRNA (CTL). SNAIL1-depleted cells were concurrently rescued with FLAG-tagged shRNA-resistant WT SNAIL1, or an S82A,S104A SNAIL1 mutant. Cells were then cultured on fibronectin-coated soft (80–120 Pa) or stiff (120 kPa) hydrogels for 12 h. Nuclear (N) and cytosolic (C) fractions were isolated and western blotting performed on extracts from each subcellular fraction. Lamin A/C served as a nuclear marker whereas β-tubulin served as a cytosolic marker. The experiment was performed twice (n=2). (F) Quantification of the relative distribution of SNAIL1 protein level between the nucleus and cytosol for experiment the described in E. Quantifications are plotted as the mean±s.d.
Fig. 7.
Fig. 7.
SNAIL1 regulates the fibrogenic response of CAFs following exposure to mechanical signals. (A) Western blotting was performed on cell extracts of normal human breast fibroblasts (NF) and human breast tumor cancer CAFs (CAF) with the indicated antibodies. (B) Q-PCR analyses for mRNA level of select fibrogenic genes in CAFs [WT, YAP-depleted (YAP KD) or SNAIL1-depleted (SN1 KD)] after plating on soft (80–120 Pa) or stiff (120 kPA) fibronectin-coated hydrogels for 12 h: collagens (COL), TGFβs, MMPs and lysyl oxidases (LOX). (C) Fibronectin immunostaining of ECM produced by human breast cancer CAFs infected with control scrambled shRNA or SNAIL1 shRNA (–SNAIL1), and normal human breast fibroblasts (NF). (D) The degree of global order of fibronectin fibers from images in C. shSCR, scrambled shRNA; shSN, SNAIL1 shRNA. This experiment was performed three times (n=3). Results are presented as mean±s.d. *P<0.05; NS, not significant (unpaired, two-tailed Student's t-tests). Scale bars: 50 μm. (E) SHG images of collagen fiber production and organization in matrix produced by cultured human breast cancer CAFs and CAFs depleted of SNAIL1 with shRNA (–SNAIL1).
Fig. 8.
Fig. 8.
SNAIL1 affects YAP activity in response to mechanical signals. (A) Human breast tumor CAFs were infected with lentiviruses expressing shRNA targeting SNAIL1 (–SNAIL1) or YAP (–YAP) or with scrambled shRNA (CTL) and then plated on fibronectin-coated soft (80–120 Pa) or stiff (120 kPa) hydrogels for 12 h. Western blotting on cell extracts was performed with the indicated antibodies. (B) Quantification of the relative protein level of SNAIL1 and YAP in the experiment described in A. Level of protein in control cells on soft substrate was arbitrarily set at 1. (C,D) Immunofluorescent staining for SNAIL1 (C) or YAP (D) (green), F-actin (Rhodamine–phalloidin, red), and nuclei (DAPI, blue) in human CAFs cells plated on fibronectin-coated soft (80–120 Pa) or stiff (120 kPa) hydrogels for 12 h. Histograms on right quantify the nuclear-to-cytosolic fluorescent intensity ratio of SNAIL1 (C) or YAP (D) using ImageJ software. Bars represent mean±s.d. More than 50 cells, in multiple fields were scored. **P<0.01 (unpaired, two-tailed Student's t-tests). Scale bars: 20 μm. (E) Summary of mechanotransduction regulation of SNAIL1 protein and resultant cellular response in CAFs. FC, focal complex; FA, focal adhesion.
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