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First published online 14 April 2009
doi: 10.1242/jcs.025957

Journal of Cell Science 122, 1441-1451 (2009)
Published by The Company of Biologists 2009
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LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor

Lihua Ying1, Agatha Lau2, Cristina M. Alvira1, Robert West3, Gordon M. Cann1, Bin Zhou2,*, Caroline Kinnear2, Eric Jan4, Peter Sarnow4, Matt Van de Rijn3 and Marlene Rabinovitch1,{ddagger}

1 Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, 94305, USA
2 Department of Pediatrics, Laboratory Medicine, Pathobiology and Medicine at the University of Toronto, Ontario, Canada M5G 1X8
3 Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305, USA
4 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, 94305, USA


Figure 1
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  		Fig. 1. Stable transfection of HT1080 cells with LC3 induces fibronectin synthesis. (A) Western immunoblot of LC3 protein expression (top) and densitometric analyses (bottom) in HT1080 fibrosarcoma cells stably transfected with wild-type LC3 (LC3-WT), R/Q mutant LC3 (LC3-R/Q) and vector alone. A doublet appears at 16 kDa in LC3-WT but a more intense slightly downward shifted upper band in LC3-R/Q transfected cells. Upper bands were measured. (B) Western immunoblot of fibronectin protein relative to β-actin (top) with densitometric analyses below in LC3-WT, LC3-R/Q and vector-transfected cells. (C) Representative autoradiographs of fibronectin (top) following [35S]methionine labeling of cells and gelatin Sepharose purification of fibronectin, followed by radioactivity measurement (bottom) in LC3-WT, LC3-R/Q and vector-transfected cells. (D) Quantitative RT-PCR for Fn1 mRNA levels in LC3-WT, LC3-R/Q and vector-transfected cells. In all panels, bars represent mean ± s.e.m. of n=3 different experiments each carried out in triplicate. *P<0.05; **P<0.01; ***P<0.005.

 

Figure 2
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  		Fig. 2. Polysome analyses of stably transfected HT1080 cells suggest that enhanced Fn1 mRNA translation is dependent on the ARE in LC3. Polysome fractionation was carried out as described in Materials and Methods. RNA was extracted from each sucrose fraction, and equal volumes of each sample were analyzed by quantitative RT-PCR. The relative expression of fibronectin was normalized to 18S of the same sample. All analyses were performed three times and representative experiments are shown. The locations of the monosomes and/or preinitiation complexes and of the polysomes were determined by ethidium bromide staining (data not shown). (A) The 260 nm absorbance profile for LC3-WT (left) and LC3-R/Q (right) polysome fractions. (B,C) Comparison of relative gene expression of human FN1 (B) or Myc (C), normalized to 18S in 11 polysome fractions of the sucrose gradient from cells transfected with LC3-WT (filled bars) and LC3-R/Q mutant (open bars). (D) HT1080 cells stably transfected with LC3-WT were transiently transfected with rat Fn1 mRNA-WT or Fn1 mRNA with ARE deleted. Levels of rat Fn1 mRNA were quantitatively assessed by quantitative RT-PCR normalized to 18S. (E) RNA-binding assay was performed using cytoplasmic extracts from HT1080 cells transfected with vector, LC3-WT or LC3-R/Q and radiolabeled single-stranded oligonucleotides containing the fibronectin ARE sequence. Arrow indicates the specific LC3 binding band. Competition was done by adding 50x excess of the unlabeled wild-type fibronectin ARE oligonucleotides (LC3-WT+cold) or 50x excess of an irrelevant oligonucleotide that contains no consensus ARE (LC3-WT+irrelevant). Densitometry analysis of the binding band volume for cells transfected with vector, LC3-WT or LC3-R/Q is shown on the right.

 

Figure 3
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  		Fig. 3. LC3 increases proliferation of HT1080 cells in culture and in SCID mice. (A) Growth curves of HT1080 cells transfected with LC3-WT, LC3-R/Q or vector alone. Cells were seeded at 10,000 cells/well in growth medium and counted at indicated time points up to 7 days. All experiments were performed three times, and an average of triplicate samples was calculated at each time point. At day 3: **P<0.05 and ***P<0.001 for LC3-WT vs vector and LC3-R/Q-transfected cells. (B) Tumor growth in SCID mice. 2x106 HT1080 cells transfected with LC3-WT, LC3-R/Q or vector were injected into SCID mice subcutaneously, and the volume of the tumor was measured starting on day 14 following injection. Bars represent mean ± s.e.m. of n=5 at each time point. **P<0.005 for LC3-WT vs LC3-R/Q or vector-transfected cells.

 

Figure 4
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  		Fig. 4. Proliferation, adhesion and invasion of LC3-WT stably transfected HT1080 cells is fibronectin dependent. HT1080 cells stably transfected with vector or LC3-WT were transiently transfected with control siRNA or fibronectin siRNA. (A) Western blot of fibronectin protein (top panel) and qRT-PCR of Fn1 mRNA (bottom panel) show that both fibronectin protein and mRNA were efficiently knocked down following fibronectin siRNA transfection for 72 hours, in both vector and LC3-WT-transfected HT1080 cells. (B) MTT proliferation assay. (C,D) Adhesion assays. Representative images of adherent cells under conditions of LC3 transfection ± fibronectin siRNA (C). A quantified adhesion assay, with the additional condition that cells were cultured in the presence or absence of fibronectin as a substrate is shown in D. (E) Results of invasion assays. Bars represent mean ± s.e.m. of triplicate assessments from three separate experiments; *P<0.05; **P<0.01; ***P<0.001.

 

Figure 5
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  		Fig. 5. The increase of CTGF in HT1080 cells stably transfected with LC3-WT is fibronectin dependent. (A) CTGF protein expression in Western immunoblots (top) and densitometry (bottom) in vector- and LC3-WT-transfected HT1080 cells. Bars represent mean ± s.e.m. of three separate experiments. (B) Western blot (top) and densitometry (bottom) for CTGF protein in vector- or LC3-WT-transfected HT1080 cells treated with control or fibronectin siRNA. Bars represent mean ± s.e.m. of three experiments; *P<0.05; ***P<0.001. (C) Immunofluorescence analyses of frozen sections of vector- and LC3-WT-transfected HT1080 fibrosarcoma tumors removed from SCID mice after 21 days. A coordinated increase in immunofluorescence for fibronectin (green; left panel) and CTGF (red; middle panel) is observed in LC3-WT-transfected vs vector-transfected cells. Parallel sections stained with immunoperoxidase using a CD-31 (PECAM) antibody (right panel) to label endothelial cells shows an increase in the number of vessels in the WT-LC3 tumor. Mean density of PECAM staining in five random fields from three sections of each tumor was quantified using ImageJ software. The bars compare the number of PECAM-positive cells per field; mean ± s.e.m.; *P<0.05.

 

Figure 6
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  		Fig. 6. CTGF is necessary for enhanced adhesion, invasion and proliferation of HT1080 cells stably transfected with LC3-WT. (A) Western blot of CTGF protein (top) and qRT-PCR of Ctgf mRNA (bottom) in vector- and LC3-WT-transfected HT1080 cells transiently transfected with control siRNA or CTGF. Both CTGF protein and mRNA were efficiently knocked down following fibronectin siRNA transfection for 72 hours, in both vector- and LC3-WT-transfected HT1080 cells. (B-D) Adhesion (B), invasion (C) and proliferation (D) of HT1080 cells stably transfected with LC3-WT (as judged by MTT assay) compared in transfected cells transiently cotransfected with control siRNA or CTGF siRNA. Cell adhesion was assessed for cells cultured with or without fibronectin coating of the dishes (B). Bars denote mean ± s.e.m. of three experiments; *P<0.05; ** P<0.01; ***P<0.005.

 

Figure 7
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  		Fig. 7. LC3 and CTGF expression in soft tissue tumors. cDNA gene-profiling analysis of MAP1LC3B (LC3), CTGF, TGFβ and TNF{alpha} expression in soft tissue tumors. Truncated heat map and expression values are shown for selected tumors. Mean centered expression is shown for representative gene spots. Red represents high expression; black represents median expression; green represents low expression; and gray represents no data. Fold change values from significant analysis of microarray (SAM) relative to controls for corresponding samples are shown below. DTF, desmoid-type fibromatosis; EHE, epithelioid hemangioendothelioma; AS, angiosarcoma; MLPS, myxoid liposarcoma; SFT, solitary fibrous tumor; SS, synovial sarcoma.

 

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© The Company of Biologists Ltd 2009