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CTGF drives autophagy, glycolysis and senescence in cancer-associated fibroblasts via HIF1 activation, metabolically promoting tumor growth.

Capparelli C, Whitaker-Menezes D, Guido C, Balliet R, Pestell TG, Howell A, Sneddon S, Pestell RG, Martinez-Outschoorn U, Lisanti MP, Sotgia F - Cell Cycle (2012)

Bottom Line: In addition, loss of stromal Cav-1 results in the metabolic reprogramming of cancer-associated fibroblasts, with the induction of autophagy and glycolysis.Here, we show that CTGF exerts compartment-specific effects on tumorigenesis, depending on the cell-type.As loss of Cav-1 is a stromal marker of poor clinical outcome in women with primary breast cancer, dissecting the downstream signaling effects of Cav-1 are important for understanding disease pathogenesis, and identifying novel therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: The Jefferson Stem Cell Biology and Regenerative Medicine Center, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA.

ABSTRACT
Previous studies have demonstrated that loss of caveolin-1 (Cav-1) in stromal cells drives the activation of the TGF-β signaling, with increased transcription of TGF-β target genes, such as connective tissue growth factor (CTGF). In addition, loss of stromal Cav-1 results in the metabolic reprogramming of cancer-associated fibroblasts, with the induction of autophagy and glycolysis. However, it remains unknown if activation of the TGF-β / CTGF pathway regulates the metabolism of cancer-associated fibroblasts. Therefore, we investigated whether CTGF modulates metabolism in the tumor microenvironment. For this purpose, CTGF was overexpressed in normal human fibroblasts or MDA-MB-231 breast cancer cells. Overexpression of CTGF induces HIF-1α-dependent metabolic alterations, with the induction of autophagy/mitophagy, senescence, and glycolysis. Here, we show that CTGF exerts compartment-specific effects on tumorigenesis, depending on the cell-type. In a xenograft model, CTGF overexpressing fibroblasts promote the growth of co-injected MDA-MB-231 cells, without any increases in angiogenesis. Conversely, CTGF overexpression in MDA-MB-231 cells dramatically inhibits tumor growth in mice. Intriguingly, increased extracellular matrix deposition was seen in tumors with either fibroblast or MDA-MB-231 overexpression of CTGF. Thus, the effects of CTGF expression on tumor formation are independent of its extracellular matrix function, but rather depend on its ability to activate catabolic metabolism. As such, CTGF-mediated induction of autophagy in fibroblasts supports tumor growth via the generation of recycled nutrients, whereas CTGF-mediated autophagy in breast cancer cells suppresses tumor growth, via tumor cell self-digestion. Our studies shed new light on the compartment-specific role of CTGF in mammary tumorigenesis, and provide novel insights into the mechanism(s) generating a lethal tumor microenvironment in patients lacking stromal Cav-1. As loss of Cav-1 is a stromal marker of poor clinical outcome in women with primary breast cancer, dissecting the downstream signaling effects of Cav-1 are important for understanding disease pathogenesis, and identifying novel therapeutic targets.

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Figure 5. CTGF overexpression in fibroblasts induces a senescence phenotype. (A) Immunoblot analysis performed on control and CTGF overexpressing fibroblasts demonstrates that CTGF expression induces the upregulation of p21 (CIP1/WAF1) and of p16Ink4A, both inhibitors of cell cycle progression. No changes were detected for p19 expression levels. Conversely, CTGF expression also induces the upregulation of cyclin D1, probably as a compensatory response against senescence. Equal loading was assessed using β-actin. (B) A β-galactosidase Assay was performed by FACS analysis on control and CTGF overexpressing fibroblasts. The number of β-Galactosidase-positive cells (left) and the β-galactosidase intensity mean (right) are both increased in CTGF overexpressing fibroblasts. (C) Conventional β-galactosidase staining was also performed on control and CTGF overexpressing fibroblasts to independently confirm that CTGF overexpression induces senescence. Note that CTGF-fibroblasts have intense blue staining, indicative of increased β-galactosidase activity.
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Figure 5: Figure 5. CTGF overexpression in fibroblasts induces a senescence phenotype. (A) Immunoblot analysis performed on control and CTGF overexpressing fibroblasts demonstrates that CTGF expression induces the upregulation of p21 (CIP1/WAF1) and of p16Ink4A, both inhibitors of cell cycle progression. No changes were detected for p19 expression levels. Conversely, CTGF expression also induces the upregulation of cyclin D1, probably as a compensatory response against senescence. Equal loading was assessed using β-actin. (B) A β-galactosidase Assay was performed by FACS analysis on control and CTGF overexpressing fibroblasts. The number of β-Galactosidase-positive cells (left) and the β-galactosidase intensity mean (right) are both increased in CTGF overexpressing fibroblasts. (C) Conventional β-galactosidase staining was also performed on control and CTGF overexpressing fibroblasts to independently confirm that CTGF overexpression induces senescence. Note that CTGF-fibroblasts have intense blue staining, indicative of increased β-galactosidase activity.

Mentions: Several studies have reported that increased intracellular ROS is involved in induction of senescence. In addition, recent evidence suggests that autophagy may also mediate the acquisition of a senescent phenotype.36,37 To verify if CTGF expression induces a senescent phenotype in fibroblasts, we next analyzed the expression of genes implicated in senescence by immunoblotting. Figure 5A shows that CTGF overexpression drives the upregulation of p21(WAF/CIP1) and p16(INK4A), both inducers of cell cycle arrest. However, no changes were observed in p19(ARF) protein expression. Conversely, CTGF induces an increase of Cyclin D1 expression, likely a compensatory response to senescence. To independently assess if CTGF induces a senescent phenotype, we next performed a β-galactosidase (β-Gal) activity assay by flow cytometry (Fig. 5B) and a β-Gal staining assay (Fig. 5C). Figure 5B shows that CTGF expression increases β-Gal activity, as judged by increased numbers β-Gal-positive cells and increased mean intensity. Similarly, conventional β-Gal staining is augmented in CTGF fibroblasts as compared with control fibroblasts (Fig. 5C), confirming the ability of CTGF to trigger a senescence phenotype.


CTGF drives autophagy, glycolysis and senescence in cancer-associated fibroblasts via HIF1 activation, metabolically promoting tumor growth.

Capparelli C, Whitaker-Menezes D, Guido C, Balliet R, Pestell TG, Howell A, Sneddon S, Pestell RG, Martinez-Outschoorn U, Lisanti MP, Sotgia F - Cell Cycle (2012)

Figure 5. CTGF overexpression in fibroblasts induces a senescence phenotype. (A) Immunoblot analysis performed on control and CTGF overexpressing fibroblasts demonstrates that CTGF expression induces the upregulation of p21 (CIP1/WAF1) and of p16Ink4A, both inhibitors of cell cycle progression. No changes were detected for p19 expression levels. Conversely, CTGF expression also induces the upregulation of cyclin D1, probably as a compensatory response against senescence. Equal loading was assessed using β-actin. (B) A β-galactosidase Assay was performed by FACS analysis on control and CTGF overexpressing fibroblasts. The number of β-Galactosidase-positive cells (left) and the β-galactosidase intensity mean (right) are both increased in CTGF overexpressing fibroblasts. (C) Conventional β-galactosidase staining was also performed on control and CTGF overexpressing fibroblasts to independently confirm that CTGF overexpression induces senescence. Note that CTGF-fibroblasts have intense blue staining, indicative of increased β-galactosidase activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Figure 5. CTGF overexpression in fibroblasts induces a senescence phenotype. (A) Immunoblot analysis performed on control and CTGF overexpressing fibroblasts demonstrates that CTGF expression induces the upregulation of p21 (CIP1/WAF1) and of p16Ink4A, both inhibitors of cell cycle progression. No changes were detected for p19 expression levels. Conversely, CTGF expression also induces the upregulation of cyclin D1, probably as a compensatory response against senescence. Equal loading was assessed using β-actin. (B) A β-galactosidase Assay was performed by FACS analysis on control and CTGF overexpressing fibroblasts. The number of β-Galactosidase-positive cells (left) and the β-galactosidase intensity mean (right) are both increased in CTGF overexpressing fibroblasts. (C) Conventional β-galactosidase staining was also performed on control and CTGF overexpressing fibroblasts to independently confirm that CTGF overexpression induces senescence. Note that CTGF-fibroblasts have intense blue staining, indicative of increased β-galactosidase activity.
Mentions: Several studies have reported that increased intracellular ROS is involved in induction of senescence. In addition, recent evidence suggests that autophagy may also mediate the acquisition of a senescent phenotype.36,37 To verify if CTGF expression induces a senescent phenotype in fibroblasts, we next analyzed the expression of genes implicated in senescence by immunoblotting. Figure 5A shows that CTGF overexpression drives the upregulation of p21(WAF/CIP1) and p16(INK4A), both inducers of cell cycle arrest. However, no changes were observed in p19(ARF) protein expression. Conversely, CTGF induces an increase of Cyclin D1 expression, likely a compensatory response to senescence. To independently assess if CTGF induces a senescent phenotype, we next performed a β-galactosidase (β-Gal) activity assay by flow cytometry (Fig. 5B) and a β-Gal staining assay (Fig. 5C). Figure 5B shows that CTGF expression increases β-Gal activity, as judged by increased numbers β-Gal-positive cells and increased mean intensity. Similarly, conventional β-Gal staining is augmented in CTGF fibroblasts as compared with control fibroblasts (Fig. 5C), confirming the ability of CTGF to trigger a senescence phenotype.

Bottom Line: In addition, loss of stromal Cav-1 results in the metabolic reprogramming of cancer-associated fibroblasts, with the induction of autophagy and glycolysis.Here, we show that CTGF exerts compartment-specific effects on tumorigenesis, depending on the cell-type.As loss of Cav-1 is a stromal marker of poor clinical outcome in women with primary breast cancer, dissecting the downstream signaling effects of Cav-1 are important for understanding disease pathogenesis, and identifying novel therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: The Jefferson Stem Cell Biology and Regenerative Medicine Center, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA.

ABSTRACT
Previous studies have demonstrated that loss of caveolin-1 (Cav-1) in stromal cells drives the activation of the TGF-β signaling, with increased transcription of TGF-β target genes, such as connective tissue growth factor (CTGF). In addition, loss of stromal Cav-1 results in the metabolic reprogramming of cancer-associated fibroblasts, with the induction of autophagy and glycolysis. However, it remains unknown if activation of the TGF-β / CTGF pathway regulates the metabolism of cancer-associated fibroblasts. Therefore, we investigated whether CTGF modulates metabolism in the tumor microenvironment. For this purpose, CTGF was overexpressed in normal human fibroblasts or MDA-MB-231 breast cancer cells. Overexpression of CTGF induces HIF-1α-dependent metabolic alterations, with the induction of autophagy/mitophagy, senescence, and glycolysis. Here, we show that CTGF exerts compartment-specific effects on tumorigenesis, depending on the cell-type. In a xenograft model, CTGF overexpressing fibroblasts promote the growth of co-injected MDA-MB-231 cells, without any increases in angiogenesis. Conversely, CTGF overexpression in MDA-MB-231 cells dramatically inhibits tumor growth in mice. Intriguingly, increased extracellular matrix deposition was seen in tumors with either fibroblast or MDA-MB-231 overexpression of CTGF. Thus, the effects of CTGF expression on tumor formation are independent of its extracellular matrix function, but rather depend on its ability to activate catabolic metabolism. As such, CTGF-mediated induction of autophagy in fibroblasts supports tumor growth via the generation of recycled nutrients, whereas CTGF-mediated autophagy in breast cancer cells suppresses tumor growth, via tumor cell self-digestion. Our studies shed new light on the compartment-specific role of CTGF in mammary tumorigenesis, and provide novel insights into the mechanism(s) generating a lethal tumor microenvironment in patients lacking stromal Cav-1. As loss of Cav-1 is a stromal marker of poor clinical outcome in women with primary breast cancer, dissecting the downstream signaling effects of Cav-1 are important for understanding disease pathogenesis, and identifying novel therapeutic targets.

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Related in: MedlinePlus