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Use of synthetic isoprenoids to target protein prenylation and Rho GTPases in breast cancer invasion.

Chen M, Knifley T, Subramanian T, Spielmann HP, O'Connor KL - PLoS ONE (2014)

Bottom Line: We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM.To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation.Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells.

View Article: PubMed Central - PubMed

Affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America ; Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH) and anilinofarnesol (AFOH), to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting prenylation by using PFIs may offer a promising mechanism for treatment of invasive breast cancer.

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AGOH inhibits 3D invasive growth of MDA-MB-231 cells.MDA-MB-231 cells were seeded in Matrigel and treated with AGOH at the indicated concentration. After culturing for 8 days, phase contrast images were taken from randomly chosen fields (A, C, E) or Matrigel containing colonies were fixed and immunostained for F-actin (phalloidin, red) and nuclei (DAPI, blue) (B, D). The representative images from three separate experiments are shown. Scale bars represent 50 µm.
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pone-0089892-g003: AGOH inhibits 3D invasive growth of MDA-MB-231 cells.MDA-MB-231 cells were seeded in Matrigel and treated with AGOH at the indicated concentration. After culturing for 8 days, phase contrast images were taken from randomly chosen fields (A, C, E) or Matrigel containing colonies were fixed and immunostained for F-actin (phalloidin, red) and nuclei (DAPI, blue) (B, D). The representative images from three separate experiments are shown. Scale bars represent 50 µm.

Mentions: Culturing cells in 3D is commonly used to assess the physiologically relevant morphogenesis and invasive potential of breast epithelial and cancer cells [18], [30]. Accordingly, we next investigated the effect of AGOH on 3D invasive growth. In contrast to the 2D invasion assay, treatment of MDA-MB-231 cells in 3D culture with 20 µM AGOH showed a significant reduction in invasive growth (Figure 3A and C). This effect is even more dramatic at 100 µM where live colonies were difficult to find (Figure 3E). The immunocytochemistry staining revealed that DMSO-treated cells developed invasive growth (Figure 3B); however, cells treated with 20 µM AGOH lost their invasive potential in 3D culture (Figure 3D). Surprisingly, Ras activities in these cells were not affected (Figure S1), suggesting that Ras was not the primary target for AGOH in these breast cancer cells.


Use of synthetic isoprenoids to target protein prenylation and Rho GTPases in breast cancer invasion.

Chen M, Knifley T, Subramanian T, Spielmann HP, O'Connor KL - PLoS ONE (2014)

AGOH inhibits 3D invasive growth of MDA-MB-231 cells.MDA-MB-231 cells were seeded in Matrigel and treated with AGOH at the indicated concentration. After culturing for 8 days, phase contrast images were taken from randomly chosen fields (A, C, E) or Matrigel containing colonies were fixed and immunostained for F-actin (phalloidin, red) and nuclei (DAPI, blue) (B, D). The representative images from three separate experiments are shown. Scale bars represent 50 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3935959&req=5

pone-0089892-g003: AGOH inhibits 3D invasive growth of MDA-MB-231 cells.MDA-MB-231 cells were seeded in Matrigel and treated with AGOH at the indicated concentration. After culturing for 8 days, phase contrast images were taken from randomly chosen fields (A, C, E) or Matrigel containing colonies were fixed and immunostained for F-actin (phalloidin, red) and nuclei (DAPI, blue) (B, D). The representative images from three separate experiments are shown. Scale bars represent 50 µm.
Mentions: Culturing cells in 3D is commonly used to assess the physiologically relevant morphogenesis and invasive potential of breast epithelial and cancer cells [18], [30]. Accordingly, we next investigated the effect of AGOH on 3D invasive growth. In contrast to the 2D invasion assay, treatment of MDA-MB-231 cells in 3D culture with 20 µM AGOH showed a significant reduction in invasive growth (Figure 3A and C). This effect is even more dramatic at 100 µM where live colonies were difficult to find (Figure 3E). The immunocytochemistry staining revealed that DMSO-treated cells developed invasive growth (Figure 3B); however, cells treated with 20 µM AGOH lost their invasive potential in 3D culture (Figure 3D). Surprisingly, Ras activities in these cells were not affected (Figure S1), suggesting that Ras was not the primary target for AGOH in these breast cancer cells.

Bottom Line: We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM.To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation.Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells.

View Article: PubMed Central - PubMed

Affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America ; Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH) and anilinofarnesol (AFOH), to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting prenylation by using PFIs may offer a promising mechanism for treatment of invasive breast cancer.

Show MeSH
Related in: MedlinePlus