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Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression.

Ventura R, Mordec K, Waszczuk J, Wang Z, Lai J, Fridlib M, Buckley D, Kemble G, Heuer TS - EBioMedicine (2015)

Bottom Line: Dose-dependent effects are observed between 20-200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays.Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K-AKT-mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific.Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses.

View Article: PubMed Central - PubMed

Affiliation: 3-V Biosciences, Menlo Park, CA, United States.

ABSTRACT

Unlabelled: Inhibition of de novo palmitate synthesis via fatty acid synthase (FASN) inhibition provides an unproven approach to cancer therapy with a strong biological rationale. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types. TVB-3166, an orally-available, reversible, potent, and selective FASN inhibitor induces apoptosis, inhibits anchorage-independent cell growth under lipid-rich conditions, and inhibits in-vivo xenograft tumor growth. Dose-dependent effects are observed between 20-200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays. Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K-AKT-mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific. Our results demonstrate that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models and provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. The reported findings inform ongoing studies to link mechanisms of action with defined tumor types and advance the discovery of biomarkers supporting development of FASN inhibitors as cancer therapeutics.

Research in context: Fatty acid synthase (FASN) is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for selecting tumors highly sensitive to FASN inhibition are identified. These preclinical data provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers.

No MeSH data available.


Related in: MedlinePlus

TVB-3166 inhibits lipid raft architecture and signal transduction of the AKT–mTOR and β-catenin pathways. In vitro immunofluorescent staining of lipid rafts (cholera toxin) and N-Ras in COLO-205 (A) and CALU-6 (B) tumor cell lines. (C) Western blot analysis of CALU6, COLO-205, OVCAR-8, and 22Rv1 tumor cell lines. Cells were treated with 0.02, 0.2, or 2.0 μM TVB-3166 for 96 h. (D) Inhibition of β-catenin pathway activity by FASN inhibition. COLO-205 and A549 cells were treated with 0.2 μM TVB-3166 for 48 h for TCF promoter-driven luciferase expression analysis (left) or 96 h for Western blot analysis (right). COLO-205 cells have constitutive pathway activity. Where indicated, cells were stimulated with 200 ng/ml Wnt3A for 18 h before treating with TVB-3166.
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f0025: TVB-3166 inhibits lipid raft architecture and signal transduction of the AKT–mTOR and β-catenin pathways. In vitro immunofluorescent staining of lipid rafts (cholera toxin) and N-Ras in COLO-205 (A) and CALU-6 (B) tumor cell lines. (C) Western blot analysis of CALU6, COLO-205, OVCAR-8, and 22Rv1 tumor cell lines. Cells were treated with 0.02, 0.2, or 2.0 μM TVB-3166 for 96 h. (D) Inhibition of β-catenin pathway activity by FASN inhibition. COLO-205 and A549 cells were treated with 0.2 μM TVB-3166 for 48 h for TCF promoter-driven luciferase expression analysis (left) or 96 h for Western blot analysis (right). COLO-205 cells have constitutive pathway activity. Where indicated, cells were stimulated with 200 ng/ml Wnt3A for 18 h before treating with TVB-3166.

Mentions: Having demonstrated that FASN inhibition inhibits tumor cell growth and survival under varied growth conditions; it was of interest to characterize the cellular mechanisms that contribute to these activities including those acting directly on lipid components. We examined the effect of FASN inhibition on lipid raft architecture and palmitoylated protein localization. CALU-6 or COLO-205 tumor cells were treated with TVB-3166 for 96 h, at which time lipid raft architecture and palmitoylated protein localization were imaged by immuno-fluorescent confocal microscopy. Lipid rafts were imaged using FITC-conjugated cholera-toxin subunit-B, which specifically binds to the cell surface raft-associated protein ganglioside GM1. As an example of palmitoylated protein localization, N-Ras localization was determined using the Alexa-fluor-conjugated antibody. FASN inhibition with TVB-3166 significantly disrupted lipid raft organization and membrane localization of N-Ras in both tumor cell types. In vehicle-treated cells lipid rafts were seen as a largely continuous ring around the cell with infrequent spots of lower intensity staining. N-Ras showed similar membrane-associated staining; however, was also found with lower intensity and partially punctate staining in the cytoplasm. Fig. 4A–B shows representative control and drug treated cells. Following FASN inhibition by 96 h of treatment with TVB-3166, lipid raft staining of the plasma membrane was discontinuous and overall had a lower intensity than that observed in control cells; sporadic staining was observed at irregular intervals. N-Ras staining also was altered following FASN inhibition. The plasma–membrane-associated pattern was discontinuous and irregular like the lipid rafts and the cytoplasmic distribution showed an increased number of regions without any staining. In addition to a greater number of cytoplasmic regions lacking N-Ras staining, these regions were larger following FASN inhibition than those in vehicle-treated cells. The effects were dose dependent; treatment with 0.1 μM TVB-3166 (near the FASN IC50) induced moderate changes that were more pronounced at 1 μM TVB-3166. Similar effects of FASN inhibition were observed in both the CALU-6 and COLO-205 tumor cells. Together the lipid raft and N-Ras immunofluorescence data show that FASN activity is required for normal lipid raft architecture and localization of membrane-associated signaling proteins such as N-Ras.


Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression.

Ventura R, Mordec K, Waszczuk J, Wang Z, Lai J, Fridlib M, Buckley D, Kemble G, Heuer TS - EBioMedicine (2015)

TVB-3166 inhibits lipid raft architecture and signal transduction of the AKT–mTOR and β-catenin pathways. In vitro immunofluorescent staining of lipid rafts (cholera toxin) and N-Ras in COLO-205 (A) and CALU-6 (B) tumor cell lines. (C) Western blot analysis of CALU6, COLO-205, OVCAR-8, and 22Rv1 tumor cell lines. Cells were treated with 0.02, 0.2, or 2.0 μM TVB-3166 for 96 h. (D) Inhibition of β-catenin pathway activity by FASN inhibition. COLO-205 and A549 cells were treated with 0.2 μM TVB-3166 for 48 h for TCF promoter-driven luciferase expression analysis (left) or 96 h for Western blot analysis (right). COLO-205 cells have constitutive pathway activity. Where indicated, cells were stimulated with 200 ng/ml Wnt3A for 18 h before treating with TVB-3166.
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Related In: Results  -  Collection

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f0025: TVB-3166 inhibits lipid raft architecture and signal transduction of the AKT–mTOR and β-catenin pathways. In vitro immunofluorescent staining of lipid rafts (cholera toxin) and N-Ras in COLO-205 (A) and CALU-6 (B) tumor cell lines. (C) Western blot analysis of CALU6, COLO-205, OVCAR-8, and 22Rv1 tumor cell lines. Cells were treated with 0.02, 0.2, or 2.0 μM TVB-3166 for 96 h. (D) Inhibition of β-catenin pathway activity by FASN inhibition. COLO-205 and A549 cells were treated with 0.2 μM TVB-3166 for 48 h for TCF promoter-driven luciferase expression analysis (left) or 96 h for Western blot analysis (right). COLO-205 cells have constitutive pathway activity. Where indicated, cells were stimulated with 200 ng/ml Wnt3A for 18 h before treating with TVB-3166.
Mentions: Having demonstrated that FASN inhibition inhibits tumor cell growth and survival under varied growth conditions; it was of interest to characterize the cellular mechanisms that contribute to these activities including those acting directly on lipid components. We examined the effect of FASN inhibition on lipid raft architecture and palmitoylated protein localization. CALU-6 or COLO-205 tumor cells were treated with TVB-3166 for 96 h, at which time lipid raft architecture and palmitoylated protein localization were imaged by immuno-fluorescent confocal microscopy. Lipid rafts were imaged using FITC-conjugated cholera-toxin subunit-B, which specifically binds to the cell surface raft-associated protein ganglioside GM1. As an example of palmitoylated protein localization, N-Ras localization was determined using the Alexa-fluor-conjugated antibody. FASN inhibition with TVB-3166 significantly disrupted lipid raft organization and membrane localization of N-Ras in both tumor cell types. In vehicle-treated cells lipid rafts were seen as a largely continuous ring around the cell with infrequent spots of lower intensity staining. N-Ras showed similar membrane-associated staining; however, was also found with lower intensity and partially punctate staining in the cytoplasm. Fig. 4A–B shows representative control and drug treated cells. Following FASN inhibition by 96 h of treatment with TVB-3166, lipid raft staining of the plasma membrane was discontinuous and overall had a lower intensity than that observed in control cells; sporadic staining was observed at irregular intervals. N-Ras staining also was altered following FASN inhibition. The plasma–membrane-associated pattern was discontinuous and irregular like the lipid rafts and the cytoplasmic distribution showed an increased number of regions without any staining. In addition to a greater number of cytoplasmic regions lacking N-Ras staining, these regions were larger following FASN inhibition than those in vehicle-treated cells. The effects were dose dependent; treatment with 0.1 μM TVB-3166 (near the FASN IC50) induced moderate changes that were more pronounced at 1 μM TVB-3166. Similar effects of FASN inhibition were observed in both the CALU-6 and COLO-205 tumor cells. Together the lipid raft and N-Ras immunofluorescence data show that FASN activity is required for normal lipid raft architecture and localization of membrane-associated signaling proteins such as N-Ras.

Bottom Line: Dose-dependent effects are observed between 20-200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays.Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K-AKT-mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific.Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses.

View Article: PubMed Central - PubMed

Affiliation: 3-V Biosciences, Menlo Park, CA, United States.

ABSTRACT

Unlabelled: Inhibition of de novo palmitate synthesis via fatty acid synthase (FASN) inhibition provides an unproven approach to cancer therapy with a strong biological rationale. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types. TVB-3166, an orally-available, reversible, potent, and selective FASN inhibitor induces apoptosis, inhibits anchorage-independent cell growth under lipid-rich conditions, and inhibits in-vivo xenograft tumor growth. Dose-dependent effects are observed between 20-200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays. Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K-AKT-mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific. Our results demonstrate that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models and provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. The reported findings inform ongoing studies to link mechanisms of action with defined tumor types and advance the discovery of biomarkers supporting development of FASN inhibitors as cancer therapeutics.

Research in context: Fatty acid synthase (FASN) is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for selecting tumors highly sensitive to FASN inhibition are identified. These preclinical data provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers.

No MeSH data available.


Related in: MedlinePlus