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Dietary restriction-resistant human tumors harboring the PIK3CA-activating mutation H1047R are sensitive to metformin.

Cufí S, Corominas-Faja B, Lopez-Bonet E, Bonavia R, Pernas S, López IÁ, Dorca J, Martínez S, López NB, Fernández SD, Cuyàs E, Visa J, Rodríguez-Gallego E, Quirantes-Piné R, Segura-Carretero A, Joven J, Martin-Castillo B, Menendez JA - Oncotarget (2013)

Bottom Line: The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects.Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues.Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing metformin significantly reduced the growth of SW48-mutated (PIK3CAH1047R/+) xenotumors by approximately 50%.

View Article: PubMed Central - PubMed

Affiliation: Metabolism and Cancer Group, Translational Research Laboratory, Catalan Institute of Oncology, Girona, Catalonia, Spain.

ABSTRACT
Cancer cells expressing constitutively active phosphatidylinositol-3 kinase (PI3K) are proliferative regardless of the absence of insulin, and they form dietary restriction (DR)-resistant tumors in vivo. Because the binding of insulin to its receptors activates the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling cascade, activating mutations in the PIK3CA oncogene may determine tumor response to DR-like pharmacological strategies targeting the insulin and mTOR pathways. The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects. However, it remains unclear whether PIK3CA-activating mutations might preclude the anti-cancer activity of metformin in vivo. To model the oncogenic PIK3CA-driven early stages of cancer, we used the clonal breast cancer cell line MCF10DCIS.com, which harbors the gain-of-function H1047R hot-spot mutation in the catalytic domain of the PI3KCA gene and has been shown to form DR-refractory xenotumors. To model PIK3CA-activating mutations in late stages of cancer, we took advantage of the isogenic conversion of a PIK3CA-wild-type tumor into a PIK3CA H1047R-mutated tumor using the highly metastatic colorectal cancer cell line SW48. MCF10DCIS.com xenotumors, although only modestly affected by treatment with oral metformin (approximately 40% tumor growth inhibition), were highly sensitive to the intraperitoneal (i.p.) administration of metformin, the anti-cancer activity of which increased in a time-dependent manner and reached >80% tumor growth inhibition by the end of the treatment. Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues. Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing metformin significantly reduced the growth of SW48-mutated (PIK3CAH1047R/+) xenotumors by approximately 50%. Thus, metformin can no longer be considered as a bona fide DR mimetic, at least in terms of anti-cancer activity, because tumors harboring the insulin-unresponsive, DR-resistant, PIK3CA-activating mutation H1047R remain sensitive to the anti-tumoral effects of the drug. Given the high prevalence of PIK3CA mutations in human carcinomas and the emerging role of PIK3CA mutation status in the treatment selection process, these findings might have a significant impact on the design of future trials evaluating the potential of combining metformin with targeted therapy.

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Metformin inhibits insulin-independent growth of PIK3CA-mutated tumor cells in vitroA. Top. Proliferation curves of MCF10DCIS.com cells cultured in the presence of increasing concentrations of insulin and/or metformin. MCF10DCIS.com cells were plated in 24-well plates at a density of 5,000 cells/well and cultured in 0.1% horse serum in the absence or presence of insulin (0, 100 and 1,000 ng/mL), metformin (1 μmol/L, 10 μmol/L, 100 μmol/L, 1 mmol/L, and 10 mmol/L), or a combination of insulin and metformin as specified. The data presented are the means of number cells × 104/well (±SD) from one representative experiment made in triplicate and obtained after 0, 2, 4, and 6 days. Bottom. MTT uptake curves of MCF10DCIS.com cells cultured in the presence of increasing concentrations of insulin and/or metformin. MCF10DCIS.com cells were plated in 96-well plates at a density of ∼2,000 cells/ well and cultured in 0.1% horse serum in the absence or presence of insulin (0, 100 and 1,000 ng/mL), metformin (1 μmol/L, 10 μmol/L, 100 μmol/L, 1 mmol/L, and 10 mmol/L), or a combination of insulin and metformin as specified. The data presented are the means ±SD of fold-increases in OD570.
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Figure 2: Metformin inhibits insulin-independent growth of PIK3CA-mutated tumor cells in vitroA. Top. Proliferation curves of MCF10DCIS.com cells cultured in the presence of increasing concentrations of insulin and/or metformin. MCF10DCIS.com cells were plated in 24-well plates at a density of 5,000 cells/well and cultured in 0.1% horse serum in the absence or presence of insulin (0, 100 and 1,000 ng/mL), metformin (1 μmol/L, 10 μmol/L, 100 μmol/L, 1 mmol/L, and 10 mmol/L), or a combination of insulin and metformin as specified. The data presented are the means of number cells × 104/well (±SD) from one representative experiment made in triplicate and obtained after 0, 2, 4, and 6 days. Bottom. MTT uptake curves of MCF10DCIS.com cells cultured in the presence of increasing concentrations of insulin and/or metformin. MCF10DCIS.com cells were plated in 96-well plates at a density of ∼2,000 cells/ well and cultured in 0.1% horse serum in the absence or presence of insulin (0, 100 and 1,000 ng/mL), metformin (1 μmol/L, 10 μmol/L, 100 μmol/L, 1 mmol/L, and 10 mmol/L), or a combination of insulin and metformin as specified. The data presented are the means ±SD of fold-increases in OD570.

Mentions: Insulin-independent growth of PIK3CA H1047R-mutated tumor cells is inhibited by metformin in vitro. Because the constitutive activation of the PI3K pathway drives MCF10DCIS.com cells to form DR-resistant tumors in vivo and to grow in an insulin-independent manner in vitro, we determined whether exogenous supplementation with metformin significantly affects tumor cell unresponsiveness to insulin in vitro. DR-resistant MCF10DCIS.com cells grew similarly in culture in an insulin-independent fashion, i.e., insulin failed to cause a dose-dependent increase in cell number. Metformin did not act as an insulin sensitizer for insulin-resistant PIK3CA H1047R-mutated MCF10DCIS.com cells; rather, it acted as a growth inhibitor in a dose-dependent manner (Fig. 2).


Dietary restriction-resistant human tumors harboring the PIK3CA-activating mutation H1047R are sensitive to metformin.

Cufí S, Corominas-Faja B, Lopez-Bonet E, Bonavia R, Pernas S, López IÁ, Dorca J, Martínez S, López NB, Fernández SD, Cuyàs E, Visa J, Rodríguez-Gallego E, Quirantes-Piné R, Segura-Carretero A, Joven J, Martin-Castillo B, Menendez JA - Oncotarget (2013)

Metformin inhibits insulin-independent growth of PIK3CA-mutated tumor cells in vitroA. Top. Proliferation curves of MCF10DCIS.com cells cultured in the presence of increasing concentrations of insulin and/or metformin. MCF10DCIS.com cells were plated in 24-well plates at a density of 5,000 cells/well and cultured in 0.1% horse serum in the absence or presence of insulin (0, 100 and 1,000 ng/mL), metformin (1 μmol/L, 10 μmol/L, 100 μmol/L, 1 mmol/L, and 10 mmol/L), or a combination of insulin and metformin as specified. The data presented are the means of number cells × 104/well (±SD) from one representative experiment made in triplicate and obtained after 0, 2, 4, and 6 days. Bottom. MTT uptake curves of MCF10DCIS.com cells cultured in the presence of increasing concentrations of insulin and/or metformin. MCF10DCIS.com cells were plated in 96-well plates at a density of ∼2,000 cells/ well and cultured in 0.1% horse serum in the absence or presence of insulin (0, 100 and 1,000 ng/mL), metformin (1 μmol/L, 10 μmol/L, 100 μmol/L, 1 mmol/L, and 10 mmol/L), or a combination of insulin and metformin as specified. The data presented are the means ±SD of fold-increases in OD570.
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Related In: Results  -  Collection

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Figure 2: Metformin inhibits insulin-independent growth of PIK3CA-mutated tumor cells in vitroA. Top. Proliferation curves of MCF10DCIS.com cells cultured in the presence of increasing concentrations of insulin and/or metformin. MCF10DCIS.com cells were plated in 24-well plates at a density of 5,000 cells/well and cultured in 0.1% horse serum in the absence or presence of insulin (0, 100 and 1,000 ng/mL), metformin (1 μmol/L, 10 μmol/L, 100 μmol/L, 1 mmol/L, and 10 mmol/L), or a combination of insulin and metformin as specified. The data presented are the means of number cells × 104/well (±SD) from one representative experiment made in triplicate and obtained after 0, 2, 4, and 6 days. Bottom. MTT uptake curves of MCF10DCIS.com cells cultured in the presence of increasing concentrations of insulin and/or metformin. MCF10DCIS.com cells were plated in 96-well plates at a density of ∼2,000 cells/ well and cultured in 0.1% horse serum in the absence or presence of insulin (0, 100 and 1,000 ng/mL), metformin (1 μmol/L, 10 μmol/L, 100 μmol/L, 1 mmol/L, and 10 mmol/L), or a combination of insulin and metformin as specified. The data presented are the means ±SD of fold-increases in OD570.
Mentions: Insulin-independent growth of PIK3CA H1047R-mutated tumor cells is inhibited by metformin in vitro. Because the constitutive activation of the PI3K pathway drives MCF10DCIS.com cells to form DR-resistant tumors in vivo and to grow in an insulin-independent manner in vitro, we determined whether exogenous supplementation with metformin significantly affects tumor cell unresponsiveness to insulin in vitro. DR-resistant MCF10DCIS.com cells grew similarly in culture in an insulin-independent fashion, i.e., insulin failed to cause a dose-dependent increase in cell number. Metformin did not act as an insulin sensitizer for insulin-resistant PIK3CA H1047R-mutated MCF10DCIS.com cells; rather, it acted as a growth inhibitor in a dose-dependent manner (Fig. 2).

Bottom Line: The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects.Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues.Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing metformin significantly reduced the growth of SW48-mutated (PIK3CAH1047R/+) xenotumors by approximately 50%.

View Article: PubMed Central - PubMed

Affiliation: Metabolism and Cancer Group, Translational Research Laboratory, Catalan Institute of Oncology, Girona, Catalonia, Spain.

ABSTRACT
Cancer cells expressing constitutively active phosphatidylinositol-3 kinase (PI3K) are proliferative regardless of the absence of insulin, and they form dietary restriction (DR)-resistant tumors in vivo. Because the binding of insulin to its receptors activates the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling cascade, activating mutations in the PIK3CA oncogene may determine tumor response to DR-like pharmacological strategies targeting the insulin and mTOR pathways. The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects. However, it remains unclear whether PIK3CA-activating mutations might preclude the anti-cancer activity of metformin in vivo. To model the oncogenic PIK3CA-driven early stages of cancer, we used the clonal breast cancer cell line MCF10DCIS.com, which harbors the gain-of-function H1047R hot-spot mutation in the catalytic domain of the PI3KCA gene and has been shown to form DR-refractory xenotumors. To model PIK3CA-activating mutations in late stages of cancer, we took advantage of the isogenic conversion of a PIK3CA-wild-type tumor into a PIK3CA H1047R-mutated tumor using the highly metastatic colorectal cancer cell line SW48. MCF10DCIS.com xenotumors, although only modestly affected by treatment with oral metformin (approximately 40% tumor growth inhibition), were highly sensitive to the intraperitoneal (i.p.) administration of metformin, the anti-cancer activity of which increased in a time-dependent manner and reached >80% tumor growth inhibition by the end of the treatment. Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues. Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing metformin significantly reduced the growth of SW48-mutated (PIK3CAH1047R/+) xenotumors by approximately 50%. Thus, metformin can no longer be considered as a bona fide DR mimetic, at least in terms of anti-cancer activity, because tumors harboring the insulin-unresponsive, DR-resistant, PIK3CA-activating mutation H1047R remain sensitive to the anti-tumoral effects of the drug. Given the high prevalence of PIK3CA mutations in human carcinomas and the emerging role of PIK3CA mutation status in the treatment selection process, these findings might have a significant impact on the design of future trials evaluating the potential of combining metformin with targeted therapy.

Show MeSH
Related in: MedlinePlus