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. 2010 Mar;3(3):359-70.
doi: 10.1158/1940-6207.CAPR-09-0068. Epub 2010 Feb 9.

Elevated phosphate activates N-ras and promotes cell transformation and skin tumorigenesis

Corinne E Camalier  1 , Matthew R Young, Gerd Bobe, Christine M Perella, Nancy H Colburn, George R Beck Jr
Affiliations

Elevated phosphate activates N-ras and promotes cell transformation and skin tumorigenesis

Corinne E Camalier et al. Cancer Prev Res (Phila). 2010 Mar.

Abstract

Recent results suggest a paradigm shift from viewing inorganic phosphate as a passive requirement for basic cell functions to an active regulator of cell behavior. We have previously shown that elevated concentrations of phosphate increased cell proliferation and expression of protumorigenic genes such as Fra-1 and osteopontin in a preosteoblast cell line. Therefore, we hypothesized that elevated phosphate concentrations would promote cell transformation in vitro and tumorigenesis in vivo. Supplementation of medium with phosphate increased anchorage-independent transformation and proliferation of BALB/c mouse JB6 epidermal cells, activation of N-ras, ERK1/2, and activator protein-1, and increased gene expression of Fra-1, COX-2, and osteopontin in a dose-dependent manner. These in vitro results led to the hypothesis that varying the levels of dietary inorganic phosphate would alter tumorigenesis in the mouse model of skin carcinogenesis. Female FVB/N mice were treated with 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate and fed high- or low-phosphate diets (1.2% versus 0.2% of the diet) for 19 weeks. The high-phosphate diet increased skin papilloma number by approximately 50% without changing feed intake and body weights. High dietary phosphate increased serum concentrations of phosphate, parathyroid hormone, and osteopontin and decreased serum concentrations of calcium. Thus, we conclude that elevated phosphate promotes cell transformation and skin tumorigenesis partly by increasing the availability of phosphate for activation of N-ras and its downstream targets, which defines reducing dietary phosphate as a novel target for chemoprevention.

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Figures

Figure 1
Figure 1. High inorganic phosphate (Pi) concentrations increase proliferation, anchorage independent growth, and transformation-responsive gene expression in transformation-sensitive JB6 cells
(A) High Pi increases proliferation of JB6 cells: cells were plated in a 96 well plate at 1×104/100 μL and incubated for 36 hr in final medium concentrations of 1, 2, and 3 mM Pi. Cell growth was measured using XTT assay and results are expressed as the mean XTT reading ± SEM (6 replicates/treatment). Columns with different characters differ at p<0.001. (B) High PI increases cell number: JB6 cells were plated at 5×104 cells/ml and grown in 1 or 3 mM phosphate (final) for 96 hr and cell number recorded. Columns with different characters differ at p<0.05. (C) High Pi increases mitotic cells: JB6 cells were grown in 1 or 3mM final Pi for 36 hr and analyzed by flow cytometry. Columns with different characters differ at p<0.05. (D) High Pi increases anchorage independent growth of JB6 cells in soft agar: JB6 cells (1×104 cells/100 μL) were plated for 14 d in the presence of DMSO (control) or 10ng/mL TPA in agar containing 1.2, 2.2, or 3.5 mM Pi. Colony number, expressed as the mean colony number ± SEM (5 replicates/treatment), was quantified by staining with neutral red and analyzed by automated microscopy. Columns with different characters differ at p<0.05. (E) High Pi increases transformation responsive gene expression in JB6 cells: cells were grown for 7 days in medium supplemented with 1.2, 2.2, or 3.7 mM Pi. The RNA was harvested for Northern blotting and probed for osteopontin (OPN), Fra-1, Cox-2, and actin (representative of multiple experiments). (F) High Pi augments TPA-induced transformation responsive gene expression: JB6 cells were grown in the presence of 10 ng/mL TPA in DMSO for 24 hr in a medium containing 0.2, 1.2, 2.4, or 4.8 mM Pi. The resulting RNA samples were analyzed by Northern blotting for OPN, 18S rRNA, and 28S rRNA. (G) High Pi increases early gene expression: JB6 cells were cultured in growth medium (1mM Pi) followed by addition of 4mM Pi (5mM final) for the indicated times. The resulting RNA samples were analyzed by Northern blotting and probed as indicated.
Figure 2
Figure 2. High inorganic phosphate (Pi) concentrations increase AP-1 activation in transformation-sensitive JB6 cells
(A) High Pi increases AP-1 transcriptional activity in JB6 cells: cells were transfected with a 4xAP-1 luciferase and Renilla reporter constructs, and after 24 hrs serum starved overnight. Medium was then supplemented to a final concentration of 1, 2, or 3mM Pi in the presence of DMSO (control) or 10ng/ml TPA in DMSO for 6 hrs. AP-1 transcriptional activity was measured using a luciferase reporter assay (6 replicates/treatment) and Renilla by Stop-and-Glo. Luciferase activity was then normalized to Renilla and averaged. Columns with different characters differ at p<0.05. (B) High Pi increases within 5 min AP-1 DNA binding in JB6 cells: cells were supplemented with 2mM Pi for 0, 5, 10, 15, 30, or 60 min and EMSA was performed using an oligo containing a consensus AP-1 binding element. (C) High Pi primarily increases within 5 min DNA binding of the AP-1 proteins Fra-2, JunB, and FosB: cells were supplemented with 2 mM Pi for 5 min and EMSA was performed using an oligo containing a consensus AP-1 element and antibodies specific to the 7 individual AP-1 family members (Fra-1, Fra-2, c-fos, FosB, JunB, c-jun, JunD) and CREB (representative of multiple experiments). (D) High Pi alters AP-1 protein forms: JB6 cells were serum starved (1mM Pi) followed by addition of 2mM Pi (3mM final) for the indicated times. Nuclear lysates were analyzed by Western blotting and probed as indicated. (E) High Pi increases Egr1 expression within 20 minutes. JB6 cells were treated as in D and cells harvested for Northern analysis. Results are representative of multiple experiments.
Figure 3
Figure 3. High inorganic phosphate (Pi) concentrations activate N-ras in transformation-sensitive JB6 cells
(A) High Pi increases within 5 min ras activity and phosphorylation of ERK1/2 in JB6 cells: cells were serum starved overnight and supplemented with 2 or 4 mM Pi for 5 min (1, 3, and 5 mM final). Ras activation of whole cell lysate blots was measured using the Ras-Binding Domain of Raf-1 and detected using pan-ras antibody. Phosphorylation of ERK1/2 was measured from a duplicate whole cell lysate blot using phospho-ERK1/2 antibody. (B) High Pi increases within 2.5 min N-ras activity and phosphorylation of ERK1/2 in JB6 cells: cells were serum starved overnight and treated with 2mM Pi (3mM final) for 0, 2.5, 5, 10, and 15 min. A ras activation assay was performed (Active ras) and whole cell lysate (WCL) from the same samples were probed with antibodies for N-ras and phospho-ERK1/2, respectively. (C) Quantitation of N-ras activity at 0, 2.5, and 5 minutes from 3 separate experiments: Results are expressed as arbitrary units obtained from densitometry analysis. Columns with different characters differ at p<0.05. (D) Phosphate transport is required for Pi to increase N-ras activity: cells were serum starved overnight and supplemented for 2.5 min with 0 or 2mM Pi and Vehicle (H2O) or 1mM of the phosphate transport inhibitor foscarnet (phosphonoformic acid). Ras activation (Active ras) and whole cell lysate (WCL) blots were probed with antibodies for N-ras. (E) Inhibition of GDP/GTP exchange blocks Pi induced ERK1/2 phosphorylation: JB6 cells were serum starved overnight (1mM Pi) and pretreated with 2.5mM GDPβS. The cells were treated with 2mM Pi (3mM final) for 10 minutes and resulting cell lysates analyzed by Western blotting.
Figure 4
Figure 4. High phosphate diet promotes skin tumorigenesis in DMBA/TPA-treated FVB/N mice
(A) A high phosphate diet increases skin papilloma number in DMBA/TPA-treated FVB/N mice: skin papilloma numbers were counted weekly from 14 mice/group, which were fed for 19 wk high (1.2% Pi, HPD) or low phosphate diets (0.2% Pi, LPD) starting with the DMBA initiation. Stars indicate differences between dietary groups at p < 0.05. (B) A high phosphate diet accelerates early skin papilloma growth: skin papilloma dimensions were measured weekly from 14 mice/group, which were fed for 19 wk HPD or LPD starting with the DMBA initiation. Stars indicate differences between dietary groups at p < 0.05. (C) A high phosphate diet accelerates first appearance of skin papilloma number in DMBA/TPA-treated FVB/N mice: skin papilloma numbers were counted weekly from 14 mice/group, which were fed for 19 wk high (1.2% Pi, HPD) or low phosphate diets (0.2% Pi, LPD) starting with the DMBA initiation. Stars indicate differences between dietary groups at p ���0.05. (D) The low phosphate diet does not affect body weight in DMBA/TPA-treated FVB/N mice: Individual body weight was measured every 3 weeks from 14 mice/group, which were fed for 19 wk HPD or LPD starting with the DMBA initiation.
Figure 5
Figure 5. High phosphate diets increase serum concentrations of parathyroid hormone (PTH) in FVB/N mice and osteopontin (OPN) in TPA-treated FVB/N mice
(A) A high phosphate diet increases serum concentrations of parathyroid hormone (PTH), but not IGF-I in FVB/N mice: serum samples were taken from 5 mice per group, which consumed for 5 wk high (1.2% Pi, HPD) or low phosphate diets (0.2% Pi, LPD), and analyzed for PTH and IGF-I. Columns with different characters differ at p < 0.05. (B) A high phosphate diet increases serum concentrations of OPN in TPA-treated FVB/N mice: serum samples were taken from 6 mice per group, which consumed for 2 wk high (1.2% Pi, HPD) or low phosphate diets (0.2% Pi, LPD), and then were treated topically 6 hr before sample collection with acetone (control) or 10nM of TPA in acetone. Columns with different characters differ at p < 0.01.
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