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TRPM5 Mediates Acidic Extracellular pH Signaling and TRPM5 Inhibition Reduces Spontaneous Metastasis in Mouse B16-BL6 Melanoma Cells

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TRPM5 Mediates Acidic Extracellular pH Signaling and TRPM5 Inhibition Reduces Spontaneous Metastasis in Mouse B16-BL6 Melanoma Cells

Toyonobu Maeda et al. Oncotarget.

Abstract

Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pH e ) has been found to increase intracellular Ca2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca2+-dependent monovalent cation channel, is associated with acidic pH e signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pH e -induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pH e -induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pH e critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pH e . Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pH e signaling and may be a promising target for preventing metastasis of some types of tumor.

Keywords: MMP-9; TRPM5; acidic extracellular pH; melanoma; metastasis.

Conflict of interest statement

CONFLICTS OF INTEREST We have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1
Expression of Trpm1 (A) and Trpm5 (B) mRNAs by confluent cultures incubated with serum-free medium at the indicated pH for 24 h. Total RNA was extracted and reverse transcribed for RT-qPCR. Data were expressed, relative to the level of F1 cells at pHe 7.4, as mean ± SE in triplicate assay. *P<0.05, **P<0.01, ***P<0.001. NS, not significant.
Figure 2
Figure 2. Introduction of Trpm5 siRNA reduces acidic pHe-induced MMP-9 production and alters morphology to fibroblastic
(A) Reduction of Trpm5 mRNA by transfection of Trpm5 siRNA. PCR product was separated on agarose gel and stained with ethidium bromide. Reduction rate by siRNA was quantified by qPCR (see Supplementary Figure 1). (B) Inhibition of acidic pHe-induced MMP-9 production by transfection of Trpm5 siRNA. Following transfection, the cells were incubated in serum-free medium at neutral and acidic pH. The MMP-9 concentration of conditioned medium was analyzed by gelatin zymography. (C) Densitometric analysis of MMP-9 activity on zymogram in B (n = 3). Data were expressed relative to maximum induction at pHe 5.9. (D) Acidic pHe induced fibroblastic shape, but this change was inhibited by introduction of Trpm5 siRNA. Representative results are shown. Bar, 50 µm. Cell shapes are highlighted in yellow line and their quantification is shown in Supplementary Figure 2.
Figure 3
Figure 3. Constitutive expression of Trpm5 mRNA does not affect cell growth but increases the induction of MMP-9 and actin reorganization by acidic pHe
(A) Western blotting of representative clones of Trpm5-expressing vector transfected cells. (B) Growth curves of the representative clones (n = 3). (C) Zymographic analysis of acidic pHe-induced MMP-9 secretion from clone #9 of mock and #16 of Trpm5 transfectants. This panel shows application of 1/4 samples to see differences in expression. (D) Densitometric analysis of the results consisting of three-clone each transfectants: Mock (# 3, #7, and #9 in (C)) and Trpm5 (#8, #10, and #16 in (C)). (E) Rhodamine-phalloidin staining. Arrowheads show actin stress fibers, whose quantification is shown in Supplementary Figure 3.
Figure 4
Figure 4. Constitutive Trpm5 mRNA expression increases experimental pulmonary metastasis through tail vein injection of B16-BL6 cells
Representative clones of cells transfected with empty vector (control) and Trpm5-expressing vector (Trpm5) were grown individually, pooled and injected into tail veins of mice. (A) Photograph of a lung 3 weeks after injection. (B) Metastasized foci at the lung surface. Data are represented as mean ± SE (n = 15). ***P < 0.01.
Figure 5
Figure 5. TPPO treatment inhibits acidic pHe-induced MMP-9 production
Cells were treated with the indicated concentration of TPPO in serum-free medium for 24 h. (A) Conditioned media were concentrated and analyzed by zymography. (B) Densitometric analysis of the results in (A). (C) Cells were cultured with the indicated concentrations of TPPO in the presence of 10% FBS for 2 days, and cell survival was measured by the CCK8 assay. (D) Cells were pre-treated for 1 h and treated for a further 24 h with 50 mM of TPPO in the absence of serum. Total RNA was extracted, reverse-transcribed, and amplified by qPCR. Data are represented as mean ± SE (n = 3). *P < 0.05, **P < 0.01.
Figure 6
Figure 6
TPPO treatment inhibits acidic-pHe-induced NF-κB activity (A) and Mmp9 (B), Vim (vimentin) (C), and Cdh2 (N-cadherin) (D) mRNA levels. (A) Cells were transfected with NF-κB-driven luciferase vector, pre-treated with 50 mM of TPPO in serum-free medium at neutral pHe for 1 h, and then treated with 50 mM of TPPO in serum-free medium at acidic pHe for 24 h. Cell lysates were collected and NF-κB-driven luciferase activity measured. (BD) Cells were pre-treated for 1 h and treated for a further 24 h with 50 mM of TPPO in the absence of serum. Total RNA was extracted, reverse-transcribed, and amplified by qPCR. Data are represented as mean ± SE (n = 3). *P < 0.05, **P < 0.01, ***P < 0.005.
Figure 7
Figure 7. TPPO administration reduces pulmonary metastasis without affecting tumor growth, body weight, or CBG
Cells were injected into the left footpads of mice. Beginning 2 days later, mice were subcutaneously injected with 10 mg/kg TPPO dissolved in DMSO every other day. Primary tumors were amputated 3 weeks later and treatment with TPPO continued for another 4 weeks. (A) Numbers of pulmonary metastasized foci (n = 17). (B) Volumes of primary tumors (n = 17). (C) Body weight (n = 17). (D) Tumor weight after the amputation of tumor tissue (n = 17). (E) CBG concentration (n = 3). Data are represented as mean ± SE. *P < 0.05.
Figure 8
Figure 8. Immunohistochemical analysis of TRPM5 expression in clinical specimens of human melanoma
Representative results of immunohistochemical analysis of TRPM5 expression in primary melanoma (skin) (A) with immunoreactivity evaluated as +++ and secondary melanoma (lung) (B) with immunoreactivity evaluated as +++. The arrow indicates endothelial cells, defined as the standard for immunoreactivity. Data are summarized in Table 1. Scale bar, 100 µm.
Figure 9
Figure 9. TRPM5 mRNA expression correlates with survival rate of patients with melanoma and gastric cancer
(A) Melanoma. (B) Gastric cancer. (C) Ovarian cancer. (D) Lung cancer. (E) Breast cancer. (F) Rectal cancer. Data were obtained from the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo) for melanoma (GSE19234) and rectal cancer (GSE87211) and from the KM-plotter database (http://kmplot.com/analysis/index.php?p=background) for gastric, ovarian, breast, and lung cancer.

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