TRPM7 silencing modulates glucose metabolic reprogramming to inhibit the growth of ovarian cancer by enhancing AMPK activation to promote HIF-1α degradation

Abstract

Background: Tumor cell metabolic reprogramming is crucial for the malignant behavior of cancer cells by promoting their proliferation. However, little is known on how transient receptor potential 7 (TRPM7) modulates metabolic reprogramming in ovarian cancer.

Methods: The effects of TRPM7 silencing on transcriptome profile, glucose uptake, lactic acid production, extracellular acidification rate (ECAR), oxygen consumption rate (OCR), intracellular ROS and ATP levels, and NAD+/NADH ratios in ovarian cancer cells were examined. The impacts of TRPM7 silencing on the levels of glycolysis-related HK2, PDK1 and oxidative phosphorylation (OXPHOS)-related IDH3B and UQCRC1, HIF-1α expression and AMPK phosphorylation were determined in ovarian cancer. The effect of AMPK activity on HIF-1α ubiquitination degradation was investigated in ovarian cancer cells.

Results: Compared with the control, TRPM7 silencing suppressed the proliferation of ovarian cancer cells by shifting preferable glycolysis to OXPHOS. In parallel, TRPM7 silencing decreased the glucose uptake of tumor-bearing mice and TRPM7 levels were negatively correlated with IDH3B and UQCRC1, but positively with HK2 and PDK1 expression in ovarian cancer tissues. Mechanistically, TRPM7 silencing significantly increased AMPK phosphorylation and decreased HIF-1α protein levels in ovarian cancer, particularly in HIF-1α silencing cells. The shifting from glycolysis to OXPHOS by TRPM7 silencing was abrogated by HIF-1α over-expression and impaired by inhibiting AMPK activity in ovarian cancer cells. Moreover, enhanced AMPK activation inhibited glycolysis, which was abrogated by HIF-1α over-expression in ovarian cancer cells. Moreover, the enhanced AMPK activation promoted HIF-1α ubiquitination degradation.

Conclusions: TRPM7 silencing enhanced AMPK activation to shift glycolysis to oxidative phosphorylation by promoting HIF-1α ubiquitination degradation in ovarian cancer. Hence, TRPM7 may be a therapeutic target for intervention of ovarian cancer.

Keywords: AMPK; Glycolysis; HIF-1α; Ovarian cancer; Oxidative phosphorylation; TRPM7; Ubiquitination.

Fig. 1

TRPM7 promotes the growth of ovarian cancer. A A volcano plot displayed the DEGs between sh-control SKOV3 and sh-TRPM7 SKOV3 cells. B GSEA analysis predicted that TRPM7 was involved in cell cycling and positive regulation of cell mitosis and acute inflammatory. C EdU assay exhibited that TRPM7 silencing inhibited cell proliferation. Scale bar = 50 μm. D TRPM7 silencing inhibited the growth of xenograft SKOV3 in vivo. E GO analysis exhibited that the DEGs were enriched in the indicated biological processes in the TRPM7 silencing SKOV3 cells. F Circos plot displayed the relationships between the DEGs and potential functions in TRPM7 silencing SKOV3 cells. (G) GSEA analysis predicted that TRPM7 was involved in the AMPK pathway

Fig. 2

TRPM7 silencing shifts glycolysis to OXPHOS in ovarian cancer cells. A The effect of TRPM7 silencing on the glucose uptake levels, (B) extracellular lactate levels, (C) ECARs, (D) OCRs, (E) NAD⁺/NADH ratios, (F) ATP levels, (G) intracellular ROS levels in SKOV3 and HO8910 cells. H IHC analysis of TRPM7 expression in SKOV3 xenograft tumors. Scale bar = 50 μm. I Representative photographs of 18F-FDG PET/CT scans of SKOV3 xenograft tumors in mice following treatment with, or without, carvacrol, a potent inhibitor of TRPM7. SKOV3 and carvacrol were injected to right forelimbs. The SUV max was lower in the carvacrol group than in the control group (n = 7 per group)

Fig. 3

TRPM7 silencing reduces the expression of glycolysis-related regulators, but enhances the OXPHOS-related regulators in ovarian cancer cells. A IHC analysis of TRPM7, HK2, PDK1, IDH3B and UQCRC1 expression in human ovarian cancer tissues and non-tumor ovarian tissues. Scale bar = 50 μm. B Correlation analysis between the levels of TRPM7 and HK2, PDK1, IDH3B, or UQCRC1 expression in ovarian cancer tissues. C-D Western blot and RT-qPCR analyses of HK2, PDK1, IDH3B and UQCRC1 expression in the indicated cells. E Immunofluorescent analysis of HK2, PDK1, IDH3B and UQCRC1 expression in ovarian cancer cells after stained with mouse anti-HK2, rabbit anti-PDK1, mouse anti-IDH3B, rabbit anti-UQCRC1 and subsequent Alexa Fluor™488-goat anti-mouse IgG and Alexa Fluor™ 594-goat anti-rabbit IgG as well as DAPI, scale bar = 50 μm. F Western blot analysis of PKM2 in the nuclear and cytoplasmic fractions following TRPM7 silencing in SKOV3 and HO8910 cells. G IHC analysis of HK2, PDK1, IDH3B and UQCRC1 expression (scale bar = 50 μm)

Fig. 4

TRPM7 silencing enhances the AMPK activation, which is associated with inhibiting glycolysis in ovarian cancer cells. A Western blot and IHC analyses of HIF-1α expression and AMPK phosphorylation in the indicated cells and xenograft SKOV3 tumors (scale bar = 50 μm). B, C, F Treatment with CC to inhibit AMPK activation enhanced glycolysis, which was mitigated by HIF-1α silencing, by measuring glucose uptake (B), lactic acid production (C) and ECAR (F) in SKOV3-sh-control, SKOV3-sh-TRPM7, HO8910-sh-control and HO8910-sh-TRPM7 cells. D, E, G Treatment with metformin to enhance the AMPK activation attenuated glycolysis, which was abrogated by HIF-1α over-expression, by measuring glucose uptake (D), lactic acid production (E) and ECAR (G) in the indicated cells

Fig. 5

The shifting glycolysis to OXPHOS by TRPM7 silencing is abrogated or mitigated by HIF-1α over-expression or AMPK inhibition in ovarian cancer cells. HIF-1α over-expression abrogated the TRPM7 silencing-promoted glycolysis in ovarian cancer cells, determined by the glucose uptake (A), lactic acid production (B) and ECAR (C). Treatment with CC (20 μM) to inhibit AMPK activation mitigated the TRPM7 silencing-induced OXPHOS in ovarian cancer cells, determined by the ATP levels (D), ROS levels (E), NAD⁺/NAPH ratios (F) and OCR (G)

Fig. 6

Altered AMPK activation or HIF-1α expression modulates the TRPM7 silencing-decreased expression of glycolysis-relative regulators and increased expression of OXPHOS-relative regulators in ovarian cancer cells. A-D Western blot analysis of IDH3B, UQCRC1, HIF-1α, HK2, PDK1 expression and AMPK phosphorylation in the indicated groups of cells

Fig. 7

TRPM7 silencing activates the AMPK signaling to promote ubiquitination and degradation of HIF-1α in ovarian cancer cells. A Western blot displayed that TRPM7 silencing decreased HIF-1 protein levels in ovarian cancer cells under a hypoxic condition. B In vivo ubiquitination assays indicated that the decreased HIF-1α protein by TRPM7 silencing was abrogated by treatment with MG132, suggesting that TRPM7 silencing promoted HIF-1α ubiquitination and degradation in SKOV3 cells. C There was no detectable HIF-1α protein in the SKOV3 cells under a normoxic condition. D Inhibition of AMPK activation by CC increased HIF-1α protein levels in both control and TRPM7 silencing SKOV3 cells under a hypoxic condition. E, F In vivo ubiquitination assays revealed that treatment with CC to inhibit AMPK activation mitigated the TRPM7 silencing-induced HIF-1α ubiquitination and degradation in ovarian cancer cells under both normoxic and hypoxic conditions

Fig. 8

TRPM7 silencing shifts glycolysis to OXPHOS by modulating the AMPK / HIF-1α signaling in ovarian cancer cells. TRPM7 silencing can enhance the AMPK activation promote HIF-1α ubiquitination proteasomal degradation that attenuates the HIF-1α-enhanced glycolysis to shift glycolysis to OXPHOS, inhibiting ovarian cancer cell proliferation and tumor growth