Transient Receptor Potential Melastatin 8 (TRPM8) Channel Regulates Proliferation and Migration of Breast Cancer Cells by Activating the AMPK-ULK1 Pathway to Enhance Basal Autophagy

Abstract

The calcium-permeable cation channel TRPM8 (transient receptor potential melastatin 8) is a member of the TRP superfamily of cation channels that is upregulated in various types of cancer with high levels of autophagy, including prostate, pancreatic, breast, lung, and colon cancers. Autophagy is closely regulated by AMP-activated protein kinase (AMPK) and plays an important role in tumor growth by generating nutrients through degradation of intracellular structures. Additionally, AMPK activity is regulated by intracellular Ca²⁺ concentration. Considering that TRPM8 is a non-selective Ca²⁺-permeable cation channel and plays a key role in calcium homoeostasis, we hypothesized that TRPM8 may control AMPK activity thus modulating cellular autophagy to regulate the proliferation and migration of breast cancer cells. In this study, overexpression of TRPM8 enhanced the level of basal autophagy, whereas TRPM8 knockdown reduced the level of basal autophagy in several types of mammalian cancer cells. Moreover, the activity of the TRPM8 channel modulated the level of basal autophagy. The mechanism of regulation of autophagy by TRPM8 involves autophagy-associated signaling pathways for activation of AMPK and ULK1 and phagophore formation. Impaired AMPK abolished TRPM8-dependent regulation of autophagy. TRPM8 interacts with AMPK in a protein complex, and cytoplasmic C-terminus of TRPM8 mediates the TRPM8-AMPK interaction. Finally, basal autophagy mediates the regulatory effects of TRPM8 on the proliferation and migration of breast cancer cells. Thus, this study identifies TRPM8 as a novel regulator of basal autophagy in cancer cells acting by interacting with AMPK, which in turn activates AMPK to activate ULK1 in a coordinated cascade of TRPM8-mediated breast cancer progression.

Keywords: AMP-activated protein kinase(AMPK); LC3B; autophagy; cancer; transient receptor potential melastatin 8 (TRPM8).

Figure 1

RPM8 expression activates basal autophagy level. (A, B) HeLa cells were incubated with 10 M CQ for the indicated times. The cell lysates were subjected to western blot (WB) analysis using indicated antibodies (N = 3). (C, D) Construct for TRPM8 expression was transfected into HeLa cells for 48 h; the cells were used for WB analysis using indicated antibodies (N = 3). (E, F) Construct for ptfLC3 expression was cotransfected with the TRPM8 or control vector into HeLa cells for 48 h, and accumulation of yellow and red puncta were observed by fluorescence microscopy; representative confocal images are shown (scale bar, 20 μm). (G, H) WB analysis was performed in the lysates of HeLa cells transfected with TRPM8-specific siRNA to decrease TRPM8 expression (N = 3). (I, J) Yellow and red puncta in HeLa cells transfected with TRPM8 siRNA. N represents the number of replicate experiments. Ctrl, control; CQ, chloroquine; LC3, microtubule-associated protein 1 light chain 3. *P < 0.05; NS, not significant.

Figure 2

Mechanism of the stimulatory effect of TRPM8 channel function on basal autophagy. (A, B) HeLa cells were incubated with menthol (10 μM) for 48 h. The cells were lysed and assayed by western blot using indicated antibodies (N = 3). (C, D) WB analysis of HeLa cell lysates after treatment with 2 μM icilin, 0.5 μM AMTB, or their combination for 48 h (N = 3). (E, F) HEK293 cells were cotransfected with EGFP and wild type TRPM8, mutant, or a control vector. After 48 h of transfection, EGFP-positive cells were selected for recording of TRPM8 channel-mediated current. The number of cells used for current recordings: Vector (6); WT: (10); V976W (12). (G, H) HeLa cells were transfected with the constructs for the expression of wild type or mutant TRPM8. After 48 h of transfection, cells were lysed and subjected to WB analysis using indicated antibodies (N = 3). N represents the number of replicate experiments. EGFP, enhanced green fluorescent protein; Ctrl, control; *P < 0.05; NS, not significant.

Figure 3

Upregulation of autophagy by TRPM8 in various cancer cell lines. (A, B) The construct for TRPM8 expression was transiently transfected into MCF7 for 48 h; the cells were used for WB analysis using indicated antibodies to detect autophagy-associated proteins (N = 3). (C, D) Similar WB analysis of lysates of HCT116 cells transfected with TRPM8 (N = 3). (E–H) WB analysis of lysates of MDA-MB-231 cells treated with 10 μM menthol, 2 μM icilin, 0.5 μM AMTB, or combined treatments for 48 h (N = 3). N represents the number of replicate experiments. *P < 0.05; NS, not significant.

Figure 4

Involvement of the AMPK-ULK1-LC3 signaling cascade in TRPM8-stimulated autophagy. (A, B) The construct for Flag-AMPK expression was transiently transfected into MCF7 cells. After 48 h of transfection, the AMPK-ULK1-LC3 signaling cascade-related proteins were detected by WB (N = 3). (C, D) MCF7 cells were transiently transfected with wild type TRPM8, mutant V976W, or control vector. After 48 h of transfection, protein lysates were used for WB analysis (N = 3). (E, F) MDA-MB-231 cells treated with 2 μM icilin, 0.5 μM AMTB, or their combination for 48 h were extracted for WB analysis (N = 3). (G, H) MCF7 cells were transfected with siRNA against human TRPM8; siRNA against TRPM8 (siTRPM8-1 and siTRPM8-2) successfully knocked down TRPM8 expression compared with that in the control scramble siRNA samples according to WB analysis using an anti-TRPM8 antibody. The AMPK-ULK1-LC3 signaling cascade-related proteins detected by WB analysis using the indicated antibodies (N = 3). N represents the number of replicate experiments. *P < 0.05; NS, not significant.

Figure 5

Influence of AMPK impairment on the stimulatory effect of TRPM8 on autophagy. (A, B) MCF7 cells were transiently transfected with siRNA against human AMPK and a Flag-TRPM8 construct. After 48 h of transfection, protein lysates were extracted for WB analysis to determine the effect of TRPM8 overexpression on basal autophagy in the presence of AMPK knockdown (N = 3). (C, D) WB analysis of cell lysates of MDA-MB-231 cells treatment with 2 μM icilin, 10 μM menthol, or a combination of 10 M compound C for 48 h (N = 3). N represents the number of replicate experiments. *P < 0.05; NS, not significant.

Figure 6

AMPK interacts with TRPM8. (A, B) Co-IP analysis. (A) Constructs for GFP-TRPM8 and Flag-AMPK expression were transiently transfected into MCF7 cells. After 48 h of transfection, protein lysates were immunoprecipitated with an anti-GFP antibody and assayed by immunoblot with an anti-Flag antibody (lower panel). Reciprocal Co-IP with an anti-Flag antibody used for immunoprecipitation and anti-GFP used for WB analysis (upper panel) (N = 3). (B) The construct for GFP-AMPK expression was cotransfected with M8-N, M8-LI, M8-LII, or M8-C into MCF7 cells. Protein lysates were immunoprecipitated with an anti-Flag antibody and assayed by immunoblot with an anti-GFP antibody (upper). The constructs for GFP-AMPK and Flag-M8-C expression were cotransfected into MCF7 cells. Protein lysates were immunoprecipitated with an anti-GFP antibody and assayed by immunoblot with an anti-Flag antibody (upper) (N = 3). (C) GST pull-down analysis. Protein lysates of MCF7 cells transiently expressing Flag-AMPK were incubated with purified cytoplasmic C-terminus of TRPM8 GST fusion protein (GST-M8C). GST-M8C, but not control GST, successfully pulled down Flag-AMPK. PD: pull-down. The lysate was used as a positive control (N = 3). N represents the number of replicate experiments. PD, pull down; WCL, whole cell lysates.

Figure 7

Autophagy is essential for the regulatory effect of TRPM8 on the proliferation and migration of breast cancer cells. (A, B) Cell proliferation experiments. MCF7 cells were transfected with TRPM8 constructs. After 24 h of transfection, cells were treated with 10 M CQ. (A) The in vitro colony formation assay. Cells were further cultured in growth media for 7-10 days to form the colonies. The colonies were washed with ice-cold PBS three times, stained with trypan blue, and counted (N = 3). (B) Ki67 expression was detected by immunostaining (N = 3). (C) MDA-MB-231 cells were transiently transfected with siRNA against human ATG7 (siATG7) and a Flag-TRPM8 construct. After 48 h of transfection, protein lysates were extracted for WB analysis to determine the effect of TRPM8 overexpression on basal autophagy in the presence of ATG7 knockdown (N =3). (D, E) Similar experiment was performed in MDA-MB-231 cells transfected with siATG7 and a Flag-TRPM8 construct. (N = 3). (F, G) Cell migration determined by wound healing assay. (F) MCF7 cells were transfected with the TRPM8 constructs for 24 h and scratched with a sterile 10 μl tip. After three washes with 1× PBS, the cells were cultured for 24–72 h in serum-free medium in the presence of 10 μM CQ (N = 3). (G) Similar wound healing assay as in (F) but with MDA-MB-231 cells transfected with siATG7 and a Flag-TRPM8 construct (N = 3). N represents the number of replicate experiments. *P < 0.05; NS, not significant.