Caveolin-1-LRP6 signaling module stimulates aerobic glycolysis in prostate cancer

Abstract

Caveolin 1 (Cav-1) is a plasma membrane-associated protein with the capacity to modulate signaling activities in a context-dependent fashion. Interactions between Cav-1 and low-density lipoprotein receptor-related protein 6 (LRP6) were reported to be important for the regulation of Wnt-β-catenin (β-cat) signaling. Cav-1 also interacts with insulin and IGF-I receptors (IGF-IR/IR) and can stimulate IR kinase activities. We found positive correlation between Cav-1 and LRP6 expression in both human primary prostate cancer and metastasis tissues and in PC-3 cells. Cav-1 stimulation of Wnt-β-cat signaling and c-Myc levels was positively associated with LRP6 expression in LNCaP, PC-3, and DU145 prostate cancer cells. Importantly, LRP6 and, to a lesser extent, Cav-1 were found to stimulate aerobic glycolysis. These activities were positively associated with the expression of HK2 and Glut3 and shown to be dependent on Akt signaling by both gene knockdown and chemical inhibition methods. We further showed that Cav-1 and LRP6 exert their effects on Akt and glycolytic activities by stimulating IGF-IR/IR signaling. Overall, our results show that Cav-1 interacts with LRP6 to generate an integrated signaling module that leads to the activation of IGF-IR/IR and results in stimulation of Akt-mTORC1 signaling and aerobic glycolysis in prostate cancer.

Conflict of interest statement

Potential conflicts of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1

Cav-1 and LRP6 interaction in PCa. A, Positive association between Cav-1 and LRP6 expression in benign prostate epithelia (Benign) adjacent to human primary PCa, and bone metastases (Mets) was shown by immunohistochemical staining. Cav-1 and LRP6 levels were both low in Benign but Cav-1+ cancer cells exhibited strong LRP6 imunostaining (second row,), whereas the Cav-1− PCa cells demonstrated minimal level of LRP6 (third row). In bone metastases, Cav-1+ cancer cells had high level of LRP6 (bottom panels). Original magnification: 200x. B, the average LRP6 immunostaining score in the Cav-1+ PCa were significantly higher than in the Cav-1−area of the same PCa tissue (means± SEM, *P<0.05). C, PC-3 cells were treated with LRPsi, Cav si, and NC si and the expression of LRP6 and Cav-1 mRNA was determined by qRT-PCR analysis after 24 hours. Data are the ratio of the means ± SD relative to that in NCsi from 3 independent experiments. **P < 0.0001,(compared to NC si). D, PC-3 cell lysates were prepared after 48 hours of transfection with LRP6 si, Cav-1si, or NCsi and analyzed by Western blotting. Cav-1– or LRP6-specific siRNA 1 and 2 are from Invitrogen and Santa Cruz respectively.

Figure 2

Cav-1 stimulation of the Wnt–β-cat signaling pathway A, Cav-1 was overexpressed in LNCaP cells after infection with AdCa . B, PC-3 cells were treated with Cavsi or control NCsi. Both LNCaP and PC-3 cells were2 incubated for 48 hours and treated with rhWnt3a (25 ng/mL) for the indicated times. C, Cav-1 knockdown (Cavsh11 and Cavsh11G) and NC stable clones of DU145 cells were treated with rhWnt3a (25 ng/mL) for 45 minutes. D, PC-3 and LNCaP cells treated with rhWnt3a (50 ng/mL) and rhDKK1 (100 ng/mL) for 1 hour.

Figure 3

LRP6 and Cav-1 stimulate glycolysis and cell proliferation by regulating the expression of Akt, c-myc, HK2, and Glut3. A, For 48 hours, PC-3 cells were transfected with Cavsi, LRPsi, or control NCsi;,LNCaP with AdCa and AdCt and then subsequently transfected with LRPsi and NCsi. β-Actin was used as the loading control. B, Lactate accumulation in the medium of treated PC-3 and LNCaP cells. The lactate concentrations were normalized to viable cell number. C, Cellular glucose uptake was determined by measuring intracellular 2-[3H]deoxyglucose at 48 hours. D, PC-3 cell proliferation after 48 hours of transfection with LRPsi, Cavsi, or NCsi. B–D, data are means ± SD from 3 independent experiments. *P < 0.05, **P < 0.01, Student’s t test.

Figure 4

LRP6 and Cav-1 stimulate glucose metabolism via the activation of Akt signaling and independent of β-cat signaling. A &C, Lactate accumulation in the medium of treated PC-3 and LNCaP cells was measured using a lactate assay kit. Lactate concentrations were normalized to viable cell number.. Data are means ± SD from 3 independent experiments. *P < 0.05, **P < 0.01, Student’s t test. In A, β-cat knockdown in PC-3 and LNCaP cells was confirmed by immunoblotting. B, PC-3 cells were treated with Akti1/2 (1 µM) and rapamycin (Rapa; 50 nM) for 1 hour, and cell lysates were analyzed by Western blotting. D, PC-3 cells were treated with LRPsi, Cavsi, and NCsi for 48 hours, and the levels of Raptor and P-Raptor were assessed by immunoblotting.

Figure 5

LRP6 and Cav-1 stimulate IGF-IR/IRmTORC1 signaling. A, PC-3 cells transfected with LRPsi, Cavsi, and NCsi..Cav-1 was overexpressed in LNCaP cells by infection with AdCa (or `AdCt) and then transfected with LRPsi or NCsi. β-Actin as the loading control. B, PC-3 cells were transfected with LRPsi, Cavsi, IRsi, IGF-IRsi, and NCsi for 40 hours were serum starved for 8 hours, and treated with Wnt3a (50 ng/mL) for 30 minutes. C, PC-3 cells were serum starved for 8 hours, treated with Wnt3a (50 ng/mL) and PPP (0.5 µM) or AG1024 (1.0 µM) for 30 minutes. D, PC-3 cells were transfected as in (B). LNCaP cells were infected with AdCa (or AdCt) and subsequently transfected with LRPsi, Cavsi, NCsi, IRsi, IGF-IRsi, or NCsi.The concentration of lactate accumulation in the medium of the treated PC-3 and LNCaP cells was measured after normalization to viable cells. Data are plotted as means ± SD from 3 independent experiments. *P < 0.05, **P < 0.005. A–D: The cells were incubated for 48 hours and analyzed by Western blotting. E, LNCaP cells were transfected with Cav-1V5His or LRP6Flag, then immunoprecipitated with anti-Flag, anti-V5 mouse mAb, anti IR, or anti–IGF-IR rabbit pAb. Anti-Flag and anti-V5 immunocomplexes were analyzed using rabbit pAb anti-Flag, anti-V5, anti-IR, and anti–IGF-IR, and the anti-IR or anti–IGF-IR immunocomplexes were analyzed using mouse mAb anti-Flag, anti-V5, anti-IR and rabbit mAb anti–IGF-IR.

Figure 6

Diagram illustrates Cav-1–LRP6 cross-talk stimulates IGF-IR/IR phosphorylation in the lipid raft, causes activation of PI3K signaling, and results in increased expression of HK2 and Glut3, which directly stimulate aerobic glycolysis.