Proprotein convertase subtilisin/kexin type 9 deficiency reduces melanoma metastasis in liver

Abstract

High circulating cholesterol is associated with hypercholesterolemia, atherosclerosis, and stroke. However, the relation between cholesterol and tumorigenesis/metastasis is controversial. The proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low-density lipoprotein cholesterol homeostasis by targeting the low-density lipoprotein receptor (LDLR) for degradation. PCSK9 is mostly expressed in liver, which is one of the most common sites for metastatic disease. To reveal the function of PCSK9 and also evaluate the impact of cholesterol in liver metastasis development, B16F1 melanoma cells were injected into wild-type (WT) and Pcsk9(-/-) mice to induce liver metastasis. On chow diet, Pcsk9(-/-) mice harbored two-fold less liver metastases than WT mice. This decrease is related to low cholesterol levels in Pcsk9(-/-) mice, as the protection was lost after normalizing Pcsk9(-/-) cholesterol levels by a 2-week high cholesterol diet. Furthermore, a prolongation of this diet strongly increased metastasis in both genotypes, suggesting that high cholesterol levels promote metastatic progression. The protective effect of the PCSK9 deficiency is also associated with increased apoptosis in liver stroma and metastases. Tumor necrosis factor.α (TNFα) mRNA and protein were, respectively, higher in liver stroma and plasma of injected mice, likely increasing the apoptotic TNFα signaling. Furthermore, the anti-apoptotic factor B-cell lymphoma 2 was downregulated. TNFα regulation is LDLR-independent, as its mRNA level was similarly upregulated in mice lacking both PCSK9 and LDLR. Our findings show that PCSK9 deficiency reduces liver metastasis by its ability to lower cholesterol levels and by possibly enhancing TNFα-mediated apoptosis.

Figure 1

PCSK9 is upregulated in intestinal tumors. An Apc^min/+ mouse small intestine section stained with hematoxylin and eosin shows the structure of an intestinal tumor (T) and its adjacent normal tissue (N). Expression of PCSK9, LDLR, and HMG-CoA reductase was assessed by qPCR in 18 couples of tumors and their adjacent normal tissues collected from three Apc^min/+ mice of 4 months of age. mRNA levels in tumors were normalized to that of their respective adjacent stromal tissues, which was set to 1. Error bars represent SEM. *P < .05; **P < .01 (Student's t test).

Figure 2

The loss of PCSK9 decreases hepatic metastasis in a cholesterol-dependent manner. (A) Mice were fed a chow diet or an HCD for 14 or 26 days before euthanasia and were injected with B16F1 cells 12 days before euthanasia. Livers were dissected and representative livers and liver sections are shown. Insets emphasize the accumulation of lipid droplets in the livers of mice fed an HCD for 26 days. (B) Tumor density (area of tumors/total area of liver) was evaluated in 12 to 18 mice per genotype and per condition. (C) Total cholesterol levels were measured in plasma collected on day of sacrifice. Error bars represent SEM. *P < .05, **P < .01, ***P < .001 (Student's t test).

Figure 3

B16F1 cells do not express PCSK9 and their LDLR is insensitive to exogenous PCSK9. (A) PCSK9 expression was quantified by qPCR in HepG2 cells, B16F1 cells, and B16F1 cell-derived liver tumors (n = 3 for each). As a control, expression of the ubiquitous furin was also quantified. Error bars represent SEM. (B) Media of HEK293 cells that express the empty pIRES2-EGFP vector, mouse (mWT) or human (hWT) WT PCSK9, or human gain-of-function PCSK9^D374Y (hD347Y), all containing a C-terminal V5 tag, were collected and transferred onto B16F1 or HepG2 cells for 12 hours. HEK293 cell media and B16F1 and HepG2 cell lysates were analyzed by Western blot analysis using a V5 Ab to reveal PCSK9 and mouse or human LDLR and β-actin Abs. Band intensities were quantified by ImageJ software and LDLR intensities were first normalized to that of β-actin (LDLR/act). Ratios were then normalized to that of the empty vector that was set to 1. A representative experiment of three is shown.

Figure 4

The adhesion of B16F1 cells to primary hepatocytes is regulated by PCSK9 and cholesterol. B16F1 cells were incubated with ³H-thymidine for 24 hours and then added onto WT, Pcsk9^-/-, Ldlr^-/-, and Ldlr^-/-Pcsk9^-/- primary hepatocytes, which were pre-incubated in media containing either 10% FBS or 10% LPDS for 3 hours. After 1 hour of incubation, unadhered B16F1 cells were washed out, the remaining cells were lysed, and radioactivity in the total lysates was counted. The error bars indicate SEM of four independent experiments. *P < .05; (Student's t test).

Figure 5

PCSK9 affects cell proliferation and apoptosis of hepatic cells but not those of B16F1 cells. (A) Proliferation of B16F1 cells incubated with or without 5 µg/ml of purified PCSK9, and grown in media containing 10% FBS, was measured using an MTS assay. (B) B16F1 cells stably expressing the empty pIRES2-EGFP vector (pIR), V5-tagged human PCSK9 (PCSK9), or its variant PCSK9^D374Y (D347Y) were grown in media containing 10% FBS or 10% LPDS and their proliferation was measured. PCSK9 expression in these cell lines was assessed by Western blot analysis of serum-free media using a V5 Ab (inset). (C) HepG2 cells stably expressing NTsh, PC9sh, or human PCSK9 were grown in media containing 10% FBS and their proliferation was measured. PCSK9 expression and efficiency of PCSK9 knockdown in these cell lines were assessed by Western blot analysis of serum-free media using a V5 Ab (inset). (D) Caspase-3 activity was measured in HepG2 cells stably expressing NTsh and PC9sh, WT and Pcsk9^-/- primary hepatocytes, and B16F1 cells pre-incubated with or without 5 µg/ml of purified PCSK9 for 24 hours, either in the absence or presence of 1 µM staurosporine. (E) Caspase-3 activity was assessed in non-injected liver extracts and in liver stroma and tumor extracts obtained from mice 12 days post-B16F1 cell injection. Liver sections from both non-injected and B16F1 cell-injected WT and Pcsk9^-/- mice were submitted to a TUNEL assay. Arrows point at labeled apoptotic nuclei and dashed lines separate tumoral (T) and stromal (S) areas. The error bars indicate SEM of three independent experiments (A–D) or data for six mice per genotype (E). *P < .05, **P < .01, ***P < .001 (Student's t test).

Figure 6

TNFα pathway is altered in Pcsk9^-/- mice. Expression of TNFα, TNFR1, TRAF2, and Bcl-2 was analyzed by qPCR in non-injected livers (n = 9/genotype) and in liver stroma and tumors (n = 5–7/genotype) collected from mice 12 days post-injection. TNFα expression was also assessed in non-injected livers (n = 12/genotype) and in liver stroma and tumors (n = 5/genotype) collected from Ldlr^-/- and Ldlr^-/-Pcsk9^-/- (dKO) mice (hatched bars). Error bars indicate SEM. *P < .05; **P < .01 (Student's t test). In the lower right panel, a scheme of the TNFα signaling pathway is shown. Binding of TNFα to TNFR1 triggers intracellular recruitment of TNF receptor-associated death domain to TNFR1, which provides an assembly platform for TRAF2 and Fas-associated protein with a death domain (FADD). The binding of TRAF2 leads to NF-κB activation that induces the transcription of survival and anti-apoptotic proteins, including Bcl-2 and TRAF2. The binding of FADD leads to caspase activation and apoptosis. The events regulated by the absence of PCSK9 are indicated by open arrows.

Figure 7

Plasma TNFα and PCSK9 are upregulated along metastasis formation. (A) TNFα was measured before injection and 2 and 12 days post-injection in the plasma of WT and Pcsk9^-/- mice (n = 12/genotype). (B) Circulating PCSK9 and plasma TC were measured before injection and 2 and 12 days post-injection in WT mice (n = 12). (C) PCSK9 and LDLR expression levels were measured by qPCR in non-injected livers and in B16F1 cell-injected livers (stroma) from WT mice (n = 5). The data are represented by the means ± SEM. *P < .05; **P < .01; ***P < .001 (Student's t test).