Enhanced hepatocarcinogenesis in mouse models and human hepatocellular carcinoma by coordinate KLF6 depletion and increased messenger RNA splicing

Abstract

KLF6-SV1 (SV1), the major splice variant of KLF6, antagonizes the KLF6 tumor suppressor by an unknown mechanism. Decreased KLF6 expression in human hepatocellular carcinoma (HCC) correlates with increased mortality, but the contribution of increased SV1 is unknown. We sought to define the impact of SV1 on human outcomes and experimental murine hepatocarcinogenesis and to elucidate its mechanism of action. In hepatitis C virus (HCV)-related HCC, an increased ratio of SV1/KLF6 within the tumor was associated with features of more advanced disease. Six months after a single injection of diethylnitrosamine (DEN), SV1 hepatocyte transgenic mice developed more histologically advanced tumors, whereas Klf6-depleted mice developed bigger tumors compared to the Klf6fl(+/+) control mice. Nine months after DEN, SV1 transgenic mice with Klf6 depletion had the greatest tumor burden. Primary mouse hepatocytes from both the SV1 transgenic animals and those with hepatocyte-specific Klf6 depletion displayed increased DNA synthesis, with an additive effect in hepatocytes harboring both SV1 overexpression and Klf6 depletion. Parallel results were obtained by viral SV1 transduction and depletion of Klf6 through adenovirus-Cre infection of primary Klf6fl(+/+) hepatocytes. Increased DNA synthesis was due to both enhanced cell proliferation and increased ploidy. Coimmunoprecipitation studies in 293T cells uncovered a direct interaction of transfected SV1 with KLF6. Accelerated KLF6 degradation in the presence of SV1 was abrogated by the proteasome inhibitor MG132.

Conclusion: An increased SV1/KLF6 ratio correlates with more aggressive HCC. In mice, an increased SV1/KLF6 ratio, generated either by increasing SV1, decreasing KLF6, or both, accelerates hepatic carcinogenesis. Moreover, SV1 binds directly to KLF6 and accelerates its degradation. These findings represent a novel mechanism underlying the antagonism of tumor suppressor gene function by a splice variant of the same gene.

Figure 1. SV1/KLF6 ratio is increased in HCV-associated hepatocellular carcinoma and is associated with aggressive clinical behavior

The SV1/KLF6 mRNA ratio in human HCV-associated liver disease samples is significantly increased in HCC compared to non-tumoral tissues (p<0.001; N: normal liver (n=9), Ci: cirrhosis (n=9), D: dysplastic nodules (n=27), VE: very early HCC (n=16), E: early HCC (n=17), A: advanced HCC (51), AA: very advanced HCC (n=20). SV1/KLF6 ratio was significantly higher in larger tumors (0.09 vs 0.13, p=0.04) and in HCC with vascular invasion (0.07, 0.12, p=0.01, Figure 1B).

Figure 2. Hepatocyte specific Klf6 depleted mice have larger tumors, and SV1 transgenic mice have more progressed tumors than controls after 6 months of DEN treatment

AlbCre Klf6fl(+/+) mice have significantly larger tumors than the Klf6fl(+/+) controls 6 months after DEN treatment (p<0.05) (2A, 2B). SV1 Klf6fl(+/+) mice have tumors with higher grading. This is even more pronounced in SV1 AlbCre Klf6fl(+/+) animals (p <0.0001) (2C). 2D shows representative H&E images of Klf6fl(+/+) livers 6 months after DEN treatment with vaguely nodular parenchyma with large cell changes (grade 0-1) (i) and tumor cells with hyperchromatic nuclei and increased nuclear/cytoplasmic ratio with microvascular invasion in SV1 AlbCre Klf6fl(+/+) livers (grade2) (ii) .

Figure 3. Hepatocyte specific SV1 transgenic mice with Klf6 depletion have a greater tumor burden than Klf6 wildtype control mice 9 months after DEN treatment

SV1 AlbCre Klf6fl(+/+) mice have significantly more (3A (p<0.001), 3B (p<0.05)) and larger tumors (3C (p<0.05)) resulting in a significantly higher tumor load (3D) than Klf6fl(+/+) controls 9 months after DEN treatment.

Figure 4. SV1 over-expression and Klf6 depletion increase proliferation in primary hepatocytes

In primary hepatocytes isolated from Klf6fl(+/+) mice with induced acute Klf6 depletion through adenoCre virus infection ex vivo, ³H-thymidine incorporation is increased significantly (p<0.05), with an additional effect when cells are treated with both adenoCre- and pBabeSV1-lentivirus (p<0.05) (4A). Correspondingly hepatocytes from AlbCre Klf6fl(+/+) (p<0.05), SV1 Klf6fl(+/+) (p=0.09) and SV1 AlbCre Klf6fl(+/+) (p=0.0005) have more ³H-thymidine incorporation than Klf6fl(+/+) control cells (4B). The cell count of SV1 Klf6fl(+/+) hepatocytes 24hours after isolation (p<0.05)- and of AlbCre Klf6fl(+/+)- (p<0.05) and SV1 AlbCre Klf6fl(+/+) (p<0.02) mice 48 hours after isolation was significantly higher than that of Klf6fl(+/+) control hepatocytes (4C).

Figure 5. SV1 over-expression and Klf6 depletion increase ploidy in primary hepatocytes

FACS analysis of freshly isolated hepatocytes from AlbCre Klf6fl(+/+), SV1 Klf6fl(+/+) and SV1 AlbCre Klf6fl(+/+) mice shows a significant increase of hepatocytes with 4N as opposed to 2N when compared to Klf6fl(+/+) hepatocytes (5A) (p<0.05) without a corresponding increase in S-phase (5A). Ploidy in hepatocytes from AlbCre Klf6fl(+/+), SV1 Klf6fl(+/+) and SV1 AlbCre Klf6fl(+/+) animals compared to Klf6fl(+/+) controls is significantly increased 5 hours-, and even more pronounced 48 hours after hepatocyte isolation. At 48hours after isolation there were significantly more hepatocytes with 3 or 4 nuclei as compared to 5 hours after isolation in SV1 AlbCre Klf6fl(+/+) hepatocytes (p<0.05). 5C shows representative phase contrast microscopic images of hepatocytes from Klf6fl(+/+) hepatocytes 5 hours (aⁱ) and 48 hours (aⁱⁱ) and from SV1 Klf6fl(+/+) again at 5- (bⁱ) and 48 hours (bⁱⁱ) after hepatocyte isolation. The arrows point out hepatocytes with one nucleus. “*” indicates a hepatocyte with 3 nuclei.

Figure 6. SV1 physically binds to KLF6

In 293T cells transfected with either empty pCI-neo, FLAG-KLF6 or FLAG-SV1 cDNA Immunoprecipitation was performed with α-FLAG, followed by an immunoblot with α-KLF6. In cells transfected with FLAG-KLF6, SV1 was pulled down and in cells transfected with FLAG-SV1, KLF6 was pulled down.

Figure 7. SV1 inhibits KLF6 transcriptional activity

Transfection of FLAG-KLF6 significantly increases p21-luciferase activity in 293T-(7A, p<0.05) and HUH7 cells (7B, p<0.01). This increase is inhibited when FLAG-SV1 is co-transfected (7A, p<0.05) (7B, p<0.05). FLAG-SV1 alone does not affect p21 luciferase activity (7A,B). Similarly in isolated primary hepatocytes from Klf6 depleted mice (AlbCre Klf6fl(+/+), SV1 AlbCre Klf6fl(+/+)) versus Klf6fl(+/+) controls (7C) as well as hepatocytes from Klf6fl(+/+) mice treated with adenoCre- versus lacZ control virus (7D) p21 protein levels were decreased.

Figure 8. SV1 accelerates KLF6 degradation

Co-transfecting increasing amounts of FLAG-SV1 to a constant amount of FLAG-KLF6 leads to a decrease in KLF6 protein levels in 293T- (8A) and HUH7 cells (8C). 8B shows the immunoblot quantification in 293T cells (p<0.05) (n=4) . 293T- (8D) and HUH7 cells (8F) were co-transfected with either FLAG-KLF6 + pCI-neo-GFP or FLAG-KLF6 + FLAG-SV1 and protein was collected 0, 15, 30 or 60 minutes after adding cycloheximide. Co-transfection of FLAG-SV1 accelerated the degradation of KLF6 in both 293T cells (8D). This degradation can be inhibited by adding the proteasome inhibitor MG132 (8D). The quantification of 6 immunoblots in 293T cells (8E) shows lower KLF6 protein levels at timepoint 0 (p<0.05) and accelerated KLF6 degradation (p<0.005) in the presence of SV1 as seen at timepoint 60minutes after cycloheximide (8E). Corresponding can be observed in protein from primary hepatocytes of Klf6fl(+/+) versus SV1 Klf6fl(+/+) mice (8F).