Hepatic prohibitin 1 and methionine adenosyltransferase α1 defend against primary and secondary liver cancer metastasis

Abstract

Background & aims: The liver is a common site of cancer metastasis, most commonly from colorectal cancer, and primary liver cancers that have metastasized are associated with poor outcomes. The underlying mechanisms by which the liver defends against these processes are largely unknown. Prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) are highly expressed in the liver. They positively regulate each other and their deletion results in primary liver cancer. Here we investigated their roles in primary and secondary liver cancer metastasis.

Methods: We identified common target genes of PHB1 and MAT1A using a metastasis array, and measured promoter activity and transcription factor binding using luciferase reporter assays and chromatin immunoprecipitation, respectively. We examined how PHB1 or MAT1A loss promotes liver cancer metastasis and whether their loss sensitizes to colorectal liver metastasis (CRLM).

Results: Matrix metalloproteinase-7 (MMP-7) is a common target of MAT1A and PHB1 and its induction is responsible for increased migration and invasion when MAT1A or PHB1 is silenced. Mechanistically, PHB1 and MAT1A negatively regulate MMP7 promoter activity via an AP-1 site by repressing the MAFG-FOSB complex. Loss of MAT1A or PHB1 also increased MMP-7 in extracellular vesicles, which were internalized by colon and pancreatic cancer cells to enhance their oncogenicity. Low hepatic MAT1A or PHB1 expression sensitized to CRLM, but not if endogenous hepatic MMP-7 was knocked down first, which lowered CD4⁺ T cells while increasing CD8⁺ T cells in the tumor microenvironment. Hepatocytes co-cultured with colorectal cancer cells express less MAT1A/PHB1 but more MMP-7. Consistently, CRLM raised distant hepatocytes' MMP-7 expression in mice and humans.

Conclusion: We have identified a PHB1/MAT1A-MAFG/FOSB-MMP-7 axis that controls primary liver cancer metastasis and sensitization to CRLM.

Impact and implications: Primary and secondary liver cancer metastasis is associated with poor outcomes but whether the liver has underlying defense mechanism(s) against metastasis is unknown. Here we examined the hypothesis that hepatic prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) cooperate to defend the liver against metastasis. Our studies found PHB1 and MAT1A form a complex that suppresses matrix metalloproteinase-7 (MMP-7) at the transcriptional level and loss of either PHB1 or MAT1A sensitizes the liver to metastasis via MMP-7 induction. Strategies that target the PHB1/MAT1A-MMP-7 axis may be a promising approach for the treatment of primary and secondary liver cancer metastasis.

Keywords: FOSB; MAFG; Prohibitin 1; cholangiocarcinoma; colorectal cancer; hepatocellular carcinoma; matrix metalloproteinase-7; metastasis; methionine adenosyltransferase 1A.

Figure 1.. PHB1 and MAT1A cooperate to negatively regulate MMP-7 expression at the transcriptional level.

Cells were transfected with MAT1A or PHB1 overexpression vector (OV) or treated with siRNA (si) for 48 hours prior to measuring MMP-7 expression in the cells and medium. A) MMP-7 mRNA levels, ANOVA test, *p = 0.01, 0.05, 0.025, 0.01, 0.04, 0.04, 0.05, and 0.014 vs EV. B) intracellular and C) secreted MMP-7 protein levels in MzChA-1 and HepG2 cells. Student’s t-test, ANOVA test, *p = 0.028, 0.013, 0.03, and 0.014. D) MMP-7 mRNA levels and E) promoter activity were examined after MAT1A or PHB1 OV alone or combined, and MAT1A or PHB1 siRNA alone or together in MzChA-1 cells. Results are expressed as mean ± SEM from at least three independent experiments, ANOVA test, *p = 0.05, 0.014, 0.01, 0.026, 0.029, and 0.011 vs. respective controls (EV+EV, and SC+SC), ^#p = 0.006, 0.037, 0.03, and 0.004 vs. single OV or si.

Figure 2.. MAFG and FOSB positively regulate MMP-7 while PHB1 and MAT1A negatively regulate FOSB expression.

A) mRNA and protein levels of MMP-7 were measured by RT-qPCR and western blot after varying MAFG and FOSB expression for 48 hours. ANOVA test, *p = 0.047, 0.018, 0.05, 0.03, 0.01, 0.04, 0.01, 0.016, 0.04, 0.028, 0.017, 0.03, 0.014, 0.02, and 0.04, ***p = 0.000087 vs. EV or SC B) FOSB expression was examined after varying PHB1 and MAT1A expression for 48 hours. ANOVA test, *p = 0.034, 0.05, 0.018, 0.028, 0.024, 0.01, 0.018, 0.013, 0.015, 0.01, 0.012, 0.045, 0.011, 0.012, 0.024, and 0.026 vs. EV or SC C) MAFG and FOSB expression were measured in SAMe-D and PHB1-D cells as compared to primary mouse hepatocytes (PMH). ANOVA test, **p = 0.00151, 0.00157, 0.00137, and 0.0049 vs. PMH D) MMP-7 mRNA levels and E) promoter activity were examined after MAFG or FOSB OV alone or combined, and MAFG or FOSB siRNA alone or together in HepG2 cells. ANOVA test, *p = 0.047, 0.031, 0.018, 0.037, 0.047, 0.029, 0.016, 0.04, 0.017, 0.013, 0.017, and 0.014 vs. EV+EV and SC+SC, ^#p = 0.037, 0.0048, 0.002, 0.0027, 0.029, 0.011, 0.0049, and 0.002 vs. single OV or si. Results are expressed as mean % of EV ± SEM from at least three independent experiments.

Figure 3.. Effects of PHB1, MAT1A, MAFG and FOSB on MMP-7 promoter activity.

A) Effects of varying PHB1, MAT1A, MAFG and FOSB expression on WT MMP-7 (−1270 to +20bp) and B) MMP-7 promoter activity with AP-1 site mutation. AP-1 site mutant MMP-7 promoter activity was ~ 20% of the WT promoter (data not shown). Results are expressed as mean % of EV or SC ± SEM from three experiments done in duplicates. C) ChIP was done using anti-c-JUN antibody followed by Seq-ChIP using antibodies to PHB1, MATα1, MAFG and FOSB to examine binding to the MMP-7 promoter (−73 to +202 bp) containing the AP-1 site. D) EMSA was done using the consensus sequence of AP-1 using recombinant proteins (1μg) alone or combined. Results are representative of three independent experiments, ANOVA test, *p = 0.01, 0.025, 0.015, 0.018, 0.02, 0.015, 0.016, 0.04, 0.05, 0.042, 0.028, 0.04, 0.015, 0.017, 0.035, and 0.038 vs. EV or SC.

Figure 4.. Effects of PHB1, MAT1A, and MMP-7 on migration and invasion.

A) Effects of varying PHB1, MAT1A and MMP-7 expressions alone or together on MzChA-1 cell migration and B) invasion. C) Results are expressed as mean % of EV+EV or SC+SC ± SEM from three experiments done in duplicates, ANOVA test, *p = 0.04, 0.01, 0.014, 0.04, 0.012, 0.017, 0.018, 0.02, 0.02, 0.01, 0.024, 0.01, 0.017, 0.014, 0.012, and 0.01,0.026, 0.019, 0.02, 0.024 vs. relative controls (EV+EV or SC+SC). D) MATα1-D and E) PHB1-D cells expressing SC or MMP-7 CRISPR stable knockdown and luciferase were injected into the liver of nude mice (MATα1-D) and WT C57BL/6 mice (PHB1-D), and tumor growth was monitored by small animal imaging as described in Methods. Representative small animal images showing luciferase measurement are shown and summarized in the graph to the right of the images. Representative H&E staining of liver, lung, and pancreas with numbers of mice that exhibited metastasis or liver necrosis are indicated below the pictures in nude mice or WT C57BL/6 mice. Results are expressed as mean ± SEM from n=7–8 per group, ANOVA test, Student’s t-test, *p = 0.05, 0.016 and 0.01, **p = 0.001 vs SC.

Figure 5.. Mice and HCC cells lacking MAT1A or PHB1 release EVs containing more MMP-7 to induce cancer cell growth, migration and invasion.

A) EVs isolated from culture media examined under electron microscopy. B) EVs were isolated from sera were analyzed by NanoSight, Student’s t-test, ^ns p = 0.319, *p = 0.043 vs. Flox from n=3 per group. C) MMP-7 and EV markers in EVs isolated from sera were evaluated by western blot. Student’s t-test, ANOVA test, *p = 0.036, 0.029, and 0.014 vs. respective controls (WT, Flox, and PMH). D) Immunofluorescence staining of MMP-7 in MC38 cells treated with EVs from sera of the Mat1a and Phb1 KO mice. E) MMP-7 and EV markers in EVs isolated from culture media were evaluated by western blot. F) BrdU incorporation, Student’s t-test, *p = 0.004, and 0.049. G) invasion and migration of MC38 cells treated with EVs from sera of Mat1a KO and WT littermates, Phb1 KO and flox littermates. Student’s t-test, *p = 0.021, **p = 0.0083, ***p = 0.00018, 0.00077. vs. respective controls (WT, and Flox) H) BrdU incorporation, I) migration (top two rows) and invasion (bottom row) of MC38 cells treated with EVs from culture media of PMH, MATα1-D and PHB1-D, MATα1-D with MMP-7 CRISPR KO and PHB1-D with MMP-7 CRISPR KO cells. Results are expressed as mean % of respective controls ± SEM from three experiments done in duplicates, ANOVA test, *p = 0.05, 0.016, 0.05, and 0.011, **p = 0.0086, ^ns p = 0.691, and 0.900 vs. PMH.

Figure 6.. Mat1a KO and liver-specific Phb1^+/− mice are sensitized to CRLM by a mechanism that requires hepatic MMP-7.

A) Representative H&E and IHC staining of MMP-7 in normal mouse liver (top) and human CRLM tissue array (bottom). B) Representative H&E and IHC staining of MMP-7 in murine CRLM model from different groups. C) Assessment of tumor growth in WT, Mat1a KO, Phb1^+/− mice with or without silencing endogenous MMP-7 by in vivo bioluminescence imaging. Results are expressed as mean ± SEM from N=8–12 per group, ANOVA test, *p = 0.05, 0.03, and 0.018, **p = 0.004, and 0.006, ^ns p = 0.51 and 0.34 vs. WT+SC/KO. D) MAT1A, PHB1 and MMP-7 expression in human hepatocytes with and without co-culture of RKO cells. Results are expressed as mean % of control expression (without co-culture) from three experiments done in duplicates, Student’s t-test, **p = 0.0024, ***p = 0.000019 and 0.000057 vs. without co-culture.

Figure 7.. Features of CD4⁺ and CD8⁺ T Cell Infiltration in CRLM.

A) flow cytometry analysis of percentage for CD4⁺ and CD8⁺ subsets of CD3⁺ T-lymphocytes in the CRLM from WT (left), Mat1a KO (middle), Phb1^+/− (right) mice with SC or MMP-7 KO adenovirus injection (N=3–5). ANOVA test, *p = 0.048, 0.019, and 0.027, ***p = 0.00098 and 0.000377 vs. respective SC. B) Percentage of T cells activation marker - Ki67⁺ in CD8⁺ T cells (left) and quantified MFI (right) for T cells activation markers - INF-γ, GrB and Perforin in CD8⁺ T cells in CRLM from WT injected with SC or MMP-7 KO adenovirus. Results are expressed as mean ± SEM from three experiments done in duplicates, Student’s t-test, *p = 0.015, 0.011, and 0.05, **p = 0.0079 vs. respective SC.