Phosphomevalonate Kinase Controls β‐Catenin Signaling via the Metabolite 5‐Diphosphomevalonate

β‐catenin signaling is abnormally activated in cancer. Here, this work screens the mevalonate metabolic pathway enzyme PMVK to stabilize β‐catenin signaling using a human genome‐wide library. On the one hand, PMVK‐produced MVA‐5PP competitively binds to CKIα to prevent β‐catenin Ser45 phosphorylation and degradation. On the other hand, PMVK functions as a protein kinase to directly phosphorylate β‐catenin Ser184 to increase its protein nuclear localization. This synergistic effect of PMVK and MVA‐5PP together promotes β‐catenin signaling. In addition, PMVK deletion impairs mouse embryonic development and causes embryonic lethal. PMVK deficiency in liver tissue alleviates DEN/CCl4‐induced hepatocarcinogenesis. Finally, the small molecule inhibitor of PMVK, PMVKi5, is developed and PMVKi5 inhibits carcinogenesis of liver and colorectal tissues. These findings reveal a non‐canonical function of a key metabolic enzyme PMVK and a novel link between the mevalonate pathway and β‐catenin signaling in carcinogenesis providing a new target for clinical cancer therapy.


Introduction
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. [1] Although the current

Genome-Wide CRISPR Screen Identifies Regulators of -Catenin Signaling in Huh7 Cells
To circumvent the difficulty of targeting the -catenin signaling pathway itself in HCC, we instead used a human genome-scale CRISPR-Cas9 knockout (GeCKO) v2 library to identify genes that support the pathway. [17] We chose the CTNNB1 gene wild-type (WT) HCC cell line Huh7 to avoid the abnormal activation of -catenin signaling caused by the gene mutations. Preliminary screening showed that the IC 50 of Huh7 cells for compound XAV-939, a tankyrase1/2 inhibitor stabilizing of both Axin1 and Axin2, which indirectly promotes the degradation of -catenin [18] (Figure S1A, Supporting Information). Huh7 cells were then infected with lentiviral stocks encoding the GeCKO v2 library, followed by selection with puromycin. The infected cells were treated with DMSO or XAV-939 for 14 days, and then analyzed by high-throughput sequencing for sgRNA enrichment of more than twofold ( Figure 1A). Pathway enrichment showed that gR-NAs targeted against the Wnt--catenin signaling pathway were significantly enriched as expected ( Figure S1B, Supporting Information). Interestingly, protein class analysis showed the highest enrichment to be for "metabolite interconversion enzyme," implying that these were involved in regulating -catenin signaling ( Figure S1C, Supporting Information). Therefore, we selected 174 significantly down-regulated enriched genes (potentially stabilizing -catenin levels, called negative gene) overlapping with metabolite interconversion enzyme, while the screened candidate genes should also be up-regulated in HCC to fit the role of an oncogene ( Figure 1B; Figure S1D and Table S1, Supporting Information). The results showed that PMVK, a metabolic enzyme of the mevalonate pathway, was significantly enriched ( Figure 1C,D). However, because the relationship between PMVK and the -catenin signaling was unclear, PMVK was further investigated as a candidate gene.

Knockout of PMVK Reduces -Catenin Protein Levels and Blocks Embryonic Development in Mice
Initial experiments showed that XAV-939 has a stronger inhibitory effect on PMVK knockdown Huh7 cell lines ( Figure 1E). To further determine the regulatory role of PMVK on -catenin, we constructed PMVK knockout mice and found that WT and PMVK +/− mice reproduced normally, but that PMVK homozygous knockout was embryonic lethal ( Figure S1E,F, Supporting Information). H&E staining showed no obvious difference in embryonic morphology in PMVK +/− mice compared to WT mice. In addition to PMVK -catenin protein levels were also significantly reduced in both PMVK +/− and PMVK −/− mice ( Figure 1F). Given that Wnt is the major intracellular modality regulatingcatenin signaling and its importance in embryonic development, we analyzed the role of PMVK in Wnt--catenin signaling and our results showed that knockdown of PMVK blocked Wnt3aactivated -catenin signaling ( Figure S1H, Supporting Information). PMVK knockdown upregulated some -catenin-dependent Wnt proteins, which may have triggered a negative feedback (Figure S1I, Supporting Information). These results first suggested that PMVK is required for mouse embryonic development, and  Table S1, Supporting Information, for details. C) Volcano map showing sgRNA library screening distribution. Blue dots indicate significant deletion of sgRNAs (negative selection). Orange dots indicate significant enrichment of sgRNAs (positive selection). Red dots show screening of positive gene representatives. FC, Fold change. D) Each PMVK sgRNA was analyzed in pairs in the DMSO and XAV-939 groups. E) Cell viability was determined by MTT after 2 μg shRNA#1 PMVK plasmid transiently transfected with Huh7, DMSO or XAV-939 (30 μm) treated for 72 h. Data are shown as mean ± SD. F) Representative IHC images of H&E, Ki67, PMVK, total -catenin and -catenin p-Ser184 in wild-type, PMVK +/− and PMVK −/− E12.5 embryos. Scale bar, 1 mm or 50 μm. The IHC staining experiment was performed twice.

Figure 2.
Knockdown of PMVK reduces -catenin signaling and inhibits tumor growth. A) -catenin, c-Myc, Cyclin D1, and PMVK protein expression in the indicated cancer cell lines. B) mRNA expression of -catenin in the indicated cancer cell lines. C) HEK293 cells were transfected with the indicated vectors. Cells were treated with CHX (50 μg mL −1 ) for the indicated time and the expression of PMVK and -catenin were analyzed by western blotting. D) The intensity of -catenin expression for each time point in (C) was quantified by densitometry, with -actin as a normalizer. E) Distribution of PMVK and -catenin protein levels in the nucleus or cytoplasm in the indicated PMVK-knockdown or control cancer cell lines. -actin as a cytoplasm normalizer and Histone H3 as a nucleus normalizer. F) qRT-PCR analysis was performed to measure the mRNA levels of PMVK and -catenin target genes in Huh7 cells. G) Cell proliferation assays were performed in Huh7 (up) and Hep3B (down) cells stably expressing the indicated plasmids. rWT, rescued wild type PMVK. H) Subcutaneous xenograft experiments were performed in Huh7 (up) and Hep3B (down) cells stably expressing the indicated plasmids. n = 5 mice per group. I) Subcutaneous xenograft tumor weight for (H). Data are shown as mean ± SD. that its knockdown PMVK significantly reduces -catenin protein levels.

PMVK Knockdown Suppresses -Catenin Signaling Pathway and Tumor Growth
To better study how PMVK regulates -catenin, we generated stable PMVK knockdowns in CTNNB1-WT Huh7 cells and AXIN1-mutant Hep3B cells. Each of these dramatically repressedcatenin protein expression and downstream signaling in a manner that could be rescued by WT PMVK (Figure 2A). The fact that PMVK knockdown did not affect -catenin mRNA levels indicates that PMVK regulates -catenin protein levels ( Figure 2B). Consistent with this, PMVK knockdown significantly reduced -catenin protein half-life while reducing its nuclear localization ( Figure 2C-E) and the expression of known -catenin target genes ( Figure 2F). Conversely, overexpression of PMVK retarded -catenin degradation while promoting the nuclear localization ( Figure S2A-C, Supporting Information). Consistent with this and the accompanying reduction in c-Myc and Cyclin D1 levels, PMVK knockdown significantly inhibited cell proliferation in a manner that could be rescued by PMVK re-expression (Figure 2G). Similar results were obtained in a mouse xenograft tumor model ( Figure 2H,I and Figure S2D, Supporting Information). Thus, PMVK appears to stabilize the -catenin protein, which is in turn necessary to maintain cell proliferation and tumor growth.
It has been shown that prenylation-modified Rho-GTPase can activate YAP/TAZ signaling, which contributes to -catenin activity. [19] To investigate whether PMVK stabilizes -catenin protein levels in relation to prenylation modification, we used shRNAs knockdown KRas protein. Results showed that knockdown of KRas did not affect -catenin levels ( Figure S2E, Supporting Information). To investigate whether PMVK-mediated regulation of -catenin is related to cholesterol levels, we supplemented PMVK-expressing HCC cells with cholesterol and showed that this only partially restored cell proliferation (Figure S2F, Supporting Information). This suggests that cholesterol may be involved in the regulation of -catenin by PMVK but is not the main cause. Combined with the results of previous experiments in which knockdown of PMVK blocked Wnt3a-activatedcatenin ( Figure S1H, Supporting Information), we hypothesized that PMVK may directly interact with and stabilize -catenin.

PMVK-Mediated Phosphorylation of -Catenin at Ser184 Enhances Its Stability and Promotes Its Nuclear Translocation
Co-immunoprecipitation (co-IP) experiments with the endogenous proteins indicated that PMVK interacts directly withcatenin ( Figure 3A). Subsequent domain mapping demonstrated that this association requires PMVK's kinase domain andcatenin's N-terminal domain ( Figure S3A,B, Supporting Information). Given that PMVK is a metabolic kinase, [21] we asked whether it can directly phosphorylate -catenin by performing phosphorylation experiments in vitro and identifying phosphorylation sites by mass spectrometry ( Figure 3B and Table S2, Supporting Information). These studies showed that the evolutionarily conserved -catenin Ser184 residue was directly phosphorylated by PMVK and this was confirmed using a pSer184-specific antibody ( Figure 3C and Figure S3C, Supporting Information).
We then asked whether PMVK-mediated -catenin phosphorylation was responsible for stabilizing -catenin. To this end, we generated S184A (phosphorylation "dead") and S184D (phosphomimetic) phosphorylation site mutants and determined their half-lives following cycloheximide (CHX) block. The S184A mutant protein showed a significant decrease in its half-life while S184D's half-life was prolonged ( Figure 3D,E). Consistently, S184A showed increased levels of ubiquitination while S184D showed the opposite ( Figure 3F). Furthermore, -catenin S184A showed markedly reduced nuclear localization ( Figure 3G). In addition, immunofluorescence experiments also showed that S184A was mainly localized in the cytoplasm while S184D was significantly localized in the nucleus compared to -catenin WT. This further suggested that PMVK-mediated phosphorylation of -catenin at Ser184 promotes its nuclear localization ( Figure S3E, Supporting Information). IHC staining of mouse embryos also showed that endogenous phosphorylated -catenin Ser184 preferentially localizes to the nucleus ( Figure 1E).
In adult hepatocytes, CKI normally phosphorylates -catenin S45, which initiates GSK3 -mediated -catenin phosphorylation as well as -TrCP-mediated -catenin ubiquitination and proteasome-mediated degradation. [20] Therefore, we asked whether PMVK-mediated -catenin Ser184 phosphorylation is regulated via changes in the above degradation complex proteins. The results showed that, whereas knockdown and overexpression of PMVK respectively decreased and increasedcatenin p-Ser184 phosphorylation, neither affected the levels of CKI , GSK3 , and -TrCP in HCC cells ( Figure S3D, Supporting Information). However, -catenin S184D showed significantly reduced association with CKI , GSK3 , and -TrCP compared with S184A, whereas the phosphorylation of both Thr41 and Ser45 was significantly increased in S184A. Overexpression of WTcatenin or S184D, but not S184A, also activated -catenin downstream signaling ( Figure 3H). Together the above results indicated that PMVK promotes -catenin protein stability and nuclear translocation by phosphorylating its Ser184 site, which in turn interferes with the de-stabilizing phosphorylation events involving -catenin residues 41 and 45.

PMVK-Generated MVA-5PP Competitively Binds CKI Causing Stabilization of -Catenin
-catenin's stability and turnover are regulated by the destruction complex. [21] Our new findings show that, by directly phosphorylating -catenin, PMVK participates directly in this regulation. We therefore next asked whether the interaction between PMVK and -catenin occurs within the destruction complex or elsewhere. Co-IP results showed that ectopic PMVK expression reduced the interaction between -catenin and several destruction complex-associated proteins ( Figure 4A). Interestingly, the overexpression of a metabolically inactive mutant of PMVK, PMVK-(17-23), [22] did not affect the interaction of -catenin with the destruction complex whereas PMVK WT did ( Figure S4A, Supporting Information). This result suggested that the PMVK-mediated stabilization of -catenin protein levels relies upon downstream metabolites. Indeed, addition of the known PMVK metabolite MVA-5PP to Huh7 and Hep3B cells increased their levels of both total and p-S184 -catenin and the down-stream -catenin targets c-Myc and Cyclin D1 in a concentration-dependent manner ( Figure 4B). Moreover, the addition of MVA-5PP to cells lacking PMVK reduced -catenin's association with the destruction complex proteins ( Figure 4C). Reverse in silico screening of the destruction complex's structure indicated a potential association between MVA-5PP and CKI (8.2 kcal mol −1 ) ( Figure S4B, Supporting Information).
Diphosphomevalonate decarboxylase (MVD) catalyzes the fourth step of mevalonate biosynthesis involving the cationdependent reaction of MVA-5PP with ATP to form isopentenyl diphosphate and ADP to release a carbon dioxide and a phosphate group, which binds to MVA-5PP as a positive control (8.6 kcal mol −1 ) ( Figure S4B, Supporting Information). [23] To verify this result, we used Microscale thermophoresis (MST) to confirm  Table S2, Supporting Information, for details. C) Recombinant purified GST--catenin-His and PMVK-His proteins were subjected to an in vitro kinase reaction. -catenin phosphorylation was identified with -catenin p-Ser184 antibody. Fast AP, Fast alkaline phosphatase. D) HEK293 cells were transfected with the indicated vectors. Cells were treated with CHX (50 μg mL −1 ) for the indicated times and the levels of different -catenin mutants were analyzed by Western blotting. E) The expression intensity of different -catenin mutants at each time point in (D) was quantified by densitometry, with -actin as a normalizer. Data are shown as mean ± SD. F) HEK293 cells were transfected with the indicated plasmids, treated with MG132 for 6 h and then lysed in RIPA buffer. Immuno-precipitated HA--catenin proteins were subjected to immunoblot assay with Myc-tag antibody. G) Distribution of different -catenin mutants protein levels in the nucleus or cytoplasm in Huh7 and Hep3B cells transfected with the indicated plasmids. -actin as a cytoplasm normalizer and Histone H3 as a nucleus normalizer. H) HEK293 cells were transfected with the indicated plasmids and then lysed in RIPA buffer. Immuno-precipitation of different -catenin mutants proteins with anti-HA magnetic beads and subjected to immunoblot assay.
binding of MVA-5PP to the K46, Y64, and D157 sites of CKI , with much less binding being seen with a mutant CKI (K46A, Y64A, and D157A) ( Figure 4D,E). In vitro phosphorylation assays also showed that MVA-5PP inhibited, in a concentrationdependent manner, the phosphorylation of -catenin S45 by CKI but not -catenin T41 by GSK3 ( Figure 4F,G). Finally, shRNA-mediated depletion of MVD, which served an independent means of increasing MVA-5PP levels, likewise increasedcatenin protein. This suggested that the stabilization of -catenin was primarily caused by MVA-5PP rather than its downstream . PMVK-produced MVA-5PP competitively binds CKI to stabilize -catenin. A) HEK293 cells were transfected with the indicated plasmids and then lysed in RIPA buffer. Immuno-precipitated HA--catenin proteins were subjected to immunoblot assay. B) MVA-5PP was added to Huh7 and Hep3B cells at the indicated final concentrations and subjected to immunoblot assay -catenin (total and p-S184 form), c-Myc and Cyclin D1 protein levels. C) HEK293 cells were transfected with the indicated plasmids, supplemented with MVA-5PP (200 μm) and then lysed in RIPA buffer. Immuno-precipitation of HA--catenin proteins were subjected to immunoblot assay. D) The molecular docking of MVA-5PP and CKI is shown based on the crystal structure of CKI (PDB code: 6GZD). Lys 46, Tyr 64, and Asp 157 indicate the bound residues. E) Kd values were determined by MST for MVA-5PP bound to purified human CKI or CKI mutant (K46A, Y64A and D157A) proteins. Data are shown as mean ± SD. F) Purified recombinant GST--catenin-His protein and FLAG-CKI protein were subjected to in vitro phosphorylation assays at different concentrations of MVA-5PP. Immunoblotting was performed using -catenin p-Thr41/Ser45 antibody. G) Recombinant GST--catenin-His and FLAG-GSK3 proteins were subjected to in vitro phosphorylation assays at different concentrations of MVA-5PP followed by immunoblotting using -catenin p-Thr41/Ser45. H) Immunoblot assay of -catenin (total and p-S184 form), c-Myc, Cyclin D1, and CKI protein levels after MVA-5PP (200 μm) treatment in the indicated PMVK-knockdown or control cancer cell lines. www.advancedsciencenews.com www.advancedscience.com metabolites ( Figure S4C, Supporting Information). In addition, the supplementation of cells with exogenous MVA-5P increased the levels of total and p-S184 -catenin as well as c-Myc and Cyclin D1, indicating that PMVK's upstream metabolites also contribute to the activation of -catenin signaling, but that the primary effector is MVA-5PP ( Figure S4D, Supporting Information).
Cholesterol deficiency stimulates that transcription factor SREBP2, which in turn increases the expression of genes involved in the cholesterol biosynthesis pathway. [24] To evaluate whether this can promote -catenin p-Ser184 and -catenin signaling, we treated HCC cells with methyl--cyclodextrin (M--CD) which depletes cholesterol. [25] Subsequent immunoblotting showed that M--CD treatment partially decreased the levels total and p-S184 -catenin. This suggested that -catenin S184 phosphorylation is dependent on certain minimal levels of MVA-5PP ( Figure S4E, Supporting Information). The data also suggest that the product of PMVK, namely MVA-5PP, rather than PMVK itself, competitively binds to CKI to prevent the phosphorylationdependent degradation of -catenin.
Finally, in vitro supplementation with MVA-5PP restoredcatenin levels in PMVK knockdown cells but failed to rescuecatenin p-Ser184 or the downstream signaling defects. This may have been due to the lack of PMVK-mediated -catenin Ser184 phosphorylation ( Figure 4H).

PMVK Knockout in Hepatocytes Blocks HCC Generation in Mice
To determine the role of PMVK in HCC development in vivo, PMVK hepatocyte-specific conditional knockout (CKO) mice were constructed ( Figure S5A, Supporting Information). Immunoblotting confirmed that these mice also had significantly reduced levels of both total and p-S184 -catenin ( Figure S5B, Supporting Information). Although the results of Ki67 staining showed that cell proliferation was suppressed in PMVK flfl Alb cre mice ( Figure S5C, Supporting Information), there were no significant differences in body or liver weights compared to WT mice at 8 weeks of age ( Figure S5D,E, Supporting Information). To evaluate PMVK's role on HCC development, we generated tumors in both groups of mice with DEN/CCl 4 ( Figure 5A). CKO mice had both significantly reduced tumor numbers and smaller tumor volumes compared to control WT mice ( Figure 5B-D). Serum TC and TG levels were also significantly reduced in the CKO group ( Figure 5E,F) and Ki-67 staining showed that tumor proliferation was significantly inhibited ( Figure S5F, Supporting Information). Adeno-associated virus 8 (AAV8) restoration of PMVK expression promoted tumor growth and increased serum levels of TC and TG in CKO mice ( Figure 5B-F). To further investigate the effects of PMVK and -catenin Ser184 phosphorylation on tumor growth, we delivered PMVK or -catenin (WT and S184A/D) via AAV8 in WT mice. Our results showed that overexpression of PMVK, -catenin WT or S184D significantly promoted HCC growth ( Figure 5G-I). S184D also significantly reduced the survival rate ( Figure 5J). Collectively, these data suggest that liverspecific PMVK deletion significantly slows HCC development by reducing cell proliferation and cholesterol and triglyceride levels whereas PMVK-mediated phosphorylation of -catenin Ser184 strongly promotes tumor growth.

PMVK Is Overexpressed in HCC Patients and Correlates with Poor Prognosis
In tumor samples from 10 HCC patients, PMVK, -catenin (total and p-S184 form), c-Myc and Cyclin D1 were significantly upregulated relative to matched normal liver (Figure 6A-F). In addition, PMVK positivity correlated with -catenin protein levels ( Figure 6G), in contrast to other enzymes of the mevalonate pathway ( Figure S6A-D, Supporting Information). PMVK also positively correlated with -catenin p-Ser184, which in turn correlated with the levels of downstream target gene proteins (Figure 6H-O). PMVK was also highly expressed in HCCs from the TCGA database ( Figure 6P). Similar findings were shown in the GEO databases (GSE45436, GSE25097, GSE102083, and GSE14520). PMVK mRNA levels also positively correlated with the expression of -catenin target genes in the TCGA-LIHC and GSE14520 dataset ( Figure S6E-K, Supporting Information). The PMVK gene also tended to be highly amplified in HCC samples, thus explaining the basis for its over-expression ( Figure 6Q,R). Finally, PMVK upregulation correlated with poor HCC patient survival and advanced clinical stage ( Figure 6S and Figure S6L, Supporting Information). Together, these data identify PMVK as a potential therapeutic target in HCC.
To further assess the role of PMVKi5 in tumorigenesis, we again utilized the DEN/CCl 4 HCC model in which mice were treated with PMVKi5 (30 mg kg −1 intraperitoneally) every 2 days for a total of 14 doses ( Figure 7F). Both prevention and treatment group mice showed significant reduced tumor burdens compared to control tumor-bearing mice ( Figure 7G-I). PMVKi5 treatment also inhibited -catenin protein levels and downstream targets in mouse tumors ( Figure 7J). Ki67 staining confirmed that PMVKi5 significantly inhibited tumor proliferation ( Figure 7K). To further evaluate PMVKi5 efficacy in other cancers, we used the AOM/DSS induced colorectal cancer (CRC) mouse model ( Figure S7G, Supporting Information). Similarly, PMVKi5 also inhibited tumorigenesis in this model ( Figure S7H-K, Supporting Information). Collectively, these data confirm our previous results by showing that PMVKi5 blocks -catenin signaling by inhibiting PMVK.
Importantly, PMVKi5 did not alter mouse body weight or serum ALS, ATL levels, indicating that PMVKi5 treatment does not cause liver injury ( Figure S7D-F,K, Supporting Information). PMVKi5 thus represents a promising lead compound that should be safe and effective for the prevention and treatment of HCC and/or CRC.

Discussion
The WNT/ -catenin signaling pathway is often activated in cancer due to the aberrant accumulation and nuclear localization www.advancedsciencenews.com www.advancedscience.com  of its effector -catenin. [10] Although many -catenin inhibitors have been screened in the past decades, the eventual development of resistance has tended to stymie such efforts. There is thus an urgent need to screen for new targets that regulatecatenin indirectly. [26] This consideration was the primary motivation for our genome-wide Crispr screen that identified PMVK as one such potential target.
Of note, metabolic enzymes were significantly enriched in the above-mentioned screen. Although metabolic reprogramming is one of the hallmarks of malignancy, its relationship to -catenin signaling has, until now, been unclear. [27] Our Crispr/Cas9 screen led us to focus our attention on the mevalonate pathway enzyme PMVK as a novel target that regulates -catenin signaling. As a result, we have found that PMVK plays key roles in embryonic development and tumor growth, while PMVK-mediatedcatenin p-Ser184 phosphorylation is also significantly upregulated in both key nodes ( Figure S5G, Supporting Information). The importance of this activity may be due to the uniqueness of the mevalonate pathway as the upstream pathway of cholesterol synthesis and the importance of -catenin signaling in embryonic development and malignancy. [28] Previous studies have also shown that the mevalonate metabolic pathway contributes prominently to prominent tumor growth, including the ability of MVA-5P to stabilize mutant p53. [7] In addition, mevalonate pathway metabolites are also important for prenylation, geranylgeranylation, and farnesylation of several oncogenic proteins, such as Rho, Rac, Rab, and Ras. [29] Although geranylgeranylation can activate the YAP/TAZ signaling pathway and thus promote the -catenin signaling pathway, [19,30] our results show that this activity is not involved in the PMVKmediated regulation of -catenin signaling ( Figure S2E, Supporting Information). In addition, knockdown of PMVK resulted in blocked synthesis of the mevalonate pathway, which also impaired cholesterol levels. Previous studies have shown that accumulated cholesterol stabilizes -catenin signaling. [31] But in our study we showed that supplementation of additional cholesterol levels in HCC cells with knockdown PMVK only partially restored cell proliferation levels, suggesting that although cholesterol is an important factor for cell proliferation, it is not a major factor in the regulation of -catenin signaling by PMVK ( Figure S2F, Supporting Information).
Interestingly, PMVK knockdown also impaired Wnt3aactivated -catenin signaling, suggesting that the mechanism of PMVK involves the disruption complex or the nuclear localization of -catenin. qRT-PCR showed that PMVK knockdown increased some Wnt family transcript levels, possibly due to a classical feedback mechanism triggered by the loss of -catenin signaling. This prompted us to explore whether PMVK interacts directly with -catenin. As a result, we determined that PMVK directly phosphorylates -catenin Ser184, thereby increasing -catenin protein stability, promoting its nuclear translocation and increasing target gene expression. Most HCCs contain mutations in CTNNB1, which stabilize -catenin by preventing its association with the destruction complex. However, we found that knockdown of PMVK still destabilized -catenin and impaired its downstream signaling in Hep3B (AXIN1 mutant) cells, in which the destruction complex is non-functional. This suggested that, by phosphorylating the critical Ser184 residue, PMVK activates -catenin via a destruction complex-independent mechanism. In the HCC mouse model, overexpression of -catenin p-Ser184 strongly promoted tumor growth. Although stabilizing -catenin indirectly promotes its nuclear localization, the actual mechanism by which -catenin Ser184 phosphorylation accomplishes this requires further clarification. Regardless of the actual means by which this is accomplished, -catenin Ser184 phosphorylation serves as a new and novel marker for -catenin pathway activation.
PMVK phosphorylation of -catenin Ser184 is dependent on its classical metabolic enzyme function. Because PMVK mutants cannot affect the interaction of -catenin with the destruction complex, this implies that the downstream metabolites of PMVK may cooperate with mutations in the destruction complex or in -catenin itself to further stabilize the protein, promote its nuclear localization and enhance it's signaling. Interestingly, the PMVK metabolite, MVA-5PP similarly inhibited the interaction of -catenin with the destruction complex with similar consequences. We found that the mechanistic underpinnings of this activity involves the binding of MVA-5PP to CKI . Genetic knockdown of MVD also increased -catenin protein level. This suggests that PMVK stabilization of -catenin is dependent on its metabolite MVA-5PP rather than on the mevalonate pathway metabolite isopentenyl diphosphate or other intermediates. In the developing chicken liver, MVK and PMVK remain highly active for the first 5 days, while the activity of MVD is low. [32] This suggests that increased MVA-5PP may be elevated in a way that satisfies the strong dependency on -catenin signaling during early development. [33] Collectively, we uncovered a non-canonical function of MVA-5PP by which it and the PMVK enzyme that generates it, play synergistic roles in regulating the -catenin signaling pathway.
Analysis of clinical samples and data from TCGA indicated that PMVK is highly expressed in HCC, likely due to its genomic amplification. PMVK levels were also found to correlate closely with clinical stage and -catenin p-Ser184 correlated with total -catenin and its downstream signals. This indicates that PMVK and -catenin p-Ser184 protein levels are potentially useful as prognostic markers in HCC. Finally PMVK inhibitors represent a novel targeted therapy directed against a previously unsuspected target that drives HCC progression and may be useful in CRC as well. We also evaluated other inhibitors of the mevalonate pathway on PMVK-regulated -catenin signaling. Simvastatin showed the same effect as PMVKi5 on -catenin, due to the fact that statins block the mevalonate pathway at its source (also blocking the synthesis of MVA-5PP). Zoledronic acid instead promotes -catenin protein levels, the target of zoledronic acid is the inhibition of farnesyl pyrophosphate synthesis which may indirectly contribute to the accumulation of upstream metabolites, similar to knockdown of MVD ( Figure  S7C, Supporting Information).
In conclusion, we have demonstrated how the mevalonate pathway enzyme PMVK controls -catenin signaling. In doing so, we have revealed a new mechanism by which metabolic reprogramming controls HCC and CRC pathogenesis. PMVK inhibition, perhaps with PMVKi5 or its analogs thus represents a promising and rational therapeutic strategy.

Experimental Section
Vectors, Reagents, Antibodies, Cell Culture, and Stable Cell Lines: Human PMVK shRNAs (Table S3, Supporting Information) were expressed in the PLKO.1 vector. The open reading frames of human PMVK, -catenin, and CKI were amplified and cloned into pHAGE-CMV-MCS-PGK-3×FLAG and pCMV-HA vectors. Mutations in the PMVK, -catenin, and CKI cDNA sequences were generated by overlap extension PCR. Primers for constructs used in this study are listed in Table S4, Supporting Information, and were synthesized by GeneCreate Biological Engineering Co. Ltd (Wuhan, China). Transfection and the establishment of stable cell lines were performed as previously described. [34] 5-diphosphomevalonate was purchased from Sigma-Aldrich. More reagent information in this study is listed in Table S5, Supporting Information. Anti-HA magnetic beads and anti-FLAG affinity agarose beads were purchased from Selleck (Houston, USA). Antibodies used in this study are listed in Table S6, Supporting Information.
Human HEK293 cells were obtained from the American Type Culture Collection in 2009. Human liver tumor-derived cell lines Huh7 and Hep3B were obtained from the China Center for Type Culture Collection and the Cell Bank of the Type Culture Collection of The Chinese Academy Sciences and cultured as previously described. [34] All cells were regularly authenticated by short tandem repeat analyses, were tested for the absence of Mycoplasma and were used within five passages after thawing.
Animal Experiments: All animal studies were approved by the Animal Care Committee of Wuhan University (No. WQ20210130). 4-week-old female BALB/c nude mice were purchased from Gempharmatech (Jiangsu, China). PMVK −/− mice were generated, bred and raised at Cyagen Biosciences with CRISPR-Cas9 technology to delete exon E1-E5 of the PMVK gene. PMVK fl/fl mice (Cyagen Biosciences) have loxP sites flanking exons 2 and 5 of the PMVK gene. Alb-Cre mice were kindly provided by Prof. Yong Liu (Wuhan University). To generate PMVK fl/fl Alb cre mice, PMVK fl/fl mice were crossed with Alb-Cre mice. All mouse genotype identification primer sequences used are shown in Table S7, Supporting Information.
For xenograft experiments, tumor growth was monitored every 3 days for a total period of 30 days. Tumor volumes were calculated by the equation V (mm 3 ) = a × b × (a + b)/2, where a is the length and b is the width.
Littermate mice were randomly divided into control and various treatment groups including: control, PMVKi5 prevention, and PMVKi5 treatment. All cohorts were maintained as blinded cohorts following these treatments.
Human Genome-Wide CRISPR Screening: Amplification of the sgRNA library followed the description of Feng Zhang et al. [17,35] Twelve T225 culture flasks were prepared for library virus generation according to the HEK293 culture conditions described above. Each flask was transfected with 20 μg of library plasmid (10 μg each of libraries A and B), 10 μg of pMD2.G and 15 μg of psPAX2 were transfected using 100 μL of Lipofectamine 2000 and 200 μL of Plus Reagent. The medium was replaced after 24 h and the cell supernatants were collected at 48 and 72 h of transfection and passed through 0.45 μm filters. The packed lentiviral library was then used to infect at least 5 × 10 7 Huh7 cells in the presence of 1 μg mL −1 polybrene, ensuring a multiplicity of infection (MOI) of 0.3 so that most cells receive only one stably integrated RNA guide. 24 h after infection, puromycin was added to select for stably transduced cells. Allow the cell count to expand to >8 × 10 7 , ensuring that the initial sieve cell count is a library coverage multiple >300×. Each group of 4 × 10 7 cells were treated with vector and XAV393 (30 μm), respectively, and cells were passaged every 2 days for a total of 14 days of drug treatment. All cells were collected and genomic DNA was extracted, enriched for fragments containing sgRNA sequences using amplification primers (forward primer, ATGGACTATCATATGCTTACCGTA; reverse primer, AGTACACGA-CATCACTTTCCC) and sequenced on an Illumina NovaSeq 6000 following a second round of PCR reactions with the addition of splice primers and indexT sequences used to identify different samples. The number of sgR-NAs of XAV-939 and vehicle was identified using the MAGeCK algorithm and analyzed using DESEq2 to obtain significantly enriched or depleted sgRNAs.
Immunohistochemistry and Immunofluorescence: Liver sections or mouse embryos were fixed in 10% formalin and embedded in paraffin according to standard protocols. Liver sections or mouse embryo were sectioned and stained with H&E, Ki67, PMVK, -catenin, and -catenin p-Ser184. Images were obtained on a Leica Aperio VERSA 8 microscope.
Immunofluorescence analysis was performed as previously described. [34] Briefly, HEK293 cells were transfected with the indicated vectors. 48 h later, they were fixed in 4% paraformaldehyde for 15 min, followed by 0.2% Triton X-100 permeabilization for 5 min and 3% BSA blocking for 1 h. After incubation with primary antibody against FLAG (1:200) overnight in 4°C, the cells were washed well with PBST and incubated with ABflo 488-conjugated goat anti-rabbit (1:500) for 1 h at room temperature. After additional extensive washing, the cells were labeled with DAPI (5 μg mL −1 ) for 5 min at room temperature. Fluorescence imaging was performed with a LSM800 confocal laser scanning microscope.
Co-IP and Immunoblotting: Transfected cells were lysed in 1 mL lysis buffer [50 mm Tris (pH 7.4), 150 mm NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 5 mm EDTA, 10 mg mL −1 aprotinin, 10 mg mL −1 leupeptin, and 1 mm PMSF]. Sepharose beads were washed three times with 1 mL lysis buffer containing 0.1% NP-40. Co-IP and immunoblot analysis were performed as described. [36] qRT-PCR: Total RNA was isolated using Trizol followed by DNase treatment. Reverse transcription was performed with a cDNA Synthesis Kit and qPCR were performed using SYBR Green master mix using standard protocols. All qPCR primer sequences used are shown in Table S8, Supporting Information. -actin was used as an internal control.
Protein Half-Life Assay: After achieving ≈70% confluence, cells in 12well plates were transfected with the indicated plasmids using Lipofectamine 2000. 36 h later, the cells were treated with cycloheximide (CHX, 50 μg mL −1 ) for the indicated times before collection for immunoblot analysis.
MTT Assay: For MTT assays, cells were seeded into 96-well plates (10 3 cells per well) and cultured in DMEM supplemented with 10% FBS. After 3-7 days of incubation, MTT was added and cells were incubated for 4 h at 37°C. At the indicated time, this was replaced with 200 μL of DMSO. Absorbance was then read at 570 nm using an ELX800 absorbance microplate reader.
In Vitro Phosphorylation Assay: Recombinant GST--catenin-His and PMVK-His were prepared and purified from Rossita. 1 μg GST--catenin-His protein was incubated with 1 μg PMVK-His in a 50 μL kinase reaction buffer (20 mm Tris-HCl (pH 7.5), 10 mm MgCl 2 , 50 mm KCl, 30 μm ATP) at 37°C for 0.5 h. Reactions were stopped by the addition of SDS sample buffer, and samples were then heated for 5 min at 95°C. Phosphorylation of -catenin Ser184 was identified by mass spectrometry and immunoblot with -catenin p-Ser184 antibody that was generated by ABclonal by immunizing rabbits with a S184-monophosphorylated peptide (KKEA(S-pho)RHA) conjugated with keyhole limpet hemocyanin.
In Vivo Ubiquitination Assay: 36 h after transfection, cells were treated with 20 μm MG132 for 6 h. They were then lysed in RIPA lysis buffer as described above. The samples were boiled for 10 min in SDS sample buffer and analyzed by immunoblot with the indicated antibodies.
Microscale Thermophoresis: A Monolith NT.115 instrument (Nan-oTemper Technologies, Germany) was used to measure the Kd of binding of MAV-5PP to CKI -GFP protein and PMVKi5 to PMVK-GFP protein.
All samples to be tested were first solubilized in RIPA buffer. The CKI -GFP or PMVK-GFP was added to each dilution and incubated at room temperature for 5 min. The samples were loaded into silica capillaries