PAR-Induced Harnessing of EZH2 to β-Catenin: Implications for Colorectal Cancer

G-protein-coupled receptors (GPCRs) are involved in a wide array of physiological and disease functions, yet knowledge of their role in colon cancer stem cell maintenance is still lacking. In addition, the molecular mechanisms underlying GPCR-induced post-translational signaling regulation are poorly understood. Here, we find that protease-activated receptor 4 (PAR4) unexpectedly acts as a potent oncogene, inducing β-catenin stability and transcriptional activity. Both PAR4 and PAR2 are able to drive the association of methyltransferase EZH2 with β-catenin, culminating in β-catenin methylation. This methylation on a lysine residue at the N-terminal portion of β-catenin suppresses the ubiquitination of β-catenin, thereby promoting PAR-induced β-catenin stability and transcriptional activity. Indeed, EZH2 is found to be directly correlated with high PAR4-driven tumors, and is abundantly expressed in large tumors, whereas very little to almost none is expressed in small tumors. A truncated form of β-catenin, ∆N133β-catenin, devoid of lysine, as well as serine/threonine residues, exhibits low levels of β-catenin and a markedly reduced transcriptional activity following PAR4 activation, in contrast to wt β-catenin. Our study demonstrates the importance of β-catenin lysine methylation in terms of its sustained expression and function. Taken together, we reveal that PAR-induced post-transcriptional regulation of β-catenin is centrally involved in colon cancer.


Introduction
Despite the emerging role of G-protein-coupled receptors (GPCRs) in a wide array of physiological and disease functions, knowledge of their regulation of post-translational signaling is still incomplete [1][2][3][4]. Whereas GPCR post-translational modification (PTM) involves phosphorylation on serine/threonine residues by a GPCR receptor kinase family (GRK), ubiquitination for targeted degradation, SUMOylation, S-nitrosylation, tyrosine sulfation, and methylation [5], our knowledge of their signaling induced post-translational involvement is lacking.
GPCRs control many aspects of tumorigenesis, including proliferation, invasion, survival at secondary sites, and several cancer-associated signaling pathways [6]. As a subfamily of GPCRs, Frizzled (FZD) receptors play a pivotal role in development, tissue patterning homeostasis, and cancer. Activation of the Wnt/β-catenin signaling pathway, also called the Wnt canonical pathway, involves the stabilization of β-catenin following ligation of Wnts to FZDs and recruitment of low-density lipoprotein-related protein 5/6 (LRP5/6) coreceptors [7]. Inactivation of β-catenin takes place following its phosphorylation tagging at serine S45 by CK1, followed by S33, S37 and T41 phosphorylation by GSK3β [8]. This phosphorylation cascade labels β-catenin for degradation. β-catenin can then be recognized by the E3 ligase βTrCP, which mediates the ubiquitination of lysine residues K19 and K49, leading to proteasome-dependent degradation [9]. Upon association of Wnts with their receptor and coreceptors, the degradation complex is inhibited. β-catenin is then released, which is translocated to the nucleus, consequently prompting the expression of Wnt-β-catenin target genes downstream [10].

PAR 4 induces β-catenin stabilization.
Previously, we have demonstrated the stabilization and TOPflash transcriptional activity of β-catenin induced either by PAR 1 or PAR 2 [16,17]. Due to the unexpected oncogenic function of PAR 4 , we set out to study the PAR 4 -induced β-catenin stabilization path. For this purpose, HEK293 cells were transiently transfected with flg-β-catenin and Par4, followed by the addition of AYPGKF, a synthetic hexapeptide exhibiting the internal PAR 4 ligand sequence for activation. A marked increase in β-catenin levels was observed after 4-5 h of PAR 4 activation ( Figure 1A). The transcriptional activity of β-catenin induced by PAR 4 was evaluated by the TOPflash luciferase assay carried out in HEK293 cells. Cell lysates were used in the Lef/Tcf Luciferase assay following the transient transfection of Par4, flg-β-catenin, β-gal and lef plasmids. This demonstrated the increased transcriptional function of β-catenin driven by PAR 4 ( Figure 1B).
Activation of PAR2 or PAR4 induces the association of EZH2 with β-catenin and methylation on lysine/s (K) residue/s. Consequently, it prolongs the stabilization of β-catenin and enhances its transcriptional activity. PAR-induced β-catenin contributes centrally to colorectal cancer growth.

Results
PAR4 induces β-catenin stabilization. Previously, we have demonstrated the stabilization and TOPflash transcriptional activity of β-catenin induced either by PAR1 or PAR2 [16,17]. Due to the unexpected oncogenic function of PAR4, we set out to study the PAR4induced β-catenin stabilization path. For this purpose, HEK293 cells were transiently transfected with flg-β-catenin and Par4, followed by the addition of AYPGKF, a synthetic hexapeptide exhibiting the internal PAR4 ligand sequence for activation. A marked increase in β-catenin levels was observed after 4-5 h of PAR4 activation ( Figure 1A). The transcriptional activity of β-catenin induced by PAR4 was evaluated by the TOPflash luciferase assay carried out in HEK293 cells. Cell lysates were used in the Lef/Tcf Luciferase assay following the transient transfection of Par4, flg-β-catenin, β-gal and lef plasmids. This demonstrated the increased transcriptional function of β-catenin driven by PAR4 ( Figure 1B). Knock down of Par4 reduces the otherwise large Par4-driven tumors in mice. The role of Par4 in tumor generation was further examined by preparing a construct of shRNA-Par4, for its silencing of aggressive colon cancer cell lines, while on the other hand, establishing Par4 clones resulted in the overexpression of the gene in parental RKO cells. HCT-116 cells were stably infected with the lentiviral construct of shRNA-Par4. The effectiveness of shRNA-Par4 silencing in HCT-116 cells following infection with viral particles was evaluated by means of RT-PCR and quantitated by real-time qPCR analyses (Figure 2A,B). Infection of shRNA-Par4 significantly reduced levels of Par4 mRNA compared to the nontreated wild-type (wt) HCT-116 cells ( Figure 2C,D). When nude mice were inoculated with Par4-silenced cells, no tumors developed. In contrast, large tumors were generated following inoculation with wt HCT-116 cells. This outcome indicates the central role played by Par4 in the HCT-116 aggressive tumor cell line. To demonstrate the direct impact of PAR 4 in tumor growth, we generated stable clones expressing Par4 in RKO cells, a colorectal cancer cell line transformed on the background of mismatch repair system (e.g., of intact β-catenin pathway). These clones were named RKO/Par4a-c. The levels of Par4 mRNA in these cells are shown for a representative clone, RKO/Par4a ( Figure 3A). When these clones were subcutaneously injected into nude mice (s.c.), large tumors were generated compared to the mice injected with parental RKO cells lacking Par4 expression ( Figure 3B,C).
lysates to detect β-catenin using anti-flag antibody (1:1000) and normalized to β-actin (1:1000) for protein loading. The figure is representative of the assay performed in triplicate. (B). Lef/Tcf transcriptional activity. HEK293 cells were transiently co-transfected with 0.2 μg Par4, 0.075 μg TOPflash, 0.125 μg LEF, and 0.2 μg β-gal plasmids. Luciferase activity was normalized to β-gal for transfection efficiency. The mean of duplicates is shown for each treatment. The results are representative of experiments performed in triplicate.
Knock down of Par4 reduces the otherwise large Par4-driven tumors in mice. The role of Par4 in tumor generation was further examined by preparing a construct of shRNA-Par4, for its silencing of aggressive colon cancer cell lines, while on the other hand, establishing Par4 clones resulted in the overexpression of the gene in parental RKO cells. HCT-116 cells were stably infected with the lentiviral construct of shRNA-Par4. The effectiveness of shRNA-Par4 silencing in HCT-116 cells following infection with viral particles was evaluated by means of RT-PCR and quantitated by real-time qPCR analyses (Figure 2A,B). Infection of shRNA-Par4 significantly reduced levels of Par4 mRNA compared to the nontreated wild-type (wt) HCT-116 cells ( Figure 2C,D). When nude mice were inoculated with Par4-silenced cells, no tumors developed. In contrast, large tumors were generated following inoculation with wt HCT-116 cells. This outcome indicates the central role played by Par4 in the HCT-116 aggressive tumor cell line. To demonstrate the direct impact of PAR4 in tumor growth, we generated stable clones expressing Par4 in RKO cells, a colorectal cancer cell line transformed on the background of mismatch repair system (e.g., of intact β-catenin pathway). These clones were named RKO/Par4a-c. The levels of Par4 mRNA in these cells are shown for a representative clone, RKO/Par4a ( Figure 3A). When these clones were subcutaneously injected into nude mice (s.c.), large tumors were generated compared to the mice injected with parental RKO cells lacking Par4 expression (  Six-week-old male nude mice were injected subcutaneously with either wt HCT116 cells or shRNA-Par4 HCT116 cells (1 × 10 6 ). The experiment was terminated after 5 weeks. These results are representative of the experiment performed three times. Number of mice per each treatment; n = 6 (2 died in the HCT116 wt-inoculated cells). Tumor volume (D) was measured as 0.5 (length) × width 2 .
stably infected with shRNA-Par4. Par4 mRNA expression was assessed by RT-PCR, normalized to GAPDH (A) and by real-time PCR normalized to HPRT (B). (C,D) Tumor generation in wt and shRNA-Par4 HCT116 cells. Six-week-old male nude mice were injected subcutaneously with either wt HCT116 cells or shRNA-Par4 HCT116 cells (1 × 10 6 ). The experiment was terminated after 5 weeks. These results are representative of the experiment performed three times. Number of mice per each treatment; n = 6 (2 died in the HCT116 wt-inoculated cells). Tumor volume (D) was measured as 0.5 (length) × width 2 . EZH2 is overexpressed in PAR-driven tumors. Next, we evaluated the levels of EZH2 in PAR-driven tumors. Western blot analyses of proteins extracted either from tumors generated by RKO/Par4 clones compared with RKO parental cells, or tumors generated by HCT116 aggressive colon cancer cells versus sh-silenced Par4 HCT116 cells were performed. Pronounced high EZH2 levels were observed in HCT116 as well as in the RKO/Par4-generated tumors. In contrast, no expression of EZH2 was observed in tumors generated by shRNA-Par4 HCT116 cells, nor in RKO non-aggressive cells ( Figure 4A,B). Immunohistological (IHC) staining of EZH2 in sections of RKO/Par4a-derived tumor tissues compared with tissues of small tumors derived by parental RKO cells resulted in the following outcome. High levels of EZH2 were observed in the tumor section derived from EZH2 is overexpressed in PAR-driven tumors. Next, we evaluated the levels of EZH2 in PAR-driven tumors. Western blot analyses of proteins extracted either from tumors generated by RKO/Par4 clones compared with RKO parental cells, or tumors generated by HCT116 aggressive colon cancer cells versus sh-silenced Par4 HCT116 cells were performed. Pronounced high EZH2 levels were observed in HCT116 as well as in the RKO/Par4-generated tumors. In contrast, no expression of EZH2 was observed in tumors generated by shRNA-Par4 HCT116 cells, nor in RKO non-aggressive cells ( Figure 4A,B). Immunohistological (IHC) staining of EZH2 in sections of RKO/Par4a-derived tumor tissues compared with tissues of small tumors derived by parental RKO cells resulted in the following outcome. High levels of EZH2 were observed in the tumor section derived from RKO/Par4a clone inoculation, while there was nearly none in the tissue sections obtained following inoculation with the RKO parental cells ( Figure 4C). RKO/Par4a clone inoculation, while there was nearly none in the tissue sections obtained following inoculation with the RKO parental cells ( Figure 4C). Representative sections of mouse-generated PAR4-driven tumors. IHC staining, using anti-EZH2 (1:50 dilution) antibodies. All images were acquired using a Nikon light microscope at magnifications of 10× and 20×. Scale bars 50 μm. EZH2 is abundantly expressed in the large tumors (of high PAR4 (RKO/Par4a cells)). Very little to almost no EZH2 was detected in the small-appearing tumors (e.g., RKO). As controls for the IHC staining, tissue sections were processed in a similar fashion, but without primary antibodies.
Inhibition of EZH2 by GSK126 inhibits HT-29 spheroid formation. Spheroid growth and maintenance indicate better cell-cell interactions in vivo [44]. Thus, HT29 cells were grown in the presence of Matrigel and appropriate medium supplements for spheroid formation. When we subjected the spheroids to GSK126, an inhibitor of EZH2 (e.g., 2 μM and 5 μM), the spheres started to undergo apoptosis ( Figure 5A). Next, we extracted proteins from the HT-29 spheroids before and after AYPGKF PAR4 activation. Western blot analyses showed an increase in the level of EZH2 upon long-term activation of PAR4 ( Figure 5B). This indicates that activation of PAR4 induces EZH2 levels towards a potent association with PAR4-induced β-catenin. Representative sections of mousegenerated PAR 4 -driven tumors. IHC staining, using anti-EZH2 (1:50 dilution) antibodies. All images were acquired using a Nikon light microscope at magnifications of 10× and 20×. Scale bars 50 µm. EZH2 is abundantly expressed in the large tumors (of high PAR 4 (RKO/Par4a cells)). Very little to almost no EZH2 was detected in the small-appearing tumors (e.g., RKO). As controls for the IHC staining, tissue sections were processed in a similar fashion, but without primary antibodies. [44]. Thus, HT29 cells were grown in the presence of Matrigel and appropriate medium supplements for spheroid formation. When we subjected the spheroids to GSK126, an inhibitor of EZH2 (e.g., 2 µM and 5 µM), the spheres started to undergo apoptosis ( Figure 5A). Next, we extracted proteins from the HT-29 spheroids before and after AYPGKF PAR 4 activation. Western blot analyses showed an increase in the level of EZH2 upon long-term activation of PAR 4 ( Figure 5B). This indicates that activation of PAR 4 induces EZH2 levels towards a potent association with PAR 4 -induced β-catenin.

Inhibition of EZH2 by GSK126 inhibits HT-29 spheroid formation. Spheroid growth and maintenance indicate better cell-cell interactions in vivo
Stem cell markers by PAR 4 . To further appreciate the significance of PAR 4 in stimulating stem cell marker expression levels, HT-29 colon cancer cells were subjected to AYPGKF for PAR 4 activation and either treated or not with tcY-NH2, a potent antagonist of PAR 4 . Next, mRNA was collected, and real-time qPCR was carried out for a panel of stem cell markers known to be elevated by PAR 4 . As can be seen ( Figure 5C), all genes analyzed were significantly upregulated by AYPGKF PAR 4 activation, especially LGR5, as well as CD44 and OCT4. In contrast, spheroids that were activated for PAR 4 and then treated with tcY-NH2 showed very low expression levels, which is similar to the effect obtained in shRNA-silenced Par4 (data not shown). Overall, this points to the powerful role played by PAR 4 as a member of the colon cancer stem cell compartment.

Stem cell markers by PAR4.
To further appreciate the significance of PAR4 in stimulating stem cell marker expression levels, HT-29 colon cancer cells were subjected to AY-PGKF for PAR4 activation and either treated or not with tcY-NH2, a potent antagonist of PAR4. Next, mRNA was collected, and real-time qPCR was carried out for a panel of stem cell markers known to be elevated by PAR4. As can be seen ( Figure 5C), all genes analyzed were significantly upregulated by AYPGKF PAR4 activation, especially LGR5, as well as CD44 and OCT4. In contrast, spheroids that were activated for PAR4 and then treated with tcY-NH2 showed very low expression levels, which is similar to the effect obtained in shRNA-silenced Par4 (data not shown). Overall, this points to the powerful role played by PAR4 as a member of the colon cancer stem cell compartment.
Activation of PAR4 or PAR2 promotes the binding of EZH2 to β-catenin. We next examined whether EZH2 interacts with β-catenin following the activation of PAR4 or PAR2. To this end, immunoprecipitation (IP) analysis between EZH2 and β-catenin was carried out following application of either SLIGKV (for PAR2 activation) or AYPGKF (for PAR4). Briefly, HEK293 cells were transfected with flg-β-catenin, ezh2 and Par4 plasmids. Cells were activated for the indicated periods of time, which were between 15 min and 4 h. High levels of β-catenin were obtained within the immune complex of EZH2 following 15 min of PAR4 (or PAR2) activation, which subsequently decreased. It was concluded that the EZH2-β-catenin association takes place early on, prior to β-catenin stabilization. Con- Activation of PAR 4 or PAR 2 promotes the binding of EZH2 to β-catenin. We next examined whether EZH2 interacts with β-catenin following the activation of PAR 4 or PAR 2 . To this end, immunoprecipitation (IP) analysis between EZH2 and β-catenin was carried out following application of either SLIGKV (for PAR 2 activation) or AYPGKF (for PAR 4 ). Briefly, HEK293 cells were transfected with flg-β-catenin, ezh2 and Par4 plasmids. Cells were activated for the indicated periods of time, which were between 15 min and 4 h. High levels of β-catenin were obtained within the immune complex of EZH2 following 15 min of PAR 4 (or PAR 2 ) activation, which subsequently decreased. It was concluded that the EZH2-β-catenin association takes place early on, prior to β-catenin stabilization. Concomitantly, lysine (K) methylation of β-catenin was obtained at the same immune complex ( Figure 6A,B). EZH2-β-catenin interaction points to a regulatory type of interaction. Notably, similar results were observed following SLIGKV PAR 2 activation, showing methylation of the β-catenin lysine and association of EZH2 with β-catenin ( Figure 6C,D). These data are in agreement with results obtained by Zhu P et al. [9] showing that Lnc-β-Catm associates with β-catenin and EZH2, as can be observed with the AYPGKF activation of PAR 4 or the SLIGKV activation of PAR 2 .  On the basis of the results indicating that activation of PAR2 or PAR4 induces the association of EZH2 with β-catenin, methylating it on a lysine residue, we next evaluated the effect of this methylation on β-catenin transcriptional activity. To this end, Lef/Tcf luciferase assay was performed in HEK293 cells that were transfected with either Par4 alone or with both Par4 and ezh2, as also with flg-β-catenin, β-gal and lef plasmids. EZH2 expression in the cells resulted in higher levels of Lef/Tcf transcriptional activity following PAR4 activation compared to cells transfected with Par4 alone. Cells transfected with both ezh2 and Par4 continued to show markedly enhanced levels of Lef/Tcf activity. These cells exhibited a higher baseline level of Lef/Tcf activity in control non-activated cells (Figure 7A). While the transcriptional activity induced by PAR4 alone began to diminish in a timeframe of 6 h following PAR4 activation, in the presence of EZH2, Lef/Tcf activity continued to increase ( Figure 6E). Similar observations were obtained when SLIGKV activa- On the basis of the results indicating that activation of PAR 2 or PAR 4 induces the association of EZH2 with βcatenin, methylating it on a lysine residue, we next evaluated the effect of this methylation on β-catenin transcriptional activity. To this end, Lef/Tcf luciferase assay was performed in HEK293 cells that were transfected with either Par4 alone or with both Par4 and ezh2, as also with flg-β-catenin, β-gal and lef plasmids. EZH2 expression in the cells resulted in higher levels of Lef/Tcf transcriptional activity following PAR 4 activation compared to cells transfected with Par4 alone. Cells transfected with both ezh2 and Par4 continued to show markedly enhanced levels of Lef/Tcf activity. These cells exhibited a higher baseline level of Lef/Tcf activity in control non-activated cells ( Figure 7A). While the transcriptional activity induced by PAR 4 alone began to diminish in a timeframe of 6 h following PAR 4 activation, in the presence of EZH2, Lef/Tcf activity continued to increase ( Figure 6E). Similar observations were obtained when SLIGKV activation of cells transfected with Par2 alone or both Par2 and ezh2 was performed. Increased levels were seen after 2 and 4 h of SLIGKV activation, which then subsequently diminished ( Figure 6F). Truncated ΔN133β-catenin is impaired in β-catenin stabilization. Three lysine residues are located in the N-terminal portion of β-catenin: K19, K49 and K133. To further evaluate the impact of EZH2 methylation on β-catenin function, a truncated form of βcatenin devoid of 133 N-terminal amino acids, including its lysine residues, was prepared. The truncated β-catenin plasmid-ΔN133β-catenin-was then used to analyze the levels of β-catenin following AYPGKF PAR4 activation. While a distinct increase in β-catenin was seen following PAR4 activation, no increase was obtained using the truncated form N133β-catenin. Similar data were obtained following PAR2 SLIGKV activation of wt βcatenin versus truncated ΔN133β-catenin forms (data not shown). This is indicative of the important role of EZH2 in mediating β-catenin methylation followed by PAR4 (or PAR2)induced stability of β-catenin ( Figure 7A,B).
Transcriptional activity of the truncated ΔN133β-catenin construct was evaluated by means of the Lef/Tcf luciferase assay. HEK293 cells were transfected with Par4 and wt βcatenin or with truncated β-catenin-the N133β-catenin construct-and analyzed for transcriptional activity following AYPGKF PAR4 activation. While the wt β-catenin plasmid induces abundant and high Lef/Tcf transcriptional activity, cells transfected with the truncated N133β-catenin construct showed a very low level of transcription ( Figure 7C). To evaluate the functionality of Δ133β-catenin, we assessed Axin-β-catenin interactions Truncated ∆N133β-catenin is impaired in β-catenin stabilization. Three lysine residues are located in the N-terminal portion of β-catenin: K19, K49 and K133. To further evaluate the impact of EZH2 methylation on β-catenin function, a truncated form of βcatenin devoid of 133 N-terminal amino acids, including its lysine residues, was prepared. The truncated β-catenin plasmid-∆N133β-catenin-was then used to analyze the levels of β-catenin following AYPGKF PAR 4 activation. While a distinct increase in β-catenin was seen following PAR 4 activation, no increase was obtained using the truncated form N133β-catenin. Similar data were obtained following PAR 2 SLIGKV activation of wt βcatenin versus truncated ∆N133β-catenin forms (data not shown). This is indicative of the important role of EZH2 in mediating β-catenin methylation followed by PAR 4 (or PAR 2 )-induced stability of β-catenin ( Figure 7A,B).
Transcriptional activity of the truncated ∆N133β-catenin construct was evaluated by means of the Lef/Tcf luciferase assay. HEK293 cells were transfected with Par4 and wt β-catenin or with truncated β-catenin-the N133β-catenin construct-and analyzed for transcriptional activity following AYPGKF PAR 4 activation. While the wt β-catenin plasmid induces abundant and high Lef/Tcf transcriptional activity, cells transfected with the truncated N133β-catenin construct showed a very low level of transcription ( Figure 7C). To evaluate the functionality of ∆133β-catenin, we assessed Axin-β-catenin interactions following PAR 2 SLIGKV activation. This is based on a publication by Li et al. [45] that indicated that Axin binds to β-catenin following Wnt activation. When we analyzed immuno complex formation between Axin and either wt β-catenin or ∆133β-catenin, we observed that the 133β-catenin is associated with Axin in a similar manner to wt β-catenin ( Figure 7D). We therefore conclude that ∆133β-catenin, while devoid of its N-terminal portion, is functional and capable of associating with Axin as part of β-catenin signaling.
Methylation by EZH2 prolongs β-catenin half-life in PAR 4 activated cells. One option to explain the distinct increase in β-catenin levels and transcriptional activity in the presence of EZH2 is the prolonged half-life of β-catenin. To further establish the impact of EZH2 on β-catenin, we analyzed the half-life of β-catenin in the presence and absence of EZH2. This was achieved by using the cycloheximide (CHX) pulse-chase assay. CHX is a known inhibitor of eukaryotic protein synthesis. In the presence of CHX, levels of unstable proteins will decrease, whereas relatively stable proteins will show little change over time [46]. HEK293 cells were transfected with either β-catenin and Par4 plasmids alone, or in combination with ezh2. Next, cells were activated with AYPGKF for PAR 4 activation to stabilize β-catenin, then CHX was added so that no additional proteins could be synthesized. In the presence of ezh2, β-catenin expression levels can be seen for longer and sustained periods of time compared to cells transfected with β-catenin and Par4 alone ( Figure 8A,B). This result suggests that EZH2-mediated lysine methylation contributes to the stability of β-catenin, keeping it from being degraded. It is also supported by the prolonged Lef/Tcf transcriptional activity in the presence of EZH2 following the addition of CHX ( Figure 8C). following PAR2 SLIGKV activation. This is based on a publication by Li et al. [45] that indicated that Axin binds to β-catenin following Wnt activation. When we analyzed immuno complex formation between Axin and either wt β-catenin or Δ133β-catenin, we observed that the 133β-catenin is associated with Axin in a similar manner to wt β-catenin ( Figure 7D). We therefore conclude that Δ133β-catenin, while devoid of its N-terminal portion, is functional and capable of associating with Axin as part of β-catenin signaling. Methylation by EZH2 prolongs β-catenin half-life in PAR4 activated cells. One option to explain the distinct increase in β-catenin levels and transcriptional activity in the presence of EZH2 is the prolonged half-life of β-catenin. To further establish the impact of EZH2 on β-catenin, we analyzed the half-life of β-catenin in the presence and absence of EZH2. This was achieved by using the cycloheximide (CHX) pulse-chase assay. CHX is a known inhibitor of eukaryotic protein synthesis. In the presence of CHX, levels of unstable proteins will decrease, whereas relatively stable proteins will show little change over time [46]. HEK293 cells were transfected with either β-catenin and Par4 plasmids alone, or in combination with ezh2. Next, cells were activated with AYPGKF for PAR4 activation to stabilize β-catenin, then CHX was added so that no additional proteins could be synthesized. In the presence of ezh2, β-catenin expression levels can be seen for longer and sustained periods of time compared to cells transfected with β-catenin and Par4 alone ( Figure 8A,B). This result suggests that EZH2-mediated lysine methylation contributes to the stability of β-catenin, keeping it from being degraded. It is also supported by the prolonged Lef/Tcf transcriptional activity in the presence of EZH2 following the addition of CHX ( Figure 8C).

In summary. Induced EZH2 association with β-catenin and methylation on K49.
The activation of either of the PARs leads to the association of EZH2 with β-catenin and methylation of β-catenin on lysine (K) 49. Consequently β-catenin is stabilized and enters the nuclei, where it acts as a co-transcription factor and generates tumors (Scheme 1). lysates were collected, and luciferase activity was normalized to β-gal activity to control for transfection efficiency. This experiment was carried out in triplicate.
In summary. Induced EZH2 association with β-catenin and methylation on K49. The activation of either of the PARs leads to the association of EZH2 with β-catenin and methylation of β-catenin on lysine (K) 49. Consequently β-catenin is stabilized and enters the nuclei, where it acts as a co-transcription factor and generates tumors (Scheme 1). Scheme 1. Depicting PAR2 or PAR4 induced EZH2 association with β-catenin.

Discussion
Here we present original data demonstrating the importance of EZH2 non-histone methylation of β-catenin, instigated by PARs. Lysine methylation of β-catenin is essential, and is required for the prolonged stabilization of β-catenin. It unravels an exclusive mode of post-translational regulation of a pivotal player in colon cancer. Supporting data were obtained from the deletion construct of β-catenin devoid of its 133 N-terminal amino acids, lacking, among other things, three lysine (K) residues: K19, K49 and K133. The truncated form of β-catenin exhibits low levels of expression and no transcriptional activity. It is proposed that, early on, methylation of β-catenin is a prerequisite for the stabilization and transcriptional activity of β-catenin. PAR-induced β-catenin stabilization plays a central role in colon cancer growth.
Truncated N133 β-catenin, in addition to lysine amino acids, is devoid of serine/threonine (S/T) residues for phosphorylation. The phosphorylation of β-catenin (on four S/T residues: S33, S37, T41 and S45) assigns it to proteasomal degradation [8,47]. Specifically, CK1 -induced Ser45 phosphorylation generates a priming site for GSK3β, which is necessary for GSK3β-mediated phosphorylation of the Thr41, Ser37, and Ser33 residues. Ser33 and Ser37 in β-catenin form docking sites for the E3 ubiquitin ligase; the β transducing repeat-containing protein (β-TRCP) that ubiquitinates β-catenin and targets it for proteasomal degradation. On the other hand, methylated lysine contributes to the prolonged half-life period for enhanced β-catenin stabilization. Once lysine methylation takes place, it inhibits the tagging of β-catenin by phosphorylation for ubiquitination [9].
Zhu P et al. discovered a long non-coding RNA called lnc-β-Catm (lncRNA for βcatenin methylation) that associates with β-catenin and EZH2, thereby promoting the methylation of β-catenin on lysine 49 (K49) by EZH2. This methylation inhibits β-catenin ubiquitination by the E3 ligase and promotes its stability, thus allowing β-catenin to activate Wnt-β-catenin signaling for a longer period of time, contributing to the sustained Scheme 1. Depicting PAR 2 or PAR 4 induced EZH2 association with β-catenin.

Discussion
Here we present original data demonstrating the importance of EZH2 non-histone methylation of β-catenin, instigated by PARs. Lysine methylation of β-catenin is essential, and is required for the prolonged stabilization of β-catenin. It unravels an exclusive mode of post-translational regulation of a pivotal player in colon cancer. Supporting data were obtained from the deletion construct of β-catenin devoid of its 133 N-terminal amino acids, lacking, among other things, three lysine (K) residues: K19, K49 and K133. The truncated form of β-catenin exhibits low levels of expression and no transcriptional activity. It is proposed that, early on, methylation of β-catenin is a prerequisite for the stabilization and transcriptional activity of β-catenin. PAR-induced β-catenin stabilization plays a central role in colon cancer growth.
Truncated N133 β-catenin, in addition to lysine amino acids, is devoid of serine/threonine (S/T) residues for phosphorylation. The phosphorylation of β-catenin (on four S/T residues: S33, S37, T41 and S45) assigns it to proteasomal degradation [8,47]. Specifically, CK1αinduced Ser45 phosphorylation generates a priming site for GSK3β, which is necessary for GSK3β-mediated phosphorylation of the Thr41, Ser37, and Ser33 residues. Ser33 and Ser37 in β-catenin form docking sites for the E3 ubiquitin ligase; the β transducing repeatcontaining protein (β-TRCP) that ubiquitinates β-catenin and targets it for proteasomal degradation. On the other hand, methylated lysine contributes to the prolonged half-life period for enhanced β-catenin stabilization. Once lysine methylation takes place, it inhibits the tagging of β-catenin by phosphorylation for ubiquitination [9].
Zhu P et al. discovered a long non-coding RNA called lnc-β-Catm (lncRNA for βcatenin methylation) that associates with β-catenin and EZH2, thereby promoting the methylation of β-catenin on lysine 49 (K49) by EZH2. This methylation inhibits β-catenin ubiquitination by the E3 ligase and promotes its stability, thus allowing β-catenin to activate Wnt-β-catenin signaling for a longer period of time, contributing to the sustained stemness of liver CSCs [9]. Our data indicate that the activation of PARs induces the methylation of β-catenin through the association of EZH2, resulting in a longer and continued stability of β-catenin. Whether PAR-induced EZH2 association leads to methylation on K49 alone or K19 in addition still remains to be established.
EZH2 also functions in a PCR2-independent manner. Emerging research has shown that EZH2 methylates non-histone targets interacting with other proteins to activate downstream genes. For example, Kim E et al. discovered that EZH2 binds to and methylates STAT3, leading to its enhanced activity through the increased tyrosine phosphorylation of STAT3 [48]. Another study showed that the phosphorylation of EZH2, mediated directly or indirectly by PI3K/AKT pathway, can switch its function from a Polycomb repressor to a transcriptional co-activator of androgen receptors, and potentially other factors as well [33,35,49].
Mutations of EZH2 are found in hematological malignancies [50]. They promote cancer cell proliferation, anchorage-independent growth, and invasiveness [39][40][41]. In vivo, the inhibition of EZH2 reduces tumor growth rates to various extents [42,43]. Focusing on breast cancer, EZH2 was found to regulate the structure of basal-like cell populations by inducing a 'bi-lineage' differentiation state. In this state, cells express both basal and luminal lineage markers [51]. In contrast, GATA3, a driver of luminal differentiation, was demonstrated to carry out a function opposite to that of EZH2, acting to diminish the bi-lineage identity and luminal progenitor gene expression [51].
In fact, EZH2 has been shown to be overexpressed in many cancers, including hepatocellular carcinoma, breast [40], bladder [52], and lung cancer [53]. In prostate cancer, EZH2 has also been used as a molecular marker for poor prognosis [35]. Overexpression of EZH2 is critical for the function of stem cell self-renewal. A mechanism has been described in which EZH2 expression-mediated downregulation of DNA damage repair leads to accumulation of recurrent RAF1 gene amplification in Cancer Initiated Cells (CIC), which activates p-ERK-β-catenin signaling and enhanced CIC growth [54]. As such, a clinical trial (e.g., AZD6244) for a drug that inhibits RAF1-ERK signaling resulted in the inhibition of the progression of breast cancer through the eradication of CICs [50]. In addition, there is a role for EZH2 in immunotherapy. It has been shown to be negatively correlated with CD8+ cytotoxic T cells in ovarian cancer [55].
Although EZH2 works in a variety of ways, including the canonical pathway of epigenetic transcription [56] and gene upregulation, it can also work as a tumor suppressor [57]. For this reason, a precise understanding of the mode by which this protein works, from a molecular level to a cluster network, is essential for developing a future clinical mode of therapy [58].
EZH2 has been shown to bind to and transactivate β-catenin, leading to increased expression of the target genes c-myc and cyclin D1 [59]. C-myc expression also leads to ezh2 expression by targeting miRNA [60]. Aberrant Wnt signaling in colorectal cancers leads to deregulation of the c-myc gene [61]. Therefore, one could assume that PAR activation inducing Lef/Tcf gene transcription could bring about c-myc expression, and thus influence EZH2 expression. This might explain why when PAR 4 cells are highly expressed, higher levels of EZH2 expression are exhibited, as indicated in our data. Along this line of evidence, we also showed that the EZH2 inhibitor GSK126 impairs PAR 4 -induced spheroid growth, as well as the growth of stem cell markers that are otherwise markedly elevated by PAR 4 activation. Taken together, these results emphasize the central role played by EZH2 in PAR 4 -induced malignancy. Elevated levels of several Lef/Tcf transcription targets, such as lgr5, cd44 and oct4, were observed in these spheroids. The expression of these genes is then quelled by inhibition of EZH2 with GSK126.
Overall, it is proposed herein that activation of the GPCR oncogenes PAR 4 and PAR 2 potently induce lysine methylation of β-catenin by EZH2, promoting its sustained stability levels in colon cancer.

Animal Models
The animals used in the experiments were treated in accordance with the guidelines of the institution ethics committee (AAALAC standard). Mice (HSD: Athymic nude-Foxn1Nu Nu/Nu mice) were kept under specific pathogen-free (SPF) conditions at the Hadassah Medical Center animal facility unit of the Hebrew University and were regularly screened for standard pathogens. All animal experiments were approved by the animal committee of the Hebrew University (MD-20-15924-5).

sh-RNA Construct Preparation and Viral Particle Generation
To prepare sh-RNA of PAR4, the sequence was successfully cloned into the plentilox3.7 (pLL3.7) lentiviral vector following the protocol provided by the website of Addgene (Cat#11795). The target sequence was as follows: sh-Par4# (5 -ATGACAGCACGCCCTCAAT -3). For the generation of shPar4 viral particles, HEK 293 cells were transfected with a three-plasmid system that included packaging CMVD R8.91, envelope (CMV-VSV-G), and shPar4-pLL3.7, using PEI as a transfection reagent. The medium was replaced with fresh medium 24 h later. On day 3 after transfection, the medium was collected, and the viral particles were concentrated 10-fold by centrifuging for 1 h at 40,000× g rpm.

Generation of Truncated β-Catenin Construct
To eliminate the methylation of β-catenin, a truncated construct of the CTNNB1 gene was prepared that was devoid of 133 a.a at its N-terminus. In brief, CTNNB1 wild-type plasmid template was amplified with Q5 high-fidelity polymerase (NEB, Ipswich, MA, USA), using forward primer (5 CATGCAGTTGTAAACTTGATTAA 3 ) with a BamH1 restriction site and reverse primer (5 CAGGTCAGTATCAAACCAG 3 ) with a Not1 restriction site. The amplified product was purified, digested, and cloned into the pcDNA3-RFP vector between the BamH1 and EcoRI sites. Sequences of the plasmid constructs ∆N133-pcDNA-RFP were confirmed by Sanger sequencing.

Generation of the Par4 Construct and Production of Viral Particles
To prepare stable clones of PAR4, the human (h)Par4 gene was amplified with Q5 highfidelity polymerase (New England Biolabs; NEB, Ipswich, MA, USA) using forward primer (5 GAATTCGCCGCCACCATG TGGGGGCG ACTGCTCC 3) with an EcoR1 restriction site (underlined) with Kozak sequences (bold) and the reverse primer (5 ACTAGTTCACTGG-AGCAAAGA GGAGTGGG 3) with an Spe1 restriction site, and cloned into the pLVX-EF1 α-IRES-Puro viral vector. For the generation of viral particles, the same procedure was applied as that described earlier.

Preparation of RKO Stable Clones Expressing Par4
RKO (0.5 × 106) cells were infected with hPar4 10× viral particles along with Polybrene infection reagent. At 72 h post transduction, cells were subjected to puromycin selection (0.5 µg/mL). Cells with puromycin resistance were grown and collected, and were either used to isolate RNA or to perform protein lysate preparation.

Quantitative Real-Time PCR (qRT PCR) and RT PCR
RNA was extracted from cells using GenElute RNA kit (Sigma-Aldrich, USA). To prepare the cDNA, 1 µg of RNA was reverse transcribed using reverse transcriptase (Promega, Madison, WI, USA). qRT PCR was conducted using specific forward and reverse primers for each gene listed in Table 1 (the Hprt gene was used as a housekeeping gene for normalization using gene-specific primers). In qRT PCR, triplicates of the 6 ng cDNA template were used with 500 nM gene-specific primers using 2× PerfeCTa SYBR Green mix (Agentek, Tel Aviv-jaffa, Israel) on an automated rotor gene system RG-3000A (Corbett research, Sydney, Australia). All of the data obtained from three independent qRT-PCR experiments were analyzed using the 2 −∆∆Ct method as described in the manufacturer's instructions, and were expressed as fold change over the indicated controls. To compare expression of Par4 in HCT116 vs. HCT116 sh-Par4, RT-PCR was performed. The PCR conditions were an initial denaturation at 98 • C for 5 min, denaturation at 98 • C for 30 s, annealing for 60 s at 60 • C, and extension for 1 min at 72 • C (31 cycles of amplification). The GAPDH gene was used as an internal control.

Cell Lysate Preparation
To prepare protein cell lysate for immunoprecipitation, cells were solubilized in CelLytic TM M buffer (Sigma-Aldrich, USA), while for the rest of the protein cell lysate preparation, we used lysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% triton X-100. All of the mentioned lysis buffers were supplemented with protease inhibitor cocktail, 1 mM phenylmethylsulfonylfluoride, PMSF, and 1 mM Naorthovanadate (Sigma, St. Louis, MO, USA) to prevent protein degradation. The protein cell lysate used for IP and BA were incubated at 4 • C for 20 min and then disrupted by sonication. Finally, soluble supernatant was collected after centrifugation at 12,000× g rpm for 20 min at 4 • C.

TOPflash Luciferase Reporter Assay
HEK-293T cells (0.2 × 10 6 ) were seeded in 6-well plates and incubated overnight at 37 • C. The cells were transfected with the desired target plasmids (PAR2/PAR4/EZH2/βcatenin wt or ∆N133) along with human Lef-1 TOPflash (Tcf Optimal Promoter + luciferase, T cell factor (Tcf) reporter plasmid containing two sets (the second set in reverse orientation) of three copies of the Tcf binding site upstream of the thymidine kinase (TK) minimal promoter and luciferase open reading frame using PEI transfection reagent (Boehringer-Mannheim). CMV/β-gal plasmid was co-transfected as an internal control for transfection efficiency. After 48 h transfection, the cells were washed and lysed, and then luciferase assay was performed with the Luciferase Reporter System (Cat# E1500; Promega, Heidelberg, Germany) according to the manufacturer's instructions, and luminescence was detected on a Tecan SparkTM10M multimode microplate detection system (Switzerland).

Cycloheximide Chase Assay
To compare the turnover of β-catenin with or without EZH2, plasmids were transfected into HEK293 cells as described above. Following transfection, cells were pre-treated with AYPGKF (200 µM) for 2 h and then exposed to cycloheximide (100 µg/mL) for 5 min to 2 h. Cells were then harvested at regular intervals, and lysates were prepared as mentioned above.

Ectopic Tumor Xenograft Mouse Model Study
To determine the in vivo tumorigenicity of the PAR4/f2rl3 gene, an ectopic tumor xenograft mouse study was performed. In brief, RKO/PAR4-RKO stable clones or HCT116/ HCT116 shPAR4 cells were starved O/N, and the next day were treated with AYPGKF (200 µM) for 4 h. After washing off the cells, 1 × 10 6 cells were injected subcutaneously into the right flank of groups (n = 6) of six-to eight-week-old Hsd: Athymic Nude-Foxn1nu mice (referred to as nude mice). Tumor volumes were monitored twice a week by caliper measurements of each dimension and calculated using the following formula: V = 4/3 π (length/2) (width/2) (depth/2). Mice were terminated by cervical dislocation under aesthetic conditions when the tumor volumes reached the volume stipulated in the Institutional Animal Committee's approval or when the animals showed distress, in order to avoid unnecessary suffering.

Immunohistochemistry
Paraffin-embedded slides derived from PAR4-RKO tumor tissue compared with small tumor tissue derived from parental RKO cells were used for IHC. After deparaffinization and rehydration, the slides were incubated with 3% H2O2 prior to antigen retrieval. Antigen unmasking was carried out by heating (20 min) in a microwave oven with 1× antigen retrieval citrate buffer (Cat# ab93678, Abcam). After blocking with CAS-Block (Cat# 008120, Invitrogen, MA, USA), the slides were incubated with EZH2 antibody (Cat# ab191080; Abcam, Cambridge, UK). Next, following washing, the slides were incubated with peroxidaseconjugated antibody (Abcam, Cambridge, UK). Color was developed using the DAB substrate kit (Cat# 34002, Thermo Scientific, Waltham, MA, USA), followed by counter staining with Mayer's hematoxylin (Cat# 3801582E, Leica, Wetzlar, Germany). Controls using only secondary antibodies (with no primary antibodies) showed low to background staining in all cases.

Statistical Analyses
All of the experiments were carried out in triplicate, whereby the data are represented as mean ± SD. Significant differences in the tested samples in comparison to control were determined by performing either Student's t test or analysis of variance (ANOVA) with Tukey's multiple comparison post test (GraphPad Prism 6.0; Bioz Stars, Los Altos, CA, USA), wherever required. The criterion for statistical significance was as follows: p < 0.05 was considered significant (*), p < 0.01 as highly significant (**), and p < 0.001 as very highly significant (***).  Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committee of Hebrew University (MD-20-15924-5, July 2020).

Data Availability Statement:
The data that support findings of this study are available at the corresponding author upon request.

Conflicts of Interest:
The authors declare no conflict of interest.