ZEB1 protein stability is regulated by MEK-ERK signaling
We examined the expression of protein products of EMT transcription factors in CRC cell lines with constitutive MEK-ERK activation. In RKO isogenic cell lines expressing BRAFV600E vs BRAF wild-type (wt) [T29], only ZEB1 and ZEB2 protein levels were increased (Fig.1A-B). Treatment of these cells with the MEK inhibitor cobimetinib suppressed expression of ZEB1 but not ZEB2 (Fig.1B). Similarly, treatment of RKO cells with the selective BRAFV600E inhibitors vemurafenib (Fig.1C) or encorafenib (Fig. 1D) were each shown to suppress ZEB1 expression. Furthermore, another MEK inhibitor trametinib was also shown to decrease ZEB1 expression (Fig. 1D). In Colo320 cells transfected with a mutant KRAS G12D plasmid, induction of ZEB1 protein expression was observed (Fig.1E). Among EMT factors, these data demonstrate preferential regulation of ZEB1 by MEK-ERK signaling.
Treatment of RKO isogenic cells with the proteasome inhibitor, MG132, enhanced ZEB1 expression in BRAFV600E and in wt cell lines (Fig.1F) indicating that ZEB1 undergoes proteasomal degradation. Cells treated with MG132 showed decreased K48-linkage polyubiquitinated ZEB1 protein in BRAFV600E cell in contrast to wt cells (Fig.1G), and this ubiquitination could be reversed by MEK inhibitor treatment (Fig.1H). Enhanced ZEB1 stability was also observed in BRAFV600E compared to BRAF wt cells by treating RKO isogenic cells with an inhibitor of protein synthesis, i.e., cycloheximide (Fig. 1D). Treatment with cobimetinib increased proteasomal degradation of ZEB1 (Fig.1J). Together, these results suggest that MEK-ERK activation can protect ZEB1 from proteasomal degradation to enhance its stability.
The functional consequence of ZEB1 stabilization was examined in a wound healing assay. Isogenic BRAFV600E cells with overexpression of ZEB1 were observed to migrate faster than did cells with BRAF wt (Fig.1K), and this result was confirmed in a transwell assay (Fig.1L-M). An inhibitor of MEK, shown to suppress ZEB1 (Fig. 1B), blocked CRC cell migration (Fig.1N-P). These data suggest that regulation of ZEB1 stability by MEK-ERK can modulate CRC cell migration.
USP10 can destabilize ZEB1 protein
Deubiquitinases (DUBs) can regulate protein stability and in a prior study, we reported that ubiquitin-specific protease 10 (USP10) could deubiquitinate and thereby activated AMPK . We examined the BioGrid database to identify potential DUBs that may interact with ZEB1 and identified USP10 as a potential candidate (supplementary table S1). As shown in Fig.2A-B, USP10 co-immunoprecipitated with ZEB1 in RKO cells. For confirmation, reciprocal immunoprecipitation using antibodies against USP10 or ZEB1 were shown to pull down ZEB1 or USP10 proteins, respectively, in Colo320 cells (Fig.2C-D). Since USP10 is a ubiquitin-specific protease, we determined if USP10 can stabilize ZEB1. Unexpectedly, knockdown of USP10 in RKO cells significantly increased the level of endogenous ZEB1 protein (Fig.2E). USP10 knockdown using a second shRNA was also shown to induce ZEB1 (Fig.2E), and consistent results were observed in Colo320 cells (Fig.2F). To determine whether USP10 can regulate ZEB1 stability, we treated cells expressing USP10 shRNA or control shRNA with cycloheximide and observed that ZEB1 induction was due to its enhanced stability in USP10 knockdown cells (Fig.2G). Furthermore, we found that K48-linkage polyubiquitination of ZEB1 was decreased in cells expressing USP10 shRNA (Fig.2H), indicating impaired proteasomal degradation. Together, these results suggest that USP10 serves to destabilize ZEB1 in human CRC cells.
Since depletion of USP10 can induce ZEB1, we then determined the effect of USP10 on the ability of ZEB1 to regulate CRC cell migration. Using a wound healing assay and a transwell assay, we found that knockdown of USP10 and its associated induction of ZEB1 (Fig. 2E,F), can enhance cell migration in both assays (Fig.2I-K). To further confirm that USP10 regulates cell migration through ZEB1, we performed a knockdown of USP10 or ZEB1 separately or together. CRC cells expressing USP10 shRNA showed faster migration whereas as cells with ZEB1 shRNA displayed slower migration (Fig. 2L). Knockdown of USP10 in cells with ZEB1 shRNA did not accelerate cell migration (Fig.2L), indicating that USP10 regulates cell migration through ZEB1.
Activated MEK-ERK signaling promotes cell migration through USP10
We demonstrated that ZEB1 induction was due to its enhanced stability in USP10 knockdown cells (Fig. 2G). To determine if ERK signaling can mediate stabilization of ZEB1 through USP10, knockdown of USP10 in isogenic cells increased ZEB1 expression in BRAF mutant vs wildtype cells to a similar level (Fig.3A) that was associated with decreased K48 ubiquitination of ZEB1 (Fig.3B). The functional consequence of this finding was shown in a wound healing assay and a transwell assay that examined cell migration. In RKO and T29 cells with control shRNA expression, RKO cells expressing BRAFV600E migrated faster than did T29 cells. However, knockdown of USP10 in both cell lines was shown to increase cell migration to a similar extent (Fig. 3C). In a transwell assay, depletion of USP10 was also shown to enhance migration of both isogenic CRC cell lines (Fig.3 D,E). Taken together, activation of MEK-ERK can stabilize ZEB1 and promote cell migration through USP10.
We then inhibited ERK signaling which was shown to suppress ZEB1 and its regulation by USP10. In RKO and Colo320 cells treated with cobimetinib, we observed a decrease in ZEB1 protein level compared to control treated cells (Fig.4 A,B). Knockdown of USP10 increased ZEB1 expression in the presence or absence of a MEK inhibitor (Fig.4 A,B) or the BRAF inhibitor, vemurafenib (Fig.4C). Enhanced K48 ubiquitination of ZEB1 was detected in both cobimetinib and vemurafenib treated cells, but knockdown of USP10 impaired these ubiquitination signals (Fig.4D-E). Cobimetinib was shown to suppress CRC cell migration whereas depletion of USP10 reversed and accelerated cell migration even in the presence of MEK inhibition (Fig.4F-H). Accordingly, suppression of MEK-ERK can destabilize ZEB1 through USP10.
USP10 destabilizes ZEB1 by editing its ubiquitination
As a DUB, USP10 can stabilize its substrate or regulate substrate activity. To elucidate the mechanism of ZEB1 protein destabilization by USP10, we transfected control and USP10 depleted RKO cells with a panel of ubiquitin mutants where only one lysine was intact. We detected ZEB1 ubiquitination with different lysine linkage. Unexpectedly, we found that K27- linkage ubiquitinated ZEB1 was decreased, whereas K48-linkage ubiquitinated ZEB1 was consistently increased in USP10 knockdown cells (Fig.5A). This finding suggests that USP10 can selectively remove K27-linkage to allow for subsequent K48-linked ubiquitination of ZEB1. USP10 knockdown was shown to increase K27 ubiquitination and inhibit K48 ubiquitination of ZEB1. To confirm this result, we re-introduced wildtype USP10 or a catalytically dead mutant of USP10 into USP10 knockdown cells. Reconstitution of wildtype USP10 was shown to reverse the ubiquitination pattern of ZEB1, but not cells with the catalytic dead mutant (Fig.5B).
To assess the functional consequence of regulation of ZEB1 by USP10, CRC cell migration was again analyzed. We observed increased cell migration in USP10 knockdown cells in presence or absence of cobimetinib (Fig.5C). Next, we assessed the effect of a USP10 catalytically dead mutant on cell migration. While re-introduction of wt USP10 into USP10 depleted cells was shown to suppress cell migration, this was not observed for the catalytically dead USP10 mutant. Together, these results suggest that USP10 can edit ZEB1 ubiquitination and thereby, influence CRC cell migration through its DUB activity.
ERK phosphorylates USP10 at S236 and promotes its disassociation from ZEB1
We determined whether the interaction of USP10 with ZEB1 is altered by MEK-ERK signaling. We found that the USP10-ZEB1 interaction was attenuated in MEK-ERK activated BRAFV600E compared to BRAF wt cells (Fig.6A), and this interaction was enhanced by treatment with cobimetinib (Fig.6B). Since ERK kinases have various cytosolic and nuclear substrates in contrast to RAF and MEK1/2 kinases that have narrow substrate specificity , we hypothesized that ERK can phosphorylate USP10 which may alter its interaction with ZEB1. ERK activated BRAFV600E cells showed enhanced phosphorylation of USP10 compared to wt cells (Fig.6C), and suppression of MEK-ERK attenuated USP10 phosphorylation shown to enhance the USP10-ZEB1 interaction (Fig.6D). Analysis of the USP10 protein sequence identified two potential ERK phosphorylation sites, T74 and S236, based on the ERK substrate motif. Whereas mutation of Thr to Ala at 74 had no effect phosphorylation of USP10 and its interaction with ZEB1, mutation of Ser to Ala at 236 suppressed ERK-mediated USP10 phosphorylation and enhanced the USP10-ZEB1 interaction. Furthermore, treatment of cells expressing this phosphorylation mutant (S236A) with cobimetinib did not alter the phosphorylation event nor the interaction between USP10 and ZEB1 (Fig.6E), and a double mutant behaved similarly to the S236A mutant (Fig.6E). Importantly, expression of the USP10 S236A mutant was shown to suppress K27 ubiquitination and to increase K48 ubiquitination of ZEB1 (Fig.6F) that resulted in its degradation (Fig. 6G) and its suppression of cell migration. Lastly, inhibition of MEK/ERK failed to impact ZEB1 ubiquitination pattern, protein stability and cell migration in cells expressing the S236A mutant (Fig.6F-H).
Targeting MEK-ERK-USP10-ZEB1 axis inhibits metastasis in vivo.
To further study the role of the MEK-ERK-USP10- ZEB1 axis in tumor metastasis, we utilized a human-mouse xenograft model. We found that knockdown of USP10 in RKO cells xenografted into mice promoted lung metastasis to a significantly greater extent compared to xenografted control cells (Fig. 7A,B). Furthermore, knockdown of ZEB1 was also shown to dramatically suppress lung tumor development, whereas knockdown of USP10 in ZEB1-depleted cells failed to further reduce the number of lung nodules (Fig.7A,B). These results demonstrate that USP10 can regulate CRC cell metastasis through the EMT transcription factor ZEB1.
We then determined whether inhibition of MEK-ERK signaling can suppress tumor metastasis through the USP10-ZEB1 axis in our CRC mouse model. We treated cells with encorafenib in combination with cetuximab since this EGFR inhibitor is needed due to rebound activation of EGRF signaling when RAF/RAS MEK-ERK signaling is inhibited . While treatment with encorafenib or cetuximab alone did not significantly suppressed tumor metastasis, we found that their combination significantly suppressed lung metastasis in our CRC murine model (Fig.7C,D). To further demonstrate the role of USP10 in MEK-ERK-mediated CRC metastasis, we performed knockdown of USP10 in xenografted CRC cells which was shown to markedly enhance lung metastasis in the presence or absence of treatment with encorafenib, cetuximab or their combination (Fig.7C,D).