Deubiquitinating Enzyme USP9X Suppresses Tumor Growth via LATS Kinase and Core Components of the Hippo Pathway

Abstract

The core LATS kinases of the Hippo tumor suppressor pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Recent studies have identified various E3 ubiquitin ligases that negatively regulate the Hippo pathway via ubiquitination, yet few deubiquitinating enzymes (DUB) have been implicated. In this study, we report the DUB USP9X is an important regulator of the core kinases of this pathway. USP9X interacted strongly with LATS kinase and to a lesser extent with WW45, KIBRA, and Angiomotin, and LATS co-migrated exclusively with USP9X during gel filtration chromatography analysis. Knockdown of USP9X significantly downregulated and destabilized LATS and resulted in enhanced nuclear translocation of YAP and TAZ, accompanied with activation of their target genes. In the absence of USP9X, cells exhibited an epithelial-to-mesenchymal transition phenotype, acquired anchorage-independent growth in soft agar, and led to enlarged, disorganized, three-dimensional acini. YAP/TAZ target gene activation in response to USP9X knockdown was suppressed by knockdown of YAP, TAZ, and TEAD2. Deletion of USP9X in mouse embryonic fibroblasts resulted in significant downregulation of LATS. Furthermore, USP9X protein expression correlated positively with LATS but negatively with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. Overall, these results strongly indicate that USP9X potentiates LATS kinase to suppress tumor growth. Cancer Res; 77(18); 4921-33. ©2017 AACR.

Figure 1. USP9X interacts with Hippo pathway components.

A, USP9X was identified as the top interactor of KIBRA. The chart for the unique peptide counts is presented (left panel). HA-tagged components of the Hippo pathway were transiently expressed in 293 cells and cell lysates were immunoprecipitated with an anti-HA antibody.The co-precipitation of endogenous USP9X with various components was done using USP9X antibody (right panel). B, FPLC analysis of 293 cell lysate.45 fractions (A11-15, B1-15 & C1-11) were collected and proteins combined from two fractions were pooled and run on each lane. Fractions starting from A11 to C11 were shown. Only LATS was found to be strongly co-migrated in the same fractions as USP9X (fractions B5-6). C, D, E, Analytic reciprocal co-immunoprecipitation experiments showing the interaction of USP9X with LATS2, WW45, and KIBRA with LATS2 being the strongest interactor of USP9X. F, Mapping of the LATS2-interacting domain of USP9X. G, Mapping of the USP9X-interacting domain of LATS2.

Figure 2. USP9X stabilizes upstream Hippo components.

A, Knockdown of USP9X by siRNA in HPDE6 (left) and MCF10A (right) cells resulted in downregulation of LATS, WW45, and phospho-YAP. Down-regulation of KIBRA was seen in MCF10A cells. B, Screening of USP9X shRNA in MCF10A cells. Co-downregulation of LATS with USP9X was seen with shRNA #3 and #4. C, MCF10A were stably knocked down with USP9X shRNA #3. LATS, WW45, KIBRA, and phospho-YAP were downregulated (left panel) and TAZ was significantly translocated to the nucleus (right panel) in response to USP9X silencing. D, LATS was downregulated in Floxed USP9X MEF cells after Cre expression. E, F, Knockdown of USP9X by siRNA (E) and shRNA (F) in various cell lines resulted in upregulation of YAP/TAZ target genes, CTGF and CYR61 as accessed by real-time PCR.

Figure 3. USP9X deubiquitinates Hippo components LATS, AMOT, KIBRA, and WW45 in 293 cells.

Expression of increasing amount of USP9X resulted in decreasing amount of endogenous ubiquitinated LATS (A), AMOT (B), exogenous Kibra (C) and WW45 (D). E, Knockdown of USP9X augmented ubiquitinated LATS2. F, Enzyme defective USP9X (Myc-USP9X-H1871A) lost the ability to protect Lats2 from ubiquitination.

Figure 4. Knockdown of USP9X leads to oncogenic transformation.

A, Knockdown of USP9X by shRNA #3 & #4 in MCF10A cells resulted in cell morphological change reminiscent of EMT. B, Increased expression of fibronectin, vimentin and decreased expression of E-Cadherin in USP9X knockdown MCF10A cells. C, Increased invasion activity of USP9X shRNA #3 MCF10A cells in matrigel transwell assays. D, USP9X shRNA #3 MCF7 cells displayed enhanced anchorage-independent growth in soft agar. E, Three-dimensional culture of USP9X shRNA #3 MCF10A in matrigel. USP9X shRNA #3 MCF10A acini have enlarged and disorganized morphology. F, USP9X and LATS expression are highly regulated by growth signal. MCF10A grown in different percentage of serum were assessed for the expression of USP9X, LATS, AMOT, YAP, and TAZ.

Figure 5. YAP/TAZ and TEAD are downstream mediators of USP9X.

A, The activation of YAP/TAZ target genes, CTGF and Cyr61, due to USP9X knockdown could be rescued by knockdown of YAP/TAZ. The mRNAs of CTGF and Cyr61 were accessed by RT-PCR. B, The enhanced anchorage-independent growth by the USP9X knockdown in MCF7 cells could be rescued by knockdown of YAP and TAZ. C, D, TEAD2 was identified as one of the major TEAD isoforms downstream of USP9X. The activation of YAP/TAZ target genes, CTGF and Cyr61, due to USP9X knockdown could be rescued by knockdown of TEAD2. siRNA of the four TEAD isoforms could be effectively knocked down in cells (C) and TEAD2 knockdown could significantly suppress increased CTGF and Cyr61 mRNA in USP9X knockdown cells (D). E, TEAD2 is able to rescue the anchorage-independent growth of USP9X knockdown cells. F, LATS2 is able to rescue the YAP and TAZ expression induced by the USP9X knockdown.

Figure 6. USP9X and LATS are positively correlated in cancer cell lines and pancreatic cancer tissues.

A, Decreased protein expression of USP9X, LATS, WW45, YAP, and TAZ was detected in pancreatic cancer cells. B, Increased mRNA levels of CTGF (left) and Cyr61 (right) in pancreatic cancer cells quantified by RT-PCR. C, USP9X, and LATS protein levels were downregulated in triple negative breast cancer cells. D, Immunohistochemical (IHC) staining of USP9X and LATS in normal and pancreatic cancer tissue microarrays (left panel). USP9X and LATS protein expression were predominantly negative in pancreatic cancer tissues (right panel). E, Characterization of USP9X, LATS and YAP/TAZ antibodies used for IHC tissue staining (top). USP9X and LATS were expressed weakly as opposed to strong YAP/TAZ expression in analogous tissue sections derived from pancreatic cancer patients (bottom). F-H, Analysis of USP9X, YAP and YAP/TAZ target genes using pancreatic cancer patient dataset, GSE21501. F, Tumors expressing USP9X lower than the lower quartile cut off had shorter life-span. G-H, Positive correlation of YAP and its target gene CTGF only observed in the low expression of USP9X.