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Targeting the E3 Ubiquitin Ligase PJA1 Enhances Tumor-Suppressing TGFβ Signaling

These findings provide a novel mechanism regulating the tumor suppressor function of TGFβ in liver carcinogenesis RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFβ/SMAD signaling. SMAD3 and its adaptors, such as β2SP, are important mediators of TGFβ signaling and regulate gene expression to suppress stem cell–like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/β2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3+/−), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFβ1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients. Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFβ in liver carcinogenesis.


Introduction
The incidence of hepatocellular carcinoma (HCC) in the United States has increased three-to four-fold in recent years, and overall 5-year survival rates are only 11% (1,2). Despite four new therapeutics and genomic analyses from The Cancer Genome Atlas (TCGA), HCC remains incurable with few effective genomic-based targeted therapeutics (3,4). Moreover, HCC is a heterogeneous cancer, and most patients present with underlying cirrhosis or decompensated liver disease; consequently, they are difficult to treat with standard doses of chemotherapeutics (5,6). Better strategies for HCC diagnosis, prevention, and therapy are urgently needed.
Liver is a regenerative organ with multiple regions in a lobule containing stem cells and niches regulating regeneration (7). The TGFb/SMAD pathway is a key regulator of liver stem cells (LSC) and liver regeneration (8)(9)(10)(11)(12)(13). Some forms of HCC have characteristics of cancer stem cells (14) and may arise through transformation of cells in the LSC population. Mechanistic insight into the pathways that drive stem cell transformation could lead to the development or identification of targeted therapeutics for these cancers. Through an analysis of 9,125 cancers, we discovered that cancers with a cancer stem cell signature have altered TGFb signaling (15). We observed that expression patterns of genes encoding components of this complex pathway are decreased in HCCs ($30%-40% of HCCs), suggesting an inhibitory role of TGFb in cancer stem cell transformation. An inhibitory role for TGFb in this process is consistent with TGFb-deficient mouse models of HCC (16)(17)(18).
TGFb represents a large family of growth and differentiation factors that mobilize a complex signaling network to control cell fate by regulating differentiation, proliferation, motility, adhesion, and apoptosis (19)(20)(21). TGFb superfamily signals are conveyed by specific intracellular mediators, the SMAD proteins. Vertebrates possess at least eight SMAD proteins, which are divided into three functional classes: (i) receptor-activated SMADs (R-SMAD): SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8 (also known as SMAD9); (ii) comediator SMAD: SMAD4; and (iii) inhibitory SMADs (I-SMAD): SMAD6 and SMAD7. Upon phosphorylation by TGFb receptors, R-SMADs translocate to the nucleus together with SMAD4 to regulate transcriptional targets. A negative feedback loop involving transcriptional upregulation of I-SMADs limits SMAD-mediated signaling (21). Transcriptional output of R-SMADs is influenced by positive and negative regulators of the TGFb-SMAD pathway, interacting proteins, and the activity of other signal transduction pathways (22,23).
Here, we focused on SMAD3-dependent signaling, because SMAD3 controls stem cell function and contributes to liver regeneration (24)(25)(26). Impaired function of SMAD3 is associated with a human stem cell syndrome (27) that has an increased risk of developing cancer (28). The adaptor protein, beta spectrin (b2SP), which is encoded by SPTBN1, genetically and biochemically interacts with SMAD3 to promote expression of tumor-suppressing genes in mice (18).
PJA1 of the PRAJA family of E3 ubiquitin ligases represents a negative regulator of SMAD3 and b2SP, because PJA1 promotes ubiquitination of SMAD3 and b2SP (29). The expression of PJA1 is increased in some cancers, including those of the gastrointestinal tract (29). Thus, we hypothesized that PJA1 impairs a tumorsuppressing TGFb response mediated by SMAD3 and b2SP, thereby functioning as an oncogene or contributor to tumorigenesis.
To investigate this hypothesis, we manipulated PJA1 abundance in cultured HCC cells and determined the effect on phenotypes associated with cancer, such as proliferation, anchorage-independent growth, and tumor growth in mouse xenograft studies. We established LSCs from mice with compromised SMAD3 activity (Smad3 þ/À ) and overexpressed PJA1 and tested their ability to form tumors when injected into mice. Because E3 ubiquitin ligases represent attractive potential therapeutic targets, we demonstrated that small-molecule inhibitors of PJA1 increased SMAD3 and b2SP abundance in cultured HCC cells, increased SMAD3-dependent reporter gene expression, reduced HCC cell viability, and impaired xenografted tumor growth. Our results indicated that PJA1 functions as a tumor-promoting E3 ubiquitin ligase by inhibiting SMAD3/b2SP-regulated genes and suggested that PJA1 represents a potentially useful therapeutic target in HCC associated with impaired TGFb signaling.

Study approval
All animal experiments were performed according to the guidelines for the care and use of laboratory animals and were approved by the institutional biomedical research ethics committee of The University of Texas MD Anderson Cancer Center (MDACC, Houston, TX).

Plasmids, reagents, and antibodies
The PCR-generated DNA fragments of the human PJA1, mouse PJA1 (30), human SMAD3, and human SPTBN1 genes (29) were subcloned into pCMV5 to generate constructs with a Flag tag or HA tag, or into pcDNA TM 6 to generate constructs with a V5 tag. Human PJA1 ring domain deletion (dR; 350-395 aa deletion) constructs were generated in a Flag-tagged or HA-tagged vector. Mouse PJA1 1 -150 aa or 150-300 aa mutants were generated in a Flag-tagged or V5-tagged vector. Mouse PJA1 cDNA was obtained from GE Dharmacon, Inc. and subcloned into the pB513B vector. Lentiviral particles containing short hairpin RNA (shRNA) of PJA1 (sc-91297) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology. The 4xSBE luciferase reporter and 3TP luciferase reporter plasmids were from Addgene, Inc., the Renilla plasmid was from Promega. MG132 (M7449, Sigma), and TGFb1 (Sigma, T1654) were purchased from Sigma. G-418 was from (4727878001, Sigma).

Cell proliferation and viability assay
PJA1-dR-c1, PJA1-dR-c2, and control cells were seeded onto 6-well plates (1 Â 10 4 cells/well). The cultures were incubated for 6 days. Cell numbers were measured daily by Trypan blue staining (0.4%; Thermo Fisher Scientific, T10282) using the Countess Automated Cell Counter (Invitrogen). All assays were performed in triplicate and repeated at least three times. PJA1/wt/LSCs and PJA1/Smad3 þ/À /LSCs were isolated from single mice and immediately seeded and cultured onto 96well plates (3 Â 10 3 cells/well) in suitable supplement-containing medium. Cell proliferation was measured using CyQUANT NF Cell Proliferation Assay reagent at day 1, 3, and 6 as described previously (32). Data are from three independent experiments. HepG2, Hep3B, and Huh7 cells were seeded onto 96-well plates (3 Â 10 3 cells/well). Twenty-four hours later, the cells were exposed to RTA402 or RTA405 at the indicated concentration in serum-free DMEM. Viability was measured by MTS assay after 24, 48, or 72 hours. Control cells were treated with DMSO. Data presents the viable cells as a percent of control cells.
In all transfections, the expression plasmid Renilla (Promega) served as an internal control to correct for transfection efficiency and samples were collected and analyzed according to the manufacturer's instructions for the Luciferase Assay System (Promega). The cells were extracted using 100 mL of luciferase cell culture lysis reagent. Ten microliters of cell extract were used for measuring Renilla enzyme activity; 20 mL were used for the luciferase assay. After subtracting the background (nontransfected cell control or DMSO treated), luciferase activity was normalized to Renilla activity (arbitrary units) for each sample.

IHC and immunofluorescence analyses
For IHC analysis, tissues were fixed in 10% formalin and embedded in paraffin in accordance with standard procedures. Cryosections (5 mm thick) were stained for caspase-3, Ki67, or PJA1. Diaminobenzidine was used as a chromogen, and hematoxylin and eosin was used for counterstaining. Quantitative analysis was performed by manually counting positive staining cells in samples from multiple tumors or normal liver tissue and HCC tissue. The researcher scoring the samples was blinded to the sample identity.
For immunofluorescence analysis of CD133 þ stem cells, cells derived from wild-type mice injected with PJA1 plasmids (PJA1/wt/ LSCs), or from Smad3 þ/À mice injected with PJA1 plasmids (PJA1/ Smad3 þ/À /LSCs) were isolated and seeded onto chamber slides at 2 Â 10 4 cells per well for 12 hours. For confocal imaging of HepG2 cells exposed to RTA402 or RTA405, 2 Â 10 4 cells were plated onto cover slips in 6-well plates. After 12 hours, the cells were exposed to TGFb1, RTA402, or RTA405 for 2 hours. Control cells were exposed to DMSO. For all immunofluorescence analysis, cells were fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and blocked in 10% normal goat serum in PBS. The cells were incubated with primary antibodies, washed 3 times in PBS, and then incubated with goat antimouse or goat anti-rabbit secondary antibodies conjugated with Alexa-488 or Alexa-555. DAPI or DRAQ5 was used for nuclear staining. For confocal analysis, slides were examined using Zeiss LSM 710. Quantitative analysis of the cells with nuclear SMAD3 was performed by manually counting the number of foci in positivestaining cells. The researcher scoring the samples was blinded to the sample identity.
Hydrodynamic tail vein injections and liver cancer stem cell formation assays in mice Ten-to 12-week-old female wild-type and Smad3 þ/À mice were prepared for hydrodynamic tail vein injection using the Sleeping Beauty (SB) transposon/transposase system. The PiggyBac Transposon System (pB513B transposon and SB transposase vector) was purchased from System Biosciences, Inc. (32). Mouse PJA1 cDNA (GE Dharmacon, Inc.) was subcloned into the pB513B vector. PJA1 plasmids or control plasmid DNA (empty pB513B; 5 mg in 1.5 mL of 0.45% saline solution per mouse) in an equivalent volume of saline solution, along with SB transposase in a ratio of 25:1, were diluted in 1.5 mL of 0.45% saline solution, filtered through 0.22 mm filter (Millipore), and injected hydrodynamically through the tail vein into wild-type or Smad3 þ/À mice twice per month for 3 months. At 3 months, the mice were humanely killed, and samples of liver tissue were fixed in 10% formalin, embedded in paraffin, and stained for PJA1. Single-cell suspensions from the mouse livers were prepared as described previously (32). CD133 þ LSCs were isolated from each liver preparation using a magnet-activated cell-sorting column with an antibody recognizing CD133. Isolated CD133 þ LSCs from each mouse were assessed for anchorage-independent growth, colony formation, and proliferation assays or were injected into mice to evaluate tumor formation.

Whole-transcriptome sequencing and database analyses
Whole-transcriptome RNA sequencing was performed and analyzed at MDACC DNA core facility. Gene expression profiling data were gathered from human liver cancer cell lines: HepG2-shRNA-Ctrl (cells stable expressing control shRNA), HepG2-shRNA-PJA1 (cells stable expressing shRNA targeting PJA1), HepG2-TGFb1 (cells were treated with TGFb1 at 200 pmol/L for 1 hour), and HepG2-Ctrl (cells were treated with vehicle). We identified differentially expressed mRNAs between the four experimental conditions using the standard comparison mode with multiple testing corrections (P adj < 0.05). Differentially regulated genes in each condition were determined and compared [Gene Expression Omnibus (GEO): GSE132723; Supplementary Table S1).
For evaluating stemness indices and TGFb pathway activity, we used TCGA pan-cancer and liver hepatocellular carcinoma transcriptome sequencing data (bam files downloaded on May 2019); their related clinical data were obtained from the Cancer Genomics Hub (https://ucscgenomics.soe.ucsc.edu/cghub-adds-childhood-cancerdata/) and TCGA Data Portal (https://tcga-data.nci.nih.gov/tcga/). Paired-end FASTQ files for each sample were extracted from bam files using bam2fastq (http://www.hudsonalpha.org/gsl/information/ software/bam2fastq). The number of fragments per kilobase of nonoverlapped exon per million fragments mapped (FPKM) was generated at The University of Texas MDACC. Stemness indices (mRNAsi score) were generated by a one-class logistic regression machinelearning algorithm program (15). TGFb pathway activity was determined as described previously (15,17). TCGA HCC samples (n ¼ 368) were stratified by their stemness index, defined as ranking the samples by their RNA-based stemness index (mRNAsi) and dividing the samples into top, intermediate, and bottom thirds.
To evaluate the relationship between PJA1 expression and gene expression in HCC, we downloaded the Affymetrix mRNA microarray data GSE9843 (n ¼ 91, HCC) from NCBI's GEO. This dataset was divided into four quartiles according to PJA1 mRNA abundance and differentially expressed genes. We used an mRNA z-scores threshold of AE2.0 to determine whether a gene significantly increased or decreased compared with those of the normal samples. Transcriptomes of the highest PJA1 quartile were compared with those of the lowest PJA1 quartile using Nexus Expression 3.0 (BioDiscovery). Upregulated or downregulated genes in samples expressing a high level of PJA1 are provided in Supplementary Table S2.

PJA1 functions as a tumor promoter and enhances the function of liver cancer stem cells
We first detected the amount of PJA1 in human liver cancer cell lines including HepG2, Hep3B, SNU387, SNU398, SNU449, SNU475, and Huh7, and compared the analyses with a normal hepatocyte cell line, THLE-3. We found that higher levels of PJA1 were present in most liver cancer cell lines, whereas PJA1 was not detected in normal hepatocytes (Fig. 1A). We inhibited PJA1 function by knocking down PJA1 with shRNAs or by expressing a dominant-negative mutant lacking the RING domain (34) in four human liver cancer cell lines (Hep3B, HepG2, SNU398, and SNU475). Each cell line overexpressing the RING domain-deleted PJA1 exhibited significantly reduced proliferation compared with control cells (Fig. 1B). Knockdown of PJA1 significantly reduced colony formation in all four liver cancer cell lines ( Fig. 1C; Supplementary Fig. S1) and anchorage-independent growth of SNU475 and HepG2 cells (Fig. 1D). Knockdown of PJA1 impaired tumor growth in a xenograft model of subcutaneously injected SNU475 cells in nude mice (Fig. 1E). Knockdown of PJA1 resulted in reduced numbers of Ki67-positive cells and increased numbers of cells positive for the apoptosis effector caspase-3, suggesting that PJA1 promoted HCC cell proliferation and protected against apoptosis (Fig. 1F). These data indicated that PJA1 functions as a tumor promoter and that reducing its activity in liver cancer cells impairs malignant phenotypes.
Previously, we identified a range of stem cell signatures in 9,125 samples from 33 tumor types, including 368 TCGA HCCs, in the TCGA databases (15). Here, we assessed the relationship between TGFb pathway activity (17) and stem cell signatures in the 33 tumor types ( Fig. 2A; Supplementary Fig. S2), which revealed that low TGFb pathway activity correlated with a higher stem cell index. In addition, we stratified the HCC samples in this dataset according to those with a high, intermediate, and low stem cell index and found a negative correlation between TGFb pathway activity and stem cell-like character (Fig. 2B). Thus, these results indicated that impairment of TGFb signaling, as would be expected if PJA1 activity is high, could contribute to stem cell-like properties of cancers, including HCC.
To explore the effect of PJA1 overexpression on stem cell properties and an interaction with TGFb signaling, we investigated whether PJA1 exhibited an interaction with SMAD3 in transforming LSCs into liver cancer stem cells by comparing the effect of PJA overexpression in wild-type or Smad3 þ/À mice on LSCs. We introduced SB transposase along with SB transposon expressing mouse PJA1 in the mouse liver using hydrodynamic tail vein injection as described in Materials and Methods. We confirmed the presence of PJA1 in mouse livers by IHC staining, and isolated LSCs at day 90, using the stem cell marker CD133 þ (Fig. 2C and D; ref. 32).
Compared with the CD133 þ cells from the PJA1-injected wild-type mice, CD133 þ cells isolated from the PJA1-injected Smad3 þ/À mice exhibited enhanced proliferation in culture and increased Ki67 staining ( Fig. 2E and F). Furthermore, the cells from the PJA1-injected Smad3 þ/À mice formed colonies of cells at 14 days in culture (Fig. 2G), indicating that individual cells within the population were responsible for increased proliferation. To assess whether the LSCs from the PJA1injected Smad3 þ/À mice had properties of cancer stem cells, we compared the anchorage-independent growth of cells from the PJA1-injected wild-type mice and the PJA1-injected Smad3 þ/À . The LSCs from the PJA1-injected Smad3 þ/À mice exhibited an increased frequency to form colonies in soft agar (Fig. 2H) and when injected subcutaneously into immune-compromised mice, 2 of 6 mice injected with cells from the PJA1-injected Smad3 þ/À mice formed tumors and liver metastases (Fig. 2I). None of the mice injected with the cells from the PJA1-injected wild-type or plasmid controlinjected Smad3 þ/À mice formed tumors. These data suggested that increased PJA1 alone did not overcome regulatory mechanisms limiting transformation of LSCs. However, in the context of compromised SMAD3 signaling, increased PJA1 activity enhanced the potential for individual LSCs to transform into highly proliferative, malignant cancer stem cells.

PJA1 promotes phosphorylated SMAD3 ubiquitination and degradation
We previously reported that PJA1 overexpression in HepG2 cells promoted SMAD3 ubiquitination (29). Here, we investigated the interaction between PJA1 and SMAD3 in terms of the regions of PJA1 involved and the subcellular location of the interactions. We found that the middle region (amino acids 150-300) of PJA1 was necessary for the interaction with SMAD3 (Fig. 3A). In HepG2 cells exposed to TGFb1, we found that TGFb1 treatment promoted the interaction of PJA1 and SMAD3 both in the cytoplasm and the nucleus (Fig. 3B, right top 2 panels). PJA1 also interacted with SMAD2 in cell cytoplasm in a TGFb-independent manner, and the two exhibited a weak interaction in the nucleus (Fig. 3B, right 2 bottom panels). Overexpression of PJA1 decreased coexpressed Flag-tagged SMAD3 but not SMAD2 abundance (Fig. 3C). PJA1-mediated polyubiquitination was only detected for phosphorylated SMAD3 (p-SMAD3) in cells exposed to TGFb1; polyubiquitin-modified forms of nonphosphorylated SMAD3 were not detected in HepG2 cells in the presence or absence of TGFb1 (Fig. 3D). Although mono-ubiquitin-modified forms were detected for nonphosphorylated SMAD2 in both presence and absence of TGFb1, no ubiquitin-modified form was detected for phosphorylated SMAD2 (Fig. 3D). Although both SMAD3 and SMAD2 may interact with PJA1, only the phosphorylated form of SMAD3 is a substrate for ubiquitination. Inhibition of the proteasome with MG132 partially prevented the reduction in p-SMAD3, but not in p-SMAD2 abundance in cells overexpressing HA-tagged PJA1 (Fig. 3E). These data indicated that PJA1 promotes the ubiquitination and proteasomal degradation of p-SMAD3, leading us to predict that PJA1 limits SMAD3-dependent signaling downstream of TGFb signaling.

PJA1 limits SMAD3-dependent gene regulation in response to TGFb1 stimulation
We analyzed the effect of knocking down PJA1 on the expression of SMAD3-regulated, cancer-related genes, and the overlap between PJA1-regulated genes and those stimulated by TGFb1. We performed transcriptome sequencing analysis of HepG2 cells in which PJA1 was knocked down or that were exposed to TGFb1 and determined the differentially regulated genes (Fig. 4A; Supplementary Table S3). Among the overlapping differentially regulated genes, 48% of the genes upregulated by PJA1 knockdown overlapped with those upregulated by TGFb1 and 52% of TGFb1upregulated genes overlapped with those upregulated by PJA1 knockdown. Similarly, 50% of the genes downregulated by PJA1 knockdown overlapped with those downregulated by TGFb1 and 62% of TGFb1-downregulated genes overlapped with those downregulated by PJA1 knockdown. The genes that showed similar regulation include: the co-upregulating genes SERPINE3 (encoding a member of the PAI1 family), RUNX2, FOS, CDKN2B, and VCAN, which are associated with cell proliferation, apoptosis, and migration, and angiogenesis; the co-downregulating genes NODAL and TGFB3 (both members of the TGFb superfamily), TERT, and MYCBP. These data suggested that PJA1 and TGFb1 reciprocally regulate overlapping genes and that PJA1 may play an oncogenic role by affecting TGFb signaling.
We verified the inverse relationship between PJA1-and TGFb1-regulated gene expression in several independent experiments. We selected two genes (SERPIN1 and FOS) induced by the TGFb1/SMAD3/bSPN pathway (35,36) and analyzed mRNA microarray data from Oncomine (Fig. 4B). For both SERPIN1 and FOS, there was a statistically significant inverse relationship between PJA1 expression and the mRNA abundances of these coregulated transcripts. Consistent with a reciprocal functional relationship with the TGFb pathway, PJA1 knockdown increased transcripts of SERPIN1 and FOS and reduced those of MYC and TERT (Fig. 4C). HepG2 cells transfected with either of 2 reporter genes controlled by TGFb signaling, one with four copies of the SMAD3 binding element (4SBE) or one with three copies of a TGFb-regulated sequence from tissue plasminogen promoter reporter (3TP), exhibited significantly reduced expression in cells coexpressing Flag-SMAD3 and HA-PJA1 in comparison with the expression in cells expressing Flag-SMAD3 alone (Fig. 4D). The SMAD3/b2SP complex binds to the TERT promoter (18). PJA1 knockdown significantly increased basal and TGFb1-induced SMAD3 and b2SP occupancy at the TERT promoter in HepG2 cells (Fig. 4E).
In summary, these results indicated that PJA1 promotes the ubiquitination of phosphorylated SMAD3, resulting in reduced activity of a TGFb/SMAD3/b2SP-dependent tumor-suppressing pathway in HCC cells (Fig. 4F). Thus, we predicted that inhibition of PJA1 may enhance TGFb signaling, increasing the activity of this tumorsuppressor pathway and thus suppressing tumor growth.
Small molecules that interfere with PJA1 enhance SMAD3 signaling and impair tumor growth E3 ubiquitin ligases are potential targets for drug development (37). We collaborated with REATA Pharmaceuticals on a screen of small molecules for inhibitors of E3 ubiquitin ligases and identified two triterpenoid compounds with PJA1 inhibitory activity: RTA402 and RTA405. Synthetic triterpenoids are a subclass of antioxidant inflammation modulators, derived from oleanolic acid found in medicinal plants (38,39). Here, we performed computational molecular docking simulations with oleanolic acid, a natural triterpenoid, or the synthetic triterpenoid (RTA402/405) to the RING domain of PJA1 (Supplementary Materials and Methods; ref. 40). Both oleanolic acid and the synthetic triterpenoid bound to a pocket in the RING domain (Fig. 5A). The docking energy of oleanolic acid for the RING domain of PJA1 was predicted to be 10.12 kcal/mol and that of the synthetic triterpenoid is predicted to be 8.66 kcal/mol, suggesting that both naturally occurring and synthetic triterpenoids bind tightly to PJA1.
To determine the effect of RTA402 and RTA405 on PJA1, we exposed HepG2 and SNU449 cells to each compound (0.25 mmol/L for 1 day) and found these compounds decreased the abundance of PJA1 (Fig. 5B). Exposure of HepG2 or SNU449 cells to RTA402 increased the abundance of b2SP and p-SMAD3 (Fig. 5B). Exposure of HepG2 cells to either compound increased the proportion of cells with nuclear SMAD3 even in the absence of added TGFb1 (Fig. 5C). Analysis of the effect of RTA402 or RTA405 on SMAD3 transcriptional activity using the SBE reporter assay revealed that these compounds enhanced SMAD3 transcriptional activity (Fig. 5D). Collectively, these data indicated that RTA402 and RTA405 enhanced SMAD3-mediated gene regulation, likely by affecting PJA1 activity.
We tested the effect of RTA402 and RTA405 on liver cancer cell line growth in culture. Both compounds inhibited the growth of three different liver cancer cell lines in culture in a dose-and time-dependent manner (Fig. 6A). To evaluate apoptotic cell death, we exposed HCC cell lines to RTA402 or RTA405 (1 mmol/L) for 2 hours (Fig. 6B and C). RTA405 increased the proportion of apoptotic cells in all five HCC cell lines, and RTA402 increased the proportion of apoptotic cells SNU449, SNU398, and SNU475 cells, but not in HepG2 or Huh7 cells. When administered to the mice at the time the tumor cells were injected, RTA405 produced a concentration-dependent reduction in growth of HepG2 tumors in immune-deficient nude mice (Fig. 6D).

PJA1 is a potential therapeutic target for HCC
We analyzed transcriptome sequencing data of 374 HCC patient samples in TCGA. The amount of PJA1 mRNA was significantly increased in HCC compared with the amount in normal liver (Fig. 7A,  left). An increase in PJA1 transcripts in patients with HCC relative to the amount in normal liver was also detected in the Roessler liver 2 data from Oncomine and the Wurmbach liver data from GEO (Fig. 7A, middle and right; ref. 41). Analysis of PJA1 protein revealed an increase in HCCs compared with its abundance in normal livers (Fig. 7B). We analyzed the transcriptomes of 91 HCC patient samples, (Continued.) Scale bars, 50 mm. E, Proliferative potential of CD133 þ cells isolated from PJA1-injected wild-type mice (PJA1/wt/LSCs) and PJA1-injected Smad3 þ/À mice (PJA1/Smad3 þ/À /LSCs). CD133 þ LSCs were cultured in liver cell medium on lysine/laminin-coated plates. Ã , P < 0.05; ÃÃ , P < 0.001, one-way ANOVA. F, Identification of proliferative LSCs by immunostaining of Ki67. Nuclei were labeled with DRAQ5. G, Differences in growth behavior of PJA1/Smad3 þ/À /LSCs and PJA1/wt/LSCs. CD133 þ LSCs from WT or Smad3 þ/À mice were plated on lysine/laminin-coated plates and grown in liver cell medium. Representative images from day 14 in culture are shown. H, Growth of LSC colonies in soft agar. Cells were grown for 4 weeks, and colonies were counted from three experiments. Ã , P < 0.05, twotailed Student t test. I, Tumor formation by PJA1/Smad3 þ/À /LSCs or PJA1/wt/LSCs. LSCs were injected subcutaneously into NOD/SCID gamma chain-knockout mice (n ¼ 6, per cell line). After 30 days, mice were sacrificed and evaluated for the formation of tumors at the site of injection and in the liver. Representative images from the two mice injected with PJA1/Smad3AE/LSCs that developed tumors are shown. Scale bar, 5 mm. E and H, Quantitative data are presented as mean AE SD.

Targeting Hepatocellular Carcinoma through PJA1
AACRJournals.org Cancer Res; 80(9) May 1, 2020     The indicated cells were treated with the indicated concentrations of RTA402 or RTA405 for various times. B, Induction of apoptosis in HCC cells exposed to RTA402 or RTA405. The indicated cells were treated with RTA402 (1 mmol/L) or RTA405 (1 mmol/L) for 2 hours. Representative results of FACS analysis for three independent experiments are shown. Percent apoptotic cells from the boxed areas are indicated beneath each FACS plot. C, Quantitative data of FACS analysis in B. Data are shown as mean AE SD of three independent experiments. Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001, one-way ANOVA with post hoc Bonferroni test. D, Effect of RTA405 on HCC tumor growth. Mice were subcutaneously injected with 5 Â 10 6 HepG2 cells and intraperitoneally injected with PBS (control, Ctrl), RTA405 (15 mg/kg), or RTA405 (30 mg/kg). Six mice were used for each treatment group. Error bars are shown as mean AE SDs. Ã , P < 0.05, one-way ANOVA.
which revealed an increase in the expression of oncogenes and genes associated with antiapoptosis and a decrease in the expression of genes associated with TGFb signaling and DNA damage repair in samples with high PJA1 expression (Fig. 7C). Thus, PJA1 is upregulated in some HCC and may promote tumorigenesis or progression. We further investigated PJA1 genetic alterations in TCGA HCC data. We found 24 patients with HCC (7%) with PJA1 genetic alterations, including 19 patients with increased PJA1 mRNA expression PJA1 is a potential target for therapeutic intervention in HCC. A, The abundance of PJA1 mRNA in HCC compared with that in normal liver tissue from three independent cancer datasets. Fold change compares the difference in the mean. B, IHC staining for PJA1 abundance in normal liver (n ¼ 10) and HCC (n ¼ 13). Blue arrowheads, negative PJA1 staining in cell nucleus; red arrowheads, positive PJA1 staining in cell nucleus. Magnification, Â10; inset magnification, Â40. Scale bar, 100 mm. A and B, Mean AE SD is indicated. Statistical analysis was performed by two-tailed Student t tests. C, Transcriptomic analyses of HCC patient datasets from GEO (GSE9843, n ¼ 91). Transcriptomic data were clustered into four quartiles according to PJA1 expression using Nexus Expression 3.0. Representative pathways and genes associated with high PJA1 expression are listed. Green boxed genes are those with a negative correlation with PJA1 expression and red boxed genes are those with a positive correlation with PJA1 expression. D, Overall survival analysis of TCGA HCC patient dataset. Overall survival according to increased or normal mRNA levels of PJA1 in patients with HCC shows statistically significant differences (Log rank test, P ¼ 0.0042; left). Median survival of these two groups is listed on the right. Table 4). We found that increased mRNA expression of PJA1 was markedly associated with reduced overall survival of patients with HCC (Fig. 7D, left). Median survival of HCC with high PJA1 mRNA expression is dramatically shorter than the patients with HCC with normal PJA1 mRNA expression (13.63 months vs. 60.84 months; Fig. 7D, right). These data suggest that high activity of PJA1 is associated with poor HCC prognosis.

Discussion
There are two major themes for roles of TGFb in human disease: one is increased TGFb activity, as occurs in patients with fibrosis and progressive cancers, and the other is decreased TGFb activity, as occurs in early tumorigenesis, hereditary hemorrhagic telangiectasia, developmental defects, and arteriosclerosis (42). Here, we explored a mechanism for impairment of the tumor-suppressing activity of TGFb signaling through the ubiquitin ligase PJA1.
Following up on our previous work showing that PJA1 interacted with b2SP and mediated both b2SP and SMAD3 ubiquitination (29), and that a TGFb pathway mediated by SMAD3/b2SP promotes tumor-suppressing gene expression (18), we focused on HCC and investigated whether PJA1 functioned as a tumor promoter. We focused on HCC because SMAD3 is a tumor suppressor that is frequently inactivated in gastrointestinal cancers including HCC (36) and because heterozygous deficiency in SPTBN results in the spontaneous development of HCC in mice between 9 and 12 months of age (18). Homozygous SPTBN deficiency is embryonic lethal (43,44).
Because SMAD3 and b2SP are implicated in the regulation of stem cell-like properties and because altered activity of this pathway may contribute to the formation of cancer stem cells, we examined the effect of PJA1 overexpression on the characteristics of LSCs from Smad3 wild-type and Smad3 þ/À mice. Excess PJA1 promoted transformation of LSCs from Smad3 þ/À mice such that the cells had increased proliferative potential, reduced apoptosis, and increased anchorageindependent growth. Although not a sufficiently large sample for statistical power, we observed that two of the mice injected with PJA-overexpressing Smad3 þ/À LSCs developed metastatic liver tumors, consistent with enhanced transformation of these cells compared with those from the PJA-overexpressing Smad3 wild-type mice. Thus, in the context of limited TGFb/SMAD3 signaling, excess PJA1 can tip the balance toward a cancer stem cell-like phenotype. Cancers associated with impaired signaling through this pathway, for example through reduced abundance of the components or decreased release of latent TGFb, may benefit from inhibition of PJA1 as long as the components of the TGFb pathway are functional.
Of potentially translational importance, we showed through molecular docking simulations that synthetic triterpenoids bound to PJA1 and that exposing HCC cells in culture to such compounds promoted SMAD3-mediated transcription, SMAD3 translocation into the nucleus, stabilized b2SP, and reduced cell viability by stimulating apoptosis.
RTA405 was the most effective in vitro and exhibited tumorinhibiting activity when administered to mice. Synthetic triterpenoids, including RTA405, are under investigation in chronic kidney disease and chronic liver disease (45,46), and studies are evaluating natural triterpenes for cardiovascular disease and inflammation (47). Although our results indicated that the synthetic triterpenes could bind to PJA1, these compounds may have multiple cellular targets (38). Future studies will need to address the specificity of such compounds for different protein targets and optimize those for targeted therapeutic applications.
In conclusion, we found that PJA1 is upregulated in some hepatocellular tumor tissues and cell lines, that PJA1 inhibits TGFb/ SMAD3 signaling, and that the synthetic triterpene RTA 405 promoted SMAD3 activity and inhibited HCC tumor growth in nude mice. Furthermore, we identified a mechanism by which increased PJA1 could contribute to the development of HCC by inhibiting tumorsuppressing and stem cell-suppressing activities of TGFb signaling. Targeting PJA1 in the context of TGFb signaling may prove efficacious in battling this lethal cancer.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.