The Crucial p53-Dependent Oncogenic Role of JAB1 in Osteosarcoma in vivo

Abstract

Osteosarcoma (OS) is the most common primary bone cancer and ranks amongst the leading causes of cancer mortality in young adults. Jun activation domain binding protein 1 (JAB1) is overexpressed in many cancers and has recently emerged as a novel target for cancer treatment. However, the role of JAB1 in osteosarcoma was virtually unknown. In this study, we demonstrate that JAB1-knockdown in malignant osteosarcoma cell lines significantly reduced their oncogenic properties, including proliferation, colony formation, and motility. We also performed RNA-sequencing analysis in JAB1-knockdown OS cells and identified 4110 genes that are significantly differentially expressed. This demonstrated for the first time that JAB1 regulates a large and specific transcriptome in cancer. We also found that JAB1 is overexpressed in human OS and correlates with a poor prognosis. Moreover, we generated a novel mouse model that overexpresses Jab1 specifically in osteoblasts upon a TP53 heterozygous sensitizing background. Interestingly, by 13 months of age, a significant proportion of these mice spontaneously developed conventional OS. Finally, we demonstrate that a novel, highly specific small molecule inhibitor of JAB1, CSN5i-3, reduces osteosarcoma cell viability and has specific effects on the ubiquitin-proteasome system in OS. Thus, we show for the first time that the overexpression of JAB1 in vivo can result in accelerated spontaneous tumor formation in a p53-dependent manner. In summary, JAB1 might be a unique target for the treatment of osteosarcoma and other cancers.

Introduction

Osteosarcoma (OS) is the most common primary bone cancer that predominately affects adolescents and causes the third most cancer-related deaths in young adults (1). Human genetic studies identified various germline mutations associated with an increased incidence of osteosarcomas, such as Li-Fraumeni TP53 mutations and Retinoblastoma RB1 mutations (2). Current osteosarcoma treatment consists of chemotherapy and aggressive surgical resection; however, the 5-year survival rate remains at only 70%, which is further reduced to as low as 20% in patients with metastases (1, 2).

Osteosarcoma is characterized by a complex karyotype with high-level genomic instability (2). Furthermore, a lack of mouse models renders the study of OS initiation and pathogenesis challenging. However, based on human and mouse genetic studies, mutant TP53 emerged as a major driver of OS formation (3, 4). Mice with a heterozygous deletion of Tp53 develop osteosarcoma with ~25% incidence rate by 18 months of age, and mouse models with the mesenchymal cell lineage-specific disruption of Tp53 and Rb results in osteosarcoma formation with a higher penetrance and a shorter latency (3–7). Thus, TP53 is the main driver of OS development.

Jun activation domain-binding protein 1 (JAB1), also known as COP9 signalosome subunit 5 (CSN5/COPS5), is the fifth and enzymatic subunit of the highly conserved macromolecular complex, the COP9 Signalosome (CSN) (8). The importance of the CSN is underscored by the fact that the deletion of any individual subunits, CSN1-8, in mice, results in early embryonic lethality (8, 9). The CSN plays an important role in the regulation of protein turnover through its ability to cleave NEDD8, a small ubiquitin-like protein, from the active form of the largest family of E3 ubiquitin ligases, the Cullin-RING ligases (CRLs), thus inactivating them (8, 10). Intriguingly, Jab1 has been shown to play an essential role in cellular differentiation, cell cycle regulation, apoptosis, and DNA damage repair (9, 11–15). Indeed, our previous studies have demonstrated that Jab1 is required for the successive stages of skeletogenesis (12, 13). Interestingly, JAB1 is also overexpressed in many human cancers, including breast and prostate cancer (9). Mechanistically, JAB1 is capable of inactivating several tumor suppressors, including p53 (16). However, the role of Jab1 in osteosarcoma pathogenesis in vivo was unknown until this study. Here, we report that JAB1 is overexpressed in human osteosarcoma patient biopsy samples, and that the knockdown of JAB1 in highly malignant human OS cancer cell lines reduces their oncogenic properties. We identified a large and specific JAB1-regulated transcriptome in OS. We also report for the first time that the in vivo overexpression of Jab1 in mice specifically in osteoblasts results in accelerated spontaneous osteosarcoma formation in a p53-dependent manner.

Results

The knockdown of JAB1 reduces osteosarcoma oncogenic properties.

To investigate the effect of the loss of JAB1 on tumorigenesis, we performed lentiviral shRNA knockdowns in 143B and U2OS cells, two highly malignant human osteosarcoma cell lines (17, 18). As for TP53 status, 143B harbors a R156P mutation and U2OS cells has wild-type TP53 (19). We performed experiments using a scrambled control shRNA and at least 2 shRNAs specifically targeting JAB1/COPS5 (Figure 1A and B). Similarly to what was reported in hepatocellular carcinoma, the loss of JAB1 in 143B cells had relatively little effect on the protein abundance of COPS3, a potential driver gene in OS (20–22), and COPS8, a subunit linked to gastric cancer (23, 24) (Figure S1). To further determine the effect of JAB1 loss on OS oncogenic properties, we performed standard functional assays in 143B and U2OS cells. Our colony formation results indicate that JAB1-knockdown cells had a significantly reduced number of crystal violet stained colonies in 143B (64.6 vs. 6.4) and in U2OS: (90.3 vs. 31.7) (Figure 1C), (Figure S2). The MTT assay demonstrated significantly reduced cell viability after 48 hours in 143B and U2OS cells (Figure 1D). Finally, the in vitro wound assay demonstrated that JAB1 loss inhibits cell migration (Figures 1E and S3). JAB1 silencing in LM7 cells, another highly metastatic OS cell line in which TP53 is deleted, resulted in a similar functional defect (Figure S3) (19).

Figure 1. The downregulation of JAB1 inhibits 143B and U2OS osteosarcoma cell growth in vitro.

(A-B) The JAB1 silencing efficiency was confirmed to be at least 80% by RT-qPCR and western blot analysis (n = 3). (C) The colony formation assay demonstrated that JAB1-knockdown significantly decreases the abilities of OS cells to form colonies (n = 3-6). (D) The MTT assay demonstrated that the JAB1-knockdown significantly decreased cell viability (n = 3-6). (E) The scrape motility assay demonstrated that JAB1-knockdown significantly inhibited cell migration (n = 3-6). Error bars represent means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.005. All controls in this experiment are a scrambled shRNA.

RNA-sequencing reveals that JAB1 regulates a unique oncogenic transcriptome in OS.

Next, we sought to obtain an unbiased JAB1-mediated transcriptome in OS to gain insights into the underlying mechanism of JAB1-mediated OS pathogenesis using RNA-sequencing in 143B JAB1-knockdown cells. Upon JAB1 depletion, there were a total of 4110 genes significantly differentially expressed, with 37.4% of those genes downregulated, and 62.6% upregulated (Figure 2A). Principal Component Analysis revealed that there is a very distinct set of genes that are dysregulated upon JAB1 silencing (Figure 2B).

Figure 2. The RNA-seq analysis of JAB1-depleted 143B human OS cells.

(A) The pie chart of significantly differentially expressed genes in JAB1-knockdown versus control 143B cells. (B) The Principal Component Analysis identified the two distinct sets of genes that are differentially expressed in Wild-Type (WT, gray dots) vs. JAB1-knockdown (KD, black dots) 143B cells. (C) Gene Ontology analysis list of the significantly downregulated genes and (D) upregulated genes using DAVID analysis. The x-axis denotes the number of genes. For all GO Terms presented here, p < 0.003.

Next, we submitted the lists of both significantly upregulated and downregulated genes to the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (25, 26). Figures 2C and 2D list the top 5 most significantly altered Gene Ontology (GO) terms regarding molecular functions, cellular components, and biological processes. Among the downregulated genes, a large number are involved in protein binding for molecular function, and their cellular components are mainly in the cytoplasm and cytosol (Figure 2C). The most significantly downregulated biological processes are involved in mitosis, cell cycle progression, and cell-cell adhesion (Figure 2C). In contrast, the cellular component of the upregulated genes is mainly localized in the nucleus, and interestingly, the molecular function of the upregulated genes is also mainly involved in protein binding (Figure 2D). This overall finding is consistent with the well-established role of JAB1 in binding to a diverse set of proteins (9, 10). Next, we performed standard Gene Set Enrichment Analysis (GSEA) to identify the key biological pathways that are regulated by JAB1 in OS (Figures 3A and 3B). GSEA using the HALLMARK gene sets of the Molecular Signature Database demonstrated that the cell cycle regulation was among the most downregulated pathways upon JAB1-knockdown (Figure 3A and Table S1). Moreover, the G2/M Checkpoint and E2F Targets pathways, which are part of the Rb/E2F network involved in controlling cell cycle progression and OS pathogenesis (27), is significantly altered. Furthermore, the DNA Repair and p53 pathways were also changed upon JAB1-knockdown (Figure 3B). Therefore, GSEA identified very similar pathways as DAVID analysis in JAB1-knockdown cells, particularly the cell cycle regulation among the downregulated genes, suggesting this is among the key JAB1 downstream pathways in OS (Tables S1 and S2).

Figure 3. The Gene Set Enrichment Analysis identifies altered oncogenic pathways upon JAB1-knockdown in 143B OS cells.

(A) HALLMARK enrichment plots of G2/M checkpoint (NES = 1.98, FDR = 0.013) and E2F Targets (NES = 2.1. FDR = 0.005) gene sets (top left and bottom left panels) with the corresponding heat maps of these pathways at the right. (B) HALLMARK enrichment plots of the DNA repair (NES = −1.91, FDR = 0.059) and p53 (NES = −1.27, FDR = 0.373) pathways gene sets (top left and bottom left panels) with the corresponding heat maps of these pathways at the right.

The knockdown of JAB1 impairs cell cycle progression, increases apoptosis, and its overexpression in human osteosarcoma is correlated with poor prognosis.

To determine the effect of the JAB1 loss on cell cycle progression, we conducted flow cytometry analysis in JAB1-knockdown 143B cells. There were significant changes in the percentage of cells at each phase of the cell cycle (Figure 4C). Cyclin A2 plays an important role in the G2/M phase transition, and is a common marker of cell proliferation (28). Cyclin B1 is important for entry into mitosis (29). The levels of these two cyclins were downregulated, indicating impaired cell cycle progression upon JAB1-knockdown (Figure 4C). The most striking change in our flow cytometry analysis was the large increase in the percentage of cells in the sub G0 phase, suggesting an increased number of apoptotic cells (Figure 4C). Indeed, our GSEA analysis also identified an increase in the apoptosis pathway (Figure 4D). Notably, the levels of BAX, a key pro-apoptotic Bcl-2 family member, was increased upon JAB1-knockdown in 143B cells (Figure 4E). Furthermore, there was no change in the expression of BAK, another key pro-apoptotic Bcl-2 family member, and an increase in anti-apoptotic BCL-2 upon JAB1-knockdown (Figure 4D). Densitometry analysis revealed that there was an increased ratio of BAX:BCL-2, a key metric of apoptotic activity, in JAB1-knockdown 143B cells, indicating increased apoptosis and that BAX might be the major effector. Next, an unbiased screening identified 28 out of 45 of the most common signaling reporters being clearly altered (Figure 4F and Supplementary Table S3). These results indicate that JAB1-knockdown likely increases apoptosis and alters multiple major signal transduction pathways in OS cells.

Figure 4. JAB1 is overexpressed in human OS biopsies, and JAB1-knockdown increases apoptosis and alters multiple major signal transduction pathways in OS cells.

(A) The representative JAB1 immunohistochemistry of a human OS biopsy sample from an array of 51 samples, with a staining intensity score of 3 (left panel) and 0 (Middle panel). Right panel shows JAB1 immunohistochemistry in a normal human adult cortical bone sample. (B) Left panel, the percentage of each staining score across all samples. Middle panel, the staining intensity score was matched to the patient outcome. Right panel, the ratio of alive patients to dead patients for each staining intensity score. A lower number indicates poorer survival. (C) Left, the flow cytometry analysis of cell cycle in control and JAB1-knockdown 143B cells. Error bars represent means ± SD (n = 3). Right, western blotting demonstrates decreased expression of Cyclin A2 and Cyclin B1. (D) Left, The GSEA identifies the apoptosis pathway (NES = −1.21, FDR = 0.421) as being upregulated in JAB1-knockdown 143B cells. Right, Western blotting demonstrates increased expressions of Bax and Bcl-2, but not Bak, in 143B JAB1-knockdown cells. Densitometry analysis of the Bax:Bcl-2 ratio. (E) The Cignal Reporter Assay in JAB1-knockdown 143B cells. Red bars indicate all of the downregulated pathways; yellow bars indicate the top 4 most upregulated pathways. A detailed list of pathways is listed in Supplementary Table S3.

JAB1 was previously reported to be overexpressed in many cancer cell types (9). Thus, we conducted JAB1 immunostaining using a tissue microarray containing human OS biopsy sections from 51 different osteosarcoma patients (Figure 4A). Interestingly, the quantification of the staining revealed that greater than 75% of the OS samples had high intensity staining (a score of 2 or 3) (Figure 4B). Of the 19 OS samples that received a staining score of 3, only 5 of them survived (Figure 4B). In contrast, JAB1 expression in normal bone was much weaker (Figure 4A). Thus, JAB1 is likely overexpressed in human osteosarcoma and might be correlated with poor survival outcomes.

The overexpression of JAB1 in mice results in accelerated spontaneous bone tumor formation in a p53-dependent manner.

To date, JAB1-mediated animal tumor models are still very much lacking. To understand the role of JAB1 in oncogenesis in vivo, we generated a novel mouse model that overexpresses Jab1 specifically in osteoblasts (Figure 5A–B) (30). The Col1a1-Jab1 transgenic mice exhibited no obvious growth abnormalities and no tumor formation (Figure 5D). Both human and mouse genetic studies have identified mutant Tp53 as the most prominent driver of OS development (2). Thus, we crossed our two Col1a1-Jab1 transgenic mouse lines (Tg#1 and Tg#2) with Tp53 heterozygous mice. The Col1a1-Jab1; p53^+/− mice were viable and indistinguishable from littermates prior to weaning age. However, by 13 months of age, these mice developed spontaneous bone tumors with a 30.4% penetrance (Figures 5C and 5D). The average age of tumor onset for Tg#1; p53^+/− and Tg#2; p53^+/− was 408 days and 392 days, respectively, with a range of 293-449 days. In contrast, only two p53^+/− mice developed osteosarcomas by 331 days and 541 days of age respectively. In both Col1a1-Jab1; p53^+/− transgenic lines, the osteosarcomas were nearly evenly distributed between the hindlimb and forelimb (Figures 5D and 6A–D). Moreover, X-Ray analysis revealed mineral deposition in the tumors dissected from these limbs (Figure 6E–I). Overall, the tumors were conventional OS with hypercellularity. They can be categorized into osteoblastic and fibroblastic, but not chondroblastic, osteosarcomas (Figure 6J–N). Interestingly, in JAB1-knockdown 143B cells, the HALLMARK Epithelial Mesenchymal Transition pathway was significantly downregulated, suggesting that JAB1 might be involved in EMT-mediated metastasis (Figure 6R). In support of this, some spontaneous osteosarcomas in Col1a1-Jab1;p53^+/− mice displayed local invasion into the surrounding tissue, including the muscle and fat, but not into the nerves (Figures 6O–6Q). This is very similar to a mouse model of NOTCH-mediated OS (31). Additionally, SNAI1, a transcription factor that induces EMT, is decreased upon JAB1-knockdown in 143B cells (Figure 6R). Thus, the osteoblast-specific overexpression of JAB1 accelerates spontaneous OS formation in mice in a p53-dependent manner, and may promote EMT.

Figure 5. The osteoblast-specific overexpression of Jab1 results in accelerated, spontaneous, p53-dependent OS formation in mice.

(A) The schematic representation of the construct used to generate Col1a1-Jab1 transgenic mice. (B) The RT-qPCR analysis of Jab1 expression from 8-week-old long bones of control and 2 independent Col1a1-Jab1 mouse transgenic lines (n = 3). (C) The representative image of a hindlimb tumor from a Col1a1-Jab1; p53^+/− transgenic mouse. (D) The summary table of the different cohorts of mice used in this study and their OS occurrence. Tg, Transgenic

Figure 6. The characterization of spontaneous OS tumors in Col1a1-Jab1; p53^+/− mice.

(A-D) Representative images of mice that formed bone tumors, with dotted lines outlining the tumors. (E-I) X-ray analysis of those tumors, and (J-N) corresponding H&E staining of the tumors at 200x magnification. Scale bars, 50 μm. Histology revealed the local invasion of OS cells into the (O) surrounding muscle and (P) adipose, but not (Q) nerve tissue. Scale bars, 100X, 100 μm, 200X, 50 μm. (R) (Left) The HALLMARK GSEA enrichment plot and the heat map of Epithelial Mesenchymal Transition pathway (NES = 1.35, FDR = 0.144), (Right) Western blot analysis of SNAI1 in JAB1-knockdown 143B OS cells.

JAB1 is a potential therapeutic target for OS treatment.

The NEDDylation pathway is known to trigger the activation of the largest family of E3 ubiquitin ligases, the Cullin-RING ligases (CRLs) (32). As illustrated in Figure 7A, the NAE1 (NEDD8-Activating Enzyme E1 Regulator Subunit) initiates the NEDDylation and activation of CRLs through the addition of an ubiquitin-like protein NEDD8, in a cascade analogous to ubiquitin transfer. On the other hand, JAB1 is solely responsible for catalyzing the removal of NEDD8 from CRLs, thus deactivating CRLs and maintaining their cellular homeostasis (Figure 7A) (32). In recent years, the NEDDylation pathway has emerged as an attractive therapeutic target for cancer treatment (Figure 7A) (33–35). Indeed, MLN4924, a specific inhibitor of NAE1, is currently in clinical trials for the treatment of various cancers (35). Moreover, recently a highly specific small molecule inhibitor of JAB1, CSN5i-3, has been developed (36). CSN5i-3-mediated JAB1 inhibition reduced cell viability in a large panel of cell lines, as well as repressed the growth of lymphoma xenografts in mice (36), but CSN5i-3’s effects in OS have not been studied. Thus, we treated 143B OS cells and, as a control, human fetal osteoblasts (hFOBs) with CSN5i-3 and MLN4924 to determine if disrupting the NEDDylation pathway can prevent OS cell growth. Indeed, both drugs inhibited OS cell growth in a dose-dependent manner, and 143B OS cells were more sensitive than hFOBs to both CSN5i-3 (3.5 uM vs 6.6 uM) and MLN4924 (0.46 uM vs. 14 uM) (Figures 7C and 7E). Additionally, we generated three-dimensional multicellular tumor spheroids (sarcospheres) and determined their IC50 after 48 hours of treatment with CSN5i-3 (Figure 7D). This clonal, non-adherent, self-renewing, cancer stem cell like-model more closely represent the in vivo microenvironment, and better correlates with the in vivo response to chemotherapy when compared with monolayer cultures (18). Interestingly, our results demonstrate that 143B sarcospheres are more sensitive to treatment with CSN5i-3, with an IC50 of 2.9 μM (Figures 7D and 7E). In CSN5i-3 treated cells, as expected (32, 36), the amount of the NEDDylated forms of CUL1 and CUL4A/B had increased, whereas in contrast, the expression of CUL1 and CUL4A/B in MLN4924-treated cells were completely abolished (Figure 7B). We also examined the expression of FBXO22, a poorly characterized F-box protein that plays a role in substrate specificity of CRL complexes (37). Similar to a previous report in a colon cancer cell line (36), FBXO22 levels were also completely abolished in CSN5i-3-treated OS cells, but unchanged in MLN4924-treated OS cells (Figure 7B). The expression of SKP2, another F-box protein that plays an important role in cell cycle progression (37), decreased in CSN5i-3-treated OS cells but increased in MLN4924-treated OS cells (Figure 7E). These data suggest that JAB1 might be a potential therapeutic target for OS, and FBXO22 might be a unique CSN5i-3 downstream target in diverse cancer cell types.

Figure 7. JAB1 is a potential therapeutic target in osteosarcoma.

(A) Schematic representation of Cullin-RING Ligase (CRL) activation by NAE1 and inactivation by JAB1, and the effects of MLN4924 and CSN5i-3 on CRL homeostasis. (B) Western blot analysis of 143B cells treated with CSN5i-3 or MLN4924 demonstrated their differential effects on representative CRL Cullin and F-Box proteins. (C) Dose-response curves showing the cell viability of 143B cells (top) and hFOB cells (bottom) treated with CSN5i-3 (left) and MLN4924 (right) for 48 hours. Each point in each graph represents mean ± SD of 6 technical replicates. Representative curves from two independent experiments with similar results are shown for each cell line and drug (D) Left, dose-response curve showing the cell viability of 143B sarcospheres treated with CSN5i-3 for 48 hours. Each point represents mean ± SD for 6 technical replicates. A representative curve from two independent experiments is shown. Right, representative pictures of 143B sarcospheres treated with 0, 5, and 20 μM CSN5i-3 for 48 hours. (E) IC50 Table.

Discussion

In this study, for the first time, we demonstrate that JAB1-knockdown in metastatic osteosarcoma cell lines led to reduced oncogenic properties, with significantly reduced proliferation, colony formation, and motility. We also showed for the first time by RNA-sequencing that there exists a large JAB1-mediated oncogenic transcriptome in OS cells. Additionally, we show for the first time that in human OS patient biopsy samples, JAB1 is overexpressed in more than 75% of patients, and that there is likely a positive correlation between JAB1 expression levels and OS mortality (Figure 4B). Most importantly, we also show for the first time that JAB1 overexpression specifically in osteoblasts in mice on a p53^+/− sensitizing background results in an accelerated spontaneous bone tumor formation. Finally, for the first time, we demonstrate that JAB1 might be a target for OS treatment using CSN5i-3, a novel, specific, and potent small molecule inhibitor of JAB1. Thus, our results strongly suggest that JAB1 might be a diagnostic and prognostic biomarker for osteosarcoma, and that JAB1 is a promising therapeutic target for treating osteosarcomas.

Gene ontology analysis of our RNA-seq dataset demonstrates that the molecular function of a significant number of the differentially expressed genes in JAB1-knockdown cells are involved in protein binding (Figures 2C and 2D), which is consistent with the well-established function of JAB1 in interacting with many other proteins (9). Moreover, GSEA analysis confirmed that JAB1 regulates important oncogenic pathways, including the p53 pathway, Rb pathway, cell cycle arrest, DNA repair, and cell-cell adhesion (Figure 3). Interestingly, JAB1 expression has previously been linked to radiation sensitivity and DNA damage repair in OS in vitro (38). However, the underlying mechanism of JAB1’s role in these processes remains to be determined.

Our results demonstrate for the first time that Jab1 overexpression results in spontaneous OS formation in a p53-dependent manner in mice. The low penetrance of tumor formation in this model is likely due to the low-level Jab1 transgene expression achieved in osteoblasts. Interestingly, a previous study also reported that it was very challenging to achieve high-level Jab1 expression in vivo (39). Therefore, generating a mouse model with high-level Jab1 expression will be essential to further address the role of Jab1 in tumorigenesis in whole animals.

In this study, we also provide evidence that JAB1 may be a suitable therapeutic target for clinical intervention in OS. The JAB1-containing COP9 Signalosome is an essential regulator of Cullin-RING Ligases (CRLs), which are central mediators of oncogenesis (8). CRL homeostasis is tightly regulated by NEDDylation (Figure 7A). A small molecule inhibitor of NAE1, MLN4924 (Figure 7A), inhibited tumor xenografts in mice, and is currently in many Phase I and II clinical trials for the treatment of hematologic and other cancers, as well as a phase III clinical trial in combination with azacitidine for the treatment of acute myeloid leukemia, but is associated with severe side effects, serious adverse events, and drug resistance (33–36, 40). CSN5i-3 is a recently developed and highly specific small molecule inhibitor of JAB1 (36). In contrast to the global inhibition of protein degradation by pan proteasome inhibitors, or the broad effect of inactivating all CRLs using MLN4924, CSN5i-3 might offer greater specificity, and therefore likely reduced side effects, due to its inactivation of only a subset of CRLs (36, 41). In this study, for the first time, we investigated the effect of both MLN4924 and CSN5i-3 on OS cells (Figure 7). We demonstrate that CSN5i-3 reduces OS cell viability, and that OS cells are more sensitive to treatment with both CSN5i-3 and MLN4924 compared with a human fetal osteoblast (hFOB) cell line (Figure 7E). Further studies are needed to determine if inhibition of JAB1 using CSN5i-3 can overcome radio and chemotherapy resistance in OS, similarly to reports of JAB1 knockdown in nasopharyngeal carcinoma and OS cells (38, 42, 43).

We also found that CSN5i-3 and MLN4924 also differentially affect two CRL F-box proteins, FBXO22 and SKP2 (Figure 7E). SKP2, as a well-studied F-Box protein, is an established oncogene that is overexpressed in OS cells, and its downregulation inhibits OS cell growth and metastasis in vitro and in vivo (37, 44). Interestingly, MLN4924 treatment in fact increased the expression of SKP2, whereas CSN5i-3 treatment decreased its expression OS cells (Figure 7E). In contrast to SKP2, much less is known about FBXO22, which has both a positive and negative role in breast cancer progression and metastasis, respectively (45). Furthermore, Fbxo22-depletion resulted in the reduced response of ER-positive breast cancer cells to tamoxifen, and the overexpression of JAB1 has been shown to confer tamoxifen resistance in ER-positive breast cancer (46, 47). In our study, CSN5i-3 treatment completely abolished FBXO22 expression (Figure 7E). Thus, our results and others indicate that FBXO22 may be a unique target of CSN5i-3 and JAB1 in OS and other cancers. The specific role of FBXO22 in cancer cells, especially identification of its downstream substrates involved in oncogenesis, remains to be elucidated to facilitate our understanding of the mechanism controlling JAB1-mediated cancer pathogenesis. While our present study demonstrates that CSN5i-3’s inhibition of JAB1 may be suitable for the treatment of OS, further studies are necessary to address its in vivo efficacy in treating OS patients.

Materials and Methods

Complete and detailed materials and methods may be found in the Supplementary Materials.

Antibodies

The antibodies and antibody dilutions used in this study are listed in Supplementary Table S4.

Transgenic Construct

The full-length FLAG-tagged Jab1 cDNA was cloned into a Col1a1-WPRE transgenic expression vector as described previously (30). Mice were maintained on a FVB/N background, and genotyping was performed as previously described (13). Tp53 heterozygous mice were maintained as previously described (4).

RNA-sequencing

Total RNAs were isolated from control and JAB-knockdown 143B cells as described (48). N = 3 each for each group. RNA-sequencing was performed at the Genomics Core at Case Western Reserve University. The dataset has been deposited into the NCBI Gene Expression Omnibus under the accession number GSE117773.