The results reported here allowed us to advance our understanding of how UBR5, as part of the N-end rule ubiquitination pathway, works to regulate basic biological processes, and what involvement it might have in cancer initiation, progression and maintenance. Importantly, cancers with mutations and/or amplification of genes involved in N-end rule ubiquitination, may represent a unique subset of cancers that are exquisitely susceptible to novel therapeutic interventions [20, 21, 22, 23]. Our data show that UBR5 is highly expressed in multiple lung adenocarcinoma cell lines but is present at reduced levels or absent in normal cell lines. Western blot analysis data further confirms high expression levels of UBR5 protein in primary resected non-small cell lung cancer samples when compared to the adjacent normal lung tissue from the same patient. Previous reports and database searches identified UBR5 as a gene that is mutated, amplified and over-expressed in many human cancers [24, 25]. Since human UBR5 was initially identified to be somatically mutated in breast cancer cell lines, several studies have subsequently shown UBR5 to be dysregulated in a wide array of other cancers. A recent study shows that UBR5 was responsible for polyubiquitination of anterior gradient 2 (AGR2), a protein that was first identified as being downregulated in breast cancer cell lines via sequencing data [4]. In support of this, proteasome inhibition also results in suppression of AGR2 transcription by downregulation of E2F1 [4, 26]. The TCGA data from lung adenocarcinoma reported in this study, suggests that UBR5 is either altered, or the gene copy number is significantly increased, in several patient samples. These amplifications are most likely in the form of allelic imbalance resulting in increased mRNA copy number of UBR5 [25]. Our findings further support previous pathological studies looking at the gene cluster on chromosome 8q22 (where UBR5 is located) showing it is amplified/mutated in lung cancer [9, 10]. The fusion of locus 8q22 and zinc finger protein 423 (ZNF423) on 16q12 was also identified in head and neck cancer primary tumors where downregulation of this fusion inhibits cell proliferation in nasopharyngeal carcinoma [27]. When considering the multiple mutations reported to date for UBR5, many of them are found to be associated with the conserved C-terminal cysteine of the HECT domain. This domain serves as a recognition site for the target protein and alteration of this domain is associated with disrupted UBR5 ligase activity [28, 29].
Over-expression of UBR5 contributes to the genesis of human cancer. Our IP/MS data further reveals UBR5 interacting with multiple proteins known to be involved in metabolism, inflammation, DNA replication, differentiation and apoptosis. For example, our data demonstrates that Annexin A1 (ANXA1) interacts with UBR5. ANXA1 is a direct regulator of NFkB signaling via binding to p65 and regulating p65-induced transcription. Furthermore, increased expression of ANXA1 has been shown to induce apoptosis in some cell types [30, 31]. Additional examples of UBR5 interacting proteins from our MS data include translational activators (GCN1L1), non-apoptotic caspase (CASP14) and mitochondrial ATP transporters (SLC25A5 and SLC25A6). The SLC25 family of proteins has been shown to be overexpressed in various cancers and involved in ADP/ATP exchange between mitochondrial matrix and cytosol [32]. UBR5 interaction with SLC25 family member proteins further suggests that disruption in UBR5 expression could result in mitochondrial dysfunction and prevention of apoptosis in disease state. Previous studies have reported that UBR5 interacts with alpha4, a component of the mTOR pathway, in human MCF-7 breast cancer cell line [33]. The mTOR/AKT pathway/signaling cascade is crucial in the regulation of cellular proliferation, inhibition of apoptosis and metabolism. Proteins in this pathway are often reported to be mutated or amplified in lung adenocarcinomas. Also, the mTOR complex is known to activate AKT through phosphorylation at Serine 473 [34, 35]. Our IP data suggests a direct interaction of UBR5 with proteins of the mTOR complex, including Raptor and Rictor. Further experiments would be needed to elucidate the nature of these interactions. We have also observed that UBR5 interacts with DNAPK, a molecule known to be activated during DNA damage responses, which further supports a role for UBR5 in ATM and DNA-PK mediated H2AX-phosphorylation, as well as cancer development and progression [11, 36].
The activation of the AKT pathway is known to play a role in cell survival and cell proliferation in various types of cancers. In a similar study, UBR5 was shown to be overexpressed in gall bladder cancer and downregulation of UBR5 inhibited the cell proliferation of relevant cancer cell lines [5]. The data reported by Zhang, et al., suggests that loss of UBR5 results in increase of PTEN, a tumor suppressor gene which acts as a negative regulator of the PI3K/AKT pathway in gall bladder cancer [5]. As reported here, we also observed a robust decrease in phosphorylated AKT at Serine 473 using multiple methods of reducing UBR5 levels in lung adenocarcinoma cell lines. Additionally, UBR5 was previously shown to interact with SOX2 and mediated its proteolytic degradation in ESC. SOX2 was shown to be overexpressed in esophageal cancer and inhibiting AKT stabilizes SOX2 expression in esophageal squamous cell carcinomas [16]. UBR5-mediated ubiquitination of citrate synthase has also been shown to play a role in AKT activation during hypoxic conditions. Loss of citrate synthase via ubiquitination by UBR5, leads to an accumulation of citrate in the cytosol during hypoxia, which leads to the activation of AKT signaling resulting in increased invasion and metastasis of breast cancer cells [37]. Excitingly, as reported here, we have found that UBR5 interacts with total and phosphorylated AKT, and the loss of UBR5 results in decreased phosphorylation of AKT. These findings suggest that post-transcriptional modification of proteins by UBR5 could be responsible for normal signaling and disruption of this UBR5-mediated regulation could lead to increases in the activation of AKT signaling, further enhancing the invasiveness and metastatic properties of cancer cells.
UBR5 has been shown to promote cell proliferation and inhibit apoptosis. Our data clearly demonstrate that loss of UBR5 leads to rapid and robust loss of cell viability and clonogenic potential in lung adenocarcinoma lines. In agreement with our results, a study by Ji, et al., showed that the loss of UBR5 in colon cancer cells was shown to decrease cell proliferation and could involve the degradation of p21, a cyclin-dependent kinase inhibitor, by polyubiquitination [38]. Similar regulations by UBR5 have been shown in gastric and colorectal cancer where increased ubiquitination by UBR5 destabilizes tumor suppressor genes leading to a reduction in the stability of these proteins [39, 40]. Collectively, these finding suggest that UBR5 disruption is involved in the genesis of human cancer through its ability to regulate the stability of multiple proteins involved in inhibiting apoptosis. As reported here, our RPPA data highlights a variety of proteins whose expression levels were altered by the loss of UBR5. This analysis reveals the critical role that UBR5 might play in maintaining extracellular matrix organization, promoting invasion and migration, and increasing cell survival in lung adenocarcinoma cells. Loss of UBR5 could lead to the destabilization of proteins which are responsible for these critical cell survival and proliferation processes. Additionally, proteins like p21 and phosphorylated S6, were found to be upregulated after loss of UBR5, which further supports a critical role for UBR5 [35, 38]. Further validation of the many proteins revealed in this study to be impacted by the loss of UBR5, has the potential to uncover novel targets for each of the signaling pathways discussed.
Loss of UBR5 has been shown to be embryonic lethal. A knock-out mouse of UBR5 was reported previously, but the mice did not survive past embryonic day 10.5 because of failed yolk sac development [14]. As multiple human cancers exhibit increased expression levels of UBR5, we were prompted to investigate what impact the loss of UBR5 might have on cancer cells that have become reliant on this protein for survival. For these experiments, we used an established xenograft immunodeficient mouse model (NRGs). Remarkably, cells lacking UBR5 resulted in a significant reduction of tumor volumes as reported here. This finding further supports previous results reported in xenograft mouse models in which subcutaneously injected breast and gallbladder cells lacking UBR5 yielded smaller tumors [4, 5]. Collectively, these data suggest that UBR5 plays an essential role in cell survival and the loss of UBR5 has a tumor suppressive role in multiple human cancers. Therefore, lung cancer patients might not be the only subset of cancer patients who would benefit from inhibition of the N-end rule ubiquitination pathway.