Michael D. Birnbaum

Arginyltransferase suppresses cell tumorigenic potential and inversely correlates with metastases in human cancers

Arginylation is an emerging post-translational modification mediated by arginyltransferase (ATE1) that is essential for mammalian embryogenesis and regulation of the cytoskeleton. Here, we discovered that Ate1-knockout (KO) embryonic fibroblasts exhibit tumorigenic properties, including abnormally rapid contact-independent growth, reduced ability to form cell–cell contacts and chromosomal aberrations. Ate1-KO fibroblasts can form large colonies in Matrigel and exhibit invasive behavior, unlike wild-type fibroblasts. Furthermore, Ate1-KO cells form tumors in subcutaneous xenograft assays in immunocompromised mice. Abnormal growth in these cells can be partially rescued by reintroduction of stably expressed specific Ate1 isoforms, which also reduce the ability of these cells to form tumors. Tumor array studies and bioinformatics analysis show that Ate1 is downregulated in several types of human cancer samples at the protein level, and that its transcription level inversely correlates with metastatic progression and patient survival. We conclude that Ate1-KO results in carcinogenic transformation of cultured fibroblasts, suggesting that in addition to its previously known activities Ate1 gene is essential for tumor suppression and also likely participates in suppression of metastatic growth.

Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response

Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis.

Reduced Arginyltransferase 1 is a driver and a potential prognostic indicator of prostate cancer metastasis

Most prostate cancer cases remain indolent for long periods of time, but metastatic progression quickly worsens the prognosis and leads to mortality. However, little is known about what promotes the metastasis of prostate cancer and there is a lack of effective prognostic indicators, making it immensely difficult to manage options for treatment or surveillance. Arginyltransferase 1 (Ate1) is the enzyme mediating post-translational protein arginylation, which has recently been identified as a master regulator affecting many cancer-relevant pathways including stress response, cell cycle checkpoints, and cell migration/adhesion. However, the precise role of Ate1 in cancer remains unknown. In this study, we found the occurrence of metastasis of prostate cancer is inversely correlated with the levels of Ate1 protein and mRNA in the primary tumor. We also found that metastatic prostate cancer cell lines have a reduced level of Ate1 protein compared to non-metastatic cell lines, and that a depletion of Ate1 drives prostate cancer cells towards more aggressive pro-metastatic phenotypes without affecting proliferation rates. Furthermore, we demonstrated that a reduction of Ate1 can result from chronic stress, and that shRNA-reduced Ate1 increases cellular resistance to stress, and drives spontaneous and stress-induced genomic mutations. Finally, by using a prostate orthotropic xenograft mouse model, we found that a reduction of Ate1 was sufficient to enhance the metastatic phenotypes of prostate cancer cell line PC-3 in vivo. Our study revealed a novel role of Ate1 in suppressing prostate cancer metastasis, which has a profound significance for establishing metastatic indicators for prostate cancer, and for finding potential treatments to prevent its metastasis.

Abstract A039: Arginyltransferase Ate1 reduction drives prostate cancer metastasis

This study demonstrates that diminished Arginyltransferase (Ate1) expression in prostate cancer is sufficient to drive progression and distal metastasis, as shown via in vitro and in vivo models, and in correlation analysis with human patient samples. While prostate cancer affects a large population of men, it is usually a dormant disease and is only lethal in the small subpopulation in which distal metastasis occurs. Unfortunately, distal metastasis is currently difficult to predict and treat. Understanding what provokes metastasis in prostate cancer is therefore clinically important. Ate1 is the enzyme solely responsible for mediating post-translational protein arginylation in most eukaryotic cells, including mammalian cells. Previous studies show that a complete loss of Ate1 in yeast and mammalian cells increases resistance to stress-induced cell death and promotes genomic instability. Ate1 loss further promotes cancer-relevant phenotypes in mouse fibroblasts, such as persisting cellular replication despite stress or cell-cell contacts. Furthermore, a lower level of Ate1 appears to correlate with shorter survival in patients with several types of cancer, including prostate cancer. However, the role of Ate1 in progression and metastasis of prostate cancer (or any cancer) remained unknown. Here we demonstrate in prostate-relevant models that a reduction of Ate1 drives prostate cancer progression and metastasis. We first examined Ate1’s impact on stress-induced cell death on prostate cancer cells in vitro in models relevant to the tumor microenvironment and cancer treatments. PC-3 and LNCaP prostate cancer cells with shRNA targeting Ate1 (or nontargeting control) were stressed with cellular oxidant hydrogen peroxide, gamma radiation, and apoptotic inducer staurosporine. Consistent with our hypothesis, the knockdown of Ate1 resulted in less cell death in these cells. Interestingly, while a reduction of Ate1 in these cell lines does not affect proliferation, it does cause an increase in anchorage-independent growth in LNCaP cells via the soft agar assay, and an increase in PC-3 cell invasion through Matrigel. Additional analysis of prostate cancer cell lines PC-3, LNCaP, metastatic PC-3-derivative PC3-ML, metastatic LNCaP-derivative C4-2B, and metastatic line DU145 showed a distinct and significant relationship between increased malignancy and decreased Ate1 expression. Next, to study the effect of Ate1 on metastasis in vivo, PC-3 cells expressing luciferase and with shRNA targeting Ate1 (or control) were injected into prostates of immune-compromised (nu/nu) mouse (orthotopic xenografts). PC3-ML, a derivative of PC-3 known to be metastasis-competent, was used as a positive metastatic control. We used luciferin luminescence to measure tumor growth and the presence of local invasions to adjacent tissues such as seminal vesicles and testicles, as well as metastasis to distal sites including lymph nodes, spleen, and lung. Sixty days after tumor inoculation, all three cell types consistently formed similar primary tumors but varied greatly in their metastatic behaviors. The PC-3 cells with nontargeting control shRNA largely failed to metastasize to distal sites and only occasionally exhibited local invasion, as expected from previous reports. However, PC-3 cells with Ate1 downregulated by shRNA consistently formed distal metastases in lungs, spleen, and lymph nodes with similar frequency to the positive control, PC3-ML. As further support for Ate1’s role in prostate cancer progression, results from data mining and immune-histologic examination of human tissue samples revealed a consistent downregulation of Ate1 protein and mRNA during prostate cancer progression and metastasis, offering promising value of predicting metastatic events before they occur. In conclusion, Ate1 reduction may be a potent indicator of prostate cancer progression, and provide insight into the molecular mechanism of prostate cancer metastasis. Citation Format: Michael D. Birnbaum, Ning Zhou, Kerry L. Burnstein, Fangliang Zhang. Arginyltransferase Ate1 reduction drives prostate cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A039.

Abstract 2801: Arginylation as a novel regulator of prostate cancer progression

Post-translational arginylation, mediated by Arginyltransferase (Ate1), is an important protein modification involved in stress response yet remains poorly studied. Our lab recently reported that a loss of Ate1 is sufficient to induce tumorigenesis and loss of contact inhibition in fibroblasts. However, the role of Ate1 in cancer initiation and progression is not clear. This study examines the effects of Ate1 loss in prostate cancer models of tumorigenesis and progression. We first show that Ate1 is required for normal cellular responses to several types of stress present in cancer. Preliminary data in fibroblasts shows that Ate1 is essential for cell death following oxidative stress, hypoxia, UV and gamma radiation, and apoptosis-inducing drugs. These results inspired further tests to examine if cancer progression may be directly stimulated by a loss of Ate1 through increased cell survival and invasiveness. To study the effects of lost Ate1, we characterize established prostate cancer cell lines with Ate1 stably knocked down. In PC-3 prostate cancer cells, a reduction of Ate1 corresponded with increased invasiveness in the Boyden Chamber invasion assay, and reduced H2O2-induced cell death. In LnCaP prostate cancer cells, reduced Ate1 induced significantly higher anchorage-independent growth during a soft-agar growth assay. In conclusion, our data suggests that the loss of Ate1 in prostate cancer serves to stimulate cancer progression, and that Ate1 protein expression levels may predict malignancy. Citation Format: Michael Birnbaum, Akhilesh Kumar, Fangliang Zhang. Arginylation as a novel regulator of prostate cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2801.

Abstract 1252: Arginylation as a new link between oxidative stress response and prostate cancer progression

In this study, we evaluate the role of post-translational arginylation in prostate cancer, and introduce its potential as a biomarker of prostate cancer progression. Arginylation has not yet been studied in cancer. Our lab has observed Aringyltransferase (Ate1) is required for oxidative stress-mediated apoptosis. Our preliminary data in multiple eukaryotic cell models demonstrate that a downregulation of Ate1 increases resistance to oxidative stress, and elevated Ate1 is sufficient to induce cell death. Preliminary data mining shows that a downregulation of Ate1 correlates with a poorer prognosis in prostate cancer. Because prostate cancer cells produce a high amount of oxidative stress, the decrease of Ate1 likely increases their survival. We hypothesize that Ate1 is essential for the normal cellular response to insurmountable oxidative stress leading to apoptosis, and that a downregulation of Ate1 will promote cancer cell progression. We found that a loss of Ate1 in a nontumorigenic fibroblast cell line promotes spontaneous tumorigenicity within immunocompromised mice, a disruption of normal cell-cell interactions, and loss of contact inhibition. Also consistent with our hypothesis, prostate cancer cell line PC-3 with stably knocked down Ate1 exhibit higher invasiveness in the Boyden Chamber invasion assay, and higher resistance to H2O2-induced cell death during a 12-hour variable dose treatment. Similarly, the highly metastatic subpopulation of PC-3, PC3-ML, has naturally reduced Ate1 and exhibits higher invasiveness and resistance to H2O2. Finally, histological analysis of primary patient prostate cancer samples show a dramatic reduction of Ate1 compared to normal prostate tissue. These findings warrant further study of Ate1 as a metastasis suppressor, as well as a potential biomarker for prostate cancer progression. Citation Format: Fangliang Zhang, Michael D. Birnbaum, Akhilesh Kumar, William M. Morgan. Arginylation as a new link between oxidative stress response and prostate cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1252. doi:10.1158/1538-7445.AM2015-1252

Abstract P2-06-04: p300 acts as a tumor suppressor by inhibiting pro-metastatic microRNA pathways

The histone acetyltransferase p300 is an important epigenetic factor known to regulate gene silencing, DNA repair, and cell cycle. Altered p300 expression is linked to cancerous phenotypes and has potential as both a biomarker and therapeutic target. Existing anti-cancer therapies such as histone deacetylase inhibitors (vorinostat, valproic acid, trichostatin A) are known to affect p300 levels and activity. Understanding how p300 affects cancerous and metastatic development is crucial to optimizing and advancing these treatments. However, whether p300 acts as a tumor suppressor or promoter is still controversial. To address this issue, we stably knocked down p300 levels in MCF-7, BT 474 and MDA MB 231 to 30-40% endogenous levels to directly examine p3009s effects on tumorigenicity. Our experiments show loss of p300 significantly increased cell motility in the scratch assay (p To identify targets of p300 that might regulate its anti-tumor effect, we observed that miR-let-7c was downregulated in KD-p300 MCF-7 by 70% (p Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-06-04.