ObjectivesTumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields with antimitotic effects on cancerous cells. TTFields concomitant with pemetrexed and a platinum agent are approved in the US and EU as first line therapy for unresectable, locally advanced or metastatic malignant pleural mesothelioma (MPM). The goal of the current study was to characterize the mechanism of action of TTFields in MPM cell lines and animal models.MethodsHuman MPM cell lines MSTO-211H and NCI-H2052 were treated with TTFields to determine the frequency that elicits maximal cytotoxicity. The effect of TTFields on DNA damage and repair, and the cytotoxic effect of TTFields in combination with cisplatin and/or pemetrexed were examined. Efficacy of TTFields concomitant with cisplatin and pemetrexed was evaluated in orthotopic IL-45 and subcutaneous RN5 murine models.ResultsTTFields at a frequency of 150 kHz demonstrated the highest cytotoxicity to MPM cells. Application of 150 kHz TTFields resulted in increased formation of DNA double strand breaks, elevated expression of DNA damage induced cell cycle arrest proteins, and reduced expression of Fanconi Anemia (FA)-BRCA DNA repair pathway proteins. Co-treatment of TTFields with cisplatin or pemetrexed significantly increased treatment efficacy versus each modality alone, with additivity and synergy exhibited by the TTFields-pemetrexed and TTFields-cisplatin combinations, respectively. In animal models, tumor volume was significantly lower for the TTFields-cisplatin-pemetrexed combination compared to control, accompanied by increased DNA damage within the tumor.ConclusionThis research demonstrated that the efficacy of TTFields for the treatment of MPM is associated with reduced expression of FA-BRCA pathway proteins and increased DNA damage. This mechanism of action is consistent with the observed synergism for TTFields-cisplatin vs additivity for TTFields-pemetrexed, as cisplatin-induced DNA damage is repaired via the FA-BRCA pathway.
Polyubiquitin chains serve a variety of physiological roles. Typically the chains are bound covalently to a protein substrate and in many cases target it for degradation by the 26S proteasome. However, several studies have demonstrated the existence of free polyubiquitin chains which are not linked to a specific substrate. Several physiological functions have been attributed to these chains, among them playing a role in signal transduction and serving as storage of ubiquitin for utilization under stress. In the present study, we have established a system for the detection of free ubiquitin chains and monitoring their level under changing conditions. Using this system, we show that UFD4 (ubiquitin fusion degradation 4), a HECT (homologous with E6-AP C-terminus) domain ubiquitin ligase, is involved in free chain generation. We also show that generation of these chains is stimulated in response to a variety of stresses, particularly those caused by DNA damage. However, it appears that the stress-induced synthesis of free chains is catalyzed by a different ligase, HUL5 (HECT ubiquitin ligase 5), which is also a HECT domain E3.
Abstract Introduction: Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer with a poor prognosis and limited treatment options. Tumor Treating Fields (TTFields) are a noninvasive, locoregionally, antineoplastic treatment, delivering low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields, that has demonstrated a promising median overall survival in patients with MPM without increases in systemic toxicity (STELLAR clinical trial). Accordingly, TTFields with pemetrexed and a platinum-based chemotherapy agent received FDA-approval as first line therapy for MPM. While efficacy of TTFields for MPM treatment is well-established, the underlying mechanism of action needs further elucidation. Methods: Human MPM cell lines (NCI-H2052 and MSTO-211H) were treated using various TTFields frequencies to assess the most effective frequency. The effect of optimal frequency TTFields on levels of DNA double strand breaks (DSB) was examined by fluorescent microscopy detection of γH2AX foci, and the levels of DNA damage repair proteins was evaluated by immunoblotting. The combined cytotoxic effect of TTFields with cisplatin or pemetrexed was tested in vitro, and efficacy of TTFields in combination with both chemotherapeutic agents was examined in C57BL/6 mice injected subcutaneously with RN-5 cells, by measuring tumor volume and through detection for DNA damage within the tumor. Results: The optimal TTFields frequency in both MPM cell lines was 150 kHz, demonstrating significant cytotoxicity and increases in formation of DNA DSB. These effects were associated with reduced expression of proteins from the Fanconi Anemia (FA) repair pathway for DNA repair - FANCA, FANCD2, FANCJ, and BRCA1. Co-treatment of TTFields with cisplatin or pemetrexed significantly increased treatment efficacy versus each treatment alone, with an additive effect shown by the TTFields-pemetrexed combination, and a tendency towards synergism displayed for TTFields-cisplatin co-administration. In animal models, tumor volume fold increase was significantly decreased for co-treatment with TTFields and chemotherapy (cisplatin + pemetrexed) versus the control, showing also increased DNA damage within the tumor bed in comparison to control or chemotherapy alone. Conclusions: The results presented here demonstrate that the efficacy of TTFields for treatment of MPM is associated with reduced expression of FA pathway proteins and increased DNA DSB. This effect may account for the synergistic effect seen for TTFields-cisplatin co-treatment, as cisplatin is known to cause DNA damage that requires the FA pathway for repair. This research provides further insights on the mechanism of action of TTFields in MPM, a treatment already approved against this malignancy. Citation Format: Helena Mumblat, Antonia Martinez, Ori Braten, Mijal Munster, Eyal Dor-On, Rosa S. Schneiderman, Yaara Porat, Tali Voloshin, Shiri Davidi, Roni Blatt, Anna Shteingauz, Catherine Tempel-Brami, Einav Zeevi, Carolina Lajterer, Yuval Shmueli, Shiri Danilov, Adi Haber, Moshe Giladi, Adrian Kinzel, Uri Weinberg, Yoram Palti. Efficacy of Tumor Treating Fields (TTFields) in mesothelioma is associated with reduced capacity for DNA damage repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1186.
Hepatocellular carcinoma (HCC), a highly aggressive liver cancer, is a leading cause of cancer-related death. Tumor Treating Fields (TTFields) are electric fields that exert antimitotic effects on cancerous cells. The aims of the current research were to test the efficacy of TTFields in HCC, explore the underlying mechanisms, and investigate the possible combination of TTFields with sorafenib, one of the few front-line treatments for patients with advanced HCC. HepG2 and Huh-7D12 human HCC cell lines were treated with TTFields at various frequencies to determine the optimal frequency eliciting maximal cell count reduction. Clonogenic, apoptotic effects, and autophagy induction were measured. The efficacy of TTFields alone and with concomitant sorafenib was tested in cell cultures and in an orthotopic N1S1 rat model. Tumor volume was examined at the beginning and following 5 days of treatment. At study cessation, tumors were weighed and examined by immunohistochemistry to assess autophagy and apoptosis. TTFields were found in vitro to exert maximal effect at 150 kHz, reducing cell count and colony formation, increasing apoptosis and autophagy, and augmenting the effects of sorafenib. In animals, TTFields concomitant with sorafenib reduced tumor weight and volume fold change, and increased cases of stable disease following treatment versus TTFields or sorafenib alone. While each treatment alone elevated levels of autophagy relative to control, TTFields concomitant with sorafenib induced a significant increase versus control in tumor ER stress and apoptosis levels, demonstrating increased stress under the multimodal treatment. Overall, TTFields treatment demonstrated efficacy and enhanced the effects of sorafenib for the treatment of HCC in vitro and in vivo, via a mechanism involving induction of autophagy.
Significance A substrate-conjugated polyubiquitin chain is accepted as the “canonical” proteasomal degradation signal. Using a cellular (human and yeast) proteomic screen in the exclusive presence of nonpolymerizable ubiquitin, we show that a large group of proteins is degraded by the proteasome following monoubiquitination. The screen also unraveled polyubiquitin-dependent substrates, as they are stabilized in the presence of this ubiquitin mutant. Notably, monoubiquitination- and polyubiquitination-dependent substrates display distinct important characteristics. Monoubiquitinated proteins are of lower molecular mass and of lesser structural disorder. The two groups can be assigned to defined cellular pathways. Furthermore, some of the characteristics are confined to either human or yeast cells, suggesting that the mechanism of action/recognition of the ubiquitin system in the two organisms are different somehow.
A polyubiquitin chain attached covalently to the target substrate has been recognized for long as the “canonical” proteasomal degradation signal. However, several proteins have been shown to be targeted for degradation following monoubiquitination, indicating that the proteasome can recognize signals other than a ubiquitin polymer. A comprehensive screen aiming at determining the extent of this mode of recognition revealed that ∼40% of mammalian and ∼20% of yeast proteins are degraded following monoubiquitination. Characterization of these proteins showed that on average, the monoubiquitinated proteins are smaller than the polyubiquitinated ones, and in humans, are less disordered. Further, proteins degraded by the two different modes belong to distinct functional groups. These findings along with detailed structural analysis of the proteasome, its ubiquitin receptors and deubiquitinating enzymes, suggest that the ubiquitin signal – its formation, recognition, editing, and removal – is far more complex and diverse than originally assumed. Also see the video abstract here: https://youtu.be/QKpN9c6Rg20
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