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Small ubiquitin-like modifier (SUMO)-targeted E3 ubiquitin ligases (STUbLs) are specialized enzymes that recognize SUMOylated proteins and attach ubiquitin to them. They therefore connect the cellular SUMOylation and ubiquitination circuits. STUbLs participate in diverse molecular processes that span cell cycle regulated events, including DNA repair, replication, mitosis, and transcription. They operate during unperturbed conditions and in response to challenges, such as genotoxic stress. These E3 ubiquitin ligases modify their target substrates by catalyzing ubiquitin chains that form different linkages, resulting in proteolytic or non-proteolytic outcomes. Often, STUbLs function in compartmentalized environments, such as the nuclear envelope or kinetochore, and actively aid in nuclear relocalization of damaged DNA and stalled replication forks to promote DNA repair or fork restart. Furthermore, STUbLs reside in the same vicinity as SUMO proteases and deubiquitinases (DUBs), providing spatiotemporal control of their targets. In this review, we focus on the molecular mechanisms by which STUbLs help to maintain genome stability across different species.

Minichromosome maintenance protein 10 (Mcm10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 mutations in patients with distinctive but overlapping clinical phenotypes – natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of Mcm10-associated disease, we modeled these mutations in human cell lines. Mcm10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of Mcm10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in Mcm10-deficient cells require endonucleolytic processing by Mus81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic mutations in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes in both NKD and CM patients.

Triple negative breast cancer (TNBC) is the most metastatic and deadliest breast cancer (BC) subtype, accounting for 20-30% of all BCs. There is a critical need to identify molecular targets that could be exploited as new biomarkers of TNBC prognosis and for improving therapies. Although TNBC lacks estrogen and progesterone receptors, 15-40% of TNBC patients express the glucocorticoid receptor (GR). Women with TNBC that express high levels of GR have poor outcomes. We hypothesize that GR is a key mediator of advanced cancer phenotypes in TNBC. Specifically, we propose that GR acts as a “sensor” for stress signaling pathways commonly activated by soluble factors that are abundant within the tumor microenvironment (TME). Using TNBC models, we showed previously that GR is phosphorylated on Ser134 in response to cellular stress stimuli such as hypoxia. Herein, we show that pS134-GR is elevated in TNBC tumor tissue samples relative to non-TNBC tissues. In vitro studies in TNBC models demonstrate that GR Ser134 phosphorylation is promoted by cytokines (TGFbeta), and growth factors (HGF) and occurs in the absence of GR ligands such as Dexamethasone or cortisol. In response to stress signaling inputs, studies with kinase inhibitors confirmed that p38 MAPK is required for GR Ser134 (pS134-GR) phosphorylation. To evaluate the functional significance of pS134-GR, we created CRISPR models of MDA-MB-231 TNBC cells expressing either wt GR or phospho-mutant GR that cannot be phosphorylated at Ser134 (S134A). RNAseq studies were performed to identify pS134-GR target genes in TNBC models. Our transcriptome data demonstrated a requirement for pS134 GR in the expression of gene sets associated with TGFβ and p38 MAPK signaling. Pathway analysis revealed that pS134-GR target genes primarily regulate cancer cell migration. In vitro assays revealed that pS134-GR is essential for inducing cell migration and anchorage-independent growth in TNBC cells, even in the absence of exogenous GR ligands. Furthermore, using co-IP assays, we identified that upon phosphorylation at Ser134, GR interacts with the scaffolding protein 14-3-3zeta. Like pS134-GR, 14-3-3zeta is highly expressed in TNBC when compared to non-TNBC patients. We observed co-recruitment of both pS134-GR and 14-3-3zeta to known pS134-GR target genes (i.e. PTK6) in TNBC cells. These data prompted us to test the requirement for 14-3-3zeta in GR-mediated phenotypes. Short hairpin RNA knock-down experiments demonstrated that expression of 14-3-3zeta is required for serum and TGFbeta-induced TNBC cell migration. We conclude that the pS134-GR/14-3-3zeta complex is a key “sensor” of local stress signals within the TME (TGFbeta) and a potent mediator of cell migration in TNBC models. Further studies are aimed at exploring pS134 GR as a biomarker and therapeutic target in TNBC.

Tobacco is a complex chemical mixture, containing many toxicants and carcinogens. Most rodent risk assessment studies have focused on single chemicals or the complicated mixtures of tobacco smoke or its fractions. There are few studies evaluating how specific chemicals interact with one another to form the potent carcinogenic mixture of tobacco smoke. We hypothesized that tobacco smoke aldehydes like formaldehyde and acetaldehyde could enhance the carcinogenic properties of the tobacco-specific nitrosamines, N’-nitrosonornicotine (NNN) and 4-methylnitrosamine-1-(3-pyridyl)-1-butanone (NNK), through a variety of mechanisms. This hypothesis was tested in two established rodent tumor models, the NNN-induced rat esophageal tumor model and the NNK-induced A/J mouse lung tumor model. In the first model, rats were exposed to 0, 4, or 8 ppm NNN in the drinking water in the presence or absence of 3000 ppm acetaldehyde for up to 100 weeks. The number of esophageal papillomas per rat was doubled in animals receiving both acetaldehyde and 8 ppm NNN (0.5 versus 1.15 tumors/rat, respectively). Acetaldehyde alone did not cause esophageal tumors. DNA adduct levels were not affected by the combination of the two chemicals. In the second model, A/J mice were exposed to NNK (i.p, 0, 2.5, or 7.5 μmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 15 ppmv) for 3 hours in a nose-only inhalation chamber. Lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had an increased number of adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase dysplasia in tumors initiated by NNK. As in the rat study, DNA adduct levels were not affected by the coexposure. In a separate experiment, we tested the hypothesis that the elevated levels of carbon dioxide in tobacco smoke could affect the carcinogenic properties of NNK in the A/J mouse; tobacco smoke contains 12.5% carbon dioxide. Mice received a 3 h nose-only carbon dioxide (0, 5, 10, or 15%) coexposure of mice receiving NNK (i.p. in saline, 0, 2.5, or 7.5 μmol). The coexposure to carbon dioxide more than doubled the number of lung adenomas induced by 2.5 μmol NNK, with the maximal effect observed with 10% carbon dioxide (0%: 1.8 ± 1.9; 5%: 3.9 ± 2.8; 10%: 7.1 ± 3.5; 15%; 5.5 ± 2.9 lung adenomas/mouse). Lung adenomas were also significantly increased in mice receiving 7.5 μmol NNK, although to a lesser extent (0%: 11 ± 6.2; 5%: 13 ± 7.9; 10%: 18 ± 6.0; 15%; 12 ± 5.3 lung adenomas/mouse). This additive and synergistic effect of carbon dioxide was highly significant (p value = 1.0 × 10-14). The mechanism of this interaction is under investigation. Collectively, these studies support the hypothesis that the aldehydes and carbon dioxide present in the tobacco mixture interact to enhance the carcinogenic potency of the tobacco specific nitrosamines. (Funded by CA-184987.) Citation Format: Lisa A. Peterson, Marissa K. Oram, Donna E. Seabloom, William E. Smith, Alessia Stornetta, Karin R. Vevang, Monica Flavin, Alexandru F. Tabaran, Ingrid Cornax, M. Gerard O’Sullivan, Pramod Upadhyaya, Lin Zhang, Stephen S. Hecht, Silvia Balbo, Timothy S. Wiedmann. Interactions between tobacco smoke chemicals in rodent tumor models [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A19.