Daniel Garama

A Synthetic Lethal Interaction between Glutathione Synthesis and Mitochondrial Reactive Oxygen Species Provides a Tumor-Specific Vulnerability Dependent on STAT3

ABSTRACT Increased production of mitochondrion-derived reactive oxygen species (ROS) is characteristic of a metabolic shift observed during malignant transformation. While the exact sources and roles of ROS in tumorigenesis remain to be defined, it has become clear that maintaining redox balance is critical for cancer cell proliferation and survival and, as such, may represent a vulnerability that can be exploited therapeutically. STAT3, a latent cytosolic transcription factor activated by diverse cytokines and growth factors, has been shown to exhibit an additional, nontranscriptional function in mitochondria, including modulation of electron transport chain activity. In particular, malignant transformation by Ras oncogenes exploits mitochondrial STAT3 functions. We used mass spectrometry-based metabolomics profiling to explore the biochemical basis for the STAT3 dependence of Ras transformation. We identified the gamma-glutamyl cycle, the production of glutathione, and the regulation of ROS as a mitochondrion-STAT3-dependent pathway in Ras-transformed cells. Experimental inhibition of key enzymes in the glutathione cycle resulted in the depletion of glutathione, accumulation of ROS, oxidative DNA damage, and cell death in an oncogenic Ras- and mitochondrial STAT3-dependent manner. These data uncover a synthetic lethal interaction involving glutathione production and mitochondrial ROS regulation in Ras-transformed cells that is governed by mitochondrial STAT3 and might be exploited therapeutically.

Mitochondrial STAT3: Powering up a potent factor

The JAK-STAT3 signaling pathway is engaged by many cytokines and growth factor stimuli to control diverse biological processes including proliferation, angiogenesis, survival, immune modulation, and metabolism. For over two decades it has been accepted that STAT3-dependent biology is due to its potency as a transcription factor capable of regulating the expression of many hundreds of genes. However, recent evidence of non-canonical and non-genomic activities of STAT3 has emerged. The most exciting of these activities is its capacity to translocate into the mitochondria where it regulates the activity of the electron transport chain and the opening of the mitochondrial permeability transition pore. These have broad consequences including cell survival and the production of reactive oxygen species and ATP in both normal tissue and under pathological conditions. Despite these fascinating observations there are many key unanswered questions about the mechanism of STAT mitochondrial activity.

Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns.

Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns.

Discovery and Validation of Novel Protein Biomarkers in Ovarian Cancer Patient Urine

For the vast majority of ovarian cancer patients, optimal surgical debulking remains a key prognostic factor associated with improved survival. A standardized, biomarker‐based test, to preoperatively discriminate benign from malignant disease and inform appropriate patient triage, is highly desirable. However, no fit‐for‐purpose biomarkers have yet been identified.

A Novel Fatty Acid-Binding Protein-Like Carotenoid- Binding Protein from the Gonad of the New Zealand Sea Urchin Evechinus chloroticus

A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9′-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid.

The tumor suppressor Hic1 maintains chromosomal stability independent of Tp53

Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.

STAT3: a multifaceted oncoprotein

Abstract Signal transducer and activator of transcription (STAT) 3 is a key signalling protein engaged by a multitude of growth factors and cytokines to elicit diverse biological outcomes including cellular growth, differentiation, and survival. The complete loss of STAT3 is not compatible with life and even partial loss of function mutations lead to debilitating pathologies like hyper IgE syndrome. Conversely, augmented STAT3 activity has been reported in as many as 50% of all human tumours. The dogma of STAT3 activity posits that it is a tyrosine phosphorylated transcription factor which modulates the expression of hundreds of genes. However, the regulation and biological consequences of STAT3 activation are far more complex. In addition to tyrosine phosphorylation, STAT3 is decorated with a plethora of post-translational modifications which regulate STAT3’s nuclear function in addition to its non-genomic activities. In addition to these emerging complexities in the biochemical regulation of STAT3 activity, recent studies reveal that STAT3 is either oncogenic or a tumour suppressor. This review will explore these complexities.