Faculty Bibliography

Increased production of mitochondrial-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, non-transcriptional 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 mitochondrial-STAT3 dependent pathway in Ras-transformed cells. Experimental inhibition of key enzymes in the glutathione cycle resulted in 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.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19ARF as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14ARF expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

Juvenile myelomonocytic leukemia, acute myeloid leukemia (AML), and other myeloproliferative neoplasms (MPNs) are genetically heterogeneous but frequently display activating mutations in Ras GTPases and activation of signal transducer and activator of transcription 3 (STAT3). Altered STAT3 activity is observed in up to 50% of AML correlating with poor prognosis. Activated STAT proteins, classically associated with tyrosine phosphorylation, support tumor development as transcription factors, but alternative STAT functions independent of tyrosine phosphorylation have been documented, including roles for serine-phosphorylated STAT3 in mitochondria supporting transformation by oncogenic Ras. We examined requirements for STAT3 in experimental murine K-Ras-dependent hematopoietic neoplasia. We show that STAT3 is phosphorylated on S727 but not Y705 in diseased animals. Moreover, a mouse with a point mutation abrogating STAT3 S727 phosphorylation displayed delayed onset and decreased disease severity with significantly extended survival. Activated K-Ras required STAT3 for cytokine-independent growth of myeloid progenitors in vitro, and mitochondrially restricted STAT3 and STAT3-Y705F, both transcriptionally inert mutants, supported factor-independent growth. STAT3 was dispensable for growth of normal or K-Ras-mutant myeloid progenitors in response to cytokines. However, abrogation of STAT3-S727 phosphorylation impaired factor-independent malignant growth. These data document that serine-phosphorylated mitochondrial STAT3 supports neoplastic hematopoietic cell growth induced by K-Ras.

Activating mutations in the RasGTPases are the most common oncogenic lesions in human cancer. Similarly, elevated STAT3 expression and/or phosphorylation are observed in the majority of human cancers. We recently found that activated Ras requires a mitochondrial rather than a nuclear activity of STAT3 to support cellular transformation. This mitochondrial activity of STAT3 was supported by phosphorylation on serine 727 (S727) in the carboxyl-terminus of STAT3. In this study we show that the H-Ras oncoprotein engages the MEK-ERK pathway to drive phosphorylation of STAT3 on S727, while phosphoinositide 3-kinase (PI3K) and mTOR activity were superfluous. Moreover, pharmacological inhibition of MEK reduced transformation by H-, K- or N-Ras. However, cells expressing a mitochondrially restricted STAT3 with a phospho-mimetic mutation at S727 were partially resistant to inhibition of the ERK pathway, exhibiting a partial rescue of anchorage-independent cell growth in the presence of MEK inhibitor. This study shows that the MEK-ERK pathway is required for activated Ras-induced phosphorylation of STAT3 on S727, that inhibition of STAT3 S727 phosphorylation contributes to the anti-oncogenic potential of MEK inhibitors, and that mitochondrial STAT3 is one of the critical substrates of the Ras-MEK-ERK- axis during cellular transformation.

Type I interferons (IFNs) stimulate transcription through a latent heterotrimeric transcription factor composed of tyrosine-phosphorylated STAT1 and STAT2 and the DNA binding partner IRF9, with STAT2 contributing a critical transactivation domain. Human RVB1 and RVB2, which are highly conserved AAA(+) ATP binding proteins contained in chromatin-remodeling complexes such as Ino80, SNF2-related CBP activator protein (SRCAP), and Tip60/NuA4, interacted with the transactivation domain of STAT2 in the nuclei of IFN-stimulated cells. RNA interference (RNAi) experiments demonstrated that RVB proteins were required for robust activation of IFN-alpha-stimulated genes (ISGs). The requirement for RVB proteins was specific to IFN-alpha/STAT2 signaling; transcription of tumor necrosis factor alpha (TNF-alpha)- and IFN-gamma-driven genes was not affected by RVB1 depletion. Using RNAi-based depletion, we assessed the involvement of catalytic subunits of the RVB-containing Tip60, BRD8, Ino80, SRCAP, and URI complexes. No component other than RVB1/2 was uniquely required for ISG induction, suggesting that RVB1/2 functions as part of an as yet unidentified complex. Chromatin immunoprecipitation assays indicated that RVB1/2 was required for recruitment of RNA polymerase II (Pol II) to ISG promoters but was dispensable for STAT2 recruitment to chromatin. We hypothesize that an RVB1/2 chromatin-remodeling complex is required for efficient Pol II recruitment and initiation at ISG promoters and is recruited through interaction with the STAT2 transactivation domain.

INTRODUCTION/BACKGROUND:Although breast cancers expressing estrogen receptor-α (ERα) and progesterone receptors (PR) are the most common form of mammary malignancy in humans, it has been difficult to develop a suitable mouse model showing similar steroid hormone responsiveness. STAT transcription factors play critical roles in mammary gland tumorigenesis, but the precise role of STAT1 remains unclear. Herein, we show that a subset of human breast cancers display reduced STAT1 expression and that mice lacking STAT1 surprisingly develop ERα+/PR+ mammary tumors. METHODS:We used a combination of approaches, including histological examination, gene targeted mice, gene expression analysis, tumor transplantaion, and immunophenotyping, to pursue this study. RESULTS:Forty-five percent (37/83) of human ERα+ and 22% (17/78) of ERα- breast cancers display undetectable or low levels of STAT1 expression in neoplastic cells. In contrast, STAT1 expression is elevated in epithelial cells of normal breast tissues adjacent to the malignant lesions, suggesting that STAT1 is selectively downregulated in the tumor cells during tumor progression. Interestingly, the expression levels of STAT1 in the tumor-infiltrating stromal cells remain elevated, indicating that single-cell resolution analysis of STAT1 level in primary breast cancer biopsies is necessary for accurate assessment. Female mice lacking functional STAT1 spontaneously develop mammary adenocarcinomas that comprise > 90% ERα+/PR+ tumor cells, and depend on estrogen for tumor engraftment and progression. Phenotypic marker analyses demonstrate that STAT1-/- mammary tumors arise from luminal epithelial cells, but not myoepithelial cells. In addition, the molecular signature of the STAT1-/- mammary tumors overlaps closely to that of human luminal breast cancers. Finally, introduction of wildtype STAT1, but not a STAT1 mutant lacking the critical Tyr701 residue, into STAT1-/- mammary tumor cells results in apoptosis, demonstrating that the tumor suppressor function of STAT1 is cell-autonomous and requires its transcriptional activity. CONCLUSIONS:Our findings demonstrate that STAT1 suppresses mammary tumor formation and its expression is frequently lost during breast cancer progression. Spontaneous mammary tumors that develop in STAT1-/- mice closely recapitulate the progression, ovarian hormone responsiveness, and molecular characteristics of human luminal breast cancer, the most common subtype of human breast neoplasms, and thus represent a valuable platform for testing novel treatments and detection modalities.

The STAT signaling pathway is one of the seven common pathways that govern cell fate decisions during animal development. Comparative genomics revealed multiple incidences of stat gene duplications throughout metazoan evolutionary history. While pseudogenization is a frequent fate of duplicated genes, many of these STAT duplications evolved into novel genes through rapid sequence diversification and neofunctionalization. Additionally, the core of STAT gene regulatory networks, comprising stat1 through 4, stat5 and stat6, arose early in vertebrate evolution, probably through the two whole genome duplication events that occurred after the split of Cephalochordates but before the rise of Chondrichthyes. While another complete genome duplication event took place during the evolution of bony fish after their separation from the tetrapods about 450 million years ago (Mya), modern fish have only one set of these core stats, suggesting the rapid loss of most duplicated stat genes. The two stat5 genes in mammals likely arose from a duplication event in early Eutherian evolution, a period from about 310 Mya at the avian-mammal divergence to the separation of marsupials from other mammals about 130 Mya. These analyses indicate that whole genome duplications and gene duplications by unequal chromosomal crossing over were likely the major mechanisms underlying the evolution of STATs.

The chemokine-mediated recruitment of effector T cells to sites of inflammation is a central feature of the immune response. The extent to which chemokine expression levels are limited by the intrinsic developmental characteristics of a tissue has remained unexplored. We show in mice that effector T cells cannot accumulate within the decidua, the specialized stromal tissue encapsulating the fetus and placenta. Impaired accumulation was in part attributable to the epigenetic silencing of key T cell-attracting inflammatory chemokine genes in decidual stromal cells, as evidenced by promoter accrual of repressive histone marks. These findings give insight into mechanisms of fetomaternal immune tolerance, as well as reveal the epigenetic modification of tissue stromal cells as a modality for limiting effector T cell trafficking.