Lidia Avalle

STAT1 and STAT3 in tumorigenesis: A matter of balance

The transcription factors STAT1 and STAT3 appear to play opposite roles in tumorigenesis. While STAT3 promotes cell survival/proliferation, motility and immune tolerance and is considered as an oncogene, STAT1 mostly triggers anti-proliferative and pro-apoptotic responses while enhancing anti-tumor immunity. Despite being activated downstream of common cytokine and growth factor receptors, their activation is reciprocally regulated and perturbation in their balanced expression or phosphorylation levels may re-direct cytokine/growth factor signals from proliferative to apoptotic, or from inflammatory to anti-inflammatory. Here we review the functional canonical and non-canonical effects of STAT1 and STAT3 activation in tumorigenesis and their potential cross-regulation mechanisms.

The immediate early genes Fos and Egr1 become STAT1 transcriptional targets in the absence of STAT3

Signal transducer and activators of transcription (STAT)1 and STAT3 cross‐regulate their activity downstream of gp130 cytokines, and eliminating STAT3 leads to IFN‐γ‐like responses to IL‐6 correlating with prolonged STAT1 phosphorylation. Here we demonstrate that the increased gp130‐mediated induction of the IFN‐γ‐responsive interferon regulatory factor 1 gene observed in STAT3−/− cells correlates with prolonged STAT1 binding to its promoter. Intriguingly, gp130‐mediated induction of the immediate early genes FBJ osteosarcoma oncogene and early growth response 1 is also prolonged in STAT3−/− cells, with STAT1 binding to their promoters. Thus the abrogation of STAT3 expression, perturbing the signaling balance, directs the STAT1 oncosuppressor to transcribe new target genes, known to drive mitogen responses and tumor transformation.

STAT3 in cancer: A double edged sword

The transcription factor signal transducer and activator of transcription (STAT) 3 is activated downstream of cytokines, growth factors and oncogenes to mediate their functions under both physiological and pathological conditions. In particular, aberrant/unrestrained STAT3 activity is detected in a wide variety of tumors, driving multiple pro-oncogenic functions. For that, STAT3 is widely considered as an oncogene and is the object of intense translational studies. One of the distinctive features of this factor is however, its ability to elicit different and sometimes contrasting effects under different conditions. In particular, STAT3 activities have been shown to be either pro-oncogenic or tumor-suppressive according to the tumor aetiology/mutational landscape, suggesting that the molecular bases underlining its functions are still incompletely understood. Here we discuss some of the properties that may provide the bases to explain STAT3 heterogeneous functions, and in particular how post-translational modifications contribute shaping its sub-cellular localization and activities, the cross talk between these activities and cell metabolic conditions, and finally how its functions can control the behaviour of both tumor and tumor microenvironment cell populations.

Constitutive STAT3 activation in epidermal keratinocytes enhances cell clonogenicity and favours spontaneous immortalization by opposing differentiation and senescence checkpoints.

STAT3, a pleiotropic transcription factor acting downstream of cytokines and growth factors, is known to enhance proliferation, migration, invasion and aerobic glycolysis in tumors upon aberrant activation. In the murine epidermis, STAT3 is necessary for experimentally induced carcinogenesis. Skin tumorigenesis is conversely enhanced by overexpression in keratinocytes of the constitutively active STAT3C mutant, which also induces robust, psoriasis‐like epidermal hyperplasia. We show here that STAT3C expression at physiological levels in knock‐in mice leads to mild epidermal hyperplasia and attenuated expression of terminal differentiation markers. Altered differentiation is confirmed in isolated primary epidermal keratinocytes in vitro, correlating with enhanced proliferative and clonogenic potential, attenuated senescence and, strikingly, high‐frequency spontaneous immortalization. These results suggest that moderate levels of continuous STAT3 activation, which closely resemble those triggered by chronic inflammation or persistent growth factor stimulation, may establish a preneoplastic state in part by promoting the escape of epidermal progenitor cells from differentiation and senescence checkpoints.

Psoriasis: A STAT3-Centric View

Signal Transducer and Activator of Transcription (STAT)3 has recently emerged as a key player in the development and pathogenesis of psoriasis and psoriatic-like inflammatory conditions. Indeed, STAT3 hyperactivation has been reported in virtually every cell type involved in disease initiation and maintenance, and this factor mediates the signal of most cytokines that are involved in disease pathogenesis, including the central Interleukin (IL)-23/IL-17/IL-22 axis. Despite the recent availability of effective biological agents (monoclonal antibodies) against IL-17 and IL-23, which have radically changed the current standard of disease management, the possibility of targeting either STAT3 itself or, even better, the family of upstream activators Janus kinases (JAK1, 2, 3, and TYK2) offers additional therapeutic options. Due to the oral/topical administration modality of these small molecule drugs, their lower cost, and the reduced risk of eliciting adverse immune responses, these compounds are being actively scrutinized in clinical settings. Here, we summarize the main pathological features of psoriatic conditions that provide the rationale for targeting the JAK/STAT3 axis in disease treatment.

Universal and Specific Functions of STAT3 in Solid Tumours

STAT3 is constitutively activated in a high percentage of tumours and tumour-derived cells of both liquid and solid origin, often correlating with aggressive disease and bad prognosis. Persistent STAT3 activity, to which tumours often become addicted, is mostly due to the aberrant activation of pro-oncogenic/pro-inflammatory signals that can trigger its phosphorylation, such as oncogenes, growth factor receptors and cytokines. Among STAT3-mediated functions are increased survival and proliferation, enhanced angiogenesis, motility and invasion, and down-modulation of anti-tumour immune responses. Moreover, STAT3 was recently shown to play unexpected roles in regulating cell metabolism and mitochondrial activity via both transcriptional and non-transcriptional mechanisms. Here, we review the main knowledge about the role of STAT3 in solid tumours, with a particular focus on breast cancer and our recent work with mouse models.

STAT3 localizes to the ER, acting as a gatekeeper for ER-mitochondrion Ca 2+ fluxes and apoptotic responses

STAT3 is an oncogenic transcription factor exerting its functions both as a canonical transcriptional activator and as a non-canonical regulator of energy metabolism and mitochondrial functions. While both activities are required for cell transformation downstream of different oncogenic stimuli, they rely on different post-translational activating events, namely phosphorylation on either Y705 (nuclear activities) or S727 (mitochondrial functions). Here, we report the discovery of the unexpected STAT3 localization to the endoplasmic reticulum (ER), from where it modulates ER-mitochondria Ca2+ release by interacting with the Ca2+ channel IP3R3 and facilitating its degradation. The release of Ca2+ is of paramount importance for life/death cell decisions, as excessive Ca2+ causes mitochondrial Ca2+ overload, the opening of the mitochondrial permeability transition pore, and the initiation of the intrinsic apoptotic program. Indeed, STAT3 silencing enhances ER Ca2+ release and sensitivity to apoptosis following oxidative stress in STAT3-dependent mammary tumor cells, correlating with increased IP3R3 levels. Accordingly, basal-like mammary tumors, which frequently display constitutively active STAT3, show an inverse correlation between IP3R3 and STAT3 protein levels. These results suggest that STAT3-mediated IP3R3 downregulation in the ER crucially contributes to its anti-apoptotic functions via modulation of Ca2+ fluxes.

MicroRNAs-143 and -145 induce epithelial to mesenchymal transition and modulate the expression of junction proteins

Transforming growth factor (TGF)-β is one of the major inducers of epithelial to mesenchymal transition (EMT), a crucial program that has a critical role in promoting carcinoma’s metastasis formation. MicroRNAs-143 and -145, which are both TGF-β direct transcriptional targets, are essential for the differentiation of vascular smooth muscle cells (VSMC) during embryogenesis, a TGF-β-dependent process reminiscent of EMT. Their role in adult tissues is however less well defined and even ambiguous, as their expression was correlated both positively and negatively with tumor progression. Here we show that high expression of both miRs-143 and -145 in mouse mammary tumor cells expressing constitutively active STAT3 (S3C) is involved in mediating their disrupted cell–cell junctions. Additionally, miR-143 appears to have a unique role in tumorigenesis by enhancing cell migration in vitro and extravasation in vivo while impairing anchorage-independent growth, which may explain the contradictory reports about its role in tumors. Accordingly, we demonstrate that overexpression of either miRNA in the non-transformed mammary epithelial NMuMG cells leads to upregulation of EMT markers and of several endogenous TGF-β targets, downmodulation of a number of junction proteins and increased motility, correlating with enhanced basal and TGF-β-induced SMAD-mediated transcription. Moreover, pervasive transcriptome perturbation consistent with the described phenotype was observed. In particular, the expression of several transcription factors involved in the mitogenic responses, of MAPK family members and, importantly, of several tight junction proteins and the SMAD co-repressor TGIF was significantly reduced. Our results provide important mechanistic insight into the non-redundant role of miRs-143 and -145 in EMT-related processes in both transformed and non-transformed cells, and suggest that their expression must be finely coordinated to warrant optimal migration/invasion while not interfering with cell growth.

SP1 and STAT3 functionally synergize to induce the RhoU small GTPase and a subclass of non-canonical WNT responsive genes correlating with poor prognosis in breast cancer.

Breast cancer is a complex disease in which heterogeneity makes clinical management very challenging. Although breast cancer subtypes classified according to specific molecular features are associated to better or worse prognosis, the identification of specific markers predicting disease outcome within the single subtypes still lags behind. Both the non-canonical WNT and the STAT3 pathways are often constitutively activated in breast tumors, and both can induce the small GTPase RhoU gene transcription. Here we show that RhoU transcription can be triggered by both canonical and non-canonical WNT ligands via the activation of JNK and the recruitment of the SP1 transcription factor to the RhoU promoter, identifying for the first time SP1 as a JNK-dependent mediator of WNT signaling. RhoU down-regulation by silencing or treatment with JNK, SP1 or STAT3 inhibitors lead to impaired cell migration in basal-like MDA-MB-231 cells, which display constitutive activation of both the non-canonical WNT and STAT3 pathways. These data suggest that STAT3 and SP1 can cooperate to induce high RhoU expression and enhance migration of breast cancer cells. In vivo binding of both factors characterizes a group of SP1/STAT3 responsive genes belonging to the non-canonical WNT and the IL-6/STAT3 pathways. High expression of this signature is significantly correlated with poor prognosis across all profiled patients. Thus, concomitant binding of both STAT3 and SP1 defines a subclass of genes contributing to breast cancer aggressiveness, suggesting the relevance of developing novel targeted therapies combining inhibitors of the STAT3 and WNT pathways or of their downstream mediators. Novelty and Impact The WNT and STAT3 pathways are often activated in breast tumors, but whether they can cooperate towards aggressiveness is not presently known. Here the authors show that WNT ligands can elicit the activation of the transcription factor SP1, which cooperates with STAT3 to induce a subset of non-canonical WNT and IL-6/STAT3 genes. Expression of this gene signature correlates with bad prognosis in breast cancer, suggesting coordinated interference with both TFs as a novel therapeutic option.

PO-237 The pro-oncogenic transcription factor stat3 regulates ca2 +release and apoptosis from the endoplasmic reticulum via interaction with the ca2 +channel ip3r3

Introduction Signal Transducer and Activator of Transcription (STAT) 3 is an oncogenic transcription factor found constitutively activated in several tumours, where it exerts its functions both as a canonical transcription factor and as a non-canonical regulator of energy metabolism and mitochondrial functions. These two activities rely on different post-translational activating events; the phosphorylation on Y705 is involved in nuclear activities, while that on S727 is relevant for mitochondrial functions. Mitochondrial STAT3 increases aerobic glycolysis and decreases ROS production, partly by interacting with the Electron Transfer Complexes (ETC). Material and methods By means of cell fractionations, we tested STAT3 localization to the Endoplasmic Reticulum (ER) in breast cancer cell lines dependent or not on STAT3 activity. We then measured Ca 2+ release and apoptotic response in the same cells. The physical interaction between inositol 1,4,5-trisphosphate receptor type 3 (IP3R3) and STAT3 was demonstrated by co-IP either of the endogenous proteins or of their truncated/mutated forms, while STAT3 role in the degradation of IP3R3 was tested by serum starvation and refeeding experiments, followed by WB. Results and discussions We describe here the previously undetected abundant localization of STAT3 also to the ER. In this cellular compartment IP3R3, a Ca 2+ channel that allows Ca 2+ release from the ER and the mitochondrial associated membranes (MAMs) in response to IP3, regulates the balance between mitochondrial activation and Ca 2+ -triggered apoptosis. We observed that STAT3 within the ER physically interacts with IP3R3 and, via its phosphorylation on S727, it down-regulates Ca 2+ release and apoptosis. Indeed, STAT3 silencing enhances both ER Ca 2+ release and sensitivity to apoptosis following oxidative stress in STAT3-dependent mammary tumour cells, correlating with increased IP3R3 levels. In line with this, basal-like breast tumours, which frequently display constitutively active STAT3, show an inverse correlation between IP3R3 and STAT3 protein levels. Conclusion Our results indicate that S727-phosphorylated STAT3 contribute to mammary tumour aggressiveness, also by localising to the ER and regulating Ca 2+ fluxes. STAT3-mediated enhanced IP3R3 degradation leads to decreased Ca 2+ release and thus to resistance to apoptosis. This new non-canonical STAT3 role appears to be particularly relevant in basal-like breast cancers, adding a new mechanisms through which STAT3 exerts its well established pro-oncogenic anti-apoptotic role.