Cell Metabolism Control Through O-GlcNAcylation of STAT5: A Full or Empty Fuel Tank Makes a Big Difference for Cancer Cell Growth and Survival.

O-GlcNAcylation is a post-translational modification that influences tyrosine phosphorylation in healthy and malignant cells. O-GlcNAc is a product of the hexosamine biosynthetic pathway, a side pathway of glucose metabolism. It is essential for cell survival and proper gene regulation, mirroring the metabolic status of a cell. STAT3 and STAT5 proteins are essential transcription factors that can act in a mutational context-dependent manner as oncogenes or tumor suppressors. They regulate gene expression for vital processes such as cell differentiation, survival, or growth, and are also critically involved in metabolic control. The role of STAT3/5 proteins in metabolic processes is partly independent of their transcriptional regulatory role, but is still poorly understood. Interestingly, STAT3 and STAT5 are modified by O-GlcNAc in response to the metabolic status of the cell. Here, we discuss and summarize evidence of O-GlcNAcylation-regulating STAT function, focusing in particular on hyperactive STAT5A transplant studies in the hematopoietic system. We emphasize that a single O-GlcNAc modification is essential to promote development of neoplastic cell growth through enhancing STAT5A tyrosine phosphorylation. Inhibition of O-GlcNAcylation of STAT5A on threonine 92 lowers tyrosine phosphorylation of oncogenic STAT5A and ablates malignant transformation. We conclude on strategies for new therapeutic options to block O-GlcNAcylation in combination with tyrosine kinase inhibitors to target neoplastic cancer cell growth and survival.

Induction of Arterial Inflammation by Immune Checkpoint Inhibitor Therapy in Lung Cancer Patients as Measured by 2-[18F]FDG Positron Emission Tomography/Computed Tomography Depends on Pre-Existing Vascular Inflammation.

BACKGROUND: Immune checkpoint inhibitors (ICI) are one of the most effective therapies in oncology, albeit associated with various immune-related adverse events also affecting the cardiovascular system. METHODS: We aimed to investigate the effect of ICI on arterial 2-[18F]FDG uptake by using 2-[18F]FDG PET/CT imaging pre/post treatment in 47 patients with lung cancer. Maximum 2-[18F]FDG standardized uptake values (SUVmax) and target-to-background ratios (TBRs) were calculated along six arterial segments. We classified the arterial PET lesions by pre-existing active inflammation (cut-off: TBRpre ≥ 1.6). 2-[18F]FDG metabolic activity pre/post treatment was also quantified in bone marrow, spleen, and liver. Circulating blood biomarkers were additionally collected at baseline and after immunotherapy. RESULTS: ICI treatment resulted in significantly increased arterial inflammatory activity, detected by increased TBRs, in all arterial PET lesions analyzed. In particular, a significant elevation of arterial 2-[18F]FDG uptake was only recorded in PET lesions without pre-existing inflammation, in calcified as well as in non-calcified lesions. Furthermore, a significant increase in arterial 2-[18F]FDG metabolic activity after immunotherapy was solely observed in patients not previously treated with chemotherapy or radiotherapy as well as in those without CV risk factors. No significant changes were recorded in either 2-[18F]FDG uptake of bone marrow, spleen and liver after treatment, or the blood biomarkers. CONCLUSIONS: ICI induces vascular inflammation in lung cancer patients lacking pre-existing arterial inflammation.

Autocrine PDGFR signaling promotes mammary cancer metastasis.

Metastasis is the major cause of cancer morbidity, but strategies for direct interference with invasion processes are lacking. Dedifferentiated, late-stage tumor cells secrete multiple factors that represent attractive targets for therapeutic intervention. Here we show that metastatic potential of oncogenic mammary epithelial cells requires an autocrine PDGF/PDGFR loop, which is established as a consequence of TGF-beta-induced epithelial-mesenchymal transition (EMT), a faithful in vitro correlate of metastasis. The cooperation of autocrine PDGFR signaling with oncogenic Ras hyperactivates PI3K and is required for survival during EMT. Autocrine PDGFR signaling also contributes to maintenance of EMT, possibly through activation of STAT1 and other distinct pathways. Inhibition of PDGFR signaling interfered with EMT and caused apoptosis in murine and human mammary carcinoma cell lines. Consequently, overexpression of a dominant-negative PDGFR or application of the established cancer drug STI571 interfered with experimental metastasis in mice. Similarly, in mouse mammary tumor virus-Neu (MMTV-Neu) transgenic mice, TGF-beta enhanced metastasis of mammary tumors, induced EMT, and elevated PDGFR signaling. Finally, expression of PDGFRalpha and -beta correlated with invasive behavior in human mammary carcinomas. Thus, autocrine PDGFR signaling plays an essential role during cancer progression, suggesting a novel application of STI571 to therapeutically interfere with metastasis.

Ras and TGF[beta] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways.

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras-transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor beta (TGFbeta) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFbeta receptor (TGFbeta-R) and oncogenic Ras signaling and is stabilized by autocrine TGFbeta production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFbeta alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFbeta-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFbeta-R and Raf/MAPK signaling.

How Do We Improve the Impact of Iodine Deficiency Disorders Prevention in Europe and Beyond?

BACKGROUND: Iodine deficiency disorders (IDD) represent a global health threat to individuals and societies. IDD prevention programmes have been introduced in many parts of the world. However, challenges remain, particularly in Europe due to fragmentation and diversity of approaches that are not harmonized. OBJECTIVES: This review is dedicated to the public-health impact of IDD prevention programmes. It sums up experiences collected by the EUthyroid consortium so far and provides information on stakeholders that should be involved in actions directed to improve the impact of IDD prevention. METHODS: A joint European database for combining registry-based outcome and monitoring data as well as tools for harmonizing study methods were established. Methods for analyzing thyroglobulin from a dried blood spot are available for assessing the iodine status in the general population and at-risk groups. Mother-child cohorts are used for in-depth analysis of the potential impact of mild-to-moderate iodine deficiency on the neurocognitive development of the offspring. A decision-analytic model has been developed to evaluate the long-term effectiveness and cost effectiveness of IDD prevention programmes. RESULTS: EUthyroid has produced tools and infrastructure to improve the quality of IDD monitoring and follows a dissemination strategy targeting policymakers and the general public. There are tight connections to major stakeholders in the field of IDD monitoring and prevention. CONCLUSIONS: EUthyroid has taken steps towards achieving a euthyroid Europe. Our challenge is to inspire a greater sense of urgency in both policymakers and the wider public to address this remediable deficit caused by IDD.

NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression.

The transcription factor NF-kappaB is activated in a range of human cancers and is thought to promote tumorigenesis, mainly due to its ability to protect transformed cells from apoptosis. To investigate the role of NF-kappaB in epithelial plasticity and metastasis, we utilized a well-characterized in vitro/in vivo model of mammary carcinogenesis that depends on the collaboration of the Ha-Ras oncoprotein and TGF-beta. We show here that the IKK-2/IkappaBalpha/NF-kappaB pathway is required for the induction and maintenance of epithelial-mesenchymal transition (EMT). Inhibition of NF-kappaB signaling prevented EMT in Ras-transformed epithelial cells, while activation of this pathway promoted the transition to a mesenchymal phenotype even in the absence of TGF-beta. Furthermore, inhibition of NF-kappaB activity in mesenchymal cells caused a reversal of EMT, suggesting that NF-kappaB is essential for both the induction and maintenance of EMT. In line with the importance of EMT for invasion, blocking of NF-kappaB activity abrogated the metastatic potential of mammary epithelial cells in a mouse model system. Collectively, these data provide evidence of an essential role for NF-kappaB during distinct steps of breast cancer progression and suggest that the cooperation of Ras- and TGF-beta-dependent signaling pathways in late-stage tumorigenesis depends critically on NF-kappaB activity.

Beta-catenin/LEF-1 signalling in breast cancer--central players activated by a plethora of inputs.

Although the role of Wnt signalling in breast cancer is far from being fully understood, in the last years its importance has been reported frequently. Besides stimulation by canonical Wnt signalling, the downstream effectors beta-catenin and the transcriptional modulators of the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) family can also be activated by other inputs including the TGF-beta pathway. Wnt and TGF-beta signalling are both major signal transduction pathways, which provide important cues during development and tumor progression. However, particularly TGF-beta has a complicated influence on oncogenesis, which ranges from suppressive to promoting activity. Signalling pathways activated in parallel with TGF-beta might determine the oncogenic influence, and therefore place signals cooperating with TGF-beta into the limelight. During early development Wnt and TGF-beta signalling collaborate extensively. Here we provide an overview of the known interactions of Wnt with TGF-beta signalling in development and metastasis, particularly in breast cancer. We want to focus on the Wnt-activated transcription factor complex beta-catenin/LEF-1, its upstream activators, its downstream targets and consequences on the cellular level in response to beta-catenin/LEF-1 activation.

Enhanced tenascin-C expression and matrix deposition during Ras/TGF-beta-induced progression of mammary tumor cells.

Overexpression of tenascin-C (TN-C) in breast carcinomas has been associated with a migratory or even invasive tumor cell phenotype. The mechanisms regulating expression and matrix deposition of TN-C in normal and cancerous breast tissues are, however, little understood. Here, we demonstrate that mouse mammary epithelial cells (EpH4) transformed by oncogenic Ha-Ras (EpRas) overexpress TN-C, which accumulates in the cytoplasm. When EpRas cells undergo epithelial-mesenchymal transition (EMT) in response to TGFbeta1, they secrete TN-C into the culture medium. In EpRas cells undergoing TGFbeta1-induced EMT in three-dimensional (3D)-collagen gel cultures, TN-C was deposited into an extracellular matrix (ECM) already containing fibronectin and perlecan. Under less physiological 2D plastic cultures, EpRas cells undergoing EMT failed to deposit TN-C into an (apparently incomplete) ECM. Ras-downstream signaling was dissected by pharmacological inhibitors and effector-specific Ras mutants (V12S35, V12C40), specifically inhibiting or activating ERK/MAPK or PI3K signaling, respectively. We showed that TN-C overexpression required a hyperactive ERK/MAPK-signaling pathway, while elevated PI3K signaling did not enhance TN-C expression. Similarly, tumors induced by cells exhibiting hyperactive ERK/MAPK signaling showed expression of TN-C in the tumor cells themselves, while only endothelial cells expressed TN-C in tumors caused by the V12C40 mutant (incapable of EMT in vivo). Taken together, our data indicate that hyperactive ERK/MAPK signaling causes enhanced expression of TN-C, while its secretion is induced by TGFbeta1 and both signals cooperate in TN-C matrix deposition. Importantly, both signals also cooperate to induce EMT in vitro and tumor progression/metastasis in vivo.

Oncogenic Ras/Her-2 mediate hyperproliferation of polarized epithelial cells in 3D cultures and rapid tumor growth via the PI3K pathway.

Carcinogenesis by oncogenic Ras and Her-2 involves enhanced proliferation of epithelial cells in vivo. However, hyperproliferation induced by these oncogenes, or their downstream pathways in vitro has mainly been studied in cultured, fibroblastic cell lines. Here, we demonstrate that oncogenic Ha-Ras or constitutively active Her-2 cause increased proliferation and cyclin D1 upregulation in fully polarized, mammary epithelial cells (EpH4), if cultivated as organotypic structures in three-dimensional collagen/matrigel matrices. Under standard culture conditions, however, these oncogenes failed to induce hyperproliferation. Using both specific low molecular weight inhibitors and Ras-effector-specific mutants, we dissected signaling pathways downstream of oncogenic Ras (PI3K, Mek1/MAPK) with respect to (i) hyperproliferation in collagen gels and tumorigenesis in mice and (ii) epithelial/mesenchymal transition (EMT). We show that the Ras-activated PI3K pathway is required to induce rapid tumor growth and enhanced proliferation of EpH4 cells in collagen gels, but fails to cause EMT in vitro and in vivo. On the other hand, Ras-dependent activation of the Mek1/MAPK pathway in EpH4 cells (previously shown to cause EMT and metastasis) did not induce hyperproliferation in collagen gels and caused only slow tumor growth. Our data thus indicate that Ras-dependent signaling through the PI3K- and MAPK pathways fulfil distinct, but complementary functions during carcinogenesis.

Expression profiling of epithelial plasticity in tumor progression.

Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and trans-differentiation to mesenchymal-like cells with invasive behavior.

Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis.

In contrast to the aberrant control of proliferation, apoptosis, angiogenesis and lifespan, the cellular mechanisms that cause local invasion and metastasis of tumour cells are still poorly understood. New experimental approaches have identified different types of epithelial-plasticity changes in tumour cells towards fibroblastoid phenotypes as crucial events that occur during metastasis, and many molecules and signalling pathways cooperate to trigger these processes.

Mechanisms in epithelial plasticity and metastasis: insights from 3D cultures and expression profiling.

Most human tumors are of epithelial origin (carcinomas) and metastases from such tumors lead to >80% of all cancer deaths. In contrast to aberrant control of proliferation, cell cycle, apoptosis, angiogenesis, and lifespan, mechanisms involved in local invasion and metastasis are still insufficiently understood. We will review a set of (often conflicting) in vitro/in vivo data that suggest the existence of several types of epithelial cell plasticity changes towards a fibroblastoid, invasive phenotype, which increasingly emerge as crucial events during metastasis. New cellular models were identified, which form organotypic structures under near-physiological 3D-culture conditions in vitro as well as tumors/metastases in vivo. In these models, key proteins and signaling pathways were identified (e.g., TGFbeta, ERK/MAPK, PI3K, and PDGF), which specify distinct types of epithelial plasticity correlated with steps in cancer progression and metastasis. The existence of several distinct epithelial plasticity phenotypes is also strongly suggested by expression profiling of polysome-bound mRNA, yielding a better representation of the proteome than conventional expression profiling.