Theresia R. Kress

Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis

The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci. Recent work suggested that rather than up- and downregulating selected groups of genes, Myc targets all active promoters and enhancers in the genome (a phenomenon termed ‘invasion’) and acts as a general amplifier of transcription. However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations, we detected general increases in total RNA or messenger RNA copies per cell (hereby termed ‘amplification’) when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome, Myc does not directly act as a global transcriptional amplifier. Instead, our results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.

Deregulated MYC expression induces dependence upon AMPK-related kinase 5

Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules. Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.

p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication

The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3′ UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.

MYC: connecting selective transcriptional control to global RNA production.

Two opposing models have been proposed to describe the function of the MYC oncoprotein in shaping cellular transcriptomes: one posits that MYC amplifies transcription at all active loci; the other that MYC differentially controls discrete sets of genes, the products of which affect global transcript levels. Here, we argue that differential gene regulation by MYC is the sole unifying model that is consistent with all available data. Among other effects, MYC endows cells with physiological and metabolic changes that have the potential to feed back on global RNA production, processing and turnover. The field is progressing steadily towards a full characterization of the MYC-regulated genes and pathways that mediate these biological effects and — by the same token — endow MYC with its pervasive oncogenic potential.

High Erk activity suppresses expression of the cell cycle inhibitor p27Kip1 in colorectal cancer cells

The molecular heterogeneity of human cancer cells at the level of signaling protein activities remains poorly understood. Using a panel of 64 colorectal (CRC) cancer cell lines the activity status of the MAP kinases Erk1 and Erk2 was investigated. Erk1/2 activity varied greatly within the CRC cell line panel and was not detectably associated with the speed of cell growth in 10 CRC lines analyzed. As expected, mutations in K-Ras or B-Raf were often, albeit not always, linked to high Erk1/2 activity. The phosphorylation of several known Erk1/2 targets investigated did not generally reflect Erk1/2 activity in the 10 CRC lines analyzed. However, the reduction of Erk1/2 activity with MEK inhibitors generally abolished cell growth but only led to an increase of cellular p27Kip1 levels in CRC cells with high Erk1/2 activity levels. The results indicate that high Erk1/2 activation is utilized by some CRC lines to override the cell cycle brake p27Kip1, while others presumably rely on different mechanisms in order to inactivate this important cell cycle brake. Such detailed knowledge of the molecular diversity of cancer cell signaling mechanisms may eventually help to develop molecularly targeted, patient-specific therapeutic strategies and treatments.

Aldosterone Stimulates Activity and Surface Expression of NHE3 in Human Primary Proximal Tubule Epithelial Cells (RPTEC)

The steroid hormone aldosterone is a major regulator of extracellular volume and blood pressure. Aldosterone effectors are for example the epithelial Na+ channel (ENaC), the Na+-K+-ATPase and the proximal tubule Na+/H+ exchanger isoform 3 (NHE3). The aim of this study was to investigate whether aldosterone acts directly on proximal tubule cells to stimulate NHE3 and if so whether the EGF-receptor (EGFR) is involved. For this purpose, primary human renal proximal tubule cells were exposed to aldosterone. NHE3 activity was determined from Na+- dependent pH-recovery, NHE3 surface expression was determined by biotinylation and immunoblotting. EGFR-expression was assessed by ELISA. pHi- measurements revealed an aldosterone-induced increase in NHE3 activity, which was inhibited by the mineralocorticoid receptor blocker spironolactone and by the EGFR-kinase inhibitor AG1478. Immunoprecipitation and immunoblot analysis showed an aldosterone-induced increase in NHE3 surface expression, which was also inhibited by spironolactone and AG1478. Furthermore, aldosterone enhanced EGFR-expression. In conclusion, aldosterone stimulates NHE3 in human proximal tubule cells. The underlying mechanisms include AG1478 inhibitable kinase and are paralleled by enhanced EGFR expression, which could be compatible with EGF-receptor-pathway-dependent surface expression and activity of NHE3 in human primary renal proximal tubule epithelial cells.

Degradation dynamics of microRNAs revealed by a novel pulse-chase approach

The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2> 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2= 4-14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions.

INSPEcT: a computational tool to infer mRNA synthesis, processing and degradation dynamics from RNA- and 4sU-seq time course experiments

MOTIVATION Cellular mRNA levels originate from the combined action of multiple regulatory processes, which can be recapitulated by the rates of pre-mRNA synthesis, pre-mRNA processing and mRNA degradation. Recent experimental and computational advances set the basis to study these intertwined levels of regulation. Nevertheless, software for the comprehensive quantification of RNA dynamics is still lacking. RESULTS INSPEcT is an R package for the integrative analysis of RNA- and 4sU-seq data to study the dynamics of transcriptional regulation. INSPEcT provides gene-level quantification of these rates, and a modeling framework to identify which of these regulatory processes are most likely to explain the observed mRNA and pre-mRNA concentrations. Software performance is tested on a synthetic dataset, instrumental to guide the choice of the modeling parameters and the experimental design. AVAILABILITY AND IMPLEMENTATION INSPEcT is submitted to Bioconductor and is currently available as Supplementary Additional File S1. CONTACT mattia.pelizzola@iit.it SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Mutual epithelium-macrophage dependency in liver carcinogenesis mediated by ST18

The ST18 gene has been proposed to act either as a tumor suppressor or as an oncogene in different human cancers, but direct evidence for its role in tumorigenesis has been lacking thus far. Here, we demonstrate that ST18 is critical for tumor progression and maintenance in a mouse model of liver cancer, based on oncogenic transformation and adoptive transfer of primary precursor cells (hepatoblasts). ST18 messenger RNA (mRNA) and protein were detectable neither in normal liver nor in cultured hepatoblasts, but were readily expressed after subcutaneous engraftment and tumor growth. ST18 expression in liver cells was induced by inflammatory cues, including acute or chronic inflammation in vivo, as well as coculture with macrophages in vitro. Knocking down the ST18 mRNA in transplanted hepatoblasts delayed tumor progression. Induction of ST18 knockdown in pre‐established tumors caused rapid tumor involution associated with pervasive morphological changes, proliferative arrest, and apoptosis in tumor cells, as well as depletion of tumor‐associated macrophages, vascular ectasia, and hemorrhage. Reciprocally, systemic depletion of macrophages in recipient animals had very similar phenotypic consequences, impairing either tumor development or maintenance, and suppressing ST18 expression in hepatoblasts. Finally, RNA sequencing of ST18‐depleted tumors before involution revealed down‐regulation of inflammatory response genes, pointing to the suppression of nuclear factor kappa B–dependent transcription. Conclusion: ST18 expression in epithelial cells is induced by tumor‐associated macrophages, contributing to the reciprocal feed‐forward loop between both cell types in liver tumorigenesis. Our findings warrant the exploration of means to interfere with ST18‐dependent epithelium–macrophage interactions in a therapeutic setting. (Hepatology 2017;65:1708‐1719).

Smyd2 is a Myc-regulated gene critical for MLL-AF9 induced leukemogenesis

The Smyd2 protein (Set- and Mynd domain containing protein 2) is a methyl-transferase that can modify both histones and cytoplasmic proteins. Smyd2 is over-expressed in several cancer types and was shown to be limiting for tumor development in the pancreas. However, genetic evidence for a role of Smyd2 in other cancers or in mouse development was missing to date. Using germ line-deleted mouse strains, we now show that Smyd2 and the related protein Smyd3 are dispensable for normal development. Ablation of Smyd2 did not affect hematopoiesis, but retarded the development of leukemia promoted by MLL-AF9, a fusion oncogene associated with acute myeloid leukemia (AML) in humans. Smyd2-deleted leukemic cells showed a competitive disadvantage relative to wild-type cells, either in vitro or in vivo. The Smyd2 gene was directly activated by the oncogenic transcription factor Myc in either MLL9-AF9-induced leukemias, Myc-induced lymphomas, or fibroblasts. However, unlike leukemias, the development of lymphomas was not dependent upon Smyd2. Our data indicate that Smyd2 has a critical role downstream of Myc in AML.