University of Groningen C/EBPβ isoforms and the regulation of metabolism Ackermann, Tobias

University of Groningen

C/EBPβ isoforms and the regulation of metabolism

Ackermann, Tobias

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C/EBPβ isoforms and the

regulation of metabolism

A fine balance between health and disease

PhD thesis

By

ISBN (book): 978-94-034-1117-0

ISBN: (e-book): 978-94-034-1116-3

Cover design and layout: Tobias Ackermann

C/EBPβ isoforms and the regulation of

metabolism

A fine balance between health and disease

PhD thesis

To obtain the degree of PhD at the

University of Groningen

on the authority of the

Rector Magnificus Prof. E. Sterken

and in accordance with

the decision by the College of Deans.

This thesis will be defended in public on

Monday 29 October 2018 at 16:15.

By

Tobias Ackermann

Born on 27 November 1987

In Jena, Germany

Supervisor

Prof. C. F. Calkhoven

Co-supervisor

Dr. G. Hartleben

Assessment committee

Prof. G. de Haan

Prof. A. J. A. van der Sluis

Prof. P. J. Coffer

Paranymphs

Gertrud Kortman

Tobias Krauße

Britt Sterken

Table of contents

Chapter I

Aim and outline of the thesis

9

Chapter II

Cancer cell metabolism and its regulators

13

Chapter III

Deficiency in mTORC1-controlled C/EBPβ-mRNA

translation improves metabolic health in mice

39

Chapter IV

C/EBPβ-LIP induces tumour metabolic reprogramming

by regulating the let-7/LIN28B circuit

77

Chapter V

Stimulation of the malate aspartate shuttle by

C/EBPβ-LIP causes glycolysis addiction

115

Chapter VI

Discussion and perspectives

143

Appendices Scientific summary

Wetenschappelijke samenvatting

159

161

Lay summaries

165

9

Chapter I

Aim and outline of the thesis

Aim of thesis

The transcription factor C/EBPβ is known to regulate metabolism, cell proliferation and differentiation. Its transactivation activity is largely determined by the ratio between the long isoform C/EBPβ-LAP and short isoform C/EBPβ-LIP. Therefore, it is essential to investigate the functions of the single isoforms to better understand the role of C/EBPβ in the cell. The aim of this thesis was to investigate upstream regulators and downstream effects of C/EBPβ isoforms in physiology and pathophysiology (cancer). The first part of the thesis answers the questions whether mTORC1 signalling stimulates C/EBPβ-LIP expression in vivo and (if yes) whether LIP is an important downstream mediator of physiological effects of mTORC1. We therefore investigated whether deficient C/EBPβ-LIP expression in mice has a similar effect on animal physiology and metabolism as was observed in other animal models with reduced mTORC1 signalling. The second objective of this thesis was to investigate effects of the single C/EBPβ isoforms LAP and LIP on cellular metabolism. Furthermore, by performing transcriptome and proteome analysis of cells expressing either LAP or LIP we aimed to identify downstream mediators of the different C/EBPβ isoforms in the regulation of cellular metabolism. As a third objective, we addressed whether the changes in cell metabolism caused by the different C/EBPβ isoforms impose metabolic vulnerability onto cancer cells that could be exploited for novel cancer therapy options by using C/EBPβ as a biomarker.

10

Outline of the thesis

Chapter II reviews the metabolism of proliferating and cancer cells and the function

of selected oncoproteins and tumour suppressors in the regulation of cellular metabolism. We describe how glucose, glutamine and mitochondrial metabolism contribute to cell growth and proliferation by supporting de novo biosynthesis processes that are altered in cancer cells. In addition, we describe regulators and regulatory mechanisms that control cell and cancer metabolism.

In chapter III, we describe the role of mTORC1-4E-BP signalling in the control of

C/EBPβ-LIP expression. We found that high mTORC1 activity stimulates LIP translation in vivo. Additional in vitro analysis revealed 4E-BPs as critical mTORC1 downstream targets in the regulation of LIP expression. Furthermore, we used a mouse model with deficiency in LIP translation (C/EBPβΔuORF mice) to investigate whether low LIP expression has similar effects on animal metabolism as found in other mouse models with reduced mTORC1 signalling. Mouse models with reduced mTORC1 signalling (e.g. 4E-BP1 overexpressing mice, S6 kinase1 knock-out mice, mTORC1 inhibition by rapamycin or calorie restriction) show a general healthy metabolism and a delay in age-associated phenotypes. We find that LIP deficient C/EBPβΔuORF mice show a lowered body weight, less steatosis and increased glucose and insulin tolerance and thereby resemble the phenotype of other mouse models with decreased mTORC1 signalling or calorie restriction.

In chapter IV, we study the function of C/EBPβ isoforms in cellular metabolism and

analyse the effects of the single isoforms on transcriptome and proteome of the cell. We describe that LIP induces energy production and metabolic reprogramming of the cell. The analysis of the transcriptome and proteome points towards a post-transcriptional mechanism induced by LIP to control cellular metabolism. Further analysis of the omics data and CRISPR/Cas9-based knockout identified LIN28B as important downstream mediator of metabolic functions of LIP. Furthermore, we provide evidence that all members of the LIN28B-regulating microRNA family let-7 are novel C/EBPβ targets that are transcriptionally downregulated by LIP resulting in the de-repression of LIN28B. Further, we present a new mouse model (R26LIP) that uses the endogenous Rosa26 locus to introduce a transgenic LIP expression cassette resulting in the ubiquitous overexpression of LIP in all investigated tissues. LIP

11

overexpressing R26LIP mice show a decreased let-7 and increased LIN28B expression in skin and bone marrow cells, which supports the results of our in vitro analysis. Furthermore, these mice develop a hyperplasia in skin and show a metabolic reprogramming in bone marrow cells thereby resembling the phenotypes of LIN28 overexpressing mice.

In chapter V, we investigate whether LIP induced metabolic changes addict cells to

specific nutrients and metabolic pathways. We see that cells with high LIP expression are sensitive to glucose starvation and inhibition of glycolysis (2-deoxyglucose). In addition, we show that LIP reprograms cytoplasmic NADH utilisation by increasing the malate-aspartate-shuttle and usage of electrons from cytoplasmic NADH for mitochondrial ATP production. We show that the increased NADH oxidising capacity of the induced malate-aspartate-shuttle causes the 2-deoxyglucose mediated cell death in cells with high LIP expression. Inhibition of NADH oxidising cellular processes (malate-aspartate-shuttle and lactate dehydrogenase) reduces the sensitivity of cells to 2-deoxyglucose while a treatment with an NADH oxidizing drug, duroquinone, increases the sensitivity to 2-deoxyglucose in resident cells. Therefore we hypothesize that the NADH/NAD+ ratio is critically involved in mediating 2-deoxyglucose caused cell death in cells with high LIP expression. This survival dependency of cells with high LIP expression on glycolysis was not only shown in cells ectopically expressing LIP but also in human triple negative breast cancer cell lines that have high endogenous LIP levels and therefore could have an implication for cancer biology.

In Chapter VI, I discuss the studies presented in this thesis in a broader context and

I suggest further experiments. In addition, I discuss a possible role of LIP in cell migration and metastasis, and in the regulation of protein translation. Furthermore, I suggest a nutrient level controlled negative feedback loop for LIP expression and a positive signalling feedback loop that involves mTORC1, LIP and let-7.

13

Chapter II

Cancer cell metabolism and its regulators

Tobias Ackermann

, Christine Müller

and Cornelis F. Calkhoven

1

European Research Institute for the Biology of Ageing (ERIBA), University

Medical Centre Groningen, University of Groningen, 9700 AD Groningen,

The Netherlands.

14

Introduction

Metabolic reprogramming is considered an hallmark in cancer development1. In proliferating cells, including cancer cells, metabolic pathways provide building blocks and energy to maintain cellular integrity and enable the cell to grow and multiply. In addition, the metabolism of cancer cells has to adapt to the changing conditions within and around a tumour caused by the growing tumour itself. Almost hundred years ago Otto Warburg discovered that cancer cells show impaired oxygen consumption while they consume high amounts of glucose and secrete lactate. He concluded that aerobic glycolysis is the main metabolic pathway within a cancer cell and the cell intrinsic switch from oxidative metabolism to aerobic glycolysis is now known as the “Warburg effect”2. However, in the past 15 years the development of new tools and methods helped to get a more refined picture of metabolic pathways in cancer cells and their regulation by oncoproteins. This has led to the discovery that, in general, cancer cells show an increase of anabolic and a restriction of catabolic reactions. Furthermore, former pathways of catabolic reactions (glycolysis or tricarboxylic acid cycle (TCA) cycle) are reprogrammed or repurposed and used to produce biomolecule intermediates for anabolic pathways3,4. This review will discuss the prominent features of cancer metabolism and some relevant underlying regulatory mechanisms.

Glucose metabolism in cancer

Glucose is one of the main nutrients for the cell and glycolysis is its central metabolic pathway. During glycolysis, glucose is structurally remodelled, cleaved and oxidised to produce energy and biomolecules for other catabolic or anabolic pathways (Fig 1). In differentiated cells, glycolysis is used to produce pyruvate that fuels the oxidative TCA cycle and respiration to produce ATP in the mitochondria3. Cancer cells show a strong increase in glucose uptake and glycolytic flux, which contributes significantly to the energy (ATP) production of the cell2,5,6. This renders the cell less dependent on the oxidative TCA cycle and respiration for ATP production and allows the cells to use the carbon of glucose for biomass production instead of energy production. Furthermore, many cancer cells limit the activity of the final step of glycolysis by expressing pyruvate kinase isoenzyme M2 (PKM2), a pyruvate kinase variant with low efficiency to phosphoenolpyruvate7. This leads to elevated levels of glycolytic intermediates in the cell and thereby increases the flux into biosynthetic pathways like serine/glycine biosynthesis (S/GB) or pentose-phosphate pathway (PPP) (Fig 1). Both pathways produce key metabolites, including

15

Figure 1: Overview of metabolic pathways in the cell fuelled by glucose (glycolysis in red, pentose phosphate pathway in blue, Serine/Glycine biosynthesis in green)

nicotinamide adenine dinucleotide phosphate hydride (NADPH), ribose-5-phosphate and 5,10-methylenetetrahydrofolate (mTHF), which are required for a diversity of de-novo biosynthesis pathways8. Furthermore, the end product of glycolysis, pyruvate, can enter the mitochondria to generate citrate or aspartate which are used for de-novo lipid and nucleotide biosynthesis in the cell9

Taken together, in a cancer cell the glycolysis is used to distribute glucose into PPP, S/GB, lipid synthesis and de-novo nucleotide biosynthesis. However, the exact distribution depends on genetic driver mutations, available nutrients and the microenvironment of the tumour. Furthermore, recent publications showed that mitochondrial respiration is necessary for the ability to use glucose in anabolic pathways10,11.

II

6-Phosphogluconolactone 6-Phosphogluconate Ribulose-5-phosphate

Ribose-5-phosphate NADPH NADP+ 3-Phosphohydroxypyruvate NADH NAD+ a-Ketoglutarate Glutamate 3-Phosphoserine Serine Glycine THF 5,10-methenyl-THF Nucleotide biosynthesis NADH NAD+ Lactate Acetyl-CoA OxaloacetateCitrate TCA cycle ATP ADP Mitochondrion Cytoplasm Acetyl-CoA Oxaloacetate Protein biosynthesis Aspartate a-Ketoglutarate Glutamate

Aspartate _{Fatty acid}

biosynthesis NADPH

16

In addition to nutrients, many metabolic reactions require co-factors (e.g. NAD[P]+) to reduce or oxidise their substrates. After these co-factors are used for the reaction their regeneration is crucial for all metabolic pathways to maintain their flux. In anaerobic glycolysis, the NAD+ that is used for glucose oxidation is regenerated as part of the pathway, which makes the pathway self-sustainable. Within other pathways like the ribose-5-phosphate biosynthesis, important co-factors like NADPH are generated and made available for glutathione reduction,

Glucose Pyruvate ATP ADP ATP ADP ADP ATP ADP ATP NAD+ NADH NADH NAD+ Lactate 2x

Glucose + 2 ADP 2 Lactate + 2 ATP Anaerobic glycolysis

A Ribose-5-phosphate biosynthesis

and NADPH production

Glucose + ATP + 2 NADP+

Ribose-5-phosphate + ADP + 2 NADPH Glucose Ribose-5-phosphate ATP ADP NADP+ NADPH NADP+ NADPH B Serine biosynthesis Glucose Serine ATP ADP ATP ADP ADP ATP NAD+ NADH 2x NAD+ NADH a-Ketoglutarate Glutamate

Glucose + 2 Glutamate + 4 NAD+ 2 Serine + 2 a-Ketoglutarate + 4 NADH C

Glucose + 2 Glutamate + 2 NAD+

2 Aspartate + 2 a-Ketoglutarate + 2 NADH Aspartate biosynthesis Glucose Aspartate ATP ADP ATP ADP ADP ATP ADP ATP NAD+ NADH 2x ATP ADP a-Ketoglutarate Glutamate D

Figure 2: Overview of metabolic pathways originating from glucose

17

fatty acid synthesis and detoxification reactions in the cell (Fig 2A and B). However, some metabolic pathways like the serine and aspartate biosynthesis need supporting metabolic reactions to regenerate the essential NAD+ co-factor (Fig 2C and D). One possibility source of NAD+ is the reduction of pyruvate into lactate by the enzyme lactate dehydrogenase (LDH) with concomitant oxidation of NADH into NAD+. However, this process only regenerates NAD+ equivalent to the before produced NADH during glycolysis. Therefore, additional pathways have to be engaged to supply sufficient NAD+ to drive for example serine and aspartate biosynthesis. In addition, lactate that is produced by the LDH reaction is secreted by the cell (Warburg effect) and therefore its carbon is lost for biosynthetic processes12. Therefore, to sustain glycolysis and to supply sufficient NAD+ for the various metabolic pathways cancer cells use oxidative phosphorylation in the mitochondria to oxidise NADH into NAD+. However, NAD+/NADH cannot pass the mitochondrial membranes and therefore mitochondria cannot directly re-oxidise cytosolic NADH. Instead cells use substrate cycles that transport electrons derived from oxidation of cytosolic NADH into NAD+ into the mitochondria where the electrons contribute to oxidative respiration and ATP production: the glycerol-phosphate-shuttle (GPS) and the malate-aspartate-shuttle (MAS). Briefly, in the GPS the electrons from cytosolic NADH are transported to mitochondrial FADH that enters the electron transport chain at complex II using glycerol-3-phosphate as intermediate electron carrier, and in the MAS electrons from cytosolic NADH are used to generate malate and transported to mitochondrial NADH that enters the electron transport chain at complex I using malate as intermediate electron carrier. Thus for NAD+ regeneration through the MAS is critical for cell growth and proliferation and it has been shown that certain cancer types like squamous cell lung cancer or diffuse large B-cell lymphoma show amplification of Malate Dehydrogenase (MDH1)14, the enzyme that reduces oxaloacetate to malate in the cytoplasm and thereby oxidises NADH to NAD+. Malate that is transported into the mitochondria is converted in the reverse reaction by mitochondrial MDH2, generating oxaloacetate with concomitant conversion of mitochondrial NAD+ into NADH by complex I in the electron transport chain (ETC) (Fig 3). Consequently, treatment with metformin that inhibits complex 1 has been shown to interfere with tumour growth in vivo13.

In summary, although glycolysis is the core driving metabolic pathway it requires additional interconnected (branching) pathways and mitochondrial respiration to optimally use the metabolic intermediates as carbon source for biomass production (Fig 1 and 2).

18

Glutamine and mitochondrial metabolism

In most differentiated cells the mitochondria generate almost all of the cellular ATP15. Otto Warburg first described that cancer cells use glycolysis for energy production under aerobic conditions and he therefore hypothesised that the metabolic shift is caused by non-functional or damaged mitochondria. However, at that time it was never measured to which extent oxygen consumption by the mitochondria contributes to ATP production in the cell. More recent data show that between 45% to 90% of cellular ATP is coupled to oxygen consumption in cancer cells15. Furthermore, cancer cells with depleted mitochondrial DNA (rho0) have a decreased capacity to form tumours in vivo and glucose starvation experiments show that cells require mitochondrial activity to survive 16,17. Therefore, mitochondria are essential for the survival of cancer cells and their oncogenic capacities.

A key metabolic pathway in the mitochondrion is the tricarboxylic acid (TCA) cycle (Fig 3). In differentiated cells, the TCA cycle oxidizes Acetyl Coenzyme A (Acetyl-CoA) generated from pyruvate or fatty acids to NADH, FADH2 and CO2, of

which NADH and FADH2 are used to produce energy via the electron transport

chain (ETC). Similar to the glycolysis, the TCA cycle connects several catabolic and anabolic pathways, thereby serves as an metabolic hub for biosynthetic reactions and supplies the cell with products for protein modifications 18. For example, citrate that is produced in the TCA cycle is essential for fatty acid synthesis, NADPH production and transport of Acetyl-CoA from the mitochondrion to the cytoplasm and nucleus 9,19. Acetyl-CoA is used by acetyltransferases to modify lysine residues within proteins and thereby change their activity as well as acetylation of histones is essential for epigenetic regulation 20. Another key metabolite is oxaloacetate, which is used for aspartate production and is thereby essential for de novo nucleotide biosynthesis 13. These cataplerotic reactions drain metabolites from the TCA cycle. Therefore, anaplerotic reactions or metabolites are required to refill the cycle 21.

Glutamine is next to glucose a major nutrient of the cell and is used to refill the TCA cycle after cataplerotic reactions have used TCA cycle metabolites 22. Glutamine is deaminized in a stepwise process to glutamate and α-ketoglutarate and the amino groups are used for de novo amino acid production (e.g. serine and aspartate) and nucleotide synthesis 23 (Fig 3). Glutamate is used for glutathione synthesis and thereby is essential for the cellular redox balance. In addition, glutamate is used for protein biosynthesis. α-Ketoglutarate enters the TCA cycle where it is oxidised to oxaloacetate. However, some cancer cells are able to reverse the flux of the TCA cycle and thereby directly generate citrate from α-ketoglutarate,

19

which is used for fatty acid synthesis or NADPH production. Thereby the cancer cells become independent from Acetyl-CoA derived from pyruvate or fatty acid oxidation 22.

The tetrahydrofolate cycle (also known as one-carbon metabolism) is another metabolic pathway that partly takes place in the mitochondrion and particularly this part is associated with cancer development and cancer cell survival

24

. The pathway produces 5,10-Methylenetetrahydrofolate (MTNF) and S-Adenosyl methionine (SAM) which are important substrates for methylation reactions within biosynthetic processes (nucleotide and amino acid synthesis) as well as for protein- and nucleic acid modification (Fig 3). Therefore the availability of SAM and MTNF within the cancer cell is crucial for gene regulation and biosynthetic capacities and a blockage of the one carbon metabolism is currently discussed as a novel cancer therapy 25.

Taken together, a lot of evidence shows that cancer cells in general require mitochondrial function to produce products/substrates for several de novo

Pyruvate Acetyl-CoA TCA cycle ATP ADP Mitochondrion Cytoplasm Acetyl-CoA Oxaloacetate Aspartate a-Ketoglutarate Glutamate Aspartate Oxaloacetate Citrate a-Ketogluterate Malate NAD+ NADH NADH NAD+ NAD+ NADH Glutamate Oxaloacetate _Malate NAD+ NADH a-Keto glutarate Glutamate Glutamine Serine Glycine THF 5,10-methenyl-THF Serine Glycine 10-formyl-THF Formate Formate 10-formyl-THF Glycine THF 5,10-methenyl-THF 5-methyl-THF Homocysteine THF Methionine S-adenosylmethionine

DNA and protein methylation protein acetylation NADP+ NADPH ATP NADPH NADP+ NADP+ NADPH protein biosynthesis 3 Pi Nucleotide biosynthesis Glutamine

Figure 3: Overview of metabolic pathways associated with the mitochondrion (glutaminolysis in red, malate-aspartate-Shuttle in blue, tetrahydrofolate cycle in green)

20

biosynthetic pathways and regulatory protein modifications (e.g. histone acetylation and methylation). However, loss-of-function mutations in some enzymes of the TCA cycle are known to drive cancer development in specific tissues like kidney. Best studied are mutations in fumarate (FDH) and succinate dehydrogenase (SDH) that result in an accumulation of the oncometabolites fumarate and succinate, which induce a hypoxia-like response by stabilizing HIF transcription factors. Furthermore, fumarate and succinate increase DNA methylation in the cell by inhibiting α-ketoglutarate dependent histone and DNA demethylases and thereby alter gene expression 26–28.

Catabolic processes in cancer cells

All cells need catabolic reactions for general maintenance and homeostasis. One type of catabolic processes converts macromolecules to its monomers (e.g. proteins to amino acids), providing substrates for other catabolic or anabolic reactions. Another type of catabolic pathways breaks down and transforms the monomers to new metabolites (e.g. glucose to pyruvate) and thereby in addition produces energy in form of ATP, NADH or NADPH. As explained before cancer cells change the balance between catabolic and anabolic reactions towards anabolic reactions. In addition, cancer cells re-purpose or remodel the flux of catabolic pathways to provide metabolites for biosynthetic pathways. Furthermore, cancer cells have to reduce certain catabolic reactions to ensure that molecules, which just got synthesised, can be used to form more cell mass and are not broken down again. For example, the synthesis of fatty acids needs 8 molecules of ATP and 14 NADPH units and is essential for the synthesis of new membranes upon cell growth and proliferation 29. Fatty acid break down would lead to a waste of energy and a shortage in biomolecules for membrane formation. Therefore, exponential proliferating cancer cells found different ways to limit their fatty acid oxidation capacities. For example in hepatocellular carcinoma it was found that acetyl-coenzyme A carboxylase alpha (ACCα) is able to form a complex with carnitine palmitoyltransferase 1A (CPT1a) and thereby reduces the transport of palmitate into the mitochondria, which inhibits fatty acid oxidation 30. However, under stress conditions (e.g. nutrient limiting conditions or cancer therapy) cancer cells can adapt their metabolism and use fatty acid oxidation for energy production. In these circumstances, the energy production via fatty acid oxidation is essential for cell survival 31,32.

21

Another major catabolic pathway is autophagy, which allows the controlled degradation and recycling of cellular macromolecules and organelles. Autophagy is activated upon cellular stress signals like starvation or oxidative stress and supplies the cell with nutrients (fatty acids, amino acids and glucose) for anabolic reactions, energy production and is required for cell survival. A detailed description of mediators and molecular mechanism of autophagy processes can be found in these reviews 33,34. It is shown that autophagy has tumour suppressive capacities. Beclin 1 and UVRAG, important mediators of autophagy, are found mutated in cancer and a heterozygous loss of Beclin 1 leads to spontaneous development of tumours in mice

35_{. Upon cell transformation and tumour induction, the production and}

accumulation of macromolecules is essential for cell growth and proliferation. Therefore, activation of autophagy would only lead to a waste of energy and resources 36. However, certain hematopoietic cancer cells show high level of autophagy and autophagy becomes important for cellular survival when cancer cells face environmental stress conditions 37,38. Furthermore, autophagy in non-tumour cells can have tumour promoting roles. Autophagy in cancer associated fibroblasts stimulates their secretion of nutrients which support cancer growth 36.

In summary, catabolic pathways like autophagy and lipolysis have different importance and functions during different phases of tumour development and there are differences between cancer cells and cancer supporting non-cancer cells regarding the tumour promoting or inhibiting functions of these pathways. Therefore, a tight regulation of catabolic pathways is necessary to ensure cancer cell proliferation and survival.

Regulation of cancer cell metabolism

Complex organisms need signal transduction networks to coordinate their development and homeostasis. Therefore, different from single cell organisms like yeast, mammalian cells don’t adopt their metabolism and start to proliferate in nutrient rich environments. Proliferation requires external growth factors or proliferation signals in form of signal transduction molecules. However, transformed cells show alterations in signal transduction pathways that renders them independent from external signals1. In this chapter the effects of selected oncogenes and tumour suppressors on cellular metabolism are described.

22

MYC

The MYC transcription factor gene belongs to the family of MYC, MYCL and MYCN proto-oncogenes. The MYC transcription factor proteins are basic helix-loop-helix proteins, which bind to E-box sequences (5’-CACGTG-3’) in the genome and regulates the transcription of target genes by recruitment of histone acetyl-transferases 39. MYC is known to directly regulate the transcription of 10 to 15% of the coding genes in humans and thereby controls cell proliferation, apoptosis, DNA damage pathways and cell metabolism. 40. MYC is known for its potent oncogenic functions and overexpression of MYC by gene amplification or genomic rearrangements as it is found in many different cancer types induces metabolic reprogramming in the cell. MYC directly stimulates the transcription of different key enzymes of glycolysis, glutaminolysis, nucleotide, amino acid and protein biosynthesis to promote cellular growth and proliferation 39,41. Furthermore, it stimulates the expression of other metabolic key regulators like LIN28 (see later) and thereby creates a feed forward

loop towards a cancer metabolic reprogramming in the cell 42. In addition to the stimulation of the metabolic flux, oncogenic MYC couples cell survival to nutrient

Figure 4: Overview of MYC regulated metabolic processes (taken from 45)

23

availability. Cells with high levels of MYC are addicted to glucose and glutamine metabolism and deprivation from these nutrients or inhibition of these pathways results in apoptosis and cell death 43,44.

In summary, MYC is able to directly regulate the expression of key enzymes of metabolic pathways to foster cell growth and proliferation; however, it also addicts the cell to these metabolic changes.

mTORC1

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase found in two complexes – mTORC1 and mTORC2. In this paragraph, we will focus on the function of mTORC1. MTORC1 is activated by Insulin-Phosphoinositide 3-kinase-AKT-Signaling, RAS-mitogen-activated protein kinase (MAPK)-Signalling and nutrient sensing pathways. AKT and MAPK phosphorylate TSC1 and TSC2 (Tuberous sclerosis) which deactivates their GTPase-activating function. As a result of TSC1/2 inhibition the small GTP binding protein RHEB is activated which stimulates mTORC1 activity. In addition RAG, another small GTP binding protein, that is downstream of the nutrient sensing pathway, is able to activate mTORC1 upon high amino acid levels 46. MTORC1 is known to be involved in the regulation of cancer cell survival, proliferation, protein biosynthesis and cellular metabolism 47.

In order to stimulate mRNA translation, mTORC1 phosphorylates the p70S6 kinase (S6K) and eIF4E-binding proteins (4E-BPs) to regulate protein biosynthesis. The S6K is activated upon phosphorylation and phosphorylates eukaryotic initiation factor 4B (eIF4B) and the ribosomal protein S6. Both proteins directly enhance the translation rate in the cell 48,49. 4E-BPs are phosphorylated at multiple sites by mTORC1 and thereby release bound eIF4E. The released eIF4E binds to the 5’cap of mRNAs as a first step in translation initiation and thereby increases protein biosynthesis in the cell 50. Apart from the global increase in protein biosynthesis upon mTORC1 activation, recent publications show that mTORC1 is involved in the differential translation of cellular mRNAs with 5’-terminal oligopyrimidine (TOP), TOP-like motives or small upstream open reading frames (uORFs) in their 5’UTR 51–

53_.

In addition to protein biosynthesis, mTORC1 is able to stimulate lipid biosynthesis via an enhanced processing and activation of SREBP1a, 1c and 2 through S6K1 and CREB regulated transcription coactivator 2 (CRTC2) 54,55. SREBPs are transcription factors that directly regulate key enzymes of lipogenesis and thereby stimulate cellular lipid synthesis. Furthermore, SREBPs are able to stimulate the

24

pentose phosphate pathway and thereby increase ribose production, which contributes to nucleotide biosynthesis. Furthermore, mTORC1 stimulates purine and pyrimidine biosynthetic processes via several mechanisms. mTORC1-S6K1 activates CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydro-orotase), which catalyses the first rate limiting steps in the de novo pyrimidine biosynthesis 56. In order to stimulate purine biosynthesis, mTORC1 regulates the serine/glycine biosynthesis and the mitochondrial tetrahydrofolate (mTHF) pathway via the translation control of the ATF4-mRNA 57. Furthermore, mTORC1 was shown to stimulate the translation of oncogenes like HIF1α and MYC to control glycolysis and glutaminolysis 46,55.

Figure 5: Overview of mTORC1 regulated metabolic processes (taken from 58)

In addition to the increase of anabolism, mTORC1 inhibits catabolic pathways in the cell like autophagy and fatty acid oxidation. mTORC1 directly phosphorylates key mediators of autophagy ULK1 and ATG13 and thereby interferes with initiation of autophagy. In order to reduce fatty acid oxidation, mTORC1 regulates involved mitochondrial transporters and key enzymes via PPARα 58.

25

In summary, mTORC1 is a central regulator of most metabolic pathways within the cell and reprograms them towards a proliferation supporting state. However, most mechanisms are indirect and rely on the regulation of other key regulators like SREBPs, HIF1α or ATF4.

LIN28

LIN28A and LIN28B are the human homologs of the Lin-28 gene that was first

discovered in C.elegans as a heterochronic gene during larval development 59,60. The LIN28A and LIN28B are RNA-binding proteins that are highly expressed during embryogenesis and in certain adult stem and progenitor cells 61. Furthermore, LIN28A/B can drive tumourigenesis and are important for tumour maintenance in certain cancer types 62–65. LIN28A/B are best known for the regulation of the let-7 microRNAs that are considered to act tumour-suppressive. LIN28A/B bind to the stem loop of most of the let-7 precursors and mature let-7 microRNAs and marks them for degradation (LIN28A) or transports them into the nucleolus preventing their maturation (LIN28B). Conversely, let-7 suppresses both LIN28A and -B-mRNAs which creates a bistable regulatory switch. Through inhibition of the let-7 function LIN28A/B increase the expression of let-7 targets in the cell that are connected to metabolism and tumour suppression (more information on let-7 function in let-7 paragraph) 65–67. Another crucial function of LIN28A/B is to directly bind mRNAs of metabolic enzymes, cell cycle regulators and proteins involved in mRNA processing and alter their translation and mRNA stability 63,68,69. Depending on the cellular context, LIN28A/B have different effects on cellular metabolism. In pluripotent stem cells, LIN28A/B bind to mRNAs of glycolytic enzymes, increase their protein levels and stimulate the glycolytic flux but at the same time LIN28A/B decreases the mRNA stability and protein levels of mitochondrial enzymes and thereby inhibit mitochondrial respiration 70. In non-pluripotent cells, LIN28A/B also binds mRNAs encoding for glycolytic and mitochondrial enzymes, but in contrast to stem cells it increases the protein levels also of the mitochondrial enzymes and thereby stimulates both the glycolytic flux and mitochondrial respiration and thus boosts the energy level in the cell 69. Co-factors or involved mediators responsible for these differences in LIN28A/B function between the cell types are so far unknown. Furthermore, LIN28A/B are able to increase the one-carbon and nucleotide metabolism to support cell proliferation and prime cells for reprogramming to pluripotency 70.

26

Figure 6: Overview of LIN28A/B regulated metabolic processes

In summary, LIN28A/B have a unique mechanism to control the cellular metabolism by directly regulating the mRNA stability and translation of key metabolic enzymes to induce a metabolism in the cell that favours biomass production and supports cell proliferation.

TP53

The transcription factor and tumour suppressor TP53 is mutated in more than 50% of all human cancers and a TP53 knock out leads to a very early onset of tumour development in mice 71,72. TP53 functions as a tetramer that binds to the DNA-recognition site 5’-RRRCWWGYYY-3’(R=purine, W= adenosine or thymidine, Y= pyrimidine) and is known as a master regulator of cell cycle, senescence, apoptosis and DNA repair. More recent work shows that TP53 can also regulate the tumour microenvironment, invasion capacity and cellular metabolism73,74.

TP53 induces metabolic changes that could be summarized as a “reverse” Warburg effect contrasting the oncogene-induced metabolic alterations described

LIN28A/B

let-7

Glucose

Glycolysis

OxPhos

Nucleotide metabolism

and methylation

II

27

earlier. For example, TP53 downregulates glycolysis by inhibiting the expression of the glucose transporters GLUT1 and GLUT4 and by upregulation of the fructose-2,5-bisphosphatase TIGAR 75,76. Dephosphorylation of fructose-2,5-bisphosphate results in reduced activity of Phosphofructokinase (PFK) and therefore in a decrease in the glycolytic flux. The reduced glycolytic flux caused by TIGAR directs glucose-6-phosphate into the pentose glucose-6-phosphate pathway (PPP) for the production of NADPH and precursors for nucleotide biosynthesis to support ROS defence and DNA repair

45,77

. However, a single study showed that TP53 binds and inhibits G6PDH, a rate limiting enzyme in PPP, which limit the flux of the PPP 78. Future studies have to clarify if either inhibition or usage of PPP is induced by P53 or under which conditions TP53 increases or inhibits PPP. Furthermore, TP53 stimulates other catabolic pathways within the cell. It upregulates the mRNA expression of the key enzymes of the glutaminolysis (GLS2), several proteins involved in β-oxidation (CPT1c, MCD, LPIN and PANK1) and proteins required for the electron transport chain (SCO2) to increase mitochondrial energy production after shutting down glycolysis 45. To cope with the increased ROS levels caused by increased respiration, TP53 elevates the ROS defence in the cell by upregulating Sestrins (SESN1 and 2) and the synthesis of GSH via glutaminolysis 79,80. In addition, it increases the mitochondrial integrity via p53R2 and Sco2 to limit the ROS production in the cell

81,82_{. Another catabolic pathway stimulated by TP53 is autophagy. TP53 increases the}

expression of initiators of autophagy - DRAM, ULK1, cathepsin D and the β1 and β2 subunits of AMPK. In addition, other TP53 targets like PUMA, BAX and BNIP3 have pro-autophagic functions. However, p53-/- cells show increased autophagy levels and experiments revealed that cytoplasmic localisation of TP53 inhibits autophagy. Therefore, the function of TP53 in autophagy is rather complex and needs more studies to clarify the relationship between TP53 and autophagy 83.

To summarize, the tumour suppressor TP53 controls the cellular metabolism in an “anti-Warburg manner”. It directly inhibits the expression of metabolic enzymes and regulates other regulators of cellular metabolism to repress anabolic pathways (e.g. glycolysis) and to increase catabolic pathways (e.g. fatty acid oxidation).

28

Figure 7: Overview of TP53 regulated metabolic processes (adapted from 45)

Let-7 family of microRNAs

Let-7 is a family of functionally redundant and tumour suppressive microRNAs. MicroRNAs are small non-coding RNAs which bind to their specific seed-sequences in the 3’-untranslated region (3’UTR) of target mRNAs and thereby decrease the translation rate and mRNA-stability 84. Targets of let-7 miRNAs are oncogenes (RAS,

MYC, LIN28A/B) and cell cycle regulators (CDK1/6 and CYCLINA2/D1/D2) 85. In humans, the let-7 family consists of the nine members let-7a, b, c, d, e, f, g, i, and mir-98. Some let-7 family members are encoded together in one cluster (e.g. let-7a-1/f-1/d cluster), other members are organised in a cluster with other miRNAs (e.g. miR-100/let-7a-2/miR-125b-1 cluster), and some are encoded alone within an intron (e.g. let-7g) 86. All family members are encoded on different chromosomes and some let-7 family members are encoded multiple times in the genome (e.g. let-7a has three copies in humans). Therefore, their high copy number within the genome and redundancy in targets is seen as a strong obstacle in tumour initialization 87,88.

?

29

Let-7 miRNAs are known to regulate the cell metabolism and energy homeostasis via the control of oncogenes LIN28A/B and MYC (see paragraphs on LIN28A/B and MYC) as well as by directly targeting the mRNAs of key enzymes of glycolytic and fat metabolism anabolic pathways (G6PDH – PPP, PDHK1 – pyruvate usage in TCA cycle, FASN – fatty acid synthesis) 85,89,90. Furthermore, let-7 miRNAs target mRNAs coding for proteins in the Insulin receptor-signalling cascade and in the amino acid sensing pathway, thereby repressing mTORC1 signalling and increasing autophagy in the cell 91,92.

In summary, let-7 miRNAs regulate cellular metabolism by a broad variety of mediators and thereby induce an anti-Warburg metabolism in the cell.

Figure 8: Overview of let-7 regulated metabolic processes

LIN28A/B

_let-7

Glucose

Glycolysis

Fatty acid biosynthesis

mTORC1

FASN _G6PDH

30

Conclusion

The knowledge about metabolism of cancer cells got more refined since Warburg’s first observation. Many mediators and molecular changes affected by cancer metabolic reprogramming were identified in the past years. In this chapter, general mechanisms and mediators which are deregulated in many cancer cells and cancer types were described. However, there are huge differences in the metabolic flux of certain metabolic pathways (e.g. lipolysis, pentose phosphate pathway and autophagy) between different cancer types. Even cancer cells within a tumour have to adopt their metabolic fluxes to changing environmental and nutrient conditions caused by its own growth and progression which could result in tumour cells with different metabolic adaptations depending on the location within the tumour 93. Therefore, more work is needed to investigate whether a cancer cell is able to adapt to the new conditions, whether new acquired mutations are necessary for the adaptation or if another pre-existing clone is responsible for the progression and survival of the tumour under for example nutrient or oxygen limiting conditions.

Many studies focus on the effects of cancer metabolic reprogramming on cell proliferation and survival. However, recent studies show that the cellular metabolism can have an influence on metastatic capacity of cancer cells as well. In breast cancer models for example, the metabolism of the parent tumour cells determines in which organs the cancer cells can form a secondary tumour. Oxidative tumour cells mainly form metastases in the lung and bone marrow, while parent cancer cells with high glycolytic flux and oxygen consumption are able to form metastases in bone marrow, lung and liver . In addition, cancer cells isolated from different metastatic sites show distinct metabolic profiles compared to the parental tumour and other metastatic sites. This indicates that the metabolism of tumour cells has to adapt to the new environment upon metastasis formation or that different clones within one tumour show different metabolic phenotypes which enables them to survive in different metastatic niches 94,95.

Most tumours consist of several clones of cancer cells and of different populations with more stem- or differentiated-like cell character each probably characterised by different metabolic networks. Therefore, cancer cells cannot be treated as a homogenous population. In addition, cancer cells interact with their surrounding cells and the host organism. Therefore, more efforts are needed to investigate how the microenvironment can influence metabolism in cancer cells.

31

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37

39

Chapter III

Deficiency

in

mTORC1-controlled

C/EBPβ-mRNA

translation improves metabolic health in mice

Laura M. Zidek

, Tobias Ackermann

, Götz Hartleben

, Sabrina Eichwald

,

Gertrud Kortman

, Michael Kiehntopf

, Achim Leutz

, Nahum Sonenberg

,

Zhao-Qi Wang

, Julia von Maltzahn

, Christine Müller

and Cornelis F.

Calkhoven

EMBO reports (2015), Volume 16, Issue 8, DOI: 10.15252/embr.201439837

Leibniz Institute for Age Research - Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany.

2

Department of Clinical Chemistry and Laboratory Diagnostics, University Hospital Jena, Erlanger Allee 101, D-07747 Jena.

3 _{Max Delbrück Center for Molecular Medicine, Robert Rössle Strasse 10, D-13092}

Berlin, Germany.

4

Department of Biochemistry & Goodman Cancer Research Center, McGill University, Montreal, Quebec H3A 1A3, Canada.

5

European Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, NL-9713 AV Groningen, the Netherlands.

40

Abstract

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1-signalling are the mRNA-translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E-binding proteins (4E-BPs). However, little is known about vertebrate mRNAs that are specifically controlled by mTORC1-signalling and are engaged in regulating mTORC1-associated physiology. Here we show that translation of the CCAAT/Enhancer Binding Protein beta (C/EBPβ) mRNA into the C/EBPβ-LIP isoform is suppressed in response to mTORC1-inhibition either through pharmacological treatment or calorie restriction. Our data indicate that the function of 4E-BPs is required for suppression of LIP. Intriguingly, mice lacking the cis-regulatory upstream open reading frame (uORF) in the C/EBPβ-mRNA, which is required for mTORC1-stimulated translation into C/EBPβ-LIP, display an improved metabolic phenotype with features also found under calorie restriction. Thus, our data suggest that translational adjustment of C/EBPβ-isoform expression is one of the key processes that direct metabolic adaptation in response to changes in mTORC1 activity.

41

Introduction

C/EBPβ is a transcriptional regulator with a broad tissue expression including liver and adipose tissue (http://www.genecards.org). It controls genes related to glucose and fat metabolism as well as other cellular processes 1,2. The Cebpb gene is intronless and from its mRNA three different protein isoforms are expressed through usage of alternative translation initiation sites (Fig EV1A). The isoforms LAP* and LAP (Liver Activating Protein) are transcriptional activators that consist of transactivation domains and a DNA-binding domain. The truncated isoform LIP (Liver Inhibitory Protein) lacks the N-terminal transactivation domains but still possesses the DNA-binding domain. LIP can therefore act as a competitive inhibitor for LAP*/LAP function 3. However, LIP may also have additional and distinct functions. Hence, the ratio between LAP and LIP is crucial for the biological functions elicited by C/EBPβ. Translation from both the LAP* and LAP AUG-codons is achieved by regular translation initiation, although translation into LAP* is often weaker since this AUG-codon lacks a Kozak-consensus sequence required for efficient recognition by the ribosome 4,5. Expression of LIP from a distal initiation codon depends on a cis-regulatory uORF located in the 5’UTR of the C/EBPβ-mRNA. The limited size of the uORF allows the small ribosomal subunit to remain attached to the mRNA after translation termination and to resume scanning along the mRNA. After reloading of the ribosomal complex with initiator tRNA translation of LIP from the downstream initiation codon can be re-initiated. Mutation of the uORF consequently results in diminished LIP expression 4,5 (see also schematic representation in Fig EV1A).

Reducing signalling through mTORC1 by pharmacological treatment, mutations, restricted calorie intake or low protein:carbohydrate macronutrient ratio enhances metabolic health and increases life span in many species up to mammals. On the contrary, hyper-activation of mTORC1 is believed to promote metabolic disorders resulting from overfeeding such as diabetes 6-15. Our earlier studies pointed to an involvement of mTORC1 signalling in the regulation of C/EBPβ-LIP expression, since mTORC1 inhibition by rapamycin reduced LIP expression in a uORF-dependent manner 4,16.

Here we show that interventions that cause a reduction in mTORC1 signalling also decrease the translation of the C/EBPβ-LIP isoform. Genetic elimination of the mTORC1-sensitive uORF of the C/EBPβ mRNA in mice similarly results in a reduction of the LIP isoform 5. Our data show that mice with this mutation display an improved metabolic phenotype, including reduced fat