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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Ackermann, T. (2018). C/EBPβ isoforms and the regulation of metabolism: A fine balance between health and disease. University of Groningen.

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Chapter VI

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The transcription factor CCAAT/ Enhancer Binding Protein beta (C/EBPβ) is expressed in every tissue of the human body (https://www.proteinatlas.org/ENSG00000172216-CEBPB/tissue) and it regulates the expression of genes that are involved in cellular processes like proliferation, differentiation, senescence, apoptosis and metabolism. The CEBPB-mRNA is translated into three different C/EBPβ protein isoforms. The two long isoforms C/EBPβ-LAP* and -LAP (liver-enriched transcriptional activator protein) are transcriptional activators while the N-terminally truncated isoform C/EBPβ-LIP (liver-enriched inhibitory protein) lacks the transactivation domains but retains the dimerization and DNA-binding domain and therefore competes with LAP/LAP* for DNA-binding sides. Consequently, the ratio between the LAP and LIP isoforms determines the transcriptional activity of C/EBPβ in the cell. All C/EBPβ isoforms are derived from the same mRNA though a translation control mechanism. Translation of LAP/LAP* is achieved through regular translation initiation using alternative AUG-codons. Translation into LIP depends on a small, upstream open reading frame (uORF) that is out of frame in regard to the main reading frame. A mutation of the initiation codon of the uORF (ΔuORF) results in deficient C/EBPβ-LIP expression 1,2.

C/EBPβ isoform ratio as downstream mediator of mTORC1 signalling

The kinase complex mechanistic target of rapamycin complex 1 (mTORC1) regulates cellular growth and metabolism in response to nutrients, energy state and growth. mTORC1 is deregulated in a wide variety of cancers 3. Pharmacological inhibition of mTORC1 or genetic alterations that mimic reduced mTORC1 signalling (S6 kinase knock out mice and 4E-BP overexpression mice) results in increased health- and lifespan in mice 4. Furthermore, mTORC1 signalling is reduced in response to calorie restriction and it is suggested that this is essential for the health and lifespan extending effects of caloric restriction in mice and other organisms. One of the major downstream effects of activated mTORC1 signalling is the stimulation of global translation rate. In addition, mTORC1 controls the specific translation of mRNAs with cis-regulatory elements in their mRNA-leader sequences, including 5’ terminal oligo pyrimidine (TOP) tracts, TOP-like sequences and uORFs 5.

The expression of C/EBPβ-LIP depends on an uORF in the CEBPB-mRNA leader and inhibition of mTORC1 by rapamycin reduces the expression of LIP in cell culture experiments 1. In chapter III, we show that C/EBPβ-LIP expression is controlled by mTORC1 signalling in vivo. Furthermore, mice with a mutation in the initiation codon of the uORF within the CEBPB-mRNA (C/EBPβΔuORF) are deficient

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in LIP expression and show a phenotype that resembles the phenotype of mice under calorie restriction, rapamycin treatment or of genetically modified mouse models with reduced mTORC1 signalling. LIP-deficiency results in an attenuation of age related lipid accumulation in white adipose tissue (WAT) and other tissues, increased fatty acid oxidation and an improved glucose tolerance and insulin sensitivity. Further experiments (which are not part of this thesis) show that LIP deficient female C/EBPβΔuORF mice have a reduced tumour incidence and an increased health and lifespan 6. However, it still has to be shown how important the expression of LIP is for effects caused by mTORC1 signalling. To answer this question the LIP deficient C/EBPβΔuORF mice should be crossed with mouse models that show increased mTORC1 signalling like the TSC1 knockout mice. On the other hand, it would be interesting to investigate if overexpression of LIP compromises the effects of calorie restriction and rapamycin treatment on animal health and lifespan. On the cellular level, one publication already shows that a maintained LIP expression can reverse anti-proliferation effects of rapamycin treatment in lymphoma cells 7. In addition, it is shown that the differential translation of mRNAs mediated by the 4E-BP pathway (not S6K pathway) is responsible for the proliferation stimulatory function of mTORC18 and we found that LIP is downstream of 4E-BPs (chapter III,

Figure 1). Taken together, the data indicate that LIP might have an important

proliferation stimulating function within the mTORC1 signalling pathway and we speculate that low LIP levels facilitate the positive effects of caloric restriction on health and lifespan. Furthermore, we hypothesize that experimentally or (patho-) physiologically high LIP expression drives ageing and age related diseases.

Further, it is shown that LIP expression can be regulated independently of mTORC1 by other pathways which regulate translation in the cell 1,9,10. A mouse model with a heterogeneous deletion of eukaryotic initiation factor 6 (eIF6+/-) has reduced levels of LIP and shows similar phenotypes as LIP deficient C/EBPβΔuORF mice 11. In addition, other mouse model like Myc+/- mice show life and health span extensions and Myc is a known regulator of eIF4e which regulates the expression of LIP 12,13. Therefore, it would be interesting to investigate whether the eIF6+/- and

Myc+/- mouse models depend on LIP deficiency to prolong health and lifespan and whether LIP is a general signalling hub for the regulation of health and lifespan in mammals.

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C/EBPβ-LIP reprograms cellular metabolism through regulation of the let-7/LIN28B circuit

In chapter III, we show that a deficiency in C/EBPβ-LIP expression alters whole body metabolism towards increased fatty acid oxidation. One of the experiments revealed that overexpression of LIP in mouse hepatoma cells (Hepa1-6) decreases their ability to oxidise palmitate (Chapter III, Fig. 4B). This experiment gives a first indication that C/EBPβ isoforms differently regulate cellular metabolism. Therefore, in connection with the lower cancer incidence in the female LIP-deficient C/EBPβΔuORF mice and the increased cancer incidence in mice with LIP overexpression, we speculate that LIP might contribute to cancer development by controlling cellular metabolism similar to what is described in chapter II for other oncoproteins 6,14.

In chapter IV, we show that LIP overexpression increases the glycolytic flux, oxygen consumption and energy level (ATP, NADH) in the cell while LIP deficiency reduces these parameters (chapter IV, Figure 1). This shows that LIP is able to induce energy production and metabolic flux in the cell, which is one key aspect of cancer metabolic reprogramming. The analysis of transcriptome and proteome data identified the RNA binding protein LIN28B and its targets as potential mediators of the LIP induced changes. Knockout of LIN28B abrogates the effects of LIP on cellular metabolism proving that LIN28B and its targets are critically involved. We discovered that several members of the microRNA family let-7, well-known inhibitors of LIN28B expression, are transcriptionally repressed by LIP indicating that downregulation of let-7 is responsible for the LIN28B upregulation in response to LIP. LIN28B in turn inhibits let-7 maturation as part of a negative regulatory circuit. The balance between let-7 and LIN28B is essential for proper regulation of proliferation, cellular metabolism and differentiation of the cell and in the context of cancer let-7 is considered to act tumour suppressive and LIN28B oncogenic. Therefore, LIN28B and let-7 are tightly regulated during development and tissue regeneration and a gain of LIN28B or loss of let-7 is reported in many different cancer types 15. Furthermore, overexpression of LIN28B in liver or intestine leads to cancer development 16,17. The data shown in chapter IV indicates that LIP is an important regulator of the LIN28B/let-7 circuit by shifting it towards the LIN28B site. In LIP overexpressing R26LIP mice the investigated tissues bone marrow and skin show alterations in cellular metabolism and hyperplasia that are reminiscent to phenotypes observed in LIN28 overexpressing mice 18. Despite these similarities, future experiments have to show whether LIP requires the upregulation of LIN28B or downregulation of let-7 microRNAs to induce the metabolic changes and induction

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of hyperplasia in vivo. Furthermore, the question is whether LIP-mediated increase in cancer incidence is accompanied and requires upregulation of LIN28B. In addition, HER2-positive or triple negative breast cancer cells have low levels of let-7 and high levels of LIN28A/B 19. Notably, LIP is also highly expressed in these types of breast cancer and LIP overexpression leads to breast cancer development 20–22. This raises the question if and to which extent LIP is contributing to the low let-7 levels and increased LIN28A/B expression in the breast tumours and if LIP activation precedes the repression of let-7 and activation of LIN28A/B during tumour development 23.

Both C/EBPβ and LIN28A/B play important roles in the skin. LIN28A overexpression or LIP overexpression (R26LIP) in mice results in a hyperplasia of the skin, and knockout of C/EBPβ in keratinocytes leads to resistance to 7,12 dimethyl-benz[a]anthracene (DMBA) induced skin tumourigenesis 24,25. Although the role of the single C/EBPβ isoforms were not addressed in this skin cancer model these published data together with our results indicate that LIP might have a role in skin cancer development and the regulation of let-7 and LIN28A/B could be an important downstream mediator.

In summary, our results and the literature strongly suggest to further examine the role and connection between LIP, let-7 and LIN28B in normal physiology and cancer biology. Furthermore, it would be interesting to investigate the expression of LIP (or the LIP/LAP ratio) in other tissues in relation to their normal differentiation state and during cancer development and metastasis in relation to patient survival.

Metabolic addiction of cells with high LIP expression

In chapter II it is described that oncogenes like MYC couple the cellular survival to the metabolic changes they induce. If nutrients become limiting, high MYC levels result in cancer cell death while untransformed cells just reduce growth and proliferation or change their energy source 26. In chapter IV, we described that LIP increases glucose metabolism and thereby increases energy production in the cell. Therefore, we asked if LIP cells are addicted to the supply of glucose.

Depletion of glucose or inhibition of glycolysis by 2-deoxyglucose (2-DG) reduces proliferation and induces apoptosis in several cell lines with high LIP expression levels. Moreover, we show that LIP reprograms the usage of cytoplasmic NADH by inducing the malate-aspartate-shuttle (MAS). This is known to support proliferation by allowing the usage of pyruvate in biosynthetic processes 27,28. We discovered that

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inhibition of glycolysis with 2-DG in cells that express high levels of LIP result in low NADH/NAD+ ratios, which is associated with reduced proliferation and increased apoptosis. That low NADH/NAD+ ratios are causative for cell death was shown by inhibition of NADH consuming processes (malate-aspartate-shuttle and lactate dehydrogenase) following 2-DG treatment, which reduces cell death in high LIP expressing cells. Furthermore, cell lines that are resistant to 2-DG treatment show synthetic lethality for additional treatment with duroquinone, a drug that decreases the NADH/NAD+ ratio. This again, suggests a critical involvement of the NAD+/NADH ratio in 2-DG mediated cell death. However, further work is needed to investigate the mechanism by which a low NADH/NAD+ ratio leads to induction of apoptosis.

Metabolome analysis has to prove if the LIP induced changes alter the usage of glucose and pyruvate in biosynthetic pathways and whether other metabolic pathways are affected by LIP. It would be interesting to investigate whether increased NADH/NAD+ ratios caused by LIP result in an increase in Hydroxyglutarate. Similar to the “oncometabolite” R-2-Hydroxyglutarate, S-2-Hydroxyglutarate inhibits dioxygenases, which increase the methylation of lysine number 9 in histone 3 and thereby interferes with expression of differentiation associated genes 29. In addition, it is known that some cancer types induce lipolysis and beta-oxidation upon glucose depletion to survive 30,31. In chapter III we show that LIP can block fatty acid (FA) oxidation. Therefore, next to the changed NADH usage the blocked FA oxidation might be another contributing factor to the LIP induced sensitivity to glucose limitation.

In summary, the data from chapter IV and V together show that LIP increases metabolic flux and energy production in the cell, and couples the cellular survival to this metabolic reprogramming. This strongly suggests that LIP alters the metabolism in a similar manner as other oncoproteins like MYC or RAS and that these changes are likely to contribute to the observed increased cancer incidence in the LIP overexpressing mice.

Effects of LIP beyond metabolism

In chapter IV we use transcriptome and proteome analysis to identify the mediators of LIP induced metabolic alterations. These unbiased approaches have generated more data than used in our manuscript that may help to understand the functions of LIP. Functional clustering analysis (DAVID) of mRNAs and proteins that are downregulated by LIP shows enrichment for cell adhesion and polysaccharide

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binding or are associated with collagen subtypes and the extracellular matrix. All these functions are related to cell-cell interactions and a downregulation might lead to an increase in cell migration. Therefore, we performed in vitro scratch assays showing that LIP-deficient C/EBPβΔuORF mouse embryonic fibroblasts (MEF) migrate significant slower than wt control MEFs (Figure 1A and B). Furthermore, LIP overexpression increases the migration rate of Cebpb knockout MEFs, while LAP overexpression has no effect (Figure 1C and D). This indicates that in addition to the regulation of cellular metabolism LIP regulates cell migration. In that context, it is intriguing that highly invasive breast cancer cells show high LIP expression. Furthermore, different studies suggested a migration stimulatory function for LIP and identified single LIP downstream targets that are involved in the migratory phenotype of LIP 20,21,32. We identified that LIP is able to repress whole clusters with functions in cell-cell interactions and extracellular matrix production that are likely to contribute to these phenotypes. Further in vivo studies are required to investigate whether LIP increases cancer cell invasion and metastasis formation.

Recent studies describe the involvement of cellular metabolism in metastatic capacities of cancer cells. In breast cancer cells, it is shown that cells with high glycolytic flux and mitochondrial respiration metastasise to bone marrow, lung and liver while cells with high mitochondrial respiration only metastasise to the lung and bone marrow. In addition, knockdown of pyruvate dehydrogenase kinase 1 (PDK1), which is found highly expressed in liver metastasis of breast cancer cells, in the parent tumour impairs the formation of liver metastasis 33. Therefore, cellular metabolism determines the site of metastasis formation in addition to its functions in proliferation and cell survival and LIP could be an important regulator that combines the regulation of proliferation, metabolism and metastasis.

In chapter IV, we describe an imbalance between transcriptome and proteome and we suggest that LIN28B is responsible for this imbalance through post-transcriptional regulation of its target mRNAs. However, LIN28B targets represent only a quarter of the proteins whose expression changed upon LIP overexpression (255 of 1069). Therefore, other factors must be involved in the post-transcriptional alterations induced by LIP. The functional clustering analysis of the proteome data shows that proteins that are involved in mRNA stability and processing as well as translation factors activity are enriched. We do not know whether the mRNAs of these proteins are elevated by LIP in the transcriptome data (similar to LIN28B) or if these changes are secondary like the LIN28B targets.

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Figure1. C/EBPβ-LIP regulates cell migration

A western blot for wt and C/EBPβΔuORF

mouse embryonic fibroblasts (MEF), B relative migration rate of wt and C/EBPβΔuORF

MEF (n=3)

C western blot for EV, LIP and LAP overexpressing C/EBPβ knock out (CBko) MEF D relative migration rate of EV control, LIP and LAP overexpressing CBko MEF (n=3)

Therefore, it would be important to more deeply analyse the transcriptome and proteome data. Moreover, it is known that microRNAs can interfere with mRNA stability as well as protein synthesis rate. LIP might repress other microRNAs in addition to let-7 and thereby contribute to the differences between transcriptome and proteome and explain upregulation of proteins.

C/EBPβ-LIP – a link in several feedback loops?

In this thesis, we show that C/EBPβ-LIP is a downstream mediator of mTORC1 signalling (chapter III) and that LIP induces the expression of LIN28B through the repression of let-7 miRNAs (chapter IV). Others showed that let-7 regulates mTORC1 signalling 34. This indicates that LIP might be a new link within a positive feedback loop between LIN28 and mTORC1 signalling where LIP induces LIN28B,

C D A LAP LIP αb-tubulin wt MEF ΔDuORF B LIP/LAP 1.9 0.53 EV LIP LAP LAP LIP βb-actin wt uORF 0.0 0.5 1.0 1.5

relative migration rate (scratch assay)

*** EV LIP LAP 0.0 0.5 1.0 1.5 2.0 2.5

relative migration rate (scratch assay)

***

CBko MEF

VI

β α

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which in turn suppresses let-7 and consequently induces mTORC1, which again induces LIP (Figure 2). LIN28B has been shown to be regulated mainly by feedback loops involving transcription factors like MYC or NF-kB 35,36.

Figure 2. Proposed model of mTORC1/LIP/let-7 feedback loop

Furthermore, LIP is produced by a translation control mechanism and its expression is not directly correlated with Cebpb-mRNA levels1. Altogether, LIP represses let-7 microRNAs, which affect stability and translation efficency of mRNAs like LIN28B, which is a RNA-binding protein that increases the translation efficency of target mRNAs. Here, three out of five regulatory events within the feedback loop are post-transcriptional. Therefore, we suggest that LIP forms a positive feedback loop which uncouples protein expression from mRNA levels (Figure 3).

mTORC1

C/EBPβ-LIP

let-7

LIN28B

let-7

mTORC1

C/EBPβ-LIP

let-7

LIN28B

let-7

Figure 3. Summary of moleculare processes within mTORC1/LIP/let-7 feedback loop

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Next to this signalling feedback loop, our data suggest that LIP is part of a feedback loop maintaining metabolic homeostasis. High nutrient levels increase LIP expression in the cell via insulin and mTORC1 signalling (chapter III). LIP induces glucose metabolism and energy production of the cell (chapter IV) and renders the cell depedent on glucose (chapter V). Under healthy homeostasis, elevated metabolism rates result in reduction of nutrient levels and reduced insulin and mTORC1 signalling (feeding-fasting-cycle) and thereby to a reduction in LIP expression (chapter III) (Figure 4A). In certain cancer types, mTORC1 signalling and insulin sig nalling are highly deregulated and independent of external signals 5,37. Therefore, the proposed negative feedback on nutrient level will not be able to reduce cellular LIP level upon nutrient deprivation. In chapter V, we show that glucose deprivation of cells with constitutively high level of LIP leads to reduced cell proliferation and the induction of apoptosis (Figure 4B). This might serve as a tumour supressive mechanism in vivo and eliminate cells which acquired mutation that make them independent of growth factor signalling. Further, it suggests the blockage of glycolysis as a potential treatment for cancer cells with high LIP expression.

In summary, this thesis focuses on the regulation and the downstream mediators of the C/EBPβ-LIP transcription factor protein isoform. Altogether we identified mTORC1 as a major upstream regulator of C/EBPβ-LIP in vivo; we show that LIP expression is reduced in calorie restricted mice and that experimental LIP-deficiency results in calorie restriction type of metabolic alterations; we reveal that LIP regulates whole body and cellular metabolism and addicts cells to the

LIP-nutrient level C/EBPβ-LIP energy production nutrient demand nutrient level C/EBPβ-LIP energy production nutrient demand apoptosis

A

B

Figure 4. Proposed model for LIP centred nutrient level based feedback loop in physiology (A) and its alteration in cancer (B)

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induced metabolic alterations; we identified the let-7/LIN28B circuit as downstream mediators of LIP. Our data suggests that LIP is involved in cellular signalling and metabolic feedback loops. In addition, our results are in line with other published data indicating that LIP has important functions in tissue maintenance and stem cell regulation, cancer development and metastasis formation 14,23,38,39. Therefore, more work is needed to further understand the importance of the C/EBPβ-LAP and -LIP isoforms in health and disease and as potential therapeutic target for cancer therapy or in metabolic diseases.

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