Functions of the C/EBPβ isoforms in breast cancer
Sterken, Britt
DOI:
10.33612/diss.172465560
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Sterken, B. (2021). Functions of the C/EBPβ isoforms in breast cancer. University of Groningen. https://doi.org/10.33612/diss.172465560
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Chapter VI
The transcription factor C/EBPβ is a member of the CCAAT/enhancer binding protein family and is expressed in all tissues in the body (https://www.proteinatlas.org/ENSG00000172216-CEBPB). It is a single exon gene, but comes in different protein isoforms (LAP*, LAP and LIP) that are alternatively translated from a single CEBPB-mRNA. Translation into LAP and LAP* requires recognition of regular translation initiation sites. However, translation into LIP requires the translation of a uORF within the CEBPB-mRNA, and genetic ablation of the uORF results in a LIP-deficiency 1. A large number of studies have demonstrated that C/EBPβ is a mediator of proliferation and differentiation in a variety of tissues 2–13. Its protein isoforms LAP and LIP are widely expressed, but structurally and functionally different, as LIP lacks the N-terminal transactivation domains but competes for the same DNA binding sites as LAP. Therefore, the transcriptional activity of C/EBPβ is determined by the ratio between the LIP and LAP isoforms.
Previously, we established a translational control model for C/EBPβ, where the genetic ablation of the uORF resulted in systemic LIP-deficiency in the mouse 12. The phenotypes of these mice resemble the phenotypes of mice upon caloric restriction or mTORC1 inhibition, showing an increased health-and lifespan and a reduced overall tumour incidence 4,14. In addition, knock-in of LIP in at the Cebpb locus results in a reduced lifespan and increased overall tumour incidence, proposing that LIP has oncogenic functions 13. Integration of LIP under the whey acidic protein (WAP) promotor results in mammary epithelial specific overexpression of LIP and development of hyperplasias and less frequently neoplasias 15. C/EBPβ is a key transcriptional regulator in mammary gland development, its absence resulting in deficient mammary gland development (reviewed in 16,17). Given the low mutation rate, copy number variation and
mRNA overexpression of CEBPB in breast cancer
(https://cancer.sanger.ac.uk/cosmic), likely, the translational control into its different isoforms is involved in mediating the oncogenic functions in cancer development. In addition, a variety of studies have linked growth signalling to a favoured translation into the LIP isoform 18–20. Moreover, Gene Set Enrichment Analysis (GSEA) identified enriched gene expression patterns in triple negative
breast cancer samples that share common promotor motifs for C/EBPβ 21. However, the exact role of C/EBPβ in TNBC remains so far unknown. In this thesis, we show that LAP inhibits cell migration and invasion of cell lines derived from TNBC. Moreover, we show a regulation of microenvironmental genes by C/EBPβ in BT20 cells and in specific a regulation of extracellular matrix genes. We show that endogenous variation of the LIP/LAP ratio in basal-like murine tumours does not correlate with tumour specific survival. In addition, we generated a translational control model for C/EBPα to study the role of the isoforms on metabolism and oncogenesis.
C/EBPβ as a potential regulator of breast cancer metastasis
Previously, it was revealed that high LIP expression in human breast cancer samples negatively correlates with estrogen receptor (ER) and progesterone (PR) expression, and correlates with proliferation, aneuploidy and poor prognosis 22. Later on, studies demonstrated that LIP is highly expressed in HER2+ cancer and that LIP overexpressing tumours showed resistance to treatment with the ERBB2 inhibitor trastuzumab 18. In chapter III, we demonstrated that cell lines derived from TNBC display a high expression of LIP and a high LIP/LAP ratio. We observe that lowering the LIP/LAP ratio by overexpression of LAP reduces the migration and invasion of TNBC cells, and that C/EBPβ regulates migration-related and microenvironment migration-related genes in BT20 cells. Therefore, the C/EBPβ isoforms are potentially an interesting therapeutic target in treating cancer cell dissemination.
Breast cancer metastasis can be summarised as a series of events including the migration, invasion, extravasation, intravasation and colonisation at distant target sites 23. The metastatic process involves a complex interplay between the cancer cells and its surrounding microenvironment. During cancer progression, tumour cells often become plastic and undergo morphological and phenotypical conversions, which are often induced by an Epithelial to Mesenchymal Transition (EMT) 24. The EMT is characterised by a downregulation of epithelial markers
and a gain of mesenchymal markers, and is a highly conserved and reversible process during which immotile epithelial cells give rise to polarised migrating mesenchymal cells. Previous studies have demonstrated a regulation of EMT genes by C/EBPβ, however, evidence is largely contradictory. One study provides evidence that LAP induces the EMT and transforms MCF10a cells 25, whereas the majority of studies reveals oncogenic functions for LIP (reviewed in Chapter II). Moreover, multiple studies have investigated the role of C/EBPβ in the EMT, but not the isoform specific functions. For instance, it was revealed that loss of C/EBPβ is required for TGFβ-induced EMT 26. In this study, we revealed a downregulation of E-cadherin and an upregulation of N-cadherin and Vimentin upon LIP overexpression in MCF10a cells, however, other mesenchymal markers were downregulated. Moreover, Gene Set Enrichment Analysis (GSEA) revealed a mixed regulation of EMT genes, indicating that C/EBPβ and its isoforms regulate EMT genes, but not in a one-directional manner. Whereas during the EMT in embryonic development epithelial cells lose their epithelial origin and become fully mesenchymal, cancer cells are often characterised by a “ hybrid” E/M phenotype. Also referred to as a partial EMT, this intermediate status between epithelial and mesenchymal phenotypes is thought to enhance invasiveness as reviewed in 27. Often, this partial EMT is characterised by collective cell migration, where the cells with epithelial phenotypes remain attached to other cells and mesenchymal leader cells interact with the ECM. Therefore, more detailed analysis of the role of the C/EBPβ isoforms in the EMT is required. Analysis of the RNA-seq data obtained from BT20 CEBPB-knockout cells revealed a downregulation of pro-oncogenic extracellular matrix genes and remodellers. Previously it was revealed that C/EBPβ drives the malignancy of mammary epithelial cells by promoting the expression of α5 integrin transcription, putting C/EBPβ forward as a regulator of integrin-dependent stromal–epithelial interactions 28. α5 integrin and its ligand fibronectin (FN) are frequently upregulated in metastatic tumours, and the study showed that inhibition of ligation of FN to α5 integrin reverted the phenotype towards non-malignant tissue. Here, we observe a downregulation of Tenascin C (TNC), Collagen 5A1 (COL5A1), Fibronectin 1 (FN1), and Thrombospondin 1 (THBS1)
(all ECM organisation) and MMP2, Lysyl Oxidase Like 1 (LOXL1) and Procollagen C-Endopeptidase Enhancer 2 (PCOLCE2) (all ECM remodelling) upon CEBPB-ko in BT20 cells. Moreover, a majority of those genes demonstrated to be upregulated by LAP and not LIP, indicating that potentially LAP promotes metastasis by contributing to remodelling of and interaction with the ECM during breast cancer progression. To understand how exactly LIP and LAP regulate cell migration and invasion, its interactions with the ECM and deposition and remodelling of the ECM should be analysed. Furthermore, expression patterns of the isoforms in different types, stages, and regions within the tumours could contribute to understanding the isoform-specific roles in breast cancer progression. One major challenge in this field is the lack of a LIP-specific antibody. Whereas LAP can LIP-specifically be targeted by the use of a N-terminal specific antibody, LIP is identical to the C-N-terminal part of LAP and even though multiple groups have put effort into generating a LIP specific antibody, it so far remained unsuccessful. Therefore, studying the expression of LIP and LAP in different types and stages of human breast tumours, and modelling of LIP and LAP in GEMM models for breast cancer, is required to establish the functions of the isoforms in breast cancer progression and metastasis.
In addition to the regulation of extracellular matrix related genes, DAVID functional clustering analysis revealed an upregulation of MHCI and MHCII and antigen presentation related genes upon CEBPB-knockout in BT20 cells. As LIP is the predominantly expressed C/EBPβ isoform in the parental BT20 cell line, one could hypothesise that LIP downregulates MHCI and MHCII molecules. Immune-escape strategies are commonly found in transformed cells, which include the loss/reduction of MHCI and MHCII expression to avoid T-cell recognition 29. Therefore, our RNA-seq data propose a potential additional role in the evasion of immune cells for the C/EBPβ-isoforms.
We previously reported that LIP reprograms cellular metabolism by inducing the stem cell factor Lin28b via repression of let-7 microRNAs. In order for cancer cells to establish uncontrolled proliferation it requires adjustments in cellular metabolism. Under aerobic conditions, cells use glucose in the glycolysis which
gets processed to pyruvate, which in the mitochondria is processed to carbon dioxide. Upon anaerobic conditions, little of this pyruvate is dispatched to the mitochondria and lactate production is favoured. Otto Warburg demonstrated that cancer cells often acquire a different metabolism known as “aerobic glycolysis”, during which the cancer cells, even upon aerobic conditions, limit their metabolism largely to glycolysis, even though oxygen is available 30,31. More recent research revealed also an important role of mitochondrial metabolism – the TCA cycle and oxidative Phosphorylation (OXPHOS) – in cancer growth, with tumour cells displaying increased flux through mitochondrial pathways 32. More recently, it was revealed that metastatic breast cancer cell lines display an enhanced metabolic activity, by upregulating both the glycolysis and OXPHOS (reviewed in 33). Moreover, derivation of metastatic breast cancer cells with preferential colonisation sites revealed different metabolic profiles, with cells prone to liver metastasis upregulating glycolysis, whereas cells prone to bone and lung metastasis favour OXPHOS 34. Interestingly, we find that LIP boosts both basal and maximal extracellular acidification rate (ECAR) as a measure for glycolysis and oxygen consumption rate (OCR) as a measure for OXPHOS, and that LAP only boosts the OCR. Furthermore, we demonstrate that this metabolic induction by LIP is dependent on RNA-binding protein Lin28b 5, which is known to regulate the translation of mRNAs for glycolytic and mitochondrial enzymes35,36. Therefore, further studies should investigate how potential metabolic alterations induced by LIP/LAP might influence where cancer cells colonise, but also whether potential regulation of translation into LIP/LAP might be a way to adjust or adapt to metastasising tumour cells’ needs. Furthermore, the regulation of metabolic enzymes by LIP was demonstrated to be regulated in a post-translational fashion. Therefore, in addition to our existing RNA-seq data obtained from BT20 CEBPB-knockout cells, proteomics might reveal a potential additional regulation level mediated by C/EBPβ in TNBC cells and could aid the search for targets that promote breast cancer progression.
Previously we have successfully reduced the LIP/LAP ratio in vitro in a screen using the ENZO library of FDA-approved drugs, and for future experiments it will be interesting to test these drugs in vivo and study their effects on breast cancer metastasis 37. The role of the LIP and LAP isoforms in cell invasion, their partial regulation of the EMT and their effect on the microenvironment have to
be further researched in in vitro and in vivo models to reveal whether the LIP/LAP ratio can be put forward as a therapeutic target in breast cancer.
Generation of C/EBPβ-isoform specific Genetically Engineered Mouse Models (GEMMs) for breast cancer
The lack of proper models to investigate the role of LIP and LAP in breast tumourigenesis is one major limitation in the field of C/EBPβ. Whereas studies previously have used allografts to maintain intact immune systems, it often does not reflect the mutational heterogeneity and intratumoural clonal heterogeneity found in mammary tumours. Given this tumour complexity, the use of genetically engineered mouse models (GEMMs) has become one of the most accurate ways to model the disease, its de novo tumourigenesis allowing analysis of cause-effect relations, rather than correlations 38. In addition, given the regulation of antigen presentation-related and extracellular matrix-like genes as described in Chapter III, modelling of the role of the C/EBPβ-isoforms in an intact breast cancer microenvironment seems essential.
For these reasons, we chose to model LIP and LAP in a GEMM model for breast cancer. Given the high mutational rate of p53 in basal-like breast cancer ( ~80 % of all basal-like cancers), we chose to make use of the existing Wap-Cre;Trp53F/F (WP) model 39–41, where we selectively overexpressed LIP-luciferase and LAP-luciferase. Based on data obtained from MMECs isolated from the WP-LIP and WP-LAP cohorts, we concluded that LIP and LAP expression is silenced by DNA-methylation, which corresponded to a lack of luminescence signal in the majority of WP-LIP and WP-LAP tumours. Whereas in previous studies c-Myc and Cas9 have been successfully integrated and overexpressed from the Col1a1 locus in the same mouse model 42, similar to our study, the overexpression of esterogen receptor (ER) α is silenced over time 43. Given previous reports that obtaining ERα positive mammary tumours in mice has proven to be difficult (most GEMMs developing ERα -negative tumours and the few cases that do develop ER+ tumours do not respond to estrogen or endocrine therapy) 44, this
indicates that transgene expression of genes that do not favour tumour growth are prone to epigenetic silencing in this mouse model. Even though LIP proves to promote hyperplasia formation in the mammary gland and predisposes to oncogenesis 13,15, this study indicates that perhaps only strong tumour drivers are successfully expressed in this p53-deficient breast cancer model.
Analysis of tumours obtained from WP, WP-LIP and WP-LAP cohorts revealed consistent expression of C/EBPβ in all tumours and a high variability in LIP/LAP ratios amongst the tumours. With an average LIP/LAP ratio of ~1 in the tumours from all cohorts, this ratio proved to be higher than in normal differentiated tissues 45, but lower than we observed in cell lines derived from TNBC (LIP:LAP ratios of ~3). This corresponds to a relatively low LIP/LAP ratio observed in the murine basal-like 4T1 cell line 26. Even though LIP transgene expression increases hyperproliferation in the mammary gland 15, the observed differences in LIP/LAP ratios between human and mouse basal-like breast cancer might involve different C/EBPβ isoform functions between the two species. The variation in the LIP/LAP ratio in the p53-deficient mouse mammary tumours allowed for correlational studies between LIP/LAP ratio and survival. Even though we found no correlation between LIP/LAP ratio and mammary tumour-specific survival, more drastic changes in LIP/LAP ratio might be required to influence tumour development, as is reported and observed in our CebpbΔuORF model 45. Given the relatively high expression of C/EBPβ in the mammary tumours obtained from our cohorts, loss-of-function studies might reveal more about the functions of the C/EBPβ-isoforms. Our existing CebpbΔuORF model drastically decreases the
LIP/LAP ratio systemically, and might be a suitable model to study the loss of function of LIP in mammary tumourigenesis. Therefore, we propose crossing of the CebpbΔuORF model with the existing Wap-Cre:Trp53F/F model, followed by surgical resection and transplantation of tumours into WT recipient mice to prevent potential systemic effects within the CebpbΔuORF mice. In addition, to
model the LIP overexpression, our R26-LIP mice can be crossed with the Wap-Cre:Trp53F/F model, as LIP transgene expression from the R26 locus has been proven not to be epigenetically silenced 5.
Generation of the C/EBPα
-∆uORF mouse
Previously, we published that mutation of the CEBPB-uORF results in a LIP-deficiency which increases health-and lifespan in mice that display a reduced overall tumour incidence, resembling the phenotype of mice kept under caloric restriction or mTORC1 inhibition4,12,14. Both C/EBPα and C/EBPβ translation is controlled via mTORC1 signalling, with mTORC1 signalling favouring translation into the truncated isoforms LIP and p30 respectively (reviewed in Chapter II, Chapter V and published in 1). To investigate the importance of uORF-mediated translational control of C/EBPα in vivo, we generated the C/EBPα-∆uORF mouse model. Both C/EBPα and C/EBPβ are established regulators of metabolism, proliferation, and differentiation. In particular, C/EBPα is known for mediating adipocyte differentiation46–49. To test the involvement of the C/EBPα uORF-mediated translational control in the regulation of energy homeostasis, mice will be subjected to a series of tests to measure insulin sensitivity, glucose tolerance, leptin sensitivity, and energy expenditure and body weight and composition. Cebpa-/- mice die a few hours after birth due to hypoglycemia, and show defects in lung and liver development and a lack of mature neutrophils and eosinophils. Crossing of heterozygous C/EBPα-∆uORF mice reveals offspring generation at regular Mendelian ratios, indicating that p30 is dispensible for normal development, or potentially another truncated isoform of CEBPs takes over during development – even though preliminary results show no upregulation of C/EBPβ-LIP upon depletion of p30. Moreover, preliminary data obtained from young cohorts (3-6 months of age) reveals no clear differences in mouse weight, which might show larger differences in weight at higher age as described for the C/EBPβ-∆uORF mice4.
In addition to the established functions in fat differentiation and metabolism, C/EBPα is a major regulator of proliferation. The p42 isoform is a well-known tumour suppressor and CEBPA is found mutated in ~15% of the acute myeloid leukemia (AML) patients, often harbouring nonsense mutations in the coding sequence specific for p42, thereby abolishing p42 expression while preserving p30 50,51. In addition, in vivo models harbouring mutations resulting in p42-deficiency
result in the development of AML 52. Next to its role in AML, C/EBPα is known to display decreased levels in solid tumours of breast, liver, skin, head and neck, lung, gastric, cervical, endometrial and liposarcoma. 53–61. Interestingly, recently C/EBPα has been reported to influence the metastatic process by regulating the EMT, where it was shown that C/EBPα is downregulated via TGFβ signalling directly via SMAD3 62. Overexpression of C/EBPα, after EMT induction by stimulation with TGFβ, induces the Mesenchymal to Epithelial Transition (MET) in breast epithelial cells, and they therefore speculate that C/EBPα is an epithelial gatekeeper and that re-expression of C/EBPα might be favourable in the prevention of breast cancer metastasis. Even though the authors do not elaborate on isoform-specific functions, there are certain parallels with our study described in Chapter III, where overexpression of LIP in untransformed mammary epithelial cells (partially) induces the EMT, hypothesising that this might inhibit the function of LAP. The redundant functions described earlier for C/EBPα and C/EBPβ might explain why LIP induces the EMT, by either inhibiting the epithelial functions of LAP and p42. However, the overexpression of LAP in TNBC cell lines (data not shown) does not result in a regulation of EMT genes, and similarly, Lourenço et al do not show the regulation of EMT markers by C/EBPα in breast cancer models 62. Possibly, the CEBP interaction partners that are required for the regulation of EMT genes, are present in untransformed epithelial cells but not cancer cells. To test the involvement of the C/EBPα and C/EBPβ isoforms in EMT, the p30-deficient C/EBPα-∆uORF model could be crossed with existing breast cancer mouse models, to study whether breast cancer cells without p30 are able to undergo EMT. Moreover, one could test whether the short isoforms of C/EBPα (p30) and C/EBPβ (LIP) inhibit the function of C/EBPα (p42) and its epithelial induction, by making a double mutant with p30 and LIP deficiency, and crossing this with an existing metastatic mouse model. Potentially, this regulation of C/EBPα and C/EBPβ influencing breast cancer progression by (partly) regulating the EMT, is regulated at later stages by translational control mediated by oncogenes, such as mTORC1, or c-MYC which is characteristically amplified in TNBC.
Overall conclusion
In this thesis, we show that LAP overexpression decreases migration and invasion of triple-negative breast cancer cells and LIP promotes migration in breast epithelial cells, which we find associated with a mixed EMT signature. We propose a role for the LIP/LAP ratio in the regulation of breast cancer cell migration and ECM remodelling, two key characteristics that are associated with the aggressive phenotype of TNBC cells (Chapter III). We followed up to study the effects of LIP and LAP in in vivo using GEMMs for basal-like breast cancer in Chapter IV, where we showed that the exogenous LIP and LAP expression are silenced by methylation, and that endogenous variation in LIP/LAP ratio does not correlate to tumour onset and survival. In chapter V, we designed a C/EBPα-∆uORF model to study the effects of p30 ablation on health, ageing and cancer development, which potentially can be used to study the EMT in breast cancer. Our studies propose that both LIP and LAP isoforms might contribute to breast tumour development by altering cell invasion, metabolism and the microenvironment. However, future GEMM models for breast cancer containing uORF mutations and LIP transgene expression will be required to investigate how LIP and LAP contribute to tumour progression and metastasis.
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