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 II
Translational regulation and functions of the
C/EBPβ-isoforms in mammary gland development, breast cancer and
the cancer microenvironment
C/EBPβ – translational control and functions
The transcription factor CCAAT/enhancer binding protein β (C/EBPβ) regulates biological processes including metabolism, immunity, differentiation and proliferation in multiple tissues, and the deregulated expression of the C/EBPβ-isoforms has been found to contribute to ageing and cancer1–9. C/EBPβ belongs
to the C/EBP family of transcription factors that consists of 6 members: C/EBPα, C/EBPβ, C/EBPγ, C/EBP∂, C/EBPε and C/EBPζ. All members share highly conserved leucine zipper dimerisation and basic DNA-binding domains in the C-terminus allowing for functional homo- and heterodimer formation between the C/EBP family members10. C/EBPβ is expressed in three different isoforms with
distinct transcriptional functions: C/EBPβ-LAP* (Liver-enriched transcriptional Activator Protein *, also known as LAP1), C/EBPβ-LAP (Liver-enriched transcriptional Activator Protein, also known as LAP2) and C/EBPβ-LIP (Liver-enriched Inhibitory Protein). The LAP* and LAP isoforms contain N-terminal transactivation domains and multiple studies demonstrated that they are activators of gene transcription11–13. The short isoform LIP lacks the
transactivation domains and competes for the same DNA binding sites as LAP, thereby suppressing LAP-mediated gene activation in a competitive manner13,14.
The deregulated expression of the C/EBPβ-isoforms has been found to be involved in different biological processes such as acute-phase response, mammary gland lactation and tumourigenesis, liver regeneration, cell metabolism, oncogenesis and health and lifespan5–9,15–17. Therefore, a balanced
expression of the C/EBPβ-isoforms is essential for the functioning of C/EBPβ as a transcriptional regulator of a variety of biological processes.
Previously, we demonstrated that the expression of the different C/EBPβ isoforms depends on an evolutionary conserved upstream open reading frame (uORF) in the CEBPB-mRNA 14. uORFs are translational control elements that regulate
expression of downstream open reading frames. uORFs have been found in 5’ leader sequences of different species, with one or more uORFs are found in 44% of mouse and 49% of human transcripts18. Because eukaryotic ribosomes usually
sites, uORFs are able to influence the translation of a downstream coding sequence. Translation initiation is characterised by a series of different steps. Briefly, it comprises of the formation of a 48S initiation complex, in which the P-site of the 40S ribosomal subunit pairs Met-tRNAiMETwith the initiation codon,
and the joining of the 48S complex with the 60S ribosomal subunit. In order for the small 40S ribosomal subunit to be recruited to the 5’ cap, it has to bind the ternary complex that consists of eIF2, Methionine loaded initiator tRNA (Met-tRNAiMET) and GTP, thereby forming the 43S pre-initiation complex (43S PIC),
which includes additional eIFs 3, 5, 1 and 1A. Even though the 43S complex could bind to unstructured 5’ UTRs, in general the binding to the 5’ cap requires eIF4F and eIF4B or eIF4H, which unwind secondary structures in the mRNA thereby allowing for ribosomal attachment and scanning along the mRNA. eIF4F is comprised of the cap-binding protein eIF4E, the helicase eIF4A and the bridging factor eIF4G. The amount of available eIF4E is a limiting factor in the translation initiation, as 4E-Binding Proteins (4E-BPs) retain eIF4E which is only released after the phosphorylation of 4E-BPs. After the binding to the capped 5′ proximal region of mRNA, the 43S complex scans from the 5’ to 3’ direction until it reaches a methionine codon in appropriate Kozak-context. After the initiation codon recognition and 48S assembly, eIF2-bound GTP is hydrolysed, initiation factors are released, and the 60S ribosomal subunit joins, after which translation initiates19,20. Upon translation termination, ribosomes are separated and recycled.
However, if a short uORF is recognised and translated, the small 40S ribosomal subunit can remain attached to the mRNA after termination at the uORF stop codon, resume scanning, acquire a new eIF2-TC, and re-initiate translation at downstream open reading frames. For instance, expression of the well-studied yeast gene Gcn-4 or the eukaryotic orthologue ATF-4 is regulated by uORFs in the corresponding mRNAs, where the translation initiation and reinitiation of the uORFs more proximal to the Gcn-4 and ATF-4 coding sequences inhibit translation of Gcn-4 and ATF421,22. Similarly, the differential isoform expression
of C/EBPβ is dependent on the recognition of distinct initiation sites. C/EBPβ is expressed in three different isoforms that can be translated simultaneously from a single CEBPB-mRNA: C/EBPβ-LAP*, C/EBPβ-LAP and C/EBPβ-LIP. The
translation of the CEBPB-mRNA into LAP* (figure 1a) and LAP (figure 1b) is regulated via leaky scanning and regular translation initiation, although the translation into LAP* is usually weak due to the AUG start codon lacking an optimal Kozak sequence14,23,24. Translation into the small inhibitory isoform LIP
(figure 1c) requires recognition and translation of the CEBPB-uORF5,14. The
recognition and translation initiation at the uORF on the CEBPB-mRNA results in translation of the uORF peptide. After translation termination at the uORF stop codon the small ribosomal subunit remains attached to the mRNA and scanning until it reinitiates at the LIP start codon. The genetic elimination of the uORF results in a LIP-deficiency5,9. For efficient reinitiation the reloading of
post-termination ribosomes with new Met-tRNAiMET is required. Due to the close
proximity (4 nucleotides) of the LAP initiation codon to the uORF stop codon the 40s ribosomal subunit is not yet reloaded with new Met-tRNAiMET when it
passes the LAP initiation site. Therefore,translation cannot reinitiate at the LAP initiation site. However, upon scanning to the downstream LIP initiation site Met-tRNAiMET reloading can occur and translation reinitiation can take place25.
Similarly to the translational regulation of C/EBPβ, the C/EBPα isoform expression is regulated in a uORF-dependent manner (further described in Chapter V).
Previously, we have demonstrated that the translation into the C/EBPβ isoforms is dependent on RNA-dependent protein kinase (PKR) and mammalian target of rapamycin complex 1 (mTORC1) signalling, through regulation of eIF2α and eIF4E respectively14. PKR affects the translation initiation via the
phosphorylation-induced inactivation of eIF2α. Constitutive activation of eIF2α by inhibition of its phosphorylation was demonstrated to favour translation into LIP. Moreover, mTORC1 signalling specifically induces the expression of LIP through regulation of 4E Binding-Proteins (4E-BP) and eIF4E9,14. Activation of
mTORC1 resulting in the phosphorylation of 4E-binding proteins (4E-BPs) and subsequent release of eIF4E promotes the LIP expression in a uORF-dependent manner26. Later studies demonstrated that genetic ablation of the
tumour incidence, resembling the phenotype of mice upon caloric restriction or inhibition of mTORC19,27–30. Therefore, it is thought that the translational control
of CEBPB plays an important role in determining the health-and lifespan and oncogenesis. This review will discuss the role of C/EBPβ and its isoforms in oncogenesis with a specific focus on breast cancer and the microenvironment. A large number of studies have demonstrated that C/EBPβ is a regulator of proliferation, differentiation, metabolism, immunity and senescence in a variety of tissues. In 1995, two independent groups generated a Cebpb-/- mouse, where both groups observed similar phenotypes in the immune system. Here, it was reported that Cebpb-/- mice display lymphoproliferative and myeloproliferative disorders, high circulating interleukin (IL)-6 levels and increased susceptibility to systemic Candida Albicans infection31. Moreover, Cebpb-/- mice displayed an
increased susceptibility to infections of salmonella and listeria, impaired Granulocyte Colony-Stimulating Factor (C-CSF) induction in macrophages, and an impairment of tumour cytotoxicity exhibited by the Cebpb-/- macrophages32.
In addition to the immunological phenotype of the Cebpb-/- mice, livers from
Cebpb-/- mice showed a proliferative deficit after partial hepatectomy, which was
associated with a prolonged period of hypoglycaemia33. In 1997, it was
demonstrated that Cebpb-/- females are sterile due to ovary-intrinsic defects. This study revealed that C/EBPβ is essential for granulosa cell differentiation in response to Luteinizing hormone (LH)3. Shortly after, C/EBPβ was characterised
as a key mediator in mammary gland development, with mammary glands from
Cebpb-/- displaying reduced growth and branching of developing ducts34.Besides
the Cebpb-/- model, different mouse models have been generated to study the C/EBPβ isoform specific functions in vivo. A mouse model containing a mono-or biallelic replacement of the wt Cebpb gene locus with a locus only expressing LIP displayed increased tumour incidence in mice and in particular a predisposition to the development of B-cell Non-Hodgkin lymphomas and histiocytic carcinomas6. Further analysis of lymphomas from wt mice and
heterozygous LIP transgene mice revealed a downregulation of cytokine and chemokine biosynthesis, Toll–like receptor pathways, and the innate immune
Figure 1: Translation initiation and re-initiation from the CEBPB-mRNA. a) Translation of C/EBPβ-LAP* by translation initiation. Ribosomes scan the mRNA from the 5’-cap to the LAP*-AUG to initiate translation. LAP* is often weakly expressed because it has no Kozak sequence. b) Translation of C/EBPβ-LAP by translation initiation. Ribosomes scan the mRNA from the 5’-cap
Poly A
cap 5’ UTR uORF 3’ UTR
LAP* LAP LIP
LAP* protein
Poly A
cap 5’ UTR uORF 3’ UTR
LAP* LAP LIP
LAP protein
Poly A
cap 5’ UTR uORF 3’ UTR
LAP* LAP LIP
LIP protein
skipped initiation reinitiation termination
AUG initiation codon stop codon
ternary coplex carrying initiator tRNA
60S ribosomal subunit 40S ribosomal subunit a. Translation initiation at the LAP*-AUG
b. Translation initiation at the LAP-AUG
omitting the LAP*-AUG and uORF-AUG in order to initiate translation at the LAP-AUG which has a suboptimal Kozak sequence. c) Translation of C/EBPβ-LIP by translation reinitiation. Ribosomes scan the mRNA from the 5’-cap omitting the LAP*-AUG to initiate the translation from the uORF-AUG. After producing a small uORF peptide, the translation is terminated and the post-termination ribosomes restart scanning the mRNA omitting the nearby (4nt downstream) LAP-AUG. During the re-scanning, the post-termination ribosomes are reloaded by a new initiator tRNA (Met-tRNAiMET) required for translation reinitiation at the LIP-AUG. Adapted from Zaini et
al 201725.
response in the lymphomas isolated from heterozygous LIP mice. Moreover, markers for activation of M1 macrophages were decreased in the lymphomas and markers for activation of M2 macrophages were upregulated, indicating the development of a more pro-tumourigenic environment in LIP knockin (ki) lymphomas. Our lab created LIP-deficient CebpbΔuORF mice through genetic
ablation of the uORF in the Cebpb gene5. Partial hepatectomy of livers of CebpbΔuORF mice revealed a reduced liver regeneration pace and demonstrated that
LIP is facilitating S-phase entry of liver cells, showing that the reduction in liver regeneration after hepatectomy in Cebpb-/- mice is due to the lack of LIP expression. Furthermore, the female CebpbΔuORF mice display an increased
lifespan and reduced overall tumour incidence7, showing a reduction in tumour
load (number of different tumour types per mouse) and tumour spread (total number of differently located tumours per mouse irrespective of the tumour type). Moreover, in a recent study we showed that LIP induces the expression of the oncofetal RNA-binding protein LIN28B, which induces glycolysis and mitochondrial respiration reminiscent of cancer metabolism and thereby possibly induces a tumour prone state8. In addition, transgenic mice
overexpressing LIP display a thickened epidermis, which resembles the phenotype of skin from mice with transgene Lin28a overexpression35. Previously,
the ectopic expression of C/EBPβ-LAP in skin was shown to reduce proliferation and induce differentiation and upregulation of differentiation markers keratin 1 and keratin 1036. Similarly, it was shown that C/EBPα and C/EBPβ are
co-expressed in keratinocytes that are undergoing terminal differentiation, whereas mice lacking expression of C/EBPα and C/EBPβ show increased proliferation and
decreased differentiation in the skin37. Conversely, the deletion of Cebpb in the
skin protects against chemically induced skin tumourigenesis. Treatment of
Cebpb-/- mice with 7,12 dimethylbenz[a]anthracene (DMBA) revealed that Cebpb-/- mice were resistant to the development of skin tumours and displayed
increased DMBA-induced apoptosis in epidermal keratinocytes38. In addition, it
was shown that C/EBPβ is also essential for the development of oncogenic HA-RAS tumours, since Cebpb deficiency resulted in rapid tumour regression and upregulation of p53 and apoptosis39. Moreover, it was shown that targeting
C/EBPβ in skin tumourigenesis has potential synergistic effects when combined with alkylating chemotherapeutics, by promoting apoptosis in tumours after DNA damage40. All in all, the above-discussed models highlight the involvement
of C/EBPβ and its isoforms in a variety of cellular functions such as differentiation, proliferation, immunity and oncogenesis in different tissues, and put C/EBPβ forward as a potential therapeutic target in cancer.
The functions of C/EBPβ in mammary gland development
As described earlier, the analysis of mammary glands from Cebpb-/- mice revealed an impaired lobuloalveolar development34, resembling mammary gland
phenotypes from mouse knockout (KO) models for the Progesterone Receptor (PR), prolactin receptor, Stat5ab, cyclin D1, Id2, p27, and Receptor activator of nuclear factor kappa-Β ligand (RANKL) (reviewed in 41,42), putting C/EBPβ
forward as one of the essential mediators of mammary gland development. The transplantation of Cebpb-/- mouse mammary epithelial cells (MMECs) into the cleared mammary fat pads of nude mice revealed that the defect in development is cell intrinsic34. Isolation of Cebpb-/- MMECs followed by mammosphere
formation assays revealed an impaired repopulation ability, and mammary glands from Cebpb-/- mice contained fewer mammary stem cells and less luminal progenitors. Moreover, Cebpb-/- mammary stem cells isolated from the luminal compartment displayed increased expression of basal markers43. 3D-cultured
mammary epithelial cells isolated from Cebpb-/- mice did not respond to lactogenic hormones and the expression of β-casein and Whey Acidic Protein (WAP), markers for mammary epithelial cell differentiation, was largely absent44.
β-casein is a classical marker for differentiation in the mammary gland and its expression is regulated by a complex regulatory region in its promotor. Previously, it was demonstrated that C/EBPβ binding sites at the β-casein gene are essential for hormone-induced β-casein transcription in mammary epithelial cells45. Later, it was revealed that C/EBPβ-LAP and –LIP isoform expression
changes significantly during mammary gland development, and that the isoform ratio is regulated by glucocorticoids. It was reported that in rats LIP is upregulated a 100-fold during pregnancy when epithelial proliferation is high. Moreover, treatment with glucocorticoids reduced the expression of LIP. Based on these results a model was proposed in which glucocorticoid treatment reduces LIP expression thereby relieving repression of β-casein transcription16. In addition, it
was found that mammary glands from Cebpb-/- mice showed an increased PR expression and altered localisation, which correlated with decreased proliferation of the mammary epithelial cells46. Another study proposed a potential link
between LIP expression and translational control in the mammary gland, as high expression of LIP was found in tumours derived from Balb/c mice in comparison to pre-neoplastic tissue, and was positively correlated to the expression of eukaryotic initiation factor eIF2α17. These studies suggested that LIP is involved
in mammary epithelial cell proliferation and likely in breast cancer. Moreover, these results pointed towards a possible link between translational control of C/EBPβ and breast cancer tumourigenesis, where later it was confirmed that high eIF2α activity stimulates translation reinitiation at the LIP start codon thereby promoting its translation14. All in all, these studies point out that C/EBPβ is
required for the development and luminal differentiation in the mammary gland and that this is largely cell intrinsically regulated. Moreover, these studies imply a role of LIP in mammary cell proliferation and oncogenesis.
The functions of C/EBPβ in breast cancer
Mutations in the CEBPB gene rarely contribute to development of solid or hematopoietic cancers47. Data from the COSMIC database
(https://cancer.sanger.ac.uk/cosmic) show that from 2575 breast cancer tumours sequenced, 3 tumours contained mutations in the CEBPB gene (0,12%), and out
of 1492 tumours 43 contained Copy Number Variations for CEBPB (2,88%) (figure 2a, b). Gene expression data revealed that 93 out of 1104 breast tumours displayed elevated expression of CEBPB-mRNA (8,24%) (figure 2c). As mentioned before, an increased expression of the protein isoform C/EBPβ-LIP in transformed mammary tissue vs pre-neoplastic tissue was reported, and later high LIP expression in human breast cancer samples was correlated with absence of estrogen (ER) and progesterone (PR) receptors, high proliferative index, poor differentiation and aneuploidy17,48. Several in vivo studies provided evidence that
high systemic LIP/LAP ratios predispose to cancer6,7. Moreover, the selective
overexpression of the short inhibitory isoform LIP in the mammary gland results in hyperplasias and less frequently in neoplasias49 (figure 2d). Given that the CEBPB-mRNA is translated into different protein isoforms, it is likely that the
translational control of C/EBPβ influences breast tumourigenesis.
Apart from the in vivo studies showing that LIP predisposes to overall and mammary gland oncogenesis, isoform-specific functions were demonstrated to be essential in the progression and treatment of breast cancer. In 2006, it was shown that C/EBPβ-LAP is essential in mediating the transforming growth factor beta (TGF-β) induced cytostatic response, by promoting expression of the cycle inhibitor p15INK4b and repression of c-Myc. Analysis of metastatic breast cancer pleural fluid samples revealed that this TGF-β induced cytostatic response is absent in half of the patients and that this was due to an upregulation of LIP, while the overexpression of LAP restored this cytostatic response50. In the human
mammary epithelial cell line MCF10a EGF receptor (EGFR) signalling was found to increase the binding activity of CUG triplet repeat RNA binding protein 1 (CUG-BP1) to the C/EBPβ mRNA, thereby favouring translation into the LIP-isoform51. Moreover, it was shown that the Erb-B2 Receptor Tyrosine Kinase 2
(ERBB2, also known as HER2) controls LIP translation via CUGBP1, and thereby ERBB2 averts TGF-β mediated growth arrest by switching the isoform ratio towards a higher LIP/LAP ratio. In addition, in an in vivo tumour model using LIP-overexpressing MCF10A (RAS/ERBB2) cells, the LIP overexpressing tumours were resistant to treatment with the ERRB2 inhibitor trastuzumab, and
therefore a role for LIP in conferring trastuzumab resistance in HER2+ breast cancers was proposed52. In addition, the LIP/LAP ratio was upregulated by
Insulin Growth Factor Receptor 1 (IGF-R1) signalling in an EGF-independent manner in mammary epithelial cells, and the increased LIP levels promoted cell survival by suppression of anoikis, thereby putting LIP forward as a potential pro-metastatic factor by promoting cell survival after detachment from surrounding extracellular matrix53. These studies illustrate how translation into LIP is often
favoured by growth signalling and provides evidence that the translational regulation of C/EBPβ into its distinct isoforms might influence therapy resistance and metastasis.
Whereas earlier studies pointed out a role of C/EBPβ in HER2+ breast cancer, recent studies have identified C/EBPβ as a potential transcriptional regulator in Triple Negative Breast Cancer (TNBC). Enriched genes in Triple Negative Breast Cancer (TNBC) vs. ER+ HER2- breast cancer samples as identified by Gene Set Enrichment Analysis (GSEA) share common promotor motifs for C/EBPβ54.
Moreover, high C/EBPβ expression correlates with expression of the TNBC markers, miRNA cluster C19MC, keratin (KRT) KRT5, KRT14, and KRT17, which might be driven through C/EBPβ-dependent promoter/enhancer activation55. Isoform specifically, it was demonstrated that preventing LIP
degradation by inhibition of the lysosome and proteasome may overcome resistance of TNBC to doxorubicin, by downregulating the P-glycoprotein multidrug (Pgp) transporter and upregulation of Calreticulin (CRT) and the subsequent immune response, suggesting LIP is essential in doxorubicin-mediated apoptosis and anti-tumour immunity in TNBC56. These studies point
towards a potential important role of C/EBPβ and its isoforms in TNBC.
C/EBPβ in the microenvironment and breast cancer metastasis
First described as Nuclear Factor for Interleukin-6 (NF-IL6), because of its binding to a response element in the Interleukin (IL)-6 promotor57,58, C/EBPβ has
been demonstrated to regulate a wide variety of cytokines and chemokines including IL-6, IL-859, IL-1β60 IL-861 and IL-462, Granulocyte-Colony Stimulating
Figure 2: a) Mutations in the CEBPB gene b) Copy Number Variations (CNVs) of the CEBPB gene and c) CEBPB mRNA expression found in human breast cancer according to the COSMIC database (https://cancer.sanger.ac.uk/cosmic). d) Timeline distribution of studies showing the importance of the translationally controlled C/EBPβ-isoform expression in oncogenesis and breast cancer.
motif chemokine 12 (CXCL12)65 and binds to a number of other acute phase
response and inflammation gene promoters. More recently, it was demonstrated that C/EBPβ regulates the M2 macrophage polarisation via β-adrenergic stimulation66,67. Macrophages found in human and mouse metastatic tumours
largely display an M2-like phenotype, which are known to contribute to angiogenesis and tissue remodelling and protumourigenic immunoregulatory functions (reviewed in 68). Although these data are solely based on analysed gene
High LIP expression in aggresive breast cancer
(Zahnow et al 1997)
WAP-LIP Tg mice predisposed to mammary
hyper-and neoplasias (Zahnow et al 2001)
Model for disrupted uORF-mediated translational control of C/EBPβ-LIP reduces tumour incidence (Muller et al 2018) Endogenous overexpression of LIP predisposes to oncogenesis (Begay et al 2014)
d
wt CEBPB mutant CEBPB
Total = 2575
samples without copy number gain samples with copy number gain
Total = 1492
Normal CEBPB expression
CEBPB overexpression
Total = 1104
expression profiles and not biological functions, it proposes that C/EBPβ could be a potential target for inhibiting tumour-promoting macrophage functions in cancer. More recently, tumour metabolism was linked to C/EBPβ-mediated cytokine production in triple negative breast cancer tumour development, where data show that the high glycolytic rate in TNBC suppresses autophagy, resulting in induced C/EBPβ-LAP expression which promotes Myeloid-Derived Suppressor Cells (MSDCs) development via G-CSF and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) expression, thereby evading tumour immunity69. A significant tumour growth reduction was observed upon
knockout of LAP in 4T1 cells, accompanied by increased Interferon gamma (IFNγ)+ and TNFα+ in CD8+ T cells in tumour tissues. In addition, knockdown of Cebpb in 4T1 cells was reported to upregulate chemokines CCL2 (MCP-1) or CCL5, which are both implicated to promote breast cancer metastasis. Knockdown of Cebpb in 4T1 murine breast cancer cells, and consequent transplantation into recipient mice, influences metastasis and tumour growth by increased major histocompatibility complex II (MHCII) expression, followed by the accumulation of CD45-, CD3- and CD4-positive (CD4+) lymphocytes in the tumours70. These studies show C/EBPβ in cancer cells elicits a cytokine response
and is able to mediate anti-tumour immunity, however, the functions of the C/EBPβ-isoforms are not yet established in much detail. In addition to its reported role in immunity, there are various studies that have focused on investigating the role of C/EBPβ in breast cancer cell migration. In a model that uses 4T1 cells for an in vivo tumour formation assay, it was observed that C/EBPβ depletion promotes invasion and metastasis of 4T1 cells by inducing the Epithelial to Mesenchymal Transition (EMT), and that the loss of C/EBPβ in human breast cancer samples is inversely correlated with EMT. Isoform specifically, it was observed that C/EBPβ is a transcriptional activator of junction proteins and in particular its long isoform LAP upregulates Cdh1 (E-cadherin) and Cxadr (Coxsackievirus and adenovirus receptor)71. In contradiction to this,
another study showed that overexpression of LAP in MCF10a cells promotes anchorage independent growth and acquisition of invasive phenotypes. Induction of this invasive phenotype was characterised by an internalisation of
cell adhesion molecule E-cadherin and establishment of an EMT signature72. In
addition, it was reported that LAP confers EGF-independent growth of MCF10a cells, speculating that compounds targeting the ErbB family could be less effective in tumours expressing high levels of C/EBPβ-LAP, thereby contradicting earlier studies showing that LIP confers resistance to trastuzumab treatment52,73.
However, the majority of currently published data implies that LIP promotes parts of the metastatic cascade. It was demonstrated that the short isoform LIP upregulates the expression of CXCR4 by inhibiting the binding of transcriptional repressor YY1 to the CXCR4 promotor, thereby promoting breast cancer cell migration of MCF7 cells, and demonstrating that despite being a transcriptional repressor, LIP can directly upregulate pro-invasive genes74. Analysis of samples
obtained from patients with advanced breast cancer revealed a correlation between LIP and CXCR4 expression. Moreover, C/EBPβ was reported to be upregulated in ErbB2- and RasV12- transformed MCF10A mammary epithelial cells (MECs), where knockdown of CEBPB resulted in decreased expression of α5 integrin. Blocking the interaction of α5 integrin with its ligand fibronectin reduced the malignant phenotype of transformed MECs, putting C/EBPβ forward as a key regulator of MEC malignancy75.
In summary, these studies point towards a potential interesting role of C/EBPβ in inflammation, tumour immunity and metastasis. However, evidence surrounding the role of the C/EBPβ-isoforms LIP and LAP in breast cancer progression and metastasis remain to a certain extend contradictory and how exactly the LIP/LAP isoform ratio affects the microenvironment and breast cancer development remains largely unknown.
Summary and future perspectives
In 2001, Hanahan and Weinberg described the hallmarks of cancer, summarising six biological capabilities that cancer cells acquire during tumourigenesis and tumour progression, namely sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis76. Based on an increasing
proposing that the deregulation of cellular energetics and avoiding immune destruction contribute as “emerging” hallmarks of cancer. In addition, they proposed genome instability and tumour-promoting inflammation as “enabling” hallmarks, that enable cancer cells to survive, proliferate, and disseminate77.
Classical oncogenes such as the transcription factor MYC are known to affect a majority of those hallmarks of cancer as reviewed in78. The identification of novel
oncogenic factors contributing to the (multiple) hallmarks of cancer is essential for our understanding and the treatment of cancer. Previous studies using genetically modified mouse models of C/EBPβ suggest that C/EBPβ-LIP acts as an oncoprotein - as its absence delays tumour onset7, and its overexpression
promotes tumour incidence6. Whereas the functions of C/EBPβ and its isoforms
in differentiation and proliferation have been established in multiple tissues, recent studies have established novel roles of C/EBPβ in the regulation of emerging hallmarks of cancer. For instance, we have established a novel role of C/EBPβ in the regulation of cellular energetics, by showing that LIP upregulates glycolysis and OXPHOS, and thereby induces a metabolic profile similar to cancer cells8. Moreover, a novel role of C/EBPβ as a regulator of MHCII
expression and consequent anti-tumour immunity was reported70. In summary,
C/EBPβ modulates five hallmarks of cancer (figure 3). In addition, C/EBPβ is a major regulator of inflammatory cytokines and despite the lack of publications which investigate the direct connections between C/EBPβ and tumour inflammation we hypothesise that C/EBPβ influences the “Tumour promoting inflammation”. Due to its involvement in multiple hallmarks of cancer, C/EBPβ could potentially be used as a therapeutic target in cancer.
However, simply putting C/EBPβ forward as an oncogenic transcription factors proves to be difficult, as also Spike and Rosen (2019) discuss in “C/EBPβ Isoform Specific Gene Regulation: It’s a Lot more Complicated than you Think!”79.
Whereas in some cases C/EBPβ promotes tumour development, in other cases it proves to be rather anti-oncogenic. As an example, C/EBPβ is required for the induction of chemically-induced skin tumourigenesis, whereas Johansson et al
Figure 3: C/EBPβ and the Hallmarks of Cancer. Summary displaying studies affecting various Hallmarks of Cancer (* same study affects multiple hallmarks). Adapted from Hanahan et al 201177.
show that loss of C/EBPβ promotes cancer progression, as it shifts the TGFβ response from growth inhibition to induction of the EMT breast cancer cells71.
Although these observations might be explained by differences in LIP/LAP ratios, it is also possible that C/EBPβ plays different roles in different types of cancer and/or different stages of cancer, and generalising its oncogenic functions therefore proves to be difficult. One problem is that many studies do not differentiate between the different C/EBPβ isoforms - whereas other studies show that LIP and LAP have distinct functions and the LIP/LAP ratio is essential for tissue homeostasis. In addition, the studies that do differentiate between the C/EBPβ-isoforms find oncogenic functions for both isoforms, or show
Deregulating cellular energetics Ackermann et al, Communications Biology (2019) Resisting cell death Ewing et al, Cell Death and Differentiation 2008 Sustaining proliferative signalling Evading growth suppressors Avoiding immune destruction Enabling replicative immortality Tumor-promoting Activating invasion & metastasis Inducing angiogenesis Genome instability & mutation
Bundy et al, molecular Cancer (2005)
Bundy et al, Oncogene (2003)* Bundy et al,
Oncogene 2003*
Park et al, The Journal of biological chemistry (2013)
Johansson et al, Oncogene (2013)
Kurzejamska et al, Oncogenesis (2014) Li et al, Molecular Cancer (2011) Li et al, Cell Metabolism (2018) Zahnow et al, Cancer Research (2001)
Arnal-Estapé et al, Cancer Research (2010)
controversial findings. As mentioned before, some studies have demonstrated LIP to be a mediator of the EMT and thereby cell invasion, while other studies have demonstrated a role for LAP in inducing EMT and invasion and even cell transformation. Moreover, recent studies have shown an involvement of C/EBPβ in recruiting tumour infiltrating lymphocytes, and a process by which tumour cell metabolism can modulate LAP to repress anti-tumour immunity, proposing LAP to be the oncogenic isoform. However, the study only analysed LAP and LAP* but LIP expression and function was not analysed. Whereas it remains possible for both isoforms to exhibit oncogenic functions, the majority of studies show oncogenic capacities for LIP, which fits to the classical existing literature describing LAP as mediator of differentiation and LIP as a mediator of proliferation. However, so far, xeno-and allograft experiments have been performed to study C/EBPβ in breast cancer, but C/EBPβ-isoform specific genetically engineered mouse models (GEMMs) in breast cancer are lacking and little remains known about the role of LIP and LAP in breast cancer development
in vivo. WAP-LIP transgene mice largely develop hyperplasias and only in some
cases neoplasias, indicating that LIP overexpression predisposes to oncogenesis but most likely requires another oncogenic hit to induce tumour development. Therefore, future studies modelling of the role of the C/EBPβ isoforms in a GEMM for breast cancer might reveal more about the roles of LIP and LAP in breast tumour development. The use of such models is especially beneficial, since previous studies revealed a major role of C/EBPβ in cytokine production and immune regulation, and therefore models with an intact microenvironment will not only reveal the cell intrinsic role of LIP and LAP in tumour development but also how this might interplay with the tumour microenvironment.
Previously, high LIP expression was correlated with absence of estrogen and progesterone receptors, high proliferative index, poor differentiation and aneuploidy in human breast tumours48. In addition, recent studies revealed
enriched gene expression patterns in samples obtained from TNBC that reveal common promoter motifs for C/EBPβ, and that C/EBPβ is potentially involved with the TNBC marker expression54,55 Even though TNBC patients can be treated
makes the search for druggable targets urgent. Using a Cebpb-uORF-reporter based cellular screening strategy we identified drugs from the ENZO library of FDA-approved drugs that specifically downregulate LIP, showing its potential value as a therapeutic target25. Further investigations into the roles of the
C/EBPβ-isoforms in different types and stages of breast cancer will be essential. One major limitation remains the absence of LIP-specific antibodies. Antibody generation has been proven to be difficult because the LIP isoform is identical to the C-terminal part of LAP. A LIP-specific antibody would be essential to establish the role of the C/EBPβ isoforms in a spatiotemporal manner in breast cancer. As some tumour suppressors act as cancer maintenance genes at later cancer stages80,81, this might also provide explanations for different reports on observed
oncogenic functions for both isoforms.
In conclusion, the transcription factor C/EBPβ exhibits a variety of oncogenic functions, however, the exact isoform-specific functions in breast cancer remain to be investigated. Future efforts should focus on modelling LIP and LAP in spontaneous in vivo breast cancer tumour models and studying its functions in breast cancer development and the microenvironment. Moreover, in addition to its role in HER2+ breast cancer, future efforts should focus on investigating the role of the C/EBPβ isoforms in TNBC, and whether the LIP/LAP isoform ratio could serve as diagnostic marker and potential therapeutic target in breast cancer.
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