Project topics and materials
DNA methylation
DNA methylation is a widespread modification in bacteria, plants and mammals, and this covalent molecular transformation is a natural modification of DNA, which takes place at position 5 of cytosine, and especially of cytosines preceding guanine (CpG) in human cells (Illingworth et al., 2008). DNA methylation produced during DNA replication is considered as a stable gene-silencing mechanism. DNA methylation is established and maintained by specific DNA methyltransferases (DNMT1, 3A and 3B) with S-adenosyl-methionine as methyl donor (Bostick et al., 2007).
DNMT3A and 3B are required for initial DNA methylation, and DNMT1 maintains the methylation status (Bernstein et al., 2007). DNA methylation inhibits transcription either “passively” by blocking the access of transcription factors to their binding sites, or “actively” through recruiting methylated binding domain proteins that inhibits gene expression (Nan et al., 1998). CpG-rich DNA fragments are called ‘CpG islands’, which are not methylated in dividing and differentiating tissues. However, in normal body cells, most of these CpG islands are methylated in a tissue-specific manner and certain subsets of methylated CpG islands at the promoter can lead to long-term silencing of transcription. In this sense, DNA methylation is formed during differentiation and can cause cells to partially or completely lose the ability to divide (Weber et al., 2007). CpG island-containing gene promoters are usually unmethylated in normal cells to maintain their euchromatic structure. Nevertheless, during cancer development, many of these CpG island-containing gene promoters are hypermethylated, changing open euchromatic structure to compact heterochromatic structure (Illingworth et al., 2008).
E-selectin as a receptor for targeted delivery
E-selectin can serve as a receptor for the delivery of anti-inflammatory drugs, anti-cancer drugs, and imaging markers in endothelial cells. For this purpose, antibodies against E-selectin, or artificial ligands of E-selectin are conjugated to the surface of polymeric particles. These immunoparticles are used to encapsulate the agent, so they can selectively bind to E-selectinexpressing endothelial cells and get internalized, together with the agent inside them. This technique allows the specific delivery of drugs to the pro-inflammatory microenvironment harboring tumor cells. Naturally, immunoparticles targeting E-selectin can also directly compete with cancer cells to bind to E-selectin. Based on these principles, intravenous injections of two E-selectin-targeting drug-carrying immunoparticles:
P-(Esbp)-DOX and P-(Esbp)-KLAK, inhibited primary tumor growth and metastasis of lung carcinoma in mice. Moreover, the “drug free” immunoparticle P-(Esbp) also exhibited anti-metastatic effects by competing with circulating lung carcinoma cells. By targeting E-selectin, we can also carry out targeted gene therapy, if viral vectors are encapsulated. ESTA-conjugated multistage vector (ESTA-MSV) can carry therapeutic anti-STAT3 siRNA to bone marrow vascular endothelium of mice, and infect breast cancer cells there. In vitro, anti-E-selectin lipoplexes can deliver anti-VE-cadherin siRNAs to inflamed primary vascular endothelial cells originating from different vascular beds, which are generally difficult to transfect (Jubeli et al., 2012). Overall, E-selectin-mediated endothelial adhesion plays a key role in metastasis, which opens new avenues for therapeutic interventions aiming at inhibiting the fatal complication of cancer.
Regulation of Argonaute protein functions
The small RNA duplex generated by Dicer cleavage is subsequently loaded in an Argonaute protein to form an RNA-induced silencing complex (RISC) (Hammond et al., 2001; Mourelatos et al., 2002; Tabara et al., 1999). Argonaute proteins are under a large number of modulations, most of which have not yet been reported to affect miRNA levels but rather miRISC functions. However, since miRNA loading and release is closely linked to miRISC functions, some prominent examples of the regulation of Argonaute protein functions are discussed below. As the key component of RISC, Argonaute proteins can be modulated by numerous modifications.
Prolyl 4-hydroxylation of human Ago2 by type I collagen prolyl 4-hydroxylase (4PH) increases the stability of Ago2 and localization within processing bodies (P-bodies) (Qi et al., 2008; Wu et al., 2011a). Poly ADP-ribosylation of Ago2 takes place upon stress or viral infection, which inhibits the targeting ability of RISC (Leung et al., 2011; Seo et al., 2013). Argonaute proteins are also subject to degradation by the ubiquitin–proteasome pathway. In mouse embryonic stem cells, LIN41 acts as an E3 ubiquitin ligase for Ago2 (Rybak et al., 2009).
Phosphorylation is the most studied type of post-translational modifications of Ago2. MAP kinase-activated protein kinase 2 (MAPKAPK2) (Zeng et al., 2008) and Akt3 (Horman et al., 2013) phosphorylate Ago2 at Ser387, resulting in its localization to P-bodies and translational repression, respectively. The importance of this phosphorylation was further validated by Bridge et al., who showed that Akt3 induces this phosphorylation to initiate LIMD1 binding, a scaffold protein recruiting TNRC6A (trinucleotide repeat-containing gene 6A) and downstream effectors for translational repression/mRNA destabilization. This Serine is conserved through Ago1-4, and Ago3 has an additional glutamic acid residue (E390) on the same interaction interface, which allows Ago3 to bind to the LIM domain containing proteins irrespective of Akt signaling (Bridge et al., 2017). Under hypoxia, epidermal growth factor receptor (EGFR) phosphorylates Ago2 at Tyr393, resulting in the dissociation of Ago2 from Dicer and the reduction of processing of some pre-miRNAs (Shen et al., 2013) (Figure 1-13).
The phosphorylation at Tyr529 on Ago2 was proposed to reduce small RNA binding and target repression, though the responsible kinase remains to be discovered (Mazumder et al., 2013; Rüdel et al., 2011). The C-terminal S824-S834 of Argonaute proteins is hyper-phosphorylated at five Serine residues in human and C.elegans. This hyper-phosphorylation does not affect miRNA binding, localization, or cleavage activity of Ago2, but rather mRNA binding, and it plays a role at late stages of gene silencing since the cluster remains unphosphorylated on Ago2 mutants that cannot bind miRNAs or mRNAs (Quévillon Huberdeau et al., 2017).
S824-S834 hyper-phosphorylation is mediated by CSNK1A1, followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Interestingly, although the hyper-phosphorylation inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. This is because non-phosphorylatable Ago2 is overall occupied by an expanded target repertoire, potently reducing the active pool of Ago2 on a per-target basis. In this sense, S824-S834 hyperphosphorylation cycle stimulated by target engagement maintains the global efficiency of miRNA-mediated silencing (Golden et al., 2017). MAP kinases also phosphorylate Ago2 at different sites (see Section 1.2.2).
Chapter 1. Introduction and reviews of the literature
1.1 Cancer
1.1.1 Causes of cancer
1.1.2 Multi-stepped carcinogenesis
1.1.3 Hallmarks of cancer
1.1.4 Endothelial cell adhesion molecules and cancer metastasis
1.2 microRNA
1.2.1 Regulation of miRNA biogenesis
1.2.2 MAP kinase pathways and microRNA pathway
1.2.3 microRNAs mediating endothelial cell adhesion molecules
1.2.4 miRNAs as the cause and consequence of cancer-related epigenetic alteration
1.3 Hypothesis and objectives
Chapter 2. p38 and JNK pathways control E-selectin-dependent extravasation of colon cancer cells by modulating miR-31 transcription
2.1 Résumé
2.2 Abstract
2.3 Introduction
2.4 Results
2.5 Discussion
2.6 Materials and methods
Chapter 3: p38 activation induces production of miR-146a and miR-31 to repress E-selectin expression and inhibit transendothelial migration of colon cancer cells
3.1 Résumé
3.2 Abstract
3.3 Introduction
3.4 Results
3.5 Discussion
3.6 Materials and methods
Chapter 4. General discussion
4.1 E-selectin-mediated metastases
4.2 miRNAs repress the expression of E-selectin and the transendothelial migration of colon cancer cells through different mechanisms
4.3 The mechanisms through which IL-1β induces the expression of the miRNAs
4.4 p38 MAP kinase represses the transcription and the translation of E-selectin through miR-146a and miR-31, respectively
Chapter 5. Conclusion and perspectives
5.1 Conclusion
5.2 Perspectives
Bibliography
