TESI SPERIMENTALE DI DOTTORATO DI RICERCA IN
IMMUNOLOGIA CLINICA E SPERIMENTALE (XXX CICLO)
TGF-β1-Mediated Regulation of the
Human NK Cells miRNome in the
Tumor Microenvironment
Coordinatrice:
Prof.ssa Maria Cristina Mingari
Relatore:
Prof.ssa Cristina Bottino
Candidato:
Fabio Caliendo
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INDEX OF CONTENTS
INTRODUCTION... 3
NK CELLS... 3
NK CELL RECEPTORS... 5
INHIBITORY RECEPTORS... 6
ACTIVATING RECEPTORS... 9
CHEMOKINE RECEPTORS... 13
IMMUNE SYSTEM RECOGNITION OF CANCER... 15
MYELOID CELLS IN THE TUMOR MICROENVIRONMENT... 17
T CELLS IN THE TUMOR MICROENVIRONMENT... 18
NK CELLS IN THE TUMOR MICROENVIRONMENT... 20
MicroRNAs... 22
NEUROBLASTOMA... 27
PUBLICATIONS... 32
TGF-β1 Downregulates the Expression of CX
3CR1 by
Inducing miR-27a-5p in Primary Human NK Cells... 33
Imatinib and Nilotinib Off-Target Effects on Human NK
Cells, Monocytes, and M2 Macrophages... 45
ACKNOWLEDGEMENTS... 57
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INTRODUCTION
NK CELLS
NK cells are lymphocytes of the innate immune system nowadays classified as member of the cytotoxic innate lymphoid cells (ILCs) family, which also encompasses lymphoid tissue inducers (LTi) cells and regulatory non-cytotoxic cells. ILCs are characterized by precise features: they have a lymphoid morphology and lack the expression of antigen specific receptors derived by the RAG-dependent genetic rearrangement. In addition, ILCs express interleukin (IL)-2 receptor-α (called CD25) and IL-7 receptor-α (called CD127).
ILCs are grouped in 3 subsets: ILC1, ILC2 and ILC3. Initially, NK cells were classified in ILC1 group (Spits, Artis et al. 2013), but lineage studies showing that NK cells and all the other ILCs develop through separate precursors (Constantinides, McDonald et al. 2014; Klose, Flach et al.
2014) led to the separation of innate lymphoid cell family in cytotoxic and non-cytotoxic ILCs. In
this new classification, NK cells are the only member of cytotoxic ILCs, whereas non-cytotoxic ILCs include the remaining ILC types.
All lymphocytes develop from a common lymphoid precursor (CLP) committed to particular cell lineages. Recently, a CLP-derived committed ILC precursor that can give rise to NK cells and to the other ILC subsets has been identified. This precursor is called α-lymphoid precursor ( αLP) and is characterized by the expression of integrin α4β7 and of the transcription factors NFIL3 and TOX. Downstream of αLP there are two precursors that express the transcriptional repressor Id2: the preNK cell precursor and a GATA-3 expressing precursor. The preNK cell precursor originates from αLP. Through the expression of the key transcription factors Eomes and T-bet, αLPs give rise to conventional NK cells, whereas the GATA-3 expressing precursors, originating LTi cells, give rise to further ILC precursors through the expression of RORγt and through the guide of the transcription factor promyeloid leukaemia zinc finger (PLZF). From this latter Id2+ PLZF+ ILC precursors originate all the ILC1, ILC2 and ILC3 cell types. Therefore, at least three ILC precursors with progressively limited precursor potential exist (Artis and Spits 2015).
Traditionally, NK cells are phenotypically defined by the absence of the T cell marker CD3 and by the expression of CD56, the 140-kDa isoform of neural cell adhesion molecule (NCAM).
Based on CD56 expression, NK cells can be divided in two subsets: CD56bright and CD56dim.
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cells are more abundant in bone marrow, peripheral blood and spleen. Is now well established that CD56bright NK cells represent an earlier stage of differentiation of peripheral NK cells that finally
progress to CD56dim NK cells. In fact, CD56bright NK cells show longer telomeres than CD56dim NK,
the length of telomeres being an important indicator of a cell maturation state (Romagnani, Juelke
et al. 2007). Moreover, CD34+ hematopoietic stem cells (HSC) infused in mice give rise initially to
CD56bright NK cells and subsequently to CD56dim NK cells (Huntington, Legrand et al. 2009).
Similarly, in haploidentical hematopoietic stem cell transplanted (haplo-HSCT) human recipient the first wave of lymphoid cells is entirely composed of CD56bright NK cells (Della Chiesa, Falco et al.
2012). Despite these evidences, the lineage relationship between the two NK cell subsets is an
unresolved issue. Recently, it has been demonstrated that activated CD56dim NK cells can
upregulate CD56 (Berrien-Elliott, Wagner et al. 2015). Another article in support of the ontogenic independency of the two subsets demonstrates that patients with GATA2 mutation are characterized by a selective loss of CD56bright NK cells, without changes in the CD56dim NK cell subset (Mace,
Hsu et al. 2013). Further studied will be required to clarify the origin of the two NK cell subsets.
Both NK cell subsets directly kill target cells (transformed or infected cells) through the release of cytotoxic granules (loaded with perforin and granzymes) and/or via death receptor pathways (Fas, TRAIL). In addition they shape the adaptive immune response through the release of cytokines and chemokines. However, differences exist between the two NK cell subsets in term of which function predominates. CD56bright NK cells produce a high amounts of cytokines and chemokines within
minutes after activation and have little ability to kill target cells. In contrast, CD56dim NK cells have
a low cytokine production activity and a prominent ability to kill target cells. The particular distribution of these two NK subsets, determined by the differential expression of chemokine receptors, mirrors distinct roles played by each one during the human immune response.
CD56dim NK cells express Chemerin-R, CXCR1 and CX3CR1, which bind ligands mainly
expressed in inflammatory peripheral tissues, whereas CD56bright NK cells express CCR7 and
CD62L and are attracted by chemokines CCL19 and CCL21, produced by cells in secondary lymphoid tissues (SLTs), where they shape the adaptive immune response interacting with dendritic cells (DCs) and T cells (Bellora, Castriconi et al. 2014).
One of the main cytokine secreted by CD56bright NK cells is INF-γ. This cytokine can shape the Th1
immune response, up-regulate MHC class I expression and activate macrophage killing. To this end, it makes sense for CD56bright NK cells to be primarily located at parafollicular T cell and APC rich
regions of SLTs. As a result of their ability to produce IFN-γ at SLTs, CD56bright NK cells link the
innate and adaptive immune response.
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high cytolitic activity against target cells. Moreover, CD56dim NK cells express high levels of CD16,
which binds to the constant (Fc) region of immunoglobulins. The engagement of CD16 by an adequate amount of immunoglobulins immobilized on the cell surface results in an NK-mediated target cell lysis event called antibody-dependent cellular cytotoxicity (ADCC) (Caligiuri 2008).
NK CELL RECEPTORS
The NK cell activation is tuned by activating and inhibitory signals mediated by receptors expressed on the cell surface (TAB-I).
RECEPTOR LIGAND SPECIFICITY FUNCTION
KIR MHC class I Activating/Inhibitory
CD94/NKG2 HLA-E Activating/inhibitory
NKG2D MICA, MICB, ULB1-6 Activating
NCR BAT-3, MLL5, PCNA, B7-H6 Activating/Inhibitory
LILR MHC class I Inhibitory
2B4 CD48 Inhibitory
DNAM-1 PVR, CD112 Activating
TIGIT PVR, CD112 Inhibitory
TACTILE PVR Activating
TAB-1: Human NK cell receptors.
Inhibitory receptors deliver inhibitory signals through intracellular immune receptor tyrosine-based inhibitory motifs (ITIMs), located at their cytoplasmic tails. Once phosphorylated, after ligand interaction, inhibitory receptors recruit Src homology 2 domain containing phosphatases (SHP1 or 2). ITIM-mediated signaling results in dephosphorylation of specific intracellular components that prevents both NK cell-mediated cytotoxicity and the adhesion between NK and target cells.
Some activating receptors deliver signals through the immune receptor tyrosine-based activating motifs (ITAMs), contained in adaptor molecules associated to the cytoplasmic short tails of these receptors. After ligand recognition, Src homology 2 domain containing kinases (Syk or ZAP70) are recruited. These kinases start a signal cascade which results in NK cell degranulation of cytotoxic granules, cytokine production and transcription of chemokines genes.
When ligands of activating receptors are expressed on healthy cells, the co-expression of inhibitory receptors ligands protects self cells from NK cell-mediated lysis. Indeed, the activating signals can be overridden by the signaling of ITIM-containing receptors in agreement with the missing self hypothesis (Fig.1) (Pegram, Andrews et al. 2011).
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INHIBITORY RECEPTORS
Human NK cells express different HLA-class I specific inhibitory receptors: inhibitory killer immunoglobulin Ig-like receptors (iKIRs), CD94/NKG2A and LILIRs. KIRs recognize different allelic group of A, B or C, CD94/NKG2A is specific for a non-classical HLA molecule, HLA-E (Moretta, Biassoni et al. 2002), LILR have broad HLA class I molecules recognition.
Figure 1: Activating ligands are constitutively expressed by normal cells, but the simultaneous presence of inhibitory
ligands on these cells prevents self-cell lysis. Infected or transformed cells generally downregulate inhibitory ligands, thus rendering these cells susceptible to cell lysis by NK cells.
KIRs
KIR family contains receptors with both inhibitory (iKIRs) or activating (aKIRs) functions. They have homologous extracellular domains but different intracellular portions, displaying cytoplasmic tails of different length (Biassoni, Cantoni et al. 1996). The human KIR family consist of 13 genes encoded by the leukocyte receptor complex (LRC) locus on chromosome 19q13.4. This locus is characterized by a broad variation in gene number and by allelic polymorphisms for each gene
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(Parham 2005).Genes encoding KIRs, and HLA class I molecules are located on different chromosomes (Vilches
and Parham 2002), therefore they can segregate independently leading to the generation of NK
cells that have at least one receptor specific for self HLA class I. This allows NK cells to sense the loss of single HLA class I, a typical event that take place in virus infected or transformed cells (Moretta, Biassoni et al. 2002).
KIR genes are tightly clustered and inherited together forming a haplotype. Two KIR haplotypes have been identified in humans: group A haplotype (found in all human populations) and group B haplotype. Haplotype A contains a defined number of inhibitory KIR genes: KIR2DL1, KIR2DL3, KIR3DL1 and KIR3DL2, specific for the four major HLA class I KIR ligands: HLA-C2, HLA-C, Bw4 and HLA-A3/A11. Haplotype A also contains the activating KIR KIR2DS4, which shows a weak specificity for HLA-A, HLA-C and HLA-F (Maxwell, Wallace et al. 2002; Manser,
Weinhold et al. 2015). Group B haplotype is highly variable in gene content and characterized by
the presence of activating KIRs (Falco, Moretta et al. 2013).
NK cells expressing one KIR receptor per cell are the most frequent ones (Yawata, Yawata et al.
2006; Andersson, Fauriat et al. 2009). Only those NK cells that express at least one KIR specific
for self HLA class I antigen are fully competent (licensed), implicating the existence of an education program which shape the NK cell repertoire. Two education mechanisms were proposed: the arming and disarming hypothesis (Fig.2). The arming hypothesis postulates that inhibitory KIRs that recognize self HLA class I ligands drive positive signal to NK cell, allowing full maturation and responsiveness to suitable stimuli. On the contrary, lack of this key interactions leaves NK cells in a hyporesponsive state. According to the disarming hypothesis, inhibitory KIRs are viewed as inhibitory receptors that counteract a still undefined activating signal. In the absence of iKIR/self-HLA class I interactions, the over stimulation of activating receptors results in NK cell anergy. Regardless of which mechanism take place, it is irrefutable that HLA class I molecules shapes KIR genotype in each individual (Elliott and Yokoyama 2011; Falco, Moretta et al. 2013).
CD94/NKG2
CD94/NKG2 receptors belong to the C-type lectin-like receptor superfamily. CD94 and NKG2A are encoded on chromosome 12 and are non-polymorphic in humans (Parham 2005). Five different molecular species of NKG2 molecules have been identified so far: NKG2A, B, C, E, and H. All these different NKG2 molecules can form disulfide-linked heterodimers with the invariant chain CD94. Each component of the dimer is composed of an extracellular C-type lectin-like domain, a transmembrane domain and a cytoplasmic domain.. All the CD94/NKG2 are specific for the
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classical class I molecule HLA-E in humans. HLA-E consist of three components: a heavy chain, a β2-microglobulin and a peptide derived from the signal sequence of MHC class I molecules, which allows NK cells to indirectly sense the presence of classical MHC class I molecules on target cells (Borrego, Masilamani et al. 2006).
Figure 2. NK cell education hypothesis. According to the arming hypothesis, only those NK cells that express at least
one KIR specific for self HLA class I molecules are fully competent (licensed). According the disarming hypothesis, KIRs are inhibitory receptor that counteract an undefined activating signal. The over stimulation of NK cells that lack a KIR specific for self HLA class I molecules results in NK hyporesponsiveness.
Of note, CD94/NKG2 receptors are expressed on NK cells during their ontogeny and are maintained into adulthood. The expression of these receptors early in NK cell development contributes to self- tolerance, even if they are not entirely responsible for self-tolerance (Vance,
Jamieson et al. 2002).
After the interaction between NKG2A and NKG2B with HLA-E the tyrosine residue in each ITIM becomes phosphorylated, facilitating the binding of SHP-1 and SHP-2 phosphatases, which in turn
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suppress NK cell activation processes (Gunturi, Berg et al. 2004; Borrego, Masilamani et al.
2005).
LILRs
Leukocyte Ig-like receptors (LILRs) constitute a group of the Immunoglobulin (Ig) superfamily which may play essential role in both innate and adaptive immunity.
Like KIRs, these receptors are encoded on chromosome 19 and can be divided in activating (LILRA) and inhibitory receptors (LILRB). LILR gene cluster can form only two haplotypes, one containing 13 loci and the other only 12 as a result of a deletion in LILRA3 gene. LILR genes are poorly polymorphic compared to KIR genes. The presence of ITAM or ITIM in the cytoplasmic tail of each individual receptor is responsible for the activating or inhibitory signals transmitted by LILRs. LILRB1 is the only receptor of LILRs family that have been studied in NK cells. The interaction of LILRB1 with its ligands inhibits CD16-dependent cell lysis (Brown, Trowsdale et al. 2004;
Thomas, Matthias et al. 2010) . Certain cancer cells also express LILRs, in particular LILRBs. It
was demonstrated that the expression of this receptors by tumor cells may contribute to tumor progression and immune evasion (Godal, Bachanova et al. 2010; Zhang, Lu et al. 2012; Zhang,
Mai et al. 2017)
ACTIVATING RECEPTORS
NKG2D
NKG2D is a member of the NK group 2 (NKG2) family of receptors expressed on NK and T cell surface. This activating receptor behaves as a homodimer that recognizes stress-induced MHC-I-like proteins. In humans the NKG2D ligands are MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB) and six members of UL16-binding proteins (ULBP) family.
The cytoplasmic tail of the NKG2D receptor is very short and lacks any activating or inhibitory motifs. Charged aminoacids in the transmembrane domain allow NKG2D to bind adaptor proteins which transduce NKG2D signaling (Jelencic, Lenartic et al. 2017).
NKG2D ligands are upregulated in response to cellular stress events such as: viral infection, malignant transformation, classical heat shock. In addition, NKG2D ligands are upregulated in normal healthy cells undergoing extensive proliferation, such as embryonic tissues. Therefore, NK and T cells can rapidly sense proliferating cells as targets for killing, perhaps as a control
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mechanism in tissues development (Raulet 2003; Lanier 2015).
In mice two isoforms of NKG2D are present. A long NKG2D-L isoform and a short NKG2D-S variant which can interact with both DAP10 and DAP12 adaptor proteins. Humans express only the long isoform that can exclusively interact with DAP10.
DAP12 possesses ITAM motifs and recruits Syk and Zap70 kinases, whereas DAP10 possesses a YxxM motif which recruits PI3K, Grb2 and Vav-1, leading to NK cell cytotoxicity. Both the adaptor proteins are inducers of cytokine release and cytotoxicity in NK and T cells.
As NKG2D recognizes self-ligands, this receptor plays a key role in an additional layer of regulation of NK cells activation, called “induced self”, that acts synchronously to the “missing self” mechanism. The signals from inhibitory KIRs and CD94/NKG2 receptors keep NK cells in a quiescent state and according to missing self mechanism, ligands of these inhibitory receptors are downregulated in stress conditions. NK cells recognize stress-induced self-ligands through NKG2D, providing an additional activating signal not counteracted by inhibitory signals, leading to NK cell activation, known as “induced-self” (Jelencic, Lenartic et al. 2017).
Finally, NKG2D is constitutively expressed on NK cells from the earliest stages of development. This receptor can regulate activation thresholds of other activating receptors during NK cell development, as was demonstrated in NKG2D-deficient mice. NK cell precursors of these mice showed altered receptor repertoire, were less responsive and more hyper-responsive to subsequent stimulation with non-NKG2D targets (Zafirova, Mandaric et al. 2009).
NCRs
Nkp30, Nkp44 and Nkp46 are transmembrane receptors, grouped in the natural cytotoxicity receptor (NCRs) family.
NCRs are type I transmembrane proteins belonging to the immunoglobulin superfamily and are characterized by one or two extracellular immunoglobulin-like domains, which are responsible for ligand binding. The transmembrane domain of NCRs possesses a charged amino acid that allows the association of ITAM-containing the adaptor molecules that lead to a signaling cascade that ultimately causes cell activation.
Nkp46 is expressed in NK cells and a subset of ILC3 identified as NCR3+ ILC3. NKp46 is the major lysis receptor of NK cells but the putative cellular ligand, expressed on the surface of tumor cells, is currently unknown (Sivori, Pende et al. 1999). It was shown that viral hemagglutinins (HAs) can be recognized by Nkp46. This interaction drives the NK cell-mediated killing of influenza virus infected cells expressing the HA proteins (Mandelboim, Lieberman et al. 2001) NKp44 is expressed only in activated NK cells, decidual NK cells and tonsil-resident plasmacytoid
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DCs. The Nkp44 gene is located on chromosome 6 and contains a single extracellular V-type Ig domain and a transmembrane domain that through a positive lysine, associates with ITAM-containing adaptor DAP12. Interestingly, Nkp44 is the only NCR with an ITIM motif in the cytoplasmic tail (Cantoni, Bottino et al. 1999; Campbell, Yusa et al. 2004) potentially transmitting inhibitory signals to NK cells.
Nkp30, expressed in all mature NK cells and in particular subset of T cells, contains a single extracellular immunoglobulin domain. Both CD3ζ and FcRγ can interact with a charged amino acid located in the NKp30 transmembrane domain. There are three different splice variants of this receptor, each one with specific effects on NK cell functions. Nkp30a and Nkp30b engagement is associated with release of INF-γ, whereas the engagement of Nkp30c results in the production of IL-10 and the release of only small amounts of INF-γ. Therefore, Nkp30a and Nkp30b transmit activating signals to NK cells, while Nkp30c mediates immunosuppressive signals (Delahaye,
Rusakiewicz et al. 2011).
2B4
2B4 is a member of the CD2 subset of the immunoglobulin (Ig) receptor superfamily that binds CD48 molecule on the surface of target cells. It is structurally characterized by a V-like and a C2-like extracellular domain and four immuno-receptor based tyrosine switch motifs (ITSM) in the cytoplasmic tail, which bind several SH2-domain containing kinases and phosphatases.
In humans 2B4 is expressed on NK cells, CD8+ T cells, γδ-T cells, monocytes, basophils and
eosinophils. Human NK cells express only the long isoform of this receptor with both activating or inhibitory functions.
Upon ligation, the ITSM motifs of 2B4 are phosphorylated by the Fyn and Lck kinases (members of the Src family kinases). Once phosphorylated, 2B4 can recruit several SH2 domain containing proteins, SHP-1, -2, EAT-2, ERT and SAP. The functional outcome of CD48-2B4 interaction depends on which protein is recruited at the cytoplasmic domains. The SAP recruitment seems to be a crucial event in the activating pathway, as it makes ITSM inaccessible to SHP-1 and SHP-2. When the expression of SAP is low, 2B4 binds the inhibitory SH2-containing phosphatases: SHP-1 and SHP-2. Therefore, the activating versus inhibitory function of 2B4 depends on the SAP concentration in the cell according to the “SAP threshold” model (McNerney, Lee et al. 2005). It’s widely accepted that 2B4 acts as inhibitory receptor in immature NK cells, contributing to MHC class I-independent tolerance during NK cells development. Indeed, Nkp30 and Nkp46 are expressed early in developing NK cells, before the MHC class I inhibitory receptors expression. In this setting, developing NK cells are potentially reactive to the surrounding normal cells. At this
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developmental stage, NK cells express 2B4 and SHP-1, but lack the expression of SAP. Thus, the absence of SAP in immature human NK cells allows 2B4 to function as a very early inhibitory receptor, ensuring self- tolerance in the absence of KIRs (Sivori, Falco et al. 2002; Vaidya and
Mathew 2006).
Nectin and nectin-lile molecule receptors family
Receptors members of the nectins and nectin-like family are important regulator of NK cell functions. The most important member of this receptor family is DNAX accessory molecule 1 (DNAM-1) that controls NK cell cytotoxicity and interferon-γ (INF-γ) production against transformed and infected cells.
Structurally, DNAM-1 is an Ig-like family glycoprotein composed of an extracellular portion with two Ig-like domains, a transmembrane domain and a cytoplasmic domain. It does not associate with ITAM-bearing molecules, but is directly phosphorylated after ligand binding.
Ligands of this receptor were first independently discovered by two laboratories in the 2000s (Bottino, Castriconi et al. 2003; Tahara-Hanaoka, Shibuya et al. 2004). Both groups identified two molecules as ligands of DNAM-1: CD155 (Poliovirus Receptor, PVR) and CD112 (Nectin-2, PRR2).
After ligand interaction, PKC phosphorylates the cytoplasmic tail of DNAM-1 at serine 329. At the same time, tyrosine 322 is phosphorylated by the LFA-1-associated Fyn kinase. The phosphorylation of both sites is required for full DNAM-1 signaling cascade activation, which results in calcium mobilization and secretion of cytotoxic granules from NK cells. However, these effects require synergistic activation of a specific pair of receptors in resting NK cells.
CD155 expression has been described in several cancers, but the presence of this ligand is not sufficient for tumor eradication. Indeed, cancer cells have developed escape mechanisms to prevent DNAM-1-mediated killing, commonly through DNAM-1 downregulation due to chronic ligand exposure on tumor cell surface (Bottino, Dondero et al. 2014).
NK cells express two additional receptors that share ligand specificity with DNAM-1, namely TIGIT and CD96 (TACTILE).
TIGIT belongs to the CD28 family and acts as an inhibitory receptor. This molecule is characterized by an extracellular immunoglobulin domain and two cytoplasmic ITIM motifs and is expressed on NK cells and other lymphoid cells such as CD45RO+ memory T cells and FOXP3+ regulatory T cells (Treg). TIGIT interacts with CD112, CD113 (PVRL3), and CD155 with affinity higher than
that of DNAM-1.
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CD155 binding. Once TIGIT-CD155 interaction takes place, TIGIT cytoplasmic ITIM motifs are phosphorylated, thus recruiting, via the adaptor molecule Grb2, SHIP-1 phosphatase, which in turn inhibits PI3K and MAPK signaling pathways. In addition, the high affinity of TIGIT for his ligand reduces CD155 molecules available for the activating interaction with DNAM-1 (de Andrade,
Smyth et al. 2014).
CD96 is composed of three Ig-like domains and a proximal stalk domain. Mice express only the long CD96 variant, whereas humans CD96 mRNA is differentially spliced at exon 4 giving rise to two receptor variants that differ in their extracellular domains. The longer variant is less abundant than the shorter one and encodes an 83 amino acid V-like domain, while the shorter variant encodes an I- or C-like domain. These differences in the extracellular second Ig-like domain may be physiologically relevant, as they impact on ligand-receptor interaction and therefore on receptor affinity. The cytoplasmic domain of human CD96 differs from the murine orthologue. In mouse the CD96 cytoplasmic tail is relatively short and contains an ITIM motif. The inhibitory role of mouse CD96 has been widely demonstrated (Seth, Maier et al. 2007; Chan, Martinet et al. 2014). Human CD96 cytoplasmic domain contains an ITIM motif as well and an additional YXXM motif, which in activating receptors CD28 and ICOS binds to PI3K. The presence of a YXXM motif in human CD96 confers to this receptor an activating potential (Fuchs, Cella et al. 2004; Dougall,
Kurtulus et al. 2017).
CHEMOKINE RECEPTORS
Chemokines are the largest known family of cytokines, including about 47 related molecules. These proteins are classified in four groups: CXC, CC, CX3C and C, according to the number and spacing of conserved cysteine residues in their amino acid sequences. In chemokine nomenclature, “C” describe the cysteine and “X” the amino acid residue placed between cysteines, if present. For example, CXC chemokine group includes all chemokines characterized by 2 conserved cysteines spaced by 1 amino acid residue.
Each chemokine can bind more than one receptor and each receptor can bind several chemokines, thus making this ligand-receptor system complex. Receptors for these proteins are G-protein coupled seven-transmembrane-spanning receptors, and, according to the chemokines that each receptor can bind, they are also classified in four sub-families.
Chemokines are fundamental in both adaptive and innate immune response. These molecules coordinate myeloid and lymphoid hematopoiesis, contributing to migration of precursor cells into the proper developmental niche, where appropriate growth factors are provided. Chemokines
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regulate NK, T and B lymphocytes recirculation and homing in peripheral tissues and control the migration of antigen-presenting cells. Other important functions of chemokines include inhibition or promotion of angiogenesis and regulation of tumor metastasis.
Human resting NK cells express different chemokine receptors respect to activated NK cells. Chemokine receptors are also differentially expressed by CD56bright and CD56dim NK cell subsets.
Resting human NK cells migrate in response to CXCR3 receptor ligands CXCL9 and CXCL10 and in response to CXCR4 receptor ligand CXCL12. After activation, human NK cells up-regulate CCR2, CCR4, CCR7 and CCR8 and respond to a different array of ligands: CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL19, CCL21 and CCL22 (Barlic, Sechler et al. 2003; Guo, Chen
et al. 2003; Griffith, Sokol et al. 2014)
CD56bright NK cells express a high level of CXCR3 and CCR7, whereas they show little or no
expression of CXCR1 and CXCR2. Thus, CD56bright NK cells preferentially respond to CXCL19
and CCL21, two chemokines responsible for NK cells localization to the lymph nodes, where they shape adaptive immune response.
NK cells themselves are able to produce several chemokines, in particular after activation, causing attraction of other immune cells at the activation site.
Chemokines produced by stimulated NK cells can participate in NK cell-mediated antiviral and antitumor effects. For example, CXCL9 and CXCL10, produced by NK cells upon INF-γ stimulation, can enhance the activation status of NK cells and inhibit tumor angiogenesis (Robertson 2002; Bernardini, Gismondi et al. 2012).
In particular, at the lymph nodes, INF-γ production by recruited NK cells has important consequences in skewing CD4+ T cell polarization towards a Th1 phenotype.
The pattern of chemokine receptor expressed on NK cell surface, in a given situation, depends on the set of chemokines expressed at the inflamed sites. In vivo, more chemokines act together to attract NK cells, moreover the optimal combination of chemokines required for NK cell recruitment can change over time. In addition, chemokines might act at the same time via different receptors or sequentially in different phases of inflammation. Therefore, the spatiotemporal control of chemokines expression is fundamental to allow the recruitment of NK cells to the proper site (Walzer and Vivier 2011).
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IMMUNE SYTEM RECOGNITION OF CANCER
Normal cells undergo neoplastic transformation through the accumulation of genetic alterations that contribute to the generation of an aberrant inflammatory microenvironment at the tumor site.
Cancer immunosurveillance, namely the process by which immune system protect the host from neoplastic disease, is universally accepted and supported by several experimental evidences in defined mouse models that demonstrated the importance of the interface between tumor cells and immune system. Despite the protection of the immune system, cancer cells still give rise to neoplastic diseases in immunocompetent hosts. Antitumor immune response results in both elimination of cancer cells and the selection of transformed cells with reduced immunogenicity in a process called “immunoediting”. Thus, the immune system has opposite effects on cancer growth: at first, it controls tumor growth through the lysis of transformed cells and, subsequently, it favors tumor growth via the immunoediting process which allows the proliferation of less immunogenic cancer cells. The immunoediting process consists of three phases: elimination, equilibrium and escape.
If the immune attack against tumor does not result in a complete tumor eradication, the surviving cancer cells can enter in the equilibrium phase wherein the rate of tumor cell elimination is counteracted by the rate of neoplastic cell division. In this phase, transformed cells accumulate further genetic alterations that lead to the evolution of less immunogenic cancer cells which evade from the immune system attack.
During the escape phase the immune system is no longer able to kill transformed cells, which in turn give rise to a tumor with different immunogenic properties respect to the same early cells. Cancer cells evade the immune system attack via different mechanisms: by MHC downregulation, resistance to immune mediated killing through the up-regulation of Fas-L, the modulation of TRAIL receptor, the induction of T cell anergy, the development of an immunosuppressive immune microenvironment and the secretion of immunosuppressive soluble factors (Lakshmi Narendra,
Eshvendar Reddy et al. 2013; Bottino, Dondero et al. 2014).
Proliferating cancer cells, along with non-malignant stromal cells, blood vessels, and infiltrating inflammatory cells are part of the tumor microenvironment (Fig.3). This is a unique environment which develops during tumor progression and is continuously shaped by cancer cells. The tumor microenvironment promotes tumor growth through either the abnormal induction of inflammatory mediators and the suppression of the antitumor immune response.
Tumors are generally viewed as “wound that do not heal” because the changes that occur in tumor microenvironment, during tumor progression, resemble that of chronic inflammation, namely ischemia followed by the recruitment of immune cells, then growth of blood vessels and tissue
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repair.The first response of the host against the tumor is inflammation with the subsequent infiltration of immune cells. The final goal of this inflammatory response is to eliminate cancer cells. As the inflammatory response progresses, different immunosuppressive immune cells are recruited at the tumor microenvironment. The recruitment of these cells is protective in physiological setting, resolving the inflammation and promoting tissue regeneration, but in the tumor microenvironment they favor tumor progression suppressing the antitumor immunity.
Immune cells in the tumor microenvironment comprise cells that mediate adaptive immunity, T lymphocytes, DCs and occasional B lymphocytes, as well as cells that mediate innate immunity such as macrophages, polymorphonuclear leukocytes and NK cells.
The majority of myeloid and lymphoid cells that infiltrate the tumor microenvironment are endowed with immunosuppressive functions, which ultimately dampen the function of cytotoxic T cells (CTLs) and NK cells recruited at the tumor site.
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MYELOID CELLS IN THE TUMOR MICROENVIRONEMNT
Among the myeloid cells that infiltrate the tumor myeloid-derived suppressor cells (MDSCs) are the major components of the immune suppressive network of the tumor microenvironment. MDSCs represents a heterogeneous group of immature myeloid cells at different stages of maturation, which suppress NK cell- and T-cell- dependent antitumor immunity through the production of several immunosuppressive cytokines and the action of specific enzymes. MDSCs produce IL-10, which decreases the levels of IL-12 in the tumor microenvironment, a pro-inflammatory cytokine involved in the activation of NK cells. Moreover, MDSCs induce anergy of NK cells and inhibit their cytotoxicity via TGF-β.
MDSCs upregulate the expression of arginase and iNOS, enzymes involved in the inhibition of CTL functions (Gabrilovich, Ostrand-Rosenberg et al. 2012).
Tumor associated macrophages (TAMs) represent another important immune cell type which infiltrate the tumor. These cells are recruited by the chemokine milieu of the tumor microenvironment and preferentially localize at the host-tumor interface in areas of low oxygen tension. The plasticity is the main feature of macrophages. Indeed, these cells can acquire different phenotypes depending on the cytokine stimulation they receive during activation (their phenotype is also shaped by the oxygen tension). Macrophages can be classically activated by INF-γ alone or in concert with bacterial products (LPS) or TNF-α that drive these cells toward the acquisition of an M1 phenotype. M1 macrophages secrete a large array of proinflammatory cytokines, such as: TNF-α, IL-1, IL-6, IL-12 and IL-23. These cytokines favor tumor elimination. By contrary, macrophages can be alternatively activated toward an M2 phenotype upon stimulation with Th2 cytokines (IL-4,
IL-13), IL-10 and TGF-β. Activated M2 macrophages secrete anti-inflammatory cytokines IL-10 and TGF-β, together with arginase-1. Therefore, M2 macrophages contribute to the evolution of an immunosuppressive tumor microenvironment favoring tumor progression (Bellora, Castriconi et
al. 2010; Galdiero, Bonavita et al. 2013) .
Differentially polarized macrophages secrete a defined set of chemokines shaping the immune infiltrate at the tumor microenvironment. M1 macrophages secrete a series of pro-inflammatory chemokines: CXCL9, CXCL10, CXCL15. These chemokines recruit CTL and NK cells at the tumor microenvironment. On the other hand, M2 macrophages produce CCL17, CCL22 and CCL24 that recruit the immunosuppressive Treg cells at the tumor site, together with anti-inflammatory Th2
polarized T cells. Moreover, M2 macrophages contribute to the modulation of both tumor angiogenesis and extracellular matrix remodeling via the production of the vascular endothelial growth factor A (VEGF-A) and matrix metalloproteinases MMP-2 and MMP-9 (Hao, Lu et al.
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At the early phases of tumor progression the cytokine milieu of tumor microenvironment favors the polarization of macrophages toward an M1 phenotype. However, the aberrant inflammatory environment at the tumor site, generated in the late phases of tumor progression, skews the polarization of macrophages toward an anti-inflammatory M2 phenotype. The cytokines released by M2 polarized macrophages induce an ineffective Th2 immune response against cancer and, at
same time, suppress the cytotoxic potential of CTL and NK cells (Allavena, Sica et al. 2008;
Bellora, Castriconi et al. 2010).
As for the tumor associated macrophages, tumor microenvironment can subvert the neutrophils toward a more immunosuppressive phenotype. This immunosuppressive neutrophils residing at the tumor site are known as tumor-associated neutrophils (TANs), which produce arginase and proangiogenic factors promoting tumor growth (Fridlender and Albelda 2012)
DCs are professional antigen presenting cells (APC) that provide a series of signals for the proper activation of T cells (Veglia and Gabrilovich 2017). Different subsets of DCs were described. These includes Langerhans cells, monocyte-derived DCs, myeloid DCs and plasmacytoid DCs (pDCs). In addition, cells of each of these subsets are simultaneously present at different maturation stages with different functionality. The percentage of tumor infiltrating DCs (TIDCs) at the tumor site is low and the majority of them exhibit an immature phenotype. The mature TIDCs with immune stimulatory functions are confined at the peritumoral sites, where they cannot mount an effective adaptive immune response against cancer. TIDCs phenotype is immature, they are unable to properly activate T cells, which divert toward a tolerogenic or anergic phenotype. Several evidence showed that DCs can actively suppress anti-tumor immune response and promote tumor progression. Indeed, DCs recruited at the tumor draining lymph nodes actively promotes the proliferation of immunosuppressive Treg cellsvia the secretion of TGF-β, IL-10 and IL-2 (Munn,
Sharma et al. 2002; Grohmann, Fallarino et al. 2003; Ghiringhelli, Puig et al. 2005; Ramos, de Moraes et al. 2013).
T CELLS IN THE TUMOR MICROENVIRONMMENT
The two major subsets of cytotoxic effector cells in the tumor microenvironment are the CTLs and NK cells. These cells are effective in killing tumor cell lines in vitro, but in vivo they result ineffective in the elimination of transformed cells.
T lymphocytes differentiate in the thymus from a common lymphoid progenitor and express a T-cell receptor (TCR) that is responsible for the recognition of antigen peptides presented by the MHC molecules on APCs. T-lymphocytes are classically divided in two subsets, CD8+ and CD4+ T cells,
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which recognize antigen peptides presented by MHC class I and MHC class II molecules, respectively.
CD8+ T-cells upon activation secrete INF-γ and release the content of cytotoxic granules (perforin
and granzymes) to directly kill virus infected or transformed cells. CTL infiltration at the tumor site has been widely described, but, as the tumor progress, these cells seem to lose their cytotoxic capabilities under the immunosuppressive pressure of the tumor microenvironment.
Naive CD4+ T-cells differentiate in different functional lineages depending on the polarizing
cytokine signals that they receive during activation. Generally, CD4+ T-cells can be polarized
toward a Th1 or Th2 phenotype (Annunziato, Romagnani et al. 2015). Th1 CD4+ T-cells secrete
high levels of INF-γ and TNF-α, that in turn up-regulates the expression of co-stimulatory and MHC molecules on APCs. Moreover, Th1 cells can directly contact CTL through the CD40/CD40L
interaction, thus promoting the expansion of antigen specific CD8+ T-cells.
Th2 cells release high amount of IL-4, IL-5, IL-6, IL-13 and IL-10, that induce T-cell anergy and
inhibit T-cell mediated cytotoxicity. Recently, other CD4+ subsets have been classified: IL-17
expressing Th17 cells, T follicular helper cells (TFH) and Treg cells.
Treg cells are immunosuppressive CD4+ T-cells which express high levels of CD25 (IL-2 receptor α
chain) and the transcription factor Foxp3 (forkhead box P3). CD4+ CD25high Foxp3+ regulatory
T-cells play a key role in maintaining peripheral tolerance in humans and mice, and defects in their number and functions contribute to the development of autoimmunity, lymphoproliferation, allergic dysregulation and inflammatory diseases.
Two Treg cell subsets have been identified: natural Treg cells (nTreg) and induced Treg cells (iTreg).
nTreg cells originates from the thymus during the normal T cell developmental process. iTreg cells
develop from conventional CD4+ T cells in the periphery and are particularly abundant at the
mucosal interface, where they are induced by tolerogenic APCs (Kawamoto, Maruya et al. 2014;
Arpaia, Green et al. 2015; Wang, Charbonnier et al. 2015).
Treg cells mediate their immunosuppressive functions via both dependent and
contact-independent mechanisms. These immunosuppressive cells constitutively express cytotoxic T-lymphocyte Antigen 4 (CTLA-4) on their surface, which compete with CD28 for the ligands CD80/CD86 on antigen presenting cells. Therefore, APCs are no longer able to properly stimulate effector cell activation upon interaction with CTLA-4 on Treg cell surface.
Treg cells can also suppress immune responses through contact-independent mechanisms that
involve the release of immunosuppressive cytokines IL-10, TGF-β and IL-35. In addition, Treg cells
can act as IL-2 sponge due to the up-regulation of CD25, that leads to IL-2 depletion in their surrounding microenvironment (Alroqi and Chatila 2016).
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Treg cells are highly enriched in the tumor microenvironment and their abundance correlate with
poor prognosis in many cancer types. TGF-β, IL-10 and IL-35, secreted from Treg cells at the tumor
site, inhibit the proliferation, cytokine elaboration and limit the survive of antitumor effector cells. TGF-β skews the APCs toward a tolerogenic phenotype responsible for the generation of anergic T-cells and for the differentiation of naïve CD4+ T-cells into iT
reg. In addition, Treg cells produce
VEGF, which favors tumor progression via the induction of cancer angiogenesis, and high levels of IDO, an enzyme well known for its immunosuppressive properties (Facciabene, Motz et al. 2012).
NK CELLS IN THE TUMOR MICROENVIRONMENT
NK cells play a crucial role in tumor immunosurveillance but they result inefficacious in advanced diseases. Tumor-infiltrating NK cells (TINKs) have distinct phenotypic and functional properties than their tissue residing and peripheral blood counterpart. Interestingly, TINKs share phenotypic properties with low cytotoxic decidual NK cells (dNKs), known to be endowed with regulatory and pro-angiogenic functions (Bruno, Ferlazzo et al. 2014; Levi, Amsalem et al. 2015).
Solid malignances are generally enriched in poorly cytotoxic CD56bright NK cells, which are mainly
localized within the stromal compartment. The accumulation of this NK cell subset within the tumor is driven by the peculiar chemokine milieu in the tumor microenvironment. The neoplastic transformation downregulates CXCL2, that specifically attracts CD56dim NK cells, while
upregulates CXCL9 and CXCL10, that attract the CD56bright NK cells. Moreover, in cancer patients,
both CD56dim and CD56bright NK cell subsets are less functional (low levels of activating receptors,
granzymes-B, perforin and lowered degranulation capability) and in a more immature state (CD117high CD27high CD57low).
Collectively, tumor microenvironment specifically recruits CD56bright NK cells to the tumor stroma
and, at the same time, impair NK cells cytotoxic functions by blocking or reversing their maturation and altering the balance between NK activating and inhibitory receptors.
Tumor-induced impairment of NK cell functions takes place through the action of soluble and cellular immunosuppressive factors in the tumor microenvironment (Fig.4). Different tumor-resident immune cells, such as M2-polarized macrophages, MDSCs, DCs and Treg cells, release
several immunosuppressive soluble factors, such as IL-10, IDO, PGE2, TGF-β, that impair NK cell cytoxicity. The presence of these soluble factors in cancer patient serum correlates with a poor prognosis. Among the immunosuppressive factors in the tumor microenvironment, TGF-β1 seems to play a pivotal role in suppressing NK cell functions. Several reports have shown that TGF-β1 reduces the level of NK cells developing from CD34+ precursors in the bone marrow (Allan,
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Rybalov et al. 2010) and promotes the acquisition of a decidual NK cell-like phenotype by
CD56bright CD16+ NK cells in peripheral blood (Keskin, Allan et al. 2007). Moreover, TGF-β1
interferes with NK cell infiltration in the tumor modulating their chemokine and activating receptors expression (Castriconi, Dondero et al. 2013; Bottino, Dondero et al. 2014). Besides direct immunosuppression of NK cells, another important mechanism to evade tumor immunosurveillance is via the elusion of NK cell recognition. Tumor cells can shed activating ligands from the cell surface through the action of metalloproteinases, exosomes or by active secretion. These soluble activating ligands released by tumor cells have a two-fold advantage for the tumor, reducing the density of activating ligands on tumor cells and the density of receptors for these ligands on NK cells. Tumor cells escape from NK cell-mediated killing up-regulating non canonical HLA molecules on the cell surface, such as HLA-E (ligand of NKG2D/CD94) (Stabile,
Fionda et al. 2017).
Figure 4. Inhibiting interactions between NK cells and both immune and non-immune cells comprised in the tumor
microenvironment. The tumor microenvironment suppresses NK cell function through soluble factors such as TGF-β1, IL-10, IL-6, IDO, PGE2 and soluble NKG2D ligands. The main contact-dependent immunosuppressive interactions in the tumor microenvironment are PD-1 - PD-L1 and CTLA-4 - CD80/CD86.
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microRNAs
MicroRNAs (miRNAs) are a family of short non coding RNAs, of 22 nucleotide in length which silence gene expression at the post-transcriptional level via deadenylation, degradation or translational repression of the target mRNA.
The motif at the 5' end of the miRNA that spans from nucleotide position 2 to 7, termed “seed region”, binds full or partially complementary sequences located at the 3' untranslated region (3’-UTR) of the target mRNA. This interaction function as a guide for the localization of the silencing complex near to the target mRNA, which ultimately results in gene silencing. The majority of eukaryotic coding RNAs contain at least one miRNA-binding site in the 3'-UTR, thus the biogenesis and function of this small non-coding RNAs must be tightly regulated (Fig.5). For these reasons, miRNAs dysregulation is generally associates with developmental disorders, neurodegenerative diseases and cancer.
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precursor called pre-miRNA. This precursor translocates to the cytoplasm, where is further cleaved by Dicer1, a ribonuclease III enzyme that produce the mature miRNA. Only the guide strand of the mature miRNA is loaded onto miRISC (miRNA-induced silencing complex), which contains AGO proteins. The miRNA guide strand directs the miRISC to target mRNAs by sequence complementarity and negatively regulates gene expression through translational repression or targeted mRNA degradation (Lin and Gregory 2015) .
MiRNAs originate from sequences located in various genomic contexts, including intergenic and intragenic regions, exons and introns. MiRNA loci can be isolated or grouped in tandem to form a miRNA cluster, whose components are co-transcribed. miRNA transcription is carried out by RNApol-II. This enzyme produces a miRNA precursor called pri-miRNA, generally more than 1 kb long, that is characterized by a stem-loop structure. This precursor is processed by the nuclear RNase-III Drosha generating a small hairpin-shaped RNA of about 65bp in length, called pre-miRNA, with two nucleotide single stranded overhangs at the 3' end.
Drosha is a nuclear protein of about 160 kDa that belongs, with Dicer1, to a family of RNase-III-type endonucleases that act specifically on double stranded RNAs. The carboxyl terminus of Drosha is fundamental for its activity, indeed these region comprises two RNase-III domains that dimerize, forming an active processing center which cleaves the pri-miRNA. The dsRNA-binding activity provided by the middle region of Drosha is necessary but not sufficient for the interaction with the pri-miRNA. Additional binding activity is provided by Dgcr8, a protein of 90 kDa, which comprises two dsRNA-binding domains and coordinates a ferric ion necessary for efficient pri-miRNA processing (Barr, Smith et al. 2012).
The pre-miRNA molecule is transported to the cytoplasm, where maturation is completed. The translocation is carried out by the Exportin-5, that forms a transport complex interacting with the 3'- overhangs of the pre-miRNA and the GTP-binding protein RAN.GTP. This complex allows the transport of the assembled pre-miRNA through the nuclear pore and his release into the cytoplasm via GTP hydrolization.
At the cytoplasm, Dicer1, another RNase III enzyme, cleaved the pre-miRNA near the terminal loop, producing the mature double stranded miRNA. Like other RNase-III enzymes, Dicer interacts with a dsRNA-binding protein, TAR RNA-binding protein (TRBP) in humans. TRBP modulates the processing efficiency of Dicer and the length of the mature miRNA, but does not seem to be essential for the pre-miRNA processing activity (Chakravarthy, Sternberg et al. 2010; Fukunaga,
Han et al. 2012; Lee and Doudna 2012).
The double stranded miRNA, generated by Dicer1, is subsequently loaded onto a particular type of Ago protein that, along with cofactors of Gw182/Tnrc6 family, form a ribonucleoprotein complex,
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called miRNA-induced silencing complex (miRISC). MiRISC assembly occurs though two sequential steps: RNA loading and its subsequent unwinding.
Once miRNA duplex is loaded onto the miRISC complex, the passenger strand is removed to generate a functional complex. The elimination of the passenger strand occurs via two different mechanisms. The passenger strand of perfectly matched miRNA duplexes is directly sliced by Ago2, whereas the passenger strand of miRNA duplexes with a single mismatch is unwinded without cleavage. As the majority of miRNA duplexes are imperfectly matched, the unwinding mechanism may represent a more general process. The selection of the guide strand is determined, during the loading phase of the miRNA onto the Ago proteins, on the basis of the thermodynamic properties of the RNA duplex ends.
However, the strand selection is not always the same for a given miRNA duplex, indeed an alternative strand selection has been observed, in which the passenger strand is loaded onto the Ago protein. This passenger strand is less abundant and less efficient in gene silencing than the canonical guide strand. Therefore, for a given miRNA duplex, two active miRISC complex can potentially generate (Chiang, Schoenfeld et al. 2010). This differential selection of the strand loaded onto the miRISC may be due to alternative processing by Drosha, which generates miRNA duplex with different 5' thermodynamic properties (Wu, Ye et al. 2009).
Mature miRNAs can interact with other Ago proteins (Ago1, Ago3 and Ago4) on the basis of structural properties of the duplex. In addition, the identity of the 5' nucleotide of the guide strand also contributes to the differential interaction with Ago proteins. This differential interaction has functional consequences on the mechanism by which the expression of a target gene is silenced. Indeed, Ago2 is the only human Ago protein that can slice perfectly matched target mRNA, whereas all the other Ago proteins can repress the translation of the target mRNA only by blocking the translational machinery and favoring the interaction with mRNA decay factors (Huntzinger and
Izaurralde 2011).
It has been demonstrated in several reports that, in the absence of functionally active Drosha or Dicer, small amount of miRNAs can be still detected. Thus, alternative pathways, Drosha-independent and Dicer-Drosha-independent, can generate miRNAs or miRNA-like small molecules.
For example, miRNAs can be produced during a common splice event which generates a small RNA called Mirtron. During the splicing, the RNA fragment sliced from the mRNA is debranched and refolded into a stem loop structure that resembles a pre-miRNA that is subsequently processed to form a mature miRNA (Flynt, Greimann et al. 2010). In addition, miRNAs can generate from other small RNAs in the cell, in the absence of Drosha processing. For example, miRNAs are produced from short hairpin RNAs (shRNAs), transfer RNAs (tRNAs) (Babiarz, Ruby et al. 2008;
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Chong, Zhang et al. 2010) and small nucelolar RNA (snoRNAs) (Ender, Krek et al. 2008). The
only documented miRNA that does not require Dicer processing is miR-451. Indeed, pri-miRNA-451 is too short for DICER processing, therefore Ago2 directly cleaves the precursor, generating a 30 nucleotide long small RNA that can be further processed and function as a miRNA (Cheloufi,
Dos Santos et al. 2010; Cifuentes, Xue et al. 2010). However, only 1% of all the miRNAs are
produced via alternative pathways and these alternative miRNAs results less efficient in silencing gene expression than miRNAs that follow the canonical biogenesis.
miRNAs abundance dynamically change over the time and under stress pressure to face new environmental conditions and, just as protein-coding oncogenes or tumor suppressor genes, miRNA dysregulation contributes to establish typical cell behaviors hallmarks of cancers, such as extensive proliferation, angiogenesis, resistance to cell death, evasion from immune cells-mediated attack and acquisition of invasive phenotype.
It has been reported that the expression of several miRNAs is dysregulated, both in cancer cells and stromal cells, as the result of genetic mutations, chromosome instability and altered transcription of miRNAs-containing sequences.
Mutations of components of the miRNAs biogenesis pathway can lead to a global miRNA up- or down-regulation in cancer cells. A global miRNA down-regulation has been reported to associate with poorly differentiated type of cancer. This association between global miRNA down-regulation and cancer may be explained by the roles that miRNAs play in maintaining lineage specific properties. Therefore, miRNAs down-regulation in cancer cells promotes their undifferentiated state and enhance their invasiveness and metastatic potential, acting as tumor-suppressing molecules (Lu, Getz et al. 2005).
Besides the direct effects that miRNAs play in promoting cell proliferation and survival of cancer cells, this small RNAs contribute to the evolution of the tumor microenvironment via the modulation of genes involved in tumor angiogenesis, tumor immune invasion and tumor-stromal interactions. Moreover, miRNA profiles of neighboring cells in the surrounding stroma of cancer cells that lack genomic defects is altered, expanding the role of microRNA in tumor microenvironment.
Several miRNAs have demonstrated the capability to modulate tumor angiogenesis, such as miR-200, miR-103/107, miR-205, let-7 and miR-9. The latter has the unique ability of simultaneously modulate tumor angiogenesis and invasiveness. Indeed, miR-9 causes the down-regulation of E-chaderin, which promotes cell motility and invasiveness, meanwhile this down-regulation leads to β-catenin activation that in turn up-regulates the major pro-angiogenic factor, namely the vascular endothelial growth factor VEGF-A (Ma, Young et al. 2010). MiR-126 has a unique property in
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suppressing breast cancer cells metastasis. Indeed, the miR-126 precursor produce 5p and 3p miRNA species at same frequency and both miRNAs cooperatively regulate the expression of stromal cell-derived factor 1 alpha (SDF-1α), CXCL12 and CCL2 in cancer cells. The diminished expression of the miR-126 targets suppresses the recruitment of mesenchymal stem cell and inflammatory monocytes into the tumor stroma, thereby inhibiting metastasis formation (Zhang,
Yang et al. 2013).
Unlike normal fibroblasts, CAFs enhance epithelial to mesenchymal transition and promote tumorigenesis via secretion of diverse cancer-activating cytokines and chemokines. MiR-214 and miR-31 are down-regulated in CAFs, leading to enhanced expression of pro-tumorigenic chemokines (CCL5, CCL20 and CXCL8) and SATB2 which promote tumor cell migration (Aprelikova, Yu et al. 2010; Mitra, Zillhardt et al. 2012). MiR-320 is another tumor suppressor miRNA regulated in CAFs, which targets ETS2, MMP9 and Emilin2. MiR-320 down-regulation is responsible for the typical CAF secretome, composed of MMP9, MMP2, BMP1 and LOXL2, that enhances angiogenesis and tumor cell invasion (Bronisz, Godlewski et al. 2011). Finally, miR-15a and miR-16-1 are down-regulated in both tumor cells and CAFs, targeting diverse molecules in each cell types. In cancer cells, miR-15a and miR-16-1 target Bcl-2, cyclin-D1 and Wnt3, whereas in CAFs the same miRNAs regulate the expression of the fibroblast growth factor 2 (FGF2) and its receptor FGFR1, thus enhancing the proliferation and migration of both cell types (Musumeci, Coppola et al. 2011).
It has been widely demonstrated that several distinct miRNAs regulate immune cell activation, maturation, differentiation and function. Thus, it’s not surprising that dysregulation of miRNAs in tumor infiltrating immune cells leads to an altered immune cell response against cancer cells. MicroRNAs modulate a multitude of physiological and pathophysiological processes in immune cells.
200 unique miRNAs are expressed in human and mouse NK cells, with a significant interspecies overlap that indicates their functional importance. Among these 200 miRNAs the highly expressed ones in NK cells are 150, 23b, 29a, 23a, 16, 21, let-7a, 24, miR-15b, miR-720, let-7g, miR-103 and miR-26a. Global miRNAs depletion experiments in mice, via Dgcr8 and Dicer depletion, led to an overall reduction of NK cells in the bone marrow and in peripheral tissues. Moreover, the remaining NK cells in these mice were mainly immature. The same developmental defects are caused by global miRNA overexpression. Therefore, a precise balance of miRNAs is required for proper NK cell development (Bezman, Cedars et al. 2010). NK and T cells share many common features. Indeed, both are endowed with cytotoxic functions and INF-γ production, they share a common lymphoid precursor and some other developmental
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features such as the reliance on the common γ-chain-dependent cytokines for survival during hematopoiesis and homeostasis and the need of an education process during the development. Based on these parallels, it can be seen that some miRNAs modulates common pathways of activation, homeostasis and development in both NK and T cells (Cobb, Nesterova et al. 2005;
Muljo, Ansel et al. 2005).
In conclusion, miRNAs are fundamental modulators of all aspects of the NK cell life, ranging from development to homeostasis and effector functions, thus highlighting the importance of post-translation regulation of gene expression for the proper function of the immune system.
NEUROBLASTOMA
Neuroblastoma (NB) is the most common extracranial tumor of childhood that originates at the adrenal gland and peripheral nerves and metastasize in the bone marrow. The etiology of neuroblastoma is currently unknown, but both somatic and germline genetic alterations seem to be important for the establishment of this cancer type in infants. Several genetic abnormalities have been described for Neuroblastoma. The most common genetic alteration is the MYCN locus amplification, which is located at the chromosome 2p24. MYCN is a transcription factor which regulates the progression through the G1 phase of the cell cycle. The expression of MYCN in Neuroblastoma cells is ~100 times higher than in non-malignant cells. This MYCN accumulation ensures that tumor cells stay in cycle and do not enter the G0 phase. MYCN amplification is associated with a poor prognosis. Generally, this DNA amplification is correlated with chromosome 1p loss of heterozygosity (LOH). It is not known what is the genetic event that contributes to both MYCN amplification and 1p LOH. Other genetic abnormalities have been identified in primary Neuroblasta, such as DNA amplification from chromosome 2p22 and 2p13, trisomy for 17q, allelic loss of 11q (which inversely correlate with MYCN amplification and 1p LOH) (Brodeur 2003). Risk group stratification is based on patient’s age and the presence/absence of these genetic alterations in Neuroblastoma cells. Low-risk patients are identified as those individual <18 months, with localized non-MYCN amplified tumors at diagnosis. High risk Neuroblastoma includes stage 4 patients <18 months with MYCN amplification or >18 months with or without MYCN amplification. Statistically, high-risk Neuroblastoma patients have a 5 year survival rate <50%, despite aggressive chemotherapeutic treatments.
Standard treatments for high risk Neuroblastoma use multi-modal therapeutic approaches comprising chemotherapy, radiotherapy, surgical debulking or excision of primary tumors, autologous bone marrow transplantation and differentiating agents, such as 13-cis-retinoic acid.
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Despite aggressive therapies, most patients present with recurrence of metastatic foci and multidrug resistance. Therefore, new and more effective therapeutic intervention are needed to ensure a better survival rate for Neuroblastoma patients, and immunotherapy strategies have been developed. These therapies relies on the use of cytokines, antibodies and cells. IL-12 is a critical activator of NK and T-lymphocytes which is really effective in the induction of antitumor immunity. Different trials showed that this cytokine is effective for the treatment of adult malignancies. IL-12 induces in xenograft models of Neuroblastoma in mice a potent antitumor response probably mediated by both NK cells and CD8+ T cells. Further studies showed that IL-12 in combination with IL-2 or IL-18 is
more effective than IL-12 alone in the induction of antitumor immunity against Neuroblastoma cells (Iinuma, Okinaga et al. 2006) .
Active vaccination is another well studied strategy for the treatment of Neuroblastoma (Fig.6).
Figure 6. Immunotherapeutic strategies to treat Neuroblastoma. IL-12 alone or in combination with IL-2 or IL-18
induces a potent antitumor response against Neuroblastoma, mediated by both NK and T-cells. A DNA vaccine that drives the expression of GD2 immunogenic peptides in dendritic cells was developed. Several anti-GD2 antibodies have been developed until now.
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Different groups developed DNA vaccines to drive the expression of immunogenic peptides in DCs or in Neuroblastoma cells to stimulate the immune system against cancer, the most effective targeting GD2. This molecule is a ganglioside expressed on the surface of Neuroblastoma cells. Its specific function is not completely understood but it seems to mediate the adhesion of Neuroblastoma cells to extracellular matrix. Because GD2 expression seems to be primarily restricted to NB, it became the major target of vaccine. Several anti-GD2 antibodies have been developed until now. Murine anti-G2D antibodies resulted in limited tumor responses and the development of human anti mouse antibodies (HAMAs), which further reduce the therapeutic benefits of the antibody treatment. Although the first clinical results for anti-G2D antibodies were not very encouraging, human-mouse chimeric anti-G2D antibodies fueled the interest into this therapeutic approach. Indeed, these humanized antibodies retained the anti-G2D specificity and were 50-100 times more efficient than the murine counterpart in mediating ADCC responses against Neuroblastoma cells in human patients. Cellular therapies for Neuroblastoma mainly rely on hematopoietic stem cell transplantation (HSCT). As for the other immune-based therapeutic approaches, the effectiveness of the graft-versus-tumor (GvT) reaction is limited and in most cases inadequate (Navid, Armstrong et al. 2009).
Treatment with anti-G2D antibodies in combination with GM-CSF and IL-2 resulted in improved clinical benefits and, recently, this treatment has been included in the standard care of high-risk Neuroblastoma patients. The therapeutic benefits of anti-G2D antibodies depends on FcγR-mediated activation of NK cells and other immune cells, such as granulocytes and macrophages. Several reports showed that NK cells recognize and kill Neuroblastoma cells both in vitro and in
vivo (Fig.7). DNAMI-1 is the main activating receptor involved in NK-mediated killing of NB cells,
whereas Nkp30 and NKp46 give only a minor contribution. Neuroblasts isolated from the BM of NB patients do not express MICA and MICB on their surface and among these cells only 50% express ULBP-2. Based on these results, it seems that NKG2D is not involved in NK-mediated cytotoxicity against NB cells (Castriconi, Dondero et al. 2004).
B7-H3 is a tumor promoting molecule expressed on NB cells. This molecule drives tumor cell development by different molecular mechanisms, such as the promotion of invasiveness and the reduction of chemotherapeutic-induced apoptosis. Moreover, B7-H3 on primary Neuroblasts reduced NK cell-mediated cytotoxicity (Castriconi, Dondero et al. 2004). B7-H3 combines immune escaping and tumor promoting functions, being an important therapeutic target for the development of new treatment for Neuroblastoma patients.
As other tumor types, Neuroblastomas induce a strong suppression of the antitumor immune response. In this regard, several molecular events dampen NK cytotoxicity against NB cells, such as
