The role of CCN family genes in haematological malignancies

Abstract

Haematological malignancies, although a broad range of specific disease types, continue to show considerable overlap in classification, and patients are treated using similar chemotherapy regimes. In this review we look at the role of the CCN family of matricellular proteins and indicate their role in nine haematological malignancies including both myeloid and lymphoid neoplasms. The potential for further haematological neoplasms with CCN family associations is argued by summarising the demonstrated role of CCN family genes in the differentiation of haematopoietic stem cells (HSC) and mesenchymal stem cells. The expanding field of knowledge encompassing CCN family genes and cancers of the HSC-lineage highlights the importance of extracellular matrix-interactions in both normal physiology and tumorigenesis of the blood, bone marrow and lymph nodes.

Introduction

Haematological malignancies represent cancers of the bone marrow, blood and lymph nodes. Trends for haematological malignancies show some variation geographically. Data from European cancer registries for the years 2000–2002 found an overall incidence rate of 39.37 per 100 000 people for all haematological malignancies (Sant et al. 2010). In the USA an incidence rate of 49.7 per 100 000 people was reported for 2011 (Surveillance, Epidemiology and End Results Program (SEER) Online Database). A comparison of the incidence of haematological malignancies between Japan and the USA from 1993 to 2008 found twice the incidence rate in the USA than Japan (Chihara et al. 2014).

Haematopoietic stem cells (HSCs) are multipotent, self-renewing progenitor cells that differentiate into all lymphoid and myeloid lineages of the blood and immune system (Fig. 1). Haematological malignancies arise from mutations that occur along the HSC differentiation pathway. Their classification has been one of the most complex as the criteria for disease definition has changed over time and across regions, along with the advances in immunophentoypic, molecular biological and cytogenetic techniques. Lymphoid neoplasms covers a large number of disease types, and prior to the early 1990s were primarily classified as leukaemia or lymphoma. However, the introduction of new techniques found significant overlap among the classification systems in place, and led to the proposal that the leukaemia/lymphoma and Hodgkin lymphoma/Non-Hodgkin lymphoma distinctions were not helpful. Further, cytogenetic studies revealed chromosomal translocations and other genetic markers could be used to further classify disease in a way that benefited clinicians and patients. Classification according to cell lineage (B-, T- or NK-cell) and precursor or mature cell status is currently implemented. Cancers of myeloid origin are largely chronic myelodysplastic or myeloproliferative diseases, and include chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML). The World Health Organisation regularly compiles and updates an international classification system for haematological malignancies. They stratify distinct diseases by cell lineage and their derivation from precursor or mature cells, and these standards have been applied to the classification of myeloid, lymphoid, mast cell and dendritic/histiocytic neoplasms (Ruhl et al. 2015; Swerdlow et al. 2008). It is a certainty, that as our knowledge of haematological malignancies increase, further important sub-divisions will be made (Campo et al. 2011).

Fig. 1.

The bone marrow niche including HSC and MSC lineages. This schematic highlights that both HSCs and MSCs are found at two sites within the bone marrow, the perivascular (near the sinusoids) and endosteal niche (near the bone). HSCs ultimately differentiate into all constituents of the immune system comprising the lymphoid and myeloid lineages. MSCs are known to differentiate in vivo into fat cells (adipocytes), cartilage (chondrocytes), bone (osteoblasts/ osteocytes) and stromal cells such as fibroblasts and adventitial reticular cells. Bone lining cells are also though to arise from MSCs. HSC haematopoietic srem cell; MSC mesenchymal stem cell; ECM extracellular matrix; RBC red blood cell; MPP multipotent progenitor; CLP common lymphoid progenitor; CMP common myeloid progenitor

A unifying feature of cancers of the haematological lineages would be that they form in highly connected bodily tissues. Thus, at diagnosis these diseases are often disseminated through the blood, bone marrow, lymph nodes or spleen. It is for this reason that haematological malignancies were traditionally viewed as “liquid” tumours in contrast to their solid tumour counterparts. Yet the differences between solid and liquid tumours continue to diminish. Haematological neoplasms have been shown to harbour all the hallmarks of cancer (Hanahan and Weinberg 2011). The bone marrow microenvironment in particular is increasingly recognised as an important determinant of tumour development, where surrounding stroma and immune cells can influence the initiation and progression of haematological malignancies (Ayala et al. 2009; Burger et al. 2009; Riether et al. 2015). This is similar to normal haematopoiesis, where the microenvironment tightly regulates the maintenance and function of HSCs through their location at two distinct niches, surrounding the bone or vasculature, providing different cues for quiescence or differentiation (Fig. 1) (Morrison and Scadden 2014). The bone marrow microenvironment is comprised of cells largely derived from mesenchymal stem cells (MSCs), also referred to as mesenchymal stromal cells or multipotent stromal cells. Differences between in vitro culture and their natural tendencies in vivo have led to multiple definitions of MSCs (Bara et al. 2014; da Silva Meirelles et al. 2008). In vivo, MSCs have the ability to self renew and differentiate into bone, cartilage, fat deposits and stromal cells (Fig. 1). Another barrier to identification is that MSCs are phenotypically similar to adventitial reticular cells and fibroblasts, both found within the bone marrow microenvironment (Haniffa et al. 2009; Hematti 2012; Jones and McGonagle 2008). The unique identification of MSCs has been obscured due to the large overlap in expression markers with these other stromal cells and their own heterogeneous marker expression within the same tissue (Bühring et al. 2009; Siclari et al. 2013; Sudo et al. 2007). Recently, new methods of MSC identification by their adhesive properties has been suggested (Roncoroni et al. 2013). Fibroblasts are a long-studied cell type, traditionally viewed as ubiquitous adherent cells that were not endothelium, epithelium or haematopoietic in origin, they secrete extracellular matrix (ECM) precursors. The ECM provides the structural scaffold and spatial framework of the bone marrow and together with signalling from matricellular proteins play a critical role in haematological malignancy development and response to therapy (Chong et al. 2012).

In this review, we collate the evidence that one such matricellular signalling family, CCN, plays an important role across a diverse range of haematological malignancies. Then we provide support that further associations will be made with as-yet unstudied haematological malignancies, by highlighting the role CCN proteins play in regulating the fate and function of HSCs, MSCs and their lineages.

CCN structure and function

The CCN genes are a family of six members containing CYR61/CCN1 (cysteine-rich 61), CTGF/CCN2 (connective tissue growth factor), NOV/CCN3 (nephroblastoma overexpressed), and WISP-1/CCN4, WISP-2/CCN5, and WISP-3/CCN6 (Wnt-inducible-secreted proteins) (Bork 1993). The family members, except CCN5, each have four conserved cysteine rich-domains, with sequence homology to the insulin-like growth factor-binding proteins (IGFBP), the von Willebrand factor C (VWC) domain, Thrombospondin type 1 repeat (TSR), and a C-terminal domain with a cysteine-knot motif. CCN5 consists of the first 3 domains omitting the C-terminal cysteine knot. CCN genes as matricellular proteins are key orchestrators of cell behaviour and act in a context- and tissue-specific manner to influence a wide range of cellular activities. Under normal physiological conditions, CCN family members are involved in regulating endochondral ossification, survival of olfactory bulb interneurons, pancreatic β-cell development, suppression of hair follicle stem cells, possibly dental mesenchymal to epithelial cross-talk, retinal vasculature around the optic nerve, cardiac development and regulation of angiogenesis (Hall-Glenn and Lyons 2011; Kubota and Takigawa 2015; Rhodes and Simons 2007). In particular, CCN1 and CCN5 null mice show embryonic lethality and CCN2 null mice show perinatal lethality, indicating these CCN genes are essential for normal embryogenesis (Ivkovic et al. 2003; Mo et al. 2002; Russo and Castellot 2010). Studies on CCN family genes have commonly focused on human pathologies, and the family of genes has been shown to play a role in inflammation, wound healing, atherosclerosis, fibrosis, arthritis, retinopathy of the eye and tumourigenesis (Jun and Lau 2011; Kubota and Takigawa 2015; Kular et al. 2011). They have been shown to be powerful regulators of inflammatory modulators, such as chemokines, cytokines and other growth factors, in a context-specific fashion. Their range of functions is due to their modular nature that allows them to interact with a multitude of extracellular signals and cellular receptors. Ligands shown to interact with the CCN family include transforming growth factor β (TGFβ), bone morphogenic proteins (BMPs), vascular endothelial growth factor (VEGF), glycosominoglycans and ECM components (Desnoyers 2004; Kular et al. 2011; Leask and Abraham 2006). CCN family members bind to a large number of cell adhesion receptors, such as tropomyosin receptor kinase A (TrkA), Notch-1, low-density lipoprotein receptor-related proteins (LRPs), several specific dimers of integrins and heparan sulfate proteoglycans (Chen and Lau 2009; Rachfal and Brigstock 2005). Though for some cell types the receptor for CCN proteins remains unknown.

Role in myeloid neoplasms

Although no mast cell or dendritic/histiocytic neoplasms have been reported to have an association with CCN family members, two CCN proteins have been associated with myeloid neoplasms, CCN1 with AML and CCN3 with CML (Table 1).

Table 1.

Summary of CCN-associated haematological malignancies

WHO classification		CCN family member	Haematological malignancy	CCN-expressing cell	Disease outcome	Reference (first cited)
Myeloid neoplasms		CCN1	Acute myelogenous leukaemia (AML)	Over-expressed in AML	Benefits neoplasm	Niu et al. 2014
		CCN3	Chronic myelogenous leukaemia (CML)	Under-expressed in BCR-ABL-positive CML	Benefits neoplasm	McCallum et al. 2006
Lymphoid neoplasms	Precursor lymphoid neoplasms	CCN2	B-cell precursor acute lymphoblastic leukaemia (ALL)	Over-expressed in B-cell precursor ALL	Benefits neoplasm	Vorwerk et al. 2000
	Mature B-cell neoplasms	CCN1	Multiple myeloma	Over-expressed in tumour-associated MSCs	Inhibits neoplasm	Santra et al. 2011
		CCN2	Multiple myeloma	CCN2 whole protein reduced and N-terminal fragment increased in serum	Inhibits neoplasm	Munemasa et al. 2007
			Diffuse large B-cell lymphoma (DLBCL)	Over-expressed in DLBCL sub-types with stromal infiltration	Inhibits neoplasm	Blenk et al. 2007
			Hodgkin lymphoma	Over-expressed in nodular sclerosis Hodgkin lymphoma	Inhibits neoplasm	Birgersdotter et al. 2010
			Mantle cell lymphoma	Under-expressed in tumour cells	Unknown	Rizzatti et al. 2005
		CCN4	Multiple myeloma	Under-expressed in tumour-associated MSCs	Unknown	Corre et al. 2007
	Mature T-cell and NK-cell neoplasms	CCN1	Peripheral T-cell lymphoma	Over-expressed in tumour cells	Unknown	Mahadevan et al. 2005
			Angioimmunoblastic lymphoma	Over-expressed in tumour cells	Unknown	Piccaluga et al. 2007b
		CCN2	Peripheral T-cell lymphoma	Over-expressed in tumour cells	Unknown	Mahadevan et al. 2005

CCN1

CCN1 was found to be over-expressed in bone marrow samples from AML patients, showing heterogenous levels of expression when compared by immunoblots with normal bone marrow samples (Niu et al. 2014). In the same study, CCN1 was reported to be over-expressed in AML cell lines U937 and Kasumi-1 compared with CML or T-cell ALL cell lines. A CCN1 monoclonal antibody or siRNA were shown to reduce proliferation and increase apoptosis in AML cell lines through up-regulation of Bcl-2-associated X protein (Bax) and down-regulation of B-cell lymphoma- extra large (Bcl-xl) and c-Myc, which were regulated through reduced phosphorylation of MEK and ERK. The CCN1 siRNA also showed increased AML cell apoptosis in combination with cytarabine treatment in vitro (Niu et al. 2014).

CCN3

The CCN3 gene has been linked to CML. Specifically, the BCR-ABL oncogene, a common feature of CML, was shown to reduce CCN3 gene and protein expression in the multipotent haematopoietic cell line FDCP-Mix (McCallum et al. 2006). After treatment with imatinib (a BCR-ABL tyrosine kinase inhibitor) or after complete remission, CML patient bone marrow samples were found with increased CCN3 expression, equivalent to normal healthy bone marrow. Further, when the K562 CML cell line was transfected with a full length CCN3 plasmid, cells were forced into growth arrest and had an enhanced susceptibility to imatinib (McCallum et al. 2009). McCallum et al. (2012) illustrated through in vitro studies that CCN3 upregulated integrin expression leading to increased cell adhesion and upregulated undetermined growth control mechanisms that prevented phosphorylation of Akt and ERK by BCR-ABL in CML cells. The ability of the BCR-ABL tyrosine kinase to downregulate CCN3 in CML cells was attributed to microRNA 130 a/b (Suresh et al. 2011). BCR-ABL was also shown to inhibit NOTCH1-CCN3 signalling contributing to the pathogenesis of CML (Suresh et al. 2013). CCN3 had previously been shown to be a non-canonical NOTCH1 ligand (Sakamoto et al. 2002), in CML the loss of CCN3 would result in a missing regulatory effect on the NOTCH1 signalling pathway.

Role in lymphoid neoplasms

In collating CCN family associations with lymphoid neoplasms we have included studies with gene expression profiling data identifying CCN family members without any subsequent validation, this is to expand on the small range of CCN-associated haematological malignancies and indicate possible avenues for future study. Only CCN2 has been associated with a precursor lymphoid neoplasm, CCN1, CCN2 and CCN4 have been associated with four mature B-cell neoplasms and CCN1 and CCN2 have been associated with two mature T-cell neoplasms (Table 1). CCN family associations with multiple myeloma, a disease of terminally differentiated plasma cells that accumulate in the bone marrow, are connected to bone osteolysis, or bone disease, which is due to the activation of osteoclasts and impairment of osteoblast differentiation by the myeloma cells.

CCN1

CCN1 is a biomarker and possible therapeutic target in multiple myeloma. Immunohistochemistry on bone marrow spicules or from bone marrow core biopsies from multiple myeloma patients found CCN1 to be expressed strongly in the cytoplasm of lymphoid and myeloid cells but not plasma cells (Santra et al. 2011). A possible role of CCN1 in the MSC response to multiple myeloma indicated the protein could reduce bone lesions (Li et al. 2012). The study used a multiple myeloma cell line (Hg), injected directly onto subcutaneously engrafted rabbit bone in untreated SCID mice. When these multiple myeloma-engrafted SCID-rab mice were given intra-bone injections of MSCs, they had absence of bone disease and reduced multiple myeloma cell growth. Weekly intravenous injections of MSCs prevented bone disease with no effect on tumour growth. MSCs were shown to contain high levels of CCN1 by microarray, among other bone matrix proteins such as Decorin and Lumican, compared with HSCs and the HG multiple myeloma cell line. MSC-derived CCN1 was found to increase transcription and splicing of CCN1 pre-mRNA in the IL-6-dependent multiple myeloma cell line, INA-6 (Dotterweich et al. 2014). Splicing of all introns required the CT domain of CCN1 and an RGD-binding site on INA-6 cells, though the specific RGD-associated integrin receptor was not discovered. Increased splicing led to increased CCN1 protein expression from INA6 cells. These results indicate CCN1 is acting as a microenvironmental-derived anti-myeloma factor. The largest study found CCN1 expression was significantly higher in bone biopsies from 246 multiple myeloma patients compared with 24 healthy controls, measured by microarray analysis and validated by qRT-PCR (Johnson et al. 2014). CCN1 was highly expressed in cultured MSCs in healthy controls and multiple myeloma patients, but not detected in osteoclasts, B-lymphocytes and normal or multiple myeloma plasma cells. Further, CCN1 correlated with microenvironmental-specific gene expression and not genes associated with multiple myeloma cells. CCN1 protein levels, as detected by enzyme-linked immunosorbent assays (ELISA), where significantly higher in a subset of patients with multiple myeloma (including patients with disease, in complete remission and at relapse) as well as patients with asymptomatic multiple myeloma and monoclonal gammopathy of undetermined significance, both benign precursors to multiple myeloma. Univariate and multivariate stepwise Cox regression analysis found high expression of CCN1 was linked to improved overall survival. Johnson et al. (2014) found low CCN1 levels may also serve as a risk marker for progression from asymptomatic multiple myeloma and monoclonal gammopathy of undetermined significance to multiple myeloma or a longer time to complete remission after treatment. In a SCID-hu mouse model engrafted with the multiple myeloma cell line H929, CCN1 delivered as a recombinant protein or by multiple myeloma cell line over-expression acted to reduce osteolysis through increasing osteoblast numbers and inhibiting osteoclast proliferation, the underlying mechanism acted at least partially through integrin α_Vβ₃ binding sites (Johnson et al. 2014).

CCN1 has been associated with T-cell lymphomas. CCN1 was found over-expressed 70-fold in unspecified peripheral T-cell lymphoma compared with normal T-cells or lymph node by gene expression array (Mahadevan et al. 2005). Two types of peripheral T-cell lymphomas, unspecified and angioimmunoblastic lymphoma, showed CCN1-positive staining in tumour cells in more than 95 % of patient tissue samples by immunohistochemistry (Piccaluga et al. 2007a, b). Some evidence of nuclear staining in addition to the cytoplasm was sporadically observed.

CCN2

CCN2 was found to be over-expressed in B-cell precursor acute lymphoblastic leukaemia (ALL) compared with normal HSCs and normal precursor B cells in both children and adult patient cohorts (Boag et al. 2007; Sala-Torra et al. 2007; Tesfai et al. 2012; Vorwerk et al. 2000). In children expression of CCN2 was part of a gene signature indicating worse outcome in high-risk stratified patients (Kang et al. 2010). CCN2 has been present in gene expression clusters for the TEL/AML1 subtype of B-cell precursor ALL (Gandemer et al. 2007) and MLL translocations in high-risk B-cell precursor ALL (Harvey et al. 2010). Silencing of CCN2 in two B-cell precursor ALL cell lines was shown to reduce proliferation in vitro through AKT/mTOR inactivation and increased cyclin independent kinase inhibitor protein 27 (CDKN1B) (Lu et al. 2013). In addition, a CCN2 monoclonal antibody showed evidence of improving chemotherapy response in a NOD/SCID xenograft mouse model of B-cell precursor ALL (Lu et al. 2013). In a study indicating CCN2 may play an inhibitory role to tumour growth, human CCN2-knockout MSCs were used to create an adipocyte-rich extramedullary bone marrow in the flanks of immune-compromised mice and led to a higher population of tail-vein injected leukaemia cell lines after 14 days compared to extramedullary bone marrow using control MSCs (Battula et al. 2013). This discordant result could be due to the mouse flank extramedullary sites lacking the full complement of microenvironment cell subsets for CCN2 to act through, CCN2 expression being lost from MSCs rather than leukaemia cells, or the function of CCN2 may be subtype-specific in B-cell precursor ALL.

CCN2 has been associated with four subtypes of mature B-cell neoplasms. These include mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma and multiple myeloma. In mantle cell lymphoma, CCN2 expression was found to be 7-fold lower compared with healthy naive B cells by gene expression analysis (Rizzatti et al. 2005). In DLBCL, CCN2 expression is associated with the less aggressive germinal centre B cell like subgroup (Blenk et al. 2007). CCN2 was also grouped in a gene set of stromal-like DLBCL, identifying tumours with high ECM deposition and infiltration of monocyte-derived cells (Lenz et al. 2008). CCN2, along with osteonectin (SPARC), was associated with hystiocytic infiltration in the lymphoma biopsy specimens. Expression of CCN2 significantly correlated with greater overall survival in DLBCL patients treated with chemotherapy, CCN2 was discovered in addition to 10 other genes by gene expression profiling on pre-treatment diagnostic biopsies (Rimsza et al. 2008). CCN2 has also been associated with classic Hodgkin lymphoma, specific to the less aggressive nodular sclerosis subtype and detected by immunohistochemistry with strong granular staining in Hodgkin-Reed-Sternberg tumour cells (Birgersdotter et al. 2010). CCN2 was also heterogeneously expressed in surrounding fibroblasts, macrophages and occasional lymphoid cells. Nodular sclerosis Hodgkins lymphoma is characterised by a higher infiltrate of activated and regulatory T cells and ECM deposits that form fibrotic bands surrounding the inflammatory and malignant cells. CCN2 is also an indicator of bone disease in multiple myeloma. The concentration of circulating full-length CCN2 protein was lower in the serum of multiple myeloma patients compared with healthy controls by sandwich ELISA (Munemasa et al. 2007). Further, the reduction of CCN2 full-length protein was accompanied by an increase in the detection of the N-terminal fragment of CCN2, indicating protease cleavage of the protein is occurring in multiple myeloma. The increase in N-terminal CCN2 fragments and decrease in full-length CCN2 in multiple myeloma patients were associated with increased bone disease, regardless of whether patients had undergone treatment. While it was suggested matrix metalloproteinases (MMPs) could be cutting the CCN2 protein at the hinge, a specific MMP was not identified (Munemasa et al. 2007). In another study, CCN2 was present in 37 % of non lineage-selected multiple myeloma bone marrow samples but not detectable in healthy donor bone marrow by gene expression array (Hose et al. 2009).

CCN2 was identified as aberrantly expressed in only one mature T-cell neoplasm, with 100-fold over-expression in unspecified peripheral T-cell lymphoma compared with normal T-cells or lymph node by gene expression array (Mahadevan et al. 2005).

Deregulated CCN2 is associated with sub-types of two mature B-cell lymphomas that have better patient outcomes, namely in DLBCL and Hodgkin lymphoma. These mature B-cell lymphomas, along with gallbladder cancer, are the only CCN2-deregulated cancers to be associated with a better survival outcome (Wells et al. 2014). There is currently no functional or patient outcome data for peripheral T-cell lymphoma or mantle cell lymphoma and CCN2. However, the role of CCN2 in multiple myeloma would depend on whether the absence of full-length protein or the increased circulation of the N-terminal fragment was contributing to decreased bone disease.

CCN4

CCN4 has been associated with one mature B-cell neoplasm, multiple myeloma. CCN4 mRNA was reported 3-fold under-expressed in bone marrow MSCs of multiple myeloma patients compared with healthy aged-matched controls (Corre et al. 2007). No further validation or functional studies were performed.

Role in bone marrow HSCs and their lineages

There is abundant evidence that CCN family proteins interact with HSC cells and their myeloid and lymphoid lineages. CCN2 and CCN3 have been associated with HSC differentiation and long-term repopulation. Further, CCN1, CCN2 and CCN3 have been shown to alter the chemotaxis of mature lymphoid and myeloid immune cells in a tissue-specific manner. There is very little overlap between studies on the interaction of CCN genes with immune cell lineages, which suggests that in the haematopoietic system, CCN members are more likely to be acting with other matricellular proteins, cytokines and chemokines than their fellow CCN family members. An exception to this would be the migration and activation of macrophages, where three family members have shown to have influence in tissue-specific contexts.

CCN1

In mice with experimental autoimmune myocarditis CCN1 expression was increased in liver-specific cells, consequently circulating CD11b⁺ macrophages and CD3⁺ T cells showed reduced migration to the heart (Rother et al. 2010). In contrast, liver cell expression of CCN1, during nonalcoholic fatty liver inflammation disease in mice, enhanced the chemotaxis of F4/80-positive macrophages through integrin α_M binding and MEK/ERK signalling pathways (Bian et al. 2013). CCN1 was also shown to increase neutrophil chemotaxis through fibroblast-like synoviocytes in a mouse model of collagen-induced arthritis (Zhu et al. 2013). Circulating human T-cells, B-cells, NK-cells and monocytes bind to CCN1 ex vivo and intracellular CCN1 expression can be increased in response to inflammatory signals (Löbel et al. 2012). Immune cell subsets present in human peripheral blood mononuclear cells where found to express intracellular CCN1, specifically CCN1 protein was evident in vesicles associated with the golgi apparatus (Löbel et al. 2012). In mice, CCN1 was found to bind to macrophages through integrin α_Mβ₂ and Syndecan 4, inducing them to a pro-inflammatory M1 phenotype (Bai et al. 2010; Thorne et al. 2014). CCN1 and CCN2 both adhere to monocytes presenting an integrin α_Mβ₂ binding site in atherosclerotic plaques (Schober et al. 2002) and also bound activated platelets through integrin α_Vβ₃ (Jedsadayanmata et al. 1999).

CCN2

Only one study to date has discovered a role for CCN2 in HSC differentiation to the mature B-cell lineage. Using a mouse chimeric foetal liver transplantation model, loss of CCN2 in the bone marrow stromal compartment resulted in reduced B-cell maturation past the pro-B cell stage and CCN2 was capable of enhancing pro-B to pre-B cell maturation in the presence of interleukin-7 in vitro (Cheung et al. 2014). There is considerably evidence for the role CCN2 plays in the regular functioning of mature immune cells. CCN2 is expressed in γδ T-cells, important in wound healing and fibrotic responses, after TGFβ/interleukin-15 stimulation, but not in CD4-positive αβ T-cells (Workalemahu et al. 2003). These γδ T-cells have been shown to have an anti-lymphoma effect (Kunzmann and Wilhelm 2005). Further, CCN2 was heterogeneously expressed by macrophages in several mature B-cell lymphomas, including Hodgkin, Burkitt and mantle cell lymphoma (Birgersdotter et al. 2010), suggesting CCN2 may be inhibiting mature B-cell lymphomas through tumour cell and immune cell secretion. CCN2 can also prolong survival of dendritic cells in vivo (Cheng et al. 2008). Megakaryocytes have been shown to secrete signals that induce CCN2 secretion by chondrocytes, secreted CCN2 is then taken up by platelets for their use in tissue regeneration and wound repair (Sumiyoshi et al. 2010).

CCN3

An integral role for CCN3 in HSC maintenance and differentiation has been outlined in several studies. CCN3 regulates cell cycling and Notch activation in CD34-positive primitive progenitor cells from cord blood (Gupta et al. 2007). CCN3 has a positive effect on mouse lineage- Sca1⁺c-kit⁺ CD150⁺CD34⁻ HSC long-term repopulation, which is dependent on integrin α_Vβ₃ activation by Thrombopoietin (Ishihara et al. 2014). CCN3 expression increases along the myeloid cell lineage in an opposing gradient to Notch expression in humans (McCallum and Irvine 2009). A role for CCN3 in mature immune cell function was also seen in prostate cancer, where CCN3 was shown to induce M2 macrophage chemotaxis and promote the M1 to M2 phenotype, with increased AKT/NFκB signalling resulting in increased VEGF expression that contributed to angiogenesis at the tumour site (Chen et al. 2014a).

Role in bone marrow MSCs and their lineages

The first comprehensive look at the CCN family of genes and their involvement in MSC differentiation was performed in primary cultures of human bone marrow MSCs (Schutze et al. 2005). Where CCN1, CCN2, CCN5 and CCN6 were expressed at detectable mRNA levels by RT-PCR, with differences in expression upon commitment to specific differentiation pathways (Table 2). CCN1 expression was reduced upon differentiation into all three examined lineages: adipogenic, chondrogenic and osteogenic. CCN2 was slightly reduced during adipogenic and chondrogenic differentiation. CCN5 expression was reduced during adipogenic differentiation and CCN6 expression during chondrogenic differentiation. The study by Schutze et al. (2005) highlighted that these four CCN genes are likely to play a role in maintenance of bone marrow MSCs, but it remained to seen whether they acted co-operatively or independently. Further studies have shown the role CCN genes play is more complex and many may play a transient role in MSC differentiation, acting between the initial and final stages of differentiation (Table 2).

Table 2.

CCN family involvement in human MSC differentiation

^aCCN expression promotes () or inhibits () MSC or committed progenitor differentiation into osteoblast, chondrocyte or adipocyte

^bLoss of CCN expression () in undifferentiated MSCs upon differentiation to osteoblast, chondrocyte or adipocyte, as described by Schutze et al. (2005)

In the developing mouse embryo, CCN1 is transiently expressed in cells of mesenchymal origin undergoing chondrogenesis. Further, CCN1 actively promotes chrondrogenesis in mouse limb bud-derived mesenchymal cells (Wong et al. 1997). CCN1 also promotes osteogenesis in human mononuclear cells in vitro by inhibiting osteoclast proliferation and increasing osteoblast proliferation through Wnt-3A signalling (Crockett et al. 2007; Si et al. 2006). CCN2 has been involved with all three MSC differentiation lineages, supporting both osteogenesis and chondrogenesis during normal skeletal development (Ivkovic et al. 2003; Kubota and Takigawa 2007; Safadi et al. 2003; Wang et al. 2015). CCN2 expression has been associated with mesenchymal progenitor cells within the bone marrow, that were also positive for the MSC markers Twist2 and Osterix (Wang et al. 2015). However, expression of CCN2 was diminished in these mesenchymal progenitor cells during osteoblast maturation suggesting its importance in osteoblast differentiation is transient. CCN2 was shown to inhibit adipogenesis in humanised extramedullary bone marrow within an immune-compromised mouse. In this model CCN2 expressed from human MSCs reduced adipocyte differentiation (Battula et al. 2013). MSCs can also differentiate into fibroblasts and recombinant CCN2 has been shown to promote human MSC differentiation to fibroblasts in vitro, when administered in combination with ascorbic acid (Lee et al. 2010). Fibroblasts derived from MSCs grown with recombinant CCN2 and ascorbic acid were identified by decreased expression of mesenchymal surface epitopes CD44 and alkaline phosphatase and increased production of collagen I, collagen III protein and increased gene expression of fibroblast-specific protein 1 (FSP1), decorin, elastin and HA synthase 3 (Tong et al. 2011). CCN3 has been shown to inhibit osteoblast differentiation though stimulation of Notch signalling and down-regulation of BMP signalling (Katsube et al. 2009; Minamizato et al. 2007). CCN3 has also been linked to chondrocyte differentiation, present in embryonic murine pre-hypertrophic and early hypertrophic chondrocytes and shown to act in opposition to CCN2 in terminal differentiation of chondrocytes (Kawaki et al. 2008; Yu et al. 2003). CCN4 was shown to prevent apoptosis in human bone marrow MSCs by inhibiting the TNF-related apoptosis-inducing ligand 1 (TRAIL) pathway (Schlegelmilch et al. 2014). Studies have also indicated that CCN4 is involved in later differentiation into mature chondrocytes and osteoblasts, in part through regulation of TGF-β1 signalling (French et al. 2004; Inkson et al. 2008; Yanagita et al. 2007). Other transmembrane signalling pathways contributing to MSC maintenance that CCN family genes could be interacting with include the Leptin, Wnt, TGFb/BMP and Delta like kinase (Dlk)/Pref-1 pathways (Gimble et al. 2006). Additional evidence of the important role CCN family genes perform in MSC maintenance and differentiation is their association with bone pathologies. All CCN family members, except CCN5, have been associated with primary bone cancers, such as osteosarcoma, Ewing’s sarcoma and chondrosarcoma, or metastatic bone cancers derived from primary breast or prostate tumours (Chen et al. 2014b).

Conclusion

The field of study for CCN family genes and haematological malignancies is currently relatively small. While the breadth of neoplasms associated with CCN is quite diverse (Wells et al. 2014), most haematological diseases still need robust patient cohort validation and insight into CCN function. Even so, some generalisations can already be made and there is room for hypothesis on future directions in the field.

The first finding relates to CCN-specificity. Inclusion of gene expression profiling studies, even in the absence of functional mechanisms or disease outcomes, highlights the independent nature of CCN proteins in the haematological system. Largely, there is one solitary CCN gene in each newly uncovered gene signature. Only one of the studies presented here, using large scale gene or protein arrays, has identified multiple CCN genes, and that was in peripheral T-cell lymphoma, with CCN1- and CCN2-positive protein expression. CCN gene association with lymphoid or myeloid neoplasms appears to have little redundancy or co-operation among CCN family members.

The second finding is that the cell type producing CCN may determine the disease outcome, with MSC-derived CCN expression inhibiting neoplastic growth and HSC-lineage expression of CCN supporting neoplastic growth. As is seen with MSC-derived CCN1 expression in multiple myeloma, which acts to reduce osteolysis. It is unknown if CCN2 proteins found in multiple myeloma patient serum was from a haematopoietic or mesenchymal cell compartment, though if CCN2 was to work in a similar way to CCN1 in this disease it is plausible mesenchymal cells are at least partly responsible as bone osteolysis is also reduced in these patients. More indirectly, MSC-lineage cells may be involved in DLBCL and Hodgkin lymphoma, where CCN2 expression was associated with stromal infiltration of the tumour and in both these cases was associated with disease subtypes with better patient outcomes. CCN4 is under-expressed in MSCs of multiple myeloma patients, and whether they follow this pattern and then promote bone osteolysis or not will be of interest in determining if all MSC-expressed CCN proteins behave similarly in haematopoietic malignancies to inhibit neoplastic growth. The CCN2 gene was shown to be involved in the normal as well as the pathological signalling of the HSC B-cell lineage. CCN2 was shown to be involved in normal early B-cell differentiation and in B-cell precursor ALL. This suggests that the functions of CCN2 in B-cell precursor ALL is a corruption of its role in normal HSC differentiation.

While the full scope of neoplasms influenced by CCN family members is yet to be fully discovered, current research indicates CCN genes have the potential to be used as biomarkers or therapeutic targets in several haematological malignancies. Future studies to uncover these potential translational benefits are much anticipated.

Abbreviations

ALL

Acute lymphoblastic leukaemia

AML

Acute myeloid leukaemia

Bax

Bcl-2-associated X protein

Bcl-xl

B-cell lymphoma- extra large

BMP

Bone morphogenic protein

CML

Chronic myeloid leukaemia

CDKN1B

Cyclin independent kinase inhibitor protein 27

CLP

Common lymphoid progenitors

CMP

Common myeloid progenitor

CTGF/CCN2

Connective tissue growth factor

CYR61/CCN1

Cysteine-rich 61

DLCBL

Diffuse large B-cell lymphoma

Dlk

Delta like kinase

ECM

Extracellular matrix

ELISA

Enzyme-linked immunosorbent assays

FSP1

Fibroblast-specific protein 1

HSC

Haematopoietic stem cell

IGFBP

Insulin-like growth factor-binding proteins

LRP

Low-density lipoprotein receptor-related protein

MCIJ

Monitoring of Cancer Incidence in Japan

MMP

Matrix metalloproteinase

MPP

Multipotent progenitor

MSC

Mesenchymal stem cell

NOV/CCN3 NOV

Nephroblastoma overexpressed

RBC

Red blood cell

SEER

Surveillance, epidemiology and end results program (SEER)

TGFβ

Transforming growth factor β

TRAIL

TNF-related apoptosis-inducing ligand 1

TrkA

Tropomyosin receptor kinase A

TSR

Thrombospondin type 1 repeat

VEGF

Vascular endothelial growth factor

VWC

Von Willebrand factor C

WISP-1/CCN4

Wnt-inducible-secreted protein 1

WISP-2/CCN5

Wnt-inducible-secreted protein 2

WISP-3/CCN6

Wnt-inducible-secreted protein 3