Association of CCN protein expression with increased proliferative activity and tumourigenicity
In agreement with the well documented angiogenic properties of CCN1, gastric adenocarcinoma cells transfected with CCN1 gave rise to larger and more vascularised tumours than parental cells when injected into nude mice [
10].
In primary human breast carcinomas elevated levels of CCN1, CCN2, and CCN4, but not CCN3 were associated with more advanced features [
11]. Increased CCN1 protein expression was observed in a large number of primary breast tumours that were progesterone receptor positive but estrogen receptor negative – suggesting that it might be a novel mediator of progesterone activity in breast cancer [
12]. Invasive breast cancer cell lines expressed high levels of CCN1 whereas less tumourigenic breast cancer cells, such as MCF-7, expressed lower; normal breast cells showed almost none. Forced expression of CCN1 in MCF-7 cells was sufficient to induce their growth without anchorage in the absence of estrogen and to form colonies in matrigel in a α
vβ
3 integrin-dependent way. The tumours induced by these cells in ovariectomised athymic nude mice resembled human invasive carcinomas and were highly vascularised [
13‐
15]. These observations suggested that CCN1 was involved in the progression to more advanced stages of breast cancer.
Similarly, serum-stimulated MCF7 cells expressed much higher levels of CCN5 than the serum-starved and CCN5 expression is highest at S-phase in murine fibroblasts. Additionally, the expression of CCN5 was upregulated in Wnt-1 transformed C57MG epithelial cells.
Elevated expression of CCN1 has been detected in pancreatic cancers [
16] and within invasive breast carcinomas [
13]. The expression of CCN1 was also found to have increased in several types of paediatric tumours including angiofibroma, malignant fibrous histiocytoma, infantile myofibromatosis, and malignant haemangiopericytoma [
4].
An elevated expression of CCN2 has also been detected by Northern blotting in human invasive mammary ductal carcinomas [
17], dermatofibromas, pyogenic granuloma, endothelial cells of angiolipomas and angioleiomyomas [
18], and in pancreatic tumours [
19]. A study performed with chondrosarcomas representative of various histological grades established that CCN2 expression was closely correlated with increasing levels of malignancy [
20].
In agreement with CCN2 playing a role in brain tumour angiogenesis, immunocytochemistry studies indicated that both glioblastoma tumour cells and proliferating endothelial cells stained positive for CCN2 [
21]. In astrocytomas, CCN2 expression was particularly elevated in high grade tumours, with a marked effect of CCN2 on cell proliferation. Downregulation of CCN2 expression in these cells was associated with a growth arrest at the G1/S transition while over-expression of CCN2 induced a two-fold increase of the number of cells in the G1 phase. Gene profiling analysis allowed to identify a set of about 50 genes whose expression might account for the proliferative activity of CCN2 in these cells [
22]. CCN2 was seen in a higher proportion of mononuclear cells of patients with acute lymphoblastic leukemia [
23].
The expression of CCN3 was correlated to increased proliferative index in the case of prostate and renal cell carcinomas [
24,
25] and higher metastatic potential of the Ewing's carcinoma cells [
26]. In addition to the labelling of CCN3 in the cytoplasm of acinar epithelial cells, prostate hyperplasia was showing an intense luminal labeling suggestive of CCN3 secretion in seminal fluid. In prostate tumour cell lines, expression of CCN3 was detected in the cytoplasm of three cell lines derived from metastasis to bone (PC3), brain (DU145) and lymph node (LNCap). As compared to the level of CCN3 in normal prostates, gene profiling and qRT-PCR studies established that the level of CCN3 mRNA was increased around 80 fold in CR2-TAG mice which express the simian virus 40 large T antigen under the control of cryptdin-2 promoter elements [
27]; these studies indicated that the expression of CCN3 was not detected in the neuroendocrine cells. No expression was detected in normal adult prostate luminal epithelial PNT1B cells immortalised by SV40-T antigen [
24,
28], whereas SV40-T immortalised prostate epithelial P69 cells were positive for CCN3 expression. These results suggest that CCN3 expression in these cells might be a marker for epithelial prostatic cell transformation, rather than a feature of increased proliferation and point out a role for CCN3 in the transition from normal to malignant behaviour.
In Renal Cell Carcinomas (RCCs) which represent 85–90% of all kidney tumours, a significantly higher level of secreted CCN3 protein was detected by Western blotting in the conditioned medium of fast growing tumours [
25]. An inverse relationship could be drawn between the amount of CCN3 secreted by the tumour cells and the time that was required to establish tumours
in vivo and for them to reach a given size.
About 60% of the osteosarcoma tumours were found positive for CCN3. Again, an inverse relationship was observed in these tumours between the levels of CCN3 and alkaline phosphatase – an early marker of osteoblastic differentiation. Consequently, as the expression of alkaline phosphatase was associated with loss of agressiveness of osteosarcoma cells, these results indicate that in this system, the expression of CCN3 is likely to be associated with osteoblasts proliferation and therefore represent a marker of bad prognosis. It is worth noting that when injected into chicken embryos, MAV also induces osteopetrosis – a proliferation of osteoblasts. Whether an increase of CCN3 expression is also associated to osteopetrosis remains to be established.
In the case of Ewing's sarcoma, the levels of CCN3 expression varied among the different samples tested and the expression of CCN3 in primary tumours was associated to a significantly higher risk of developing lung and bone metastasis. Patients with primary tumours negative for CCN3 expression did not develop metastasis whereas 50% of the patients with primary tumours staining positive for CCN3 developed metastasis [
26]. The expression of CCN3 in Ewing's transfected cells was shown to decrease cellular adhesivity and increase cell motility [
29], two observations that are fully consistent with CCN3 being associated with a high metastatic potential.
CCN4 and CCN6 expression was significantly increased in most colon adenocarcinomas [
30]. Overexpression of CCN4 induced an accelerated growth and morphological transformation of NRK-49F fibroblastic cells and cell lines established after infection by retrovirus contructs expressing CCN4 was showing increased tumourigenicity. When tested
ex vivo however, forced expression of CCN4 induced only a slight increase of doubling time and slight decrease of saturation density on the same cells. Inasmuch as these cells did not grow without anchorage, the over expression of CCN4 was not sufficient enough to allieviate the need for proper interactions with the ECM and attachment.
Altogether, these observations are in favour of CCN proteins playing a positive role in tumourigenesis by providing the stimulatory effects on cell growth that are required for the increased lifespan of tumour cells. Whether these effects are involving a partial or complete abrogation of apoptotic pathways is an interesting question that deserves attention. The relationship that was drawn between increased expression of CCN proteins and tumourigenicity might also affect the communication of tumour cells with the surrounding medium, as suggested by the effects of CCN3 on Ewing's sarcoma cells. By decreasing the adhesivity of the cells and by providing an increased ability to migrate and invade surrounding tissues, the CCN proteins might be key factors participating to the metastatic potential of tumour cells. The identification of CCN proteins partners should be very helpful in establishing whether abnormal interactions with physiological targets are involved in these processes.
Association of CCN protein expression with reduced proliferative activity and tumourigenicity
The first evidence indicating that the expression of CCN proteins in tumours could be associated to differentiation and growth arrest came from our studies performed with MAV-induced nephroblastomas and Wilm's Tumours. The Myeloblastosis Associated Virus (MAV)-induced avian nephroblastomas constitute a unique model of the Wilm's Tumour [
31], a paediatric tumour affecting approximately one in 10,000 children [
32]. Large quantities of CCN3 transcripts were detected in all MAV-induced tumours, whereas in normal post-natal kidney the level of CCN3 expression was low [
5].
Considering that non-acute retrovirus had been reported to induce tumours by integrating nearby growth regulatory genes in the host genome, we postulated that a) the CCN3 gene was a prefered integration site of MAV within the host genome and b) in these tumours, the expression of CCN3 was stimulated by MAV-LTR enhancer sequences integrated in the vicinity of the CCN3 locus. The use of Bacterial Artificial Chromosomes (BACs) and Fluorescent In Situ Hybridization (FISH) allowed us to recently establish that the CCN3 DNA region is not a preferential site of integration for MAV (C.L. Li, P. Coullin, C. Auffray, A. Bernheim, V. Joliot, R. Zoroob and B. Perbal, manuscript in preparation). Furthermore, the histological analysis of avian nephroblastomas and the expression pattern of CCN3 in the developing avian kidney, established that the MAV target cells express high levels of CCN3 and that elevated levels of CCN3 were detected in the more differentiated tumours (Cherel et al., manuscript in preparation; Chevalier and Perbal, unpublished data).
Analysis of Wilm's Tumours established that blastemal cells committed to abnormal differentiation were positive for CCN3 expression. Tumours with a high degree of stromal components contained the highest levels of CCN3 RNA. In some cases, the quantities of CCN3 RNA were inversely related to the amount of WT1 RNAs detected in the same samples [
33]; an observation which, at first glance, was in agreement with the
ex vivo downregulation of CCN3 promoter activity by WT1 [
34]. However, studies performed with a larger panel of samples [
35] representative of sporadic, WAGR and DDS histological types of tumours raised the possibility that the variations of CCN3 expression resulted from different relative amounts of WT1 isoforms within these tumours. Similarly, CCN2 – whose expression is also decreased in Wilm's Tumours, was reported to be a target of WT1 [
36].
In the Wilm's Tumours, expression of CCN3 was also shown to match striated muscular differentiation. During the heterotypic differentiation of the blastemal cells that takes place in these tumours, CCN3 expression was detected at an earlier developmental stage than desmin and confocal microscopy also indicated that the CCN3 protein was colocalised with desmin in heterotypic muscular fibres [
35]. These observations established the level of CCN3 expression in these tumours as a marker of heterotypic differentiation [
35] and suggested for the first time that overexpression of a CCN protein could also be associated with tumour cell growth arrest. A strong association between CCN3 expression and tumour differentiation was also observed in the case of neuroblastomas, chondrosarcomas, rhabdomyosarcomas and other musculoskeletal tumours [
4,
24,
37].
In neuroblastomas with poor pronostic features, CCN3 staining was low moderate within the tumour cells, whereas in tumours with favourable pronostic and no N-myc amplification, the CCN3 staining was strongly detected in the cytoplasm of differentiated ganglion-type cells [
4]. Immunohistochemistry, Western blotting analysis, and real-time RT-PCR performed on samples from enchondromas and chondrosarcomas of various grades also the highest levels of CCN3 expression were detected in benign enchondromas, and the lowest levels of CCN3 were detected in the tumours of higher grade. Rhabdomyosarcomas generally expressed significant amounts of CCN3, with the largest quantities being detected in the most differentiated cells [
37], an observation in agreement with CCN3 expressed in the developing skeletal muscle and fusing myoloblasts [
38].
A reduction of CCN3 expression in tumour cells was also observed in the case of human chronic myeloid leukemia tumours and in murine multipotent haematopoietic stem cells transfected with a thermo sensitive mutant of the bcr-abl gene fusion expressed in chronic myeloid leukaemia (CML) [
39]. These results indicated that the tumour-associated reduction of CCN3 expression was not a characteristic specific to solid tumours.
More recently evidence was also obtained for decreased levels of other CCN proteins in tumours. For example, the expression of CCN1 was shown to be downregulated in uterine leiomyomas [
40], rhabdomyosarcomas [
41], and in 50% of prostate carcinomas [
42]. In non-small cell lung cancer (NSCLC), the expression of CCN1 was also decreased markedly, compared to the matched normal samples [
43]. In opposition to the stimulatory effect of CCN2 on human embryonal carcinoma cell growth, its expression was increased when these cells were treated by all-trans retinoic acid and were undergoing neuronal differentiation with concomitant loss of tumourigenicity [
44].
Originally reported to be highly expressed in murine cells with a low metastatic potential, CCN4 was detected at similar levels in normal breast epithelial cells and breast tumour cell lines [
45]. In primary human colon adenocarcinomas, the expression of CCN5 was significantly decreased and it was not detected in the epithelial tumour cells of mammary carcinoma obtained from Wnt-1 transgenic mice [
30]. CCN5 was also downregulated upon transformation of BALB/c3T3 fibroblastic cells. The expression of CCN6 was impaired in most infammatory breast cancer [
46] and a relationship was observed between loss of function mutations in CCN6 and progressive pseudorheumatoid dysplasia [
47].
CCN proteins may act as antiproliferative agents
Not only the amounts of CCN proteins were low in some tumours, but an increasing body of evidence indicates that overexpression of CCN proteins in tumour cells may result in growth arrest and/or reduced tumourigenicity.
For example, early studies indicated that high levels of CCN3 expression were correlated with reduced tumourigencity and low metastatic potential of glioblastoma cells [
48]. By using a series of stable transfectants, we established that CCN3 had a marked antiproliferative effect on glioblastoma cells [
49]. Furthermore, the cells expressing CCN3 showed a significant reduction in their ability to induce tumours in nude mice. Inasmuch as the expression of CCN3 was interfering with tumour expansion, but not with early stages of tumour developement, we hypothesised that CCN3 might be cooperating with other proteins at the cell surface or in the ECM to regulate cell proliferation and invasiveness.
In agreement with these observations, confocal analysis performed on the transfected cells established that CCN3 and Connexin-43 (Cx-43) were colocalised [
49], an observation raising the possibility that they may physically interact. Similarly, Cx-43 and CCN3 proteins were colocalized at the membrane of choriocarcinoma cells [
50]. Inasmuch as an aberrant Gap Junctional Intercellular Communication (GJIC) was associated to tumourigenesis, these results suggested that CCN3 is involved in the control of cell growth at least through its relationship with GJIC. Upon restoration of cell to cell communication via Cx-43 protein channels, human choriocarcinoma cells lost their ability to induce tumour growth
in vitro, and strongly upregulated the expression of CCN3.
Along the same line, the isolation of stable transfectants expressing CCN3 under the control of constitutive and inducible promoters also permitted to establish that CCN3 was able to significantly reduce the growth rate of EWS transfectants
ex vivo [
29]. Interestingly, transfectants showed reduced adhesivity and increased motility – two features in agreement with CCN3 expression associated with higher metastatic potential (see above).
The expression of CCN1 in NSCLC stable transfectants induced a significant reduction of the proliferation rate that could be partially rescued after addition CCN1 antibodies. The CCN1 transfected cells where arrested in the G1 phase of the cell cycle, showed a decreased activity of CDK2 and an upregulation of p53, p21(WAF1), and pRB2/p130. Interestingly, the transfected cells expressing CCN1 gave rise to smaller tumours than those induced by the parental NSCLC cells [
43].
The CCN4 protein was also reported to have marked inhibitory effects on the tumourigenic properties of tumour cells
in vivo. The absolute number of tumours obtained was dramatically reduced by the expression of CCN4 and the time needed for tumours to reach a given size was considerably higher as a result of the marked inhibitory effect of CCN4 on the growth properties of transfected cells
in vivo. Furthermore, the transfectants which expressed high levels of CCN4 gave rise to a much smaller number of lung metastasis than those expressing low levels of CCN4. Recently, it was shown that overexpression of CCN4 in lung cancer cells reduced their ability to induce metastasis in nude mice, and impaired their migration within Boyden chambers in response to serum [
51].
Early results established the antiproliferative activity of CCN5. Repressed in several transformed derivatives, retroviral-driven ectopic expression of CCN5 induced a negative effect on the growth of rat transformed cells and was accompanied by an enrichement in subG1 DNA content [
52]. These observations therefore suggested that overexpression of CCN5 might induce cell death. Furthermore, CCN5 overexpression in stably-transfected rat cells transformed by p53 and H-ras, significantly reduced their tumourigenicity when tested by subcutaneous injection in athymic mice. The expression of CCN5 was detected only after repeated passages of rat and mouse embryo fibroblasts, but not in primary fibroblasts. More recently, overexpression of CCN5 was reported to inhibit both proliferation and motility of myometrial, leiomyoma smooth muscle cells and vascular smooth muscle cells [
53]. High levels of CCN5 expression were also detected in quiescent and heparin-treated vascular smooth muscle cells. [
53].
In the same light, restoration of CCN6 expression in Inflammatory Breast Cancer (IBC) cells resulted in the induction of morphological changes and a decreased ability to grow in soft agar. Importantly, the restoration of CCN6 expression resulted in tumour growth suppression, but with a decrease of tumour cell growth in grafted nude mice [
54], an observation reminiscent to the effects of CCN3 on glioblastoma cells (see above).
Collectively, these results indicated that the expression of CCN proteins was altered in various types of tumours; depending upon the type of cells and tissues, high levels of CCN expression could be associated to either differentiation or increased proliferation and metastasis. They also suggested that CCN proteins may be acting as a tumour suppressor, irrespective of their pattern of expression upon serum stimulation. The differential expression of CCN genes upon serum stimulation previously led to the conclusion that immediate early genes such as CCN1 and CCN2 were encoding positive growth regulators; whereas, genes whose expression was not induced or were repressed by serum were encoding growth suppressors.