Background
A better understanding of the molecular mechanisms involved in melanoma and cancer progression in general is undoubtedly a major challenge in the development of new diagnostic and therapeutic approaches, underlying the necessity to identify new molecular targets. During the past decade, global gene expression profiling studies on various human cancer types, mainly relying on cDNA microarray technology, led to the identification of new candidate genes involved in cancer progression. These included periostin (
POSTN), a gene encoding a secreted 90 kDa protein initially identified in a mouse osteoblastic library as a putative bone adhesion protein [
1]. This protein shows sequence similarity to fasciclin I, an insect cell adhesion protein involved in central nervous system development [
2], and human β IgH3, a TGF-β 1-induced protein promoting adhesion and spreading of dermal fibroblasts [
3]. Binding of periostin to α
Vβ
3 and α
Vβ
5 integrins has been reported to promote cell adhesion and spreading and to activate the Akt/PKB signaling pathway leading to increased cellular survival and angiogenesis [
4‐
6]. In pancreatic cancer cells, periostin was shown to bind to α
6β
4 integrin, thereby promoting phosphorylation of focal adhesion kinase and PKB through activation of the PI3 kinase pathway [
7].
Over the past seven years, periostin was proposed to be a novel therapeutic target for cancer [
8]. Indeed,
POSTN gene was found to be overexpressed in various human cancers such as ovary [
4,
9], colon [
6], pancreas [
7,
10], thyroid [
11], oral squamous cell carcinoma [
12,
13], breast [
5], lung [
14] and neuroblastoma [
15] and higher
POSTN expression levels were correlated with increased tumor aggressiveness and/or poorer survival in NSLC [
14,
16], SCLC [
17], neuroblastoma [
15], colon cancers [
6], thyroid carcinomas [
11], oral squamous cell carcinoma [
12] and pancreatic ductal adenocarcinoma [
10]. However, other studies reported a down-regulation of
POSTN transcription in bladder [
18] and lung [
19] cancer. The functional role of periostin in cancer is also under debate as both tumor-promoting [
5‐
7,
10,
20,
21] and tumor-suppressing activities [
18,
19] have been reported: on one hand, periostin was reported to increase invasiveness of tumor cell lines
in vitro [
7,
12,
20] but, on the other hand, periostin expression reduced invasiveness of bladder cancer cells [
18] and decreased anchorage-independent growth of T24 bladder cancer cells and SaOS-2 osteosarcoma cell line [
19].
In vivo, two reports demonstrated that
POSTN overexpression in tumor cell lines increases metastasis and angiogenesis in nude mice and reduces stress-induced apoptosis [
5,
6] while another report provided evidence that periostin suppresses lung metastasis of mouse melanoma cell line B16-F10 [
18].
Although
POSTN overexpression does not seem to be systematic in human tumors, studies agree on the low level of periostin expression in most tumor cell lines [
4,
7,
9,
13,
18,
19,
21]. Lower levels of
POSTN expression in tumor cell lines compared to tumor tissues are in agreement with studies showing a production of periostin by stromal cells -and not cancer cells- in tumors [
10,
16,
17,
22]. However, the nature of periostin-producing cells in tumors is another matter of controversy as separate
in situ hybridization experiments suggested that
POSTN mRNA is detected in the cytoplasm of cancer cells [
5,
7].
In this study, we relied on quantitative RT-PCR to investigate POSTN expression in a series of human normal tissues and tumors. We next focused on cutaneous melanoma to quantify periostin transcripts in a total of 113 tumor samples, including primary and metastatic lesions, and we correlated periostin expression with Breslow thickness of melanoma primary tumors. Finally, to investigate the source of periostin production in melanoma, we analyzed POSTN transcription level in 23 newly-established melanoma cell lines and matched tumors and compared the results with the expression level of COL6A3, a melanoma-associated stromal marker encoding the α3 chain of collagen VI.
Discussion
The discrepancy between different studies regarding the expression level of
POSTN in normal tissues and the recent observation that transcription of the gene is up-regulated in various tumors [
8] prompted us to set up a quantitative RT-PCR assay to measure
POSTN mRNA in human normal tissues and tumors.
In normal tissues, previous Northen blot experiments demonstrated that periostin is not expressed in PBLs, highly expressed in fetal tissues or serum but studies disagreed on the extent of
POSTN expression in a series of other tissues [
4,
19,
21]. Our data confirm the absence of
POSTN transcription in both PBLs and spleen, in agreement with previous observations [
4,
19]. Salivary gland, thymus and embryonic stem cells were also characterized by very low expression levels. The highest
POSTN cDNA levels were measured in colon, small intestine, breast and skin. However, in the three latter organs, as well as in kidney and ovaries, expression levels were highly fluctuating between tissue samples, providing a possible explanation for the discrepancies between previous studies. The diversity in cell type content among samples may possibly account for this variability. In that respect, skin samples contain different cell types including fibroblasts, keratinocytes and melanocytes for which we have measured
POSTN expression levels of, respectively, about 1500, 50 (data not shown) and 0.
In tumors, leukemia and myeloma showed negligible levels of periostin expression whereas
POSTN transcripts were detected in all solid tumors. We detected a 72-fold increase of
POSTN transcription in pancreatic adenocarcinoma compared to normal tissues (
P = 0.06), in line with previous studies reporting a 42- to more than 100-fold increase of
POSTN mRNA level in pancreatic tumors [
7,
10]. In liver and ovarian tumors, average expression levels were increased by 65- and 15-fold respectively. The only evidences for increased periostin expression in liver cancer reported so far come from immunohistochemical analysis of tumor tissues [
7]. In ovarian tumors, tumor-derived epithelial cells and ascites, previous semi-quantitative studies reported elevated levels of periostin mRNA and protein [
4,
9]. Previous studies reported
POSTN up-regulation in about half of the NSCLC tumors [
14] and a slight increase in periostin serum levels from NSCLC patients [
16]. Higher periostin serum levels were also correlated with increasing T- or N-stage in SCLC patients [
17] but, on the other hand,
in situ mRNA hybridization suggested a down-regulation of
POSTN gene transcription in SCLC tumors [
19]. Our data suggest that
POSTN transcription is increased by 5-fold in NSCLC tumors. No significant difference was measured for SCLC and NET tumors although more samples should be analyzed. Analysis of six colorectal tumors and three normal colon samples revealed only a 2-fold increase in
POSTN expression (
P = 0.16). The modest increase in
POSTN transcription that we measured in colorectal tumor samples does not match previous data from a semi-quantitative study reporting considerable periostin overexpression in 25/29 pairs of matched normal colon tissue and colon tumor samples [
6]. The reason for this is unclear but may be related to the fact that Bao
et al. mainly focused on colon cancers with hepatic metastases. In breast and kidney tumors, the difference in periostin expression was not significant due to the highly variable
POSTN expression levels measured in both normal and tumoral samples. The absence of significant increase in
POSTN expression in breast tumors contrasts with the conclusions from a previous study in which expression in tumors was compared to the low level of
POSTN mRNA in primary mammary epithelial cultures as reference for normal breast tissue [
5]. Overall, for most tumor types tested in this study, our data revealed highly variable
POSTN expression levels in both tumors and normal tissues, suggesting that larger numbers of samples should be tested to address periostin expression in tumors more significantly.
In normal skin, melanocytes do not express POSTN but fibroblasts express the gene at high level. Investigation of 46 primary cutaneous melanoma lesions did not reveal any significant difference in average POSTN expression compared to normal tissues although we found that, in primary melanoma, thicker tumors (> 4 mm) may be correlated with increased periostin expression (P = 0.07). However, melanoma cell content of tumor samples is difficult to estimate and may vary with tumor thickness, leading to possible distortion of the data. In metastatic melanoma lesions, POSTN expression ranged from very low to very high levels compared to normal skin. Classification of melanoma metastases showed that very low POSTN expression levels are found in metastases located in organs with low endogenous periostin expression, like the liver or lymph nodes. In these organs, low POSTN expression levels are expected if melanoma cells do not produce periostin. Conversely, periostin levels should be high if melanoma metastases overexpress POSTN either through acquisition of POSTN expression by melanoma cells or to increased periostin expression in tumor-associated stromal cells. In that respect, about 60% of melanoma metastatic tumors in the liver or lymph nodes showed a clear POSTN overexpression compared to normal organs.
The nature of periostin-producing cells in tumors is still under debate as separate studies reported a production of periostin by stromal cells [
10,
16,
17,
22] whereas other experiments suggested that
POSTN mRNA is detected in cancer cells [
5,
7]. To investigate the nature of periostin-producing cells in melanoma, periostin expression was measured in newly established melanoma cell lines and matched tumors. In cell lines, expression of
COL6A3 stromal cell marker was reduced to less than 1% of the value measured in matched tumors whereas periostin was expressed (at either low or high level) in about half of the melanoma cell lines. Given that normal melanocytes do not express periostin, this study suggests that melanoma cells sometimes acquire the ability to express the gene during tumorigenesis. Our data also indicate that cell lines isolated from distinct metastases of the same patient may be characterized by either negligible or high
POSTN expression, suggesting that periostin expression is acquired by melanocytes during the metastatic process. Previous studies reported low
POSTN expression in most tumor-derived cell lines, whatever their origin [
4,
7,
9,
13,
18,
19,
21] but most cancer cell lines tested had been established a long time before and subjected to extensive culture, making it difficult to establish whether the decreased expression in cancer cell lines was reflecting a loss of stromal-associated
POSTN expression as suggested by some authors [
10,
16,
17,
22] or artifacts due to prolonged cell culture. The induction of periostin expression in melanoma cells may be a consequence of mutagenic events occuring during the tumorigenic processes or may be the result of interactions with stromal components previously reported to influence development and progression of carcinomas [
26]. In that respect, earlier experiments showed an induction of
POSTN expression in tumors recovered from nude mice injected with periostin-negative transformed cell lines [
21] (although one cannot rule out the possibility that stromal cells themselves may be the source of periostin expression in these tumors). On the other hand, our data indicate that stromal cells are an important source of periostin production in melanoma tumors. In some instances, periostin expression levels were higher than 3000 in the tumor but negligible in matching cell lines, suggesting that periostin expression may be very high in tumor-associated stromal cells. In line with this, a significant increase in periostin expression was measured in fibroblast-like stellate cells from pancreatic ductal adenocarcinoma tumors, a cancer characterized by excessive desmoplasia [
10]. Stromal cells have a prominent role in cancer progression and cancer-associated fibroblasts are believed to play a crucial role by producing growth factors, chemokines and extracellular matrix components that promote tumor angiogenesis [
27].
The increased expression of periostin in melanoma metastatic lesions that we identified in this study is in agreement with
in vivo studies revealing that periostin overexpression promotes metastatic growth of cancer cells [
5,
6,
20,
28]. Hence, this work suggests that, in melanoma, periostin overexpression may also be involved in the process of metastasis. In line with this, two previous studies identified an up-regulation of integrin αV (ITGAV), one of the subunits of periostin receptor, as a predictive marker for melanoma metastasis in primary tumors [
27,
29,
30].
Methods
Chemicals
Minimum Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle Medium (DMEM), trypsin-EDTA and essential amino acids were purchased from GIBCO (Invitrogen, Merelbeke, Belgium); fetal calf serum was from HyClone (Perbio Science, Aalst, Belgium); TriPure reagent from Roche Applied Science Diagnostics (Mannheim, Germany) and all other reagents were from Sigma Aldrich (Bornem, Belgium).
Primary cultures, normal tissues, tumors and cell lines
IMR90 fetal lung fibroblasts were kindly provided by M. Ricchetti (Institut Pasteur, Paris, France); HFF-2 newborn foreskin fibroblasts were purchased from ATCC (Rockville, MD); LB2924 fibroblasts were isolated from adult abdominal skin in our laboratory and normal human epidermal melanocytes NHEM derived from foreskin were purchased from PromoCell GmbH (Heidelberg, Germany). Normal tissue and tumor samples were obtained from patients undergoing surgery or tumor resection between 1991 and 2006. Experimental procedures involving the use of biological material were approved by our Institutional Review Board. All patients gave informed consent. In general, tumor biopsies were obtained as part of screening procedures for participation in clinical immunotherapy trials. The informed consent mentioned that part of the tumor samples could be used for research purposes. In a few cases, anterior to 2002, oral informed consent was obtained. Surgical specimens were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Cell lines derived from melanoma, rhabdomyosarcoma, NSCLC, SCLC, renal cell carcinoma, bladder carcinoma and larynx epidermoid carcinoma were derived from patient specimens in our laboratory. HeLa, PA-1, A172, A375, Hs578T, MCF-7 and Capan-1 cell lines were purchased from ATCC. Cell lines derived from osteosarcoma (U2OS), hepatocarcinoma (Huh-7), stomach tumor (MZGC3) and pancreatic carcinoma (Panc-1) were kindly provided by, respectively, F. Fuks (Université Libre de Bruxelles, Brussels, Belgium), A.H. Patel (University of Oxford, Oxford, UK), P. Coulie (Université Catholique de Louvain, Brussels, Belgium) and C. Hill (Cancer Research UK, London, UK). Fibroblasts were maintained as a monolayer in MEM media supplemented with 1% essential amino acids at 37°C in a humidified atmosphere saturated with 5% CO2. All cancer cell lines were grown at 37°C in a humidified atmosphere saturated with 8% CO2 in IMDM except for U2OS and HeLa (DMEM) and MZ2 and LB23-1 (DMEM/Hepes/glucose) cell lines. All media were supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin.
RNA extraction and reverse transcription
Total cellular RNA was extracted with TriPure reagent or by the guanidine-iosthiocyanate/cesium-chloride procedure [
31]. RNA samples from 18 normal human tissues were purchased from either Clontech (Mountain View, CA) or AMBION (Austin, TX). RNA from LB656 melanocytes was kindly provided by L. Old (Ludwig Institute for Cancer Research, New-York). cDNA synthesis from 2 μg of total RNA was accomplished by extension with dT
18 in the presence of 200 U M-MLV reverse transcriptase (Invitrogen, Merelbeke, Belgium) for 1 h at 42°C in a final volume of 20 μl. Reaction volume was then adjusted to 100 μl with water. cDNA from HUES human embryonic stem cells at day 5, synthesized as described above, was a kind gift of A. Loriot and C. de Smet (Ludwig Institute for Cancer Research, Brussels).
Real-time quantitative PCR
Expression levels of β-actin (
ACTB) [
32],
POSTN and
COL6A3 were measured by qRT-PCR based on TaqMan technology using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Primers, probes and qPCR Core Kit reagent were purchased from Eurogentec (Seraing, Belgium). Sequences of primers and probes are described in Table
2. POSTN primers were chosen in exon 3 and 5 respectively, allowing the amplification of all nine
POSTN splicing variants. Reactions were done in a final volume of 25 μl with 200 nM primers, 100 nM probe, 200 μM dNTPs, 5 mM MgCl
2, 0.625 U Hot Gold Star polymerase and 2.5 μl cDNA in 1× buffer. Annealing temperature was of 60°C for
POSTN and
COL6A3 and of 62°C for
ACTB [
32]. Amplicon sizes were of 186 bp (
POSTN), 142 bp (
COL6A3) and 613 bp (
ACTB). Standard curve equations were established by serial dilutions of PCR-amplified cDNA fragments of
POSTN (602 bp) and
COL6A3 (304 bp). cDNA copy numbers were calculated using the following equations: log(cDNA
ACTB
) = (Ct-38.5)/3.7; log(cDNA
POSTN
) = (Ct-36.9)/3.35 and log(cDNA
COL 6A 3) = (Ct-37.2)/3.4.
Table 2
Primers and probes used in qRT-PCR
POSTN
| 5'-TGCCCAGCAGTTTTGCCCAT 5'-CGTTGCTCTCCAAACCTCTA | 5'-TCCCACGATGCCCAGAGTGCCA | This study |
ACTB
| 5'-GGCATCGTGATGGACTCCG 5'-GCTGGAAGGTGGACAGCGA | 5'-TCAAGATCATTGCTCCTCCTGAGCGC | [32] |
COL6A3
| 5'-GAAGACCGGCAGCTCATCAA 5'-CGATGTTGCAGATGTCCAAGCA | 5'-CACAGCAGTGGGGCATGCGCTT | This study |
Immunoblotting analysis
To detect secreted periostin in the supernatants, LB2077-1, LB2730-1 melanoma cells were grown in IMDM medium until 90–100% confluency and thereafter kept in serum-free IMDM for 48 hours. LB2181-2 and LB2667-1a cell lines were used as negative controls. Four ml-supernatants from 400 000 cells were collected after 72 hours and concentrated 10 times using the Microsep centrifugal devices 10 K (Pall Life Sciences, Ann Arbor, MI). Equal volumes of concentrated supernatants were subjected to 7% SDS-PAGE and electroblotted onto a PVDF membrane (Millipore). The membrane was incubated successively with rabbit polyclonal antibody (ab14041, Abcam, Cambridge, UK) specific for periostin and then horseradish peroxidase-conjugated anti-rabbit IgG (Stressgen, Ann Arbor, MI). Protein bands were visualized using the Luminol Reagent (Santa Cruz Biotechnology).
Statistical analysis
Student t-test was applied to compare POSTN expression in different sample series.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
GT and MM carried out the quantitative RT-PCR analysis for POSTN, COL6A3 and ACTB genes. MM performed the immunoblot analysis. FB collected tumor samples, established cancer cell lines, synthesised cDNA from a series of samples and helped to draft the manuscript. NVB participated in the melanoma fibroblast marker selection and helped to draft the manuscript. AD conceived the study, participated in its design and coordination, performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.