Gene expression profiling of cholangiocarcinoma-derived fibroblast reveals alterations related to tumor progression and indicates periostin as a poor prognostic marker

Cholangiocarcinoma (CCA) originates from biliary epithelial cells and is a unique cancer in northeastern Thailand where the prevalence of a liver fluke, Opisthorchis viverrini infection is higher than elsewhere in the country. A recent study showed a strong positive correlation of CCA incidence and the prevalence of O. viverrini infection [1]. In other countries, CCA has been shown to correlate with Clonorchis sinesis[2, 3], and chronic biliary diseases [4]. Even though CCA is caused from the different etiologies, it is well recognized to contain an abundant fibrous stroma that is mainly composed of α-smooth muscle actin (SMA) positive fibroblasts [5, 6]. In addition, the degree of α-SMA expression has been shown to correlate with the survival of patients, in part, via the ability of these cancer-associated fibroblasts to induce proliferation of bile duct epithelial and cancer cells [6].

The ability of stromal fibroblasts to generate a favorable microenvironment for cancer cells leading to cancer development, invasion and metastasis has been summarized [7, 8]. Mitotic substances have been produced from stromal fibroblasts to promote tumor growth in many cancers [9, 10]. In addition, some matrix metalloproteinases which are often mentioned as proteolytic substances of the extracellular matrix (ECM) and have been reported to show increased production from cancer stromal fibroblasts. These proteolytic substances appear to help to promote cancer cell invasion and metastasis [11]. Specifically, in CCA, stromal-derived factor 1 (SDF-1) has been secreted from stromal fibroblasts into the microenvironment in which it was located at the edge of cancer masses and was proposed to play important role in induction of CCA cell invasion and metastasis [12].

To understand the roles of fibroblasts in carcinogenesis, cancer promotion and progression, gene profiling of cancer fibroblasts have been studied in many cancers [13‐15]. Stromal cancer fibroblasts from breast cancer with invasion were compared with the expression profiles of fibroblasts in benign breast disorders. HYL (Csk-homologous kinase CHK) involving in regulation of Src kinase, GM CSF-1 ( granulocyte monocyte colony stimulating factor-1) and osteopontin were up-regulated which may result in induction of tumor growth and metastasis [13]. Among genes encoded for secreted proteins over-expressed in fibroblasts of human basal cell carcinoma [14], genes including CTSK (cathepsin K), SFRP2 (secreted frizzled-related protein 2), PDGFRL (platelet-derived growth factor receptor-like protein), and DCN (decorin) were shown to be up-regulated in non-epithelial cells of breast cancer [16]. In contrast, these genes could not be detected in fibroblasts isolated from cancers of pancreas [17], and liver metastases of colon cancer [18]. Taken together, it is possible to say that differential gene expression profile of cancer fibroblasts is partly similar but actually unique for each cancer type. This supports the importance of specific recognition of the concerted performance between fibroblasts and epithelial cells in carcinogenesis and progression in different organs of origin. So it is of great value to investigate the specific gene expression profile of the CCA-derived fibroblasts to help us better understand the molecular mechanisms that fibroblasts use to promote cancer.

Periostin (PN) is a secreted protein which was first identified in bone and implicated in regulating adhesion and differentiation of osteoblasts. The cancer biology role of PN has been investigated in a wide range of cancers including cell proliferation [19, 20], migration [21], invasion/metastasis [22, 23], and angiogenesis [23, 24]. When not regarding the specific sources, either from cancer cells or fibroblasts, secreted PN has been reported to induce tumorigenic properties of epithelial cells via the activation of integrins (ITGs) receptors [21].

Even though substantial evidence has shown that cancer-associated fibroblasts are involved in tumor promotion and with the evidence that fibroblasts in CCA induce more aggressive tumorigenic properties of cancer cells [6], the role of CCA-derived fibroblasts in this cancer is yet to be determined. In the present study, fibroblasts isolated from CCA tissues or CCA-associated fibroblasts (Cfs) which were already characterized by the present group [6], were explored. The genome wide expressions of these Cfs were determined and compared to non-tumorigenic liver fibroblasts (Lfs). The altered expression of genes focusing on the impact of soluble products from Cfs on the promotion and progression of CCA was investigated. Interestingly, PN, which has never been reported in CCA was found at a high level whereas no-to-low PN was detected in non-tumor liver tissues and cancer of hepatocytes. The overexpression of PN in CCA tissues was detected solely in fibroblasts and associated with poor prognosis and short survival of the patients. The effect of PN to induce cell proliferation and invasion has been examined.

Cancer-associated fibroblasts have been recognized for their impact in the genesis, promotion and progression of many carcinomas and highlighted in several reviews [8, 25]. CCA is notoriously associated with dense desmoplastic stroma with activated fibroblasts [5, 6]. Relatively little, however, is known about the contribution of the stromal fibroblasts to CCA. The authors in the present group have recently shown that CCA stromal fibroblasts, with and without direct interaction with cancer cells could induce cancer cell proliferation [6]. Herein, this study focused on the gene expression profile of CCA-derived fibroblasts in order to investigate the molecular mechanism of how fibroblasts induce a favorable microenvironment to promote cancer. Even though the current study is limited to a single cancer fibroblast line isolated from a single CCA patient, the validity of array results was strengthened by comparing gene expression levels in cancer fibroblasts to the two lines of normal fibroblasts; one isolated from the same CCA patient and the other from a second patient. Only genes in cancer fibroblasts altered from both normal fibroblast lines were investigated as the common up- or down-regulated genes. This is to provide evidence that the fibroblasts used in our study are valid representatives of fibroblasts found in CCA.

By comparing gene profiles in fibroblasts from CCA with those of other tumor types, it is suggested that CCA fibroblasts display not only common genotypes for activated cells but also unique characteristics. Genes involved in metabolism of cells needed to be up-regulated in order to support the active function of CCA stromal fibroblasts to produce many supporting proteins in the cancer environment. Neuropeptide Y receptor Y1 has been indicated to receive the activation signal to induce neuroproliferation [26] and doublecortin-like kinase 1, a microtubule-associated active protein kinase expressed in growth cones of postmitotic neurons [27] may help facilitate fibroblast proliferation. In similar to human basal cell carcinoma fibroblasts [14], SPARC or osteonectin, was also over-expressed in CCA-derived fibroblasts. SPARC-null mice were recently demonstrated to resist UV-induced squamous cell carcinoma, suggesting a tumor-promoting role of SPARC[28]. In contrast to the cancer-associated fibroblasts in metastatic colon cancer to the liver which showed down-regulation of SDF-1[18], CCA-derived fibroblasts had up-regulated SDF-1 (data not shown).

The highly up-regulated genes in CCA-derived fibroblasts showed several interesting functions involved in cancer progression. Serpin peptidase inhibitor, clade B member 2 (SERPINE2) or plasminogen activator inhibitor type 2 (PAI2) is involved in cancer invasion and metastasis by controlling serine protease urokinase plasminogen activator. In a recent review, several studies led to the suggestion that the significance of PAI2 expression on prognosis of cancers is organ context-dependent [29]. In breast cancer, PAI2 was expressed in both stromal and tumor cells and associated with prolonged disease-free survival [30]. In contrast, high levels of PAI2 in endometrial cancer were reported to correlate with the invasion potential of the cancer [31]. S100 calcium binding protein A4 (S100A4) has been revealed as the metastasis-inducing protein [32]. Genes such as procollagen C-endopeptidase enhancer 2 (PCPE2) were also detected which may involve in collagen synthesis [33]. These results support the function of fibroblasts in CCA to promote a desmoplastic reaction. For down-regulated genes, bone morphogenetic protein 2 (BMP2A), a multi-functional growth factor belonging to the transforming growth factor-β superfamily was decreased in CCA fibroblasts as reported in breast cancer-derived fibroblasts [13]. BMP2A-encoding protein has been elucidated to induce hypophosphorylation of retinoblastoma protein causing cell cycle arrest [34]. Hence, decreased BMP2A in the CCA microenvironment may promote cancer cells to enter the cell cycle. Moreover, a decreased level of interleukin 24 (IL-24), an apoptotic inducible cytokine [35], in cancer tissues, attenuates cancer cells from undergoing apoptosis. The response gene to complement 32 (RGC32), a novel p53-inducible gene, and bradykinin receptor B1 (BRADYB1) decreased expression in CCA-derived fibroblasts. Being intracellular protein and membrane receptor, respectively, RGC32 and BRADYB1 have elucidated the function of inhibition of fibroblast cell proliferation [36, 37]. It seems possible to conclude that down-regulated genes in fibroblasts encode proteins, if acting in the intracellular region, can inhibit the proliferation of fibroblasts themselves, but if they exist in the extracellular region, they may involve inhibition of cancer cell proliferation. This evidence strengthens the roles of fibroblast-derived proteins released into a tumor environment to induce a high proliferative capability of cancer cells.

Fibroblasts have been proposed the bipolar effects in cancers [38]. In our microarray results, ADAMTS-like 1 (ADAMTSR1) was over-expressed in CCA-derived fibroblasts. The ADAMTS-like proteins have been discussed as the enhancers of ADAMTS proteases [39]. Since some ADAMTS have been proven to be anti-angiogenic factors [40] partly via the trapping of vascular endothelial growth factor by thrombospondin motifs of ADAMTS [41]. So up-regulation of ADAMTSR1 in fibroblasts may inhibit angiogenesis. Moreover, stromelysin-1 or MMP-3 which can degrade ECM and induce cancer invasion and metastasis, showed the decreased expression in fibroblasts. Taken together, the increased expression of ADAMTSR1 and the decreased expression of MMP-3 may highlight fibroblasts in term of suppressing CCA progression.

Theoretically, proteins secreted from fibroblasts having interplay with cancer cells could be detected in the extracellular region and be involved in ECM organization and biosynthesis. Within these 2 groups of genes, we focused our interest on genes encoded secreted proteins and their products have been previously reported of their tumorigenic effects. ADAM12, AREG, AGN2, ER, JAGL1, LAMA5, NOV, PDGF-A, PN, RL, and SCG2 were selected to explore. AREG, ER, JAGL1, and LAMA5 are predominantly reported for proliferation induction in cancer cells [42‐45]. PDGF-A, NOV, AGN2, and SCG2 are involved in angiogenesis [46‐49], whereas ADAM12 and RL play an important role in cell motility, invasion and metastasis [50, 51]. For PN, many carcinogenic functions including cell proliferation, invasion, metastasis and angiogenesis have been demonstrated [20‐24]. This study employed real time PCR to verify the up-regulation of these genes, and found that only ADAM12, AREG, ER, JAGL1, PDGF-A, PN and SCG2 were significantly increased in their expression levels in CCA-derived fibroblasts and may promote CCA progression through activation of cancer growth, invasion and angiogenesis.

Herein PN was chosen to deeply explore since its well accepted multifunction in cancer as mentioned above. Moreover, the result from our group about the expressions of ADAM12, AREG, ER, JAGL1, PDGF-A, PN and SCG2 in whole CCA tissues (n = 20) showed that only AREG, PDGF-A and PN had higher level in cancer than those in benign liver tissues with statistical significance (data not shown). AREG and PDGF-A could be detected in not only fibroblasts but also in cancer and endothelial cells, however PN expressed exclusively in CCA fibroblasts. In order to demonstrate role of fibroblast-derived proteins in CCA, we determined that PN should be the first target to explore.

The strong evidence using different biological preparations of CCA-derived fibroblasts and CCA tissues confirmed the increased levels of PN at both mRNA and protein. Most of CCA tissues of all differentiated types had high levels of PN and expressed exclusively in α-SMA positive fibroblasts. In the same direction, the findings showed no expression of PN in CCA cell lines when compared to the high level expressed in the fibroblasts. This may strengthen the results of the absence of PN in cancer cells in CCA tissues. From these results taken together, it can be concluded that PN detected in CCA tissues is only of fibroblast origin as reported in some cancers [52‐54]. In cancers of head and neck, ovary, and colon, PN was found in cancer cells and has been proposed to induce tumorigenic properties of cancer cells via an autocrine mechanism [21, 22]. Hence results from the present study allow the speculation to propose a phenomenon that fibroblast-derived PN in CCA may affect cancer cells by a paracrine mode and has a promising role in cancer promotion. These results revealed that a high PN level in fibroblasts was an independent risk factor in CCA patients and those having high PN had significantly low cumulative survival time after surgery. PN might therefore be used as a poor prognostic marker in patients suffering from CCA. Detections of PN at both mRNA and encoding protein in CCA tissues are in the same direction to distinguish CCA from non-cancer syndromes of bile ducts. In addition, most benign liver tissues and hepatocellular carcinoma showed no to only a minimal expression of PN when compared to the high level detected in CCA tissues. Hence, serum PN may help to distinguish CCA from benign conditions and closely-related liver cancer and may use as the prognostic or predictive marker as previously reported [52, 55].

To show the tumorigenic impacts of PN on CCA cells, recombinant PN was employed as extracellular PN to mimic the paracrine effect of PN produced from cancer stromal fibroblasts to induce CCA cell proliferation and invasion. Though receptors ITGα_vβ₃ and ITGα_vβ₅ have been shown to be the receptors for PN in several cancer cells [21], PN promoted invasiveness of pancreatic cancer cells via the β₄ integrin [56]. This suggests the cell type dependent on a specific ITG responded to PN. The study herein reveals that PN-induced cell proliferation and invasion could be inhibited by RNAi against ITGα_5. Hence, ITGα₅ is a potentially promising receptor for PN in CCA cells. As the well known receptor for fibronectin, the apparent reason for ITGα₅ production in CCA cells is to support the abundance of fibronectin found in CCA [57]. In addition, ITGα₅ can only be from dimerization with the β₁ subunit and activation of ITGα₅β₁ has been revealed to support cell survival [58] and induce invasion and angiogenesis [59, 60]. Though further studies need to be performed before such a conclusion is valid in CCA, this work highlights the PN-induced-ITGα₅ pathway as one of the activated pathways to induce an aggressive CCA.

TGF-β has been proposed to induce the expression of PN[61]. O. viverrini excretory/secretory product has also recently been shown to be the stimulator of fibroblast proliferation via the TGF-β-mediated signal transduction pathway [62] and this pathway seems likely to be the cause of PN expression in CCA-derived fibroblasts. The authors' laboratory has checked the effect of parasitic product-treated fibroblasts and found that these fibroblasts increased PN expression compared to the normal liver fibroblasts without exposure (unpublished data). It is interesting to propose that in CCA cells; the expression of fibroblast-derived PN could be induced by TGF-β produced from infected parasites since the early stage of carcinogenesis and may be in concert with TGF-β produced from CCA cells in a late stage of cancer [12]. Though in vivo experiments are needed to confirm, fibroblast-derived PN may influence O. viverrini-associated CCA at the early stage of cancer as well as to promote cancer progression in the later time. With this information, targeting the stroma in CCA may not only be effective in treatment of primary, invasive and metastatic tumors, but may also play role in prevention of tumor development.

To the authors' knowledge, this study is the first to describe the gene expression profile of CCA-derived fibroblasts. Molecular understanding of fibroblasts in CCA by the functions of certain up- and down-regulated genes has been revealed and has suggested certain groups of genes in controlling cancer cell proliferation, invasion, metastasis and angiogenesis (Fig 8). These findings provide evidence that fibroblasts are important sources of tumorigenic substances, particularly PN, when produced into the microenvironment of CCA. High levels of PN are found in most CCA patients and can be used as a poor prognostic marker. In addition, the level of PN can be used to distinguish CCA from other benign liver conditions and hepatocellular carcinoma. The interaction of fibroblast-derived PN and CCA cells helps to promote cell proliferation and invasion probably via ITGα₅. Though further investigations are needed, this study suggests promising evidence of the value of using serum PN as a prognostic marker of poor survival in CCA patients. Moreover, targeting fibroblasts or fibroblast-derived-PN-stimulated pathways in cancer cells to attenuate the tumorigenic induction of PN is a further challenge to inhibit CCA progression in the patients.