Division of Gastroenterology, University of British Columbia; Genome Sciences Centre, B.C. Cancer Agency, Vancouver, Canada and 1 Department of Cancer Biology, Dana Farber Cancer Institute, Boston, USA
* To whom correspondence should be addressed at: University of British Columbia, Division of Gastroenterology, 100-2647 Willow Street, Vancouver, BC, V5Z 3P1 Canada. Tel: +1 604 875 5039; Fax: +1 604 875 5447; Email: itai{at}bcgsc.ca
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Abstract |
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Abbreviations: APC, adenomatous polyposis coli; 5-FU, 5-fluorouracil; HNSCC, head and neck squamous cell cancer; mAb, monoclonal antibody
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Introduction |
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Periostin was originally identified in a mouse osteoblastic library (2). Its sequence similarity to the fasciclin family of proteins suggested a role in cell adhesion and migration. Its role in tumorigenesis is still unclear; however, studies indicate that serum levels of periostin may have prognostic relevance in non-small cell lung cancers (3), neuroblastoma (4) and thymoma (5). Elevated serum levels of this protein appear to be associated with a poorer prognosis. At the molecular level, there is a high degree of similarity of periostin to ßig-h3, a transforming growth factor ß (TGFß) inducible gene. Recent studies have also shown that TGFß can induce the expression of periostin protein, and its mRNA level can be downregulated by Wnt-3 and GSK-3ß in the mammary epithelial cells (6). Given the involvement of Wnt-signaling in the pathogenesis of colorectal cancer, we hypothesized that periostin may play an important role in colon cancer tumorigenesis. In this study, we have characterized the expression of periostin in colorectal cancer and provide evidence of its potential role in contributing to tumorigenesis in colorectal cancers and metastasis.
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Materials and methods |
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Detection of periostin and TGF-ß mRNA expression levels
(A) Northern blot analysis of normal tissues. Total cellular RNA (15 µg) or mRNA (1 µg) was electrophoresed on a 1.0% agarose formaldehyde gel, then transferred onto Hybond-N nylon membrane (Amersham Life Science, Piscataway, NJ). RNA membranes were baked at 80°C for 2 h, then prehybridized and hybridized in Church's solution (0.5 M Na2HPO4, 7.5% SDS, 1 mM EDTA, pH 7.2) with salmon sperm DNA at 62°C overnight. RNA blots were washed with 0.1x Church's solution at 50°C for 2 x 10 min. The cDNA inserts for probes were purified by agarose gel electrophoresis, recovered using Gene Clean (Bio 101) or Qiaex purification (Qiagen, Valencia, CA) and labeled with [-32P] dCTP using a random primed DNA labeling kit (Boehringer Mannheim, Germany).
(B) Semi-quantitative RTPCR of cancer cell lines. Total RNA was isolated from various cancer cell lines using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Total RNA of 0.5 µg was used for quantitation using a one-step RTPCR kit (BD Biosciences, Palo Alto, CA) with MJ Research thermocycler and the following settings: periostin forward primer (5'-caccacaacgcagcgctatt-3') and reverse primer (5'-tggaagtttctcaaaagcct-3'); TGF-ß forward primer (5'-ccagatcctgtccaagctg-3')and reverse primer (5'-ctccacggctcagccact-3') at 94°C for 5 min, with 35 cycles of the following: 94°C x 1 min, 60°C x 1 min, 72°C x 1 min, followed by 72°C x 10 min, and 4°C.
Purification of periostin protein
Human Fchuman periostin fusion protein was generated as described previously (7). For purification, periostinFc protein was captured on Protein A Sepharose, eluted in a sodium citrate buffer (100 mM, pH 4), followed by neutralization with 0.1 volume of 1 M TrisHCl, pH 8.
Anti-periostin monoclonal antibody (-periostin mAb) production
The immunogen was prepared by harvesting human tumor cell culture medium in which Fcperiostin was secreted into media and then purified by affinity column. Three 6-8-week-old female BALB/c mice were immunized six times with 0.1 mg periostin-Fc/mouse plus Freund's adjuvant subcutaneously. Hybridomas were produced using polyethylene glycol-based cell fusion techniques (8) and cell cultures were maintained in DMEM (Life Technologies, Rockville, MD), supplemented with HAT, 10% FBS (Invitrogen). One hundred clones were screened against His-periostin (His-tagged protein expressed in baculovirus expression system, protein harvested by His-column purification). Screening was performed by ELISA and positive cell lines were cloned three times by limiting dilution. Stable hybridomas were grown in bulk in culture plates and monoclonal antibodies purified by affinity column.
Colon tissue samples and immunohistochemistry
Sections of paraffin-embedded colon cancer tissues and their liver metastasis were processed for immunohistochemistry using the ABC avidinbiotin peroxidase method with a mouse monoclonal antibody directed against periostin (-periostin mAb, clone 8H11) and a secondary biotinylated rabbit anti-mouse antibody (Sigma-Aldrich, St Louis, MO).
Growth assays
MIP 101 cells were seeded at 1000 cells/well in 96-well plates and incubated in DMEM (2% FBS) containing incremental concentrations of periostin, -periostin mAb, or a non-
-periostin antibody (
-cardiotrophin antibody, R & D Systems, Inc., Minneapolis, MN) for 3 days. Cell proliferation was assessed by BrdU incorporation based on the manufacturer's protocol (Roche Applied Sciences, Indianapolis, IN) following a 12-h overnight incubation with the substrate. All experiments were performed in triplicate (n = 3), with each experiment in triplicate wells.
Colony-forming assays
For clonogenic cell survival studies, MIP101 cells were seeded in 48-well plates at 100 000 cells/well and incubated in DMEM supplemented with 2% FBS containing incremental concentrations of periostin (5, 10, 50 µg/ml), -periostin mAb (0.5, 1.0, 1.5 µg/µl) or 5-fluorouracil (5-FU, 125 µM, 250 µM, 500 µM) for 7 days. Cells were washed with PBS and re-incubated in fresh medium containing the appropriate substrate, for an additional 7 days. Cells were stained with crystal violet for the visualization and assessment of colony formation.
Western blot analysis
Total protein was extracted from tissue culture cells using CHAPS lysis buffer. Total proteins from normal human organ-specific sites were obtained from a commercial source (Protein Medley, BD Biosciences, Palo Alto, CA). Ten to thirty micrograms of total protein were resolved on 412% SDSPAGE gels and then transferred onto PVDF membranes. For the detection of periostin in cell lines, tissue culture cell lysates (250 µg) were incubated overnight with -periostin mAb (clone 8H11), followed by immunoprecipitation with 50% bead slurry of Protein A/G agarose beads (Sigma-Aldrich) for 4 h at 4°C. The pellets were washed five times with 500 µl of cell lysis buffer, then resuspended with 20 µl of 4x SDS sample buffer followed by resolution on 412% SDSPAGE gels and a transfer onto the PVDF membranes. All membranes were blocked for 1 h at room temperature with 5% skim milk in TBS-T (20 mM TrisHCl, 137 mM NaCl, 0.05% Tween-20, pH 7.2). Following washes with TBS-T (1 x 15 min, 2 x 10 min at room temperature), the membranes were incubated with the appropriate primary antibodies (1:1000) overnight at 4°C. Membranes were washed again with TBS-T (1 x 15 min, 2 x 10 min) and then incubated with the appropriate horseradish peroxidase conjugated secondary antibody (1:5000) for 1 h at room temperature. After a final wash with TBS-T (1 x 15 min, 2 x 10 min), proteins were detected by enhanced chemiluminescence using Hyperfilm. The intensity of the bands were quantified by densitometry using a Hewlett Packard 6100 scanner and Image Software, version 1.52 (National Institutes of Health, Bethesda, MD). Primary antibodies to caspase 9, caspase 3 and PARP were purchased from Cell Signaling Technology, Beverly, MA.
Animal studies
Tumor xenografts were used to assess the level of periostin expression following an implantation of the breast cancer cell line MCF-7 and the colorectal cancer cell lines SW-480, MIP101 and HT-29. NIH Swiss nude mice (Taconic Laboratories, Germantown, NY) were injected with 2 million cancer cells subcutaneously. Tumors were then harvested within 3 weeks of growth and processed for northern blot analysis or semi-quantitative RTPCR for periostin and TGF-ß. All studies were approved by the Animal Ethics Committee.
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Results |
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If there was significant upregulation of periostin mRNA and protein in primary colorectal cancers and those at secondary sites (liver metastasis), then one would presume that similar levels of expression would be noted in colorectal cancer cell lines. The results of the expression of periostin in colorectal cell lines were surprising. Human periostin mRNA was not present in cell lines that represented colon cancer (HCT 116, RKO, SW-620, HT 29), breast cancer (MDA 435, MCF-7), uterine sarcoma (MES-SA, MES-SA/DX5) and pancreatic cancer (MIA PaCa-2). The only periostin mRNA transcripts that were detected, belonged to a normal colon cell line (CCD-112CoN) and a mesothelioma cell line (JMN1B) (Figure 2A). This expression was supported at the protein level, where periostin was also observed in the JMN1B cancer cell line (Figure 2B). Despite the absence of periostin in cancer cell lines in vitro, its expression could be stimulated by TGF-ß, as there was a significant increase in periostin following an incubation of the MIP101 colorectal cancer cell lines with TGF-ß1 (Figure 2C). Interestingly, periostin expression could be induced in transformed cell lines with low periostin transcripts in vitro (MCF-7, SW-480, MIP101 and HT-29) following a xenograft implantation into NIH Swiss nude mice (Figure 2D). However, unlike the in vitro results, this change in the periostin mRNA level following xenograft implantation does not appear to correlate with the TGF-ß mRNA levels in these tumors in vivo (Figure 2E). These results not only support the initial findings of an upregulation of periostin protein and gene expression in colorectal cancers, but also bring into question the significance of this dramatic change in expression between the in vitro and in vivo model systems.
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These results provide further evidence of a growth-promoting effect of periostin, and bring into light the possibility of targeting this protein to inhibit colon cancer growth.
Antibodies to periostin contributes to apoptosis in colorectal cancer cells
The results of BrdU cell proliferation study following the incubation of cells with antibodies to periostin in vitro were intriguing and raised the question of whether this effect was secondary to the activation of apoptosis. We investigated this possibility by assessing the cells with TUNEL assay and noted a significantly greater number of cells undergoing apoptosis following an incubation of MIP101 with -periostin mAb, in comparison with 5-FU alone, while no significant increase in apoptosis was detected in the cells incubated with periostin or a non-periostin antibody (Figure 4A and B). This increase in the percentage of cells undergoing apoptosis following an incubation with
-periostin mAb at concentrations >1.0 µg/µl correlated with the activation of caspases 9 and 3 and cleavage of PARP (Figure 4C). Therefore, the results seem to support our hypothesis that
-periostin mAb confers its effect by activating the apoptotic mechanism.
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Discussion |
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The results of the current study provide evidence that supports a role for periostin in colorectal tumorigenesis. Exposure of MIP101 colorectal cancer cells to incremental concentrations of periostin induced a dramatic increase in cell proliferation in a dose-dependent manner. This effect was seen following 72 h of incubation, where an increase in cell proliferation by >70% in comparison with control, untreated cells was seen. This growth-inducing effect could be completely abrogated by incubating MIP101 cells with an antibody targeting this protein at concentrations >1 µg/µl. However, it must be noted that low concentrations of -periostin mAb (0.5 µg/µl) had a paradoxical effect and was able to induce the cell proliferation of MIP101 cells. Although the mechanism of cell proliferation at this low antibody concentration is unclear, there appears to be an upregulation in TGF-ß mRNA following an incubation with
-periostin mAb at 0.5 µg/µl, while it is inhibited at 1.0 µg/µl. These in vitro studies suggest that periostin may be contributing to the growth of colorectal cancers, given the dramatic cell-proliferating effect on cells incubated in the presence of this protein.
The expression of periostin is variable throughout the gastrointestinal tract. Stomach and colon have the highest expressions of periostin. This basal level of protein expression suggests that it may play a role in the normal physiology of the gastrointestinal epithelium. In fact, the relatively higher levels of expression noted in colonic crypts follows our observations that periostin is involved in cell proliferation, as this is the site of active proliferation in the normal epithelium. Moreover, it also follows that there should be a loss of polarity and diffuse expression of this protein in colorectal cancers, as it would again correlate well with the growth-potentiating effect of this protein in malignancy. This was the histologic observation made of the periostin expression in primary tumors and secondary metastasis to the liver. In fact, a dramatic increase in the periostin mRNA expression has also been noted in head and neck squamous cell carcinoma (HNSCC) (12). Even more importantly, similar to our own observations that periostin mRNA expression was low in colorectal cancer cell lines, Gonzalez et al. (12) also observed very low levels of periostin mRNA in HNSCC cell lines, which contrasted with the levels found in primary tumors. This divergent expression in periostin mRNA between primary tumors and their respective cell lines invokes the possibility that stromal components play an integral role in stimulating the periostin expression in malignancy. Another possibility is that, as most malignant cell lines in culture are transformed and do not require a growth stimulus, these cells no longer require periostin for maintaining growth and proliferation. However, in an environment of relative nutrient deficiency, as one would find in xenograft tumor models, the need to maintain adequate growth and proliferation is again required, which results in the upregulation of periostin in cancer cell lines following an implantation into animals. This may partially explain our observation of the periostin mRNA upregulation in MCF-7 breast cancer cell lines and colorectal cancer cell lines SW-480, MIP101 and HT-29 following an implantation into nude mice, while no such expression was seen when these cells were maintained in tissue culture.
Studies have shown that periostin can be upregulated by TGFß1 (13). TGFß1 is well known for its association with the development of colon cancer. High serum levels of this protein correlate with a poor prognosis, conferring an 18-fold increase in the risk of tumor recurrence in such patients (14). This loss of sensitivity to TGFß1-induced tumor suppression is associated with TGFßRII mutations, but TGFß1 also mediates tumor aggressiveness and invasiveness in an autocrine fashion (15). It is also possible that the upregulation of periostin in colorectal cancer may be in response to TGFß1. Our in vitro studies confirmed previous observations that periostin can be induced by TGF-ß1, and although this also raised the possibility of whether a higher TGF-ß expression in tumor xenografts was responsible for upregulating periostin in vivo, this did not appear to be the case. Our results suggest that additional factors may be influencing periostin levels in vivo.
Not only does it appear that periostin is important in cell growth and proliferation, but it also appears to contribute to cell motility and migration. Recent studies revealed periostin to be a ligand for vß3 and
vß5 integrin in ovarian carcinomas and that this interaction could be abrogated by antibodies to
vß3 and
vß5 integrin (16). Recently, Bao et al. (17) showed that periostin interacts with
vß3 to activate the Akt/PKB survival pathway, to facilitate metastasis. These observations correlate well with our observations of an increased and diffuse expression of this protein in colorectal cancers that have metastasized to the liver and provide additional supporting evidence for the role of periostin in cancer cell migration and metastasis.
The results showing that exposure of colorectal cancer cells to antibodies to periostin can, not only reverse the growth promoting potential of periostin, but also induce the apoptotic pathway, was intriguing. Significantly greater number of cells were induced to undergo apoptosis (as denoted by TUNEL positivity) following an incubation with -periostin mAb than with 5-FU alone. Our results showing the involvement of the extrinsic pathway in the signaling cascade for apoptosis, with the activation of caspases 9 and 3 following an incubation with
-periostin antibody, fits nicely with the recent findings that periostin activates the Akt/PKB survival signaling pathway (17). Inhibition of the Akt/PKB pathway would promote apoptosis through Bad inactivation and caspase 9 activation. Further studies are needed to evaluate the exact mechanisms involved in activating apoptosis following the exposure to
-periostin mAb.
Our current observations of the growth-promoting potential of periostin in cancer cells and its upregulation following an implantation in tumor xenograft animal models are intriguing and supports a possible role of this gene and protein in promoting colon cancer growth and metastasis. These, along with current findings that targeting of this protein with antibodies promotes apoptosis, highlight its potential role as a therapeutic target.
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Acknowledgments |
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References |
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