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Department of Pharmacology and Cancer Biology,1 Department of Experimental Surgery, Duke University Medical Center, Durham, North Carolina 27710,2 SUGEN, Inc., South San Francisco, California 940803
Received 5 November 2003/ Returned for modification 16 December 2003/ Accepted 27 January 2004
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ABSTRACT |
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vß3-focal adhesion kinase-mediated signaling pathway. These findings demonstrate the presence of a novel mechanism by which tumor angiogenesis is acquired with the expression of a mesenchyme-specific gene as a crucial step in late stages of tumorigenesis. |
INTRODUCTION |
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Recently, large-scale efforts have been made to determine gene expression pattern differences between various types of human cancers and their corresponding normal tissues by using the serial analysis of gene expression (SAGE) and gene array analyses (14, 33-35, 37). Indeed, significant differences in gene expression patterns have been revealed by these studies. In breast cancer, for example, such investigations have led to the application of gene array analysis in the diagnosis, prognosis, and design of rational treatment of patients according to the molecular signatures of the individual tumors (21, 22, 32, 35). In the meantime, although the alterations of oncogenes and tumor suppressor genes have shown a close association with the progression of human cancers based on their defined functions, less is known about the specific contributions of a large number of genes whose expression patterns are also significantly changed during the tumorigenic process. Particularly interesting is the observation that mesenchyme-specific genes, normally associated with osteoblasts, are highly expressed by various types of human cancers (17, 31). However, the expression of mesenchyme-specific genes has not been functionally linked to the development of specific tumor phenotypes.
To address this question, we sought to determine the potential contributions of such candidate genes to specific phenotypic changes associated with the progression of late-stage tumorigenesis and identified a mesenchyme-specific gene product, periostin, as a novel angiogenic factor whose overexpression by human breast cancers leads to the significant enhancement of angiogenesis. The angiogenic activity of periostin correlated with the increased expression of the VEGF receptor Flk-1/KDR by endothelial cells through an integrin vß3-focal adhesion kinase (FAK)-mediated signaling pathway. These findings indicate that epithelial cell-derived tumors may gain the capabilities to generate more blood vessels, invade, and metastasize during late stages of tumorigenesis by the acquired expression of genes whose functions are normally associated only with mesenchymal cells.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of periostin-producing cells. Full-length human periostin cDNA (MluI-XhoI) was subcloned into a retroviral pCMV-neo-vector. 293T retroviral packaging cells were transfected with the periostin construct or vector control in the presence of pCL 10A1 vector by using Fugene 6 as the delivery vehicle. Forty-eight hours after transfection, the supernatant was harvested and filtered through a 0.4-µm-pore-size filter, and the virus-containing medium was used to infect cells. Selection with 800 µg of G418/ml was started 48 h after infection. For 293T, B16F1, and MDA-MB-231 cell lines, the drug-resistant cell populations were used for subsequent studies of tumor formation. For MCF-7 cells, a single stable clone that expresses periostin was isolated.
Tumor xenograft analysis in mice. Four-week-old female SCID-Beige mice (Charles River, Wilmington, Mass.) were subcutaneously injected with control or periostin-producing 293T (2.5 x 107), MDA-MB-231 (1 x 107), or B16F1 (0.4 x 107) cells in 0.2 ml of Hank's balanced buffer without calcium and magnesium. The growth of solid tumors from the injected cells was monitored daily for up to 2 or 4 weeks before the animals were sacrificed to remove tumors for analysis. For testing the effect of Flk-1/KDR inhibitor SU5416 on tumor growth, mice were injected subcutaneously to the opposite flank of the tumor cell injection with SU5416 (20 mg/kg) in suspension of a diluent (0.5% carboxymethylcellulose sodium, 0.9% sodium chloride, 0.4% polysorbate 80, 0.9% benzyl alcohol) every other day for 5 weeks. Control mice were treated with the diluent alone. The removed tumors were measured and calculated as follows: volume = length x width2 x 0.52.
Hemoglobin content measurement. Tumor tissue (0.25 g) was removed immediately after animal sacrifice, and blood was mechanically extracted in phosphate-buffered saline containing heparin. Hemoglobin concentration was determined as previously described (2).
Immunohistochemical analysis. For frozen tissue samples, tumor slides (thickness, 6 to 10 µm) were fixed in 2% paraformaldehyde. Sections were stained for the presence of CD31 (Becton Dickinson Labware, Bedford, Mass.), Flk-1/KDR (Research Santa Cruz, Santa Cruz, Calif.), or periostin according to the manufacturer's instructions (Vector Laboratories, Burlingame, Calif.).
Generation of recombinant periostin. Full-length human periostin cDNA with a His tag was subcloned into pFastBac1 vector (Life Technologies, Rockville, Md.). Following transformation and amplification in Escherichia coli DH10Bac, bacmid DNA containing periostin was transfected into Sf9 insect cells (Invitrogen, Carlsbad, Calif.) by using Cellfectin reagent (Life Technologies), and baculoviral medium was produced. Recombinant periostin was generated 48 h after infection of High-5 cells with viral medium. A Ni-nitrilotriacetic acid column was used to purify recombinant periostin according to the manufacturer's instructions (Life Technologies), and pure periostin was finally produced through a PD-10 column (Amersham Pharmacia Biotech, Piscataway, N.J.).
Cell migration assay. Human microvascular endothelial cells (HMVEC) (2 x 105) were preincubated with serum-free medium for 12 h and transferred onto transwells (24-well plates) for migration assays as previously described (30).
[3H]thymidine incorporation assay. HMVEC were grown in 12-well plates to subconfluence, and the culture medium was changed to conditioned medium from the parental, control, and periostin-producing MCF-7 cells for 12 h. After washing, VEGF (10 ng/ml) was added for 12 h. A total of 2 µCi of [3H]thymidine was then added to each well for 6 h. After being thoroughly washed with phosphate-buffered saline, the cells were scraped and precipitated with 200 µl of 10% trichloroacetic acid. 3H radioactivity was solubilized in 0.3 ml of 0.3 M NaOH and quantitated by liquid scintillation count.
Western blot analysis. Serum-free media from cultured confluent cells were collected, and the presence of secreted periostin was determined by immunoblotting with a polyclonal antiperiostin antibody. The antibody was generated by immunizing the rabbits with recombinant periostin protein and was purified through an affinity column. For the measurement of Flk-1/KDR and FAK activation, an anti-phospho-Tyr 951 antibody (Research Santa Cruz) and an anti-phospho-Tyr 681 FAK antibody (Biosource, Camarillo, Calif.) were used to detect phosphorylated Flk-1/KDR or FAK in cell lysates.
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RESULTS |
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Overexpression of periostin is associated with enhanced tumor growth and angiogenesis. The acquired expression of periostin by various types of cancers including breast cancer suggests that periostin may be intimately associated with the progression of tumor development. To test this hypothesis, tumor cells were engineered to produce periostin and injected into immunocompromised animals, and the growth characteristics of the resulting solid tumors were examined. Specifically, we used three cell lines that do not express endogenous periostin at a detectable level: the 293T cell line derived from human kidney epithelial cells, the highly invasive mouse melanoma cell line B16F1, and the metastatic human breast cancer line MDA-MB-231. After introducing the periostin gene into these cells by means of a retroviral vector infection system, we examined the expression of periostin by performing Western blot analysis on the conditioned media harvested from the stably infected cell populations.
As shown in Fig. 2A, all three cell populations, compared to control vector-infected cells, secreted significant amounts of periostin to the media. Interestingly, the proliferation rate of the periostin-producing cells was found to be noticeably slower than that of the control cells in culture (data not shown), suggesting that periostin does not confer a proliferation-promoting effect on tumor cells in vitro. These cell populations were then injected subcutaneously into SCID-Beige mice, and the growth characteristics of resulting tumors were analyzed over a period of 14 or 28 days. For 293T cells, the volume of tumors derived from periostin-producing cells was two- to fourfold higher than that of tumors from control cells on day 28 (Fig. 2B). Strikingly, expression of periostin also enhanced the growth of tumors derived from the B16F1 and MDA-MB-231 cells (Fig. 2B), despite the fact that those cell lines are known to be among the most aggressive types in tumor formation when they are grown as xenografts. Interestingly, we found that the growth of tumors derived from the vector-transfected control cells was not affected by the presence of periostin-producing tumors in the same animal when the two cell populations were injected into opposite flanks (data not shown), suggesting that periostin exerts its effect locally rather than systematically.
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To probe this possibility further, we performed an immunohistochemical analysis on tumor sections by utilizing an antibody against vascular endothelial cell marker CD31, an assay commonly used to detect the presence of vascular endothelial cells. Consistent with the results of the hemoglobin content measurement, we found that tumors derived from periostin-producing cells were much more intensively vascularized than control tumors as determined by the more intense staining of the marker CD31 (Fig. 2E and F). This result firmly established that the presence of periostin is intimately associated with the presence of a higher density of vasculature and endothelial cells in tumors grown as xenografts. Taken together, our data strongly suggest that periostin may act to promote tumor growth by inducing tumor angiogenesis.
Periostin promotes angiogenesis via up-regulation of VEGF receptor 2 expression in endothelial cells through an integrin vß3-FAK-mediated signaling pathway.
To explore the underlying mechanism by which periostin promotes tumor angiogenesis, we examined the effect of periostin on the activity of HMVEC that are known to play an essential role in the formation of new blood vessels. Since primary HMVEC can grow in culture for a very limited number of passages before adapting a senescence phenotype, hampering our analysis of periostin on HMVEC biology, we introduced the human telomerase catalytic subunit (hTERT) into the primary HMVEC to extend their life span in culture. As with other cell types (11, 20), hTERT expression extended the life span but had no effect on the proliferative and migration profiles of HMVEC (data not shown), and we have consequently used them in all of our subsequent experiments.
To test the possibility that periostin derived from the tumor tissue could exert angiogenic effects on vascular endothelial cells in vivo via a paracrine loop, we developed an in vitro coculture system to evaluate the angiogenic activity of periostin secreted by tumor cells. Cells from a stable clone of the MCF-7 human breast cancer cell line expressing high levels of periostin (Fig. 3A) were grown to reach confluence in the bottom chamber of transwells, and then HMVEC were plated onto the upper chamber. After incubation for 4 h, we found that the number of migrated HMVEC in the well containing periostin-producing MCF-7 cells was fivefold higher than that in the wells containing either parental or vector control MCF-7 cells (Fig. 3B). Consistent with the positive activity of periostin on endothelial cells observed with enhancement in cell migration, the proliferation of HMVEC was also stimulated by culturing the cells in periostin-containing conditioned media (Fig. 3C).
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In contrast, incubation of HMVEC with recombinant periostin or the conditioned medium from periostin-producing MCF-7 cells resulted in up-regulation of Flk-1/KDR expression in a dose- and time-dependent manner (Fig. 4A and B). Consistent with the results shown in Fig. 2E, immunostaining of tumor sections with the anti-Flk-1/KDR antibody also confirmed the presence of a higher level of presence of this VEGF receptor associated with the higher density of blood vessels in tumors derived from periostin-producing cells (Fig. 4C). To determine if up-regulation of Flk-1/KDR expression leads to an increase in the sensitivity of endothelial cells to VEGF, we pretreated HMVEC with recombinant periostin and subsequently measured the cellular proliferative response to VEGF. As shown in Fig. 4D, the proliferation of HMVEC was increased in response to VEGF in comparison to untreated control cells, and this stimulatory response was further potentiated by the periostin pretreatment. To confirm that the significant enhancement in response to VEGF by the periostin-pretreated HMVEC was the result of the increased activity of Flk-1/KDR due to its up-regulated expression, we examined the potential changes in the kinase autophosphorylation activity of Flk-1/KDR by using a specific anti-phospho-Tyr 951 antibody (3). As shown in Fig. 4E, periostin pretreatment of HMVEC led to a significant increase in the amount of phosphorylated Flk-1/KDR as a direct reflection of the increase in Flk-1/KDR expression and, consequently, enhancement in its activation in response to VEGF.
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vß3 integrins (data not shown). Treatment of HMVEC with recombinant periostin did not alter the expression profile of the integrins (data not shown). We next probed if interference with the function of integrins by specific anti-integrin antibodies has an effect on the ability of periostin to mediate cell adhesion and induction of Flk-1/KDR in HMVEC. As shown in Fig. 5A, treatment of HMVEC with a specific anti-vß3 integrin antibody inhibited adhesion of these cells to the culture wells precoated with periostin. Consistent with this result, treatment of HMVEC with periostin in the presence of the anti-vß3 integrin antibody prevented the induction of Flk-1/KDR (Fig. 5B). The specificity of the blockage of periostin activity achieved by interfering with the function of vß3 integrin was demonstrated by the lack of an effect on the periostin-mediated cell adhesion and induction of Flk-1/KDR expression when a specific anti-vß5 integrin antibody was used in the same assays (Fig. 5). The initial step of integrin signaling involves the activation of FAK. Consistent with this notion, we found that transient stimulation of HMVEC with periostin augmented the phosphorylation of FAK on tyrosine 681 (Fig. 5B), an event indicative of the activation of FAK (1). The increase in FAK phosphorylation on Tyr 681 was reversed to the basal level by the presence of the anti-vß3 integrin antibody but not the anti-vß5 integrin antibody. Taken together, these results strongly suggest that the vß3 integrin-FAK signaling pathway plays an essential role in mediating the effect of periostin on the up-regulation of Flk-1/KDR expression in HMVEC.
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DISCUSSION |
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Importantly, the acquired expression of periostin by tumors of epithelial origin described here is certainly not an isolated and rare event during tumorigenesis, since a number of other examples of acquired expression of mesenchymal genes by epithelial-cell-derived tumors can be found in the literature (17, 18, 23). For example, osteonectin, also termed SPARC, whose physiological activity is believed to be associated mainly with osteoblast function, has been found to be overexpressed by a wide range of human cancer types (15, 24, 31). As a secreted polypeptide without sequence homology with periostin, osteonectin has been implicated to promote tumor progression and angiogenesis (10). Thus, the common feature for this group of structurally unrelated and functionally diverse molecules, either as secreted or extracellular matrix-associated proteins, is that their physiological functions are normally associated strictly with cells that derive from mesenchymal origins such as osteoblasts. By acquiring the expression of such mesenchymal genes, the epithelial carcinoma cells gain the abilities that are normally associated with mesenchymal cells, an event which appears to correlate with the progression into a more aggressive cancer phenotype.
Our immunohistochemical staining data suggest that periostin is present predominantly in areas containing cancer cells within the tumor mass. Based on the result of RNA in situ hybridization, the source of periostin production was determined to be the carcinoma cells, a notion that is consistent with the conclusion of a recent study on the production of periostin by human ovarian cancer cells (6). However, another recent report suggested that the localization of periostin mRNA was mainly associated with the stromal portion of the tumor tissue sample isolated from a breast cancer patient (27). In any event, periostin produced by either cell type within the tumor tissue could exert a similar paracrine effect on the endothelial cells recruited to the tumor mass to promote angiogenesis. As a related matter, we have also observed that the tumor cells engineered to produce periostin in culture containing a normal level of fetal bovine serum had a growth disadvantage in comparison to control cells (data not shown). In fact, we had to resort to the use of tumor cell populations in which ectopic periostin expression was engineered by retroviral infection and transient drug selection since we could not obtain multiple lines of stable clones that overexpress periostin; the exception was a single stable clone from MCF7 cells which was used as a source of periostin production in a number of the experiments described above. Thus, although periostin may confer an advantage for the growth of breast tumors in vivo by altering the microenvironment through the induction of angiogenesis, its overexpression may impose a growth disadvantage when the tumor cells are grown in culture.
These observations reveal the vital importance of relying on evidence derived from primary human cancer samples rather than established cell lines to draw major conclusions on the mechanism and involvement of specific genes in tumorigenesis. Furthermore, the tumor-promoting effect of molecules such as periostin can only be revealed by in vivo analysis in animal models, rather than solely by in vitro studies in cell culture. The normal functions of this type of gene are often not associated with the promotion of cell proliferation, in contrast to the roles of many defined oncogenes. Instead, this group of proteins may exert their influence on tumorigenesis by changing the microenvironment of tumor growth through the regulation or alteration of cell adhesion, composition of the extracellular matrix, and the activities of stromal cells within and surrounding the tumor mass.
Based on the results of this study with periostin as an example, we suspect that different types of human cancers derived from epithelial origins, and even cancers from the same tissue type but derived from different individual patients, may acquire the expression of different sets of mesenchymal-specific genes to gain different mesenchymal-associated capabilities during late stages of tumorigenesis. In other words, the heterogeneity in the functions of this group of genes may confer different tumorigenic capabilities on the cancer cells that acquire the expression of such genes, creating another layer of heterogeneity and complexity for each type and even each case of cancer development. Thus, identification and characterization of such genes will become crucial for a full understanding of the molecular events associated with late stages of tumorigenesis, particularly angiogenesis and metastasis, and for the future development of specific and effective regimens for a cancer treatment tailored to each individual patient. To this end, the availability of a vast amount of data on gene expression profiles derived from the SAGE library and gene array analyses of a broad range of human cancer types in comparison to their counterparts of normal tissue has provided us with a golden opportunity through functional genomics to search for and identify candidate genes that fit this profile. Since the activities of these molecules may not be associated with the promotion of cell proliferation, the foremost criterion for evaluating the potential contribution of any candidate genes to the progression of tumorigenesis will have to be based on an assessment of the ability of those genes to promote tumorigenesis in in vivo studies of xenografts or transgenic animal model systems.
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ACKNOWLEDGMENTS |
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This study was supported in part by NIH grant CA 83770 and DOD grant BC-980188 to X.-F.W. and by DOD postdoctoral fellowship grant DAMD17-00-100228 to R.S.
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FOOTNOTES |
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