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Cell Biology International (2011) 35, 10851088 (Printed in Great Britain)
Periostin: a putative mediator involved in tumour resistance to anti-angiogenic therapy?
Wei Wang1,2, Jin‑Liang Ma1, Wei‑Dong Jia and Ge‑Liang Xu2
Center for the Study of Liver Cancer and Department of Hepatic Surgery, Anhui Provincial Hospital, Anhui Medical University, Hefei, Peoples Republic of China
Despite advances in the development of anti-angiogenic agents for cancer treatment, the increase in the survival duration of cancer patients is still rather modest. One major obstacle in anti-angiogenic therapy is the emergence of drug resistance. Understanding the molecular mechanisms that enable a tumour to evade anti-angiogenic treatment is valuable to improve therapeutic efficacy. Targeting blood supply usually causes hypoxic responses of tumours that trigger a series of adaptive changes leading to a resistant phenotype. Periostin, a secreted ECM (extracellular matrix) protein, is mainly produced by CAFs (cancer-associated fibroblasts) on hypoxic stress. As CAFs have been casually linked to tumour resistance to angiogenesis blockade and periostin can influence many aspects of tumour biology, we hypothesized that periostin might be a crucial mediator involved anti-angiogenic resistance in cancer treatment. This hypothesis is indirectly supported by the following facts: (a) high levels of periostin promote tumour angiogenesis; (b) periostin improves cancer cell survival under hypoxic conditions; and (c) genetic modulation of periostin induces EMT (epithelial–mesenchymal transition) and enhances cancer cell invasion and metastasis, which represents an escape mechanism from anticancer treatment. Testing and confirmation of this hypothesis will give more insight into the resistance mechanisms and provide the rationale for improvement of therapeutic outcome of anti-angiogenic therapy.
Key words: angiogenesis, cancer, epithelial–mesenchymal transition (EMT), metastasis, periostin, resistance
Abbreviations: CAF, cancer-associated fibroblast, ECM, extracellular matrix, EMT, epithelial–mesenchymal transition, HEK-293T cells, HEK-293 cells expressing the large T-antigen of SV40 (simian virus 40), IL-8, interleukin-8, HNSCC, head and neck squamous cell carcinoma, PKB, protein kinase B, VEGF, vascular endothelial growth factor, VEGFR, VEGF receptor
1These authors contributed equally to this work.
2Correspondence may be addressed to either of these authors (email firstname.lastname@example.org or email@example.com).
Angiogenesis involves a complex cascade of events, including activation, proliferation, and migration of endothelial cells, degradation of vascular basement membranes and formation of new vascular tubes from pre-existing vessels. This process is required for tumour growth, providing cancer cells with oxygen and nutrients (Folkman, 1990). As cancerous tumours outgrow their blood supply, a hostile microenvironment characterized by hypoxia and nutrient shortage develops. Cancer cells commonly undergo a series of adaptive changes that stimulate de novo vessel formation and thus enable them to circumvent the environmental stress. Initiation of the ‘angiogenic switch’, a rate-limiting event of tumour progression, involves the release of pro-angiogenic factors such as VEGF (vascular endothelial growth factor), IL-8 (interleukin-8) and platelet-derived endothelial cell growth factor (Relf et al., 1997), as well as down-regulation of angiogenesis suppressors such as thrombospondin (Dameron et al., 1994).
With advances in the understanding of the biology of angiogenesis, various angiogenesis inhibitors have been explored for cancer therapy, aiming to target vascular endothelial cells and impair the tumour blood supply. Given the key importance of the VEGF ligand–receptor pathway in angiogenesis, most of the anti-angiogenic agents currently being tested in clinical trials have focused on targeting this pathway. They include the VEGFR (VEGF receptor) tyrosine kinase inhibitors sunitinib and sorafenib, as well as the anti-VEGF monoclonal antibody bevacizumab. These VEGF-targeted agents have exhibited therapeutic efficacy in mouse models of cancer and in an increasing number of human cancers. However, their clinical benefits are transitory, without providing enduring cure. Clinical experience shows that the therapies targeting the VEGF pathway give a prolongation of overall survival in cancer patients by only months (Kerbel, 2008). Understanding the mechanistic basis of resistance to anti-angiogenic therapy will aid the development of more potent treatments for solid tumours.
Periostin is a 90-kDa ECM (extracellular matrix) protein containing a tandem repeat of four fascilin-like domains (Horiuchi et al., 1999; Norris et al., 2009). It was initially identified in a mouse osteoblastic library as a putative bone adhesion protein (Takeshita et al., 1993). Functional studies using periostin-null mice reveal that periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues and functions as a regulator of cardiac remodelling and hypertrophy (Norris et al., 2007; Oka et al., 2007). Periostin is able to interact with multiple integrins to co-ordinate a variety of cellular processes, including cell proliferation, EMT (epithelial–mesenchymal transition) and cell migration (Gillan et al., 2002; Butcher et al., 2007; Wallace et al., 2008). Recently, a large body of evidence indicates that periostin is aberrantly overexpressed in various human cancers and correlates with a more aggressive phenotype (Kudo et al., 2006; Kikuchi et al., 2008; Riener et al., 2010). Baril et al. (2007) reported that periostin enhances invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death. Acquired expression of periostin has been documented to promote tumour angiogenesis through up-regulation of VEGFR2 expression (Shao et al., 2004). These observations led us to hypothesize that periostin might contribute to the anti-angiogenic therapy-resistant phenotype of malignant tumours.
2. Acquired expression of periostin in response to a hypoxic tumour milieu
Hypoxia is a hallmark of solid tumours, which appears to have paradoxical effects on cancer cells: on the one hand, it inhibits cell proliferation and eventually induces apoptosis/necrosis; on the other hand, it selects for more aggressive cells that can facilitate tumour progression and dissemination (Box et al., 2010). Interactions of tumour cells with surrounding stromal cells and ECM components are thought to be crucial for adaptive responses to hypoxia (Box et al., 2010). Accumulating evidence reveals that CAFs (cancer-associated fibroblasts), key components of the tumour stroma, acquire new characteristics distinct from their normal counterparts, and contribute to tumorigenesis by stimulating angiogenesis, cancer cell proliferation and invasion (Kalluri and Zeisberg, 2006). Trimboli et al. (2009) reported that activation of stromal fibroblasts of mouse mammary glands through genetic depletion of Pten accelerates the initiation and malignant transformation of mammary epithelial tumours, which is coupled with the ECM remodelling, innate immune cell infiltration and increased angiogenesis. Wang et al. (2009) found that co-culture with fibroblasts renders lung cancer cells (PC-9 and HCC827) resistant to EGFR (epidermal growth factor receptor) tyrosine kinase inhibitors. Importantly, CAFs are capable of inducing VEGF-independent angiogenesis and can support tumour growth even in the setting of VEGF blockade, which provides an important mechanism of resistance to anti-angiogenic cancer therapy (Crawford et al., 2009).
The tumour-promoting effects of CAFs are largely mediated by the production of a broad range of soluble factors, which act in a paracrine manner and thus affect not only cancer cells but also other cell types present in the tumour stroma (Kalluri and Zeisberg, 2006). A hypoxic microenvironment is believed to shape the phenotype of CAFs. Pilch et al. (2001) reported that hypoxic stimulation promotes the release of angiogenic growth factors from cervical cancer-derived fibroblasts. Among the bioactive factors released from CAFs, periostin is attracting more and more attention. Periostin is implicated in multiple biological processes related to malignant tumour progression, including growth, invasion, EMT, angiogenesis and metastasis (Baril et al., 2007; Zhu et al., 2010; Ben et al., 2011). CAFs are the major source of periostin, although some types of carcinoma cells produce this protein as well. Most interestingly, periostin can be up-regulated by hypoxia in distinct cellular contexts (Erkan et al., 2009; Ouyang et al., 2009). These findings collectively suggest that acquired expression of periostin in cancer may be a consequence of a hypoxic microenvironment contributing to tumour progression.
3. Does periostin foster resistance to anti-angiogenic cancer therapy?
It is well accepted that hypoxia-induced adaptive changes endow cancer cells with a surviving phenotype, thereby blunting the efficacy of vessel-targeting agents. Given the frequent up-regulation of periostin under hypoxic conditions, it can be hypothesized that periostin might play a feedback role in inducing the resistance to anti-angiogenic therapy. Herein we thoroughly reviewed the literature to summarize the evidence supporting this hypothesis.
There is a strong association between periostin expression and tumour angiogenesis. Periostin correlates with increased microvessel density and lymphatic microvessel density in patients with non-small cell lung cancer (Takanami et al., 2008). In oral cancer, an enhanced blood vessel density was observed in periostin-positive cases compared with those of periostin-negative cases (Siriwardena et al., 2006). Moreover, recombinant periostin augments capillary formation in vitro in a concentration-dependent manner, providing a direct evidence for the pro-angiogenic activity of periostin. Using tumour cell lines overexpressing ectopic periostin, Shao et al. (2004) found that xenografts of these engineered cells have increased angiogenesis, which is largely mediated by the up-regulation of VEGFR2 on endothelial cells. These studies strongly point towards a critical role for periostin in the regulation of endothelial cell behaviour. However, periostin seems not to directly promote endothelial cell proliferation but rather increases the sensitivity of endothelial cells to VEGF through the induction of VEGFR2 expression (Shao et al., 2004). Therefore it should be checked whether or to what extent the angiogenesis-promoting effect of periostin could be independent on VEGF signalling.
Emerging evidence shows that VEGF signalling-targeted agents can increase invasiveness and metastasis in some tumour types (Ebos et al., 2009; Pàez-Ribes et al., 2009). It is believed that increased tumour hypoxia contributes to the development of these phenomena. Metastasis is a complex process consisting of sequential steps that involve detachment from the primary tumour, intravasation of the tumour vasculature and extravasation at distant sites to form metastases. Enhanced metastasis represents an evasion mechanism in cancer therapy and has been proposed to be partially responsible for the modest efficacy of anti-angiogenic therapy. Interestingly, periostin influences many aspects of metastasis, including survival, morphological change, growth, migration and invasion. Several studies have demonstrated that periostin benefits cancer cell survival under hypoxic stress conditions through the Akt/PKB (protein kinase B) pathway (Bao et al., 2004; Baril et al., 2007). Moreover, periostin gives rise to a more invasive phenotype of cancer cells. Gillan et al. (2002) showed that periostin secreted by epithelial ovarian carcinoma functions as a ligand for αVβ3 and αVβ5 integrins to support cell adhesion and migration. Using gain- and loss-of-function approaches, Michaylira et al. (2010) revealed that genetic modulation of periostin in oesophageal cancer promotes tumour cell migration and invasion. Also, periostin overexpression enhances invasion- and anchorage-independent growth of HNSCC (head and neck squamous cell carcinoma) cells, and results in spontaneous metastasis to the lung in an orthotopic mouse model of HNSCC (Kudo et al., 2006).
EMT is a biological process whereby epithelial cells acquire a mesenchymal phenotype, often leading to enhanced migratory capacity and invasiveness. Accumulating evidence suggests that EMT of tumour cells not only causes increased metastasis, but also confers drug resistance, which is likely attributed to the generation of cancer stem-like cells (Ahmed et al., 2010). There is a high expression of periostin during EMT of cancer cells in non-small cell lung cancer (Soltermann et al., 2008). Yan and Shao (2006) reported that forced expression of periostin induces an EMT program in tumorigenic but non-metastatic HEK-293T cells [HEK-293 cells expressing the large T-antigen of SV40 (simian virus 40)], endowing cells with a more aggressive phenotype. Importantly, periostin-engineered HEK-293T cells formed metastases in immunodeficient mice following either cardiac inoculation or injection into the mammary fat pad. These observations, combined with the findings regarding the effects of periostin on tumour angiogenesis and invasiveness, suggest a potential role for periostin in inducing anti-angiogenic resistance. On one hand, periostin contributes to establishment of a tumour-supporting microenvironment by orchestrating endothelial cell behaviour and stimulating angiogenesis. On the other hand, periostin exerts direct roles on cancer cells: improving cell survival and inducing EMT program that selects more resistant cancer cells and/or enables cells to escape from hypoxic insults of angiogenesis inhibitors. The multi-faceted functions of periostin make it an attractive target for improving the efficacy of anti-angiogenic therapy.
4. Testing the hypothesis
Xenograft models of tumour cells were generated which mimicked clinical resistance to anti-angiogenic agents such as sunitinib. Pathological analyses were conducted to assess the associations of the expression and distribution of periostin with anti-angiogenic resistance. If periostin were actually involved in an alternative pathway of tumour survival and dissemination in the setting of angiogenesis inhibition, targeting periostin would resensitize tumours to anti-angiogenic therapy and restrain tumour metastasis. Taking advantage of neutralizing antibody and/or siRNA (small interfering RNA), we could confirm the requirement of periostin for the acquisition of anti-angiogenic resistance in cancer. Conversely, the administration of purified periostin in sensitive tumours was used to investigate the effects of periostin on the sensitivity to anti-angiogenic agents and tumour malignant progression. Since periostin is a secreted protein, it may have an influence on multiple types of cells in tumours. In vitro assays using purified periostin or neutralizing antibody were carried out to examine the effects of periostin on distinct target cells, especially endothelial cells and cancer cells. Once the involvement of periostin is confirmed in the resistance-to-anti-angiogenic therapy, then the underlying molecular mechanism for periostin action should be investigated. Several previous studies have causally linked the periostin function with alteration of the integrin signalling and PI3K (phosphoinositide 3-kinase) signalling pathways (Gillan et al., 2002; Baril et al., 2007). Microarray technology will be useful to identify novel targets and signalling pathway components controlled by periostin, thus providing a better understanding of the action of periostin. Kim et al. (2005) reported that ectopic expression of periostin by a retrovirus vector suppressed in vitro cell invasiveness of the bladder cancer cells and in vivo lung metastasis of the mouse melanoma cell line B16-F10. Although the observed suppressive effects of periostin may reflect non-physiological conditions, it should be checked whether the function of periostin depended on the dose and cellular context.
5. Implications of the hypothesis
In principle, targeting tumour vascularization has several potential advantages over attacking cancer cells themselves, because the targeted vascular endothelial cells are easily accessible through the bloodstream and genetically stable and are therefore less likely to develop drug resistance. Importantly, agents directed against tumour vasculature may be applicable to a wide range of solid tumour types. However, the successful application of anti-angiogenic therapy for cancer is impeded by the emergence of drug resistance. Since periostin is up-regulated in hypoxic tumour microenvironment and has multi-faceted roles in tumour progression, it is interesting and reasonable to check for its role in the development of resistance to angiogenesis inhibitors. The proposed hypothesis suggests that periostin may contribute to anti-angiogenic resistance through modulating endothelial cell behaviour, promoting cell survival under hypoxic stress, and inducing EMT and a more invasive phenotype in cancer cells. Dissecting the function and mechanism of periostin would offer an attractive therapeutic target in circumventing anti-angiogenic resistance and improving survival benefits of conventional cancer therapy.
This research was supported by the
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Received 18 March 2011/29 April 2011; accepted 31 May 2011
Published online 27 September 2011, doi:10.1042/CBI20110171
© The Author(s) Journal compilation © 2011 Portland Press Limited