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Currently available data indicate the potential application of rapamycin and its analogues in the clinic as anticancer therapeutic agents through inhibiting tumor cell growth and tumor angiogenesis. However, whether rapamycin can directly suppress tumor metastasis remains unclear. In the present study, we demonstrated that rapamycin treatment results in reduced formation of metastatic nodules in the lung by B16 cells. This is due to two mechanisms. First, the expression of αv integrin is down-regulated by rapamycin treatment, and subsequently, the phosphorylation of focal adhesion kinase (FAK) is reduced. Second, rapamycin promotes apoptosis by up-regulating the proapoptotic molecules Bid and Bax and down-regulating Bcl-xL. Blocking the apoptosis pathway by pan-caspase inhibitor zVAD partially reversed the suppression of rapamycin in B16 metastasis. Interestingly, rapamycin up-regulates Bax and Bid in B16 cells via the S6K1 pathway and down-regulates the expression of αv integrin via other pathway(s). In addition, our data showed that autophagy was not involved in the mechanisms of rapamycin-mediated metastasis suppression. Our findings demonstrate a potential anti-metastatic effect of rapamycin via down-regulating αv integrin expression and up-regulating apoptosis signaling, suggesting that rapamycin might be worthy of clinical evaluation as an antimetastatic agent. (Cancer Sci 2009)
The development of metastases is the major cause of death for cancer patients. However, effective treatment and prevention of metastases remains unavailable. The molecular mechanisms involved in tumor metastases are incompletely understood, although it is generally accepted that tumor metastases are associated with tumor cell-matrix adhesion, with the degradation of the extracellular matrix (ECM), and with the survival of tumor cells in blood stream.(1,2) Hence, these steps provide potential targets against tumor metastasis.
Integrin signaling pathways play a fundamental role in tumor cell-matrix adhesion and metastasis.(3–5) The engagement of integrins with ECM components such as fibronectin, collagen, and laminin results in tumor cell adhesion, mobility and migration, proliferation, and survival. Integrins are membrane-spanning heterodimers composed of α and β subunits. Tumor cells commonly express altered integrins on their surface. For instance, αvβ3 integrin is not expressed in normal mammary epithelium; however, altered expression of αvβ3 integrin is frequently observed in various types of tumors, including breast,(6,7) prostate,(8) ovary,(9) melanomas,(10,11) and gliomas.(12) Notably, αv integrin is required for melanoma metastasis and targeting the αv integrin signaling pathway inhibits the metastatic potential of melanoma. (13–18) It has been shown that the engagement of αvβ3 integrin with its ligand can alter cellular behavior through the recruitment and activation of signaling protein such as focal adhesion kinase (FAK), leading to tumor cell migration and invasion.(19,20) In addition, up-regulation of cdc2, a downstream target molecule of the αvβ3 signaling pathway, also promotes tumor cell migration.(20–22)
ECM degradation, mediated by matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9,(23,24) is another prerequisite for tumor metastasis. As a consequence of ECM degradation, tumor cells have the chance to interact with and enter into blood vessels. In the bloodstream, which is different from the primary tumor microenvironment, the dynamic flow conditions are dangerous for circulating tumor cells. Such tumor cells have to be subjected to shear forces and most of them are cleared from circulation through the apoptosis pathway.(25) Therefore, apoptosis-resistance is critical for the survival of circulating tumor cells.
Rapamycin, a lipophilic macrolide antibiotic, was originally identified as a fungicide and immunosuppressant.(26) However, studies have revealed that rapamycin can potently arrest the growth of cells derived from a broad spectrum of cancers.(27–32) Rapamycin has been shown to specifically inhibit its target mTOR (mammalian target of rapamycin), which is a key player in tumor development and progression.(33) Rapamycin binds the immunophilin FK506 binding protein (FKBP12) to form the FKBP12-rapamycin complex, which then interacts with mTOR and inhibits the mTOR-mediated phosphorylation of S6K1 and 4E-BP1.(33) In addition, rapamycin is the best characterized drug that enhances autophagy,(34–36) a process of “self-eating” that involves both cancer cell death and survival. Therefore, rapamycin might interfere with different aspects of the tumor.
It has been noted that rapamycin can impede tumor metastasis by suppressing tumor angiogenesis and lymphangiogenesis.(37–39) However, the direct effects of rapamycin on tumor metastasis remain unclear. In this study, we demonstrate that rapamycin directly suppresses tumor metastasis through down-regulating αv integrin and up-regulating apoptosis signaling in a mouse model of melanoma lung metastasis. Our data also indicate that autophagy is not involved in the rapamycin-mediated suppression of metastasis.
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The major finding in the present study is that rapamycin directly suppresses the lung metastasis of circulating B16 melanoma cells through mechanisms of down-regulating αv integrin expression and up-regulating apoptosis signaling. Our data also indicate that autophagy is not involved in rapamycin-mediated metastasis suppression.
Intense studies on the antitumor effects of rapamycin have been mainly focused on the inhibition of tumor cell proliferation and tumor angiogenesis. Here, we provide additional evidence that rapamycin can directly suppress tumor metastasis. Even though it has been shown that mTOR has versatile effects on cancer development and progression, we first identified that rapamycin targets integrin, the critical signaling pathway required for tumor cell migration and metastasis. Our convincing evidence includes (i) both mRNA and protein levels of αv integrin were decreased in B16 cells after rapamycin treatment; (ii) the phosphorylation of downstream signal molecule FAK declined; and (iii) the mRNA level of effector molecule cdc2 was decreased. The members of the αv integrin family are widely expressed in various types of tumor cells, which involve tumor angiogenesis and metastasis. There are five recognized αv integrin receptors: αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8.(48) Among them, agents targeting the αvβ3 integrin receptor are now in clinical development for treating solid tumors.(49–51) Therefore, targeting αv integrin by rapamycin may have important clinical significance. In addition, although we confirm here that αv integrin is targeted by rapamycin, our ongoing study is trying to elucidate whether other integrin(s) is also targeted by rapamycin and the underlying mechanism(s).
By complexing with FKBP-12, rapamycin binds to mTOR and inhibits the mTOR-mediated phosphorylation of downstream targets S6K1 and 4E-BP1. However, S6K1 and 4E-BP1 seem not to regulate integrin expression, considering that the activities of S6K1 and 4E-BP1 are related to cell growth and cell cycle progression.(52,53) In support of this, our data show that the knockdown of S6K1 by specific siRNA does not decrease the expression of αv integrin in B16 cells (Fig. 6d), suggesting that other target(s) of mTOR might be involved in the regulation of integrin expression. Therefore, a closer examination of the regulation of integrin by mTOR signaling should be useful for the comprehensive evaluation of the tumor therapeutic value of rapamycin.
Besides the inhibition of metastasis through down-regulating integrin signaling, the present study also reveals that rapamycin can suppress metastasis through up-regulating apoptosis signaling. To succeed in metastasis, circulating cancer cells have to pass through several stressful and highly selective steps, including survival in the bloodstream, arrest in the capillary bed, and resumption of proliferation in distant organs. The acquirement of apoptosis resistance is a prerequisite for tumor cells to realize these goals. It has been reported that Bcl2 overexpression is associated with a 10-fold decrease in the number of apoptotic tumor cells at the secondary site 1 h after intravenous injection,(54) and with an increase in metastasis formation.(55) Therefore, the increase of apoptosis potential by rapamycin may play an important role in rapamycin-mediated metastasis suppression. Actually, our in vivo data clearly show that blockade of apoptosis pathway by pan-caspase inhibitor zVAD effectively declines the suppression of rapamycin in B16 lung metastasis (Fig. 5a).
The occurrence of metastasis may be unpredictable after surgical excision of primary tumor. Therefore the prevention of metastasis should be a long-term event. Considering the multiple effects on tumor cell proliferation, apoptosis, migration, and tumor angiogenesis, and the safety of long-term oral administration, rapamycin might be a good candidate agent for tumor metastasis prevention. However, the immunosuppressive property of rapamycin should be considered. Rapamycin exerts its potent immunosuppressive effects in part through direct effects on antigen-specific lymphocytes; however, rapamycin also modulates adaptive immunity through its effects on innate immune cells, including dendritic cells and macrophages, and others.(56,57) Although clinical sequels of immunosuppression do not seem to be a major concern in the current clinical evaluation of rapamycin in cancer, the long-term effects might have a negative consequence. In this context, combining other immune modulators to overcome such pitfalls may be of benefit when using rapamycin in the prevention of tumor metastasis.
The regulation of autophagy by rapamycin hints at a possible mechanism involving rapamycin-mediated metastasis suppression. However, the inhibition of autophagosome formation by Beclin1 knockdown did not change the inhibitory effect of rapamycin on metastasis, suggesting that autophagy is not involved in rapamycin-mediated suppression of B16 metastasis. However, considering the role of autophagy in energy metabolism and the requirement of energy for cell mobility, it is still possible that autophagy participates in tumor cell migration and metastasis by increasing the energy supply of tumor cells.
In summary, a novel finding in the present study is that rapamycin may directly effect tumor cells and suppresses their metastasis by regulating integrin expression and apoptosis signaling. Our data and others(31–33) together suggest that rapamycin and its analogues are worthy of clinical evaluation as an antimetastatic agent.