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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Cell. Author manuscript; available in PMC Jan 19, 2011.
Published in final edited form as:
PMCID: PMC2818597
NIHMSID: NIHMS163306

IAP REGULATION OF METASTASIS

SUMMARY

Inhibitor-of-Apoptosis (IAP) proteins contribute to tumor progression, but the requirements of this pathway are not understood. Here, we show that intermolecular cooperation between XIAP and survivin stimulates tumor cell invasion and promotes metastasis. This pathway is independent of IAP inhibition of cell death. Instead, a survivin-XIAP complex activates NFκB, which in turn leads to increased fibronectin gene expression, signaling by β1 integrin(s), and activation of cell motility kinases, FAK and Src. Therefore, IAPs are direct metastasis genes, and their antagonists could provide anti-metastatic therapies in cancer patients.

Keywords: Inhibitor of apoptosis, survivin, XIAP, fibronectin, metastasis, NFκB

INTRODUCTION

The dissemination of tumor cells to distant organs, i.e. metastasis (Nguyen and Massague, 2007), heralds a nearly invariably fatal phase of epithelial malignancies, with few, if any, therapeutic options. This process reflects the acquisition of multiple molecular traits by tumor cells, including the ability to counteract the cell death program initiated by detachment from the extracellular matrix, or anoikis (Frisch and Screaton, 2001), proteolyze and migrate through basement membrane(s), invade blood or lymphatic vessels, and proliferate in unrelated microenvironments (Weigelt et al., 2005). The exact timing of these processes is unknown, and whether they reflect sequential adaptive changes (Scheel et al., 2007), or selection of specially endowed clones (Talmadge, 2007), has been debated. However, one invariable feature of the metastatic process is deregulated gene expression, which affects sequential stages of tumor cell invasion, organ tropism, and growth at distant sites (Nguyen and Massague, 2007). A potential loss of ‘metastasis-suppressor’ genes may also contribute to this pathway (Stupack et al., 2006).

Although the molecular requirements of metastasis remain largely elusive, ‘outside-in’ signaling initiated by ligation of integrin cell surface receptors (Ginsberg et al., 2005) with extracellular matrix proteins (Juliano et al., 2004), likely plays a pivotal role in this process. As an abundant constituents of the extracellular matrix, fibronectin binds multiple integrins (Cukierman et al., 2002), resulting in the activation of Focal Adhesion Kinase (FAK) (Sieg et al., 2000), Src (Yeatman, 2004), Akt (Irie et al., 2005), as well as modulation of small GTPases of the Rho family (Nobes and Hall, 1995). In response to these signals, cells remodel their actin cytoskeleton (Juliano et al., 2004), express matrix metalloproteinases (Han et al., 2006), become migratory (Livant et al., 2000), invade basement membranes (Gaggioli et al., 2007), and acquire the ability to resist apoptosis (Fornaro et al., 2003).

In this context, aberrantly increased cell survival is an invariable requirement of metastasis (Mehlen and Puisieux, 2006), and is typically contributed via deregulated expression of Bcl-2 (Cory and Adams, 2002) or Inhibitor-of-Apoptosis (IAP) (Srinivasula and Ashwell, 2008) cytoprotective proteins. However, some of these molecules, especially IAPs, have recently emerged as broader regulators of cellular homeostasis, with functions extending beyond apoptosis inhibition (Srinivasula and Ashwell, 2008). For instance, IAP family protein XIAP has been linked to the activation of multiple gene expression networks, including Smad/TGFβ (Birkey Reffey et al., 2001), JNK (Sanna et al., 1998), or NFκB (Hofer-Warbinek et al., 2000; Lu et al., 2007), whereas survivin plays essential roles in mitosis, the cellular stress response, and developmental pathways of gene expression (Altieri, 2008). How these multiple functions of IAPs work together in cellular homeostasis is unclear, and the exact contribution of these non-cytoprotective mechanisms, if any, to tumor progression has not been investigated.

In this study, we asked whether IAP signaling affected metastasis as a key determinant of unfavorable disease outcome.

RESULTS

IAP-mediated tumor cell invasion

To begin investigating a role of IAPs in tumor progression, we first silenced the expression of XIAP or survivin in invasive breast adenocarcinoma MDA-MB-231 (Figure 1A), or prostate adenocarcinoma PC3 (Figure 1B) cells. Transfection of these cells with XIAP- or survivin-directed small interfering RNA (siRNA) suppressed the expression of the intended IAP protein, but not vice versa, whereas a non-targeting siRNA had no effect (Figure 1A, B). Under these conditions, survivin or XIAP knockdown inhibited MDA-MB-231 or PC3 cell invasion through Matrigel inserts, as compared with control transfectants (Figure 1C, D). Similar results were obtained with siRNA silencing of these IAPs in colorectal adenocarcinoma HCT116 cells, whereas a non-targeting siRNA had no effect (Figure 1E). In addition, siRNA knockdown of a different IAP, cIAP1, also abolished Matrigel invasion of PC3 cells (Figure 1F), suggesting that tumor cell invasion was a general property of multiple IAPs.

Figure 1
IAP-mediated tumor cell invasion

Next, we examined the specificity of this pathway, and we used clones of non-invasive breast adenocarcinoma MCF-7 cells stably transfected with survivin. These cells, designated MCF-7 SVV, express a 2- to 3-fold increased survivin levels compared to MCF-7 cells (Figure S1A), thus similar to more invasive cell types (Figure 1A). Compared to parental cultures, MCF-7 SVV cells did not exhibit changes in cell proliferation (Figure S1B), adhesion to fibronectin- or collagen-containing substrates (Figure S1C), remodeling of the actin cytoskeleton (Figure S1D), or expression of various integrins (Figure S1E). Instead, MCF-7 SVV cells became highly migratory on collagen-coated inserts, compared to parental cultures (Figure S1F), or MCF-7 cells transfected with empty plasmid (not shown). Consistent with this, two independent clones of MCF-7 SVV cells showed markedly enhanced invasion through Matrigel-coated inserts, whereas non-transfected MCF-7 cells were not invasive (Figure 1G).

Confirming the specificity of IAPs in this response, siRNA knockdown of XIAP or survivin in MCF-7 SVV cells (Figure 2A) suppressed Matrigel invasion, whereas a non-targeting siRNA had no effect (Figure 2B). Conversely, cell viability was only minimally affected after XIAP (7%) or survivin (14.2%) silencing, compared to control transfectants (4.8%). cIAP1 knockdown also reduced Matrigel invasion of MCF-7 SVV cells from 61.5±11.1 (control siRNA) to 11.3±1.6 invaded cells per field (p=0.0003, n=10). As an independent approach, we transduced MCF-7 SVV cells with an adenovirus (pAd) encoding a dominant negative survivin Cys84Ala/Thr34Ala mutant (pAd-T34A/C84A). Although expressed at comparable levels as pAd-GFP by fluorescence microscopy (Figure 2C, top), this survivin mutant abolished Matrigel invasion (Figure 2C), whereas cell cycle kinetics or cell viability were not affected within the same time interval (Figure S2A). Finally, we stably silenced the expression of XIAP in MCF-7 SVV cells using a lentivirus construct encoding XIAP-directed short hairpin RNA (Figure 2D). Independently established clones of these cells, designated MCF-7 SVV-XIAP KD, showed marked inhibition of Matrigel invasion, as compared with control cultures (Figure 2E).

Figure 2
IAP targeting inhibits tumor cell invasion

To independently validate these findings, we next examined a second tumor model, HCT116 cells, in which siRNA silencing of XIAP or survivin suppressed Matrigel invasion (Figure 1E). Consistent with this, homozygous deletion of XIAP (XIAP−/−) in HCT116 cells (Figure 2F) completely abolished tumor cell invasion, whereas wild type HCT116 cells (XIAP+/+) were highly invasive (Figure 2G).

IAP anti-apoptotic functions are not required for tumor cell invasion

To test whether IAP cytoprotection contributed to tumor cell invasion, we next used a model of rat insulinoma INS-1 cells. Previous studies showed that stable transfection of survivin in these cells (INS-1 SVV) (Figure S2B) does not inhibit apoptosis, due to lack of mitochondrial import (Dohi et al., 2007). INS-1 SVV cells readily invaded through Matrigel inserts whereas control INS-1 transfectants were not invasive (Figure S2C). In addition, stable transfection of anti-apoptotic Bcl-2 in INS-1 cells (Figure S2B), which promoted exponential tumor growth in vivo (Dohi et al., 2007), did not mediate tumor cell invasion (Figure S2C). We next asked whether a similar paradigm applied to XIAP, and we stably transfected XIAP−/− HCT116 cells with an Asp143Ala/Trp310Ala XIAP double mutant (D143A/W310A) that does not bind caspase 3 (D143A mutation) or caspase 9 (W310A mutation), and is thus devoid of anti-apoptotic functions (Lewis et al., 2004) (Figure 2H). Independent clones of this cell line robustly invaded through Matrigel inserts, quantitatively indistinguishably from XIAP−/− cells reconstituted with wild type, i.e. anti-apoptotic, XIAP (Figure 2I).

IAP induction of fibronectin regulates tumor cell invasion

To begin understanding how IAPs promote tumor cell invasion, we next looked at the gene expression profile of model MCF-7 SVV cells, in which expression of survivin, alone, was sufficient to confer an invasive phenotype (Figure 1F). Microarray analysis of these cells revealed a significant upregulation of multiple extracellular matrix proteins implicated in cell invasion, including lumican (4106-fold), fibronectin (334-fold), and laminin α4 (105-fold). Accordingly, MCF-7 SVV cells exhibited a >120-fold upregulation of fibronectin mRNA, by real-time PCR (Figure 3A), and increased fibronectin promoter activity, by luciferase reporter assay (Figure 3B), compared to parental MCF-7 cells. Collagen type 1 α1 and collagen type 5 α2 mRNAs were also increased in MCF-7 SVV cells, albeit less prominently, whereas expression of laminin 5 was not significantly different, compared to parental cultures (Figure 3A). Consistent with these data, MCF-7 SVV cells exhibited dramatically increased endogenous fibronectin protein content, by fluorescence microscopy (not shown), and Western blotting (Figure 3C). This newly produced fibronectin was released in the conditioned medium of MCF-7 SVV cells (Figure 3D), and was sufficient to support tumor cell migration on Transwell inserts in the absence of exogenous substrate (Figure 3E). In contrast, parental MCF-7 cells did not migrate in the absence of substrate (Figure 3E). Similar results were obtained with an unrelated cell type, as INS-1 cells stably transfected with survivin (INS-1 SVV) also exhibited a 7- to 8-fold increased fibronectin mRNA (Figure S3A), and protein (Figure S3B). In contrast, INS-1 Bcl-2 transfectants, or cells transfected with a control plasmid, showed no modulation of fibronectin levels (Figure S3A, B).

Figure 3
IAP induction of fibronectin

Using breast cancer as a model, we next asked whether a relationship between IAP and fibronectin expression existed in human tumors. Analysis of two independent patient cohorts revealed that survivin and fibronectin were coordinately increased in tumor versus normal tissues (Figure 3F). Similarly, invasive breast adenocarcinoma cell lines SUM159 and HBL100 contained high levels of endogenous fibronectin (Figure S3C), suggesting that its expression segregated with the tumor cell population, rather than stromal cells.

Finally, we tested whether fibronectin produced and released by IAP-expressing cells contributed to tumor cell invasion. Here, siRNA knockdown of fibronectin in MCF-7 SVV cells (Figure 3G) significantly inhibited Matrigel invasion, as compared with control transfectants (Figure 3H). Similarly, preincubation of MCF-7 SVV cells with a function-blocking antibody to β1 integrin(s), which comprise the main fibronectin receptor on these cells, abolished tumor cell invasion, whereas non-binding IgG was ineffective (Figure 3I).

IAP intermolecular cooperation activates NFκB

We next asked how IAP may transcriptionally upregulate fibronectin, and we focused on a potential role of NFκB in this response. For these experiments, we stably transfected survivin in wild type (WT) or XIAP−/− mouse embryonic fibroblasts (MEF) that have very low levels of endogenous survivin (Figure S4A). Expression of survivin in XIAP+/+ cells (clones #44 and #68) resulted in nuclear translocation of p65 NFκB (Figure 4A), and increased NFκB promoter activity, quantitatively similar to TNFα stimulation (Figure 4B). In contrast, survivin expression in XIAP−/− cells (clones #2 and #5) had no effect on NFκB translocation (Figure 4A), or NFκB promoter activity (Figure 4B). By EMSA, a radiolabeled NFκB probe bound to nuclear extracts in XIAP+/+ cells transfected with survivin, which was supershifted by an antibody to p65 NFκB, but not IgG (Figure 4C). Conversely, expression of survivin in a XIAP−/− background had minimal effect on NFκB-protein complexes (Figure 4C). In ‘add back’ experiments, transfection of survivin-expressing XIAP−/− cells with wild type XIAP restored the formation of NFκB complexes, whereas a RING-less XIAP mutant (RING-Δ) was ineffective (Figure 4C). Similarly, INS-1 cells stably transfected with a survivin S20A mutant, which constitutively binds XIAP (Dohi et al., 2007), strongly activated NFκB promoter activity, with or without TNFα (Figure 4D). In contrast, INS-1 cells expressing a survivin S20E mutant that does not bind XIAP (Dohi et al., 2007), showed no NFκB promoter activity (Figure 4D). Finally, siRNA knockdown of survivin in MCF-7 SVV cells significantly reduced NFκB promoter activity, compared to control transfectants (Figure S4B). Therefore, a survivin-XIAP complex (Dohi et al., 2007) activates NFκB.

Figure 4
NFκB induction of fibronectin mediates IAP tumor cell invasion

As far as the mechanism(s) of NFκB activation under these conditions, addition of recombinant XIAP to MCF-7 cell extracts promoted the phosphorylation of the negative NFκB regulator, IκBα (Figure 4E). However, the combination of survivin plus XIAP further enhanced IκBα phosphorylation (Figure 4E), in a reaction stabilized by the proteasome inhibitor, lactacystin (Figure S3C). Conversely, a Val80Asp (V80D) XIAP mutant that does not activate NFκB (Lu et al., 2007), had no effect on IκBα phosphorylation, with or without lactacystin (Figure S4C). Accordingly, reconstitution of XIAP−/− cells with wild type XIAP, but not V80D XIAP mutant, stimulated NFκB promoter activity with and without TNFα (Figure S4D).

NFκB-induction of fibronectin contributes to IAP-mediated tumor cell invasion

Consistent with a bona fide NFκB target gene, TNFα stimulation of MCF-7 or MCF-7 SVV cells resulted in increased fibronectin expression, albeit more prominently in survivin transfectants (Figure 4F). Conversely, siRNA knockdown of p65 NFκB (Figure S4E) suppressed endogenous fibronectin expression in MCF-7 SVV cells, as compared with control siRNA (Figure 4G). Similarly, siRNA silencing of survivin or XIAP also comparably suppressed fibronectin expression in MCF-7 SVV cells (Figure 4H). Functionally, siRNA knockdown of p65 NFκB (Figure S4E), or expression of a phosphorylation-defective IκBα ‘super-repressor’ mutant (Figure S4F), inhibited Matrigel invasion of MCF-7 SVV cells, as compared with control transfectants (Figure 4I).

Signaling requirements of IAP-mediated tumor cell invasion

To identify downstream effectors of IAP-directed tumor cell invasion, we next focused on kinase cascades implicated in cell motility. When attached to substrate, MCF-7 SVV cells exhibited constitutive phosphorylation of FAK on Tyr397 (Figure 5A), and Src on Tyr418 (Figure 5B), compared to parental MCF-7 cells. In contrast, phosphorylated Akt (Figure 5C), or ERK1,2 (Figure 5D) was comparable in the two cell types, and total kinase protein content was unchanged (Figure 5A–D). Stable knockdown of XIAP in MCF-7 SVV cells (MCF-7 SVV-XIAP KD) abolished FAK and Src phosphorylation, and significantly reduced endogenous fibronectin content in these cells (Figure 5E), reinforcing a role of IAP intermolecular cooperation in this response. Similarly, Src was constitutively phosphorylated in INS-1 cells expressing wild type survivin, but not a survivin S20E mutant (Figure 5F) that does not bind XIAP (Dohi et al., 2007).

Figure 5
IAP activation of cell motility kinases

Functionally, targeting FAK with a truncated dominant negative FAK protein, FRNK (Figure S5A), inhibited MCF-7 SVV cell invasion through Matrigel (Figure 5G). Similarly, two unrelated pharmacologic antagonists of Src, PP2 (Figure 5H), or SU6656 (Figure 5I), suppressed Matrigel invasion of MCF-7 SVV (Figure 5H, I), or MDA-MB-231 (Figure S5B) cells, without affecting cell viability or cell cycle kinetics (Figure 5J). In contrast, pharmacologic antagonists of MEK (PD98059 or U0126), or PI3 kinase (LY294002) had no effect on IAP-mediated tumor cell invasion (Figure 5H). Confirming their activity, all inhibitors abolished the phosphorylation of their intended target kinase (Figure S5C).

IAP protection from anoikis

The ability of tumor cells to remain viable when detached from the extracellular matrix may contribute to metastasis, and a role of IAP in this response was next investigated. MCF-7 cells forced to remain in suspension showed time-dependent anoikis (Figure 6A), and release of apoptogenic Smac from mitochondria (Figure 6B). In contrast, MCF-7 SVV cells were protected from anoikis (Figure 6A), and exhibited reduced release of Smac from mitochondria (Figure 6B). When attached to substrate, no differences were observed in background levels of Smac release between MCF-7 and MCF-7 SVV cells (Figure 6B). Survivin silencing by siRNA reversed cytoprotection of MCF-7 SVV cells in suspension (Figure S6), and strongly enhanced anoikis (Figure 6B). Conversely, survivin knockdown in attached MCF-7 SVV cells (Figure S6) did not significantly reduce cell viability within the same time interval (Figure 6C).

Figure 6
IAP suppression of anoikis

Although survivin inhibited anoikis (Figure 6C), MCF-7 and MCF-7 SVV cells grew comparably as colonies in soft agar (Figure 6D), and MCF-7 SVV cells in suspension did not release survivin from mitochondria into the cytosol (Figure 6E), which is required for cytoprotection (Dohi et al., 2007). Instead, MCF-7 SVV cells in suspension, but not parental MCF-7 cells, exhibited constitutive phosphorylation of FAK and Src (Figure 6F), which has been implicated in protection from anoikis (Bouchard et al., 2008). In addition, siRNA knockdown of fibronectin (Figure 6G), or its receptor, β1 integrin(s) (Figure 6H), suppressed FAK and Src phosphorylation in MCF-7 SVV cells in suspension.

IAP-dependent metastasis

For these studies, we used a liver metastasis model, in which tumor cells are injected directly into the spleen of immunocompromised SCID/beige mice, followed by splenectomy after 24 h to avoid potential interference of primary tumor growth on liver metastasis. Splenic injection of MCF-7 cells did not result in significant liver metastasis (Figure 7A, Figure S7A), and organs in this group were histologically normal (Figure 7B). In contrast, MCF-7 SVV cells reconstituted in the same model gave rise to extensive metastatic localization of the liver (Figure 7A, Figure S7A), with foci of viable epithelial cells in the liver parenchyma (Figure 7B, Figure S7B). Liver metastases of MCF-7 SVV cells stained for phosphorylated FAK (Figure S7C) consistent with a potential role of activated FAK in this pathway, in vivo. In contrast, stable knockdown of XIAP in MCF-7 SVV cells (MCF-7 SVV-XIAP KD) completely suppressed their ability to metastasize to the liver (Figure 7A, Figure S7A).

Figure 7
IAP-mediated metastasis, in vivo

As an independent tumor model, intrasplenic injection of wild type XIAP+/+ HCT116 cells resulted in massive metastatic colonization of the liver, with nearly complete substitution of the hepatic parenchyma by the tumor cell population (Figure 7C). In contrast, XIAP−/− cells did not significantly metastasize to the liver (Figure 7C). Quantification of liver metastasis revealed that HCT116 cells expressing wild type XIAP produced a >2-fold increase in liver weight, as compared with non-injected animals or mice reconstituted with XIAP−/− cells (Figure S7D).

We have shown above that IAP cytoprotection is not required for tumor cell invasion, in vitro, and we next asked whether a similar paradigm applied to metastatic dissemination, in vivo. XIAP−/− HCT116 cells stably transfected with D143A/W310A XIAP double mutant devoid of anti-apoptotic function (Lewis et al., 2004), produced extensive liver metastasis (p=0.038 compared to normal liver, Figure S7D), quantitatively indistinguishable from XIAP+/+ HCT116 cells (Figure 7C; not significant, Figure S7D). Similarly, INS-1 clones stably transfected with wild type survivin, which is not cytoprotective in these cells (Dohi et al., 2007), gave rise to systemic disease in SCID/beige mice, as judged by time-dependent decrease in blood glucose (Figure 7D), a biomarker of aberrant insulin production by insulinoma cells. This was associated with prominent metastatic dissemination of insulin-producing INS-1 cells to the liver (Figure 7E, F). In contrast, animals injected with INS-1 cells expressing anti-apoptotic Bcl-2 did not show changes in blood glucose levels (Figure 7D), and generated negligible liver metastasis (Figure 7F, Figure S7E). Previously, these cells promoted exponential growth of superficial tumors in SCID/beige mice, validating the activity of Bcl-2 (Dohi et al., 2007).

DISCUSSION

In this study, we have shown that intermolecular cooperation between IAP proteins, XIAP and survivin, promotes tumor cell invasion in vitro and metastatic dissemination in vivo. This pathway is independent of the role of IAPs in cell survival. Instead, these molecules orchestrate a cellular network in disparate tumor cell types centered on NFκB activation, transcriptional upregulation of fibronectin, autocrine/paracrine signaling by β1 integrins, and constitutive phosphorylation, i.e. activation, of cell motility kinases, FAK and Src (Figure 8). These data identify IAPs as direct metastasis genes, opening prospects for therapeutic intervention against these targets in patients with advanced and disseminated disease.

Figure 8
Schematic mechanistic model of IAP-induced metastasis

Beyond their roles in cytoprotection, it is now clear that IAPs function as broader regulators of cellular homeostasis, intercalated in cell division, metabolism and activation of multiple intracellular signaling pathways, including NFκB, TGFβ or JNK (Srinivasula and Ashwell, 2008). The data presented here position IAP signaling in the control of multiple, independent stages of the metastatic cascade conferring a general motility phenotype to tumor cells, unaffected by substrate attachment, competent to withstand anoikis, and ideal to support colonization of distant organs (Figure 8). In this context, a role of IAPs in cell motility may be evolutionary conserved, as a Drosophila IAP homolog has been implicated in cytoskeletal rearrangement and cell migration (Geisbrecht and Montell, 2004), via activation of small GTPase signaling (Oshima et al., 2006). However, whether a similar paradigm extended to mammalian cells, especially tumor cells, has been controversial, and a recent report suggested that silencing of IAPs actually increased tumor cell motility, potentially through enhanced c-Raf stability (Dogan et al., 2008). It is possible that the discrepancy between these results (Dogan et al., 2008) and the data presented here may reflect context-specific utilization of activated c-Raf for cell motility, as opposed to cell invasion. On the other hand, c-Raf has been known to inhibit (Slack et al., 1999), rather than promote (Dogan et al., 2008) cell migration, potentially by suppressing integrin activation (Hughes et al., 1997).

Here, the upstream step in IAP-mediated metastasis involved activation of NFκB-dependent gene expression. A link between IAPs, mostly XIAP, and NFκB activity (Srinivasula and Ashwell, 2008) has been previously associated with ubiquitination by RING-associated XIAP E3 ligase (Lewis et al., 2004), Smad4 signaling (Birkey Reffey et al., 2001), negative modulation by XIAP-binding protein(s) (Resch et al., 2009), or phosphorylation-induced degradation of IκBα, at least in endothelial cells (Hofer-Warbinek et al., 2000). Here, reconstitution experiments in XIAP−/− cells, together with ‘add-back’ studies using wild type or mutant recombinant proteins, demonstrated that NFκB activation requires the assembly of a survivin-XIAP intermolecular complex. Structurally, this may favor the recruitment of the adapter protein, TAB1 (Lu et al., 2007), resulting in downstream activation of the IKK kinase, TAK1, and IκBα phosphorylation (Hofer-Warbinek et al., 2000). Accordingly, a V80D XIAP mutant defective in TAB1 binding (Lu et al., 2007) did not cooperate with survivin in NFκB activation. However, additional requirements of a survivin-XIAP complex likely contribute to this response, as RING-less XIAP, or a Lys467Ala XIAP mutant that abolishes E3 ligase activity also failed to synergize with survivin in stimulating NFκB (T.D. and D.C.A., unpublished observations), in agreement with previous findings (Lewis et al., 2004). Broadly, IAP heterodimerization may function as general mechanism of cellular homeostasis (Srinivasula and Ashwell, 2008), as a survivin-BRUCE complex may regulate cytokinesis (Pohl and Jentsch, 2008), and a survivin-XIAP interaction (Dohi et al., 2007) opposes tumor cell apoptosis, by enhancing XIAP stability and synergistically inhibiting caspase(s) (Dohi et al., 2004).

Previously, a link between NFκB activity and metastasis has been tied to stimulation of epithelial-mesenchymal transition (EMT), expression of matrix metalloproteinase-9 (MMP-9), or repression of putative metastasis-suppressor genes (Naugler and Karin, 2008). Here, IAP activation of NFκB did not result in morphological features of EMT, as judged by the unchanged levels of E-cadherin or MMP-2 or -9 (S.M. and D.C.A., unpublished observations). Conversely, NFκB activity under these conditions contributed to a broad ‘adhesion gene signature’ in tumor cells, characterized by a several hundred fold upregulation of fibronectin. There is evidence that this pathway may occur during tumor progression in vivo, as transgenic mice expressing survivin in the urinary bladder exhibited a similar ‘adhesion gene signature’, linked to aggressive tumor behavior and abbreviated survival (Salz et al., 2005). Correlative observations in humans seem consistent with this scenario, as fibronectin is over-expressed in various epithelial malignancies (Bittner et al., 2000), and linked to disease dissemination (Clark et al., 2000), and shortened overall and disease-free survival (Yao et al., 2007). Several mechanistic models have been proposed to explain these findings, and elevated levels of fibronectin have been linked to loss of epithelial polarity (Nelson and Bissell, 2006), increased tissue rigidity (Paszek et al., 2005), disruption of mammary acinar morphogenesis (Williams et al., 2008), resistance to hormonal therapy (Helleman et al., 2008), and formation of a metastatic niche (Kaplan et al., 2005).

The data presented here suggest a broader scenario, establishing fibronectin as a direct mediator of tumor cell invasion. This process is postulated to involve a paracrine/autocrine signaling circuitry (Figure 8), in which fibronectin over-produced and released extracellularly by IAP-expressing tumor cells binds back to the cell surface, engaging cognate β1 integrins, and triggering cell- and matrix-autonomous activation of cell motility kinases, FAK and Src (Figure 8). Accordingly, outside-in signaling mediated by β1 integrins (Ginsberg et al., 2005), leads to phosphorylation of Src (Mitra and Schlaepfer, 2006), FAK (Sieg et al., 2000), and mediates tumor cell invasion, in vivo (Parsons et al., 2008). Although antibody inhibition studies demonstrated that β1 integrins are essential for IAP-mediated tumor cell invasion, immunologic or molecular targeting of fibronectin was only partially, i.e. 50%, effective, suggesting that other β1 integrin ligands upregulated in IAP-expressing cells, for instance collagen isoforms, cooperate in this response.

The ability of this autocrine/paracrine signaling circuit (Figure 8) to counter anoikis, likely provides an additional advantage for metastasis. Detachment of epithelial cells from the extracellular matrix (Frisch and Screaton, 2001), as well as unligation of so-called dependence receptors (Bredesen et al., 2005), is normally followed by apoptosis, but many tumor cell types manage to escape this response, and their ability to remain viable in suspension is thought to favor hematogenous or lymphatic dissemination (Nguyen and Massague, 2007). The requirements of transformed cells to counter anoikis have not been completely elucidated, and a potential protective role of IAPs in this process, mostly XIAP, has been linked to caspase inhibition (Liu et al., 2006). At variance with this paradigm, IAP inhibition of anoikis did not involve release of survivin from mitochondria, which is required for cytoprotection (Dohi et al., 2007), but rather β1 integrin/fibronectin-mediated phosphorylation of FAK and Src kinases, thus similar to the paradigm of IAP-mediated tumor cell motility (Figure 8). This is in line with a protective role of these molecules against mitochondrial dysfunction during anoikis, potentially via activation of PI3 kinase/Akt-mediated cell survival (Bouchard et al., 2008). This pathway was selective for anoikis, as over-expression of survivin in transformed cells did not confer a further advantage for anchorage-independent cell growth, an observation consistent with the ability of activated FAK to attenuate oncogene-mediated colony formation in soft agar (Moissoglu and Gelman, 2003).

When tested in an in vivo model that recapitulates many of the steps of the metastatic process, unaffected by primary tumor growth, IAP signaling promoted rapid and extensive metastatic dissemination to the liver. Retrospective analysis of patient series identified IAPs, especially survivin, as contributors to tumor progression (Hinnis et al., 2007), but a role for these molecules as metastasis genes had not been previously postulated. Although metastasis of Ras-transformed fibroblasts has been recently linked to FAK dephosphorylation (Zheng et al., 2009), the IAP pathway of tumor cell dissemination occurred indistinguishably in tumor types with wild type or mutant Ras, and was associated with phosphorylation of FAK in metastatic foci in vivo. Importantly, the cytoprotective functions of survivin or XIAP were not required for IAP-mediated metastasis. Accordingly, over-expression of non-cytoprotective survivin in INS-1 cells (Dohi et al., 2007), or reconstitution of XIAP-deficient HCT116 cells with a D143A/W310A XIAP mutant that does not bind caspases (Lewis et al., 2004), mediated tumor cell invasion in vitro and promoted extensive liver metastasis in vivo. In addition, expression of anti-apoptotic Bcl-2 could not substitute for IAP signaling in metastasis, suggesting that suppression of cell death, alone, is insufficient to confer a successful metastatic phenotype in vivo. These data are consistent with previous observations that IAP activation of NFκB (Lu et al., 2007) is separable from its role in caspase inhibition (Lewis et al., 2004), and that metastatic dissemination in a transgenic model of melanoma does not require survivin cytoprotection (Thomas et al., 2007).

In summary, we identified IAPs as direct metastasis genes, orchestrating a cellular network of NFκB-dependent gene expression, β1 integrin signaling and activation of FAK and Src kinases required for metastatic dissemination (Figure 8). Although IAPs are not easily ‘drugable’ targets in cancer, molecular antagonists of both XIAP and survivin have been developed, and produced some encouraging patient responses in early-phase clinical trials (Altieri, 2008). The data presented here suggest that these agents may not have overlapping specificities, and could be differentially utilized as individualized therapeutic regimens in cancer patients. Accordingly, Smac mimetics XIAP antagonists may be predicted to activate apoptosis in tumors (Varfolomeev et al., 2007) but would not interfere with IAP signaling, which plays a pivotal role in tumor progression and metastasis (this study). Conversely, agents that directly suppress the levels of XIAP and survivin in tumors, including antisense molecules and transcriptional repressors of the survivin gene, which are also being tested in early phase clinical trials (Altieri, 2008), could be rationally tested as first-in-class anti-metastatic regimens in patients with advanced and metastatic disease.

EXPERIMENTAL PROCEDURES

Cell migration and invasion

Analysis of cell migration was carried out using 6.5-mm Transwell chambers (8- μm pore size; Costar). Inserts were prepared by coating the upper and lower surfaces with 15 μg/ml collagen (Cohesion, Palo Alto, CA) for 18 h at 4°C, followed by a blocking step with DMEM containing 0.25% heat-inactivated BSA for 1 h at 37°C. In some experiments, Transwell inserts were left uncoated. The various cell types were harvested, suspended in DMEM containing 0.25% heat-inactivated BSA, and added (1×105) to the upper chamber, with aliquots of conditioned medium collected from NIH3T3 fibroblasts placed in the lower chamber as chemoattractant. After 1 h incubation, non-migrating cells were removed mechanically from the upper chamber using a cotton swab. Cells migrated to the lower surface of the Transwell membrane were fixed in methanol for 10 min at 22°C, and membranes were mounted on glass slides using Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Cell migration was quantified by counting the number of stained nuclei in five individual fields in each Transwell membrane, by fluorescence microscopy, in duplicate.

For analysis of cell invasion, the upper Transwell chamber (8- μm pore size; Costar) was coated with 0.5 μg Matrigel (Collaborative Research, Bedford, MA) diluted in cold water, and allowed to air dry. After 1 h incubation with DMEM, the various cell types (1×105) were added to the upper chamber for 6–24 h at 37°C. Cells that had invaded the lower surface of the membrane were fixed with methanol, stained with DAPI, and quantified by fluorescence microscopy. In some experiments, cells were incubated with the following pharmacologic inhibitors of Src (PP-2, 50 μM; SU6656, 25–50 μM), MEK (PD98059, 50 μM, or U0126, 25 μM), PI3 kinase (LY290042, 50 μM), or vehicle (DMSO) for 1 h at 37°C, added to Matrigel-coated membranes, and analyzed for cell invasion by fluorescence microscopy.

In vivo metastasis model

All experiments involving animals were approved by an Institutional Animal Care and Use Committee. Female SCID/beige mice (6–8 wk of age) were anesthetized with ketamine hydrochloride, the abdominal cavity was exposed by laparotomy, and animals were injected in the spleen with 2×106 MCF-7, MCF-7 SVV or MCF-7 SVV cells carrying stable shRNA knockdown of XIAP (MCF-7 SVV-XIAP KD) stably transfected with a luciferase cDNA. To avoid potential confounding effects on metastasis due to variable growth of a primary tumor, the spleen was removed 24 h after injection of the tumor cells. The incision was closed in two layers with vicryl 5/0 and wound clips. On d 1, 3, 7, and 11 after injection, animals were analyzed for metastatic disease by bioluminescence imaging using an IVIS-100 camera system for detection of luciferase expression (Xenogen, Alameda, CA). Briefly, mice were anesthetized with isoflurane and intraperitoneally injected with 2.2 mg luciferin sodium salt (GOLD Bio Technology, Inc) in PBS, pH 7.4. During image acquisition, isoflurane anesthesia was maintained using a nose cone delivery system. Both supine and prone images were scanned for a 3 min acquisition interval. Each image was acquired sequentially three to four times, and data were collected at the time of peak luminescence. The bioluminescence images were overlaid on black and white photographs of the mice collected at the same time. Signal intensity was quantified as the sum of all detected photon counts within a region of interest using Living image software (Xenogen, version 2.50). On d 11, all mice in the various groups were sacrificed and their livers were resected, and quantified for bioluminescence intensity, ex vivo. In some experiments, wild type (XIAP+/+), XIAP−/− or XIAP−/− HCT116 cells stably transfected with D143A/W341A XIAP mutant (Lewis et al., 2004) lacking anti-apoptotic function (5 animals/group), were injected (5×106 cells) in the spleen of SCID/beige mice, followed by splenectomy as described above. Animals were sacrificed after 3 weeks, and livers were analyzed histologically. In other experiments, wild type INS-1 cells (4 animals), or INS-1 stably transfected with survivin (6 animals) that is not cytoprotective in this cell type (Dohi et al., 2007) or Bcl-2 (5 animals) were injected (5×106 cells) in the spleen of SCID/beige mice, followed by splenectomy 1 d after reconstitution. Mice were monitored for blood glucose content twice weekly, and sacrificed after 3 weeks for histologic examination of livers.

Histology

Livers from the various animal groups were fixed in buffered formalin, and embedded in paraffin. For insulin staining, sections were deparaffinized, rehydrated in water, and quenched for endogenous peroxidase. Epiope heat retrieval was carried out by steaming the slides in 10% sodium citrate for 20 min. Processed slides were rinsed in PBS, pH 7.4, and stained with an antibody to insulin using standard avidin-biotin-peroxidase technique (Histostain-plus, Zymed Laboratories). Slides were incubated with DAB as a chromogen and counterstained with hematoxylin. Control sections were processed as above with non binding IgG, and resulted in no detectable staining.

Statistical analysis

Data were analyzed using the unpaired t test on a GraphPad software package for Windows (Prism 4.0). A p-value of 0.05 was considered as statistically significant.

Supplementary Material

01

Acknowledgments

We thank Drs. Leslie Shaw for discussion, I.-S. Kim for a fibronectin promoter reporter construct, Michelle Kelliher for FSIPPW vector, Bert Vogelstein for HCT116 cells, Colin Duckett for XIAP−/− MEFs and D148A/W310A XIAP cDNA, and Robert Sherwin for INS-1 cells. This work was supported by National Institutes of Health grants CA89720 (LRL), CA107548 (AMM), CA78810, CA90917, CA118005 and HL54131 (DCA).

Footnotes

SIGNIFICANCE

Metastasis is a hallmark of tumor progression, characterized by the dissemination of cancer cells to distant organs. Despite a better understanding of this process, often characterized by deregulated gene expression, anti-metastatic therapies do not presently exist, and patients with disseminated disease have limited options. We now show that Inhibitor-of-Apoptosis (IAP) molecules function as direct activators of tumor cell motility and metastasis genes independently of their roles in cytoprotection. IAP antagonists are now being tested in early phase clinical trials, and these agents may provide first-in-class anti-metastatic therapies in cancer patients.

SUPPLEMENTAL INFORMATION

Supplemental material (Supplemental Data, Experimental Procedures, and References) accompanies this paper.

ACCESSION NUMBERS

The analysis of microarray datasets is available at ku.ca.ibe@sserpxemaim under accession number E-MEXP-2184.

CONFLICT OF INTEREST

The authors declare that they have no competing interest.

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Contributor Information

Swarna Mehrotra, Prostate Cancer Discovery and Development Program, University of Massachusetts Medical School, Worcester, MA 01605.

Lucia R. Languino, Prostate Cancer Discovery and Development Program, University of Massachusetts Medical School, Worcester, MA 01605.

Christopher M. Raskett, Prostate Cancer Discovery and Development Program, University of Massachusetts Medical School, Worcester, MA 01605.

Arthur M. Mercurio, Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605.

Takehiko Dohi, Prostate Cancer Discovery and Development Program, University of Massachusetts Medical School, Worcester, MA 01605.

Dario C. Altieri, Prostate Cancer Discovery and Development Program, University of Massachusetts Medical School, Worcester, MA 01605.

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