Anoikis and Survival Connections in the Tumor Microenvironment: Is There a Role in Prostate Cancer Metastasis?

Introduction

Prostate cancer is a heterogeneous disease that progresses from prostatic intraepithelial neoplasia to locally invasive to hormone-refractory metastatic carcinoma. In 2005, a total of 232,090 new cases of prostate cancer will be diagnosed in the United States. One in six men will be diagnosed with prostate cancer during their lifetime, and 1 in 33 men will die of the disease, accounting for about 10% of cancer-related deaths in American men. In tumors confined to the prostate, radical prostatectomy, radiotherapy, and androgen ablation have been proven effective; however, no effective treatments are available for metastatic disease (1, 2). During prostate cancer progression, prostate tumors undergo conversion from androgen-dependent to androgen-independent state and metastasis. In this review, we submit that one of the mechanisms underlying manifestation of prostate metastatic phenotype is anoikis resistance.

Anoikis, a Greek word meaning loss of “home” or “homelessness,” was originally defined by Frisch a decade ago as a unique phenomenon reflecting apoptotic cell death consequential to insufficient cell-matrix interactions (3, 4) and was later recognized as a potentially significant player in tumor angiogenesis and metastasis (4–6). As a functional phenomenon, anoikis can suppress expansion of oncogenically transformed cells by preventing proliferation at migrating locations, whereas migrating tumor cells that are resistant to anoikis induction can grow at inappropriate locations (7, 8). Resistance to anoikis is thus emerging as a hallmark of metastatic cancer cells, especially because anchorage-independent growth of tumor cells is a classic characteristic of different types of human malignancies, including prostate cancer (4, 7, 8). The underlying mechanisms rendering tumor cells resistant to anoikis are not fully understood, but it has been postulated that it may comprise the stimulation of survival signals that are not extracellular matrix (ECM) contact dependent and inhibition of apoptotic pathways.

Targeting of tumor cell metastasis holds considerable therapeutic promise, and its exploitation may lead to the identification of effective new modulations, such as (a) reversing the ability of tumor cells of becoming resistant to anoikis, therefore making them more susceptible to anoikis-inducing agents; (b) interfering with the seeding process of tumor cells into secondary places by making tumor cells nonsensitive to the chemotactic and environmental cues of the new target organ; and (c) making these secondary targets less “appealing” to the cancer cells by blocking key molecules promoting cancer cell seeding and survival. In this review, we discuss the possibilities of a functional relationship of anoikis resistance to reactive tumor stroma formation and epithelial/mesenchymal transition (EMT) in prostate cancer progression.

Tumor Microenvironment

Tumorigenesis is a multistep process involving multiple genetic alterations that drive normal cells into a highly malignant phenotype with metastasis being the final stage (6). The ECM itself is a dynamic and complex network of collagens, laminin, fibronectin, and proteoglycans, which also harbors soluble growth factors (9). ECM provides cells the information regarding its context within a tissue, which can also regulate cell proliferation, migration, differentiation, and survival. Endothelial and epithelial cells are dependent upon the ECM for survival and proliferation. Disruption of the cell-matrix contacts can trigger anoikis both in vitro and in vivo, ultimately leading to caspase-dependent apoptosis (10).

During prostate tumor progression, two important changes affect the dynamics of tissue plasticity: the EMT and the formation of a reactive stroma, reflecting intense structural rearrangement of the ECM in the spatial confines of the tumor cell cytoskeleton.

Epithelial/mesenchymal transition: the key players. EMT is a fundamental process that occurs during both physiologic and pathologic conditions (11, 12). Such transitions necessary for proper embryonic development also provide a convenient venue for epithelium-derived tumors to become highly invasive and rapidly metastasize (12, 13), following a mechanism resembling a reawakening/reactivation of the embryonic program of EMT. In both embryonic and tumorigenic EMT, migrating cells “reevaluate” their surroundings, change their relationship with the ECM, and can evade apoptosis. Even if there is no histologic evidence of cellular intermediates during the transition from epithelial to mesenchymal phenotype (14), activation of mesenchymal genes in epithelial cells is critical for EMT success (15). Recently, molecules, such as Snail, Twist, Par6, and nuclear factor-κB (NF-κB), have been implicating in coordinating transforming growth factor-β's (TGF-β) participation in the EMT process.

Snail belongs to a zinc-finger family of transcriptional factors that are essential in embryonic development, more specifically during EMT of mesoderm specification (16). Snail binds to E-cadherin promoter and represses its transcription during EMT. Loss of E-cadherin is a critical step towards the malignant development and tumor progression in prostate cancer (17). Snail activates the transcription of genes that are associated with mesenchymal differentiation, such as vimentin and fibronectin (16), and during EMT, Snail blocks cell cycle progression. In addition, Snail confers apoptosis resistance to these cells by activating survival genes, such as the phosphatidylinositol 3 (PI3)/Akt cascade, and by inhibiting caspase-3 activation through tumor necrosis factor-α (TNF-α; refs. 18, 19). Elevated Snail mRNA expression is found in a variety of tumors, with only modest increase in the level of Snail protein. Snail is phosphorylated by GSK-3β kinase, and this phosphorylation is responsible for regulating the rate of Snail degradation via the proteasome (20). During EMT, GSK-3β needs to be inhibited; thus, Snail can perform its functions. Indeed, signaling through epidermal growth factor, insulin-like growth factor (IGF), and hepatocyte growth factor (HGF) causes phosphorylation of GSK-3β, allowing nuclear translocation of Snail and subsequently activating its transcriptional repressor/activator function and promote EMT (21). Therefore, it might not be coincidental to the cellular fate that growth factor signaling pathways regulating GSK-3β activity are also induced during the reactive stroma formation.

Twist, a helix-loop-helix transcriptional factor initially described in Drosophila (22), was subsequently implicated in limb morphogenesis and EMT during embryonic development (23). Twist functions as an antiapoptotic factor but also serves a role as a regulator of the EMT by inducing the expression of mesenchymal markers such as fibronectin and N-cadherin (24). Twist function is considerably up-regulated in prostate tumors (as well as other malignancies), and Twist induction is linked to poor prognosis, high Gleason grade, and decreased E-cadherin expression in prostate cancer (25). Significantly enough, down-regulation or loss of function of Twist in androgen-independent prostate cancer cell lines leads to decreased cancer cell migration and invasion capabilities (25). Twist signaling can modulate the apoptotic machinery by increasing the bcl-2/bax ratio (24) that may provide a molecular basis for the ability of Twist to confer therapeutic resistance to taxol and vincrestine in bladder, ovarian, prostate, and nasopharyngeal tumors (25). Loss of Twist expression renders the cancer cells to become more sensitive to anoikis induction and TNF-α-induced apoptosis (26, 27).

Activation of NF-κB signaling, a major activator of immune and inflammatory functions (28), is heavily associated with tumorigenesis mainly by conferring apoptosis resistance to tumor cells (29). Indeed, NF-κB is constitutively activated in human breast, prostate, colorectal, and ovarian tumors (30) and is capable of maintaining these cells in the mesenchymal state (31). Considering that retention of the mesenchymal phenotype is crucial for acquisition of migratory and invasive characteristics by the cell, one has to recognize an important functional contribution to the process of metastasis by this leading transcription factor. Moreover, NF-κB has been indirectly linked to tumor metastasis via its ability to regulate the expression of matrix metalloproteinases (MMP), interleukin-8, vascular endothelial growth factor, and CXCR4 (32, 33). Significantly enough, several lines of evidence point to the NF-κB signaling as a contributor to anoikis outcomes within the tumor microenvironment, with key anoikis regulators suppressing apoptosis via activation of the PI3/Akt signaling cascade (34–37).

In summary, during the EMT process, epithelial cells acquire mesenchymal characteristics, and survival pathways, such as the PI3/Akt signaling cascade, are activated to insure tumor mesenchymal cell successfully migrate in the stroma.

The reactive stroma. Tumor cell proliferation and invasion through the basement membrane into the stromal compartment causes a stromal response that generates a new stromal microenvironment that is different than the native one because it shows ECM remodeling, elevated protease activity, increased growth factor bioavailability, increased angiogenesis, and influx of inflammatory cells (38). The term “reactive stroma” is used to describe this support system that provides a highly nurturing environment for the invading tumor cells (39, 40).

The solid hallmark characteristic of a reactive stroma is the presence of myofibroblasts. A cellular intermediate between a fibroblast and a smooth muscle cell, a myofibroblast, is characterized by its unique cytoskeletal protein expression profile and its ultrastructural features (41). A direct correlation between myofibroblasts and invasive metastatic carcinomas has been reported in several human malignancies, including breast, lung, colon stomach, and prostate cancers (42). Myofibroblasts are directly involved in ECM remodeling because they secrete key ECM molecules, such as fibronectin (embryonic fibronectins), collagen I and III, glycoproteins (tenascin and thrombospondin-1), and proteoglycans (versican; refs. 43–47). During prostate tumorigenesis, the progression to a highly aggressive malignant state is intimately linked to the myofibroblast component of the gland (39, 40, 48–51). TGF-β actively participates in the process by inducing the expression of two growth factors (IGF-1 and HGF) that are of major significance in EMT.

Remodeling of the ECM by proteases is also important in the reactive stroma formation. Serine proteases and MMPs have their expression increased, correlating positively with the tumor metastatic potential (52). The increased proteolytic activity seen in tumor tissues is a reflection of an imbalance between the proteases (MMPs) and their inhibitors (tissue inhibitor of metalloproteases, TIMP). In prostate tumors, of low-grade Gleason scores, expression of TIMPs is elevated in comparison to MMPs' expression, whereas in higher Gleason tumors, the reverse correlation is observed (53).

One could therefore easily argue that the reactive stroma can be responsible for the production of key molecules that not only assist in the EMT process but are also tightly involved in prostate tumor neovascularization. In both processes, EMT and reactive stroma, activation of survival pathways, and suppression of apoptosis occur in a concomitant fashion, making the biochemical dissection of the precise role of anoikis resistance as an independent event in carcinogenesis a rather challenging task.

Significance of Anoikis in Tumor Metastasis

Compelling evidence suggests that the presence of a reactive stroma and EMT are critical steps in prostate cancer progression. As anoikis is structurally dependent on the cellular interactions with the ECM and the cytoskeleton rearrangement, development of anoikis resistance might be associated with these processes.

Anoikis Players: New Targets for Prostate Cancer Therapy?

The close functional links between prostate cancer progression and loss of apoptosis provides an ever-growing number of molecular insights into the development and clinical formulation of novel apoptosis-targeted cancer therapeutic approaches (54), as reinstating apoptosis represents a critical defense strategy against tumor cell chemoresistance (55).

Multiple factors with frequently overlapping functions have been reported to confer anoikis suppression to tumor cells by stimulating survival pathways. One, however, could propose that anoikis resistance not only confers the cell with the ability to survive without attachment to the ECM but may also provide a molecular signature that secures their successful migration, invasion, and metastasis to distant sites. Thus, key players emerging as novel therapeutic targets might not only participate in the development of anoikis resistance but might also serve as molecular “ambassadors” of cell migration, seeding, and proliferation of prostate tumor cells to the secondary targets (Fig. 1).

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Figure 1.

Mechanistic link of anoikis resistance to tumor cell metastasis. Two important cellular processes coordinate this cellular “connection”: EMT and reactive stroma formation. Caveolin-1, Galectin-3, and TrkB emerge as the critical contributors to apoptosis resistance, angiogenesis, and tumor cell migration. In this schematic diagram, these molecules are identified in the context of their contribution to the cellular connection driving the EMT and reactive stroma.

Galectins: changing (intracellular) places, changing (growth) phases. Galectins belong to a highly conserved family of animal lectins with members present in organisms from nematodes to mammals (56). There is strong evidence to suggest galectin overexpression in malignant epithelial cells, as well as tumor-associated stromal cells (57, 58), and a close correlation with acquisition of a metastatic phenotype. The effectiveness of galectin on malignant transformation probably resides in a swift change of its subcellular localization (cytosolic translocation). Cytoplasmic Gal-3 is antiapoptotic, whereas the nuclear presence of Gal-3 exerts proapoptotic properties. Opposite functions of Gal-3 have been documented in prostate cancer cells: cytoplasmic gal-3 induces cell migration and anchorage-independent cell growth, whereas nuclear Gal-3 exerted the opposite effect blocking cellular migration (59). The dynamics of Gal-3 cellular localization are mechanistically intriguing in response to apoptotic stimuli. The translocation from the perinuclear membrane to the mitochondria is dependent on synexin (Annexin VII), a phospholipid and Ca²⁺ binding protein (60). This dynamic subcellular localization suggests an additional level of molecular regulation that can exploited for therapeutic targeting. The precise mechanism via which Gal-3 regulates cell proliferation is not well understood, but Gal-3 can regulate cell cycle progression by blocking cyclins A and E and by stimulating p27 and p21 (cell cycle–dependent kinase inhibitors), resulting in cell cycle arrest (9). In addition to suppression of anoikis and cell cycle arrest, Gal-3 has in vitro angiogenic activity that might induce the migration of endothelial cells (61). Galectins may thus contribute not only during the early stages of malignancy by targeting cell cycle progression but also in advanced disease by promoting angiogenesis.

Trk-B. The function of TrkB, a neurotrophic tyrosine kinase, has been well described in the nervous systems. This unique receptor upon binding to its ligand, brain-derived neurotrophic factor (BDNF), is activated and ultimately responsible for the proliferation, differentiation, and survival of retinal and glial cells (62). Recent evidence has implicated activation of Trk receptors by their neurotrophin ligands (nerve growth factor, BDNF, and neurothrophins NT-3 and NT-4/NT-5 in the initiation of survival mechanisms in prostate tumor cells but not in normal cells; refs. 63–65). In prostate carcinomas, TrkB and BDNF are frequently overexpressed, and there is a close correlation with aggressive behavior and poor prognosis (66). Recent elegant genetic studies in rat intestinal epithelial cells identified TrkB as a key suppressor of anoikis, conferring anoikis resistance in soft agar assays (67). Mechanistic analysis revealed that TrkB pathway does not activate known downstream targets of Akt, such as p70S6 and Rac, suggesting that alternative molecules downstream of Akt may perform such function (66). TrkB can convert nonmalignant cells into highly tumorigenic cells (67), thus becoming a key player in the early steps of tumorigenesis.

Caveolin-1: the transporter delivers. Caveolin (cav-1) has been implicated in a variety of cellular processes ranging from signal transduction up to cholesterol homeostasis and lipid transport (68). Rapidly growing evidence directly links caveolins to human cancer development and progression (69–71). Mutations in the Cav-1 gene have been shown in breast and squamous cell carcinomas (72, 73), and the genomic location of Cav-1 on chromosome 7q31.1, a suspected tumor suppressor region, strongly supports the notion that Cav-1 is a tumor suppressor gene (74). There has been evidence, however, challenging the role Cav-1 as a tumor suppressor and consequently its potential value as a marker of tumor progression. Expression of this membrane protein is linked to increasing tumor grade and stage, thus identifying Cav-1 as a promising predictor of poor disease prognosis. Cav-1 can function as a tumor promoter in bladder, esophageal, and prostate tumors (75–77). In prostate cancer, up-regulation of cav-1 has been correlated with increased cell survival, androgen independence, and enhanced metastatic potential (78–81). Recent studies in the transgenic adenocarcinoma mouse prostate model confirm that in contrast to mammary tumors, in prostate cancer, Cav-1 is a tumor promoter, and loss of Cav-1 results in apoptosis of prostate epithelial cells via up-regulation of proapoptotic genes PTEN and Par4 (82). cav-1 is overexpressed in primary tumors, leading to increased cell proliferation, but there is reversal in the cellular demands during tumor cell migration, as survival requires reduction of cav-1 expression. Evidence on the mechanistic connection of Cav-1 in prostate cancer metastasis, points to the ability of Cav-1 to suppress anoikis by activating Akt and to also block two Akt inhibitors, PP1 and PP2A (Fig. 1; ref. 83). Perhaps the most interesting aspect of Cav-1 signaling is the relationship between Cav-1 and IGF-1 signaling. Cav-1 is a known mediator of the insulin and IGF-I signaling pathway and that has been shown to enhance matrix-independent cell survival by up-regulating IGF-IR (a mitogenic antiapoptotic receptor), expression, and signaling (84). In vivo studies revealed that targeting cav-1 expression in prostate cancer cells results in significant enhancement of androgen deprivation–induced apoptosis (83).

Insulin-like growth factor-I and the integrin connection. There is compelling evidence to support IGF signaling as a critical contributor to the development and progression of prostate cancer, independent of caveolin. The oncogenic effect of IGF resides mainly in its function as a potent inhibitor of apoptosis by blocking induction of apoptosis by TGF-β and by activation of Akt (85). IGF-I is normally secreted by the prostate stroma, and its bioavailability is mostly regulated by IGF binding protein 3. This protein, together with other six family members, is responsible for modulating the bioavailability of IGF-I. Elevated levels of IGF-1 directly correlate with prostate cancer development and, in transgenic mice, enforced overexpression of IGF-1 promotes prostate carcinogenesis (86). Increased IGF-I signaling promotes cell growth and survival via the activation of the Akt survival pathway. Moreover, IGF signaling pathway can apparently confer resistance to anoikis in a variety of cancer cells (87, 88). Valuable mechanistic insights into a crucial cross-talk between the IGF and integrin signaling pathways towards anoikis resistance stem from recent evidence, indicating that in prostate cancer cells IGF-R directly interacts with integrins to modulate the cell localization to focal contacts and cellular response to environmental cues (89). Goel et al. showed that a close association of IGF-R with integrin αβ_1C leads to a decrease in prostate tumor cell proliferation and an increase in cell adhesion. In contrast, IGF-R interaction with integrin αβ_1A causes a reverse effect, increasing the proliferation of tumor cells, and a decrease in cell adhesion (90). Of direct clinical significance is a recent report that laminin and integrin αβ_1C expression decreases during prostate cancer progression to metastatic disease (91). Furthermore, IGF-IR mediates resistance to anti-androgen therapy by up-regulating survivin. Survivin belongs to the inhibitor of apoptosis protein family of apoptosis suppressor proteins, and as such, its association with tumor progression and drug resistance is not surprising (92). The contribution of IGF-I signaling in terms of high expression IGF-I and IGF-IR in the bone is also vital in the successful metastatic bone deposits of prostate cancer cells, as well as osteoblast proliferation (93). Unlike other tumors that metastasize to the bone (e.g., breast and lung), prostate cancer causes mostly osteoblastic instead of osteolytic lesions (94). Therefore, targeting the IGF-I signaling in prostate cancer provides unique opportunities for formulating new therapeutic strategies by affecting tumor cell resistance to anoikis and metastatic deposits to the bone.

Summary

Anoikis is a specific mode of apoptosis that results from insufficient cell-matrix interactions. Although normal cells use anoikis to prevent cell proliferation at inappropriate locations, tumor cells develop means to evade this process, acquire the ability to detach from the ECM, and migrate to new sites that provide a nurturing microenvironment for their aberrant growth. The question remains if prostate tumor cells have an inherent capacity to block anoikis or develop mechanisms of anoikis evasion? Is this a mere morphologic reflection of the cellular changes taking place in the tumor microenvironment or rather a spatial rearrangement in the interaction of tumor epithelial and endothelial cells with the ECM? Specific histologic, molecular, and transcriptional events are commonly associated with prostate cancer progression, leading to the obviously attractive possibility of EMT as a part of the metastatic process. The cytoskeletal rearrangements that tumor cells go through during the process of EMT and during the migration and invasion of blood vessels may determine the plasticity of the tumor cells and their sensitivity to anoikis. Molecules that change in expression, distribution, and function during the EMT and are causally involved in the process include growth factors (such as TGF-β and IGF-1), transcription factors (Smads and Snail), cell adhesion molecules to the ECM (integrins and ECM proteins), and cell-to-cell adhesion molecules (E-cadherin), as well as extracellular proteases (MMPs and caveolin). Three proteins emerge as leading molecular candidates as players in the development of anoikis resistance during cancer progression: galectins, caveolin, and TrkB. All three present intriguing therapeutic possibilities for direct targeting for metastatic disease. One, however, has to also consider that the development of EMT behavior by prostate cancer cells is only one hurdle in achieving metastatic “success.” As the majority of human prostate tumors are vastly heterogeneous, one would expect only a few will show the molecular EMT-like signature towards an invasive front.