Clinical significance of the integrin α6β4 in human malignancies

Abstract

Integrin α6β4 is a cellular adhesion molecule that binds to laminins in the extracellular matrix and nucleates the formation of hemidesmosomes. During carcinoma progression, integrin α6β4 is released from hemidesmosomes, where it can then signal to facilitate multiple aspects of tumor progression including sustaining proliferative signaling, tumor invasion and metastasis, evasion of apoptosis, and stimulation of angiogenesis. The integrin achieves these ends by cooperating with growth factor receptors including EGFR, ErbB-2, and c-Met to amplify downstream pathways such as PI3K, AKT, MAPK and the Rho family small GTPases. Furthermore, it dramatically alters the transcriptome toward a more invasive phenotype by controlling promoter DNA demethylation of invasion and metastasis-associated proteins, such as S100A4 and autotaxin, and upregulates and activates key tumor promoting transcription factors such as the NFATs and NFkB. Expression of integrin α6β4 has been studied in many human malignancies where its overexpression is associated with aggressive behavior and a poor prognosis. This review provides an assessment of integrin α6β4 expression patterns and their prognostic significance in human malignancies, and describes key signaling functions of integrin α6β4 that contribute to tumor progression.

Introduction

Integrins are cellular adhesion molecules that serve as receptors for extracellular matrix (ECM) components and select cell adhesion molecules. Named for their ability to integrate signals from the extracellular environment to the inside of the cell, integrins are responsible for securing cells to the surrounding adhesion molecules while amplifying and potentiating signals from growth factor receptors and other extracellular stimuli (1). These transmembrane proteins permit cells to sense and respond to their environment and thus play critical roles in maintaining normal cellular functions, yet have also been implicated in promoting invasion and metastasis in human malignancies (2).

Integrins are heterodimeric receptors that consist of paired α and β subunits. In the human genome, there are 18 α and 8 β subunits that combine in a limited combination to provide 24 integrin receptors, each with its own specificity for select ECM or cellular adhesion proteins (for review, see (1, 3)). Integrins containing the α6 subunit are laminin receptors in which the α6 subunit can pair with either the β1 or β4 subunit. In contrast, the integrin β4 subunit can only pair with the α6 subunit (1, 2, 4), thus making β4 subunit expression predictive of integrin α6β4 expression.

Integrin α6β4 is predominantly expressed on epithelial cells where it is present at the basal surface adjacent to the basement membrane and nucleates the formation of hemidesmosomes. These stable adhesions are critical for the integrity of epithelial monolayers. In contrast to this function, integrin α6β4 signaling in various cancers promotes an invasive and metastatic phenotype. This functional change is mediated by phosphorylation of the cytoplasmic tail of the integrin β4 subunit that releases integrin α6β4 from hemidesmosomes and allows the integrin to promote invasive signaling through cooperation with growth factor receptors and alteration of the transcriptome, which in turn facilitates tumor progression (2, 4–9). Below, we highlight key signaling functions of integrin α6β4 and provide a review of its expression patterns and their prognostic significance in human malignancies.

Structure and normal function of integrin α6β4

The α6β4 integrin is a specialized integrin that is expressed in various normal epithelia, Schwann cells and endothelial cells. The integrin β4 subunit is distinct from other integrin subunits in that it has a particularly long cytoplasmic signaling domain. Whereas the cytoplasmic domains of other integrin subunits are less than 50 amino acids in length, the integrin β4 subunit is over 1000 amino acids in length (10, 11). As depicted in Figure 1, the cytoplasmic domain of the integrin β4 subunit is characterized by two pairs of fibronectin type III domains, a Calxβ domain and a connecting segment (12). The fibronectin repeats and the connecting segment are necessary for hemidesmosome assembly (13–15).

Figure 1. Integrin α6β4 structure and hemidesmosome assembly.

A) The integrin β4 subunit only pairs with the α6 subunit. The long cytoplasmic domain of β4 is structurally distinct from other known receptors but contains several distinct domains including a Calx-β domain, four fibronectin type III repeats, a connecting segment and C-terminal tail. B) Integrin α6β4 nucleates hemidesmosomes by binding to multiple hemidesmosomal associated proteins including Plectin (HD1), BP180 (also known as BPAG2 or collagen XVII) and BP230 (also known as BPAG1).

At the basal surface of normal cells adjacent to the basement membrane, integrin α6β4 binds to laminins in the ECM and facilitates stable adhesion through the formation of hemidesmosomes. Hemidesmosomes are large adhesion complexes that anchor the basal layer of epithelial cells to the basement membrane (13–15). In these junctions, the α6β4 integrin nucleates the connection between cytokeratin intermediate filaments in the cell and laminins in the basement membrane through its interactions with plectin, collagen XVII/BP180 and BP230, as depicted in Figure 1B (16, 17). The importance of these junctions is highlighted by the fact that mutations in the integrin β4 gene (ITGB4) can cause lethal forms of epidermolysis bullosa with pyloric atresia, a disorder characterized by blistering and ulceration of the skin and mucosal tissues (18). While it has been suggested that other integrins may be able to compensate for a loss of integrin α6β4, studies in mouse models have demonstrated that this is not the case (19). Integrin β4 knockout mice (−/−) are born with severe epidermal blistering, exhibit widespread separation of the epithelial-mesenchymal junction, and die shortly after birth. These observations emphasize the importance of the integrin β4 subunit in maintaining the integrity of the epithelial-ECM junction (19).

Hemidesmosomes are dynamic structures. During wound healing, hemidesmosomes must be dismantled to allow the leading edge cells to migrate into the wound (14, 20). This functional change is mediated by phosphorylation of the cytoplasmic tail of the integrin β4 subunit that releases integrin α6β4 from hemidesmosomes. This process occurs through stimulation by growth factor receptors such as the epidermal growth factor receptor (EGFR), and by direct phosphorylation of the integrin β4 cytoplasmic tail by protein kinase C (7, 8, 21). Upon release from hemidesmosomes, integrin α6β4 relocalizes from the keratin cytoskeleton to the actin cytoskeleton (22). When bound to F-actin, the integrin α6β4 signaling domain promotes the formation of motility structures, such as filopodia and lamellae, by cooperating with growth factor receptors and stimulating key signaling pathways (6, 7, 22–24) that in turn facilitate migration and wound closure.

Integrin α6β4 and hemidesmosomes are also suggested to play a role in the contextual orientation of cells. When cells are not adhered to the proper extracellular matrices, they will undergo a specialized form of apoptosis known as anoikis. Notably, when caspases are activated, the β4 cytoplasmic tail is cleaved leading to apoptosis (25, 26).

Integrin α6β4 signaling in malignant cells

Release of integrin α6β4 from hemidesmosomes can lead to altered signals that promote tumor cell growth, invasion and metastasis (2, 4–9). Under conditions where hemidesmosomes are disassembled, integrin α6β4 binding directly to laminin has been shown to activate both phosphoinositide 3-OH kinase (PI3K) and RhoA small GTPases (6, 24). Alternatively, the integrin can cooperate with multiple different growth factor receptors including those in the EGF receptor family (ErbB-1,2,3), c-Met, Ron, LPA1 and LPA2 (23, 27–30) to enhance signaling through PI3K, AKT, MAPK and the Rho small GTPases (6, 23, 24, 31, 32), as depicted in Figure 2. In cells with mutant p53, the α6β4 integrin promotes cell survival through activation of AKT/PKB (31), and stimulates cell cycle progression and proliferation by interacting with Shc to activate the Ras-MAPK pathway (32). As shown in breast and pancreatic cancers, this integrin can promote tumor progression through transcriptional regulation and has been shown to increase the expression of invasive and metastatic proteins such as the epithelial to mesenchymal transition (EMT)-associated protein S100A4 (also known as metastasin/FSP) (33–35). In the next several sections, we discuss our current understanding of how integrin α6β4 signaling promotes a malignant phenotype with an emphasis on its impact on invasion, cell survival and angiogenesis.

Figure 2. Cancer progression-associated signaling pathways activated by integrin α6β4.

Integrin α6β4 can activate multiple signal transduction cascades either directly by binding its ligand laminin or by amplifying signals from multiple growth factors. Enhanced signaling through these pathways contributes to tumor progression in terms of enhanced proliferation, cell survival, invasion and metastasis. Notably, integrin α6β4 can act as a tumor suppressor by promoting apoptosis in the presence of a wild-type p53. For this reason, integrin α6β4 is often overexpressed primarily in tumor types where p53 mutations are prominent such as pancreatic and basal-like breast cancers.

Invasive signaling functions

The finding that integrin α6β4 mediates stable adhesive complexes that anchor cells to the basement membrane would seem to argue against the participation of integrin α6β4 in cell migration. Despite this apparent contradiction, numerous studies have confirmed that integrin α6β4 is responsible for promoting migratory and invasive behavior in carcinoma cells. A potential explanation for this phenomenon comes from its normal role in wound healing (14, 20, 36), as described above.

Early studies in colon and breast carcinoma lines demonstrated that expression of integrin α6β4 contributes to tumor cell invasiveness (6, 9, 37). These studies lead to the pivotal discovery that integrin α6β4 can promote invasive properties in carcinoma cells by activating the PI3K pathway (6), a signaling pathway that is now well known for its role in promoting carcinoma progression (38). Notably, this was the first time that PI3K had been implicated in carcinoma invasion. This finding was subsequently confirmed in a number of additional reports (39–41), although the exact mechanism by which integrin α6β4 activates PI3K initially remained elusive. The cytoplasmic domain of the integrin β4 subunit does not contain a consensus-binding motif for the regulatory subunit of PI3K, making direct activation of this pathway by integrin β4 subunit unlikely (4, 6, 42). One mechanism for integrin β4-mediated activation of PI3K was found to involve insulin receptor substrate-1 and −2 (IRS-1 and IRS-2), which act as signaling intermediates that facilitate integrin α6β4-mediated PI3K activation (42). Ligation of integrin α6β4 promotes phosphorylation of IRS-1 and IRS-2, leading to subsequent activation of PI3K (42). An additional mechanism has been described wherein integrin α6β4 cooperates with ErbB-2 to promote PI3K-dependent invasion (39). Finally, integrin α6β4 has been shown to localize to lipid rafts in the plasma membrane, which may allow it to activate PI3K by facilitating close interactions with other receptor tyrosine kinases (4, 40).

The involvement of integrin α6β4 in cell polarization and the formation of F-actin rich motility structures such as filopodia and lamellae lead to investigations into the Rho family of small GTPases. The Rho family of small GTPases largely control the reorganization of the actin cytoskeleton needed for cell motility (43). Initial studies by Shaw and colleagues on the activation and function of PI3K in invasion found that the small GTPase Rac1 was required for invasion downstream of PI3K (6), a finding confirmed by others (44). Notably, integrin α6β4 can cooperate with growth factor receptors (45) and other integrins (23) to activate Rac1. Further studies examining the impact of integrin α6β4 on migration and invasion found that integrin α6β4 increased the activity of cAMP-specific phosphodiesterase, thereby resulting in decreased cAMP concentrations and subsequent RhoA activation (9, 24). The activation of RhoA downstream of integrin α6β4 subsequently leads to the formation of RhoA-dependent membrane ruffling and lamellae formation, as well as the generation of contraction forces that enable cell migration (24, 46). Our group found that the metastasis associated protein S100A4, which is regulated by integrin α6β4 (35), interacts with Rho effector Rhotekin to promote cell membrane ruffling (47). Notably, the traditional function for RhoA is the generation of stress fibers rather than membrane ruffling, suggesting that integrin α6β4 can change the function of RhoA to facilitate tumor invasion.

Integrin α6β4 amplifies signaling through multiple receptor tyrosine kinases and G-protein coupled receptors in order to promote tumor cell proliferation and invasion. Notably, cooperative signaling has been identified between integrin α6β4 and multiple members of the EGFR family. EGFR and integrin α6β4 have also been shown to co-localize at the leading edge of carcinoma cells subjected to EGF stimulation, and their interaction was inhibited by curcumin, a compound present in turmeric (48). Integrin α6β4 has also been shown to associate with ErbB-2 in multiple breast carcinoma cell lines (30, 49), though reports are conflicting as to whether integrin α6β4 expression correlates with ErbB-2 protein overexpression in patient-derived carcinoma tissues (50–53). Using a mouse model of ErbB-2 mediated tumorigenesis, loss of integrin β4 signaling was shown to reduce breast tumor invasive growth and metastasis, and deletion of the integrin β4 signaling domain was shown to enhance the efficacy of ErbB2-targeted therapy. This study also demonstrated that the integrin β4 subunit forms a complex with ErbB-2 and amplifies ErbB-2 signaling (27). In addition, integrin α6β4 has been shown to regulate the expression of ErbB-2 by influencing its translation (54). These findings are particularly notable as ErbB-2 amplification promotes invasion in human malignancies such as breast carcinoma, and is associated with aggressive behavior (55, 56).

While an interaction with ErbB-2 may be necessary in order for integrin α6β4 to activate PI3K mediated invasion in select cell types, ErbB-2 lacks a consensus-binding site for the regulatory subunit of PI3K. Furthermore, ErbB-2 must dimerize with an EGFR family member in order to function (30, 57). A potential solution to this issue was suggested by the finding that integrin α6β4-mediated PI3K activation is dependent on ErbB-2/ErbB-3 heterodimerization (30). The ErbB-2/ErbB-3 heterodimer is a strong activator of PI3K (30, 58), and the ErbB-3 cytoplasmic domain contains binding sites for the regulatory subunit of PI3K (59). Integrin α6β4 can regulate the expression of ErbB-3, leading to an increase in ErbB-2/ErbB-3 heterodimerization and subsequent PI3K activation (30), and notably, a positive association has been identified between integrin α6β4 and ErbB-3 expression in patient-derived tumors (51).

Integrin α6β4 can also cooperate with c-Met, a receptor tyrosine kinase activated by the hepatocyte growth factor (HGF) (28, 29). In one study, integrin α6β4 was shown to form a direct complex with c-Met that promotes HGF dependent invasion (28). Additional studies have shown that while an interaction with integrin α6β4 may contribute to c-Met dependent invasion, integrin α6β4 and c-Met are also able to promote invasion independently (29). Evidence for a physical association between integrin α6β4 and c-Met is controversial (4, 29); however, this does not preclude the probability that integrin α6β4 can cooperate with c-Met without physical association.

Ron (“recepteur d'origine nantais”), a tyrosine kinase receptor closely related to c-Met, has been shown to form a complex with integrin α6β4 that induces hemidesmosome disassembly and the relocation of integrin α6β4 to motility structures (36, 60). Further studies have shown that Ron activation is important in pancreatic carcinoma progression (61, 62), and have confirmed that Ron interacts with the integrin β4 subunit in this setting to disrupt the association between integrin β4 and plectin (60).

Cell survival and apoptosis

Integrin a6β4 promotes either cell survival or apoptosis, depending on the cellular context. In normal epithelia, integrins are critical to maintaining cellular growth and survival as long as they maintain proper contact with the ECM (63). If anchorage to the ECM is lost, the subsequent loss of integrin signaling can inhibit cell growth and promote a specialized form of apoptosis referred to as anoikis (63). While tumor suppressive functions of the integrin β4 subunit have been identified in bladder (64), colon (65), and breast carcinoma (66) cell lines, a number of additional studies have indicated that the β4 integrin promotes cell survival (19, 41, 66–68). This dichotomy appears to hinge on the expression and mutation status of the p53 tumor suppressor.

Early experiments demonstrated that expression of the integrin β4 subunit in the colon cancer cell line RKO led to increased apoptosis, thus lending support to the notion that integrin α6β4 functions as a tumor suppressor (65). Conversely, expression of the β4 subunit was unable to induce apoptosis in the cell line MDA-MB-435 (69). The Mercurio group investigated the mechanism underlying this apparent contradiction and observed that the RKO and MDA-MB-435 cell lines differed in their p53 mutation status. While RKO cells harbor a wild-type p53, the MDA-MB-435 cell line expresses mutant p53 (69). This group demonstrated that integrin α6β4 can trigger apoptosis through p53 activation in cells harboring wild-type p53 (69); however, in carcinoma cells deficient in p53, integrin α6β4 promotes cell survival by activating AKT/PKB (31, 69) and through translational regulation of VEGF expression (68). These findings suggest that tumors expressing high levels of integrin α6β4 in conjunction with loss of p53 function are resistant to apoptosis and will therefore display a more aggressive clinical course. Interestingly, an association between p53 mutations and integrin α6β4 overexpression is present in a number of aggressive human malignancies, including basal-like breast cancer, head and neck squamous cell carcinoma, and pancreatic ductal adenocarcinoma (50, 70–76).

Angiogenesis

In addition to promoting invasive properties in carcinoma cells, the integrin α6β4 can stimulate invasion and migration of endothelial cells, processes that are necessary for pathologic angiogenesis (5) (for review, see (77, 78)). Studies using knockout mice carrying a deletion in the signaling domain of the integrin β4 subunit displayed reduced angiogenesis in a retinal neovascularization model, and developed smaller and less vascularized tumors after subcutaneous implantation (5). The same study demonstrated that the integrin β4 subunit could promote both bFGF- and VEGF-induced angiogenesis by enhancing signaling through ERK and NF-κB.

The role of α6β4 in transcriptional regulation

Integrin α6β4 regulates the expression of molecules important for carcinoma invasion and metastasis. The best studied of these include NFAT1, NFAT5, S100A4, ErbB-2, ErbB-3 and autotaxin. The NFATs, or Nuclear Factors of Activated T-cells, are transcriptionally regulated by the α6β4 integrin and drive carcinoma invasion (33). As shown in breast cancer, integrin α6β4-mediated upregulation of NFAT1 leads to increased expression of autotaxin (ENPP2), an enzyme that acts as a motility factor by promoting LPA production (34, 79). Integrin α6β4 can also regulate the expression of ErbB-2 and ErbB-3 (30, 54) as mentioned above.

Interestingly, expression of the α6β4 integrin in MDA-MB-435 cells leads to altered expression of over 500 genes (35). One of the most regulated and clinically relevant of these genes is S100A4, a calcium binding protein also known as metastasin-1 (80). S100A4 promotes tumor metastases (80) and is regulated by integrin α6β4 through NFAT5 activation in conjunction with DNA demethylation of the S100A4 promoter (35). S100A4 interacts with Rhotekin to promote the formation of an S100A4/Rhotekin/RhoA complex, thus allowing RhoA to promote invasion through membrane ruffling (47). Integrin α6β4 has also been shown to negatively regulate the expression of miR-92ab and miR-99ab/100 miRNA families that impact target genes implicated in promoting cell motility (81).

Integrin α6β4 expression in human malignancies

Integrin α6β4 was originally identified as a tumor progression antigen by two separate groups, one who termed it tumor-specific antigen-180 (TSP-180) (82) and the other who referred to it as the A9 complex (83). Subsequently, the TSP-180 and A9 complexes were shown to be identical to integrin α6β4 (84, 85). During the invasive process, integrin α6β4 is released from hemidesmosomes where it can then participate in many of the most aggressive properties of advanced carcinomas. Immunohistochemical staining in patient-derived tissues confirms that in many cancers, expression and localization of integrin α6β4 are altered (as demonstrated in Fig. 3).

Figure 3. Altered localization of integrin β4 expression in invasive breast carcinoma.

In a dilated duct with benign columnar cell change (left), integrin β4 is expressed in myoepithelial cells surrounding the duct, but is absent in luminal cells. Adjacent nests of invading carcinoma cells (right) display elevated expression of the integrin β4. Immunohistochemical staining was performed using the 439-9B antibody as previously described (72). Brown staining represents positive expression of the integrin β4.

Studies examining integrin β4 expression in patient-derived tissue, in some cancer types, have obtained conflicting results. It is unclear whether these contradictory findings relate to sample size, cancer subtype examined or the method of detection. Some investigators have reported inconsistent immunohistochemical staining for integrin β4 in formalin-fixed, paraffin embedded tissue sections (53). Our group found that immunohistochemistry for the integrin β4 subunit is particularly sensitive to the antigen retrieval process (72). In addition, different studies have used a variety of clonal antibodies to detect integrin β4 expression, which may partially explain the variability in staining patterns that has been reported. Below, we discuss the clinical associations of integrin α6β4 in various human malignancies, noting where there is disagreement in the literature.

Malignancies with strong evidence that integrin β4 expression is pathologically significant

Breast cancer

In breast cancer, integrin β4 overexpression is associated with aggressive behavior and poor prognosis. Given the challenges described with immunohistochemistry, gene expression profiling provides an excellent alternative that allows for quantitative assessment of integrin β4 expression. One study used gene expression profiling and immunohistochemistry of tissue microarray (TMA) sections to demonstrate that integrin β4 is overexpressed in basal-like breast cancer (50). This finding is particularly notable as basal-like breast cancer is an aggressive subtype that is associated with a notoriously poor prognosis (71). This group further developed a 65-gene signature that included the top genes whose expression was found to correlate with that of integrin β4. This integrin “β4 signature” was shown to be a prognostic indicator that could predict both decreased survival and decreased time to recurrence in four breast cancer cohorts (50). In other studies, integrin β4 mRNA expression was found to positively correlate with nuclear grade and tumor size (53), and elevated integrin α6β4 protein expression has been found to associate with decreased survival (86). Co-expression of integrin α6β4 and Net1, a RhoA guanine nucleotide exchange factor, has also been associated with decreased distant metastasis-free survival (52).

In contrast to the findings described above, a number of early reports indicated that integrin β4 expression is absent or rare in breast cancers. This observation may be due to difficulties with immunohistochemistry on frozen specimens, or may be related to the fact that these studies were performed before the modern sub-classification of breast cancers was developed. As integrin β4 overexpression is more frequent in triple negative breast cancers and triple negative tumors represent a minority of breast cancers, these studies may not have included an adequate number of triple negative cases to detect integrin β4 overexpression. Two of these early investigations report that integrin β4 expression was absent in all invasive breast carcinomas examined (82, 87), while another found strong integrin β4 staining in only a small subset of breast tumors (88). Others have reported that integrin β4 is redistributed over the cell surface in select breast carcinomas (89). Integrin β4 expression in ductal carcinoma in situ (DCIS) is also reportedly rare, with expression identified in only 20% of cases in one study (90). According to another report, integrin β4 expression was absent in the neoplastic cells of DCIS and detected only in residual myoepithelium (87). Given more recent evidence using gene expression profiling, it is reasonable to conclude that at least a certain subset of breast tumors overexpress integrin β4, including basal-like breast cancers.

Bladder cancer

Studies investigating integrin β4 expression in bladder cancer demonstrate that it is overexpressed in a proportion of transitional cell carcinomas, and suggest its use as a prognostic marker. An early study reported that in normal urothelium integrin α6β4 is expressed in the basal layer of urothelial cells where this expression is highly polarized and localized to the lamina propria junction. The authors then examined integrin α6β4 expression in ten low stage bladder cancers, where they found increased, non-polarized expression in 80% of tumors (91). A subsequent study by the same group examined integrin α6β4 expression in bladder tumors from 57 patients; each case was categorized as having negative, weak, or strong expression of integrin α6β4, where weak was defined as expression that most closely resembled that of normal urothelium (92). They found that patients with weak integrin α6β4 expression had improved survival compared to patients with either strong or negative expression (92). Another study examined integrin β4 expression in a cohort of patients with non-muscle invasive bladder cancer and found that integrin β4 expression levels were an independent predictor of intravesical recurrence after transurethral resection (93).

Cervical cancer

Integrin β4 expression in cervical lesions has been examined primarily in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma. Multiple reports have confirmed that the integrin β4 is strongly expressed in invasive squamous cell carcinomas of the cervix (94–96). Interestingly, integrin β4 expression positively correlates with the degree of squamous atypia in CIN (94, 95). In a study of cervical biopsies from 35 patients, integrin β4 expression was present only in cells of the basal and parabasal layers of normal ectocervical mucosa, and this pattern was maintained in flat condylomas, hyperplastic epithelium, and in CIN I lesions (94). Interestingly, in CIN II-III, integrin β4 expression was present throughout the entire thickness of the epithelium, with strong staining observed toward the superficial surface. Furthermore, expression of integrin β4 was identified in 90% of cervical carcinomas studied, where expression was diffusely present in the invasive nests (94).

A larger study investigated integrin β4 expression in 40 cervical biopsies. The authors describe that in normal ectocervical mucosa, integrin β4 expression was localized to the basal aspect of cells in the basal layer of epithelium (95). They found that in CIN, expression of the integrin β4 followed the distribution of atypia: for example, in CIN II, β4 was expressed throughout the lower 2/3 of the epithelial thickness, while in CIN III, β4 was expressed throughout the full epithelial thickness. These findings suggest that the integrin β4 may play an early role in promoting the survival and growth of pre-invasive neoplasms.

A third study examined expression of the integrin β4 in 20 cases of invasive cervical carcinoma and 23 cases of CIN III (96). The invasive cervical carcinomas included examples of well, moderately, and poorly differentiated squamous cell carcinoma, as well as three endocervical adenocarcinomas. Diffuse expression of the integrin β4 was identified in all 20 cases of invasive carcinoma. In addition, integrin β4 expression was identified in all epithelial layers in 65% of CIN III lesions (96).

Head and neck cancer

In squamous cell carcinomas of the head and neck, integrin β4 is commonly overexpressed. An early study of 82 patients with SCC of the head and neck found that strong expression of the UM-A9 antigen (later identified as the α6β4 integrin) was associated with early relapse and decreased patient survival (97). In another study, integrin β4 expression was found to be upregulated in SCCs when compared to normal squamous mucosa, although an association was found between loss of integrin β4 expression and the presence of nodal metastases (98).

Multiple studies using gene expression profiling have confirmed that integrin β4 gene expression levels are prognostically significant in SCCs of the head and neck. One of these reports demonstrated that integrin β4 gene expression was associated with decreased survival in a cohort of 66 patients (99). In a larger study, increased integrin β4 gene expression was associated with the presence of lymph node metastases, distant metastases and patient death on univariate analysis, and was an independent predictor of distant metastases on multivariate analysis (100).

Lung cancer

Integrin β4 is overexpressed in non-small cell lung cancers, and expression is particularly high in pulmonary squamous cell carcinomas. An early study investigated expression of the integrin α6β4 in a series of patient-derived lung cancers and found moderate to strong expression in all of the squamous cell carcinomas (N = 36) and adenocarcinomas (N = 23) tested, though expression was notably absent in all (N =10) of the small cell carcinomas examined (101). Integrin β4 expression was identified in a number of non-small cell carcinoma cell lines (A431, A549, DG3), but was absent in the single small cell carcinoma cell line tested (AE2) (101).

In a different study that included uninvolved normal lung tissue, normal alveolar epithelial cells were found to be negative for integrin β4 expression, and instead exhibited expression of the α1β1 and α3β1 laminin receptors. They further found that bronchial and bronchiolar epithelium exhibited weak and inconsistent integrin β4 expression that was localized to the basement membrane interface. In squamous cell carcinomas of the lung, integrin β4 expression was intense and localized to the tumor-stroma interface (102). In this same study, integrin β4 expression was identified in large cell carcinomas of the lung, but was found to be absent in neuroendocrine carcinomas. Patriarca et al. (103) also found that in normal bronchial epithelium, integrin β4 expression was localized to the basal surface of cells in a linear pattern. They found strong and extensive staining for the integrin β4 in 85% of squamous cell carcinomas (N = 20), but found positive staining in only 25% pulmonary adenocarcinomas studied (N = 20). In a complementary study using molecular profiling, integrin β4 was upregulated in lung squamous cell carcinomas when compared to adenocarcinomas, and this was confirmed using both immunohistochemistry and in-situ hybridization (104).

Pancreatic cancer

Integrin β4 is overexpressed in pancreatic carcinomas, and is also a marker of poor prognosis. Using gene expression profiling, Logsdon et al. first determined that integrin β4 is upregulated in pancreatic adenocarcinoma when compared to normal pancreatic tissue and chronic pancreatitis tissue samples (105), a finding that was confirmed by others (106). In order to validate these findings, the Logsdon group performed immunohistochemistry for the integrin β4 subunit and a number of other candidate genes in 28 pancreatic adenocarcinomas, where they found that all cases had strong integrin β4 expression (105). In another report, Gleason et al. found moderate to strong integrin β4 staining in 92% (N = 48) of pancreatic adenocarcinomas that were evaluated using immunohistochemistry; they also found that in normal pancreas, integrin β4 staining was weak and expressed only along the basement membranes of large ducts (107).

Integrin β4 expression has also been studied in pancreatic intraepithelial neoplasia, a non-invasive precursor lesion to pancreatic adenocarcinoma (108). In a comprehensive study of pancreatic lesions, Cruz-Monserrate et al. (72) determined that integrin β4 overexpression is present in the early stages of pancreatic adenocarcinoma development. As reported previously, they found that in normal pancreas, integrin β4 expression is localized to the interface between ductal epithelial cells and the basement membrane (72). Upregulation of integrin β4 expression was observed in 92% (N = 113) of pancreatic adenocarcinomas studied, and distinguished pancreatic cancer from pancreatitis. Furthermore, overexpression and altered localization of the integrin β4 was identified in all pancreatic intraepithelial neoplasia lesions ranging from Grade 1A to Grade 3 (72).

Recently, elevated integrin β4 expression was shown to associate with reduced overall survival among pancreatic adenocarcinoma patients (N = 134), where it was found to have independent prognostic significance on multivariate analysis. Interestingly, elevated integrin β4 expression was also found to correlate with a number of EMT hallmarks, including solitary cell infiltration, reduced expression of E-cadherin, and increased expression of vimentin (109). Pancreatic adenocarcinoma has one of the poorest prognoses of all epithelial malignancies; the fact that integrin β4 is highly expressed in these tumors provides further evidence for its role in aggressive neoplasms.

Thyroid cancer

Thyroid carcinomas are unique in that they are one of the few malignancies to exhibit neoexpression of integrin α6β4 during cancer progression. While expression of integrin α6β4 is absent in normal and adenomatous follicular cells, strong expression has been observed in both follicular and papillary thyroid carcinomas (110). Similar findings have been reported using flow cytofluorometry, where expression of integrins such as α1β1 and α6β1 was found in normal thyroid and tumor specimens, integrin α6β4 expression was found only in thyroid carcinomas and carcinoma cell lines (111). Others have confirmed neoexpression of integrin α6β4 in thyroid carcinoma tissue (112, 113) and have also found that it is expressed in anaplastic thyroid carcinoma, an aggressive and poorly differentiated malignancy (112).

Malignancies in which integrin β4 expression has controversial or undetermined significance

Colon cancer

One of the earliest studies to investigate α6β4 expression in human malignancies found that the α6β4 integrin was expressed in colon cancer (82). However, additional reports have been controversial. One study investigating integrin β4 expression in colorectal carcinomas reported that integrin β4 expression was reduced during malignant transformation (114). This group found that while expression of the integrin β4 subunit was maintained at the basal epithelial cell membrane in normal colonic mucosa and in colonic adenomas, expression of the integrin β4 subunit was reduced or absent in most colorectal carcinomas, but was maintained in well-differentiated colon cancer (114). Similar findings were reported in another study, where expression of the integrin β4 subunit was reduced or absent in most colon carcinomas examined (115).

A contrasting study demonstrated that integrin β4 is overexpressed in a majority of colon carcinomas and that its expression is elevated in high stage, poorly differentiated cancers (116). These findings are supported by subsequent work examining integrin β4 expression in colorectal carcinomas using double immunofluorescence and RT-QPCR, where integrin β4 protein and transcript levels were increased in colorectal carcinoma when compared to normal tissue (117). Data from our laboratory also confirms the observation that integrin β4 levels are particularly high in colon cancer cell lines and patient-derived tissues (unpublished observation). Additional studies are needed to confirm conclusively that integrin β4 is overexpressed in patient-derived colorectal carcinoma tissues. Gene expression profiling or analysis of existing datasets will help to clarify this issue.

Ovarian cancer

In benign ovary, integrin β4 is basally located in surface and cyst lining epithelium (75). One of the few studies investigating integrin β4 expression in ovarian cancer found strong basal expression in all of the epithelial ovarian tumors studied, and also found that integrin β4 was expressed in malignant cells within the ascitic fluid in three out of nine cases (75). A second report found basally polarized integrin β4 expression in normal ovary and in 40% of serous ovarian carcinomas examined. The authors further noted that expression of both integrin α6 and β4 subunits were positively correlated with laminin expression (74). Interestingly, serous ovarian cancer has a similar genomic profile to basal-like breast cancer, with both subtypes displaying frequent loss of TP53, BRCA1, and RB1, suggesting that integrin β4 may play an important role in both types of cancer (70). Further work will be needed to fully characterize integrin β4 expression in ovarian neoplasia and to determine how expression associates with prognosis.

Prostate cancer

Early reports indicated that integrin β4 is downregulated in prostatic adenocarcinoma, and in one investigation, expression of integrin β4 was absent in all prostate cancers examined (N = 20) (118). Multiple additional studies have reported that integrin β4 expression is lost during the transition from benign epithelium to prostatic adenocarcinoma (119–122). A potential explanation for this phenomenon is that androgen receptor expression has been reported to cause downregulation of integrin α6β4 (123). The assertion that integrin β4 is downregulated in prostate cancer was challenged by a report demonstrating that integrin β4 mRNA is overexpressed in a subset of prostate carcinomas using gene expression datasets and a DNA microarray (124). The authors of this study also investigated integrin β4 protein expression using immunohistochemistry and found overexpression in 35% of invasive cancers and in a number of metastatic lesions (124). More recently, a population of integrin β4 positive circulating tumor cells was identified in the peripheral blood of patients with castration resistant prostate cancer (125). Overall, prostate cancer is one cancer in which integrin α6β4 is suggested to be downregulated. However, given recent findings, it will be important to determine how residual or enhanced integrin α6β4 expression, in the minority of cases that overexpress it, associates with clinical parameters.

Tumors of the central nervous system

Expression of integrin α6β4 has not been extensively studied in glial tumors; however, there is evidence demonstrating that integrin α6β4 is expressed in astrocytomas, oligodendrogliomas, glioblastomas, and a number of glioma cell lines. Integrin β4 expression has been identified in reactive astrocytes (126) as well as in subependymal glia, choroid plexus and meningothelial cells (127). One study found that integrin β4 expression is higher in astrocytomas and glioblastomas when compared to benign astrocytes (126). A larger study investigated expression of the integrin β4 subunit in a series of astrocytomas and oligodendrogliomas where they found that integrin β4 expression was slightly higher in oligodendrogliomas (128). Further studies will be needed to determine how integrin α6β4 relates to glioma stage and prognosis.

Sarcomas

Integrin β4 overexpression has been described in high grade osteosarcomas and there is evidence it may play a role in promoting a metastatic phenotype by interacting with ezrin (129). While integrin β4 is expressed in benign endothelial cells, its expression appears to be reduced in angiosarcomas and other vascular tumors (130). Integrin β4 staining is also reportedly absent in rhabdomyosarcomas, ganglioneuroblastomas, primitive peripheral neuroectodermal tumors and Ewing's sarcomas (131).

Conclusions

Integrin β4 is commonly overexpressed in high-grade malignancies. Notably, there is strong evidence that integrin β4 is overexpressed in tumors of the bladder, cervix, lung, pancreas and thyroid, and in basal-like breast cancer. In addition, integrin β4 overexpression has been identified as an adverse prognostic marker in tumors of the breast, pancreas, and in squamous cell carcinomas of the head and neck. The reason for these poor prognoses stems from the ability of the integrin α6β4 to promote several key hallmarks of cancer, including the capacity to sustain proliferative signaling, evade apoptosis, promote tissue invasion and metastasis, and stimulate angiogenesis. Notably, mutation or inactivation of p53 is one mechanism that allows integrin α6β4 to promote cell survival and amplify signaling through a number of invasive and proliferative pathways. Integrin α6β4 can trigger apoptosis in cells harboring wild-type p53; however, in carcinoma cells deficient in p53, integrin α6β4 promotes cell survival (31, 68, 69). Interestingly, tumors with a high frequency of p53 mutations (pancreatic adenocarcinoma, basal-like breast cancer, squamous cell carcinomas of the head and neck) tend to also display integrin β4 overexpression. In these tumor types, there is evidence that integrin β4 expression is clinically significant and correlates with a poor prognosis. This observation may partially explain why integrin β4 expression is prognostically significant in some, but not all tumor types.

In a number of malignancies, it is still unclear whether expression of integrin β4 is elevated or reduced. In tumors of the breast, prostate and colon, a number of studies examining integrin β4 expression have obtained conflicting results, with some authors reporting that integrin β4 is overexpressed and others reporting a reduction in integrin β4 expression. These disparate findings may relate to differences in sample size, antibody usage, tissue processing or antigen retrieval process. In breast cancer, use of gene expression databanks and modern sub-classifications have helped to clarify the association of integrin β4 with basal-like breast cancer. The integrin β4 is highly expressed in the basal cell layer in many benign epithelial tissues. During carcinoma progression, localization of the integrin α6β4 is altered, as has been described in tumors of the breast, bladder, cervix, and pancreas (72, 89, 91, 94, 95). It is likely that altered localization of the α6β4 integrin and its concurrent release from hemidesmosomes are as important in carcinoma progression as overexpression, thus altered integrin α6β4 localization should be studied carefully in these malignancies. Additional investigations using tissue microarrays or larger patient cohorts will be needed to determine whether integrin β4 associates with progression in these cancers. Gene expression profiling will provide another mechanism that allows for more in-depth investigation of integrin β4 expression and its prognostic significance in human malignancies, and will allow for validation of immunohistochemical findings.