Roles of integrins in prostate cancer progression

Integrins are expressed in normal prostate basal cells and are required for the interaction of the cells with surrounding stroma which influences their growth, survival and differentiation potential. These integrins include a2p1, a3p1, a5p1 and a6p4 [29-33]. Altered expression of integrins affects cell adhesion to adjacent cells and to the ECM and such affects have been observed in solid tumours and prostate cancer cell lines. Table 2 highlights the most well characterised integrins involved in prostate cancer progression, migration and invasion, and these integrins are discussed below.

Bonkhoff et al. (1993) investigated the expression of integrin a2p1 in normal, hyperplastic and neoplastic human prostate tissue as well as lymph node metastases samples. Results showed downregulated a2p1 in 70% of the hyperplastic samples compared to normal prostate tissues. However, a2p1 was upregulated in the lymph node metastases compared to primary lesions. In another study, the role of integrin a2 in prostate cancer metastasis was investigated [34]. Immunofluorescence staining showed the presence of a2 and p1 subunit clusters in bone metastatic prostate cancer cells (C4-2B) and not in the lymph node metastat-ic prostate cancer cells (LNCaP), in contrast to the findings of Bonkhoff et al. which reported a2p1 upregulation in lymph node metastasis. The functional blocking of the integrin a2 subunit with antibodies in the C4-2B bone metastatic prostate cancer cell line resulted in reduced adhesion and inhibiton of invasion to collagen I [34]. The role of a2p1 in bone metastasis is further supported by a study by Hall et al. (2006). A collagen-binding LNCaP cell line was derived (LNCaPcol) and showed increased levels of a2p1 with associated increased migration towards collagen I [35]. In an in vivo analysis of these cells, in which LNCaPcol was injected into the tibia of nude mice, the LNCaPcol injected mice developed bone tumours. A follow-on study was conducted to investigate the signalling pathways involved in a2p1 stimulated migration [25]. RhoC guanosine triphosphatase activity was increased by five to eight fold in collagen binding cell lines, CB-2B and LNCaPcol compared to non-collagen binding LNCaP. These results support the idea that ligation of collagen I to a2p1 activates the RhoC signalling pathway, which mediates prostate cancer invasion and metastasis to the bone.

A microarray study was conducted on 111 individuals with localised prostate cancer who had undergone radical prostectomy, including 60 individuals who had tumour recurrence after a follow-up of 123 months [36]. In this study increased integrin a3 and a3p1 expression were found to be related to worse outcome with strong a3 and a3p1 expression associated with higher incidence of recurrence. In another microarray study performed on five prostate cancer cell lines (LNCaP, DU145, PC3, LAPC-4 and 22Rv1) and 13 prostate cancer xenografts, integrin a4 showed decreased expression associated with deletion of the integrin a4 locus [26]. Since all samples were derived from metastases, it suggests that integrin a4 could be a tumour suppressor. Interestingly, integrin a7 has also been identified as a tumour suppressor [13]. The prostate cancer cell lines, PC3 and DU145 were transfected with integrin a7 expression vector and implanted in SCID mice. After six weeks, the volume of the tumours were measured and compared to mice transfected with control vector. Results showed re duced tumour volume and fewer metastases in the integrin a7 vector transfected mice. Further analysis of metastasic potential using a wound-healing assay showed reduced rates of migration in both PC3 and DU145 cells overexpressing integrin a7. Thus, these studies support the notion that integrin a7 inhibits cell migration and acts as a tumour suppressor.

An early study using DU145 and PC3 cells, which express integrin aIIbp3, suggested that integrin aIIbp3 is also involved in prostate cancer metastasis [37]. Although both cell lines express integrin aIIbp3, immunofluorescence data showed different localisation patterns of the integrin. In DU145 cells the integrin localizes to focal contact sites whereas in PC3 cells, it is mainly intracellular. Interestingly, when both the tumourigenic cell lines were injected intra-prostatically into SCID mice, only the DU145 cells metastasized. Further analysis by flow cytometry with an antibody to aIIbp3 showed higher expression of aIIbp3 in DU145 cells isolated from the prostate when compared to DU145 cells from the subcutaneous tissue. Therefore, the data suggests that integrin aIIbp3 is involved in the metastatic progression of prostate tumours. Recently, integrin a5p1 also has been found to be important in cell adhesion in prostate cancer cells [38]. When integrin a5p1 was blocked with an antibody, a decrease in the number of adherent PC3 cells to fibronectin was observed. Partial inhibition of the PC3 cell migration and the formation of quasi-spherical cell shape changes were observed, suggesting a reversal to a less mesenchymal phenotype. In addition, the blocking of a5p1 resulted in weak expression of the cytoskeletal proteins F-actin and a-actinin suggesting a weak cell-fibronectin interaction. Thus, these results support the idea that integrin a5p1 plays an important role in the adhesion of PC3 cells to fibronectin and the migration of PC3 cells.

Integrin avp3 has also been identified to be involved in prostate cancer metastasis. Zheng et al. (1999), found expression of integrin avp3 in 16 prostate cancer specimens but not in normal prostate epithelial cells. The highly metastatic and invasive PC3 cell line also expresses integrin avp3 but not the non-invasive LNCaP cell line [39]. These avp3 expressing PC3 cells and the primary prostate cancer cells were found to adhere and migrate on vitronectin. When LNCaP cells were transfected with a avp3 expression plasmid to induce avp3 expression, LNCaP cells also adhered to and migrated on vitronectin. Thus, this study suggests that avp3 is potentially involved in prostate cancer invasion and metastasis. A following study found integrin avp3 to be involved in bone metabolism and angiogenesis [40]. To investigate how inhibition of integrin avp3 in cells native to the bone would affect prostate cancer bone metastasis, a prostate cancer cell line that expresses little or no integrin avp3 was chosen. Interestingly, in this study, PC3 cells were used as they found undetectable levels of avp3 by FACS analysis and by using antibody staining. This is conflicting with the previous study which reported expression of avp3 in PC3 cells and it is possible that this is due to the use of different types of antibodies. Regardless, PC3 cells were injected directly into human bone fragments which were previously implanted subcutaneously in SCID mice and the mice were treated with anti-p3 antibody fragment (m7E3 F(ab')2). This antibody only blocks the human bone-derived avp3. After two weeks of treatment, inhibition of integrin avp3 resulted in a reduced proportion of antigenically-human blood vessels within tumour-bearing bone implants. In addition, a reduction in the rate of tumour cell proliferation with in the bone implants, reduced osteoclast number and degradation of calcified bone tissue were observed.

The integrin a6 can pair with either p1 or p4 subunits and it binds to laminin. The integrin a6p4 is a laminin receptor and is known as a hemidesmosome complex, mediating cell attachment to the ECM. It acts as the junctional complex on the basal cell surface and is involved in the attachment of epithelial cells to the adjacent basement membrane. In contrast, integrin a6p1 has been found to be involved in the cell migratory phenotype. The expression and distribution of integrin a6p1 in normal, hyperplastic and neoplastic prostate tissue and lymph node metastases was examined [33]. Approximately 85% of the grade I and grade II tumours and also the lymph node metastases showed upregulation of integrin a6p1, compared to normal and hyperplastic samples. Staining showed clusters of a6p1 receptors in acinar basement membranes which suggests integrin a6p1 is important in mediating cell attachment to the basement membrane. Then, Nagle et al. (1994), found that while most of the prostate carcinoma tissues they tested displayed downregulation of integrins, the majority of these samples expressed a6p1 [41]. This is consistent with the loss of integrin p4 in the carcinoma samples. In a separate study, integrin p4 was found to be absent in prostate carcinoma tissues and only present in normal prostate glands and PIN lesions [42], supporting the previous study. Therefore, these data suggest that integrin p4 is lost during cancer progression and therefore, integrin a6 is preferentially paired with the p1 subunit, forming a6p1. A following study found a variant form of integrin a6, a6p which was expressed in DU145, LNCaP and PC3 prostate cancer cell lines but not expressed in the normal prostate cells, PrEC [32]. This a6p variant also binds to both the p1 and the p4 subunits and has three times longer half-life than a6. Recently, King et al. (2008) investigated the role of integrin a6p1 in prostate cancer migration and bone pain in a novel xenograft mouse model [43]. The human prostate cancer cells (PC3N), were stably transfected to overexpress either the cleavable wild type (PC3N-a6-WT) which forms the a6p variant or the uncleavable (PC3N-a6-RR) form of integrin a6. The a6 subunit can be cleaved via Urokinase-type Plas-minogen Activator (uPA) treatment and the cells were directly injected and sealed into the femur of a mouse. After 21 days, tumour cells expressing wild-type integrin a6 (non-cleava-ble) showed a significant decrease in bone loss, unicortical or bicortical fractures and decreased ability of tumour cells to reach the epiphyseal plate of bone and prevented movement evoked pain, compared to the cleavable a6 integrin. Thus, these results suggest that blocking of integrin a6 cleavage in prostate tumour cells results in decreased tumour cell migration within the bone and reduced bone fractures and pain.

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