Targeting Constitutively Activated β1 Integrins inhibits Prostate Cancer Metastasis

Keywords: prostate cancer, metastasis, integrin, extracellular matrix, collagen, fibronectin

Cell lines and reagents

PC3 and LNCaP cell lines were obtained from ATCC. The PC3-mm2 cell line is a highly metastatic cell line derived from PC3 cells through in vivo passages in mice (21) (gift of Dr. Isaiah J. Fidler, MD Anderson Cancer Center). The C4-2B4 is a LNCaP subline (22), kindly provided by Dr. Robert Sikes, University of Delaware. PC3-mm2, LNCaP, and C4-2B4 cell lines were confirmed by fingerprinting. The anti-β₁ integrin monoclonal antibody mAb 33B6 was generated previously (23). Anti-β₁ integrin antibodies mAb MAR4, 9EG7, and 7F10 were purchased from Chemicon, BD Pharmingen and Novocastra, respectively. Anti-pFAK-Y397 and Alexa Fluor 647 conjugated anti-mouse or anti-rat antibodies were purchased from Invitrogen. Anti-total FAK, AKT, pAKT (Ser473), PARP and cleaved PARP antibodies were purchased from Cell Signaling. SCID mice were purchased from Jackson Laboratory (Bar Harbor, ME). All experimental procedures involving animals were performed in compliance with institutional and governmental requirements and approved by M. D. Anderson’s Animal Care and Use Committee.

Flow cytometry for β₁ integrin expression

Cells (1×10⁶) were plated on fibronectin-coated plates for 15 min, detached with trypsin, and incubated with β₁ integrin antibodies mAB, MAR4 or 9EG7 for 1 hour at 4°C, followed with Alexa Fluor 647-conjugated anti-mouse or rat antibody for 1 hr, respectively. The fluorescence intensity of the cells was measured by a BD FACSCantoII flow cytometer (BD Biosciences). Cells incubated with mouse or rat normal IgG were used as controls.

FAK and AKT phosphorylation, PARP cleavage, and apoptosis assays

PC3-mm2 cells plated on collagen-coated plate were treated with control IgG or mAb 33B6 for 24 h. The apoptotic PC3-mm2 cells were detected with sulforhodamine 101-annexin V and DEVD-NucView 488 caspase-3 substrate using NucView^™ 488 dual apoptosis assay kit (Biotium). Cell lysates were used in immunoblots with antibodies against total and or cleaved PARP (Cell Signaling), phospho-FAK-Y397-the autophosphorylation site resulting from activated integrins-Cell Signaling), or phospho-AKT (Ser473) and total AKT (Cell Signaling).

Anoikis assay

LNCaP, C4-2B4, PC3, and PC3mm2 cells were detached by 2mM EDTA, washed with medium, and resuspended in serum-free medium at 8×10⁴ cells/ml. Fourteen ml of cell suspension was added in a 15 ml conical tube with vent cap, and rotated in an incubator to prevent cell aggregation. For counting live and dead cells, 500 μl of cell suspension was incubated with 2μM Calcein AM (BD Biosciences) for 30 min at 37°C followed with 4μM propidium iodide (BD Biosciences) for 30 min at 37°C. Cells were counted using a fluorescence microscope. To detect apoptotic cells, the cell suspension was centrifuged and washed with PBS, fixed by cold methanol for 30 min on ice and washed again. Cells then were resuspended in PBS with 2 mg/ml Ribonuclease A (Sigma, St. Louis, MO) and 50 μM propidium iodide for 30 min on ice, and analyzed by flow cytometry.

β1-integrin knockdown

A β₁ integrin viral plasmid (pLKO.1-puro (Sigma, St. Louis, MO) harboring shRNA 5′-GCCCTCCAGATGACATAGAAA-3′) was used to produce lentivirus. A second, non-targeting sequence 5′-GCGCGATAGCGCTAATAATTT-3′ cloned in the same vector (Sigma) was used as a control plasmid. To make lentivirus, the viral plasmid was co-transfected with the packaging plasmid pCMV-dR8.2 dvpr and the envelope plasmid pCMV-VSVG (gifts from Dr. Yutong Sun, MD Anderson Cancer Center) into 293FT cells using Lipofectamine 2000 (Invitrogen). Viral supernatant was collected after 48–72 hrs and centrifuged at 20,000 rpm for 2hrs to pellet virus particles, which were then resuspended in RPMI medium. PC3-MM2 cells were infected by incubating them with virus particles in the presence of 8 ug/ml of polybrene (Sigma) for 24 hrs, and then medium was replaced with 5μg/ml puromycin for one week to select stable β1 integrin knockdown cells. Cells with decreased β₁ integrins have remained stable for five passages.

Adhesion assay

PC3-mm2 cells (5×10⁴ cells/100 μL) pre-labeled with 1 μM calcein AM (Invitrogen) were mixed with equal volume of 20 μg/mL mouse IgG, the anti-β1 integrin mAb 33B6, or medium only, and seeded onto 96-well plates that were pre-coated with 20μg/mL collagen-I, fibronectin, or BSA. The plates were incubated for 30 min at 37°C; the wells were washed with PBS twice and the cells that adhered to the plate were quantified by measuring the fluorescence intensity at 485/528 nm in each well on a Synergy HT fluorescent plate reader (BioTek).

Parallel plate flow chamber detachment assay

The parallel plate flow detachment assay was performed as described previously (24). In brief, fibronectin (10 μg/ml) or collagen-I (50 μg/ml in 0.1M NaHCO₃) was immobilized onto plastic slides. The slides were blocked with 2% BSA before placing into a parallel plate flow chamber. PC3-mm2 cells (2×10⁶) treated with or without mAb 33B6 (5 μg/ml) for 5 min at 37°C were injected into the flow chamber and allowed to settle on the slide for 10 min. A computer controlled syringe pump (Harvard Apparatus) was used to apply an increasing linear gradient of shear flow to the adhered cells for 300 seconds and the number of cells remaining on the slide was recorded. Shear stress calculations were determined as described (24).

In vitro spreading assay

PC3-mm2 cells (5×10⁴) were treated with 20 μg/mL mouse IgG, or 20 μg/mL anti-β1 integrin antibody, or control buffer, seeded onto coverslips coated with collagen-I (20 μg/mL), fibronectin or BSA (20 μg/mL), incubated for 1h, and fixed with 4% formaldehyde. Cell morphology was examined under a microscope.

Migration assay

Falcon HTS FluoroBlok inserts were coated with collagen-1, fibronectin or BSA at a density of 5 μg/cm² overnight. Cells (10⁵ cells in 0.3 mL/insert) in serum free RPMI medium containing 20 μg/mL mouse IgG or mAb 33B6 were seeded on the inserts and incubated at 37°C for 24 h. The migrated cells were labeled with 1μM calcein AM and counted.

Apoptosis Assay

PC3-mm2 cells plated on collagen-coated plate were treated with control IgG or mAb 33B6 for 24 h. The apoptotic PC3-mm2 cells were detected with sulforhodamine 101-annexin V and DEVD-NucView 488 caspase-3 substrate using NucView^™ 488 dual apoptosis assay kit (Biotium, Hayward, CA). Cell lysates were used in immunoblots with antibody against cleaved PARP.

Proliferation assay

Cells were mixed with 20 μg/mL mAb 33B6, mouse IgG, or buffer and plated onto the collagen, fibronectin, or BSA-coated plate. Cells were incubated at 37°C for 1–4 days. Cell proliferation was determined by viable cell counting.

Intracardiac, intra-prostatic injection and bioluminescence imaging of mice

PC3-mm2 cells were transduced with a retrovirus containing luciferase (Luc) and GFP genes as described previously (25). PC3-mm2-Luc cells were mixed with control IgG or mAb 33B6 at 30 μg/mouse for 30 min before injecting into the left ventricle (1×10⁶ cells/mouse) or the prostate (0.5×10⁶ cells/mouse) of male SCID mice. Mice were injected with control or anti-β₁ integrin antibody (30 μg/mouse or 1 mg/kg) before cell inoculation and twice per week thereafter. Tumor growth was monitored weekly using bioluminescence imaging. Images were acquired and analyzed with an IVIS Imaging System (Xenogen).

Immunohistochemical analysis

Formalin-fixed, paraffin-embedded PCa tissue samples from prostate and lymph node metastases obtained from the PCa tissue bank (supported by a SPORE award to The University of Texas M.D. Anderson Cancer Center) were immunostained with mAb 7F10 or anti-pFAKY397 according to the manufacturer’s recommended procedures. The immunostaining was considered positive when more than 10% of the tumor cells were immunoreactive.

Statistical analyses

Two-tailed, paired Student’s t test was used for statistical analyses. A P-value of less than 0.05 was considered statistically significant. Data are expressed as the means±SD unless otherwise specified. The Chi-square test was used for immunohistochemical analysis.

β₁ integrin expression and activation in human PCa specimens

To assess whether integrin activation occurs in metastatic progression in human PCa, we examined the expression of β₁ integrins in human specimens from normal prostate glands, localized PCa, and lymph node metastases. Immunohistochemical analysis showed that in normal prostate glands, β₁-integrins are expressed on the basal cell layer and localized at the basal cell/stromal interface, where integrins interact with ECM (Fig. 1A). In PCa specimens, where basal cells are absent, β₁ integrins were detected in the membranes in 13 of 20 (65%) primary PCa (Fig. 1A, and Table 1A), suggesting that β₁ integrins were upregulated in PCa cells. In lymph node metastases, β₁ integrins were detected in PCa cells in 13 of 18 (72%) lymph node specimens (Fig. 1A and Table 1A). The difference in expression between the normal prostate and primary PCa or lymph node metastasis is significant (p<0.001 by Chi-square test). However, there was no correlation between expression of β₁ integrins and the Gleason score, and no significant difference in expression of β₁ integrins between primary PCa and lymph node metastasis was observed (Table 1A).

To examine whether integrin activation occurs in PCa progression, human PCa specimens were examined for the autophosphorylation of FAK at Y397, which results from integrin activation (9). Of the 16 PCa specimens that were evaluated, seven showed positive staining for pFAK-Y397 (Fig. 1B and Table 1B). Of the 12 lymph node metastases, seven showed positive staining for pFAK-Y397 (Fig. 1B and Table 1B). Together, these results suggest that activation of β₁ integrins occurs during PCa progression.

β₁ integrin activation in PCa cell lines

To examine whether activation of β₁ integrins plays a role in PCa metastasis, we determined β₁ integrin expression and activation in several available PCa cell lines, including the lymph node derived LNCaP and its castration-resistant variant C4-2B4, as well as the bone-derived PC3 and its metastatic variant PC3-mm2 cells. LNCaP and C4-2B4 are tumorigenic, however, with low metastatic potential when implanted orthotopically or intracardially. PC3 and PC3-mm2 are tumorigenic and highly metastatic, exhibiting high incidence of metastasis in both spontaneous and experimental metastasis models (25, 26). Using anti-β₁ integrin mAb MAR4, FACS analysis showed that all four cell lines expressed high levels of β₁ integrins (Fig. 2A). Next, we examined integrin activation in these cell lines. Western blot showed that the levels of phosphorylation of pFAK397 are higher in PC3 and PC3-mm2 than LNCaP and C4-2B4 (Fig. 2B), suggesting that integrin signaling is activated in PC3 and PC3-mm2 cells. We further used the conformation-sensitive antibody 9EG7 (27–29) to examine whether β₁ integrins in these cells were present in the activated conformation. 9EG7 bound to PC3 and PC3-mm2 strongly (Fig. 2C). In contrast, 9EG7 showed modest binding to LNCaP and C4-2B4 (Fig. 2C). These observations suggest that β₁ integrins are present in an activated conformation only in cell lines with high metastatic potential.

β₁ integrin activation correlates with anoikis resistance in PCa cell lines

Next, we examined whether increased β₁ integrin activation in PCa cells correlated with cell survival by using an anoikis assay. Single cells from PCa cell lines were placed in suspension with constant rotation for 24 and 48 hrs to prevent aggregation-induced outside to inside integrin aggregation and the number of viable and dead cells determined. As shown in Fig. 2D, PC3-mm2 has the highest number of viable cells at 48h, while LNCaP and C4-2B4 cells did not survive in these anoikis conditions. The decreases in viable cell number corresponded to the increases in dead cell number (Fig. 2D). Analysis of nuclear integrity by propidium iodide staining showed that the increased dead cell number in anoikis sensitive cell lines correlated with an increase in the sub-G1 fractions (Fig. 2E), suggesting that these cells had undergone apoptosis. Consistent with this result, western blot analysis showed a correlation between anoikis sensitivity and increases in PARP cleavage (Fig. 2F). These observations indicate that the ability of PCa cells to survive in anoikis conditions correlates with the activation status of β₁ integrins and also the metastatic potential of these cell lines.

Silencing of β1 integrins in PC3-mm2 cells by expression of shRNA abrogates anoikis resistance

To examine the causal effect of β₁ integrins on PCa cell survival, we used stable expression of shRNA to knockdown β₁ integrins in PC3-mm2 cells. As shown in Fig. 3A, transfection of β₁ integrin-specific shRNA reduced the expression of β₁ integrins by more than 90%, while control shRNA did not have significant effects on β₁ integrin expression. To examine the effects on integrin-mediated survival pathways, phosphorylation of FAK397 and AKT473 were examined. Knockdown of integrin greatly reduces pFAK397 and pAKT473 phosphorylation (Fig. 3A). Further, the β₁ integrin knockdown cells in suspension are sensitized to anoikis (Fig. 3B), with a concomitant increase in PARP cleavage (Fig. 3C). These results suggest that β₁ integrins play a critical role in the survival of highly metastatic PC3-mm2 cells.

Effects of mAb 33B6 on β₁ integrin-mediated cell adhesion and migration

We reasoned that PC3 and PC3-mm2 cells have increased inside-to-outside activation of β₁ integrins, leading to increased ECM binding affinity, which increases the adhesion, migration, and survival of the tumor cells during extravasation. Thus, we examined the effects of mAb 33B6, a β₁ integrin-neutralizing antibody, on ECM-mediated adhesion, migration, and apoptosis of PC3-mm2 cells. In a static binding assay, PC3-mm2 adhered to type I collagen or fibronectin coated dishes efficiently compared to non-coated or BSA-coated plates (Fig. 4A) and the adhesion was inhibited by mAb 33B6 but not by control IgG (Fig. 4A). The β₁ integrin-mediated adhesion was further examined under flow conditions using the parallel plate flow detachment assay that mimics tumor cells in circulation. mAb 33B6 led to the detachment of PC3-mm2 cells from collagen or fibronectin-coated surface when compared to control IgG (Fig. 4B). When plated on type I collagen or fibronectin coated plates, PC3-mm2 cells showed a flattened morphology with lamellipodia-like projections, but remained rounded when plated on BSA coated plates (Fig. 4C). The cell spreading on collagen or fibronectin-coated plates was inhibited by mAb 33B6 (Fig. 4C). We further examined the effect of mAb 33B6 on the migration of PC3-mm2 cells. Little migration of PC3-mm2 cells was observed on the control BSA-coated wells, but the migratory activity was significantly stimulated by type 1 collagen or fibronectin (Fig. 4D). The ECM-induced PC3-mm2 migration was significantly inhibited by mAb 33B6 but not by control IgG (P<0.05). In addition, treatment of PC3-mm2 cells with mAb 33B6 led to the inhibition of FAK and AKT phosphorylation (Fig. 4E). This treatment led to an approximate 3-fold increase in annexin V staining and caspase 3 activation compared to IgG-treated control cells (Fig. 4F). Western blotting showed a 2–3 fold increase in PARP cleavage upon treatment with mAb 33B6 (Fig. 4F), suggesting that blocking PC3-mm2 interaction with ECM resulted in an increase in PC3-mm2 cell apoptosis. Because mAb 33B6 induced ~3% apoptosis in PC3-mm2 cells (Fig. 4F) and reduced migration of these cells to ~ 10–20% relative to that of untreated control cells, the effects of mAb 33B6 on PC3-mm2 cell migration are not due to its effect on cell viability. However, ECM inhibition did not affect the proliferation of PC3-mm2 cells on type I collagen, fibronectin or BSA-coated plates (data not shown). mAb 33B6 also did not have significant effect on the proliferation of PC3-mm2 cells in serum-free medium (data not shown). Together, these results demonstrate that mAb 33B6 inhibits ECM-mediated binding, spreading, migration, and survival of PC3-mm2 cells.

Inhibition of PC3-mm2/ECM interaction decreases lymph node metastasis in vivo

Next, we examined the role of PCa/ECM interaction in the ability of PCa cells to metastasize to lymph nodes. Dislodged PCa cells that survive in the circulation usually metastasize first to lymph nodes adjacent to the prostate, then to distant lymph nodes (30, 31). We used the intra-prostatic injection model that mimics spontaneous metastasis in vivo and examined whether inhibition of PC3-mm2/ECM interaction would block the metastasis of PC3-mm2 cells from the prostate to the lymph node. Luciferase-labeled PC3-mm2 cells were implanted intraprostatically into SCID mice and the mice were treated with or without mAb 33B6 twice per week. Tumor growth in the prostate and its metastases in the lymph nodes were monitored using bioluminescence. At 2-weeks post-injection, we observed that PC3-mm2 had metastasized from the prostate to multiple lymph nodes in the control IgG injected mice (Fig. 5A). In contrast, no obvious metastases in distant lymph nodes were detected in mice treated with mAb 33B6 (Fig. 5A). At 3-weeks post-injection, mice were killed and tumors were harvested from the primary (prostate) and metastatic (lymph nodes) sites. In the control IgG-treated mice, PC3-mm2 cells metastasized to both regional and distant lymph nodes, based on bioluminescence imaging and post-mortem tissue harvesting (Fig. 5A). In the mAb 33B6-treated mice, PC3-mm2 cells mainly metastasized to regional lymph nodes (Fig. 5A). Upon postmortem dissection, the presence of tumor cells in the lymph nodes was further confirmed by histology. The incidence of lymph node metastasis from intraprostatic injection of PC3-mm2 cells was 100%. An average of 7.2 ± 2.6 lymph node metastases versus 2.3 ± 1.8 (p < 0.001) was detected in the IgG-treated and mAb 33B6-treated mice, respectively (Fig. 5B). The decreases in tumor metastases to distant lymph nodes in mAb 33B6-treated group are not due to tumor size as one of the mice in mAb 33B6-treated group produced a large tumor but exhibited metastasis only to the local lumbar lymph node (data not shown). In addition, the average tumor sizes were similar between the control and mAb 33B6-treated groups (Fig. 5C). These results indicate that inhibition of interaction of PC3-mm2 cells with the ECM limits the ability of tumor cells to metastasize to distant lymph nodes.

Inhibition of PC3-mm2/ECM interaction on disseminated PC3-mm2 cells in vivo

Next, we examined whether blocking PC3-mm2/ECM interaction by mAb 33B6 affects the metastatic growth of disseminated PC3-mm2 cells. We mimicked the hematogenous dissemination of cancer cells by injecting luciferase-labeled PC3-mm2 cells intracardially. Bioluminescence imaging was used to examine the metastases of PC3-mm2 in vivo. PC3-mm2 cells were considered disseminated into the circulation if bioluminescence signals were detected throughout the whole-body (Fig. 6A). Bioluminescence signals subsided one day post-injection (data not shown) and the signals started to appear at multiple organ sites at 7 days post injection (Fig. 6A). Tumor volumes in the control IgG-treated group exhibited an exponential growth at multiple organ sites at 2- and 3-weeks post injection (Fig. 6A). In contrast, the growth of tumors in the mAb 33B6-treated mice was significantly decreased (Fig. 6A). Quantification of the tumor sizes based on bioluminescence intensity showed no significant difference in tumor volume between the control and mAb 33B6-treated mice at 7 days post tumor injection; however, the average tumor size was significantly smaller at 14 days, with mAb 33B6-treated tumor burden only 7% of that in the control group (p < 0.003). At 20 days, the average tumor burden of anti-β₁ integrin-treated mice was 6% of those of control mice (p < 5 × 10⁻⁵) (Fig. 6B). We also examined the tumor burden in femurs/tibias and found that the tumor sizes were also significantly reduced in the mice treated with mAb 33B6 (Fig. 6C). These in vivo data are consistent with the in vitro data, which together indicate that activation of β₁-integrins increases tumor cell survival and extravasation, resulting in increased tumor metastasis to distant metastatic sites.

β1 integrin isoforms

β1 integrins contain several alternatively spliced variants. For the human β1 subunit, at least five different cytoplasmic variants, i.e. β1_A, β1_B, β1_C, β1_C-2, β1_D have been identified (33). Among the five known β1 integrin cytoplasmic variants, β1_A and β1_C have been shown to differentially affect prostate cell functions (34). The β1_A variant was shown to act as a stimulator of cell proliferation, whereas the β1_C variant acted as an inhibitor of proliferation. Goel et al. (35) further demonstrated that β1_C expression increases the levels of an extracellular matrix protein thrombospondin 1, which inhibits angiogenesis. While β1_A protein is ubiquitously expressed, β1_C protein levels are reduced in neoplastic prostate epithelium (34). PCa cell lines, including PC3, LNCaP and DU145, mainly express β1_A integrins (35). Thus, the β1 integrins activated in PC3-mm2 cells are the β1_A variants.

Inside-out integrin activation

The mechanism(s) by which β1 integrins are activated in PC3 and PC3-mm2 cells are not known. Integrins are normally expressed in an inactive state and can be activated through inside-out or outside-in mechanisms. While the ECM ligand-induced outside-in integrin activation increases the avidity (36, 37), inside-out integrin activation increases its ligand binding affinity, cell adhesion, migration, and ECM assembly (8, 18–20). The activation of β₁ integrins in PC3-mm2 cells is likely through an inside-out mechanism, which induces conformational changes of β₁ integrins in the absence of ECM ligands (17). Diverse mechanisms have been shown to contribute to the inside-out activation, which include interaction of the integrin cytoplasmic domain with its regulatory molecules (20, 38), biochemical modification of the integrin molecule, and regulation by extracellular growth factors (39). Talin binding to the cytoplasmic domain of integrins is one of the mechanisms that leads to inside-out integrin activation (20, 38). Sakamoto et al. (40) showed that talin1 overexpression increased PCa cell adhesion, migration and resistance to anoikis, and talin1 levels were higher in metastatic tissue compared with primary prostate tumors. Talin1 overexpression was also observed in aggressive oral squamous cell carcinomas (41). These observations suggest that increase in talin1 levels may play a role in the inside-out activation of β₁ integrins in PC3-mm2 cells. However, we did not detect differences in talin1 expression among C4-2B4 and PC3-mm2 cells by Western blot (data not shown). Extracellular growth factors have been shown to activate integrins (39). In breast cancer cells, Chandrasekaran et al. (42) showed that insulin-like growth factor-1 and CD98 regulate α3β1 integrin activation. Interestingly, Chatterjee et al. (43) showed that the chemokine CCL2 increased β1-integrin activation in PC3 cells. Both IGF-1 and CCL2 have been shown to be upregulated during PCa progression (44, 45), suggesting that these or other growth factors may regulate activation of β₁ integrins in PC3-mm2 cells. Delineating the mechanisms that lead to inside-out activation of β₁ integrins in PC3-mm2 cells may provide additional strategies for metastasis prevention. Further study on the mechanisms of integrin activation and mechanisms by which β₁ integrins become constitutively activated in highly metastatic PCa cells are warranted.

Targeting activated β1-integrins as anti-metastatic therapy

While our study and several other studies showed that blocking integrin/ECM interactions is effective in inhibiting tumor growth (35, 46, 47) or metastasis (11, 48) in mouse tumor models, ECM proteins are also involved in many cellular activities essential for normal cell functions, raising the possibility of high toxicity with anti-integrin therapy. However, our studies showed that β1 integrins are activated in highly metastatic but not in low metastatic PCa cells or normal prostate epithelial cells. Thus, an anti-β1 antibody that specifically inhibits the “activated conformation” of β1 integrins may increase target specificity in selectively targeting only activated integrins. Consistent with such an idea, a monoclonal antibody that targets a tumor-specific, conformationally exposed epitope of wild-type EGFR (mAb 806) has recently been developed for the purpose of reducing the side-effect from anti-EGFR treatments (49). Whether it is possible to generate such a conformation-specific antibody for therapeutic application remains to be seen. On the other hand, an antibody against α_v integrin (DI17E6) is being tested in a clinical trial for the treatment of progressive castrate-resistant PCa with bone metastasis (50). α_v integrin is expressed in a number of human cancers, including PCa (11), as well as cells in the tumor environment, e.g. osteoclasts, osteoblasts, and angiogenic blood vessels. A preliminary report suggests that DI17E6 was well tolerated (50). Completion of this study will reveal whether blocking tumor cell/ECM interaction is feasible for cancer treatments. Because metastasis is the major cause of cancer mortality, there is a strong interest in developing “anti-metastatic” therapies, especially in PCa, as the disease is often caught early and there is nearly a 10-year “window” during which anti-metastatic therapy would be useful as “secondary prevention.” In addition to anti-integrin antibodies, several “anti-metastatic” therapies are also being developed, including the use of anti N-cadherin (51), anti-cadherin 11 (25) antibodies.

PCa cell/ECM interactions at distant metastatic sites

Besides occurring in circulation, PCa/ECM interactions have been shown to be involved in the progression of cancer in their metastatic sites (4, 52, 53). Type I collagen is the most abundant ECM in bone, raising the possibility that β₁ integrins may play a role in metastatic progression of PCa in bone. Indeed, Kostenuik et al. (54) showed that bone cell matrix promotes the adhesion of human PC3 cells via α₂β₁ integrins. Hall et al. (55) showed that LNCaP cells selected for binding to collagen exhibited increased levels of the α₂β₁ integrins and increased tumorigenesis in bone when the cells were injected into mouse tibia. In this study, we found that mAb 33B6 reduced tumor volume in bone when PC3-mm2 cells were injected intracardially. Whether mAb 33B6 also inhibits PC3-mm2 cell/bone matrix interaction requires further investigation.

Metastatic progression of PCa significantly impacts the survival of men with PCa. Thus, there is an urgent need to identify strategies for the prevention or treatment of metastasis. β₁ integrin antibody has been previously shown to inhibit breast (46) or prostate (35) tumor growth at subcutaneous sites. Our studies provide new evidence that constitutively activated β1 integrins play a role in the metastasis process and our pre-clinical studies suggest that strategies that inhibit activation of β₁ integrins may be developed for preventing PCa metastasis.

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