The Role of Alpha 6 Integrin in Prostate Cancer Migration and Bone Pain in a Novel Xenograft Model

Of the estimated 565,650 people in the U.S. who will die of cancer in 2008, almost all will have metastasis. Breast, prostate, kidney, thyroid and lung cancers metastasize to the bone. Tumor cells reside within the bone using integrin type cell adhesion receptors and elicit incapacitating bone pain and fractures. In particular, metastatic human prostate tumors express and cleave the integrin A6, a receptor for extracellular matrix components of the bone, i.e., laminin 332 and laminin 511. More than 50% of all prostate cancer patients develop severe bone pain during their remaining lifetime. One major goal is to prevent or delay cancer induced bone pain. We used a novel xenograft mouse model to directly determine if bone pain could be prevented by blocking the known cleavage of the A6 integrin adhesion receptor. Human tumor cells expressing either the wildtype or mutated A6 integrin were placed within the living bone matrix and 21 days later, integrin expression was confirmed by RT-PCR, radiographs were collected and behavioral measurements of spontaneous and evoked pain performed. All animals independent of integrin status had indistinguishable tumor burden and developed bone loss 21 days after surgery. A comparison of animals containing the wild type or mutated integrin revealed that tumor cells expressing the mutated integrin resulted in a dramatic decrease in bone loss, unicortical or bicortical fractures and a decrease in the ability of tumor cells to reach the epiphyseal plate of the bone. Further, tumor cells within the bone expressing the integrin mutation prevented cancer induced spontaneous flinching, tactile allodynia, and movement evoked pain. Preventing A6 integrin cleavage on the prostate tumor cell surface decreased the migration of tumor cells within the bone and the onset and degree of bone pain and fractures. These results suggest that strategies for blocking the cleavage of the adhesion receptors on the tumor cell surface can significantly prevent cancer induced bone pain and slow disease progression within the bone. Since integrin cleavage is mediated by Urokinase-type Plasminogen Activator (uPA), further work is warranted to test the efficacy of uPA inhibitors for prevention or delay of cancer induced bone pain

Integrin clipping: A novel adhesion switch

We previously identified a novel structural variant of the alpha6 integrin called alpha6p. This variant was produced on the cell surface and was missing the β-barrel extracellular domain. Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen Activator (uPA) function blocking antibody (3689) we showed that uPA, acting as a protease, is responsible for production of α6p. We also showed that addition of uPA in the culture media of cells that do not produce α6p, resulted in a dose dependent α6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using α2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the alpha6 integrin directly. Further, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of alpha6p, and this induction was abolished by PAI-1 but not α2-antiplasmin. Using site directed mutagenesis we have identified the site of cleavage to be at arginines 594 and 595. We have also shown that while a fraction of α6 integrin is normally associated with CD151, the α6p form is not. In order to determine whether α6 integrin clipping occurs in tissue, we have found that α6p is present in human prostate cancer tissue, in normal mouse epidermis, in mouse papillomas and squamous cell carcinomas induced by DMBA, TPA and MNNG treatments and in mouse melanomas induced by activated ras. Interestingly, subcutaneous injection into athymic nude mice of a malignant mouse keratinocyte derived cell line (6M90) that is α6p negative, results in the development of tumors that contain α6p integrin. Furthermore, we have shown that PC3N cells transfected with an uncleavable mutant of the α6 integrin grew smaller tumors when injected subcutaneously in SCID mice compared to wildtype α6 transfected cells. In addition, the tumors from the uncleavable mutant alpha6 transfected PC3N cells had higher levels of activated caspase 3 indicating higher levels of apoptosis. This finding suggests that the α6 integrin clipping is important for integrin signaling for survival. Collectively, all these data suggest that the cell surface clipping of the α6 integrin is a novel mechanism for altering integrin-laminin interactions during skin tissue remodeling and carcinogenesis

Histological examination of bone destruction, tumor cell distribution and verification of mutated integrin expression after injection of prostate tumor cells.

<p>(A) Hematoxylin-eosin staining of the normal bone (control) or bone injected with PC3N-A6-WT cells (WT, middle panel and inset) and PC3N-A6-RR cells (RR, bottom panel and inset). The growth plate of the bone (epiphyseal plate) is oriented at the top left of each panel for comparison purposes. (B) RT-PCR analysis to detect expression of the mutated 6 integrin in the bone marrow. Twenty one days following injection, bone marrow was harvested, RNA was extracted and analyzed. RNA from PC3N-A6-WT cells and PC3N-A6-RR cells growing in tissue culture was compared to the bone marrow isolated from mice injected with PC3N-A6-WT cells (Bone marrow-WT cells) or PC3NA6- RR cells (bone marrow-RR cells). GAPDH amplification was carried out as control and the kB markers are as shown.</p

Development of cancer-induced spontaneous pain, tactile allodynia, and movement-evoked pain in animals 21 days after surgery.

<p>(A) Spontaneous pain as measured by flinching of the ipsilateral hindlimb was determined in sham injected animals (control) or those injected with tumor cells expressing the wild type integrin (PC3N-A6-WT) or those injected with tumor cells expressing the mutated integrin (PC3N-A6-RR). Elevation in flinching behavior is indicative of an increased pain response. (B) Tactile allodynia as measured by paw-withdrawal from von Frey filaments in sham injected animals (Control) or those injected with tumor cells expressing the wild type integrin (PC3N-A6-WT) or those injected with tumor cells expressing the mutated integrin (PC3N-A6-RR). A decrease in the withdrawal threshold is indicative of an increased pain response. (C) Movement evoked pain was observed in sham injected animals (control) or those injected with tumor cells expressing the wild type integrin (PC3N-A6-WT) or those injected with tumor cells expressing the mutated integrin (PC3N-A6-RR).</p

Biochemical and migration phenotype of PC3N-A6-WT and PC3N-A6- RR cells expressing the wildtype(cleavable) and RR(uncleavable) integrin A6, respectively.

<p>(A) The expression of the full length 6 integrin ( 6) and uPA dependent production of the 6p variant ( 6p) was determined by western blot analysis. Integrin status within total cell lysates from doxycycline (Dox) or urokinase (uPA) untreated (−) and treated (+) PC3N-A6-WT and PC3N-A6-RR cell lines was determined. (B) Surface expression of wild type and mutated integrin 6 in doxycycline induced PC3N-A6-WT and PC3N-A6-RR cells was determined by flow cytometry. PC3N-A6-WT and PC3NA6- RR cells were incubated with primary Rat anti-integrin A6 antibody J1B5 followed by Alexa 488 anti-rat antibody and visualized using the BD FACScan. The grey peak indicates fluorescence signal from secondary antibody only. (C) Schematic to illustrate the cleavage of the full length form of the 6 integrin to yield the 6p variant. The definition of the PC3N-A6-WT and PC3N-A6-RR cells with regard to integrin status is shown. (D) Integrin mediated migration of PC3N-A6-WT cells (top panels) and PC3NA6- RR cells (bottom panels) on matrigel. The cells were placed on matrigel in the presence of a coverslip to create a cell free zone on the matrigel surface. After cell adhesion was complete, the coverslips were removed from the matrigel surface to allow migration into the cell free zone indicated by white or black curved line. Cells migration occurred under optimal growth conditions at 37 for approximately 18 hours. Cells were either allowed to migrate in the absence (left panels) or presence (right panels) of integrin blocking antibody AIIB2. Images were collected using a Zeiss Axiovert microscope equipped with a 2.5X objective.</p