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

Immunohistochemical localization of nerve growth factor, tropomyosin receptor kinase A, and p75 in the bone and articular cartilage of the mouse femur

Sequestration of nerve growth factor (NGF) significantly attenuates skeletal pain in both animals and humans. However, relatively little is known about the specific cell types that express NGF or its cognate receptors tropomyosin receptor kinase A (TrkA) and p75 in the intact bone and articular cartilage. In the present study, antibodies raised against NGF, TrkA, and p75 (also known as CD271) were used to explore the expression of these antigens in the non-decalcified young mouse femur. In general, all three antigens displayed a remarkably restricted expression in bone and cartilage with less than 2% of all DAPI+ cells in the femur displaying expression of any one of the three antigens. Robust NGF immunoreactivity was found in mostly CD-31- blood vessel-associated cells, a small subset of CD-31+ endothelial cells, an unidentified group of cells located at the subchondral bone/articular cartilage interface, and a few isolated, single cells in the bone marrow. In contrast, p75 and TrkA were almost exclusively expressed by nerve fibers located nearby NGF+ blood vessels. The only non-neuronal expression of either p75 or TrkA in the femur was the expression of p75 by a subset of cells located in the deep and middle zone of the articular cartilage. Understanding the factors that tightly regulate the basal level of expression in normal bone and how the expression of NGF, TrkA, and p75 change in injury, disease, and aging may provide insights into novel therapies that can reduce skeletal pain and improve skeletal health.NIH [CA154550, CA157449, NS023970]Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Mice with cancer-induced bone pain show a marked decline in day/night activity

Abstract. Introduction:. Cancer-induced bone pain (CIBP) is the most common type of pain with cancer. In humans, this pain can be difficult to control and highly disabling. A major problem with CIBP in humans is that it increases on weight-bearing and/or movement of a tumor-bearing bone limiting the activity and functional status of the patient. Currently, there is less data concerning whether similar negative changes in activity occur in rodent models of CIBP. Objectives:. To determine whether there are marked changes in activity in a rodent model of CIBP and compare this to changes in skin hypersensitivity. Methods:. Osteosarcoma cells were injected and confined to 1 femur of the adult male mouse. Every 7 days, spontaneous horizontal and vertical activities were assessed over a 20-hour day and night period using automated activity boxes. Mechanical hypersensitivity of the hind paw skin was assessed using von Frey testing. Results:. As the tumor cells grew within the femur, there was a significant decline in horizontal and vertical activity during the times of the day/night when the mice are normally most active. Mice also developed significant hypersensitivity in the skin of the hind paw in the tumor-bearing limb. Conclusion:. Even when the tumor is confined to a single load-bearing bone, CIBP drives a significant loss of activity, which increases with disease progression. Understanding the mechanisms that drive this reduction in activity may allow the development of therapies that allow CIBP patients to better maintain their activity and functional status

Targeting 5-HT receptors and Kv7 channels in PFC to attenuate chronic neuropathic pain in rats using a spared nerve injury model

Chronic pain remains a disabling disease with limited therapeutic options. Pyramidal neurons in the prefrontal cortex (PFC) express excitatory Gq-coupled 5-HT2A receptors (5-HT2AR) and their effector system, the inhibitory Kv7 ion channel. While recent publications show these cells innervate brainstem regions important for regulating pain, the cellular mechanisms underlying the transition to chronic pain are not well understood. The present study examined whether local blockade of 5-HT2AR or enhanced Kv7 ion channel activity in the PFC would attenuate mechanical allodynia associated with spared nerve injury (SNI) in rats. Following SNI, we show that inhibition of PFC 5-HT2ARs with M100907 or opening of PFC Kv7 channels with retigabine reduced mechanical allodynia. Parallel proteomic and RNAScope experiments evaluated 5-HT2AR/Kv7 channel protein and mRNA. Our results support the role of 5-HT2ARs and Kv7 channels in the PFC in the maintenance of chronic pain.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

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