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

Membrane-Type 1 Matrix Metal loproteinase Is Regulated by Sp1 through the Differential Activation of AKT, JNK, and ERK Pathways in Human Prostate Tumor Cells

We and other investigators have previously shown that membrane-type 1 matrix metalloproteinase (MT1-MMP) is overexpressed in invasive prostate cancer cells. However, the mechanism for this expression is not known. Here, we show that MT1-MMP is minimally expressed in nonmalignant primary prostate cells, moderately expressed in DU-145 cells, and highly expressed in invasive PC-3 and PC-3N cells. Using human MT1-MMP promoter reporter plasmids and mobility shift assays, we show that Spi regulates MT1-MMP expression in DU-145, PC-3, and PC-3N cells and in PC3-N cells using chromatin immunoprecipitation analysis and silencing RNA. Investigation of signaling pathway showed that DU-145 cells express constitutively phosphorylated extracellular stress-regulated kinase (ERK), whereas PC-3 and PC-3N cells express constitutively phosphorylated AKT/PKB and c-Jun NH2 terminal kinase (JNK). We show that MT1-MMP and Spi levels are decreased in PC-3 and PC-3N cells when phosphatidylinositol-3 kinase and JNK are inhibited, and that MT1-MMP levels are decreased in DU-145 cells when MEK is inhibited. Transient transfection of PC-3 and PC-3N cells with a dominant-negative JNK or p85, and of DU-145 cells with a dominant negative ERK, reduces MT1-MMP promoter activity. These results indicate differential signaling control of Spi-mediated transcriptional regulation of MT1-MMP in prostate cancer cell lines

Membrane-Type 1 Matrix Metalloproteinase Is Regulated by Sp1 through the Differential Activation of AKT, JNK, and ERK Pathways in Human Prostate Tumor Cells1

We and other investigators have previously shown that membrane-type 1 matrix metalloproteinase (MT1-MMP) is overexpressed in invasive prostate cancer cells. However, the mechanism for this expression is not known. Here, we show that MT1-MMP is minimally expressed in nonmalignant primary prostate cells, moderately expressed in DU-145 cells, and highly expressed in invasive PC-3 and PC-3N cells. Using human MT1-MMP promoter reporter plasmids and mobility shift assays, we show that Sp1 regulates MT1-MMP expression in DU-145, PC-3, and PC-3N cells and in PC3-N cells using chromatin immunoprecipitation analysis and silencing RNA. Investigation of signaling pathway showed that DU-145 cells express constitutively phosphorylated extracellular stress-regulated kinase (ERK), whereas PC-3 and PC-3N cells express constitutively phosphorylated AKT/PKB and c-Jun NH2 terminal kinase (JNK). We show that MT1-MMP and Sp1 levels are decreased in PC-3 and PC-3N cells when phosphatidylinositol-3 kinase and JNK are inhibited, and that MT1-MMP levels are decreased in DU-145 cells when MEK is inhibited. Transient transfection of PC-3 and PC-3N cells with a dominant-negative JNK or p85, and of DU-145 cells with a dominant negative ERK, reduces MT1-MMP promoter activity. These results indicate differential signaling control of Sp1-mediated transcriptional regulation of MT1-MMP in prostate cancer cell lines

Schwann Cells Promote Tumor Cell Invasion Through Regulation of the Laminin Receptor α6β1

Neurotropic cancers including pancreatic and prostate cancer utilize nerves as a major route of metastasis in a process known as nerve invasion (NI). During NI, tumor cells invade the perineurium of nerves and into the endoneural space where tumor cells contact Schwann cells responsible for secretion of laminin extracellular matrix proteins and myelination of peripheral nerve axons. The laminin binding cell adhesion receptor α6β1 is expressed on tumor cells undergoing NI and cleavage of α6β1 by urokinase plasminogen activator to form the variant α6β1 promotes tumor cell invasion. Here, we demonstrate that conditioned media from immortalized S16 and S16Y Schwann cells, which are of the myelinating and non-myelinating morphology respectively, regulate α6pβ1 formation and tumor invasion of neurotropic tumor cell lines. The results demonstrate that S16Y conditioned medium inhibited invasion and 6 conversion to α6p in DU145, CFPAC1 and PC3 cancer cells while S16 conditioned medium increased α6p expression and tumor cell invasion. The increased invasion mediated by the S16 cells was dependent on α6pβ1 since a function-blocking antibody, targeting α6pβ1 formation inhibited invasion. Taken together these results suggest that myelinating Schwann cells in the nerve environment promote α6β1 receptor dependent tumor cell nerve invasion

Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells

Laminin binding integrins 6 (CD49f) and 3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, 6 integrin internalized with a rate constant (k(actual)) of 3.25min(-1), threefold faster than 3 integrin (1.0min(-1)), 1.5-fold faster than the vitronectin binding v integrin (CD51) (2.2min(-1)), and significantly slower than the unrelated transferrin receptor (CD71) (15min(-1)). Silencing of 3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in k(actual) for 6 integrin. The internalized 6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of 3 integrin expression resulted in redistribution of 64 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of 3 integrin increased cell migration by 1.8-fold, which was dependent on 61 integrin. Silencing of 6 integrin expression however, had no significant effect on the k(actual) of 3 integrin or its distribution in early endosomes. These results indicate that 3 and 6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017.NIH [R01 CA 159406, CA 23074, CA 09213]12 month embargo; Accepted manuscript online: 10 August 2016This 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]

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

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