Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

Laminin binding integrins α6 (CD49f) and α3 (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, α6 integrin internalized with a rate constant (kactual ) of 3.25 min-1 , threefold faster than α3 integrin (1.0 min-1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min-1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min-1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6ß4 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 α3 integrin increased cell migration by 1.8-fold, which was dependent on α6ß1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc.

Schwann Cells Increase Prostate and Pancreatic Tumor Cell Invasion Using Laminin Binding A6 Integrin.

Human pancreatic and prostate cancers metastasize along nerve axons during perineural invasion. The extracellular matrix laminin class of proteins is an abundant component of both myelinated and non-myelinated nerves. Analysis of human pancreatic and prostate tissue revealed both perineural and endoneural invasion with Schwann cells surrounded or disrupted by tumor, respectively. Tumor and nerve cell co-culture conditions were used to determine if myelinating or non-myelinating Schwann cell (S16 and S16Y, respectively) phenotype was equally likely to promote integrin-dependent cancer cell invasion and migration on laminin. Conditioned medium from S16 cells increased tumor cell (DU145, PC3, and CFPAC1) invasion into laminin approximately 1.3-2.0 fold compared to fetal bovine serum (FBS) treated cells. Integrin function (e.g., ITGA6p formation) increased up to 1.5 fold in prostate (DU145, PC3, RWPE-1) and pancreatic (CFPAC1) cells, and invasion was dependent on ITGA6p formation and ITGB1 as determined by function-blocking antibodies. In contrast, conditioned medium isolated from S16Y cells (non-myelinating phenotype) decreased constitutive levels of ITGA6p in the tumor cells by 50% compared to untreated cells and decreased ITGA6p formation 3.0 fold compared to S16 treated cells. Flow cytometry and western blot analysis revealed loss of ITGA6p formation as reversible and independent of overall loss of ITGA6 expression. These results suggest that the myelinating phenotype of Schwann cells within the tumor microenvironment increased integrin-dependent tumor invasion on laminin.

Combined micro CT and histopathology for evaluation of skeletal metastasis in live animals.

Bone is a favored site for solid tumor metastasis, especially among patients with breast, lung or prostate carcinomas. Micro CT is a powerful and inexpensive tool that can be used to investigate tumor progression in xenograft models of human disease. Many previous studies have relied on terminal analysis of harvested bones to document metastatic tumor activity. The current protocol uses live animals and combines sequential micro CT evaluation of lesion development with matched histopathology at the end of the study. The approach allows for both rapid detection and evaluation of bone lesion progression in live animals. Bone resident tumors are established either by direct (intraosseous) or arterial (intracardiac) injection, and lesion development is evaluated for up to eight weeks. This protocol provides a clinically relevant method for investigating bone metastasis progression and the development of osteotropic therapeutic strategies for the treatment of bone metastases.

Intracellular modifiers of integrin alpha 6p production in aggressive prostate and breast cancer cell lines.

Cancer metastasis is a multi-step process in which tumor cells gain the ability to invade beyond the primary tumor and colonize distant sites. The mechanisms regulating the metastatic process confer changes to cell adhesion receptors including the integrin family of receptors. Our group previously discovered that the α6 integrin (ITGA6/CD49f) is post translationally modified by urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (uPAR), to form the variant ITGA6p. This variant of ITGA6 is a cleaved form of the receptor that lacks the ligand-binding domain. Although it is established that the uPA/uPAR axis drives ITGA6 cleavage, the mechanisms regulating cleavage have not been defined. Intracellular integrin dependent "inside-out" signaling is a major regulator of integrin function and the uPA/uPAR axis. We hypothesized that intracellular signaling molecules play a role in formation of ITGA6p to promote cell migration during cancer metastasis. In order to test our hypothesis, DU145 and PC3B1 prostate cancer and MDA-MB-231 breast cancer cell lines were treated with small interfering RNA targeting actin and the intracellular signaling regulators focal adhesion kinase (FAK), integrin linked kinase (ILK), and paxillin. The results demonstrated that inhibition of actin, FAK, and ILK expression resulted in significantly increased uPAR expression and ITGA6p production. Inhibition of actin increased ITGA6p, although inhibition of paxillin did not affect ITGA6p formation. Taken together, these results suggest that FAK and ILK dependent "inside-out" signaling, and actin dynamics regulate extracellular production of ITGA6p and the aggressive phenotype.

Macrophage-dependent cleavage of the laminin receptor α6ß1 in prostate cancer.

The laminin-binding integrin α6ß1 plays a major role in determining the aggressive phenotype of tumor cells during metastasis. Our previous work has shown that cleavage of the α6ß1 integrin to produce the structural variant α6pß1 on tumor cell surfaces is mediated by the serine protease urokinase plasminogen activator (uPA). Cleavage of α6ß1 increases tumor cell motility, invasion, and prostate cancer metastasis, and blockage of uPA inhibits α6pß1 production. In human tumors, uPA and uPAR are expressed in tumor cells and tumor-associated macrophages (TAM). TAMs localize to solid tumors and contribute to increased tumor growth and the metastatic phenotype. In this study, we utilized a coculture system of PC-3 prostate tumor cells and macrophages [12-O-tetradecanoylphorbol-13-acetate (TPA)-differentiated human leukemia HL-60 cells] to investigate the hypothesis that macrophages stimulate the production of the prometastatic variant α6pß1 on human prostate cancer cells via the uPA/uPAR axis. Our results indicate that adherent macrophages cocultured with PC-3 cells increased PC-3 uPAR mRNA, uPAR cell surface protein expression and α6 integrin cleavage. The stimulation does not require macrophage/tumor cell contact because macrophage conditioned medium is sufficient for increased uPAR transcription and α6 cleavage-dependent PC-3 cell invasion. The increased cleavage was dependent on uPAR because production was blocked by silencing RNA-targeting uPAR. These results indicate that macrophages can stimulate uPA/uPAR production in tumor cells which results in α6 integrin cleavage. These data suggest that TAMs promote prometastatic integrin-dependent pericellular proteolysis.

The laminin binding integrin alpha6beta1 in prostate cancer perineural invasion.

Metastasizing prostate tumor cells invade along nerves innervating the encapsulated human prostate gland in a process known as perineural invasion. The extracellular matrix laminin class of proteins line the neural route and tumor cells escaping from the gland express the laminin binding integrin alpha6beta1 as a prominent cell surface receptor. Integrin alpha6beta1 promotes aggressive disease and supports prostate tumor cell metastasis to bone. Laminins and their integrin receptors are necessary for the development and maintenance of the peripheral nervous system, indicating the potential role for integrin receptors in directing prostate tumor cell invasion on nerves during perineural invasion.

Human Cell Surface Receptors as Molecular Imaging Candidates for Metastatic Prostate Cancer.

Existing clinical imaging procedures lack sensitivity and specificity in detecting early prostate cancer bone metastatic lesions. In this study, we developed a highly reproducible bone metastasis xenograft model and identified possible molecular imaging candidates for detecting early bone metastatic lesions. Bone trophic human prostate cells (PC-3B1) were isolated and characterized for their ability to reach bone after intracardiac injection into SCID mice. The appearances of skeletal metastases were evaluated using digital radiographic imaging and confirmed by necropsy and histology. The PC-3B1 cells retain a bone homing phenotype after long term propagation in tissue culture and exhibit progressive bone lesions within 3 weeks following intracardiac injection. Comparative transcription signatures of PC-3 and PC-3B1 cells were determined using a cancer specific microarray and confirmed by RT-PCR analysis. The analysis identified increased expression of four cell surface molecules in PC-3B1 cells that may be suitable as molecular imaging candidates to detect bone micro metastases.

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.

Simplified purification procedure of laminin-332 and laminin-511 from human cell lines.

Laminins are glycoproteins expressed in the basement membrane of multiple epithelial tissues. Previously described purification procedures for the human laminin variants laminin-5 (LN-332) and laminin-10 (LN-511) use tissue as starting material and have multiple steps. We demonstrate a two-step laminin immunoaffinity purification method to produce consistent quantities of intact and biologically active LN-332 and LN-511 from human keratinocyte (HaCaT) and human lung carcinoma (A549) cell lines, respectively. The purification of LN-332 and LN-551 was demonstrated by PAGE analysis, silver staining and Western blot analysis. The purification procedure includes instruction on removing a cell adhesion contaminant known as galectin-3 binding protein from purified LN-511. The biological activity of purified laminin was tested in a standard cell adhesion assay and compared to commercially available LN-111. This rapid and reproducible purification method will contribute to understanding the role of LN-332 and LN-511 in cell behavior, signaling, and gene expression.

Differential localization of MT1-MMP in human prostate cancer tissue: role of IGF-1R in MT1-MMP expression.

BACKGROUND: MT1-MMP is a metalloproteinase involved in prostate cancer metastasis. The IGF-1R is a tyrosine kinase receptor involved with tumor progression and metastasis. The purpose of this investigation was to examine MT1-MMP and IGF-1R expression and localization in prostate cancer tissues and explore the role of IGF-1R in regulating MT1-MMP in prostate cancer cell lines. METHODS: Immunohistochemistry was utilized to study MT1-MMP and IGF-1R expression in human prostate tissues. IGF-1R regulation of MT1-MMP expression was determined by gene promoter analysis, quantitative RT-PCR and Western blot analysis following pharmacological inhibition of the receptor in PC-3N cells and treatment of LNCaP cells with androgen and IGF-1. RESULTS: MT1-MMP expression was high in the apical regions of the luminal cells in PIN and prostate cancer and less intense in the basalateral regions of benign tissues. IGF-1R was expressed primarily in the basal cells of normal glands and highly expressed in prostate cancer. Inhibition of IGF-1R in PC-3N cells decreased MT1-MMP expression and treatment of LNCaP cells with a synthetic androgen and IGF-1 increased MT1-MMP expression. CONCLUSIONS: These data demonstrate that MT1-MMP is highly expressed in the apical cytoplasmic regions of the luminal cells in PIN and prostate cancer when compared to basalateral cytoplasmic membrane staining in benign glands. Additionally, we demonstrate that IGF-1R is highly expressed in human prostate carcinoma. These findings suggest that MT1-MMP localization and IGF-1R expression in prostate carcinoma could be predictive biomarkers for aggressive disease and support IGF-1R as a promising therapeutic target to decrease processes of prostate cancer metastasis.

Membrane-type 1 matrix metalloproteinase 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 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.