Isis C. Sroka

The Laminin Binding Integrin α6β1 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 extacellular matrix laminin class of proteins line the neural route and tumor cells escaping from the gland express the laminin binding integrin α6β1 as a prominent cell surface receptor. Integrin α6β1 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. J. Cell. Physiol. 224: 283–288, 2010.

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.

Macrophage-Dependent Cleavage of the Laminin Receptor α6β1 in Prostate Cancer

The laminin-binding integrin α6β1 plays a major role in determining the aggressive phenotype of tumor cells during metastasis. Our previous work has shown that cleavage of the α6β1 integrin to produce the structural variant α6pβ1 on tumor cell surfaces is mediated by the serine protease urokinase plasminogen activator (uPA). Cleavage of α6β1 increases tumor cell motility, invasion, and prostate cancer metastasis, and blockage of uPA inhibits α6pβ1 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 α6pβ1 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. Mol Cancer Res; 9(10); 1319–28. ©2011 AACR.

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

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.

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.

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 α6β4 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 α6β1 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.

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.

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. J. Cell. Biochem. 117: 491–499, 2016.

Abstract 4979: A6B1 integrin internalization is increased by loss of A3B1 expression and promotes migration of human prostate cancer cells

Laminin binding integrins, A3B1 and A6B1 drive in part, cancer cell migration and metastasis. Cell migration requires dynamic internalization and recycling of surface integrins. However, whether laminin binding integrin internalization is coordinately or independently controlled is not known. In this study, we quantitated the internalization of integrin A3 and A6 and whether crosstalk influences their respective internalization and subsequent cancer cell migration. We quantitated the internalization of A6 and A3 integrins, using fluorophore conjugated antibody based internalization assays and flow cytometry. In the prostate cancer cell line DU145, we observed constitutive internalization of integrin A6 with a surface half-life of approximately 30 minutes as compared to >70 minutes for integrin A3, 45 minutes for unrelated integrin Av or 10 minutes for the unrelated transferrin receptor. Silencing A3 integrin expression resulted in a 10 percent increase in the total amount of internalized A6 integrin, with a decreased surface half-life of 20 minutes. The internalized A6 integrin showed intracellular punctate staining colocalized with the early endosome marker EEA1 and Rab4 containing vesicles. Increased integrin A6 internalization led to 40% increase in migration which was dependent on A6B1 integrin. Silencing integrin A6 expression, however, did not affect the internalization of A3 integrin indicating a unidirectional regulation of integrin internalization. The changes in the integrin internalization could be clearly ascribed to alpha subunits, as the obtained half-life of A6 and A3 integrins were independent of antibody used or integrin engagement by ligand mimetic peptides. Taken together, these data indicate that A6B1 and A3B1 integrins have different internalization kinetics and the presence of integrin A3 decreased integrin A6 internalization and ensuing cell migration. These data are consistent with previous observations in normal systems suggesting that A3B1 provides a provisional matrix during early stages of wound healing, requiring subsequent stable adhesion complexes and integrin A6 function. In cancer, the loss of A3 integrin and the increased internalization of A6 integrin may account for the “wounds that won9t heal” phenotype and provide a strategy for biological intervention to block tumor progression. (Supported in part by NIH Grants CA23074 and CA159406 and the TACMASS core service of the Arizona Cancer Center) Citation Format: Lipsa Das, Todd A. Anderson, Jaime M.c. Gard, Isis C. Sroka, Stephanie R. Strautman, Raymond B. Nagle, Beatrice S. Knudsen, Anne Cress. A6B1 integrin internalization is increased by loss of A3B1 expression and promotes migration of human prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2014-4979

Abstract 502: Schwann cells promote tumor cell invasion through regulation of the laminin receptorA6B1

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tumor cell migration and invasion of nerves, a process known as perineural nerve invasion (PNI), is a major metastatic route for neurotropic cancers including prostate and pancreatic cancer. The laminin family of extracellular matrix glycoproteins derived from myelinating Schwann cells coat nerve axons and play a prominent role in the development and maintenance of the peripheral nervous system. Tumor cells undergoing PNI utilize the laminin rich nerves as they migrate and invade during metastasis. Evidence has shown that the A6B1 laminin receptor promotes tumor cell migration and invasion following cleavage of the receptor to form the variant A6pB1 by the serine protease urokinase plasminogen activator (uPA). Here we demonstrate Schwann cell regulation of A6pB1 formation on prostate and pancreatic tumor cell lines. Conditioned medium from the S16 and S16Y cell lines, which are immortalized Schwann cells of the myelinating and non-myelinating phenotype, respectively was provided to the prostate and pancreatic tumor cell lines. It was observed that S16Y conditioned medium inhibited A6 conversion to A6p while the myelinating S16 cells increased cleavage of the receptor in DU-145, CF-PAC1 and PC-3 cancer cells. This effect was reversible as replenishing standard media conditions following a 24 hour treatment with S16 or S16Y media brought integrin cleavage levels back to steady state levels by 24 hours. Further, conditioned medium from S16 cells significantly increased pancreatic tumor cell invasion while S16Y conditioned medium decreased invasion in a modified Boyden chamber Matrigel assay. The increased invasion mediated by myelinating Schwann cells was dependent on A6 since a function blocking antibody targeting A6 inhibited invasion. Taken together these results suggest that myelinating Schwann cells (S16Y) in the pancreatic and prostatic nerve environment promote A6B1 and A6pB1 receptor dependent tumor cell invasion during PNI. (Supported in part by NIH CA23074 and CA159406 and HHMI Grant 52005889) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 502. doi:1538-7445.AM2012-502