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Human prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding α6β1 integrin was required for smooth muscle invasion both in vitro and in vivo . Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of α6 integrin and E-cadherin in a cell-cell location and α6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing α6 integrin (DU145 α6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo . In contrast, cells without α6 integrin (DU145 α6KO) and cells expressing an integrin mutant (DU145 α6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo , and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 α6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 α6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1 μmol/L S1P challenge as compared with the DU145 α6WT cells. The results suggest that gene editing of a specific α6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. SIGNIFICANCE: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific α6 integrin extracellular region., (©2019 American Association for Cancer Research.)

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 (k actual ) 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 k actual 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 k actual 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., (© 2016 Wiley Periodicals, Inc.)

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., (© 2015 Wiley Periodicals, Inc.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Laminin-binding integrin receptors are key mediators of epithelial cell migration and tumor metastasis. Recent studies have demonstrated a role for the α6 integrin (ITGA6/CD49f) in maintaining stem cell compartments within normal bone marrow and in residency of tumors metastatic to bone. In this study, we tested a function-blocking antibody specific for ITGA6, called J8H, to determine if preexisting cancer lesions in bone could be slowed and/or animal survival improved. Human prostate tumors were established by intracardiac injection into male SCID mice and treatment with J8H antibody was initiated after 1 week. Tumor progression was monitored by micro-computed tomography (CT) imaging of skeletal lesions. Animals that received weekly injections of the anti-ITGA6 antibody showed radiographic progression in only 40% of osseous tumors (femur or tibia), compared with control animals, where 80% of the lesions (femur or tibia) showed progression at 5 weeks. Kaplan-Meier survival analysis demonstrated a significant survival advantage for J8H-treated animals. Unexpectedly, CT image analysis revealed an increased proportion of bone lesions displaying a sclerotic rim of new bone formation, encapsulating the arrested lytic lesions in animals that received the anti-ITGA6 antibody treatment. Histopathology of the sclerotic lesions demonstrated well-circumscribed tumor within bone, surrounded by fibrosis. These data suggest that systemic targeting of the ITGA6-dependent function of established tumors in bone may offer a noncytotoxic approach to arrest the osteolytic progression of metastatic prostate cancer, thereby providing a new therapeutic strategy for advanced disease., (©2014 American Association for Cancer Research.)

Expression of alpha(6) integrin, a laminin receptor, on tumor cell surfaces is associated with reduced patient survival and increased metastasis in a variety of tumors. In prostate cancer, tumor extracapsular escape occurs in part via laminin-coated nerves and vascular dissemination, resulting in clinically significant bone metastases. We previously identified a novel form of alpha(6) integrin, called alpha(6)p, generated by urokinase-type plasminogen activator-dependent cleavage of the laminin-binding domain from the tumor cell surface. Cleavage increased laminin-dependent migration. Currently, we used the known conformation sensitivity of integrin function to determine if engagement of the extracellular domain inhibited integrin cleavage and the extravasation step of metastasis. We show that alpha(6) integrin was present on prostate carcinoma escaping the gland via nerves. Both endogenous and inducible levels of alpha(6)p were inhibited by engaging the extracellular domain of alpha(6) with monoclonal antibody J8H. J8H inhibited tumor cell invasion through Matrigel. A severe combined immunodeficient mouse model of extravasation and bone metastasis produced detectable, progressive osteolytic lesions within 3 weeks of intracardiac injections. Injection of tumor cells, pretreated with J8H, delayed the appearance of metastases. Validation of the alpha(6) cleavage effect on extravasation was confirmed through a genetic approach using tumor cells transfected with uncleavable alpha(6) integrin. Uncleavable alpha(6) integrin significantly delayed the onset and progression of osseous metastases out to six weeks post-injection. The results suggest that alpha(6) integrin cleavage permits extravasation of human prostate cancer cells from circulation to bone and can be manipulated to prevent metastasis.

The alpha6 integrin is essential for early nervous system development in Xenopus laevis. We have previously reported a uPA cleaved form of integrin alpha6 (alpha6p), in invasive human prostate cancer tissue, whose presence correlates with increased migration and invasive capacity. We now report that alpha6 is cleaved during the normal development of Xenopus in a spatially and temporally controlled manner. In addition, unlike normal mammalian tissues, which lack alpha6p, the major form of the alpha6 integrin present in adult Xenopus is alpha6p. The protease responsible for the cleavage in mammals, uPA, is not involved in the cleavage of Xenopus alpha6. Finally, overexpression of a mammalian alpha6 mutant which cannot be cleaved leads to developmental abnormalities suggesting a potential role for the cleavage in development.

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.

Purpose: The goal was to determine if prostate tumor cells containing a mutant alpha6 integrin would be defective in tumor re-population following clinically relevant fractionated ionizing radiation (IR) treatments., Material and Methods: Human prostate cancer cells derived from PC3N cells were used which conditionally expressed a cleavable, wild type form of alpha6 integrin (PC3N-alpha6-WT) or a mutated non-cleavable form of alpha6 integrin (PC3N-alpha6-RR). The resulting tumor growth before, during and after fractionated doses of IR (3 Gyx10 days) was analyzed using the endpoints of tumor growth inhibition (T/C), tumor growth delay (T-C), tumor doubling time (Td) and tumor cell kill (Log(10) cell kill)., Results: The T/C values were 36.1% and 39.5%, the T-C values were 20.5 days and 28.5 days and the Td values were 5.5 and 10.5 days for the irradiated PC3N-alpha6-WT and PC3N-alpha6-RR cells, respectively. The Log(10) was 1.1 for the PC3N-alpha6-WT cells and 0.8 for the PC3N-alpha6-RR cells. The tumor response to IR was altered in tumors expressing the mutant alpha6 integrin as indicated by a significant increase in tumor growth inhibition, an increase in tumor growth delay, an increase in tumor doubling time and an increase in tumor cell kill., Conclusions: Blocking integrin cleavage in vivo may be efficacious for increasing the IR responsiveness of slow growing, pro-metastatic human prostate cancer.

Integrins play a major role in cell adhesion and migration. Previous work reported that a cleaved form of integrin alpha6 (alpha6p) was detected in invasive human prostate cancer tissue, absent in normal prostate tissue and was produced by urokinase-type Plasminogen Activator (uPA) in a plasmin-independent manner. Using site-directed mutagenesis we identified amino acid residues R594 and R595, located in the "stalk" region of integrin alpha6, as essential for cleavage. The cleavage site is located on the extracellular region of the protein between the beta-barrel domain and the thigh domain. Prostate cancer cells (PC3N) were stably transfected to overexpress the cleavable, wild-type (PC3N-alpha6-WT) or the non-cleavable form of integrin alpha6 (PC3N-alpha6-RR). The number of cells invading laminin 111- and laminin 332-coated filters by PC3N-alpha6-WT cells increased by threefold as compared to PC3N-alpha6-RR cells. Plasminogen activator inhibitor-1 (PAI-1) reduced the invasion of PC3N-alpha6-WT cells by approximately 42% through laminin 332-coated filters and plasmin inhibitor aprotinin had no significant effect. Linear cell migration increased production of integrin alpha6p in the PC3N-alpha6-WT cells and not in the PC3N-alpha6-RR cells and 32% of the PC3N-alpha6-WT cells migrated on laminin 111 in the linear migration assay as compared to the 5% PC3N-alpha6-RR cells. These data taken together suggest that the uPA-mediated cell surface cleavage of the alpha6 integrin extracellular domain is involved in tumor cell invasion and migration on laminin.

During human prostate cancer progression, the integrin alpha6beta1 (laminin receptor) is expressed on the cancer cell surface during invasion and in lymph node metastases. 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 beta-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 alpha6p. We also showed that addition of uPA in the culture media of cells that do not produce alpha6p, resulted in a dose-dependent alpha6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using alpha2-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 alpha2-antiplasmin. Finally, the alpha6p integrin variant was detected in invasive human prostate carcinoma tissue indicating that this is not a tissue culture phenomenon. These data, taken together, suggest that this is a novel function of uPA, that is, to remove the beta-barrel ligand-binding domain of the integrin while preserving its heterodimer association.

During human prostate cancer progression, the majority of normally expressed integrins are suppressed with the exception of the alpha6, alpha3, and beta1 integrins. We have shown that in prostate cancer, the alpha6 integrin is found paired with the beta1 integrin and that a novel form of the alpha6 integrin that lacks a large portion of the extracellular domain (alpha6p) exists. The alpha6pbeta1 integrin is found in human prostate cancer tissue specimens as well as tissue culture cell lines and is formed on the cell surface. This review discusses the mechanism of alpha6pbeta1 production and the potential functions of this integrin variant. Our current working model predicts that the alpha6pbeta1 integrin maintains the intracellular cytoskeletal connections associated with the heterodimer while allowing for an alteration in cell adhesion. The mechanism provides a selective advantage for cancer cell metastasis., (Copyright 2003 Wiley-Liss, Inc.)