BMP-7 inhibits TGF-β-induced invasion of breast cancer cells through inhibition of integrin β3 expression

BACKGROUND The transforming growth factor (TGF)-β superfamily comprises cytokines such as TGF-β and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-β are associated with poor outcome, whereas inhibition of TGF-β-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells. METHODS In this study, we investigated the effect of BMP-7 on TGF-β-induced invasion in a 3 dimensional invasion assay. RESULTS BMP-7 inhibited TGF-β-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-β-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-β-induced expression of integrin α(v)β(3) in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin β(3) negatively affected TGF-β-induced invasion. On the other hand, overexpression of integrin β(3) counteracted the inhibitory effect of BMP7 on TGF-β-induced invasion. CONCLUSION Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-β-induced expression of integrin β(3).Prostatic carcinom

Anterolateral Ligament Expert Group consensus paper on the management of internal rotation and instability of the anterior cruciate ligament - deficient knee

Purpose of this paper is to provide an overview of the latest research on the anterolateral ligament (ALL) and present the consensus of the ALL Expert Group on the anatomy, radiographic landmarks, biomechanics, clinical and radiographic diagnosis, lesion classification, surgical technique and clinical outcomes. A consensus on controversial subjects surrounding the ALL and anterolateral knee instability has been established based on the opinion of experts, the latest publications on the subject and an exchange of experiences during the ALL Experts Meeting (November 2015, Lyon, France). The ALL is found deep to the iliotibial band. The femoral origin is just posterior and proximal to the lateral epicondyle; the tibial attachment is 21.6 mm posterior to Gerdy's tubercle and 4-10 mm below the tibial joint line. On a lateral radiographic view the femoral origin is located in the postero-inferior quadrant and the tibial attachment is close to the centre of the proximal tibial plateau. Favourable isometry of an ALL reconstruction is seen when the femoral position is proximal and posterior to the lateral epicondyle, with the ALL being tight upon extension and lax upon flexion. The ALL can be visualised on ultrasound, or on T2-weighted coronal MRI scans with proton density fat-suppressed evaluation. The ALL injury is associated with a Segond fracture, and often occurs in conjunction with acute anterior cruciate ligament (ACL) injury. Recognition and repair of the ALL lesions should be considered to improve the control of rotational stability provided by ACL reconstruction. For high-risk patients, a combined ACL and ALL reconstruction improves rotational control and reduces the rate of re-rupture, without increased postoperative complication rates compared to ACL-only reconstruction. In conclusion this paper provides a contemporary consensus on all studied features of the ALL. The findings warrant future research in order to further test these early observations, with the ultimate goal of improving the long-term outcomes of ACL-injured patients. Level of evidence Level V-Expert opinion

Targeting of αv-Integrins in Stem/Progenitor Cells and Supportive Microenvironment Impairs Bone Metastasis in Human Prostate Cancer12

Acquisition of an invasive phenotype by cancer cells is a requirement for bone metastasis. Transformed epithelial cells can switch to a motile, mesenchymal phenotype by epithelial-mesenchymal transition (EMT). Recently, it has been shown that EMT is functionally linked to prostate cancer stem cells, which are not only critically involved in prostate cancer maintenance but also in bone metastasis. We showed that treatment with the non-peptide αv-integrin antagonist GLPG0187 dose-dependently increased the E-cadherin/vimentin ratio, rendering the cells a more epithelial, sessile phenotype. In addition, GLPG0187 dose-dependently diminished the size of the aldehyde dehydrogenase high subpopulation of prostate cancer cells, suggesting that αv-integrin plays an important role in maintaining the prostate cancer stem/progenitor pool. Our data show that GLPG0187 is a potent inhibitor of osteoclastic bone resorption and angiogenesis in vitro and in vivo. Real-time bioluminescent imaging in preclinical models of prostate cancer demonstrated that blocking αv-integrins by GLPG0187 markedly reduced their metastatic tumor growth according to preventive and curative protocols. Bone tumor burden was significantly lower in the preventive protocol. In addition, the number of bone metastases/mouse was significantly inhibited. In the curative protocol, the progression of bone metastases and the formation of new bone metastases during the treatment period was significantly inhibited. In conclusion, we demonstrate that targeting of integrins by GLPG0187 can inhibit the de novo formation and progression of bone metastases in prostate cancer by antitumor (including inhibition of EMT and the size of the prostate cancer stem cell population), antiresorptive, and antiangiogenic mechanisms

Effect of systemic administration of GLPG0187 on tumor growth and metastasis in a preventive and curative protocol.

<p>A) Schematic representation of the <i>preventive</i> protocol. Mice were treated daily with either IP administrated vehicle or GLPG0187 (100 mg/kg/day) from day -1 onwards. At day 0, 100,000 UM-UC-3luc2 cells were inoculated into the left heart ventricle and once a week BLI images were taken. B) Number of metastasis per mouse. C) Total tumor burden for the mice treated with 100 mg/kg/day GLPG0187 (closed circles) or vehicle (open circles). In the insert, the first 14 days are shown. D) Representative images of mice treated with vehicle or 100 mg/kg/day GLPG0187 taken at day 28 after inoculation. E) Schematic representation of the <i>curative</i> protocol. At day -21, 100,000 UM-UC-3luc2 cells were injected into the left heart ventricle and once a week BLI images were taken. At day 0, mice were divided into groups with equal total tumor burden. Mice were daily treated with an IP dosage of either vehicle or GLPG0187 (100 mg/kg/day) from day 0 onwards. F) Number of metastasis per mouse. G) Total tumor burden for the mice treated with 100 mg/kg/day GLPG0187 (closed circles) or vehicle (open circles). H) Representative images of mice treated with vehicle or 100 mg/kg/day GLPG0187 taken at day 15 after start of treatment.</p

Effects of α_v integrin on clonogenicity and stem cell/metastasis markers.

<p>The relative percentage and size distribution of colony-forming cells in a 96-wells plate clonogenic assay of single-cell diluted cultures after 2 weeks in the α_v kd or NT cells and cells treated with a dose range of GLPG0187 for 48 hrs and plated afterwards. The area of the colonies was measured with Image J software and divided according to size. Small colonies are between 0.5 and 1.5 mm², medium sized colonies are between 1.5 and 4 mm² and large colonies are bigger than 4 mm². Data were normalized to the NT or control conditions and are presented as mean ± SEM. Percentage of colony-forming cells in the control NT cells are depicted above the respective bars (A). UM-UC-3 cells were seeded 100 cells/cm² in an ultra-low attachment plate in serum-starved conditions. The percentage of cells with sphere forming capacity (P0) was measured after 10 days of culture (B). P0 spheres were dissociated into single cells and seeded in ultra-low attachment 96 wells. The percentage of cells with sphere forming capacity (P1) was measured after 10 days of culture (B). The area of the spheres was measured with Image J software (D). Percentage of cells with high ALDH activity (ALDH^hi) as measured with Aldefluor assay. Data are normalized to the NT or vehicle treated cells. Percentages of ALDH^hi cells in the NT and control cells are depicted above the respective bars (E). qPCR analysis of NANOG (F) qPCR analysis of BMI1 (G). Relative expression levels are shown compared to respectively NT or vehicle-treated cells. All values were normalized for GAPDH and presented as mean ± SEM.</p

Effect of α_v integrin on expression levels of CD24 and urothelial differentiation markers and on senescence.

<p>Relative expression levels of CD24 (A) and CD227 (B). Relative expression levels (% of positive cells * Mean fluorescence intensity) were measured by flow cytometry and normalized to the NT or vehicle treated cells. Data are represented as mean ± SEM. Percentages of positive cells are depicted above the respective bars. qPCR analysis of KRT20. Relative expression levels are shown compared to respectively NT or non-treated cells. All values were normalized for GAPDH and presented as mean ± SEM (C). UM-UC-3 luc2 and RT-4 cells were seeded into a six-well plate and exposed to a concentration series of GLPG0187 (0–500 ng/ml). 48 h after incubation, cells were harvested and senescence associated acid β-galactosidase activity was measured. Data are represented as fold change in fluorescence intensity of the signal (D).</p

ITGAV knockdown in UM-UC-3luc2 cells affects intra-bone growth in a preclinical model.

<p>A) Percentage of mice with tumors after intra-bone inoculation of either α_v-kd-UM-UC-3luc2 or NT-UM-UC-3luc2 cells B) Total tumor burden of the mice injected with α_v-kd-UM-UC-3luc2 (<i>closed circles</i>) or NT-UM-UC-3luc2 cells (<i>open circles</i>). C) Representative images of mice intra-osseously inoculated with either αv-kd-UM-UC-3luc2 or NT-UM-UC-3luc2 cells 7 days after inoculation (<i>n</i> = 10/group *<i>P</i><0.05).</p

Effects of GLPG0187 and ITGAV knockdown on adherence to tissue culture plastic.

<p>Representative images of cells treated for 24 hours with a concentration series of GLPG0187 (dosage between 0–500 ng/ml, indicated underneath the images). Treatment resulted in a dose-dependent loss of adherence to tissue culture plastic in both UM-UC-3luc2 cells (A) and RT-4 cells (C). After 48 hours of GLPG0187 treatment, cells cultured for 4 days in GLPG0187-free medium regained their adherence to the tissue culture plastic in UM-UC-3luc2 cells (B) and RT-4 cells (D). Loss of adherence was also observed in UM-UC-3luc2 and RT4 cells stably transduced with a short hairpin targeted against ITGAV (respectively F–G for UMUC3luc2 sh ITGAV clones 1 and 2 and J–K for RT4 shITGAV clones 1 and 2). As a control, cells stably transduced with a non-targeting short hairpin (NT) were used (UMUC3 (E) and RT4 (I)). Flow cytometric analysis of relative ITGAV expression levels in UM-UC-3luc2 (H) and RT4 (L) cells (% of positive cells * mean fluorescence intensity). Data are presented as mean ± SEM, n = 3, the percentage of ITGAV positive cells is indicated above the bars.</p

Effects of α_v integrin on migration and EMT.

<p>Effects of knockdown of ITGAV and 48 hrs of GLPG0187 treatment on migratory capacity of UM-UC-3luc2 and RT-4 cells as determined by Transwell Boyden chamber migration assays. Mean numbers of migrated cells per area were measured. Mean number of migrated cells of the control NT cells are depicted above the respective bars (A). Effects of ITGAV knockdown and 48 hrs of GLPG0187 treatment on the CDH1/CDH2 ratio (B). Data were normalized to the NT or control conditions (n = 3) and are presented as mean ± SEM. qPCR (C) and protein analysis of SNAI1 (D). qPCR analysis (E) and protein analysis of SNAI2 (F). qPCR analysis (G) and protein analysis of ZEB1 (H). qPCR analysis (I) and protein analysis of ZEB2 (J). Relative expression levels are shown compared to respectively NT or vehicle treated cells. All qPCR values were normalized for GAPDH and presented as mean ± SEM.</p