Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into multiple mesoderm lineages in the course of normal tissue homeostasis or during injury. We have previously shown that MSCs migrate to sites of tumorigenesis, where they become activated by cancer cells to promote metastasis. However, the molecular and phenotypic attributes of the MSC-induced metastatic state of the cancer cells remained undetermined. Here, we show that bone marrow-derived human MSCs promote de novo production of lysyl oxidase (LOX) from human breast carcinoma cells, which is sufficient to enhance the metastasis of otherwise weakly metastatic cancer cells to the lungs and bones. We also show that LOX is an essential component of the CD44-Twist signaling axis, in which extracellular hyaluronan causes nuclear translocation of CD44 in the cancer cells, thus triggering LOX transcription by associating with its promoter. Processed and enzymatically active LOX, in turn, stimulates Twist transcription, which mediates the MSC-triggered epithelial-to-mesenchymal transition (EMT) of carcinoma cells. Surprisingly, although induction of EMT in breast cancer cells has been tightly associated with the generation of cancer stem cells, we find that LOX, despite being critical for EMT, does not contribute to the ability of MSCs to promote the formation of cancer stem cells in the carcinoma cell populations. Collectively, our studies highlight a critical role for LOX in cancer metastasis and indicate that the signaling pathways controlling stroma-induced EMT are distinct from pathways regulating the development of cancer stem cells.

Differential G protein subunit expression by prostate cancer cells and their interaction with CXCR5.

BACKGROUND: Prostate cancer (PCa) cell lines and tissues differentially express CXCR5, which positively correlate with PCa progression, and mediate PCa cell migration and invasion following interaction with CXCL13. However, the differential expression of G protein α, ß, and γ subunits by PCa cell lines and the precise combination of these proteins with CXCR5 has not been elucidated. METHODS: We examined differences in G protein expression of normal prostate (RWPE-1) and PCa cell lines (LNCaP, C4-2B, and PC3) by western blot analysis. Further, we immunoprecipitated CXCR5 with different G protein subunits, and CXCR4, following CXCL13 stimulation. To investigate constitutive coupling of CXCR5 with CXCR4 and PAR-1 we performed invasion assay in PCa cells transfected with Gαq/i2 or Gα13 siRNA, following CXCL13 treatment. We also investigated Rac and RhoA activity by G-LISA activation assay in PCa cells following CXCL13/thrombin stimulation. RESULT: Of the 22 G proteins studied, Gαi1-3, Gß1-4, Gγ5, Gγ7, and Gγ10 were expressed by both normal and PCa cell lines. Gαs was moderately expressed in C4-2B and PC3 cell lines, Gαq/11 was only present in RWPE-1 and LNCaP cell lines, while Gα12 and Gα13 were expressed in C4-2B and PC3 cell lines. Gγ9 was expressed only in PCa cell lines. Gα16, Gß5, Gγ1-4, and Gγ13 were not detected in any of the cell lines studied. Surprisingly, CXCR4 co-immunoprecipitated with CXCR5 in PCa cell lines irrespective of CXCL13 treatment. We also identified specific G protein isoforms coupled to CXCR5 in its resting and active states. Gαq/11/Gß3/Gγ9 in LNCaP and Gαi2/Gß3/Gγ9 in C4-2B and PC3 cell lines, were coupled to CXCR5 and disassociated following CXCL13 stimulation. Interestingly, Gα13 co-immunoprecipitated with CXCR5 in CXCL13-treated, but not in untreated PCa cell lines. Inhibition of Gαq/i2 significantly decreased the ability of cells to invade, whereas silencing Gα13 did not affect CXCL13-dependent cell invasion. Finally, CXCL13 treatment significantly increased Rac activity in Gαq/i2 dependent manner, but not RhoA activity, in PCa cell lines. CONCLUSIONS: These findings offer insight into molecular mechanisms of PCa progression and can help to design some therapeutic strategies involving CXCR5 and/or CXCL13 blockade and specific G protein inhibition to abrogate PCa metastasis.

Mesenchymal stem cells in the pathogenesis and therapy of breast cancer.

Mesenchymal stem cells (MSCs) are a heterogeneous mix of stromal stem cells that can give rise to cells of mesodermal lineages, namely adipocytes, osteocytes and chondrocytes. They can home to sites of injury where they promote the repair and regeneration of damaged tissues. MSCs also home to sites of tumorigenesis, and as such, are utilized as efficient cellular vehicles for the delivery of anti-neoplastic therapeutics. Recently, MSCs within the tumor microenvironment have been shown to contribute to the desmoplastic reaction and to facilitate tumor formation and progression, sparking renewed interest in their pro-tumorigenic attributes and their roles as tumor stromal cells. Here, we describe the evidence linking MSCs to inflammatory processes and breast cancer development, and discuss their newly discovered physiological roles in the context of the tumor microenvironment.

PI3Kp110-, Src-, FAK-dependent and DOCK2-independent migration and invasion of CXCL13-stimulated prostate cancer cells.

BACKGROUND: Most prostate cancer (PCa)-related deaths are due to metastasis, which is mediated in part by chemokine receptor and corresponding ligand interaction. We have previously shown that PCa tissue and cell lines express high levels of the chemokine receptor CXCR5, than compared to their normal counterparts, and interaction of CXCR5 with its specific ligand (CXCL13) promoted PCa cell invasion, migration, and differential matrix metalloproteinase (MMP) expression. This study dissects some of the molecular mechanisms following CXCL13-CXCR5 interaction that mediate PCa cell migration and invasion. RESULTS: Using Western blot analysis, kinase-specific cell-based ELISAs, and migration and invasion assays, we show that PCa cell lines differentially express phosphoinositide-3 kinase (PI3K) catalytic subunit isoforms and dedicator of cytokinesis 2 (DOCK2). Specifically, we show that PC3 and normal prostatic epithelial (RWPE-1), but not LNCaP cell lines expressed DOCK2, while RWPE, PC3, and LNCaP cell lines expressed PI3K-p110alpha and -p110beta. Moreover, PC3 selectively expressed PI3K-p110gamma, but LNCaP and RWPE cell lines expressed PI3Kp110delta. CXCL13 caused CXCR5-dependent activation of the PI3Kp85alpha in LNCaP cells, and p85alpha as well as -p101 in PC3 cells. CXCL13-CXCR5 interaction regulated LNCaP and PC3 cell migration and invasion through extracellular signal-regulated kinase 1/2 (ERK1/2) activation that was primarily dependent on the PI3Kp110 isoform(s), Src, and focal adhesion kinase (FAK), but not DOCK2. CONCLUSIONS: While additional studies will be needed to determine the PI3K-independent (i.e., DOCK2-mediated) and -dependent events that dictate PCa cell responsiveness to CXCL13, these data provide evidence of the existence of cell type- and stimulus-specific signaling events that support migration and invasion of PCa cells.

Intravitreal bevacizumab vs verteporfin photodynamic therapy for neovascular age-related macular degeneration.

OBJECTIVE: To compare verteporfin photodynamic therapy (PDT) with intravitreal bevacizumab for management of choroidal neovascularization (CNV) associated with age-related macular degeneration. METHODS: Patients with predominantly classic CNV were prospectively randomized to receive standard PDT or intravitreal bevacizumab injections (2.5 mg). Best-corrected visual acuity (BCVA) measured by Snellen charts, central retinal thickness by optical coherence tomography, and greatest linear dimension of the CNV by fluorescein angiography were compared between the groups at baseline and at 3 and 6 months. Main outcome measures were stability or improvement in BCVA, decrease in central retinal thickness, and stability in greatest linear dimension. RESULTS: Mean baseline BCVA, central retinal thickness, and greatest linear dimension were not statistically different between the bevacizumab (n = 32) and PDT (n = 30) groups. Mean central retinal thickness was significantly better at 3 and 6 months in the bevacizumab group vs the PDT group (P = .04 and P = .002, respectively). At 3 months, mean BCVA and greatest linear dimension were not significantly different between the 2 groups. At 6 months, mean BCVA and greatest linear dimension were significantly better in the bevacizumab group (P < .001 and P = .02, respectively). CONCLUSION: During 6 months, intravitreal bevacizumab was superior to PDT in controlling predominantly classic CNV in age-related macular degeneration. Additional randomized clinical trials are necessary to determine if this benefit will remain with longer follow-up.

Corneal light shield as a delivery system for standardized application of mitomycin C in excimer surface ablation.

PURPOSE: To develop a simple, reproducible method of applying intraoperative mitomycin C (MMC) in excimer surface ablation surgery. METHODS: A two-part protocol was developed to study several properties of corneal light shields. Part A tested the amount of MMC (0.2 mg/mL) absorbed, expansion dimension attained, and amount released to filter paper. Part B examined in vitro release of MMC to a photorefractive keratectomy (PRK)-treated enucleated pig eye. RESULTS: Mean MMC absorbed by corneal light shields was 63.7 +/- 5.0 mg (range: 57.4 to 72.8 mg); coefficient of variability was 7.8%. Expansion diameter and thickness were constant at 9.0 mm and 1.1 mm, respectively. Mean solution released to filter paper was 55.4 +/- 3.9 mg (range: 51.3 to 63.5 mg); coefficient of variability was 7.1%. Mean solution transferred to the PRK-treated pig eye was 2.3 +/- 0.7 mg (range: 1.1 to 3.5 mg); coefficient of variability was 33%. CONCLUSIONS: Mitomycin C contact surface area, amount absorbed, and amount released by each corneal light shield were reproducible. Uniform dimensions theoretically provide uniform distribution of MMC. This method may allow standardization of intraoperative MMC application in excimer surface ablation.

Intravitreal bevacizumab for the management of choroidal neovascularization in age-related macular degeneration.

PURPOSE: To investigate the efficacy and safety of intravitreal bevacizumab for managing choroidal neovascularization (CNV) due to age-related macular degeneration (AMD). DESIGN: Prospective interventional case series. METHODS: Seventeen eyes of 17 patients with subfoveal CNV due to AMD participated in this study at the American University of Beirut Ophthalmology Clinics. All patients had failed, refused, or were not eligible for photodynamic therapy. All eyes received a baseline eye examination, which included best-corrected visual acuity (BCVA), dilated fundus examination, ocular coherence tomography (OCT) imaging, and fluorescein angiography. An intravitreal injection of bevacizumab (2.5 mg/0.1 ml) was given at baseline and followed by two additional injections at four-week intervals. BCVA, OCT, and fluorescein angiography were repeated four weeks after each injection. Main outcome measures were improvement in BCVA and central retinal thickness (CRT). RESULTS: Mean baseline BCVA was 20/252 (median 20/200), and baseline CRT was 362 microm (median 350 microm). Improvement in VA and CRT occurred by the fourth week. At 12 weeks, mean BCVA was 20/76 (P < .001) and median BCVA was 20/50 (P < .001). Both mean and median CRT decreased to 211 microm (P < .001). Thirteen (76%) of 17 eyes had total resolution of subretinal fluid, and four eyes (24%) had BCVA better than 20/50. No systemic or ocular side effects were noted at any time. CONCLUSION: Eyes with CNV due to AMD treated with intravitreal bevacizumab had marked anatomic and visual improvement. Further studies are necessary to confirm the long-term efficacy and safety of this treatment.

Antibody Microarray Analysis of Signaling Networks Regulated by Cxcl13 and Cxcr5 in Prostate Cancer.

Advanced prostate cancer (PCa) often spreads to distant organs, leading to increased morbidity and mortality. It is now well established that chemokines and their cognate receptors play a crucial role in the multi-step process of metastasis. We have previously identified CXCR5 to be highly expressed by PCa tissues and cell lines and its specific ligand, CXCL13, is significantly elevated in the serum of patients with PCa and differentiated PCa cases with other benign prostatic diseases. CXCR5:CXCL13 interactions promote PCa cell invasion, migration, and differential matrix metalloproteinase (MMP) expression. Thus, it is important to understand the molecular and cellular processes that mediate these events. In this study, we quantified changes in apoptosis, cell cycle, and cytoskeleton rearrangement biological pathways from CXCL13-treated hormone refractory PCa cell line (PC3) to better elucidate the signaling pathways activated by CXCL13:CXCR5 interaction. Using antibody arrays that displayed 343 different protein- and phosphorylation-specific antibodies, regulatory networks that control cancer progression signaling cascades were identified. Three regulatory networks were dramatically induced by CXCL13: Akt1/2-cyclin-dependent kinases (Cdk1/2)-Cdk inhibitor 1B (CDKN1B), Integrinß3-focal adhesion kinase (Fak)/Src-Paxillin(PXN), and Akt-Jun-cAMP response-element binding protein (CREB1). In general, phosphoinositide-3 kinase (PI3K)/Akt and stress-activated protein kinase (SAPK)/c-jun kinase (JNK) were the major signaling pathways modulated by CXCL13 in PCa cells. This cluster analysis revealed proteins whose activation patterns can be attributed to CXCL13:CXCR5 interaction in the androgen-independent PC3 cell line. Taken together, these results suggest that CXCL13 contributes to cell-signaling cascades that regulate advanced PCa cell invasion, growth, and/or survival.