Bisphosphonate-Functionalized Gold Nanoparticles for Contrast-Enhanced X-Ray Detection of Breast Microcalcifications

Microcalcifications are one of the most common abnormalities detected by mammography for the diagnosis of breast cancer. However, the detection of microcalcifications and correct diagnosis of breast cancer are limited by the sensitivity and specificity of mammography. Therefore, the objective of this study was to investigate the potential of bisphosphonate-functionalized gold nanoparticles (BP-Au NPs) for contrast-enhanced radiographic detection of breast microcalcifications using two models of breast microcalcifications which allowed for precise control over levels of hydroxyapatite (HA) mineral within a low attenuating matrix. First, an in vitro imaging phantom was prepared with varying concentrations of HA uniformly dispersed in an agarose hydrogel. The X-ray attenuation of HA-agarose compositions labeled by BP-Au NPs was increased by up to 26 HU compared to unlabeled compositions for HA concentrations ranging from 1–10 mg/mL. Second, an ex vivo tissue model was developed to more closely mimic the heterogeneity of breast tissue by injecting varying concentrations of HA in a Matrigel carrier into murine mammary glands. The X-ray attenuation of HA-Matrigel compositions labeled by BP-Au NPs was increased by up to 289 HU compared to unlabeled compositions for HA concentrations ranging from 0.5–25 mg/mL, which included an HA concentration (0.5 mg/mL) that was otherwise undetectable by micro-computed tomography. Cumulatively, both models demonstrated the ability of BP-Au NPs to enhance contrast for radiographic detection of microcalcifications, including at a clinically-relevant imaging resolution. Therefore, BP-Au NPs may have potential to improve clinical detection of breast microcalcifications by mammography

Cleavage of E-Cadherin by Matrix Metalloproteinase-7 Promotes Cellular Proliferation in Nontransformed Cell Lines via Activation of RhoA

Perturbations in cell-cell contact machinery occur frequently in epithelial cancers and result in increased cancer cell migration and invasion. Previously, we demonstrated that MMP-7, a protease implicated in mammary and intestinal tumor growth, can process the adherens junction component E-cadherin. This observation leads us to test whether MMP-7 processing of E-cadherin could directly impact cell proliferation in nontransformed epithelial cell lines (MDCK and C57MG). Our goal was to investigate the possibility that MMP-7 produced by cancer cells may have effects on adjacent normal epithelium. Here, we show that MMP-7 processing of E-cadherin mediates, (1) loss of cell-cell contact, (2) increased cell migration, (3) a loss of epithelial cell polarization and (4) increased cell proliferation via RhoA activation. These data demonstrate that MMP-7 promotes epithelial cell proliferation via the processing of E-cadherin and provide insights into the molecular mechanisms that govern epithelial cell growth

Pleiotropic effects of FGFR1 on cell proliferation, survival, and migration in a 3D mammary epithelial cell model

Members of the fibroblast growth factor (FGF) family and the FGF receptors (FGFRs) have been implicated in mediating various aspects of mammary gland development and transformation. To elucidate the molecular mechanisms of FGFR1 action in a context that mimics polarized epithelial cells, we have developed an in vitro three-dimensional HC11 mouse mammary epithelial cell culture model expressing a drug-inducible FGFR1 (iFGFR1). Using this conditional model, iFGFR1 activation in these growth-arrested and polarized mammary acini initially led to reinitiation of cell proliferation, increased survival of luminal cells, and loss of cell polarity, resulting in the disruption of acinar structures characterized by the absence of an empty lumen. iFGFR1 activation also resulted in a gain of invasive properties and the induction of matrix metalloproteinase 3 (MMP-3), causing the cleavage of E-cadherin and increased expression of smooth muscle actin and vimentin. The addition of a pan MMP inhibitor abolished these phenotypes but did not prevent the effects of iFGFR1 on cell proliferation or survival

Effects of bisphosphonate ligands and PEGylation on targeted delivery of gold nanoparticles for contrast-enhanced radiographic detection of breast microcalcifications

A preclinical murine model of hydroxyapatite (HA) breast microcalcifications (µcals), which are an important clinical biomarker for breast cancer detection, was used to investigate the independent effects of high affinity bisphosphonate (BP) ligands and a polyethylene glycol (PEG) spacer on targeted delivery of gold nanoparticles (Au NPs) for contrast-enhanced radiographic detection. The addition of BP ligands to PEGylated Au NPs (BP-PEG-Au NPs) resulted in five-fold greater binding affinity for targeting HA µcals, as expected, due to the strong binding affinity of BP ligands for calcium. Therefore, BP-PEG-Au NPs were able to target HA µcals in vivo after intramammary delivery, which enabled contrast-enhanced radiographic detection of µcals in both normal and radiographically dense mammary tissues similar to previous results for BP-Au NPs, while PEG-Au NPs did not. The addition of a PEG spacer between the BP targeting ligand and Au NP surface enabled improved in vivo clearance. PEG-Au NPs and BP-PEG-Au NPs were cleared from all mammary glands (MGs) and control MGs, respectively, within 24–48 h after intramammary delivery, while BP-Au NPs were not. PEGylated Au NPs were slowly cleared from MGs by lymphatic drainage and accumulated in the spleen. Histopathology revealed uptake of PEG-Au NPs and BP-PEG-Au NPs by macrophages in the spleen, liver, and MGs; there was no evidence of toxicity due to the accumulation of NPs in organs and tissues compared with untreated controls for up to 28 days after delivery

The Role of MMP7 and Its Cross-Talk with the FAS/FASL System during the Acquisition of Chemoresistance to Oxaliplatin

Background: The efficacy of oxaliplatin in cancer chemotherapy is limited by the development of drug resistance. MMP7 has been related to the loss of tumor cell response to cytotoxic agents although the exact mechanism is not fully understood. Moreover, MMP7 is an independent prognosis factor for survival in patients with colorectal cancer. The aim of the present study was to analyze the role of MMP7 and its cross-talk with the Fas/FasL system during the acquisition of oxaliplatin resistance in colon cancer cells. Principal Findings: For this purpose we have developed three different oxaliplatin-resistant cell lines (RHT29, RHCT116 p53+/+, RHCT116 p53−/−) from the parental HT29, HCT116 p53+/+ and HCT116 p53−/− colon cancer cells. MMP7 basal expression was higher in the resistant compared to the parental cell lines. MMP7 was also upregulated by oxaliplatin in both HT29 (p53 mutant) and RHCT116 p53−/− but not in the RHCT116 p53+/+. Inhibition of MMP by 1,10-phenantroline monohydrate or siRNA of MMP7 restores cell sensitivity to oxaliplatin-induced apoptosis in both HT29 and RHCT116 p53−/− but not in the RHCT116 p53+/+. Some of these effects are caused by alterations in Fas receptor. Fas is upregulated by oxaliplatin in colon cancer cells, however the RHT29 cells treated with oxaliplatin showed a 3.8-fold lower Fas expression at the cell surface than the HT29 cells. Decrease of Fas at the plasma membrane seems to be caused by MMP7 since its inhibition restores Fas levels. Moreover, functional analysis of Fas demonstrates that this receptor was less potent in inducing apoptosis in RHT29 cells and that its activation induces MAPK signaling in resistant cells. Conclusions: Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor. Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals

Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression

Introduction: Rho signaling regulates key cellular processes including proliferation, survival, and migration, and it has been implicated in the development of many types of cancer including breast cancer. P190B Rho GTPase activating protein (RhoGAP) functions as a major inhibitor of the Rho GTPases. P190B is required for mammary gland morphogenesis, and overexpression of p190B in the mammary gland induces hyperplastic lesions. Hence, we hypothesized that p190B may play a pivotal role in mammary tumorigenesis. Methods: To investigate the effects of loss of p190B function on mammary tumor progression, p190B heterozygous mice were crossed with an MMTV-Neu breast cancer model. Effects of p190B deficiency on tumor latency, multiplicity, growth, preneoplastic progression and metastasis were evaluated. To investigate potential differences in tumor angiogenesis between the two groups, immunohistochemistry to detect von Willebrand factor was performed and quantified. To examine gene expression of potential mediators of the angiogenic switch, an angiogenesis PCR array was utilized and results were confirmed using immunohistochemistry. Finally, reciprocal transplantation of tumor fragments was performed to determine the impact of stromal deficiency of p190B on tumor angiogenesis. Results: P190B deficiency reduced tumor penetrance (53% of $p190B^{+/-}Neu$ mice vs. 100% of $p190B^{+/+}Neu$ mice formed tumors) and markedly delayed tumor onset by an average of 46 weeks. Tumor multiplicity was also decreased, but an increase in the number of preneoplastic lesions was detected indicating that p190B deficiency inhibited preneoplastic progression. Angiogenesis was decreased in the p190B heterozygous tumors, and expression of a potent angiogenic inhibitor, thrombospondin-1, was elevated in $p190B^{+/-}Neu$ mammary glands. Transplantation of $p190B^{+/-}Neu$ tumor fragments into wild-type recipients restored tumor angiogenesis. Strikingly, $p190B^{+/+}Neu$ tumor fragments were unable to grow when transplanted into $p190B^{+/-}Neu$ recipients. Conclusions: These data suggest that p190B haploinsufficiency in the epithelium inhibits MMTV-Neu tumor initiation. Furthermore, p190B deficiency in the vasculature is responsible, in part, for the inhibition of MMTV-Neu tumor progression

Death receptor pathways mediate targeted and non-targeted effects of ionizing radiations in breast cancer cells

Delayed cell death by mitotic catastrophe is a frequent mode of solid tumor cell death after γ-irradiation, a widely used treatment of cancer. Whereas the mechanisms that underlie the early γ-irradiation-induced cell death are well documented, those that drive the delayed cell death are largely unknown. Here we show that the Fas, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and tumor necrosis factor (TNF)-α death receptor pathways mediate the delayed cell death observed after γ-irradiation of breast cancer cells. Early after irradiation, we observe the increased expression of Fas, TRAIL-R and TNF-R that first sensitizes cells to apoptosis. Later, the increased expression of FasL, TRAIL and TNF-α permit the apoptosis engagement linked to mitotic catastrophe. Treatments with TNF-α, TRAIL or anti-Fas antibody, early after radiation exposure, induce apoptosis, whereas the neutralization of the three death receptors pathways impairs the delayed cell death. We also show for the first time that irradiated breast cancer cells excrete soluble forms of the three ligands that can induce the death of sensitive bystander cells. Overall, these results define the molecular basis of the delayed cell death of irradiated cancer cells and identify the death receptors pathways as crucial actors in apoptosis induced by targeted as well as non-targeted effects of ionizing radiation

Matrix metalloproteinases and soluble Fas/FasL system as novel regulators of apoptosis in children and young adults on chronic dialysis

The system of membrane receptor Fas and its ligand FasL compose one of the main pathways triggering apoptosis. However, the role of their soluble forms has not been clarified yet. Although sFasL can be converted from the membrane-bound form by matrix metalloproteinases (MMPs), there are no data on relations between sFas/sFasL, MMPs and their tissue inhibitors (TIMPs) in patients on chronic dialysis—neither children nor adults. The aim of our study was to evaluate serum concentrations of sFas, sFasL, and their potential regulators (MMP-2, MMP-7, MMP-9, TIMP-1, TIMP-2), in children and young adults chronically dialyzed. Twenty-two children on automated peritoneal dialysis (APD), 19 patients on hemodialysis (HD) and 30 controls were examined. Serum concentrations of sFas, sFasL, MMPs and TIMPs were assessed by ELISA. Median values of sFas, sFasL, sFas/sFasL ratio, MMP-2, MMP-7, MMP-9, TIMP-1 and TIMP-2 were significantly elevated in all dialyzed patients vs. controls, the highest values being observed in subjects on HD. A single HD session caused the decrease in values of all parameters to the levels below those seen in children on APD. Regression analysis revealed that MMP-7 and TIMP-1 were the best predictors of sFas and sFasL concentrations. Children and young adults on chronic dialysis are prone to sFas/sFasL system dysfunction, more pronounced in patients on hemodialysis. The correlations between sFas/sFasL and examined enzymes suggest that MMPs and TIMPs take part in the regulation of cell death in the pediatric population on chronic dialysis, triggering both anti- (sFas) and pro-apoptotic (sFasL) mechanisms

Opposing Roles for Membrane Bound and Soluble Fas Ligand in Glaucoma-Associated Retinal Ganglion Cell Death

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma