The expression and regulation of matrilysin (MMP-7) in human colon cancer and leukaemia cell lines

Matrilysin (MMP-7, EC 3.4.24.23) is the smallest member o f the matrix metalloproteinase (MMP) family and has been shown to be overexpressed in various tumours including breast and colon cancers. Matrilysin has also been shown to play an important role in several aspects of tumour biology including growth, progression, invasion and metastasis. With respect to colon cancer, matrilysin is unique in that it is the only MMP expressed exclusively by the malignant epithelia o f colonic adenocarcinomas. These facts combine to make matrilysin a promising therapeutic target. However, in order to develop drugs which specifically inhibit matrilysin it is important to understand how matrilysin gene expression is controlled, something which to date remains poorly understood. We have examined a panel of human colon tumour cell lines and have shown that matrilysin expression can be upregulated by a number of cytokines including EGF, IL-6 and bFGF. Analysis of the matrilysin promoter revealed the presence of a number of potential transcription factor binding sites including three ETS sites. We have shown that EGF treatment increased matrilysin gene expression by activation of PEA3 transcription factors using artificial promoter, western blot and EMSA analysis. ‘Supershift’ EMSA analysis showed that other PEA3 subfamily members such as ERM and ER81 may also be involved which is in agreement with other studies. In addition, we have found that EGF increased cellular levels o f [3-catenin through destabilisation of the E-cadherin/catenin complex which resulted in increased binding to the T c f site within the matrilysin promoter. We also examined the expression and regulation of matrilysin in the K562 and HL-60 myeloid leukaemia cell lines. Results showed that only the K562 cell line expressed matrilysin and in vitro invasion assays showed that the K562 cells were up to 4 times more invasive than the HL- 60 cell line. Matrilysin antibody blocking experiments showed a significant decrease in invasion in the K562 cell line suggesting a role for matrilysin in leukaemia invasion. The MMP and TIMP profiles o f these cell lines were also examined. Our data suggests that EGF plays an important role in the regulation of matrilysin gene expression via a number of new mechanisms. Furthermore, we have shown that matrilysin plays an important role in leukaemia cell line invasion. These findings have identified possible new drug targets that will inhibit matrilysin expression which in turn should lead to decreased tumourigenesis and invasion and metastasis

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

Bone seeking matrix metalloproteinase-2 inhibitors prevent bone metastatic breast cancer growth

Bone metastasis is common during breast cancer progression. Matrix metalloproteinase-2 (MMP-2) is significantly associated with aggressive breast cancer and poorer overall survival. In bone, tumor or host derived MMP-2 contributes to breast cancer growth and does so by processing substrates including type I collagen and transforming growth factorβ (TGFβ) latency proteins. These data provide strong rationale for the application of MMP-2 inhibitors to treat the disease. However, in vivo, MMP-2 is systemically expressed. Therefore, to overcome potential toxicities noted with previous broad-spectrum MMP inhibitors (MMPIs), we used highly selective bisphosphonic based MMP-2 inhibitors (BMMPIs) that allowed for specific bone targeting. In vitro, BMMPIs impacted the viability of breast cancer cell lines and osteoclast precursors but not osteoblasts. In vivo, we demonstrated using two bone metastatic models (PyMT-R221A and 4T1) that BMMPI treatment significantly reduced tumor growth and tumor associated bone destruction. Additionally, BMMPIs are superior in promoting tumor apoptosis compared to the standard of care bisphosphonate, zoledronate. We demonstrated MMP-2 selective inhibition in the bone microenvironment using specific and broad spectrum MMP probes. Further, compared to zoledronate, BMMPI treated mice had significantly lower levels of TGFβ signaling and MMP generated type I collagen carboxy-terminal (ICTP) fragments. Taken together, our data show the feasibility of selective inhibition of MMPs in the bone metastatic breast cancer microenvironment. We posit that BMMPIs could be easily translated to the clinical setting for the treatment of bone metastases given the well-tolerated nature of bisphosphonates

LTP Induction Translocates Cortactin at Distant Synapses in Wild-Type But Not Fmr1 Knock-Out Mice

Stabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present study tested whether this involves activity-driven effects on the actin-regulatory protein cortactin, and whether such effects are disturbed in the Fmr1 knock-out (KO) model of fragile X syndrome, in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin, while basal levels were reduced by nocodazole, indicating that cortactin's movements into and away from synapses are regulated by microtubule and actomyosin motors, respectively. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1 KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactin's interactions with actin, and coprecipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter protein's use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites

Lateral Lumbar Interbody Fusion in the Outpatient Setting with Multimodal Analgesic Protocol: Clinical Case Series

Objective Minimally invasive techniques and multimodal analgesia protocols have made spine surgery in the outpatient setting increasingly feasible. A number of spinal procedures have been documented in the outpatient setting, though the feasibility of lateral lumbar interbody fusion (LLIF) on an ambulatory basis has not been thoroughly assessed. To present a clinical case series of patients undergoing LLIF in the outpatient setting. Methods A prospectively maintained surgical database was retrospectively reviewed to identify patients undergoing outpatient spine procedures with an enhanced multimodal analgesia protocol from October 2016 to February 2021. Patient demographics, medical and spinal diagnoses, procedural characteristics, operative duration, estimated blood loss (EBL), postoperative length of stay (LOS), postoperative pain scores, postoperative narcotic consumption, and incidence of any intra- or postoperative complications were collected. The state’s prescription monitoring program was queried to assess rates of filling narcotic prescriptions >6 weeks following surgery. Results A total of 24 LLIF patients were included. Mean postoperative pain score was 5.8, and mean postoperative narcotic consumption was 26.8 oral morphine equivalents. All patients were discharged on the same day of surgery. No postoperative complications were observed. After the 6-week postoperative timepoint, 16.7% of patients filled a prescription for tramadol, 8.3% for hydrocodone, 4.2% for hydromorphone, 4.2% for cyclobenzaprine, and 4.2% for alprazolam. Conclusion This clinical case series demonstrates that LLIF can be both safe and feasible in the outpatient setting, with minimal narcotic medication dependence in the postoperative period

Do Patient Expectations Represent a More Important Clinical Difference? A Study of Surgical Outcomes in the Cervical Spine

Objective This study aims to compare the impact of achieving an MCID or meeting preoperative expectations on patient satisfaction following cervical spine procedures. Methods A surgical database was retrospectively reviewed for cervical spine surgery patients from 2016 to 2020. Inclusion criteria were primary or revision, single- or multilevel cervical disc arthroplasty or anterior cervical discectomy and fusions (ACDF). Visual analogue scale (VAS) neck and arm pain was assessed preoperatively and postoperatively (6-week, 12-weeks, 6-months, 1-year). Preoperative patient expectation and postoperative satisfaction were recorded. MCID achievement was determined using previously established values. Expectations met and MCID achievement were compared as possible predictors of satisfaction. Results One hundred and six cervical spine patients were included. Both meeting expectations and achieving MCID were significant predictors of satisfaction for arm pain at 6-weeks and 12-weeks (all p≤0.007). Achieving MCID significantly predicted satisfaction for neck pain at all timepoints (all p≤0.007) and meeting expectations predicted satisfaction for neck pain at 6-weeks, 12-weeks, and 1-year (all p≤0.003). Comparison of coefficients revealed no significant difference in effect size between meeting expectations and achievement of MCID as predictors of patient satisfaction (all p>0.050). Conclusion MCID achievement and meeting expectations were significant predictors of satisfaction for neck pain and short-term arm pain. Both measures may be similarly useful for interpretation of patient outcomes and the optimal choice of metric may depend on practice-specific factors

Using Swallowing Quality of Life to Compare Oropharyngeal Dysphagia Following Cervical Disc Arthroplasty or Anterior Cervical Discectomy and Fusion

Objective To evaluate dysphagia outcomes using the swallowing quality of life (SWAL-QOL) questionnaire between patients undergoing cervical disk arthroplasty (CDA) or anterior cervical discectomy and fusion (ACDF). Methods Patient-reported outcome measures (PROMs) were collected using SWAL-QOL, VAS, NDI, and SF-12 PCS. All measures were recorded preoperatively to 6-month postoperatively. Patients were grouped according to cervical procedure and instrumentation used. Differences in PROMs and SWAL-QOL domains were evaluated by t-test and one-way ANOVA with post-hoc testing, respectively. Simple linear regression was employed to evaluate the relationship between number of levels operated on and postoperative outcomes. Results 161 patients were included. CDA patients had significantly worse SWAL-QOL scores at 6-months. Preoperative VAS neck was significantly worse for patients who underwent either an ACDF procedure with a stand-alone cage or CDA as compared to patients who underwent an ACDF with anterior plating. At 6-months postoperatively, CDA patients reported a significantly worse “fatigue” score compared to ACDF patients. At 6-months postoperatively, ACDF patients reported a significantly better “sleep” scores compared to CDA patients with both recipients of an anterior plate and stand-alone cage reporting significantly better scores compared to the CDA cohort (p=0.024; p<0.001). The SWAL-QOL domain of symptom frequency at 6-weeks postoperatively was significantly associated with number of levels operated (p=0.032). Conclusion Patients undergoing either an ACDF or CDA procedure largely did not demonstrate differences in pain, disability, and dysphagia scores. However, at more longitudinal timepoints CDA patients reported worse fatigue and sleep scores compared to ACDF patients

Sward composition and soil moisture conditions affect nitrous oxide emissions and soil nitrogen dynamics following urea-nitrogen application

peer-reviewedIncreased emissions of N2O, a potent greenhouse gas (GHG), from agricultural soils is a major concern for the sustainability of grassland agriculture. Emissions of N2O are closely associated with the rates and forms of N fertilisers applied as well as prevailing weather and soil conditions. Evidence suggests that multispecies swards require less fertiliser N input, and may cycle N differently, thus reducing N loss to the environment. This study used a restricted simplex-centroid experimental design to investigate N2O emissions and soil N cycling following application of urea-N (40 kg N ha−1) to eight experimental swards (7.8 m2) with differing proportions of three plant functional groups (grass, legume, herb) represented by perennial ryegrass (PRG, Lolium perenne), white clover (WC, Trifolium repens) and ribwort plantain (PLAN, Plantago lanceolata), respectively. Swards were maintained under two contrasting soil moisture conditions to examine the balance between nitrification and denitrification. Two N2O peaks coincided with fertiliser application and heavy rainfall events; 13.4 and 17.7 g N2O-N ha−1 day−1 (ambient soil moisture) and 39.8 and 86.9 g N2O-N ha−1 day−1 (wet soil moisture). Overall, cumulative N2O emissions post-fertiliser application were higher under wet soil conditions. Increasing legume (WC) proportions from 0% to 60% in multispecies swards resulted in model predicted N2O emissions increasing from 22.3 to 96.2 g N2O-N ha−1 (ambient soil conditions) and from 59.0 to 219.3 g N2O-N ha−1 (wet soil conditions), after a uniform N application rate. Soil N dynamics support denitrification as the dominant source of N2O especially under wet soil conditions. Significant interactions of PRG or WC with PLAN on soil mineral N concentrations indicated that multispecies swards containing PLAN potentially inhibit nitrification and could be a useful mitigation strategy for N loss to the environment from grassland agriculture.Teagas

An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment

Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment.To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays).Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases

Assessment and Etiology of Attention Deficit Hyperactivity Disorder and Oppositional Defiant Disorder in Boys and Girls

Attention deficit hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD) are more common in boys than girls. In this paper, we investigated whether the prevalence differences are attributable to measurement bias. In addition, we examined sex differences in the genetic and environmental influences on variation in these behaviors. Teachers completed the Conners Teacher Rating Scale-Revised:Short version (CTRS-R:S) in a sample of 800 male and 851 female 7-year-old Dutch twins. No sex differences in the factor structure of the CTRS-R:S were found, implying the absence of measurement bias. The heritabilities for both ADHD and ODD were high and were the same in boys and girls. However, partly different genes are expressed in boys and girls