Lysophosphatidic Acid Receptor Signaling in the Human Breast Cancer Tumor Microenvironment Elicits Receptor-Dependent Effects on Tumor Progression.

Lysophosphatidic acid receptors (LPARs) are six G-protein-coupled receptors that mediate LPA signaling to promote tumorigenesis and therapy resistance in many cancer subtypes, including breast cancer. Individual-receptor-targeted monotherapies are under investigation, but receptor agonism or antagonism effects within the tumor microenvironment following treatment are minimally understood. In this study, we used three large, independent breast cancer patient cohorts (TCGA, METABRIC, and GSE96058) and single-cell RNA-sequencing data to show that increased tumor LPAR1, LPAR4, and LPAR6 expression correlated with a less aggressive phenotype, while high LPAR2 expression was particularly associated with increased tumor grade and mutational burden and decreased survival. Through gene set enrichment analysis, it was determined that cell cycling pathways were enriched in tumors with low LPAR1, LPAR4, and LPAR6 expression and high LPAR2 expression. LPAR levels were lower in tumors over normal breast tissue for LPAR1, LPAR3, LPAR4, and LPAR6, while the opposite was observed for LPAR2 and LPAR5. LPAR1 and LPAR4 were highest in cancer-associated fibroblasts, while LPAR6 was highest in endothelial cells, and LPAR2 was highest in cancer epithelial cells. Tumors high in LPAR5 and LPAR6 had the highest cytolytic activity scores, indicating decreased immune system evasion. Overall, our findings suggest that potential compensatory signaling via competing receptors must be considered in LPAR inhibitor therapy.

Positron Emission Tomography Imaging of Autotaxin in Thyroid and Breast Cancer Models Using [18F]PRIMATX.

Autotaxin (ATX) is a secreted enzyme responsible for producing lysophosphatidic acid (LPA). The ATX/LPA signaling axis is typically activated in wound healing and tissue repair processes. The ATX/LPA axis is highjacked and upregulated in the progression and persistence of several chronic inflammatory diseases, including cancer. As ATX inhibitors are now progressing to clinical testing, innovative diagnostic tools such as positron emission tomography (PET) are needed to measure ATX expression in vivo accurately. The radiotracer, [18F]PRIMATX, was recently developed and tested for PET imaging of ATX in vivo in a murine melanoma model. The goal of the present work was to further validate [18F]PRIMATX as a PET imaging agent by analyzing its in vivo metabolic stability and suitability for PET imaging of ATX in models of human 8305C thyroid tumor and murine 4T1 breast cancer. [18F]PRIMATX displayed favorable metabolic stability in vivo (65% of intact radiotracer after 60 min p.i.) and provided sufficient tumor uptake profiles in both tumor models. Radiotracer uptake could be blocked by 8-12% in 8305C thyroid tumors in the presence of ATX inhibitor AE-32-NZ70 as determined by PET and ex vivo biodistribution analyses. [18F]PRIMATX also showed high brain uptake, which was reduced by 50% through the administration of ATX inhibitor AE-32-NZ70. [18F]PRIMATX is a suitable radiotracer for PET imaging of ATX in the brain and peripheral tumor tissues.

PDGFRα Enhanced Infection of Breast Cancer Cells with Human Cytomegalovirus but Infection of Fibroblasts Increased Prometastatic Inflammation Involving Lysophosphatidate Signaling.

Human cytomegalovirus (HCMV) infects 40-70% of adults in developed countries. HCMV proteins and DNA are detected in tumors and metastases, suggesting an association with increased invasion. We investigated HCMV infection in human breast cancer cell lines compared to fibroblasts, a component of tumors, and the role of platelet-derived growth factor receptor-α (PDGFRα). HCMV productively infected HEL299 fibroblasts and, to a lesser extent, Hs578T breast cancer cells. Infection of another triple-negative cell line, MDA-MB-231, and also MCF-7 cells, was extremely low. These disparate infection rates correlated with expression of PDGFRA, which facilitates HCMV uptake. Increasing PDGFRA expression in T-47D breast cancer and BCPAP thyroid cancer cells markedly increased HCMV infection. Conversely, HCMV infection decreased PDGFRA expression, potentially attenuating signaling through this receptor. HCMV infection of fibroblasts promoted the secretion of proinflammatory factors, whereas an overall decreased secretion of inflammatory factors was observed in infected Hs578T cells. We conclude that HCMV infection in tumors will preferentially target tumor-associated fibroblasts and breast cancer cells expressing PDGFRα. HCMV infection in the tumor microenvironment, rather than cancer cells, will increase the inflammatory milieu that could enhance metastasis involving lysophosphatidate.

Dexamethasone decreases the autotaxin-lysophosphatidate-inflammatory axis in adipose tissue: implications for the metabolic syndrome and breast cancer.

Lysophosphatidate (LPA) signaling through 6 receptors is regulated by the balance of LPA production by autotaxin (ATX) vs. LPA degradation by lipid phosphate phosphatases (LPPs). LPA promotes an inflammatory cycle by increasing the synthesis of cyclooxygenase-2 and multiple inflammatory cytokines that stimulate further ATX production. We aimed to determine whether the anti-inflammatory glucocorticoid (GC) dexamethasone (Dex) functions partly by decreasing the ATX-LPA inflammatory cycle in adipose tissue, a major site of ATX secretion. Treatment of human adipose tissue with 10-1000 nM Dex decreased ATX secretion, increased LPP1 expression, and decreased mRNA expressions of IL-6, TNF-α, peroxisome proliferator-activated receptor (PPAR)-γ, and adiponectin. Cotreatment with rosiglitazone (an insulin sensitizer), insulin, or both abolished Dex-induced decreases in ATX and adiponectin secretion, but did not reverse Dex-induced decreases in secretions of 20 inflammatory cytokines and chemokines. Dex-treated mice exhibited lower ATX activity in plasma, brain, and adipose tissue; decreased mRNA levels for LPA and sphingosine 1-phosphate (S1P) receptors in brain; and decreased plasma concentrations of LPA and S1P. Our results establish a novel mechanism for the anti-inflammatory effects of Dex through decreased signaling by the ATX-LPA-inflammatory axis. The GC action in adipose tissue has implications for the pathogenesis of insulin resistance and obesity in metabolic syndrome and breast cancer treatment.-Meng, G., Tang, X., Yang, Z., Zhao, Y., Curtis, J. M., McMullen, T. P. W., Brindley, D. N. Dexamethasone decreases the autotaxin-lysophosphatidate-inflammatory axis in adipose tissue: implications for the metabolic syndrome and breast cancer.

Role of Adipose Tissue-Derived Autotaxin, Lysophosphatidate Signaling, and Inflammation in the Progression and Treatment of Breast Cancer.

Autotaxin (ATX) is a secreted enzyme that produces lysophosphatidate (LPA), which signals through six G-protein coupled receptors, promoting tumor growth, metastasis, and survival from chemotherapy and radiotherapy. Many cancer cells produce ATX, but breast cancer cells express little ATX. In breast tumors, ATX is produced by tumor-associated stroma. Breast tumors are also surrounded by adipose tissue, which is a major bodily source of ATX. In mice, a high-fat diet increases adipocyte ATX production. ATX production in obesity is also increased because of low-level inflammation in the expanded adipose tissue. This increased ATX secretion and consequent LPA signaling is associated with decreased adiponectin production, which results in adverse metabolic profiles and glucose homeostasis. Increased ATX production by inflamed adipose tissue may explain the obesity-breast cancer association. Breast tumors produce inflammatory mediators that stimulate ATX transcription in tumor-adjacent adipose tissue. This drives a feedforward inflammatory cycle since increased LPA signaling increases production of more inflammatory mediators and cyclooxygenase-2. Inhibiting ATX activity, which has implications in breast cancer adjuvant treatments, attenuates this cycle. Targeting ATX activity and LPA signaling may potentially increase chemotherapy and radiotherapy efficacy, and decrease radiation-induced fibrosis morbidity independently of breast cancer type because most ATX is not derived from breast cancer cells.

Bioprocess decision support tool for scalable manufacture of extracellular vesicles.

Newly recognized as natural nanocarriers that deliver biological information between cells, extracellular vesicles (EVs), including exosomes and microvesicles, provide unprecedented therapeutic opportunities. Large-scale and cost-effective manufacturing is imperative for EV products to meet commercial and clinical demands; successful translation requires careful decisions that minimize financial and technological risks. Here, we develop a decision support tool (DST) that computes the most cost-effective technologies for manufacturing EVs at different scales, by examining the costs of goods associated with using published protocols. The DST identifies costs of labor and consumables during EV harvest as key cost drivers, substantiating a need for larger-scale, higher-throughput, and automated technologies for harvesting EVs. Importantly, we highlight a lack of appropriate technologies for meeting clinical demands, and propose a potentially cost-effective solution. This DST can facilitate decision-making very early on in development and be used to predict, and better manage, the risk of process changes when commercializing EV products.

CAR-T Cells: A Systematic Review and Mixed Methods Analysis of the Clinical Trial Landscape.

CAR-T cells are a promising new therapy that offer significant advantages compared with conventional immunotherapies. This systematic review and clinical trial landscape identifies and critiques published CAR-T cell clinical trials and examines the critical factors required to enable CAR-T cells to become a standard therapy. A review of the literature was conducted to identify suitable studies from the MEDLINE and Ovid bibliographic databases. The literature and database searches identified 20 studies for inclusion. The average number of participants per clinical trial examined was 11 patients. All studies included in this systematic review investigated CAR-T cells and were prospective, uncontrolled clinical studies. Leukemia is the most common cancer subtype and accounts for 57.4% (n = 120) of disease indications. The majority of studies used an autologous cell source (85%, n = 17) rather than an allogeneic cell source. Translational challenges encompass technical considerations relating to CAR-T cell development, manufacturing practicability, clinical trial approaches, CAR-T cell quality and persistence, and patient management.

Design Choices and Trade-Offs in Health Care Blockchain Implementations: Systematic Review.

BACKGROUND: A blockchain is a list of records that uses cryptography to make stored data immutable; their use has recently been proposed for electronic medical record (EMR) systems. This paper details a systematic review of trade-offs in blockchain technologies that are relevant to EMRs. Trade-offs are defined as "a compromise between two desirable but incompatible features." OBJECTIVE: This review's primary research question was: "What are the trade-offs involved in different blockchain designs that are relevant to the creation of blockchain-based electronic medical records systems?" METHODS: Seven databases were systematically searched for relevant articles using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Papers published from January 1, 2017 to June 15, 2018 were selected. Quality assessments of papers were performed using the Risk Of Bias In Non-randomized Studies-of Interventions (ROBINS-I) tool and the Critical Assessment Skills Programme (CASP) tool. Database searches identified 2885 articles, of which 15 were ultimately included for analysis. RESULTS: A total of 17 trade-offs were identified impacting the design, development, and implementation of blockchain systems; these trade-offs are organized into themes, including business, application, data, and technology architecture. CONCLUSIONS: The key findings concluded the following: (1) multiple trade-offs can be managed adaptively to improve EMR utility; (2) multiple trade-offs involve improving the security of blockchain systems at the cost of other features, meaning EMR efficacy highly depends on data protection standards; and (3) multiple trade-offs result in improved blockchain scalability. Consideration of these trade-offs will be important to the specific environment in which electronic medical records are being developed. This review also uses its findings to suggest useful design choices for a hypothetical National Health Service blockchain. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/10994.

Examining Cost Measurements in Production and Delivery of Three Case Studies Using E-Learning for Applied Health Sciences: Cross-Case Synthesis.

BACKGROUND: The World Health Report (2006) by the World Health Organization conveys that a significant increase is needed in global health care resourcing to meet the current and future demand for health professionals. Electronic learning (e-Learning) presents a possible opportunity to change and optimize training by providing a scalable means for instruction, thus reducing the costs for training health professionals and providing patient education. Research literature often suggests that a benefit of e-Learning is its cost-effectiveness compared with face-to-face instruction, yet there is limited evidence with respect to the comparison of design and production costs with other forms of instruction or the establishment of standards pertaining to budgeting for these costs. OBJECTIVE: To determine the potential cost favorability of e-Learning in contrast to other forms of learning, there must first be an understanding of the components and elements for building an e-Learning course. Without first taking this step, studies lack the essential financial accounting rigor for course planning and have an inconsistent basis for comparison. This study aimed to (1) establish standard ingredients for the cost of e-Learning course production and (2) determine the variance instructional design has on the production costs of e-Learning courses. METHODS: This study made use of a cross-case method among 3 case studies using mixed methods, including horizontal budget variance calculation and qualitative interpretation of responses from course designers for budget variance using total quality management themes. The different implementation-specific aspects of these cases were used to establish common principles in the composition of budgets in the production and delivery of an applied health professional e-Learning course. RESULTS: A total of 2 case studies reported significant negative budget variances caused by issues surrounding underreporting of personnel costs, inaccurate resource task estimation, lack of contingency planning, challenges in third-party resource management, and the need to update health-related materials that became outdated during course production. The third study reported a positive budget variance because of the cost efficiency derived from previous implementation, the strong working relationship of the course project team, and the use of iterative project management methods. CONCLUSIONS: This research suggests that the delivery costs of an e-Learning course could be underestimated or underreported and identifies factors that could be used to better control budgets. Through consistent management of factors affecting the cost of course production, further research could be undertaken using standard economic evaluation methods to evaluate the advantages of using e-Learning.

Implementing Blockchains for Efficient Health Care: Systematic Review.

BACKGROUND: The decentralized nature of sensitive health information can bring about situations where timely information is unavailable, worsening health outcomes. Furthermore, as patient involvement in health care increases, there is a growing need for patients to access and control their data. Blockchain is a secure, decentralized online ledger that could be used to manage electronic health records (EHRs) efficiently, therefore with the potential to improve health outcomes by creating a conduit for interoperability. OBJECTIVE: This study aimed to perform a systematic review to assess the feasibility of blockchain as a method of managing health care records efficiently. METHODS: Reviewers identified studies via systematic searches of databases including PubMed, MEDLINE, Scopus, EMBASE, ProQuest, and Cochrane Library. Suitability for inclusion of each was assessed independently. RESULTS: Of the 71 included studies, the majority discuss potential benefits and limitations without evaluation of their effectiveness, although some systems were tested on live data. CONCLUSIONS: Blockchain could create a mechanism to manage access to EHRs stored on the cloud. Using a blockchain can increase interoperability while maintaining privacy and security of data. It contains inherent integrity and conforms to strict legal regulations. Increased interoperability would be beneficial for health outcomes. Although this technology is currently unfamiliar to most, investments into creating a sufficiently user-friendly interface and educating users on how best to take advantage of it would lead to improved health outcomes. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/10994.

Bioprocessing automation in cell therapy manufacturing: Outcomes of special interest group automation workshop.

Phacilitate held a Special Interest Group workshop event in Edinburgh, UK, in May 2017. The event brought together leading stakeholders in the cell therapy bioprocessing field to identify present and future challenges and propose potential solutions to automation in cell therapy bioprocessing. Here, we review and summarize discussions from the event. Deep biological understanding of a product, its mechanism of action and indication pathogenesis underpin many factors relating to bioprocessing and automation. To fully exploit the opportunities of bioprocess automation, therapeutics developers must closely consider whether an automation strategy is applicable, how to design an 'automatable' bioprocess and how to implement process modifications with minimal disruption. Major decisions around bioprocess automation strategy should involve all relevant stakeholders; communication between technical and business strategy decision-makers is of particular importance. Developers should leverage automation to implement in-process testing, in turn applicable to process optimization, quality assurance (QA)/ quality control (QC), batch failure control, adaptive manufacturing and regulatory demands, but a lack of precedent and technical opportunities can complicate such efforts. Sparse standardization across product characterization, hardware components and software platforms is perceived to complicate efforts to implement automation. The use of advanced algorithmic approaches such as machine learning may have application to bioprocess and supply chain optimization. Automation can substantially de-risk the wider supply chain, including tracking and traceability, cryopreservation and thawing and logistics. The regulatory implications of automation are currently unclear because few hardware options exist and novel solutions require case-by-case validation, but automation can present attractive regulatory incentives.

Implications for breast cancer treatment from increased autotaxin production in adipose tissue after radiotherapy.

We have previously established that adipose tissue adjacent to breast tumors becomes inflamed by tumor-derived cytokines. This stimulates autotaxin (ATX) secretion from adipocytes, whereas breast cancer cells produce insignificant ATX. Lysophosphatidate produced by ATX promotes inflammatory cytokine secretion in a vicious inflammatory cycle, which increases tumor growth and metastasis and decreases response to chemotherapy. We hypothesized that damage to adipose tissue during radiotherapy for breast cancer should promote lysophosphatidic acid (LPA) signaling and further inflammatory signaling, which could potentially protect cancer cells from subsequent fractions of radiation therapy. To test this hypothesis, we exposed rat and human adipose tissue to radiation doses (0.25-5 Gy) that were expected during radiotherapy. This exposure increased mRNA levels for ATX, cyclooxygenase-2, IL-1ß, IL-6, IL-10, TNF-α, and LPA1 and LPA2 receptors by 1.8- to 5.1-fold after 4 to 48 h. There were also 1.5- to 2.5-fold increases in the secretion of ATX and 14 inflammatory mediators after irradiating at 1 Gy. Inhibition of the radiation-induced activation of NF-κB, cyclooxygenase-2, poly (ADP-ribose) polymerase-1, or ataxia telangiectasia and Rad3-related protein blocked inflammatory responses to γ-radiation. Consequently, collateral damage to adipose tissue during radiotherapy could establish a comprehensive wound-healing response that involves increased signaling by LPA, cyclooxygenase-2, and other inflammatory mediators that could decrease the efficacy of further radiotherapy or chemotherapy.-Meng, G., Tang, X., Yang, Z., Benesch, M. G. K., Marshall, A., Murray, D., Hemmings, D. G., Wuest, F., McMullen, T. P. W., Brindley, D. N. Implications for breast cancer treatment from increased autotaxin production in adipose tissue after radiotherapy.

Decision Support Tools for Regenerative Medicine: Systematic Review.

BACKGROUND: Decisional tools have demonstrated their importance in informing manufacturing and commercial decisions in the monoclonal antibody domain. Recent approved therapies in regenerative medicine have shown great clinical benefits to patients. OBJECTIVE: The objective of this review was to investigate what decisional tools are available and what issues and gaps have been raised for their use in regenerative medicine. METHODS: We systematically searched MEDLINE to identify articles on decision support tools relevant to tissue engineering, and cell and gene therapy, with the aim of identifying gaps for future decisional tool development. We included published studies in English including a description of decisional tools in regenerative medicines. We extracted data using a predesigned Excel table and assessed the data both quantitatively and qualitatively. RESULTS: We identified 9 articles addressing key decisions in manufacturing and product development challenges in cell therapies. The decision objectives, parameters, assumptions, and solution methods were analyzed in detail. We found that all decisional tools focused on cell therapies, and 6 of the 9 reviews focused on allogeneic cell therapy products. We identified no available tools on tissue-engineering and gene therapy products. These studies addressed key decisions in manufacturing and product development challenges in cell therapies, such as choice of technology, through modeling. CONCLUSIONS: Our review identified a limited number of decisional tools. While the monoclonal antibodies and biologics decisional tool domain has been well developed and has shown great importance in driving more cost-effective manufacturing processes and better investment decisions, there is a lot to be learned in the regenerative medicine domain. There is ample space for expansion, especially with regard to autologous cell therapies, tissue engineering, and gene therapies. To consider the problem more comprehensively, the full needle-to-needle process should be modeled and evaluated.

Weighing benefits and risks in aspects of security, privacy and adoption of technology in a value-based healthcare system.

BACKGROUND: Technology can potentially enable the implementation of a value-based healthcare system, where the impact of quality of care is offered at optimised cost for maximised patient benefit. Technology can deliver value by aiding in data collection to evaluate outcomes and measure costs on a patient and population level. Healthcare organisations, however, face several challenges and risks that result almost exclusively from the use of these technologies. DISCUSSION: Some challenges associated with healthcare technology include their unsustainability, due to lack of scale-up plans and timely evaluations. Other risks include noncompliance with data protection policies, inadequate data governance, and overestimated expectations resulting from the rapid introduction of new technologies. CONCLUSION: Organisations need to consider the risks and challenges associated with the use of technology and develop comprehensive strategies that mitigate factors leading to non-adoption and to realise benefits for achieving a value-based healthcare system.

Adipose-Derived Stem Cells in Aesthetic Surgery: A Mixed Methods Evaluation of the Current Clinical Trial, Intellectual Property, and Regulatory Landscape.

BACKGROUND: Adipose tissue, which can be readily harvested via a number of liposuction techniques, offers an easily accessible and abundant source of adipose-derived stem cells (ASCs). Consequently, ASCs have become an increasingly popular reconstructive option and a novel means of aesthetic soft tissue augmentation. OBJECTIVES: This paper examines recent advances in the aesthetic surgery field, extending beyond traditional review formats to incorporate a comprehensive analysis of current clinical trials, adoption status, and the commercialization pathway. METHODS: Keyword searches were carried out on clinical trial databases to search for trials using ASCs for aesthetic indications. An intellectual property landscape was created using commercial software (Thomson Reuters Thomson Innovation, New York, NY). Analysis of who is claiming what in respect of ASC use in aesthetic surgery for commercial purposes was analyzed by reviewing the patent landscape in relation to these techniques. Key international regulatory guidelines were also summarized. RESULTS: Completed clinical trials lacked robust controls, employed small sample sizes, and lacked long-term follow-up data. Ongoing clinical trials still do not address such issues. In recent years, claims to intellectual property ownership have increased in the "aesthetic stem cell" domain, reflecting commercial interest in the area. However, significant translational barriers remain including regulatory challenges and ethical considerations. CONCLUSIONS: Further rigorous randomized controlled trials are required to delineate long-term clinical efficacy and safety. Providers should consider the introduction of patient reported outcome metrics to facilitate clinical adoption. Robust regulatory and ethical policies concerning stem cells and aesthetic surgery should be devised to discourage further growth of "stem cell tourism."

Normal human adipose tissue functions and differentiation in patients with biallelic LPIN1 inactivating mutations.

Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase (PAP) that in mice is necessary for normal glycerolipid biosynthesis, controlling adipocyte metabolism, and adipogenic differentiation. Mice carrying inactivating mutations in the Lpin1 gene display the characteristic features of human familial lipodystrophy. Very little is known about the roles of lipin-1 in human adipocyte physiology. Apparently, fat distribution and weight is normal in humans carrying LPIN1 inactivating mutations, but a detailed analysis of adipose tissue appearance and functions in these patients has not been available so far. In this study, we performed a systematic histopathological, biochemical, and gene expression analysis of adipose tissue biopsies from human patients harboring LPIN1 biallelic inactivating mutations and affected by recurrent episodes of severe rhabdomyolysis. We also explored the adipogenic differentiation potential of human mesenchymal cell populations derived from lipin-1 defective patients. White adipose tissue from human LPIN1 mutant patients displayed a dramatic decrease in lipin-1 protein levels and PAP activity, with a concomitant moderate reduction of adipocyte size. Nevertheless, the adipose tissue develops without obvious histological signs of lipodystrophy and with normal qualitative composition of storage lipids. The increased expression of key adipogenic determinants such as SREBP1, PPARG, and PGC1A shows that specific compensatory phenomena can be activated in vivo in human adipocytes with deficiency of functional lipin-1.

Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB activation.

BACKGROUND: We previously discovered that tetracyclines increase the expression of lipid phosphate phosphatases at the surface of cells. These enzymes degrade circulating lysophosphatidate and therefore doxycycline increases the turnover of plasma lysophosphatidate and decreases its concentration. Extracellular lysophosphatidate signals through six G protein-coupled receptors and it is a potent promoter of tumor growth, metastasis and chemo-resistance. These effects depend partly on the stimulation of inflammation that lysophosphatidate produces. METHODS: In this work, we used a syngeneic orthotopic mouse model of breast cancer to determine the impact of doxycycline on circulating lysophosphatidate concentrations and tumor growth. Cytokine/chemokine concentrations in tumor tissue and plasma were measured by multiplexing laser bead technology. Leukocyte infiltration in tumors was analyzed by immunohistochemistry. The expression of IL-6 in breast cancer cell lines was determined by RT-PCR. Cell growth was measured in Matrigel™ 3D culture. The effects of doxycycline on NF-κB-dependent signaling were analyzed by Western blotting. RESULTS: Doxycycline decreased plasma lysophosphatidate concentrations, delayed tumor growth and decreased the concentrations of several cytokines/chemokines (IL-1ß, IL-6, IL-9, CCL2, CCL11, CXCL1, CXCL2, CXCL9, G-CSF, LIF, VEGF) in the tumor. These results were compatible with the effects of doxycycline in decreasing the numbers of F4/80+ macrophages and CD31+ blood vessel endothelial cells in the tumor. Doxycycline also decreased the lysophosphatidate-induced growth of breast cancer cells in three-dimensional culture. Lysophosphatidate-induced Ki-67 expression was inhibited by doxycycline. NF-κB activity in HEK293 cells transiently expressing a NF-κB-luciferase reporter vectors was also inhibited by doxycycline. Treatment of breast cancer cells with doxycycline also decreased the translocation of NF-κB to the nucleus and the mRNA levels for IL-6 in the presence or absence of lysophosphatidate. CONCLUSION: These results contribute a new dimension for understanding the anti-inflammatory effects of tetracyclines, which make them potential candidates for adjuvant therapy of cancers and other inflammatory diseases.

Regulatory Considerations for Gene Therapy Products in the US, EU, and Japan.

Developers of gene therapy products (GTPs) must adhere to additional regulation beyond that of traditional small-molecule therapeutics, due to the unique mechanism-of-action of GTPs and the subsequent novel risks arisen. We have provided herein a summary of the regulatory structure under which GTPs fall in the United States, the European Union, and Japan, and a comprehensive overview of the regulatory guidance applicable to the developer of GTP. Understanding the regulatory requirements for seeking GTP market approval in these major jurisdictions is crucial for an effective and expedient path to market. The novel challenges facing GTP developers is highlighted by a case study of alipogene tiparvovec (Glybera).

Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate.

Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 µg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs.

Lysophosphatidate signaling stabilizes Nrf2 and increases the expression of genes involved in drug resistance and oxidative stress responses: implications for cancer treatment.

The present work elucidates novel mechanisms for lysophosphatidate (LPA)-induced chemoresistance using human breast, lung, liver, and thyroid cancer cells. LPA (0.5-10 µM) increased Nrf2 transcription factor stability and nuclear localization by ≤5-fold. This involved lysophosphatidate type 1 (LPA1) receptors as identified with 1 µM wls-31 (LPA1/2 receptor agonist) and blocking this effect with 20 µM Ki16425 (LPA1-3 antagonist, Ki = 0.34 µM). Knockdown of LPA1 by 50% to 60% with siRNA decreased Nrf2 stability and expressing LPA1, but not LPA2/3, in human HepG2 cells increased Nrf2 stabilization. LPA-induced Nrf2 expression increased transcription of multidrug-resistant transporters and antioxidant genes by 2- to 4-fold through the antioxidant response element. This protected cells from doxorubicin-induced death. This pathway was verified in vivo by orthotopic injection of 20,000 mouse 4T1 breast cancer cells into syngeneic mice. Blocking LPA production with 10 mg/kg per d ONO-8430506 (competitive autotaxin inhibitor, IC90 = 100 nM) decreased expression of Nrf2, multidrug-resistant transporters, and antioxidant genes in breast tumors by ≤90%. Combining 4 mg/kg doxorubicin every third day with ONO-8430506 synergistically decreased tumor growth and metastasis to lungs and liver by >70%, whereas doxorubicin alone had no significant effect. This study provides the first evidence that LPA increases antioxidant gene and multidrug-resistant transporter expression. Blocking this aspect of LPA signaling provides a novel strategy for improving chemotherapy.