The role of mTOR in SAF-1-mediated VEGF expression and breast cancer progression

Breast cancer is a heterogeneous disease with a huge impact on the community evident from its high indices of morbidity and mortality worldwide. In recent years, research has been extensively directed toward unravelling the molecular basis of the pathogenesis in different molecular subtypes of breast cancer. Better understanding of the different breast cancer cells and the subsequent improvement of diagnostic and therapeutic modalities of the disease resulted in a promising decline in its morbidity and mortality. In addition to surgery, radiotherapy, and conventional chemotherapy, treatment of breast cancer, nowadays, involves more targeted therapeutic options. Treatment options of breast cancer are determined mainly by the tumor stage and molecular subtypes of the tumor cells. Invasive potentials of a tumor involve also its ability to recruit blood supply through a process known as angiogenesis. Angiogenesis is an intricate process mediated by the interaction of many players including the tumor cells and stroma as well as the endothelial cells in adjacent blood vessels. Vascular endothelial growth factor (VEGF) is one of the main proangiogenic molecules secreted by breast cancer cells to promote angiogenesis through binding to the vascular endothelial growth factor receptors (VEGFR) on target cells. Although angiogenesis is a hallmark of invasive solid tumors, it is also required in some normal physiological conditions. Targeting angiogenesis indiscriminately imposes a huge risk of enormous adverse effects. Therefore, the goal of research today is to explore potential mechanisms to counteract angiogenesis selectively in the tumor cells. Secretion of VEGF by breast cancer cells is activated by several stimuli including hypoxia, metabolic stress and inflammatory conditions. Hypoxia is one of the main drivers for both physiological and pathological angiogenesis. Hypoxia induces VEGF expression in hypoxic cells through the action of hypoxia inducible factor-1 (HIF-1). However, evidence indicates that breast cancer cells are capable of secreting VEGF at early stages before hypoxia ensues in the tumor mass. Evidence shows also that breast cancer cells secrete VEGF even under normoxic conditions, which suggests that VEGF expression in breast cancer, particularly at early stages, is mediated by a mechanism(s) other than hypoxia. The goal of our research is to investigate some of these potential mechanism(s). Unravelling these mechanism(s) could pave the road for potentially novel therapeutic modalities in the treatment of breast cancer. Previous work in our lab has identified a role of serum amyloid activating factor-1 (SAF-1), a transcription factor, in overexpression of VEGF in some breast cancer cells. Here, we report that the SAF-1-mediated VEGF expression in breast cancer cells is repressed by Kruppel like factor-4 (KLF-4) transcription factor. Our findings suggest also that KLF-4 is potentially involved in the repression of the VEGF expression in a SAF-1-independent manner. We found that the level of KLF-4 is lower in breast cancer cells compared to normal breast cells. Therefore, we further investigated possible mechanism(s) for upregulation of KLF-4 or downregulation of SAF-1 in breast cancer cells to curtail VEGF expression and counteract angiogenesis and thence progression of breast cancer. There is evidence in literature that inhibition of a serine-threonine kinase called mammalian target of rapamycin (mTOR) exerts antiproliferative effect in vascular smooth muscle cells (VSMC). Evidence showed that the effect of mTOR inhibition on VSMC is mediated by upregulation of KLF-4 through a yet unknown mechanism. Thus, we have explored a potential role of mTOR inhibition in upregulation of KLF-4 in breast cancer cells and inhibition of VEGF expression. VEGF secreted by breast cancer cells not only stimulates proliferation and migration of the endothelial cells, but also promotes proliferation and migration of the secreting breast cancer cells themselves. Mounting evidence substantiates the beneficial role of mTOR inhibition in breast cancer. Our results show a novel mechanism of upregulating KLF-4 and inhibition of VEGF expression in breast cancer cells through inhibition of mTOR. Nonetheless, we noted that the effects of mTOR inhibition on breast cancer cells, including upregulation of KLF-4 and inhibition of proliferation and migration, are variable among different breast cell lines as well as among different mTOR inhibitors. One of the possible explanation of the variable response to mTOR inhibitors is the rebound upregulation of proteins in the mTOR pathway which imposes a risk of emergence of resistance or refractoriness to treatment with mTOR inhibition. The role of active mTOR in the invasiveness of breast cancer is well known, yet relatively little is known about the role and impact of total mTOR protein. We found that total mTOR protein level is higher in breast cancer cells compared to their noncancerous counterparts. mTOR protein is particularly high in the ER+ breast cancer cells which constitute the majority of breast cancer cells subtypes. High mTOR protein level in breast cancer cells could be attributed to decreased mTOR protein degradation, increased mTOR gene expression, or both. Our results indicate a defective degradation of mTOR in breast cancer cells compared to normal cells, which could, at least in part, explain why mTOR protein is high in breast cancer cells. Our results show also that transcription of mTOR gene is elevated in the ER+ breast cancer cells. Our study revealed that promoter region of mTOR in the ER+ breast cancer cells has a truncated dinucleotide tandem repeat region. Tandem repeats are a potential site for regulation of transcription of genes. Shortening of this region could be a possible mechanism of increased transcription of mTOR gene in the ER+ breast cancer cells. Moreover, we have revealed a novel mechanism of increasing mTOR degradation as well as inhibition of MTOR transcription in the ER+ breast cancer cells by treatment with metformin the antidiabetic mTOR inhibitor. In correlation, metformin treatment induced a profound effect on upregulation of KLF-4 and inhibition of proliferation and migration of the breast cancer cells, particularly, the ER+ subtype. These findings could be utilized in the optimization of chemotherapeutic regimens of breast cancer

Involvement of People in the Design of Community Building in Developing Countries

Although public participation concept or technique to enhance and /or influence decision making is a fact of life in many governments nowadays and has been used by many industries to improve their products and increase end-users satisfaction, it is still new to the Architectural, Engineering and Construction (AEC) industry. The extent to which participation should occur and the role that it plays still remains uncertain in the AEC industry. Participation has been the predominant conception for many years by many researchers. In practice, this has meant that public participation often has occurred late in the planning process. This research will focus on the involvement of people in the design process of community buildings in developing countries. During the last five decade, many community buildings were built and many of these buildings did not performed well as planned. Many of these problems in were related to design, hence the choice of architectural design of these buildings for this research. If the end-users of these buildings were involved in the planning and design stages, many of these problems would not occur. Thus, the aim of this research is to develop a conceptual framework to involve the public in the design of community buildings in developing countries. A thorough review of previous works related to the public participation in the design of community buildings has been conducted as part of this research. The review revealed most of these works in this area were done on developed countries and no research works were conducted on developing countries so far

DNAJA1- and conformational mutant p53-dependent inhibition of cancer cell migration by a novel compound identified through a virtual screen

Cancers are frequently addicted to oncogenic missense mutant p53 (mutp53). DNAJA1, a member of heat shock protein 40 (HSP40), also known as J-domain proteins (JDPs), plays a crucial role in the stabilization and oncogenic activity of misfolded or conformational mutp53 by binding to and preventing mutp53 from proteasomal degradation. However, strategies to deplete mutp53 are not well-established, and no HSP40/JDPs inhibitors are clinically available. To identify compounds that bind to DNAJA1 and induce mutp53 degradation, we performed an in silico docking study of ~10 million of compounds from the ZINC database for the J-domain of DNAJA1. A compound 7-3 was identified, and its analogue A11 effectively reduced the levels of DNAJA1 and conformational mutp53 with minimal effects on the levels of wild-type p53 and DNA-contact mutp53. A11 suppressed migration and filopodia formation in a manner dependent on DNAJA1 and conformational mutp53. A mutant DNAJA1 with alanine mutations at predicted amino acids (tyrosine 7, lysine 44, and glutamine 47) failed to bind to A11. Cells expressing the mutant DNAJA1 became insensitive to A11-mediated depletion of DNAJA1 and mutp53 as well as A11-mediated inhibition of cell migration. Thus, A11 is the first HSP40/JDP inhibitor that has not been previously characterized for depleting DNAJA1 and subsequently conformational mutp53, leading to inhibition of cancer cell migration. A11 can be exploited for a novel treatment against cancers expressing conformational mutp53

Insulin Signaling Network in Cancer

493-498The primary function of insulin is viewed as a hormone that controls blood glucose level. However, there is growing evidence that aberrant insulin level and insulin-mediated signaling can lead to cancer development and progression. The insulin-cancer relationship has stemmed from various observational and epidemiological studies, which linked higher incidence of cancer with central obesity, type II diabetes and other conditions associated with increased levels of circulating insulin, insulin resistance and hyperinsulinemic states. Increased risk of developing a range of cancers is also seen with a certain treatment options used to lower blood glucose level in diabetic patients. While metformin monotherapy has the lowest risk of developing cancer, in comparison, treatment with insulin or insulin secretagogues shows more likelihood to develop solid cancers. Cellular signaling initiated by insulin provides a clue regarding these diverse cellular outcomes. This review discusses how the insulin enacts such diverse physiological effects and the insulin-cancer relationship, with focus on the role of insulin signaling in cancer. </span

MicroRNA-141-regulated KLK10 and TNFSF-15 gene expression in hepatoblastoma cells as a novel mechanism in liver carcinogenesis

Abstract Liver cancer is one of the most pivotal global health problems, leading hepatocellular carcinoma (HCC) with a significant increase in cases worldwide. The role of non-coding-RNA in cancer proliferation and carcinogenesis has attracted much attention in the last decade; however, microRNAs (miRNAs), as non-coding RNA, are considered master mediators in various cancer progressions. Yet the role of miR-141 as a modulator for specific cellular processes in liver cancer cell proliferation is still unclear. This study identified the role of miR-141 and its potential functions in liver carcinogenesis. The level of miR-141 in HepG2 and HuH7 cells was assessed using quantitative real-time PCR (qRT-PCR) and compared with its expression in normal hepatocytes. A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HepG2 cells. The protein profile of the kallikrein-related peptidase 10 (KLK10) and tumor necrosis factor TNFSF-15 was investigated in HepG2 cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced inflammatory cytokines from transfected HepG2 cells. Our findings showed that the expression of miR-141 significantly increased in HepG2 and HuH7 cells compared to the normal hepatocytes. Transfection of HepG2 cells with an inhibitor, antagonist miR-141, showed a significant reduction of HepG2 cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HepG2 cells overexpressed miR-141 provided a hundred downregulated genes, including KLK10 and TNFSF-15. Furthermore, the expression profile of KLK10 and TNFSF-15 markedly depleted in HepG2 cells transfected with miR-141 overexpression accompanied by a decreasing level of interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α), indicating the role of miR-141 in HepG2 cell proliferation and programmed cell death. Interestingly, the experimental rats with liver cancer induced by Diethylnitrosamine injection further confirmed the upregulation of miR-141 level, IL-10, and TNF-α and the disturbance in KLK10 and TNFSF-15 gene expression compared with their expression in normal rats. The in-silico online tools, IntaRNA and miRWalk were used to confirm the direct interaction and potential binding sites between miR-141 and identified genes. Thus, the seeding regions of potential targeted sequences was cloned upstream of luciferase reporter gene in pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HepG2 cells pre-transfected with miR-141 overexpression vector, while increasing in cells pre-transfected with miR-141 specific inhibitor. In summary, these data suggest the crucial role of miR-141 in liver cancer development via targeting KLK10 and TNFSF-15 and provide miR-141 as an attractive candidate in liver cancer treatment and protection

Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

Accumulation of missense mutant p53 (mutp53) in cancers promotes malignant progression. DNAJA1, a member of HSP40 (also known as J-domain proteins: JDPs), is shown to prevent misfolded or conformational mutp53 from proteasomal degradation. Given frequent addiction of cancers to oncogenic mutp53, depleting mutp53 by DNAJA1 inhibition is a promising approach for cancer therapy. However, there is no clinically available inhibitor for DNAJA1. Our in silico molecular docking study with a natural compound-derived small molecule library identified a plumbagin derivative, PLIHZ (plumbagin&ndash;isoniazid analog), as a potential compound binding to the J domain of DNAJA1. PLIHZ efficiently reduced the levels of DNAJA1 and several conformational mutp53 with minimal impact on DNA contact mutp53 and wild-type p53 (wtp53). An analog, called PLTFBH, which showed a similar activity to PLIHZ in reducing DNAJA1 and mutp53 levels, inhibited migration of cancer cells specifically carrying conformational mutp53, but not DNA contact mutp53, p53 null, and wtp53, which was attenuated by depletion of DNAJA1 or mutp53. Moreover, PLTFBH reduced levels of multiple other HSP40/JDPs with tyrosine 7 (Y7) and/or tyrosine 8 (Y8) but failed to deplete DNAJA1 mutants with alanine substitution of these amino acids. Our study suggests PLTFBH as a potential inhibitor for multiple HSP40/JDPs

The ASOS Surgical Risk Calculator: development and validation of a tool for identifying African surgical patients at risk of severe postoperative complications

Background: The African Surgical Outcomes Study (ASOS) showed that surgical patients in Africa have a mortality twice the global average. Existing risk assessment tools are not valid for use in this population because the pattern of risk for poor outcomes differs from high-income countries. The objective of this study was to derive and validate a simple, preoperative risk stratification tool to identify African surgical patients at risk for in-hospital postoperative mortality and severe complications. Methods: ASOS was a 7-day prospective cohort study of adult patients undergoing surgery in Africa. The ASOS Surgical Risk Calculator was constructed with a multivariable logistic regression model for the outcome of in-hospital mortality and severe postoperative complications. The following preoperative risk factors were entered into the model; age, sex, smoking status, ASA physical status, preoperative chronic comorbid conditions, indication for surgery, urgency, severity, and type of surgery. Results: The model was derived from 8799 patients from 168 African hospitals. The composite outcome of severe postoperative complications and death occurred in 423/8799 (4.8%) patients. The ASOS Surgical Risk Calculator includes the following risk factors: age, ASA physical status, indication for surgery, urgency, severity, and type of surgery. The model showed good discrimination with an area under the receiver operating characteristic curve of 0.805 and good calibration with c-statistic corrected for optimism of 0.784. Conclusions: This simple preoperative risk calculator could be used to identify high-risk surgical patients in African hospitals and facilitate increased postoperative surveillance. © 2018 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.Medical Research Council of South Africa gran

Maternal and neonatal outcomes after caesarean delivery in the African Surgical Outcomes Study: a 7-day prospective observational cohort study.

BACKGROUND: Maternal and neonatal mortality is high in Africa, but few large, prospective studies have been done to investigate the risk factors associated with these poor maternal and neonatal outcomes. METHODS: A 7-day, international, prospective, observational cohort study was done in patients having caesarean delivery in 183 hospitals across 22 countries in Africa. The inclusion criteria were all consecutive patients (aged ≥18 years) admitted to participating centres having elective and non-elective caesarean delivery during the 7-day study cohort period. To ensure a representative sample, each hospital had to provide data for 90% of the eligible patients during the recruitment week. The primary outcome was in-hospital maternal mortality and complications, which were assessed by local investigators. The study was registered on the South African National Health Research Database, number KZ_2015RP7_22, and on ClinicalTrials.gov, number NCT03044899. FINDINGS: Between February, 2016, and May, 2016, 3792 patients were recruited from hospitals across Africa. 3685 were included in the postoperative complications analysis (107 missing data) and 3684 were included in the maternal mortality analysis (108 missing data). These hospitals had a combined number of specialist surgeons, obstetricians, and anaesthetists totalling 0·7 per 100 000 population (IQR 0·2-2·0). Maternal mortality was 20 (0·5%) of 3684 patients (95% CI 0·3-0·8). Complications occurred in 633 (17·4%) of 3636 mothers (16·2-18·6), which were predominantly severe intraoperative and postoperative bleeding (136 [3·8%] of 3612 mothers). Maternal mortality was independently associated with a preoperative presentation of placenta praevia, placental abruption, ruptured uterus, antepartum haemorrhage (odds ratio 4·47 [95% CI 1·46-13·65]), and perioperative severe obstetric haemorrhage (5·87 [1·99-17·34]) or anaesthesia complications (11·47 (1·20-109·20]). Neonatal mortality was 153 (4·4%) of 3506 infants (95% CI 3·7-5·0). INTERPRETATION: Maternal mortality after caesarean delivery in Africa is 50 times higher than that of high-income countries and is driven by peripartum haemorrhage and anaesthesia complications. Neonatal mortality is double the global average. Early identification and appropriate management of mothers at risk of peripartum haemorrhage might improve maternal and neonatal outcomes in Africa. FUNDING: Medical Research Council of South Africa.Medical Research Council of South Africa