: A Model System for Anti-Cancer Drug Discovery and Therapeutic Target Identification

The nematode Caenorhabditis elegans (C. elegans) offers a unique opportunity for biological and basic medical researches due to its genetic tractability and well-defined developmental lineage. It also provides an exceptional model for genetic, molecular, and cellular analysis of human disease-related genes. Recently, C. elegans has been used as an ideal model for the identification and functional analysis of drugs (or small-molecules) in vivo. In this review, we describe conserved oncogenic signaling pathways (Wnt, Notch, and Ras) and their potential roles in the development of cancer stem cells. During C. elegans germline development, these signaling pathways regulate multiple cellular processes such as germline stem cell niche specification, germline stem cell maintenance, and germ cell fate specification. Therefore, the aberrant regulations of these signaling pathways can cause either loss of germline stem cells or overproliferation of a specific cell type, resulting in sterility. This sterility phenotype allows us to identify drugs that can modulate the oncogenic signaling pathways directly or indirectly through a high-throughput screening. Current in vivo or in vitro screening methods are largely focused on the specific core signaling components. However, this phenotype-based screening will identify drugs that possibly target upstream or downstream of core signaling pathways as well as exclude toxic effects. Although phenotype-based drug screening is ideal, the identification of drug targets is a major challenge. We here introduce a new technique, called Drug Affinity Responsive Target Stability (DARTS). This innovative method is able to identify the target of the identified drug. Importantly, signaling pathways and their regulators in C. elegans are highly conserved in most vertebrates, including humans. Therefore, C. elegans will provide a great opportunity to identify therapeutic drugs and their targets, as well as to understand mechanisms underlying the formation of cancer

Activation of GPR4 by Acidosis Increases Endothelial Cell Adhesion through the cAMP/Epac Pathway

Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2′,5′-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a Gi signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the Gs/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the Gs/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells

A TMEFF2-regulated cell cycle derived gene signature is prognostic of recurrence risk in prostate cancer

Background: The clinical behavior of prostate cancer (PCa) is variable, and while the majority of cases remain indolent, 10% of patients progress to deadly forms of the disease. Current clinical predictors used at the time of diagnosis have limitations to accurately establish progression risk. Here we describe the development of a tumor suppressor regulated, cell-cycle gene expression based prognostic signature for PCa, and validate its independent contribution to risk stratification in several radical prostatectomy (RP) patient cohorts. Methods: We used RNA interference experiments in PCa cell lines to identify a gene expression based gene signature associated with Tmeff2, an androgen regulated, tumor suppressor gene whose expression shows remarkable heterogeneity in PCa. Gene expression was confirmed by qRT-PCR. Correlation of the signature with disease outcome (time to recurrence) was retrospectively evaluated in four geographically different cohorts of patients that underwent RP (834 samples), using multivariate logistical regression analysis. Multivariate analyses were adjusted for standard clinicopathological variables. Performance of the signature was compared to previously described gene expression based signatures using the SigCheck software. Results: Low levels of TMEFF2 mRNA significantly (p \u3c 0.0001) correlated with reduced disease-free survival (DFS) in patients from the Memorial Sloan Kettering Cancer Center (MSKCC) dataset. We identified a panel of 11 TMEFF2 regulated cell cycle related genes (TMCC11), with strong prognostic value. TMCC11 expression was significantly associated with time to recurrence after prostatectomy in four geographically different patient cohorts (2.9 ≤ HR ≥ 4.1; p ≤ 0.002), served as an independent indicator of poor prognosis in the four RP cohorts (1.96 ≤ HR ≥ 4.28; p ≤ 0.032) and improved the prognostic value of standard clinicopathological markers. The prognostic ability of TMCC11 panel exceeded previously published oncogenic gene signatures (p = 0.00017). Conclusions: This study provides evidence that the TMCC11 gene signature is a robust independent prognostic marker for PCa, reveals the value of using highly heterogeneously expressed genes, like Tmeff2, as guides to discover prognostic indicators, and suggests the possibility that low Tmeff2 expression marks a distinct subclass of PCa

A phase II trial of bendamustine in combination with rituximab in older patients with previously untreated diffuse large B-cell lymphoma

Bendamustine in combination with rituximab (BR) has been associated with high response rates and acceptable toxicity in older patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Evaluation of BR is warranted in the front-line setting for DLBCL patients not eligible for anthracyclines or for the elderly. In this phase II study, we enrolled DLBCL patients aged ≥65 years who were poor candidates for R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) to determine the efficacy and safety of BR in previously untreated stage II–IV DLBCL. Twenty-three patients were enrolled with a median age of 80 years. 52% of patients presented with poor functional status (Eastern Cooperative Oncology Group performance score of ≥2). The overall response rate was 78% with 12 complete responses (52%). At a median follow up of 29 months, the median overall survival was 10.2 months and the median progression-free survival was 5.4 months. The most common grade 3/4 adverse events were haematological. Combination therapy with BR demonstrates high response rates as front-line therapy in frail older patients with DLBCL, but survival rates were low. BR should be used with caution in future clinical trials involving older DLBCL patients with poor functional status

The TMEFF2 tumor suppressor modulates integrin expression, RhoA activation and migration of prostate cancer cells

Cell adhesion and migration play important roles in physiological and pathological states, including embryonic development and cancer invasion and metastasis. The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed mainly in brain and prostate and its expression is deregulated in prostate cancer. We have previously shown that TMEFF2 can function as a tumor suppressor by inhibiting cell migration and invasion of prostate cells. However, the molecular mechanisms involved in this inhibition are not clear. In this study we demonstrate that TMEFF2 affects cell adhesion and migration of prostate cancer cells and that this effect correlates with changes in integrin expression and RhoA activation. Deletion of a 13 basic-rich amino acid region in the cytoplasmic domain of TMEFF2 prevented these effects. Overexpression of TMEFF2 reduced cell attachment and migration on vitronectin and caused a concomitant decrease in RhoA activation, stress fiber formation and expression of αv, β1 and β3 integrin subunits. Conversely, TMEFF2 interference in 22Rv1 prostate cancer cells resulted in increased integrin expression. Results obtained with a double TRAMP/TMEFF2 transgenic mouse also indicated that TMEFF2 expression reduced integrin expression in the mouse prostate. In summary, the data presented here indicate an important role of TMEFF2 in regulating cell adhesion and migration that involves integrin signaling and is mediated by its cytoplasmic domain

A rapid assessment of co-benefits and trade-offs among Sustainable Development Goals

Achieving the United Nations’ 17 Sustainable Development Goals (SDGs) results in many ecological, social, and economic consequences that are inter-related. Understanding relationships between sustainability goals and determining their interactions can help prioritize effective and efficient policy options. This paper presents a framework that integrates existing knowledge from literature and expert opinions to rapidly assess the relationships between one SDG goal and another. Specifically, given the important role of the oceans in the world's social-ecological systems, this study focuses on how SDG 14 (Life Below Water), and the targets within that goal, contributes to other SDG goals. This framework differentiates relationships based on compatibility (co-benefit, trade-off, neutral), the optional nature of achieving one goal in attaining another, and whether these relationships are context dependent. The results from applying this framework indicate that oceans SDG targets are related to all other SDG goals, with two ocean targets (of seven in total) most related across all other SDG goals. Firstly, the ocean SDG target to increase economic benefits to Small Island Developing States (SIDS) and least developed countries for sustainable marine uses has positive relationships across all SDGs. Secondly, the ocean SDG target to eliminate overfishing, illegal and destructive fishing practices is a necessary pre-condition for achieving the largest number of other SDG targets. This study highlights the importance of the oceans in achieving sustainable development. The rapid assessment framework can be applied to other SDGs to comprehensively map out the subset of targets that are also pivotal in achieving sustainable development

Results from the national sepsis practice survey: predictions about mortality and morbidity and recommendations for limitation of care orders

Introduction: Critically ill patients and families rely upon physicians to provide estimates of prognosis and recommendations for care. Little is known about patient and clinician factors which influence these predictions. The association between these predictions and recommendations for continued aggressive care is also understudied. Methods: We administered a mail-based survey with simulated clinical vignettes to a random sample of the Critical Care Assembly of the American Thoracic Society. Vignettes represented a patient with septic shock with multi-organ failure with identical APACHE II scores and sepsis-associated organ failures. Vignettes varied by age (50 or 70 years old), body mass index (BMI) (normal or obese) and co-morbidities (none or recently diagnosed stage IIA lung cancer). All subjects received the vignettes with the highest and lowest mortality predictions from pilot testing and two additional, randomly selected vignettes. Respondents estimated outcomes and selected care for each hypothetical patient. Results: Despite identical severity of illness, the range of estimates for hospital mortality (5th to 95th percentile range, 17% to 78%) and for problems with self-care (5th to 95th percentile range, 2% to 74%) was wide. Similar variation was observed when clinical factors (age, BMI, and co-morbidities) were identical. Estimates of hospital mortality and problems with self-care among survivors were significantly higher in vignettes with obese BMIs (4.3% and 5.3% higher, respectively), older age (8.2% and 11.6% higher, respectively), and cancer diagnosis (5.9% and 6.9% higher, respectively). Higher estimates of mortality (adjusted odds ratio 1.29 per 10% increase in predicted mortality), perceived problems with self-care (adjusted odds ratio 1.26 per 10% increase in predicted problems with self-care), and early-stage lung cancer (adjusted odds ratio 5.82) were independently associated with recommendations to limit care. Conclusions: The studied clinical factors were consistently associated with poorer outcome predictions but did not explain the variation in prognoses offered by experienced physicians. These observations raise concern that provided information and the resulting decisions about continued aggressive care may be influenced by individual physician perception. To provide more reliable and accurate estimates of outcomes, tools are needed which incorporate patient characteristics and preferences with physician predictions and practices

Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by GPR4 small molecule inhibitors and hold potential therapeutic value

Isolation of the thrombospondin membrane receptor

Abstract Thrombospondin (TSP), a 450-kD multifunctional glycoprotein with a broad tissue distribution, is secreted upon platelet stimulation, binds to the activated platelet surface, and supports platelet aggregation. We have identified and isolated an 88-kD membrane glycoprotein present in platelets, endothelial cells, monocytes, and a variety of human tumor cell lines that is the membrane binding site for TSP. Endogenous platelet TSP binding to thrombin-and ionophore-stimulated human platelets was inhibited in the presence of the monoclonal antibody OKM5. TSP binding to C32 melanoma cells and HT1080 fibrosarcoma cells was specific and also inhibitable with OKM5 Mab. Cell labeling followed by specific immnunoprecipitation demonstrated biosynthesis of a single 88-kD glycoprotein. Binding of TSP to the isolated membrane protein was specific and saturable. These studies identify an 88-kD membrane glycoprotein that reacts with the monoclonal antibody, OKM5, and may function as the cellular TSP receptor