Radiotheranostics in oncology: Making precision medicine possible.

A quintessential setting for precision medicine, theranostics refers to a rapidly evolving field of medicine in which disease is diagnosed followed by treatment of disease-positive patients using tools for the therapy identical or similar to those used for the diagnosis. Against the backdrop of only-treat-when-visualized, the goal is a high therapeutic index with efficacy markedly surpassing toxicity. Oncology leads the way in theranostics innovation, where the approach has become possible with the identification of unique proteins and other factors selectively expressed in cancer versus healthy tissue, advances in imaging technology able to report these tissue factors, and major understanding of targeting chemicals and nanodevices together with methods to attach labels or warheads for imaging and therapy. Radiotheranostics-using radiopharmaceuticals-is becoming routine in patients with prostate cancer and neuroendocrine tumors who express the proteins PSMA (prostate-specific membrane antigen) and SSTR2 (somatostatin receptor 2), respectively, on their cancer. The palpable excitement in the field stems from the finding that a proportion of patients with large metastatic burden show complete and partial responses, and this outcome is catalyzing the search for more radiotheranostics approaches. Not every patient will benefit from radiotheranostics; but, for those who cross the target-detected line, the likelihood of response is very high.

AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System.

AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.

TSC2 regulates tumor susceptibility to TRAIL-mediated T-cell killing by orchestrating mTOR signaling.

Resistance to cancer immunotherapy continues to impair common clinical benefit. Here, we use whole-genome CRISPR-Cas9 knockout data to uncover an important role for Tuberous Sclerosis Complex 2 (TSC2) in determining tumor susceptibility to cytotoxic T lymphocyte (CTL) killing in human melanoma cells. TSC2-depleted tumor cells had disrupted mTOR regulation following CTL attack, which was associated with enhanced cell death. Wild-type tumor cells adapted to CTL attack by shifting their mTOR signaling balance toward increased mTORC2 activity, circumventing apoptosis, and necroptosis. TSC2 ablation strongly augmented tumor cell sensitivity to CTL attack in vitro and in vivo, suggesting one of its functions is to critically protect tumor cells. Mechanistically, TSC2 inactivation caused elevation of TRAIL receptor expression, cooperating with mTORC1-S6 signaling to induce tumor cell death. Clinically, we found a negative correlation between TSC2 expression and TRAIL signaling in TCGA patient cohorts. Moreover, a lower TSC2 immune response signature was observed in melanomas from patients responding to immune checkpoint blockade. Our study uncovers a pivotal role for TSC2 in the cancer immune response by governing crosstalk between TSC2-mTOR and TRAIL signaling, aiding future therapeutic exploration of this pathway in immuno-oncology.

HCMV-secreted glycoprotein gpUL4 inhibits TRAIL-mediated apoptosis and NK cell activation.

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.

ASB3 promotes hepatocellular carcinoma progression by mediating DR5 ubiquitination in TRAIL resistance.

Ankyrin-repeat proteins with a suppressor of cytokine signaling box (ASB) proteins belong to the E3 ubiquitin ligase family. 18 ASB members have been identified whose biological functions are mostly unexplored. Here, we discovered that ASB3 was essential for hepatocellular carcinoma (HCC) development and high ASB3 expression predicted poor clinical outcomes. ASB3 silencing induced HCC cell growth arrest and apoptosis in vitro and in vivo. Liver-specific deletion of Asb3 gene suppressed diethylnitrosamine (DEN)-induced liver cancer development. Mechanistically, ASB3 interacted with death receptor 5 (DR5), which promoted ubiquitination and degradation of DR5. We further showed that ASB3 knockdown stabilized DR5 and increased the sensitivity of liver cancer cells to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a DR5-dependent manner in cellular and in animal models. In summary, we demonstrated that ASB3 promoted ubiquitination and degradation of DR5 in HCC, suggesting the potential of targeting ASB3 to HCC treatment and overcome TRAIL resistance.

The TRAIL-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for specifically killing cancer cells, whereas in resistant cancers, TRAIL/TRAIL-R can promote metastasis via Rac1 and PI3K. It remains unknown, however, whether and to what extent TRAIL/TRAIL-R signaling in cancer cells can affect the immune microenvironment. Here we show that TRAIL-triggered cytokine secretion from TRAIL-resistant cancer cells is FADD dependent and identify the TRAIL-induced secretome to drive monocyte polarization to myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. TRAIL-R suppression in tumor cells impaired CCL2 production and diminished both lung MDSC presence and tumor growth. In accordance, the receptor of CCL2, CCR2, is required to facilitate increased MDSC presence and tumor growth. Finally, TRAIL and CCL2 are co-regulated with MDSC/M2 markers in lung adenocarcinoma patients. Collectively, endogenous TRAIL/TRAIL-R-mediated CCL2 secretion promotes accumulation of tumor-supportive immune cells in the cancer microenvironment, thereby revealing a tumor-supportive immune-modulatory role of the TRAIL/TRAIL-R system in cancer biology.

Caspase-8 Acts in a Non-enzymatic Role as a Scaffold for Assembly of a Pro-inflammatory "FADDosome" Complex upon TRAIL Stimulation.

TRAIL is a potent inducer of apoptosis and has been studied almost exclusively in this context. However, TRAIL can also induce NFκB-dependent expression of multiple pro-inflammatory cytokines and chemokines. Surprisingly, whereas inhibition of caspase activity blocked TRAIL-induced apoptosis, but not cytokine production, knock down or deletion of caspase-8 suppressed both outcomes, suggesting that caspase-8 participates in TRAIL-induced inflammatory signaling in a scaffold role. Consistent with this, introduction of a catalytically inactive caspase-8 mutant into CASP-8 null cells restored TRAIL-induced cytokine production, but not cell death. Furthermore, affinity precipitation of the native TRAIL receptor complex revealed that pro-caspase-8 was required for recruitment of RIPK1, via FADD, to promote NFκB activation and pro-inflammatory cytokine production downstream. Thus, caspase-8 can serve in two distinct roles in response to TRAIL receptor engagement, as a scaffold for assembly of a Caspase-8-FADD-RIPK1 "FADDosome" complex, leading to NFκB-dependent inflammation, or as a protease that promotes apoptosis.

TRAIL-driven targeting and reversing cervical cancer radioresistance by seleno-nanotherapeutics through regulating cell metabolism.

Recently, radioresistance has become a major obstacle in the radiotherapy of cervical cancer. To demonstrate enhanced radiosensitization against radioresistant cervical cancer, radioresistant cervical cancer cell line was developed and the mechanism of radioresistance was explored. Due to the overexpression of (death receptor 5, DR5) in cervical cancer, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-overexpressed cervical cancer cell membrane-camouflaged Cu2-xSe nanomedicine (CCMT) was designed. Since the CCMT was encapsulated with TRAIL-modified cell membrane, it represented high target to cervical cancer cell and immune evasion. Furthermore, Cu2-xSe had the ability to scavenge glutathione (GSH) and produce ·OH with excess H2O2 in the tumor microenvironment. The presence of CCMT combined with radiation therapy could effectively increase the 1O2 produced by X-rays. In vitro and in vivo studies elaborated that CCMT exhibited excellent radiosensitization properties to reverse radiotolerance by scavenging GSH and promoting DNA damage, apoptosis, mitochondrial membrane potential damage and metabolic disruption. Collectively, this study suggested that the development of TRAIL-overexpressed cell membrane-camouflaged Cu2-xSe nanomedicine could advance future cervical cancer treatment and minimize the disadvantages associated with radiation treatment.

The Identification of New c-FLIP Inhibitors for Restoring Apoptosis in TRAIL-Resistant Cancer Cells.

The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.

Inhibition of Chk1 stimulates cytotoxic action of platinum-based drugs and TRAIL combination in human prostate cancer cells.

Checkpoint kinase 1 (Chk1) plays an important role in regulation of the cell cycle, DNA damage response and cell death, and represents an attractive target in anticancer therapy. Small-molecule inhibitors of Chk1 have been intensively investigated either as single agents or in combination with various chemotherapeutic drugs and they can enhance the chemosensitivity of numerous tumor types. Here we newly demonstrate that pharmacological inhibition of Chk1 using potent and selective inhibitor SCH900776, currently profiled in phase II clinical trials, significantly enhances cytotoxic effects of the combination of platinum-based drugs (cisplatin or LA-12) and TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in human prostate cancer cells. The specific role of Chk1 in the drug combination-induced cytotoxicity was confirmed by siRNA-mediated silencing of this kinase. Using RNAi-based methods we also showed the importance of Bak-dependent mitochondrial apoptotic pathway in the combined anticancer action of SCH900776, cisplatin and TRAIL. The triple drug combination-induced cytotoxicity was partially enhanced by siRNA-mediated Mcl-1 silencing. Our findings suggest that targeting Chk1 may be used as an efficient strategy for sensitization of prostate cancer cells to killing action of platinum-based chemotherapeutic drugs and TRAIL.

Molecular signatures in prion disease: altered death receptor pathways in a mouse model.

BACKGROUND: Prion diseases are transmissible and fatal neurodegenerative diseases characterized by accumulation of misfolded prion protein isoform (PrPSc), astrocytosis, microgliosis, spongiosis, and neurodegeneration. Elevated levels of cell membrane associated PrPSc protein and inflammatory cytokines hint towards the activation of death receptor (DR) pathway/s in prion diseases. Activation of DRs regulate, either cell survival or apoptosis, autophagy and necroptosis based on the adaptors they interact. Very little is known about the DR pathways activation in prion disease. DR3 and DR5 that are expressed in normal mouse brain were never studied in prion disease, so also their ligands and any DR adaptors. This research gap is notable and investigated in the present study. METHODS: C57BL/6J mice were infected with Rocky Mountain Laboratory scrapie mouse prion strain. The progression of prion disease was examined by observing morphological and behavioural abnormalities. The levels of PrP isoforms and GFAP were measured as the marker of PrPSc accumulation and astrocytosis respectively using antibody-based techniques that detect proteins on blot and brain section. The levels of DRs, their glycosylation and ectodomain shedding, and associated factors warrant their examination at protein level, hence western blot analysis was employed in this study. RESULTS: Prion-infected mice developed motor deficits and neuropathology like PrPSc accumulation and astrocytosis similar to other prion diseases. Results from this research show higher expression of all DR ligands, TNFR1, Fas and p75NTR but decreased levels DR3 and DR5. The levels of DR adaptor proteins like TRADD and TRAF2 (primarily regulate pro-survival pathways) are reduced. FADD, which primarily regulate cell death, its level remains unchanged. RIPK1, which regulate pro-survival, apoptosis and necroptosis, its expression and proteolysis (inhibits necroptosis but activates apoptosis) are increased. CONCLUSIONS: The findings from the present study provide evidence towards the involvement of DR3, DR5, DR6, TL1A, TRAIL, TRADD, TRAF2, FADD and RIPK1 for the first time in prion diseases. The knowledge obtained from this research discuss the possible impacts of these 16 differentially expressed DR factors on our understanding towards the multifaceted neuropathology of prion diseases and towards future explorations into potential targeted therapeutic interventions for prion disease specific neuropathology.

Tumor micro-environment induced TRAIL secretion from engineered macrophages for anti-tumor therapy.

The high plasticity and long-term persistency make macrophages excellent vehicles for delivering anti-tumor cytokines. Macrophage delivery of chemokines and cytokines shows potential in tumor therapy. TRAIL, a promising anti-tumor cytokine, induces apoptosis in tumor cells with low toxicity to normal cells. However, its off-target toxicity and limited stability have limited its clinical progress. Here, we engineered macrophages with Mono-TRAIL and Tri-TRAIL and found that Tri-TRAIL had higher cytotoxic activity against tumor cells than Mono-TRAIL in vitro. To target the tumor microenvironment (TME), we generated macrophages secreting trimeric TRAIL (Tri-TRAIL-iM) induced by the TME-specific promoter Arg1. The Tri-TRAIL-iM cells displayed high specific activatable activity in cell-based co-culture assay and tumor-baring mice models. In addition, we demonstrated that compared to macrophages over-expressing TRAIL under a non-inducible promoter, Tri-TRAIL-iM could more effectively induce apoptosis in cancer cells, inhibit tumor growth, and reduce systemic side effects. This strategy of inducing TRAIL delivery holds great potential for cancer therapy. It is promising to be combined with other engineering methods to maximize the therapeutic effects of solid tumors.

Natural variability of TRAIL, IP-10, and CRP in healthy adults - The "HERACLES" study.

A novel host-protein score (called MMBV) helps to distinguish bacterial from viral infection by combining the blood concentrations of three biomarkers: tumour necrosis factor related apoptosis inducing ligand (TRAIL), interferon gamma induced protein 10 (IP-10), and C-reactive protein (CRP). These host biomarkers are differentially expressed in response to bacterial versus viral acute infection. We conducted a prospective study, with a time series design, in healthy adult volunteers in the Netherlands. The aim was to determine the variability of TRAIL, IP-10, and CRP and the MMBV score in healthy adults across time. Up to six blood samples were taken from each healthy volunteer over a period of up to four weeks. In 77 healthy participants without recent or current symptoms, MMBV scores (maximal) were bacterial in 1.3 % and viral (or other non-infectious etiology) in 93.5 % of participants. There was little variation in the mean concentrations of TRAIL (74.5 pg/ml), IP-10 (113.6 pg/ml), and CRP (1.90 mg/L) as well as the MMBV score. The variability of biomarker measurement was comparable to the precision of the measurement platform for TRAIL, IP-10, and CRP. Our findings establish the mean values of these biomarkers and MMBV in healthy individuals and indicate little variability between and within individuals over time, supporting the potential utility of this novel diagnostic to detect infection-induced changes.

Multiple mechanisms contribute to acquired TRAIL resistance in multiple myeloma.

Multiple Myeloma (MM) prognosis has recently improved thanks to the incorporation of new therapies to the clinic. Nonetheless, it is still a non-curable malignancy. Targeting cancer cells with agents inducing cell death has been an appealing alternative investigated over the years, as is the case of TRAIL, an agonist of DR4 and DR5 death receptors. This pathway, involved in apoptosis triggering, has demonstrated efficacy on MM cells. In this research, we have investigated the sensitivity of a panel of MM cells to this agent and generated TRAIL-resistant models by continuous culture of sensitive cells with this peptide. Using genomic and biochemical approaches, the mechanisms underlying resistance were investigated. In TRAIL-resistant cells, a strong reduction in cell-surface receptor levels was detected and impaired the apoptotic machinery to respond to the treatment, enabling cells to efficiently form the Death Inducing Signalling Complex. In addition, an upregulation of the inhibitory protein c-FLIP was detected. Even though the manipulation of these proteins was able to modify cellular responses to TRAIL, it was not complete, pointing to other mechanisms involved in TRAIL resistance.

A Genome-Wide Interaction Study of Erythrocyte ω-3 Polyunsaturated Fatty Acid Species and Memory in the Framingham Heart Study Offspring Cohort.

BACKGROUND: Cognitive decline, and more specifically Alzheimer's disease, continues to increase in prevalence globally, with few, if any, adequate preventative approaches. Several tests of cognition are utilized in the diagnosis of cognitive decline that assess executive function, short- and long-term memory, cognitive flexibility, and speech and motor control. Recent studies have separately investigated the genetic component of both cognitive health, using these measures, and circulating fatty acids. OBJECTIVES: We aimed to examine the potential moderating effect of main species of ω-3 polyunsaturated fatty acids (PUFAs) on an individual's genetically conferred risk of cognitive decline. METHODS: The Offspring cohort from the Framingham Heart Study was cross-sectionally analyzed in this genome-wide interaction study (GWIS). Our sample included all individuals with red blood cell ω-3 PUFA, genetic, cognitive testing (via Trail Making Tests [TMTs]), and covariate data (N = 1620). We used linear mixed effects models to predict each of the 3 cognitive measures (TMT A, TMT B, and TMT D) by each ω-3 PUFA, single nucleotide polymorphism (SNP) (0, 1, or 2 minor alleles), ω-3 PUFA by SNP interaction term, and adjusting for sex, age, education, APOE ε4 genotype status, and kinship (relatedness). RESULTS: Our analysis identified 31 unique SNPs from 24 genes reaching an exploratory significance threshold of 1×10-5. Fourteen of the 24 genes have been previously associated with the brain/cognition, and 5 genes have been previously associated with circulating lipids. Importantly, 8 of the genes we identified, DAB1, SORCS2, SERINC5, OSBPL3, CPA6, DLG2, MUC19, and RGMA, have been associated with both cognition and circulating lipids. We identified 22 unique SNPs for which individuals with the minor alleles benefit substantially from increased ω-3 fatty acid concentrations and 9 unique SNPs for which the common homozygote benefits. CONCLUSIONS: In this GWIS of ω-3 PUFA species on cognitive outcomes, we identified 8 unique genes with plausible biology suggesting individuals with specific polymorphisms may have greater potential to benefit from increased ω-3 PUFA intake. Additional replication in prospective settings with more diverse samples is needed.

Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck.

BACKGROUND: T cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation. METHODS: Whole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells. RESULTS: TRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation. CONCLUSIONS: TRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.

Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells.

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus-infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV-1-infected cells. By combining an unbiased large-scale screening approach with a functional assay, we identify TRAIL to be associated with NK cell degranulation against HIV-1-infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor-mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFNγ production. Moreover, TRAIL-mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL-mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti-HIV-1 activity of NK cells but also possesses a multifunctional role beyond receptor-mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.

A randomized controlled study of neoadjuvant metformin with chemotherapy in nondiabetic breast cancer women: The METNEO study.

AIMS: Clinical data demonstrate that metformin exhibits antiproliferative, proapoptotic and antimetastatic actions. Here, correlative molecular studies were undertaken to determine the roles of transmembrane tumour necrosis factor-related apoptosis-inducing ligand death receptors (DRs) and CD133, a glycoprotein biomarker of breast cancer (BC) stem cells, in the advantageous action of metformin on pathological and clinical outcomes in BC patients on neoadjuvant chemotherapy. METHODS: We randomly assigned 70 nondiabetic BC patients in a 1:1 ratio to either neoadjuvant AC-T chemotherapy (4 cycles of adriamycin 60 mg/m2 and cyclophosphamide 600 mg/m2, followed by 12 cycles of weekly paclitaxel 80 mg/m2) or AC-T with adjunct metformin (850 mg twice/day). The expressions of DR4, DR5 and CD133 were quantified in excised tissue samples with residual tumour cells. RESULTS: The overall clinical response (odds ratio: 22.67 [2.77-185.18], P = .004), breast-conserving surgery (odds ratio: 3.67 [1.303-10.321], P = .014) and pathological complete response (ß = 2.49 ± 1.13 [0.274-4.712], P = .028) rates were significantly improved in the metformin arm. Tissues obtained from the metformin arm had upregulated mRNA expression of DR4 (Mean delta cycle thresholds ± standard error of the mean: 2.68 ± 0.25 vs. 4.87 ± 0.53, P = .0003) and DR5 (0.21 ± 0.25 vs. 4.29 ± 0.95, P = .0004) compared to control arm. The enhanced DR expression negatively correlated with that of CD133 + BC stem cells, which was significantly reduced by metformin at both cytoplasmic/membranous (43.48 vs. 100.00%, P < .0001) and nuclear sites (4.35 vs. 95.00%, P < .0001). CONCLUSION: Metformin improves clinical and pathological responses to neoadjuvant AC-T chemotherapy in BC via prompting directionally opposite changes in DRs (increments) and CD133 + (decrements) expressions. This study was registered in ClinicalTrials.gov (registration number: NCT04170465, https://clinicaltrials.gov/ct2/show/NCT04170465).

Evaluation of a walking school bus program: a cluster randomized controlled trial.

BACKGROUND: The purpose of this study was to investigate the effects of a walking school bus intervention on children's active commuting to school. METHODS: We conducted a randomized controlled trial (RCT) in Houston, Texas (Year 1) and Seattle, Washington (Years 2-4) from 2012 to 2016. The study had a two-arm, cluster randomized design comparing the intervention (walking school bus and education materials) to the control (education materials) over one school year October/November - May/June). Twenty-two schools that served lower income families participated. Outcomes included percentage of days students' active commuting to school (primary, measured via survey) and moderate-to-vigorous physical activity (MVPA, measured via accelerometry). Follow-up took place in May or June. We used linear mixed-effects models to estimate the association between the intervention and outcomes of interest. RESULTS: Total sample was 418 students [Mage=9.2 (SD = 0.9) years; 46% female], 197 (47%) in the intervention group. The intervention group showed a significant increase compared with the control group over time in percentage of days active commuting (ß = 9.04; 95% CI: 1.10, 16.98; p = 0.015) and MVPA minutes/day (ß = 4.31; 95% CI: 0.70, 7.91; p = 0.02). CONCLUSIONS: These findings support implementation of walking school bus programs that are inclusive of school-age children from lower income families to support active commuting to school and improve physical activity. TRAIL REGISTRATION: This RCT is registered at clinicaltrials.gov (NCT01626807).

Daurisoline inhibits proliferation, induces apoptosis, and enhances TRAIL sensitivity of breast cancer cells by upregulating DR5.

Daurisoline (DS) is an isoquinoline alkaloid that exerts anticancer activities in various cancer cells. However, the underlying mechanisms through which DS affects the survival of breast cancer cells remain poorly understood. Therefore, the present study was undertaken to investigate the potential anticancer effect of DS on breast cancer cells and reveal the mechanism underlying the enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by DS. Cell counting kit-8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU) assay were used to evaluate the ability of cell proliferation. Flow cytometry was selected to examine the cell cycle distribution. TUNEL assay was used to detect the cell apoptosis. The protein expression was measured by Western blot analysis. DS was found to reduce the cell viability and suppress the proliferation of MCF-7 and MDA-MB-231 cells by causing G1 phase cell cycle arrest. DS could trigger apoptosis by promoting the cleavage of caspase-8 and PARP. The phosphorylation of ERK, JNK, and p38MAPK was upregulated clearly following DS treatment. Notably, SP600125 (JNK inhibitor) pretreatment significantly abrogated DS-induced PARP cleavage. DS inactivated Akt/mTOR and Wnt/ß-catenin signaling pathway and upregulated the expression of ER stress-related proteins. Additionally, DS amplified TRAIL-caused viability reduction and apoptosis in breast cancer cells. Mechanismly, DS upregulated the protein level of DR4 and DR5, and knockdown of DR5 attenuated the cotreatment-induced cleavage of PARP. Inhibition of JNK could block DS-induced upregulation of DR5. This study provides valuable insights into the mechanisms of DS inhibiting cell proliferation, triggering apoptosis, and enhancing TRAIL sensitivity of breast cancer cells.