Activation Of Signal Transduction Pathways Research Articles

Thyroid Dysfunction of Zebrafish (Danio rerio) after Early-Life Exposure and Discontinued Exposure to Tetrabromobiphenyl (BB-80) and OH-BB-80.

3,3',5,5'-Tetrabromobiphenyl (BB-80) was once used as additive flame retardants. Whether its early exposure and discontinued exposure alter thyroid function remains unknown. We investigate adverse effects after early-life exposure and discontinued exposure to BB-80 and hydroxylated BB-80 (OH-BB-80) on thyroid hormone (TH) levels, thyroid tissue, and transcriptome profiles in zebrafish larvae. BB-80 at 10 μg/L induces pathological changes of thyroid with reduced thyroid follicles in larvae (P < 0.05), whereas OH-BB-80 significantly increases T4 and T3 contents (1.8 and 2.5 times of the control, P < 0.05) at 14 days postfertilization (dpf) without morphological thyroid alterations. BB-80 and OH-BB-80 cause transcriptome aberrations with key differentially expressed genes involved in the disruption of TH synthesis and signal transduction (BB-80 at 14 dpf) or TH pathway activation (OH-BB-80 at 21 dpf). After 7 days of discontinued exposure, thyroglobulin (tg) and thyroid peroxidase (tpo) genes are downregulated (P < 0.05) by 52 and 48% for BB-80 and by 49 and 39% for OH-BB-80, respectively; however, the whole-body TH levels fail to fully recover, and the locomotor activity is impaired more by BB-80. Our results indicate significant adverse impacts of BB-80 and OH-BB-80 on TH homeostasis and thyroid function of zebrafish.

Characterization of Immunoactive and Immunotolerant CD4+ T Cells in Breast Cancer by Measuring Activity of Signaling Pathways That Determine Immune Cell Function.

Simple SummaryImmunotherapy enhances the immune response against cancer and is potentially curative. Unfortunately, few patients with breast cancer benefit from this therapy. It is not possible to predict which patients will benefit. A blood cell, called CD4+ T-cell, plays a role in the immune response and in resistance to immunotherapy. Its function is determined by activity of biochemical processes, called signal transduction pathways (STPs). We developed a new technology to measure activity of these STPs, which was used to investigate whether CD4+ T cells function abnormally in breast cancer patients. We show that in CD4+ T-cells from most of the investigated breast cancer patients a number of these STPs are overactive. The abnormal activity of a few notable STPs (Notch and TGFβ) suggests that CD4+ T-cells have changed into regulatory T-cells, which inhibit the immune response against cancer and have been associated with resistance to immunotherapy. We also provide evidence that this change in the CD4+ T- cells is caused by a factor produced by breast cancer cells. We conclude that this new technology can be used to measure STP activity in blood of patients with cancer and has the potential to better identify patients who will benefit from immunotherapy. Cancer immunotolerance may be reversed by checkpoint inhibitor immunotherapy; however, only a subset of patients responds to immunotherapy. The prediction of clinical response in the individual patient remains a challenge. CD4+ T cells play a role in activating adaptive immune responses against cancer, while the conversion to immunosuppression is mainly caused by CD4+ regulatory T cell (Treg) cells. Signal transduction pathways (STPs) control the main functions of immune cells. A novel previously described assay technology enables the quantitative measurement of activity of multiple STPs in individual cell and tissue samples. The activities of the TGFβ, NFκB, PI3K-FOXO, JAK-STAT1/2, JAK-STAT3, and Notch STPs were measured in CD4+ T cell subsets and used to investigate cellular mechanisms underlying breast cancer-induced immunotolerance. Methods: STP activity scores were measured on Affymetrix expression microarray data of the following: (1) resting and immune-activated CD4+ T cells; (2) CD4+ T-helper 1 (Th1) and T-helper 2 (Th2) cells; (3) CD4+ Treg cells; (4) immune-activated CD4+ T cells incubated with breast cancer tissue supernatants; and (5) CD4+ T cells from blood, lymph nodes, and cancer tissue of 10 primary breast cancer patients. Results: CD4+ T cell activation induced PI3K, NFκB, JAK-STAT1/2, and JAK-STAT3 STP activities. Th1, Th2, and Treg cells each showed a typical pathway activity profile. The incubation of activated CD4+ T cells with cancer supernatants reduced the PI3K, NFκB, and JAK-STAT3 pathway activities and increased the TGFβ pathway activity, characteristic of an immunotolerant state. Immunosuppressive Treg cells were characterized by high NFκB, JAK-STAT3, TGFβ, and Notch pathway activity scores. An immunotolerant pathway activity profile was identified in CD4+ T cells from tumor infiltrate and blood of a subset of primary breast cancer patients, which was most similar to the pathway activity profile in immunosuppressive Treg cells. Conclusion: Signaling pathway assays can be used to quantitatively measure the functional immune response state of lymphocyte subsets in vitro and in vivo. Clinical results suggest that, in primary breast cancer, the adaptive immune response of CD4+ T cells may be frequently replaced by immunosuppressive Treg cells, potentially causing resistance to checkpoint inhibition. In vitro study results suggest that this is mediated by soluble factors from cancer tissue. Signaling pathway activity analysis on TIL and/or blood samples may improve response prediction and monitoring response to checkpoint inhibitors and may provide new therapeutic targets (e.g., the Notch pathway) to reduce resistance to immunotherapy.

Classic Hodgkin Lymphoma Refractory for ABVD Treatment Is Characterized by Pathologically Activated Signal Transduction Pathways as Revealed by Proteomic Profiling.

Simple SummaryClassic Hodgkin lymphoma (cHL) patients refractory to standard ABVD chemo-therapy are known to have a dismal prognosis. This has led to the hypothesis that ABVD treatment-sensitive and ABVD treatment-refractory tumours are biologically distinct. In this study, cHL patients refractory to standard ABVD treatment show subtle but significant differences in protein expression that enable clustering of the two response groups, thus indicating differences between ABVD sensitive and refractory patients at the molecular level, and thereby strengthening the hypothesis that ABVD sensitive and ABVD refractory tumours may be biologically distinct.In classic Hodgkin lymphoma (cHL), the tumour microenvironment (TME) is of major pathological relevance. The paucity of neoplastic cells makes it important to study the entire TME when searching for prognostic biomarkers. Cure rates in cHL have improved markedly over the last several decades, but patients with primary refractory disease still show inferior survival. We performed a proteomic comparison of pretreatment tumour tissue from ABVD treatment-refractory versus ABVD treatment-sensitive cHL patients, in order to identify biological differences correlating with treatment outcome. Formalin-fixed paraffin-embedded tumour tissues from 36 patients with cHL, 15 with treatment-refractory disease, and 21 with treatment-sensitive disease, were processed for proteomic investigation. Label-free quantification nano liquid chromatography tandem mass spectrometry was performed on the tissues. A total of 3920 proteins were detected and quantified between the refractory and sensitive groups. This comparison revealed several subtle but significant differences in protein expression which could identify subcluster characteristics of the refractory group. Bioinformatic analysis of the biological differences indicated that a number of pathologically activated signal transduction pathways are disturbed in ABVD treatment-refractory cHL.

Rotenone, an environmental toxin, causes abnormal methylation of the mouse brain organoid's genome and ferroptosis.

More and more reports have pointed out that rotenone, as an insecticide, has high neurotoxicity and reproductive toxicity to livestock and mammals. As a highly physiological correlation system of internal organs, quasi-organs have great potential in the fields of drug toxicity and efficacy test, toxicology research, developmental biology and so on. In this study, brain organs (mBOs) derived from mouse neural stem cells were used to investigate the effects of rotenone on the physiological activity and epigenetic modification of mBOs. At the same time, Rotenone could significantly stimulate the increase of the concentration of LPO, lactic acid and hydroxyl radical in mBOs, and inhibit the expression of neuronal marker Tuj1, CHAT, PAX6 and so on. Further analysis showed that Rotenonem could induce mitochondrial damage in mBOs. The results of qPCR and Western blot showed that Rotenone could up-regulate the expressions of ferroptosis promoting protein p53, Cox2 and so on, while inhibit the expressions of negative regulatory protein of ferroptosis GPX4, FTH1, SLC7A11. In addition, the results of RRBS-Seq sequencing showed that the methylation modification at DMR level in Rotenone-treated mBOs group was significantly higher than that in Ctrl group. The results of KEGG analysis showed that compared with Ctrl, the genes with hypermethylation of promoter and Genebody in Rotenone-treated mBOs were mainly located in the Neuro active ligand-receptor interaction signal transduction pathway. In summary, rotenone can significantly lead to abnormal methylation of mouse brain organs, and lead to the loss of normal physiological function of neurons by inducing ferroptosis.

Cyclic activity of signal transduction pathways in fimbrial epithelium of the human fallopian tube.

The local environment of the fallopian tube represents the optimal conditions for reproductive processes. To maintain tissue homeostasis, signal transduction pathways are thought to play a pivotal role. Enhancing our understanding of functional signal transduction pathway activity is important to be able to clarify the role of aberrant signal transduction pathway activity leading to female subfertility and other tubal diseases. Therefore, in this study we investigate the influence of the hormonal cycle on the activity of key signal transduction pathways in the fimbrial epithelium of morphologically normal fallopian tubes. We included healthy pre- (n=17) and postmenopausal (n=8) patients who had surgical interventions for benign gynecologic conditions. Histologic sections of the fallopian tubes were reviewed by two pathologists and, for the premenopausal patients, hormone serum levels and sections of the endometrium were examined to determine the hormonal phase (early follicular [n=4], late follicular [n=3], early luteal [n=5], late luteal [n=5]). After laser capture microdissection, total mRNA was extracted from the fimbrial epithelium and real-time quantitative reverse transcription-PCR was performed to determine functional signal transduction pathway activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor-beta (TGF-β) and canonical wingless-type MMTV integration site (Wnt) pathways. The early luteal phase demonstrated high AR and ER pathway activity in comparison with the late luteal phase (p=0.016 and p=0.032, respectively) and low PI3K activity compared with the late follicular phase (p=0.036), whereas the late luteal phase showed low activity of HH and Wnt compared with the early follicular phase (both p=0.016). Signal transduction pathway activity in fimbrial epithelium from postmenopausal patients was most similar to the early follicular and/or late luteal phase with regard to the AR, ER and PI3K pathways. Wnt pathway activity in postmenopausal patients was comparable to the late follicular and early luteal phase. We observed no differences in HH and TGF-β pathway activity between pre- and postmenopausal samples. The cyclic changes in signal transduction pathway activity suggest a stage-specific function which may affect the morphology and physiology of the human fallopian tube. We demonstrated cyclic changes in activity of the AR, ER, PI3K, HH and Wnt pathways throughout the hormonal cycle.

Transcriptomic and metabolomic changes triggered by Macrosiphum rosivorum in rose (Rosa longicuspis)

BackgroundRose is one of the most popular flowers in the wold. Its field growth and quality are negatively affected by aphids. However, the defence mechanisms used by rose plants against aphids are unclear. Therefore, to understand the defence mechanism of rose under aphid stress, transcriptome and metabolome techniques were used to investigate the regulation mechanism in R. longicuspis infected with M. rosivorum.ResultIn our study, after inoculation with M. rosivorum, M. rosivorum quickly colonized R. longicuspis. A total of 34,202 genes and 758 metabolites were detected in all samples. Under M. rosivorum stress, R. longicuspis responded by MAPK cascades, plant hormone signal transduction pathway activation, RlMYBs and RlERFs transcription factors expression and ROS production. Interestingly, the ‘brassinosteroid biosynthesis’ pathway was significantly enriched in A3 d-vs.-A5 d. Further analysis showed that M. rosivorum induced the biosynthesis of secondary metabolites such as terpenoids, tannins and phenolic acids, among others. Importantly, the ‘glutathione metabolic’ and ‘glucosinolate biosynthesis’ pathways were significantly enriched, which involved in the rose against aphids.ConclusionOur study provides candidate genes and metabolites for Rosa defence against aphids. This study provides a theoretical basis for further exploring the molecular regulation mechanism of rose aphid resistance and aphid resistance breeding in the future.

Sensory Afferent Renal Nerve Activated Gαi2 Subunit Proteins Mediate the Natriuretic, Sympathoinhibitory and Normotensive Responses to Peripheral Sodium Challenges.

We have previously reported that brain Gαi2 subunit proteins are required to maintain sodium homeostasis and are endogenously upregulated in the hypothalamic paraventricular nucleus (PVN) in response to increased dietary salt intake to maintain a salt resistant phenotype in rats. However, the origin of the signal that drives the endogenous activation and up-regulation of PVN Gαi2 subunit protein signal transduction pathways is unknown. By central oligodeoxynucleotide (ODN) administration we show that the pressor responses to central acute administration and central infusion of sodium chloride occur independently of brain Gαi2 protein pathways. In response to an acute volume expansion, we demonstrate, via the use of selective afferent renal denervation (ADNX) and anteroventral third ventricle (AV3V) lesions, that the sensory afferent renal nerves, but not the sodium sensitive AV3V region, are mechanistically involved in Gαi2 protein mediated natriuresis to an acute volume expansion [peak natriuresis (μeq/min) sham AV3V: 43 ± 4 vs. AV3V 45 ± 4 vs. AV3V + Gαi2 ODN 25 ± 4, p < 0.05; sham ADNX: 43 ± 4 vs. ADNX 23 ± 6, AV3V + Gαi2 ODN 25 ± 3, p < 0.05]. Furthermore, in response to chronically elevated dietary sodium intake, endogenous up-regulation of PVN specific Gαi2 proteins does not involve the AV3V region and is mediated by the sensory afferent renal nerves to counter the development of the salt sensitivity of blood pressure (MAP [mmHg] 4% NaCl; Sham ADNX 124 ± 4 vs. ADNX 145 ± 4, p < 0.05; Sham AV3V 125 ± 4 vs. AV3V 121 ± 5). Additionally, the development of the salt sensitivity of blood pressure following central ODN-mediated Gαi2 protein down-regulation occurs independently of the actions of the brain angiotensin II type 1 receptor. Collectively, our data suggest that in response to alterations in whole body sodium the peripheral sensory afferent renal nerves, but not the central AV3V sodium sensitive region, evoke the up-regulation and activation of PVN Gαi2 protein gated pathways to maintain a salt resistant phenotype. As such, both the sensory afferent renal nerves and PVN Gαi2 protein gated pathways, represent potential targets for the treatment of the salt sensitivity of blood pressure.

Androgen Receptor Pathway Activity Assay for Sepsis Diagnosis and Prediction of Favorable Prognosis.

Introduction: Sepsis is a life-threatening complication of a bacterial infection. It is hard to predict which patients with a bacterial infection will develop sepsis, and accurate and timely diagnosis as well as assessment of prognosis is difficult. Aside from antibiotics-based treatment of the causative infection and supportive measures, treatment options have remained limited. Better understanding of the immuno-pathophysiology of sepsis is expected to lead to improved diagnostic and therapeutic solutions.Functional activity of the innate (inflammatory) and adaptive immune response is controlled by a dedicated set of cellular signal transduction pathways, that are active in the various immune cell types. To develop an immune response-based diagnostic assay for sepsis and provide novel therapeutic targets, signal transduction pathway activities have been analyzed in whole blood samples from patients with sepsis.Methods: A validated and previously published set of signal transduction pathway (STP) assays, enabling determination of immune cell function, was used to analyze public Affymetrix expression microarray data from clinical studies containing data from pediatric and adult patients with sepsis. STP assays enable quantitative measurement of STP activity on individual patient sample data, and were used to calculate activity of androgen receptor (AR), estrogen receptor (ER), JAK-STAT1/2, JAK-STAT3, Notch, Hedgehog, TGFβ, FOXO-PI3K, MAPK-AP1, and NFκB signal transduction pathways.Results: Activity of AR and TGFβ pathways was increased in children and adults with sepsis. Using the mean plus two standard deviations of normal pathway activity (in healthy individuals) as threshold for abnormal STP activity, diagnostic assay parameters were determined. For diagnosis of pediatric sepsis, the AR pathway assay showed high sensitivity (77%) and specificity (97%), with a positive prediction value (PPV) of 99% and negative prediction value (NPV) of 50%. For prediction of favorable prognosis (survival), PPV was 95%, NPV was 21%. The TGFβ pathway activity assay performed slightly less for diagnosing sepsis, with a sensitivity of 64% and specificity of 98% (PPV 99%, NPV 39%).Conclusion: The AR and TGFβ pathways have an immunosuppressive role, suggesting a causal relation between increased pathway activity and sepsis immunopathology. STP assays have been converted to qPCR assays for further evaluation of clinical utility for sepsis diagnosis and prediction of prognosis, as well as for prediction of risk at developing sepsis in patients with a bacterial infection. STPs may present novel therapeutic targets in sepsis.

Discovery and Functional Study of a Novel Genomic Locus Homologous to Bα-Mating-Type Sublocus of Lentinula edodes

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of Bα and Bβ subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (Bα-N) was homologous to the Bα sublocus, but unlike the multiallelic Bα sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and Bα-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the Bα-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.

Mechanisms of Resistance to CDK4/6 Blockade in Advanced Hormone Receptor-positive, HER2-negative Breast Cancer and Emerging Therapeutic Opportunities.

The cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have become the standard of care, in combination with antiestrogen therapy, for patients with hormone receptor-positive (HR+)/HER2- advanced breast cancer. Various preclinical and translational research efforts have begun to shed light on the genomic and molecular landscape of resistance to these agents. Drivers of resistance to CDK4/6i therapy can be broadly subdivided into alterations impacting cell-cycle mediators and activation of oncogenic signal transduction pathways. The resistance drivers with the best translational evidence supporting their putative role have been identified via next-generation sequencing of resistant tumor biopsies in the clinic and validated in laboratory models of HR+ breast cancer. Despite the diverse landscape of resistance, several common, therapeutically actionable resistance nodes have been identified, including the mitotic spindle regulator Aurora Kinase A, as well as the AKT and MAPK signaling pathways. Based upon these insights, precision-guided therapeutic strategies are under active clinical development. This review will highlight the emerging evidence, in the clinic and in the laboratory, implicating this diverse spectrum of molecular resistance drivers.

Studies on Screening of MicroRNA Related to Irradiation Response and the Regulation Mechanism of miRNA-96-5p on Irradiation Resistance in Rectal Cancer Cells

<h3>Purpose/Objective(s)</h3> To screen microRNAs that are positively correlated with radiation resistance in rectal cancer, and to investigate their regulatory mechanism. <h3>Materials/Methods</h3> Fresh rectal cancer tissues were obtained intraoperatively and divided into two equal parts, one of which was pre-stored in a refrigerator at -150℃, and the other was constructed with Patients Derived Xenograft (PDX) models for rectal cancer and irradiated to transplantation tumor screen out radiation-sensitive tumor tissues and radiation-resistant tumor tissues. According to the screening results, the pre-stored samples were screened for differential microRNAs in radiation reactivity, and the microRNAs that were positively correlated with irradiation resistance were obtained. On the base of screening results, the pre-stored samples were screened for differential microRNAs in irradiation reactivity by miRNAs microarrays, and the microRNAs that were positively correlated with irradiation resistance were obtained. Biological prediction software was used to predict the target genes regulated by microRNA, and the prediction results were verified in radiation-resistant rectal cancer cell lines. Lentivirus plasmid transfection technique was used to construct rectal cancer cell lines with down-regulated target microRNA expression, in order to further verify the effects of the changes in the expression of the microRNA on the radiation reactivity, proliferation, invasion and migration of rectal cancer cells. The target genes regulated by microRNAs were screened and verified, and the expression of the key genes in the signal transduction pathways involved in the regulation were detected. <h3>Results</h3> The results of microRNA screening and validation showed that there were 14 microRNAs with fold change > 1.5. Among them, miRNA-552-3p, miRNA-96-5p, miRNA-182-5p, and miRNA-183-5p were up-regulated in irradiation-resistant tumor tissues. The enrichment signaling pathway analysis showed that the Wnt signal transduction pathway was highly active in irradiation-resistant tumor tissues. The clone formation experiment under radiation interference showed that HR-8348 was the most radiation-resistant cell line among the five cell lines, and the expression level of miRNA-96-5p was positively correlated with the radiation-resistant ability of rectal cancer cell lines. The results of prediction and validation showed that the expression of GPC3 gene was directly regulated by miRNA-96-5p, and the down-regulated expression of miRNA-96-5p inhibited the activity of Wnt signal transduction pathway. <h3>Conclusion</h3> The high expression of miRNA-96-5p enhanced the radiation resistance of rectal cancer cells, and its effect may be related to the down-regulation of GPC3 expression and the abnormal activation of Wnt signal transduction pathway.

Microglial NLRP3 Inflammasome Activation upon TLR2 and TLR5 Ligation by Distinct α-Synuclein Assemblies.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder and is characterized by the formation of cellular inclusions inside neurons that are rich in an abnormal form of the protein α-synuclein (α-syn). Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of signaling transduction pathways. Here, we studied activation of primary microglia isolated from wild-type mouse by distinct α-syn forms and their clearance. Internalization of α-syn monomers and oligomers efficiently activated the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome via TLR2 and TLR5 ligation, thereby acting on different signaling checkpoints. We found that primary microglia effectively engulf α-syn but hesitate in its degradation. NLRP3 inhibition by the selective inhibitor CRID3 sodium salt and NLRP3 deficiency improved the overall clearance of α-syn oligomers. Together, these data show that distinct α-syn forms exert different microglial NLRP3 inflammasome activation properties, thereby compromising its degradation, which can be prevented by NLRP3 inhibition.

Survival Is Related to Estrogen Signal Transduction Pathway Activity in Postmenopausal Women Diagnosed with High-Grade Serous Ovarian Carcinoma.

Simple SummaryAll cells have a complex internal network of ‘communication chains’ called signal transduction pathways (STPs). Through interaction of different proteins in STPs, they are partly responsible for the behavior of a cell. In our study, we investigated the activity of six STPs in 85 women with advanced stage high-grade serous ovarian cancer (HGSC). To investigate the relation between and differences in survival and STP activity, women with a short disease-free survival (below 12 months) and a long disease-free survival (above 24 months) were included. We found no differences in mean STP activity between short-term survivors (52 women) and long-term survivors (33 women). However, when we analyzed postmenopausal women, we found that both disease-free and overall survival were related to estrogen receptor (ER) pathway signaling. This indicates that a better survival outcome was related to a more active ER pathway in this subgroup.High-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, has a high mortality rate. Although there are some factors associated with survival, such as stage of disease, there are remarkable differences in survival among women diagnosed with advanced stage disease. In this study, we investigate possible relations between survival and signal transduction pathway (STP) activity. We assessed the functional activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor beta (TGF-β) and canonical wingless-type MMTV integration site (Wnt) pathway in 85 primary tumor samples of patients with FIGO stage IIIC to IVB HGSC and disease-free survival (DFS) below 12 (n = 52) or over 24 months (n = 33). There were no significant differences in median pathway activity between patients with a short and long DFS. In univariate Cox proportional hazards analysis, ER pathway activity was related to a favorable DFS and overall survival (OS) in postmenopausal women (p = 0.033 and p = 0.041, respectively), but not in premenopausal women. We divided the postmenopausal group into subgroups based on ER pathway activity quartiles. Survival analysis revealed that postmenopausal women in the lowest ER quartile had a shorter DFS and OS (log-rank p = 0.006 and p < 0.001, respectively). Furthermore, we were able to form subgroups of patients based on an inverse relation between ER and PI3K pathway activity. In conclusion, in postmenopausal patients with advanced stage HGSC, a poorer survival outcome was associated with low functional ER pathway activity.

Activation of canine neutrophils by platelet-activating factor

Neutrophils are essential for innate immunity as the first line of defence. Neutrophils act as phagocytic white blood cells to kill bacteria and other microorganisms. A strong respiratory burst of neutrophils, dependent on reactive oxygen species, is produced during phagocytosis. Platelet-activating factor (PAF) is a signalling molecule with several prominent roles in tissue injury, inflammation, and platelet aggregation. However, the detailed mechanisms and intracellular signalling pathways involved in PAF-mediated neutrophil activation remain unclear. Here, we investigated the effect of PAF on changes in calcium concentration ([Ca2+]i) and oxygen radical (O2−) generation in activating canine neutrophils. We further evaluated these effects of PAF with inhibition of G protein-coupled receptors using the specific inhibitor suramin. Blood samples were collected from a total of five dogs and neutrophils were isolated. PAF stimulation of canine neutrophils caused an increase in [Ca2+]i as well as the generation of O2−, and the PAF receptor was sensitive to suramin. The results suggested that PAF stimulation of canine neutrophils may cause Ca2+ influx from the endoplasmic reticulum into the cytoplasm (as the first wave) and then trigger store-operated Ca2+ entry (as the second wave), which is an important intracellular signal transduction pathway for neutrophil activation. Furthermore, O2− generation by PAF stimulation may depend on the intracellular signalling pathway, with increasing inositol trisphosphate levels and [Ca2+]i via G protein-coupled receptors. The finding that PAF-activating platelet aggregation is involved in canine neutrophil activation suggests a close relationship between haemostasis and neutrophil activation in dogs, offering new insight into the response to infection.

Prec-O1-04 - CXCR4/CXCL12 axis as a potential therapeutic target in mycosis fungoides: an in-vitro study

The C-X-C chemokine receptor 4 (CXCR4) is overexpressed in the tumor cells of many solid and hematologic neoplasms. Its ligand, C-X-C chemokine ligand 12 (CXCL12), is secreted by cells in the tumor microenvironment, mostly by stromal fibroblasts. The engagement of CXCL12 to CXCR4 results in activation of several intracellular signal transduction pathways, including, the proapoptotic p38, the prosurvival AKT, and the chemotaxis mediator ERK. The balance between activation of AKT and activation of p38 will determine the fate of cell viability. Plerixafor, AMD3100 (AMD), is a CXCR4 antagonist that reversibly blocks the binding of CXCR4 to CXCL12. AMD is already in clinical use as bone marrow stem cell or progenitor cell mobilizers. AMD and other CXCR4 inhibitors are investigated either alone or in combination with other systemic treatments in numerous clinical trials in cancer. CXCR4 and CXCL12 are known to be overexpressed in MF skin biopsies, but the exact differential contribution of the lymphoma vs the reactive T-cells is not clear. We previously have shown that primary MF-fibroblast culture (MF-Fs) established from MF biopsies, secrete high CXCL12 which promotes the migration of MyLa (MF cell line), protect them from chemotherapy and both effects were suppressed by AMD. We studied the role of CXCR4 as essential key protein in the survival of MF, and the effect and mechanism of CXCR4 antagonist on MyLa cell viability. We found that MyLa cells express higher CXCR4 vs CD3+CD4+ PBMCs from healthy donor (FACS). Single cell analysis of skin MF biopsy by flow mass cytometry (CyTOF) revealed sub-population of T- cells expressing the highest intensity of CXCR4, CD30, PD-1, and PDL-1 compared to all other immune cells, and therefore are suspected as the lymphoma cells overexpressing CXCR4. AMD was toxic to MyLa cells with higher toxicity to MyLa co-cultured with MF-Fs than MyLa alone (MTT viability assay, trypan blue staining), and promotes their death through apoptosis (FACS). AMD-induced death was selective to MyLa cells compared to healthy PBMCs, when both were co-cultured with MF-Fs. The phosphorylation of p38 and AKT in MyLa co-cultured with MF-Fs were reduced upon AMD treatment (Western blot). Spheroids of MyLa cells with MF-Fs were established to show the toxic effect of AMD in 3D co-culture model. The intracellular signaling pathways mediated by AMD was studied by phospho-proteomic analysis of MyLa cells co-cultured with MF-Fs. In sum, we showed the overexpression of CXCR4 in subpopulation of T cells suspected as the lymphoma cells in skin biopsy, and the pivotal role of overexpressed CXCR4 in the survival of MF cell line. The apoptotic effect mediated by AMD indicates that disrupting the interaction between stroma derived CXCL12 and MF derived CXCR4 might be a new therapeutic option in MF.

Multiple Myeloma Relapse Is Associated with Increased NFκB Pathway Activity and Upregulation of the Pro-Survival BCL-2 Protein BFL-1

Simple SummaryMultiple myeloma (MM) is a blood cancer that is still incurable because patients become insensitive to available treatments. The cancerous cells in MM misuse a signaling route, called the NFκB pathway, to make MM more difficult to treat. In our study, we used a new method to measure NFκB pathway activity in cancer cells of MM patients at different stages of disease. We found that NFκB pathway activity remains unchanged during disease development, is independent of the life expectancy of MM patients, and does not predict how well the cancer cells will respond to treatment. However, the cancer cells that survive after treatment of MM patients do show higher NFκB pathway activity. In a subgroup of these patients, the cancer cells also showed higher BFL-1 gene expression. BFL-1 can enhance cancer cell survival after treatment and may therefore be a potential new candidate to target in these patients.Multiple myeloma (MM) is a hematological malignancy that is still considered incurable due to the development of therapy resistance and subsequent relapse of disease. MM plasma cells (PC) use NFκB signaling to stimulate cell growth and disease progression, and for protection against therapy-induced apoptosis. Amongst its diverse array of target genes, NFκB regulates the expression of pro-survival BCL-2 proteins BCL-XL, BFL-1, and BCL-2. A possible role for BFL-1 in MM is controversial, since BFL-1, encoded by BCL2A1, is downregulated when mature B cells differentiate into antibody-secreting PC. NFκB signaling can be activated by many factors in the bone marrow microenvironment and/or induced by genetic lesions in MM PC. We used the novel signal transduction pathway activity (STA) computational model to quantify the functional NFκB pathway output in primary MM PC from diverse patient subsets at multiple stages of disease. We found that NFκB pathway activity is not altered during disease development, is irrespective of patient prognosis, and does not predict therapy outcome. However, disease relapse after treatment resulted in increased NFκB pathway activity in surviving MM PC, which correlated with increased BCL2A1 expression in a subset of patients. This suggests that BFL-1 upregulation, in addition to BCL-XL and BCL-2, may render MM PC resistant to therapy-induced apoptosis, and that BFL-1 targeting could provide a new approach to reduce therapy resistance in a subset of relapsed/refractory MM patients.

A CRISPR knockout screen reveals new regulators of canonical Wnt signaling

The Wnt signaling pathways play fundamental roles during both development and adult homeostasis. Aberrant activation of the canonical Wnt signal transduction pathway is involved in many diseases including cancer, and is especially implicated in the development and progression of colorectal cancer. Although extensively studied, new genes, mechanisms and regulatory modulators involved in Wnt signaling activation or silencing are still being discovered. Here we applied a genome-scale CRISPR-Cas9 knockout (KO) screen based on Wnt signaling induced cell survival to reveal new inhibitors of the oncogenic, canonical Wnt pathway. We have identified several potential Wnt signaling inhibitors and have characterized the effects of the initiation factor DExH-box protein 29 (DHX29) on the Wnt cascade. We show that KO of DHX29 activates the Wnt pathway leading to upregulation of the Wnt target gene cyclin-D1, while overexpression of DHX29 inhibits the pathway. Together, our data indicate that DHX29 may function as a new canonical Wnt signaling tumor suppressor and demonstrates that this screening approach can be used as a strategy for rapid identification of novel Wnt signaling modulators.

Screening of MicroRNA Related to Irradiation Response and the Regulation Mechanism of miRNA-96-5p in Rectal Cancer Cells.

Neoadjuvant chemoradiotherapy has been widely used in the treatment of locally advanced rectal cancer due to the excellent advantages of irradiation in cancer therapy. Unfortunately, not every patient can benefit from this treatment, therefore, it is of great significance to explore biomarkers that can predict irradiation sensitivity. In this study, we screened microRNAs (miRNAs) which were positively correlated with irradiation resistance and found that miRNA-552 and miRNA-183 families were positively correlated with the irradiation resistance of rectal cancer, and found that high expression of miRNA-96-5p enhanced the irradiation resistance of rectal cancer cells through direct regulation of the GPC3 gene and abnormal activation of the canonical Wnt signal transduction pathway. Based on the radioreactivity results of patient-derived xenograft models, this is the first screening report for radio-resistant biomarkers in rectal cancer. Our results suggest that miRNA-96-5p expression is an important factor affecting the radiation response of colorectal cancer cells.

Abstract 741: Characterization of high-grade serous ovarian carcinoma by measuring functional signal transduction pathway activity

Abstract Introduction: The majority of advanced stage high-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, recurs within the first 24 months despite complete remission after initial treatment. Yet there are remarkable differences in disease-free (DFS) and overall survival (OS). We hypothesize that differences in signal transduction pathway (STP) activity may relate to survival and may also improve targeted therapy selection. We investigate the STP activity in relation to survival in patients diagnosed with advanced stage HGSC, as well as identify STPs that may be targeted in alternative treatment strategies. Methods: We included 85 HGSC patients (DFS &amp;lt;12 months, n=52 and DFS &amp;gt;24 months, n=33) who achieved complete remission after treatment with debulking surgery and chemotherapy. Total mRNA was extracted, and RT-qPCR was performed to determine expression levels of pathway-specific target genes in primary HGSC samples taken prior to start of chemotherapy. OncoSignal pathway assays were used to quantitatively measure functional STP activity of the androgen and estrogen receptor (ER), PI3K, Hedgehog, TGF-β and Wnt pathways. Survival analysis in relation to STP activity was performed by means of Cox regression analysis and differences between groups were analyzed with Mann-Whitney U tests. Results: Differences in PI3K, Hedgehog and TGF-β pathway activity were observed across the HGSC cohort, suggesting unique tumor activation patterns. Median STP activity was not discernably different between the short and long DFS groups. Significantly higher ER pathway activity was found in premenopausal women (n=16) compared to postmenopausal women (n=67) (p=0.002). Cox regression analysis in postmenopausal women (n=67) showed that ER pathway activity was positively associated with DFS (HR=0.943, p=0.033) and OS (HR=0.930, p=0.041) in univariate analysis. None of the pathways were significantly related to DFS or OS in premenopausal women (n=16). Conclusion: We observed a positive association between ER pathway activity and survival in postmenopausal women but not in premenopausal women. Also, differences in activation levels of the other STPs were observed in individual patients. STP analysis may be used to aid targeted therapy selection, such as anti-hormonal therapy, in HGSC patients. Citation Format: Phyllis van der Ploeg, Laura van Lieshout, Yvonne Wesseling-Rozendaal, Anja van de Stolpe, Diederick Keizer, Steven Bosch, Marjolein Lentjes-Beer, Caroline Vos, Joanne de Hullu, Ruud Bekkers, Jurgen Piek. Characterization of high-grade serous ovarian carcinoma by measuring functional signal transduction pathway activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 741.

Abstract 740: A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology

Abstract Introduction: OncoSignal test was developed to determine and quantify functional signal transduction pathway activity levels of 4 key oncogenic signaling pathways (ER, AR, PI3K and MAPK), based on the quantitative measurement of mRNA expression levels of the direct target genes. Here we present data of a 4 pathway qPCR test, that can be used in external labs and is able to measure simultaneously the 4 pathway activities in tumor tissue samples possibly providing important information for optimal therapy selection for treatment of breast cancer. Materials and methods: OncoSignal 4 pathway test was compared between 2 reference laboratories; the Philips service lab in The Netherlands and at Protean BioDiagnostics, a US CAP-certified CLIA service lab. mRNA was isolated from unstained FFPE tissue sections of well characterized breast tissues obtained from 65 patients (retrospective). Annotated areas with at least 50% tumor cells were used (total volume about 0.25mm3). The OncoSignal test was used to measure the signal transduction pathway activity of AR, ER, PI3K and MAPK pathways. IHC staining for ER, PR, Ki67 and HER2 was also available for all samples enabling tumor categorization into conventional breast cancer subtypes. Results: Excellent inter-lab concordance was found with correlation coefficients of 0.99, 0.99, 0.98 and 0.99 for ER, AR, PI3K and MAPK, respectively. Average absolute difference between pathway activities was 3.6 activity points on a 0-100 activity score scale. All 32 cases passed the criteria set for reproducibility. Of the 65 tested breast cancer samples, 58 could be subtyped based on IHC. Five of the 58 samples failed QC. Of interest, ER IHC positive luminal A (41%) and B (28%) breast cancer showed high variation in ER pathway activity between cases, suggesting that the ER signaling pathway is not the tumor driving pathway in a significant subset of IHC ER positive tumors. As expected the Her2-enriched (17%) and TNBC (17%) sub-groups predominantly had a very low ER pathway activity score. AR pathway activity was most prominent in the HER2 enriched tumor groups, while in TNBC the MAPK pathway was most active. Conclusion: The OncoSignal 4 pathway test is robust and can be implemented in external (local) labs as shown by the excellent correlation results. The results of the signal pathway activity analysis correlate well with breast subtype classification, however, the assay has the potential to uncover specific pathway activation independent of subtype which may have significant value for precision oncology and targeted therapy selection. The test has potential for wide utilization as it can be performed on conventional FFPE prepared breast specimens, and may be complementary to conventional mutational and IHC analysis for classifying breast cancer and selecting appropriate therapy. Citation Format: Eveline den Biezen, Saskia Vermeer, Diederick Keizer, Martijn Akse, Martijn van Zelst, Dianne van Strijp, Hannah Park, Anthony Magliocco. A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 740.