Expression Microarray Analysis Research Articles

Long-term transcriptomic and proteomic effects in Sprague Dawley rat thyroid and plasma after internal low dose 131I exposure.

BackgroundRadioiodide (131I) is commonly used to treat thyroid cancer and hyperthyroidis.131I released during nuclear accidents, have resulted in increased incidence of thyroid cancer in children. Therefore, a better understanding of underlying cellular mechanisms behind 131I exposure is of great clinical and radiation protection interest. The aim of this work was to study the long-term dose-related effects of 131I exposure in thyroid tissue and plasma in young rats and identify potential biomarkers.Materials and methodsMale Sprague Dawley rats (5-week-old) were i.v. injected with 0.5, 5.0, 50 or 500 kBq 131I (Dthyroid ca 1–1000 mGy), and killed after nine months at which time the thyroid and blood samples were collected. Gene expression microarray analysis (thyroid samples) and LC-MS/MS analysis (thyroid and plasma samples) were performed to assess differential gene and protein expression profiles in treated and corresponding untreated control samples. Bioinformatics analyses were performed using the DAVID functional annotation tool and Ingenuity Pathway Analysis (IPA). The gene expression microarray data and LC-MS/MS data were validated using qRT-PCR and ELISA, respectively.ResultsNine 131I exposure-related candidate biomarkers (transcripts: Afp and RT1-Bb, and proteins: ARF3, DLD, IKBKB, NONO, RAB6A, RPN2, and SLC25A5) were identified in thyroid tissue. Two dose-related protein candidate biomarkers were identified in thyroid (APRT and LDHA) and two in plasma (DSG4 and TGM3). Candidate biomarkers for thyroid function included the ACADL and SORBS2 (all activities), TPO and TG proteins (low activities). 131I exposure was shown to have a profound effect on metabolism, immune system, apoptosis and cell death. Furthermore, several signalling pathways essential for normal cellular function (actin cytoskeleton signalling, HGF signalling, NRF2-mediated oxidative stress, integrin signalling, calcium signalling) were also significantly regulated.ConclusionExposure-related and dose-related effects on gene and protein expression generated few expression patterns useful as biomarkers for thyroid function and cancer.

MicroRNA-21 facilitates osteoblast activity

MicroRNAs are emerging as critical post-transcriptional modulators in bone remodeling, regulating the functions of osteoblasts and osteoclasts. Intercellular crosstalk between osteoblasts and osteoclasts is mediated by miR-21 that controls the bone homeostasis response, providing potential targets for the maintenance of osteoblast function. The aim of this study was to investigate the effects of miR-21 on osteoblast function, and to explore the underlying mechanism. Increased alkaline phosphatase (ALP) activity and accelerated matrix mineralization was observed in mouse pre-osteoblast MC3T3-E1 cells compared with the non-induction (control) group. MiR-21 positively regulates osteogenic differentiation and mineralization by facilitating the expression of key osteogenic factors (ALP, Runx2, Osteopontin (OPN), Osterix (OSX) and Mef2c) in MC3T3-E1 cells. Furthermore, a deficiency of miR-21 suppresses the expression of those factors at both the mRNA and protein levels, indicating that miR-21 is a positive regulator of osteoblastic differentiation. H-E staining, Azan staining, Masson's Trichrome staining and Toluidine blue staining were performed in jaw and femur tissues of miR-21 knockout (miR-21KO) and wild-type (WT) mice. Immunohistochemical staining revealed substantially lower levels of ALP, Runx2 and OSX expression in jaw and femur tissues of miR-21KO mice. A similar trend was observed in femur tissues using quantitative real-time (RT) PCR. A total of 17 osteogenesis-related mRNAs were found to be differentially expressed in miR-21KO femur tissues using Mouse Gene Expression Microarray analysis. GeneSpring and Ingenuity Pathway Analysis revealed several potential target genes that are involved in bone remodeling, such as IL-1β and HIF-1α. Several important pathways were determined to be facilitators of miR-21, which provides a reliable reference for future studies to elucidate the biological mechanisms of osteoblast function. Taken together, these results lead us to hypothesize a potential role for miR-21 in regulating osteoblast function, thus representing a potential biomarker of osteogenesis.

Epigenetic Silencing of Tumor Suppressor miR-124 Directly Supports STAT3 Activation in Cutaneous T-Cell Lymphoma.

Increasing evidence supports a potential role for STAT3 as a tumor driver in cutaneous T-cell lymphomas (CTCL). The mechanisms leading to STAT3 activation are not fully understood; however, we recently found that miR-124, a known STAT3 regulator, is robustly silenced in MF tumor-stage and CTCL cells. Objective: We studied here whether deregulation of miR-124 contributes to STAT3 pathway activation in CTCL. Methods: We measured the effect of ectopic mir-124 expression in active phosphorylated STAT3 (p-STAT3) levels and evaluated the transcriptional impact of miR-124-dependent STAT3 pathway regulation by expression microarray analysis. Results: We found that ectopic expression of miR-124 results in massive downregulation of activated STAT3 in different CTCL lines, which resulted in a significant alteration of genetic signatures related with gene transcription and proliferation such as MYC and E2F. Conclusions: Our study highlights the importance of the miR-124/STAT3 axis in CTCL and demonstrates that the STAT3 pathway is regulated through epigenetic mechanisms in these cells. Since deregulated STAT3 signaling has a major impact on CTCL initiation and progression, a better understanding of the molecular basis of the miR-124/STAT3 axis may provide useful information for future personalized therapies.

Arsenic nano complex induced degradation of YAP sensitized ESCC cancer cells to radiation and chemotherapy

BackgroundIncreased reactive oxygen species (ROS) production by arsenic treatment in solid tumors showed to be effective to sensitize cancer cells to chemotherapies. Arsenic nano compounds are known to increase the ROS production in solid tumors.MethodsIn this study we developed arsenic–ferrosoferric oxide conjugated Nano Complex (As2S2–Fe3O4, AFCNC) to further promote the ROS induction ability of arsenic reagent in solid tumors. We screen for the molecular pathways that are affect by arsenic treatment in ESCC cancer cells. And explored the underlying molecular mechanism for the arsenic mediated degradations of the key transcription factor we identified in the gene microarray screen. Mouse xenograft model were used to further verify the synthetic effects of AFCNC with chemo and radiation therapies, and the molecular target of arsenic treatment is verified with IHC analysis.ResultsWith gene expression microarray analysis we found Hippo signaling pathway is specifically affected by arsenic treatment, and induced ubiquitination mediated degradation of YAP in KYSE-450 esophageal squamous cell carcinoma (ESCC) cells. Mechanistically we proved PML physically interacted with YAP, and arsenic induced degradation PML mediated the degradation of YAP in ESCC cells. As a cancer stem cell related transcription factor, YAP 5SA over expressions in cancer cells are correlated with resistance to chemo and radiation therapies. We found AFCNC treatment inhibited the increased invasion and migration ability of YAP 5SA overexpressing KYSE-450 cells. AFCNC treatment also effectively reversed protective effects of YAP 5SA overexpression against cisplatin induced apoptosis in KYSE-450 cells. Lastly, with ESCC mouse xenograft model we found AFCNC combined with cisplatin treatment or radiation therapy significantly reduced the tumor volumes in vivo in the xenograft ESCC tumors.ConclusionsTogether, these findings suggested besides ROS, YAP is a potential target for arsenic based therapy in ESCC, which should play an important role in the synthetic effects of arsenic nano complex with chemo and radiation therapy.

Abstract PO-004: Identification of MLL4 as a novel therapeutic target of pancreatic ductal adenocarcinoma by in vivo genome-wide RNAi screening

Abstract The prognosis of pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to the lack of effective therapeutics. It is urgent to identify novel therapeutic target for PDAC. We performed in vivo genome-wide RNAi screening to identify cancer-associated candidate genes with critical roles in the tumorigenesis of PDAC. We identify mixed-lineage leukaemia 4 (MLL4 also known as KMT2B), which belongs to histone H3 lysine 4 (H3K4) methyltransferase of MLL family, as one of the cancer-associated gene to PDAC tumorigenesis. Here, we firstly report that MLL4 expression was frequently upregulated in PDAC cell lines as well as human PDAC tissue, depicting its potential role in PDAC tumorigenesis. Lysine methylation of different position of Histone is performed by SET (suppressor of variegation) domain of MLL family, aberrant expression of SET domain results in disruption of epigenetic gene regulation and leads to cancer. To characterize the in vitro tumorigenic role of SET domain of MLL4 in the development of PDAC, gain-of-function and loss-of-function approaches of SET domain of MLL4 and MLL4 were performed in PDAC cells, respectively. Ectopic SET expression promotes cell proliferation, colony formation, cell migration as well as invasion, demonstrating its oncogenic role in PDAC cells. Moreover, MLL4-depleted PDAC cells exhibited significantly reduction of tumor growth in nude mice xenograft model. Putative MLL4 activating and repressive targets were identified using gene expression microarray analysis in PDAC cell lines Capan2 and Panc-1. Expression alteration of these genes by regulated by MLL4 contribute to PDAC development and progression. To further delineate whether MLL4 regulates expression of the activating and repressing targets via H3K4 methylation, activity of H3K4 methylation was investigated in PDAC cells with MLL4 knockdown. Suppression of MLL4 reduced the H3K4Me2 and H3K4Me3 occupancies at promoter of MLL4 target genes, suggesting that MLL4 regulates downstream genes expression through H3K4 methylation. Taken together, our findings demonstrate that aberrant overexpression of MLL4 in PDAC cells alters its downstream oncogene and tumor suppressor genes, contributing to PDAC tumorigenesis. Thus, targeting MLL4 may serve as a potential therapeutic modality in the management of PDAC. Citation Format: Frederic K. C. Fung, Yinxin Zhu, Chi Hin Wong, Yangchao Chen. Identification of MLL4 as a novel therapeutic target of pancreatic ductal adenocarcinoma by in vivo genome-wide RNAi screening [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-004.

Sirtuin 6 deficiency induces endothelial cell senescence via downregulation of forkhead box M1 expression.

Cellular senescence of endothelial cells causes vascular dysfunction, promotes atherosclerosis, and contributes to the development of age-related vascular diseases. Sirtuin 6 (SIRT6), a conserved NAD+-dependent protein deacetylase, has beneficial effects against aging, despite the fact that its functional mechanisms are largely uncharacterized. Here, we show that SIRT6 protects endothelial cells from senescence. SIRT6 expression is progressively decreased during both oxidative stress-induced senescence and replicative senescence. SIRT6 deficiency leads to endothelial dysfunction, growth arrest, and premature senescence. Using genetically engineered endothelial cell-specific SIRT6 knockout mice, we also show that down-regulation of SIRT6 expression in endothelial cells exacerbates vascular aging. Expression microarray analysis demonstrated that SIRT6 modulates the expression of multiple genes involved in cell cycle regulation. Specifically, SIRT6 appears to regulate the expression of forkhead box M1 (FOXM1), a critical transcription factor for cell cycle progression and senescence. Overexpression of FOXM1 ameliorates SIRT6 deficiency-induced endothelial cell senescence. In this work, we demonstrate the role of SIRT6 as an anti-aging factor in the vasculature. These data may provide the basis for future novel therapeutic approaches against age-related vascular disorders.

Striking Association of Lymphoid Enhancing Factor (LEF1) Overexpression and DUSP22 rearrangements in Anaplastic Large Cell Lymphoma

Introduction: Anaplastic large cell lymphomas (ALCLs) are CD30-positive T-cell lymphomas classified into ALK-positive and ALK-negative. The ALK-negative ALCLs are a genetically heterogeneous group that includes DUSP22-rearranged, TP63-rearranged and triple-negative cases. The DUSP22-rearranged ALCLs characteristically grow in a diffuse sheet-like pattern and are composed of intermediate-sized monomorphous hallmark cells, admixed with smaller cells with prominent nuclear indentations and occasional central nuclear pseudo inclusions (King RL et al, Am J Surg Pathol. 2016). Further, DUSP22-rearranged ALCL is a molecularly distinct subset of ALCL with a unique immunogenic phenotype that portends a favorable prognosis (Luchtel RA et al. Blood 2018). Lymphoid enhancer binding factor (LEF1) is a crucial nuclear mediator of the Wnt/ β-catenin pathway and a transcription factor involved in T-cell maturation. However, limited data exists on the association of LEF1 in T-cell lymphomas, including ALCL. The aims of this study were to explore LEF1 expression by immunohistochemistry in different subsets of ALCLs and to analyze the association of Wnt/ β-catenin pathway in this cohort. Methods: After approval by the institutional review board, we screened pathology reports from January 1, 1995 to December 31, 2019 and identified patients with a diagnosis of ALCL using our institutional database. Details on fluorescence in situ hybridization studies in ALK-negative cases were documented. Immunostaining for LEF1 and β catenin was performed on all cases where formalin fixed paraffin embedded tissue was available. The presence or absence of β catenin nuclear staining was recorded. LEF1 nuclear staining in the neoplastic ALCL cells was graded as negative (0), weak (1+), intermediate (2+) or strong (3+) and the percentage of LEF1-positive neoplastic cells was detailed (Figure 1). LEF1 scoring was performed by manual counting of the stained nuclei out of all tumor nuclei by two pathologists (A.R. and M.S.) and the grading was reviewed by another pathologist (P.J.K.). Results: We evaluated 45 ALCL cases, including 41 (91.1%) ALK-negative and 4 (8.9%) ALK-positive ALCLs. Among the ALK-negative ALCLs, there were16 DUSP22-rearranged, and 25 non-DUSP22-rearanged (7 TP63-rearranged and 18 triple-negative) cases. Fifteen (94%) of 16 DUSP22-rearranged ALCLs showed strong expression of LEF1 in >75% tumor cells (Figure 2). In contrast, this strong and uniform LEF1 overexpression was only detected in 1 of 29 (3%) of the remaining ALCL cases (P <.0001) (Table 1). We previously characterized a distinctive molecular signature in ALK-negative ALCLs with DUSP22-rearrangements using gene expression microarray analysis on frozen ALCL tissue samples (Luchtel RA et al. Blood 2018). On examining LEF1 gene expression in this previously published dataset, DUSP22 -rearranged cases had significantly higher LEF1 expression than any other genetic subtype (triple-negative, TP63-rearranged, or ALK-positive). Overall, mean LEF1 expression was 6.3-fold higher in ALCLs with DUSP22 rearrangements than in ALCLs without DUSP22-rearrangements (P=0.0001). LEF1 is a coactivator of the Wnt/β-catenin pathway; therefore, it is reasonable to hypothesize the overexpression of LEF1 may promote Wnt/β-catenin signaling. However, all LEF1-positive cases were negative for β-catenin by immunohistochemistry, suggesting that LEF1 overexpression is independent of the Wnt/β-catenin pathway signaling in these cases. Conclusions: There is a striking association between high LEF1 expression and DUSP22-rearrangements in ALCLs. The strong and uniform LEF1 expression pattern has a high positive predictive value (94%) and high negative predictive value (96%) for DUSP22- rearrangement in ALK-negative ALCLs (Table 2). The combination of characteristic morphologic and molecular features of DUSP22-rearranged cases with the unique LEF1 expression further emphasizes that DUSP22-rearranged ALCL represents a distinct clinicopathologic subset of ALCL. Disclosures No relevant conflicts of interest to declare.

A comparative PET imaging study of 44gSc- and 68Ga-labeled bombesin antagonist BBN2 derivatives in breast and prostate cancer models

IntroductionRadiolabeled peptides play a central role in nuclear medicine as radiotheranostics for targeted imaging and therapy of cancer. We have recently proposed the use of metabolically stabilized GRPR antagonist BBN2 for radiolabeling with 18F and 68Ga and subsequent PET imaging of GRPRs in prostate cancer. The present work studied the impact of 44gSc- and 68Ga-labeled DOTA complexes attached to GRPR antagonist BBN2 on the in vitro GRPR binding affinity, and their biodistribution and tumor uptake profiles in MCF7 breast and PC3 prostate cancer models. MethodsDOTA-Ava-BBN2 was radiolabeled with radiometals 68Ga and 44gSc. Gastrin-releasing peptide receptor (GRPR) affinities of peptides were assessed in PC3 prostate cancer cells. GRPR expression profiles were studied in human breast cancer tissue samples and MCF7 breast cancer cells. PET imaging of 68Ga- and 44gSc-labeled peptides was performed in MCF7 and PC3 xenografts as breast and prostate cancer models. ResultsRadiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava BBN2 were prepared in radiochemical yields of 70–80% (decay-corrected), respectively. High binding affinities were found for both peptides (IC50 = 15 nM (natGa) and 5 nM (natSc)). Gene expression microarray analysis revealed high GRPR mRNA expression levels in estrogen receptor (ER)-positive breast cancer, which was further confirmed with Western blot and immunohistochemistry. However, PET imaging showed only low tumor uptake of both radiotracers in MCF7 xenografts ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.27 ± 0.06); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.20 ± 0.03)). In contrast, high tumor uptake and retention were found for both radiopeptides in PC3 tumors ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.46 ± 0.07); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.51 ± 0.11)). ConclusionsComparison of 68Ga- and 44gSc-labeled DOTA-Ava-BBN2 peptides revealed slight but noticeable differences of the radiometal with an impact on the in vitro GRPR receptor binding properties in PC3 cells. No differences were found in their in vivo biodistribution profiles in MCF7 and PC3 xenografts. Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava-BBN2 displayed comparable tumor uptake and retention profiles with rapid blood and renal clearance profiles in both tumor models. Advances in knowledge and implications for patient careThe favorable PET imaging performance of [44gSc]Sc-DOTA-Ava-BBN2 in prostate cancer should warrant the development of an [43Sc]Sc-DOTA-Ava-BBN2 analog for clinical translation which comes with a main γ-line of much lower energy and intensity compared to 44gSc.

Usefulness of serum miR-1246/miR-106b ratio in patients with esophageal squamous cell carcinoma.

The function of microRNAs (miRs) is associated with the development and progression of various malignancies, with miRs presenting stably in the serum. The current study assessed the role of miR-1246 and miR-106b in the serum of patients with esophageal squamous cell carcinoma (ESCC). A comprehensive microarray analysis of miR expression was performed using the serum of patients with ESCC, which were subsequently validated via reverse transcription-quantitative PCR. A total of 55 test samples were obtained from Chiba University and 101 validation samples were gained from Chiba Cancer Center. The results revealed that miR-1246 expression significantly increased and miR-106b expression significantly decreased in each cohort. Receiver operating characteristic analysis revealed that the area under the curve (AUC) value of miR-1246 was 0.816 (sensitivity, 72.7%; specificity, 69.2%) and 0.779 (sensitivity, 71.3%; specificity, 70.6%) for the test and validation cohorts, respectively. The AUC of miR-106b was 0.716 (sensitivity, 65.5%; specificity, 61.6%) and 0.815 (sensitivity, 74.3%; specificity, 73.5%), respectively. In addition, the AUC of the miR-1246/miR-106b ratio was 0.901 (sensitivity, 80.0%; specificity, 80.0%) and 0.903 (sensitivity, 82.1%; specificity, 82.3%), respectively, which indicated a higher diagnostic ability compared with that of miR-1246 or miR-106b alone. The high miR-1246/miR-106b ratio group was associated with clinicopathological factors such as depth of invasion, progression, lymph node metastasis, and poor prognosis. Therefore, effective biomarkers may be generated by combining individual miRs obtained by comprehensive analysis of ESCC patient sera.

P. gingivalis Lipopolysaccharide Stimulates the Upregulated Expression of the Pancreatic Cancer-Related Genes Regenerating Islet-Derived 3 A/G in Mouse Pancreas.

Although epidemiological studies have shown a relationship between periodontal disease and pancreatic cancer, the molecular mechanisms involved remain unclear. In this study, the effects of systemic administration of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on gene expression were comprehensively explored in mouse pancreas that did not demonstrate any signs of inflammation. PG-LPS was prepared in physiological saline and intraperitoneally administered to male mice at a concentration of 5 mg/kg every 3 days for 1 month. After extracting total RNA from the excised mice pancreas, a comprehensive DNA microarray analysis of gene expression was performed. Tissue specimens were also subjected to hematoxylin–eosin staining and immunohistochemistry using anti-regenerating islet-derived 3A and G (Reg3A/G) antibody. ImageJ software was used to quantify the area of Reg3A/G positive cells in pancreatic islets by binarizing image date followed by area extraction. The results were compared using Mann–Whitney U test. Data are presented as mean ± standard deviation (SD) with p < 0.05 considered as significant. Reg3G, a gene related to pancreatic cancer, was one of the 10 genes with the highest levels of expression in the pancreas stimulated with PG-LPS. The comprehensive analysis revealed a 73-fold increase in Reg3G expression level in the PG-LPS group when compared with the control group; in addition, the expression level of Reg3A was increased by 11-fold in the PG-LPS group. Image analysis showed that the ratio of Reg3A/G positive cells was higher in the PG-LPS group than the control. Immunostaining showed the presence of Reg3A/G-positive cells in the alpha-cell equivalent areas around the islets of Langerhans in the PG-LPS group. These results support the notion that periodontal disease may be a risk factor for pancreatic cancer.

Neuropilin-1 is up-regulated by cancer-associated fibroblast-secreted IL-8 and associated with cell proliferation of gallbladder cancer.

We previously demonstrated that cancer‐associated fibroblasts (CAFs) promoted the proliferation of gallbladder cancer (GBC) cells, but the mechanism is not clear. Neuropilin‐1 (NRP‐1) plays an important role in various malignancies as transmembrane glycoprotein. Our goal was to reveal the relationship between CAFs and NRP‐1 and their potential functions in GBC. In this study, we found NRP‐1 was overexpressed in GBC tissue, associated with poor survival and was up‐regulated by CAFs. The cytokine array cluster analysis revealed IL‐8 secreted by CAFs facilitated the up‐regulation of NRP‐1 in tumour cells. NRP‐1 knockdown suppressed tumour growth in vivo. Gene expression microarray analysis showed 581 differentially regulated genes under NRP‐1 knockdown conditions. Ingenuity pathway analysis demonstrated that NRP‐1 knockdown may inhibit tumour progression by affecting cell proliferation. We then confirmed that NRP‐1 knockdown in NOZ and GBC‐SD cells significantly inhibited cell proliferation. Additionally, the IL‐8 mediated MDM2 and CCNA2 expression were affected by NRP‐1 knockdown. Our findings suggested that NRP‐1 was up‐regulated by CAF‐secreted IL‐8, which subsequently promoted GBC cell proliferation, and these molecules may serve as useful prognostic biomarkers and therapeutic targets for GBC.

Alteration of gene expression in mice after glaucoma filtration surgery

To clarify the early alterations of gene expression using a mouse model of glaucoma filtration surgery, we carried out microarray expression analysis. Using BALB/c mice, a filtration surgery model was made by incision of the limbal conjunctiva, followed by the insertion of a 33G needle tip into the anterior chamber, and 11-0 nylon sutures. Subgroups of mice were treated intraoperatively with 0.4 mg/ml mitomycin-C (MMC). At day 3 after surgery the bleb was maintained. The bleb region tissue was sampled 3 days after the filtration surgery, and gene expression analysis was carried out using a mouse Agilent 8 × 60 K array. We found 755 hyperexpressed transcripts in the bleb region compared to control conjunctiva. The hyperexpressed transcripts included epithelial cell metaplasia-related (Il1b, Krt16, Sprr1b), inflammation-related (Ccl2, Il6) and wound healing-related (Lox, Timp1) genes. We also found downregulation of a goblet cell marker gene (Gp2) in the bleb conjunctiva. MMC treatment suppressed elastin (Eln) gene expression and enhanced keratinization-related gene expression (Krt1, Lor) in the bleb region. Our results suggest the importance of epithelial wound healing after filtration surgery, and this filtration surgery model will be a useful tool for further pathophysiological analysis.

Are different markers of endometrial receptivity telling us different things about endometrial function?

To what extent do endocrine, immunological, gene expression and histological markers of endometrial receptivity correlate? Between November 2017 and September 2019, 121 women referred to a University Hospitals Fertility Clinic consented to inclusion in this cohort study. The women underwent timed endometrial biopsy followed by blood samples in a hormone-substituted cycle. Of these, 37 women had just started IVF treatment, and the remaining 84 had experienced recurrent implantation failure following IVF/ICSI. The hormone-substituted cycle consisted of initiation with oral oestradiol followed by addition of vaginal progesterone treatment for five full days. Endometrial biopsies were subject to histological examination, immune cell markers by immunohistochemistry (CD56+ , CD16+ , CD163+ , FoxP3) and gene expression microarray analyses with the endometrial receptivity array (ERA® ) test (Igenomix). Plasma progesterone and oestradiol were measured on the day of biopsy. CD56+ uterine natural killer (uNK) cell counts correlate with transcriptional markers of endometrial receptivity assessed by the ERA test. Endometrial maturation, receptivity and immunological markers were not correlated with mid-luteal blood plasma progesterone level. Mid-luteal serum oestradiol level correlated with markers of endometrial maturation and receptivity. The tests were carried out during a standard hormone substitution cycle, and the findings may not apply in the natural cycle. CD56+ uNK cell counts and endometrial receptivity assessed by the ERA test appear to be linked. Mid-luteal progesterone levels were not correlated to the tested markers of endometrial receptivity. In contrast, mid-luteal oestradiol level was inversely related to markers of endometrial receptivity and maturation.

Interleukin 3-induced GITR promotes the activation of human basophils

Human basophils regulate allergic reactions by secreting histamine, interleukin 4 (IL-4) and IL-13 through key surface receptors FcεRI as well as IL-3R, which are constitutively expressed on basophils. IL-3/IL-3R signaling axis plays key roles in regulating the development and activation of basophils. We and others have shown that IL-3-induced surface receptors e.g. ST2, IL-17RB and IL-2 receptors regulate the biology of basophils. However, the expression and function of IL-3-induced surface proteins on human basophils remain to be elucidated. We in this study aimed to identify new basophil activation regulators by transcriptomic analysis of IL-3-stimulated basophils. Gene expression microarray analysis of IL-3-treated basophils revealed 2050 differentially expressed genes, of which 323 genes encoded surface proteins including GITR. We identified that GITR was preferentially induced by IL-3 rather than anti-IgE, IL-33, fMLP and C5a. IL-3-induced GITR was suppressed by inhibitors targeting JAK2, PI3K and MEK1/2. Stimulation of IL-3-treated basophils by GITR enhanced the expression of IL-4 and IL-13. Moreover, IgE-mediated degranulation was enhanced by GITRL in the presence of IL-3. This transcriptomic analysis of IL-3-activated basophils helps to identify novel activation regulator. IL-3-induced GITR promoted the activation of basophils, adding new evidence supporting GITR as an important player in Th2-associated immune responses.

Abstract 5970: Function and regulatory mechanism of GPM6A in hepatocellular carcinoma development and progression

Abstract BACKGROUND: GPM6A is a glycoprotein found in endothelial cells and is considered to be a malignant lymphoma oncogene. However, the role of GPM6A in the development of hepatocellular carcinoma (HCC) is unknown. Through Affymetrix gene expression microarray and bioinformatics analysis, very low GPM6A expression was found in HCC tissue. The present study aims to explore the function and regulatory mechanism of GPM6A in HCC development and progression. METHODS: Levels of GPM6A expression was determined in various hepatoma cell lines and HCC specimens from different base disorders, and its role on cell proliferation was investigated in GPM6A stably over-expressing cell lines. Modulating effects of a specific miRNA were evaluated with miRNA mimetic transfection. RESULTS: Much lower GPM6A levels were found in HCC occuring in HBV-infection or nonalcoholic steatohepatitis. TCGA data confirmed that low GPM6A expression in HCC correlates with a poor overall survival and progression-free survival rate (p=0.018 and 0.004). Over-expression of GPM6A inhibited proliferation in SMMC-7721 and MHCC-97H cells by 31% and 30%, respectively (p&amp;lt;0.01), and led to decreased colony formation by 40%. Overexpression of GPM6A was found to significantly reduce wound healing and migration rates in these two cell types. miR-96-5p was expressed at 6 to 61-fold higher in HCC tissues than adjacent tissues (p&amp;lt;0.01); whereas GPM6A gene expression was at 3 to 55-fold lower (p&amp;lt;0.01) in a negative correlation between miR-96-5p levels and GPM6A levels in HCC specimens (r=-0.64, p&amp;lt;0.05). Further experiments demonstrated that miR-96-5p acted directly on the 3'UTR of the GPM6A gene, and transfection of miR-96-5p significantly decreased GPM6A mRNA expression by 50% in two hepatoma cell lines. Moreover, MiR-96-5p transfection promoted proliferation (66%, 59%, p&amp;lt;0.01), migration (10%, 9%, p&amp;lt;0.05) and invasion (557±66 vs. 226±19; 235±36 vs. 153±17, p&amp;lt;0.05-0.01) of SMMC-7721 and MHCC-97H hepatoma cells; whereas the function of oncogenic microRNA-96 was significantly inhibited in GPM6A-overexpressed hepatoma cells. CONCLUSIONS: GPM6A expression in HCC is commonly suppressed, and correlated to a poor prognosis. Its low expression in HCC tissues is most likely attributed to upregulated miR-96-5p, which may function as an oncolytic regulator through suppressed GMP6A that may block proliferation, cloning formation, migration and invasion of hepatoma cells. Citation Format: Rui Zong Li, Gang Xu, Min Ji Zhu, Jian Wu, Jian Wu. Function and regulatory mechanism of GPM6A in hepatocellular carcinoma development and progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5970.

Abstract 1709: New targeted therapy on SOCS1-related immune-response in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) includes heterogenous molecular subtypes, e.g., basal-like, immunomodulatory, mesenchymal, mesenchymal stem cell and luminal androgen receptor subtypes. Chemotherapy is the standard care for patients with early TNBC, but advanced or metastatic disease has unfavorable prognosis. PARP inhibitors and immune checkpoint inhibitors are effective for patients with BRCA gene mutation and PD-L1-positive TNBC, respectively. However, their clinical benefits seem to be limited. Thus, we are investigating new therapy targeted on immune modulation in TNBC. Materials and methods: First, we performed microarray expression analysis (Agilent Technologies, Inc.) of small RNA extracted from 11 breast cancer specimens. Second, miRNA subset was compared in TNBC and non-TNBC, and several genes were identified as predicted targets for interaction of miRNA in TNBC. Third, we examined a targeted gene and protein expressions in cancer tissues by real-time qPCR and immunohistochemical staining. Tissues samples were obtained from breast cancer patients with TNBC and non-TNBC, who were scheduled to receive neoadjuvant chemotherapy (NAC). Statistical significance is evaluated by chi-squared test. This study is approved at IRB in Kyorin University School of Medicine and also supported by Grant-in-Aid for Scientific Research C of Japan Society for the Promotion of Science. Results: From comprehensive analysis, SOCS1 (suppressor of cytokine signaling 1), IL-13 and several genes were predicted as interactive targets of miRNA in TNBC. We focused on SOCS1 gene amplification and its protein overexpression in breast cancer. Most patients were treated with NAC of anthracycline- and/or taxane-based regimens. Their pathological response was graded in surgical specimens according to classification of the Japanese Breast Cancer Society. From 55 tissue specimens stained by anti-SOCS1 antibody, there was no difference among staining intensity graded at score 0-3 and pathological response of chemotherapy. However, gene amplification of SOCS1 was not statistically different but relatively lower in 6 cases with pathologically partial or complete response (grade 2 or 3) than in 11 cases with no pathological response (grade 1a or 1b). Discussions: SOCS1 is well known as a negative regulator of JAK-STAT pathway. SOCS1 is also related to suppression of T cell activation and modulation of M1 macrophage. Our data suggest that down regulation of SOCS1 gene influences clinical benefit of chemotherapy as well as host immune response like tumor-infiltrating lymphocytes (TIL). We will examine relationship between SOCS1 protein expression and TIL in breast cancer and also examine cell proliferation and T cell activation in TNBC and non-TNBC cell lines under presence and absence of SOCS1 gene expression. Conclusion: SOCS1 is a promising molecular target to modulate immune editing in TNBC. Citation Format: Shigeru Imoto, Tomohiro Chiba, Sota Asaga, Hirotsugu Isaka, Shigehiro Yokoi, Yoshiharu Ishizaka, Ai Tsuchiya, Hiroshi Kamma. New targeted therapy on SOCS1-related immune-response in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1709.

Abstract 2012: Recurrent rearrangements of NAALADL2 in prostate, breast, cervical, head and neck and lung squamous cell carcinoma

Abstract Prostate cancer is a heterogeneous disease with unique molecular aberrations present in patient sub-groups. Distinct prostate cancer molecular changes have been shown to associate with specific clinical outcomes, suggesting the potential of molecular markers as diagnostic and prognostic biomarkers for prostate cancer. Recurrent ETS family gene fusions, BRAF and SPINK1 overexpression account for about 50-60% of the prostate cancer cases. Genetic aberrations in the remaining 40-50% of the cases is not known. Our attempt to identify new molecular markers in prostate cancer led to the identification of NAALADL2 gene shown to be associated with aggressive prostate cancer. In this study, we carried out a comprehensive analysis of genomic changes of NAALADL2 in prostate and other solid cancers by analyzing copy number changes and gene expression using TCGA next generation RNA sequencing data. We observed recurrent amplification, rearrangement, deletion, mutations and over-expression of NAALADL2 in prostate cancer cases. Notably, we observed mutually exclusive aberrations in NAALADL2 when compared to other cases with known prostate cancer aberrations including ETS gene fusions, indicating NAALADL2 as a distinct molecular sub-set of prostate cancer. Independent validation by fluorescent in situ hybridization (FISH) analysis using break apart probe for NAALADL2 on 874 prostate cancer revealed recurrent amplification and rearrangements in about 8% (71/874) [PN1] of the cases with higher prevalence in Caucasian American than African American cases. Based on these results, we explored prostate cancer TGCA gene fusion database and identified additional cases with gene fusions involving NAALADL2. We selected one of the gene fusions identified in the TCGA database involving NAALADL2-PIK3CA and conducted in vitro functional characterization studies and showed its oncogenic properties. Gene expression microarray analysis of RWPE1 cells transfected with NAALADL2-PIK3CA showed dysregulation of genes involved in cancer related pathways, further suggesting a role for NAALADL2-PIK3CA in prostate cancer development. Based on these studies, we explored the incidence of NAALADL2 gene fusion in other solid cancers including lung squamous cell cancer (LUSC), ovarian cancer, head and neck cancer, cervical cancer and breast cancer shown to have recurrent gene fusions identified in the TCGA gene fusion database. Notably, recurrent rearrangements and amplification are seen in a large subset (45%) of LUSC patients, but not in lung adenocarcinoma, suggesting that NAALADL2 could be developed as a novel biomarker in LUSC. Further validation studies using FISH in our independent cohort of LUSC and breast cancer patients including 79 patients from Ghana confirmed recurrent rearrangements and amplification in a subset of cases. In conclusion, similar to ERG, BRAF and FGFR genes, we show recurrent gene fusions of NAALADL2 across multiple solid cancer with potential applications as a pan cancer molecular marker for cancer diagnosis and a potential target for drug development. Citation Format: Pavithra D. Arachchige, Shannon Carskadon, James Hu, Justin Fernando, Darshan S. Chandrashekar, Nilesh S. Gupta, Sean R. Williamson, Dhananjay A. Chitale, Craig G. Rogers, James O. Peabody, Mani Menon, Tarek A. Bismar, Evelyn Jiagge, Sooryanarayana Varambally, Nallasivam Palanisamy. Recurrent rearrangements of NAALADL2 in prostate, breast, cervical, head and neck and lung squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2012.

Abstract 3971: The role of obesity-associated adipose tissue inflammation in breast cancer metastasis

Abstract Although obesity is a known risk factor for breast cancer incidence, metastasis, and mortality, the mechanisms underlying this link remain elusive. This study therefore aims to uncover the relationship between obesity-associated adipose tissue and breast cancer metastasis by identifying specific inflammatory mediators in the adipose secretome. It is well known that obesity induces hypoxic conditions in the adipose tissue, stimulating an inflammatory response that ultimately creates a favorable environment for cancer cell growth and survival. The link between obesity-associated adipose inflammation and cancer metastasis, however, remains poorly described. We have previously shown that injection of basal-like E0771's, and claudin-low metM-Wnt-Lung and metM-Wnt-Liver metastatic breast cancer cell lines in mice fed a diet-induced obesity (DIO, 60% kcal from fat) diet displayed larger tumors with increased metastatic potential compared to lean mice fed a control (10% kcal from fat) diet. When diets were supplemented with Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), the enhancing effects of DIO on tumor growth and metastasis were mitigated. Gene expression microarray analysis of non-tumor bearing mammary fat pads from these mice revealed increased mammary expression of inflammation-related genes in tissues from DIO relative to control mice. Moreover, gene expression analysis of the tumor microenvironment from these mice revealed a signature of DIO-induced immunosuppression that was normalized by Sulindac supplementation. To further investigate the relationship between obesity-associated inflammation and breast cancer metastasis, we cultured our metastatic metM-Wnt-Lung, and metM-Wnt-Liver, and claudin-low, non-metastatic M-Wnt breast cancer cell lines with conditioned media (FCM) from the mammary fat pads of mice fed control, DIO, or DIO+Sulindac diets. We then conducted phenotypic assays to characterize differences in cell growth, proliferation, and metastatic potential. After normalizing the FCM concentrations to fat pad mass, we saw increased cell viability and migration in cells treated with FCM from DIO relative to control mice. Luminex assays identified several pro-inflammatory factors associated with the pro-cancer effects of FCM from DIO versus control mice, and additional characterization studies utilizing high-throughput proteomic screening and immune-depletion approaches are on-going. Taken together, our findings suggest an important role for the adipose secretome, and specifically inflammatory mediators, in obesity driven breast cancer progression. Citation Format: Lydia K. Eisenbeis, Shannon B. McDonell, Laura A. Smith, Alyssa J. Cozzo, Michael F. Coleman, Stephen D. Hursting. The role of obesity-associated adipose tissue inflammation in breast cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3971.

Thyroid hormones suppress FOXM1 expression to reduce liver cancer progression.

Thyroid hormones (TH) are multifunctional mediators that fine‑tune several physiological processes, including metabolic rate, digestive function and tissue development via interactions with type II nuclear thyroid hormone receptors (TR). Upon binding of TH, TRs interact specifically with thyroid hormone response elements of target gene promoter regions to regulate their transcription. Earlier studies suggested a correlation between aberrant TR regulation and hepatocellular carcinoma (HCC). THs are involved in a crosstalk between hepatoma and stromal cells, and disruption of TH signaling is associated with tumorigenesis. Previous cDNA microarray analysis of target gene expression following T3 treatment of wild‑type TR‑expressing hepatoma cells led to the identification of forkhead box M1 (FOXM1) as a factor negatively regulated by T3 and associated with poor prognosis in several cancer types. Increased FOXM1 expression during late stages of HCC was associated with poorer overall and recurrence‑free survival in patients with HCC. However, the specific mechanisms underlying FOXM1 activity in liver cancer progression remain to be elucidated. Experiments from the present study showed that TH/TR signaling suppresses FOXM1 mRNA and protein expression. Depletion of FOXM1 induced inhibition of the cell growth rate and a decline in oncogenic cyclin D1, cyclin E and CDK2 expression. Conversely, overexpression of FOXM1 enhanced cell proliferation and expression of oncogenic factors, which was decreased upon FOXM1 depletion. Re‑expression of FOXM1 partially rescued suppression of cell proliferation induced by T3. Collectively, the present findings suggest that TH/TR participates in HCC progression via modulation of FOXM1 expression.

Overexpressed microRNA-140 inhibits pulmonary fibrosis in interstitial lung disease via the Wnt signaling pathway by downregulating osteoglycin.

Interstitial lung disease (ILD) comprises of a group of diffuse parenchymal lung disorders that are strongly associated with substantial morbidity and mortality. Previous studies have highlighted the therapeutic significance of microRNAs (miRNAs) in the treatment of ILD. Thus this study aims to investigate the mechanism by which miR-140 affects ILD through the regulation of osteoglycin (OGN)-Wnt signaling pathway. Gene expression microarray analysis was performed to screen ILD-related differentially expressed genes and miRNAs that regulated OGN. The targeting relationship between miR-140 and OGN was verified. Ectopic expression and knockdown experiments were performed in lung fibroblasts to explore the potential mechanism of action of miR-140 in ILD. The expression of miR-140, OGN, as well as Wnt- and pulmonary fibrosis-related factors, was determined by RT-qPCR and Western blot analysis. In addition, cell viability and apoptosis were examined. OGN was found to be negatively regulated by miR-140. The ectopic expression of miR-140 and OGN silencing resulted in increased lung fibroblast apoptosis and Wnt3a expression, along with reduced proliferation and pulmonary fibrosis. Our results also revealed that miR-140 decreased OGN, thereby activating the Wnt signaling pathway, which was observed to further affect the expression of genes associated with the progression of pulmonary fibrosis in mouse fibroblasts. In conclusion, the key findings from our study suggest that overexpressed miR-140 suppresses ILD development via the Wnt signaling pathway by downregulating OGN, which could potentially be used as a therapeutic target for ILD.