Expression Of Several Oncogenes Research Articles

PRDM1 promotes the ferroptosis and immune escape of thyroid cancer by regulating USP15-mediated SELENBP1 deubiquitination.

The deubiquitinating enzyme Ubiquitin-specific peptidase 15 (USP15) is upregulated in various cancers and promotes tumor progression by increasing the expression of several oncogenes. This project is designed to explore the role and mechanism of USP15 in thyroid cancer (TC) progression. Selenium-binding protein 1 (SELENBP1), USP15, CCL2/5, CXCL10/11, IL-4, and TGF-β1 mRNA levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). SELENBP1, USP15, GPX4, IL-10, Arg-1, Granzyme B, TNF-α, and PR domain zinc finger protein 1 (PRDM1) protein levels were examined by western blot assay. Fe+ level, malondialdehyde (MDA), and lipid-ROS levels were determined using special kits. The proportion of CD11b+CD206+ positive cells was detected using a flow cytometry assay. The role of SELENBP1 on TC cell growth was examined using a xenograft tumor model in vivo. After GeneMANIA prediction, the interaction between USP15 and SELENBP1 was verified using Co-immunoprecipitation (CoIP) assay. The binding between PRDM1 and USP15 promoter was predicted by JASPAR and validated using Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. SELENBP1 was increased in TC subjects and cell lines, and its knockdown repressed TC cell proliferation, migration, invasion, immune escape, and induced ferroptosis in vitro, as well as blocked tumor growth in vivo. In mechanism, USP15 interacted with SELENBP1 and maintained its stabilization by removing ubiquitin. Meanwhile, the upregulation of USP15 was induced by the transcription factor PRDM1. USP15 transcriptionally mediated by PRDM1 might boost TC cell malignant behaviors through deubiquitinating SELENBP1, providing a promising therapeutic target for TC treatment.

ALKBH5 enhances lipid metabolism reprogramming by increasing stability of FABP5 to promote pancreatic neuroendocrine neoplasms progression in an m6A-IGF2BP2-dependent manner

The process of post-transcriptional regulation has been recognized to be significantly impacted by the presence of N6-methyladenosine (m6A) modification. As an m6A demethylase, ALKBH5 has been shown to contribute to the progression of different cancers by increasing expression of several oncogenes. Hence, a better understanding of the key targets of ALKBH5 in cancer cells could potentially lead to the development of new therapeutic targets. However, the specific role of ALKBH5 in pancreatic neuroendocrine neoplasms (pNENs) remains largely unknown. Here, we demonstrated that ALKBH5 was up-regulated in pNENs and played a critical role in tumor growth and lipid metabolism. Mechanistically, ALKBH5 over-expression was found to increase the expression of FABP5 in an m6A-IGF2BP2 dependent manner, leading to disorders in lipid metabolism. Additionally, ALKBH5 was found to activate PI3K/Akt/mTOR signaling pathway, resulting in enhanced lipid metabolism and proliferation abilities. In conclusion, our study uncovers the ALKBH5/IGF2BP2/FABP5/mTOR axis as a mechanism for aberrant m6A modification in lipid metabolism and highlights a new molecular basis for the development of therapeutic strategies for pNENs treatment.Graphical

Differential Gene Expression of Checkpoint Markers and Cancer Markers in Mouse Models of Spontaneous Chronic Colitis.

The presence of checkpoint markers in cancer cells aids in immune escape. The identification of checkpoint markers and early cancer markers is of utmost importance to gain clarity regarding the relationship between colitis and progressive inflammation leading to cancer. Herein, the gene expression levels of checkpoint makers, cancer-related pathways, and cancer genes in colon tissues of mouse models of chronic colitis (Winnie and Winnie-Prolapse mice) using next-generation sequencing are determined. Winnie mice are a result of a Muc2 missense mutation. The identification of such genes and their subsequent expression and role at the protein level would enable novel markers for the early diagnosis of cancer in IBD patients. The differentially expressed genes in the colonic transcriptome were analysed based on the Kyoto Encyclopedia of Genes and Genomes pathway. The expression of several oncogenes is associated with the severity of IBD, with Winnie-Prolapse mice expressing a large number of key genes associated with development of cancer. This research presents a number of new targets to evaluate for the development of biomarkers and therapeutics.

The Prognostic Role of HuR Varies Between Different Subtypes of Breast Cancer Patients: Data Mining and Retrospective Analysis.

Human-antigen R (HuR) is an RNA-binding protein, which regulates the expression of several oncogenes and tumor suppressor genes through post-transcriptional mechanisms. But the role of HuR in breast cancer remains controversial. The aim of this study was to verify the association between cytoplasmic HuR level and prognosis of breast cancer patients. Data mining from the Human Protein Atlas (HPA) and Kaplan-Meier Plotter (KMP) databases was performed. Then, 394 patients with stage I-III primary breast cancer were enrolled between January 2005 and December 2016. We investigated the association between cytoplasmic HuR level and clinicopathological characteristics or survival of these patients. Immunohistochemical analysis was performed to determine HuR expression level. SPSS 21.0 statistical software was used for analysis. In the HPA and KMP datasets, HuR protein and mRNA expression level were not significantly associated with overall survival of all breast cancer patients enrolled. Results from our 394 patients indicated that higher expression level of cytoplasmic HuR was associated with larger tumor size, lymph node positive, ER negative and triple-negative subtype. For all patients enrolled, the results indicated that compared with HuR negative patients, the DFS (disease-free survival) of HuR 1+ was longer (60.5% vs 78.8, P=0.053, HR=0.616, 95% CI: 0.378-1.005), the P value was borderline. In the triple-negative breast cancer (TNBC) subgroup, HuR positive patients had significantly longer DFS than HuR negative patients (65.5% vs 30.8%, P=0.001, HR=0.345, 95% CI: 0.180-0.658). In the HR+HER2- subgroup, HuR low (0~1+) patients had significantly longer OS than HuR high (2+~3+) patients (97.0% vs 89.5%, P=0.033, HR=2.482, 95% CI: 1.074-5.736). In conclusion, our results revealed that higher expression level of HuR was related to aggressive biological characteristics which supported the findings from previous researches. In the HR+HER2- subgroup, lower HuR expression level patients had better survival time, while in the TNBC subgroup we got the opposite results. Our work indicated that HuR might play different roles in different breast cancer subtypes.

The role of lymphoid tissue SPARC in the pathogenesis and response to treatment of multiple myeloma.

Despite the significant progress in the treatment of multiple myeloma (MM), the disease remains untreatable and its cure is still an unmet clinical need. Neoplastic transformation in MM is initiated in the germinal centers (GCs) of secondary lymphoid tissue (SLT) where B cells experience extensive somatic hypermutation induced by follicular dendritic cells (FDCs) and T-cell signals. We reason that secreted protein acidic and rich in cysteine (SPARC), a common stromal motif expressed by FDCs at the origin (SLTs) and the destination (BM) of MM, plays a role in the pathogenesis of MM, and, here, we sought to investigate this role. There were 107 BM biopsies from 57 MM patients (taken at different time points) together with 13 control specimens assessed for SPARC gene and protein expression and compared with tonsillar tissues. In addition, regulation of myeloma-promoting genes by SPARC-secreting FDCs was assessed in in vitro GC reactions (GCRs). SPARC gene expression was confirmed in both human primary (BM) and secondary (tonsils) lymphoid tissues, and the expression was significantly higher in the BM. Sparc was detectable in the BM and tonsillar lysates, co-localized with the FDC markers in both tissues, and stimulation of FDCs in vitro induced significantly higher levels of SPARC expression than unstimulated controls. In addition, SPARC inversely correlated with BM PC infiltration, ISS staging, and ECOG performance of the MM patients, and in vitro addition of FDCs to lymphocytes inhibited the expression of several oncogenes associated with malignant transformation of PCs. FDC-SPARC inhibits several myelomagenic gene expression and inversely correlates with PC infiltration and MM progression. Therapeutic induction of SPARC expression through combinations of the current MM drugs, repositioning of non-MM drugs, or novel drug discovery could pave the way to better control MM in clinically severe and drug-resistant patients.

Exosome-Mediated Response to Cancer Therapy: Modulation of Epigenetic Machinery.

Exosomes, the extracellular vesicles produced in the endosomal compartments, facilitate the transportation of proteins as well as nucleic acids. Epigenetic modifications are now considered important for fine-tuning the response of cancer cells to various therapies, and the acquired resistance against targeted therapies often involves dysregulated epigenetic modifications. Depending on the constitution of their cargo, exosomes can affect several epigenetic events, thus impacting post-transcriptional regulations. Thus, a role of exosomes as facilitators of epigenetic modifications has come under increased scrutiny in recent years. Exosomes can deliver methyltransferases to recipient cells and, more importantly, non-coding RNAs, particularly microRNAs (miRNAs), represent an important exosome cargo that can affect the expression of several oncogenes and tumor suppressors, with a resulting impact on cancer therapy resistance. Exosomes often harbor other non-coding RNAs, such as long non-coding RNAs and circular RNAs that support resistance. The exosome-mediated transfer of all this cargo between cancer cells and their surrounding cells, especially tumor-associated macrophages and cancer-associated fibroblasts, has a profound effect on the sensitivity of cancer cells to several chemotherapeutics. This review focuses on the exosome-induced modulation of epigenetic events with resulting impact on sensitivity of cancer cells to various therapies, such as, tamoxifen, cisplatin, gemcitabine and tyrosine kinase inhibitors. A better understanding of the mechanisms by which exosomes can modulate response to therapy in cancer cells is critical for the development of novel therapeutic strategies to target cancer drug resistance.

JNK Signaling Promotes Bladder Cancer Immune Escape by Regulating METTL3-Mediated m6A Modification of PD-L1 mRNA.

The identification of a novel m6A-dependent mechanism underlying immune system evasion by bladder cancer cells reveals JNK signaling as a potential target for bladder cancer immunotherapy.

Oncogenesis induced by combined Phf6 and Idh2 mutations through increased oncometabolites and impaired DNA repair.

The pathogenesis of acute leukemia involves interaction among genetic alterations. Mutations of IDH1/2 and PHF6 are common and co-exist in some patients of hematopoietic malignancies, but their cooperative effects remain unexplored. In this study, we addressed the question by characterizing the hematopoietic phenotypes of mice harboring neither, Phf6 knockout, Idh2 R172K, or combined mutations. We found that the combined Phf6KOIdh2R172K mice showed biased hematopoietic differentiation toward myeloid lineages and reduced long-term hematopoietic stem cells. They rapidly developed neoplasms of myeloid and lymphoid lineages, with much shorter survival compared with single mutated and wild-type mice. The marrow and spleen cells of the combined mutated mice produced a drastically increased amount of 2-hydroxyglutarate compared with mice harboring Idh2 R172K. Single-cell RNA sequencing revealed distinct patterns of transcriptome of the hematopoietic stem/progenitor cells from the combined mutated mice, including aberrant expression of metabolic enzymes, increased expression of several oncogenes, and impairment of DNA repairs, as confirmed by the enhanced γH2AX expression in the marrow and spleen cells. We conclude that Idh2 and Phf6 mutations are synergistic in leukemogenesis, at least through overproduction of 2-hydroxyglutarate and impairment of DNA repairs.

PHF14 knockdown causes apoptosis by inducing DNA damage and impairing the activity of the damage response complex in colorectal cancer

The abnormal expression or mutation of the plant homeodomain finger protein 14 (PHF14), a recently discovered PHD finger protein, has been reported to link to a wide range of disorders, like the aetiology and pathophysiology of multiple malignancies. Its detailed biological functions, however, still remain unclear. Herein, we discovered that PHF14 expression is strongly associated with the gastrointestinal tumor grade and gastrointestinal disorders, especially colorectal cancer (CRC), with high PHF14 expressions indicating a poor prognosis. Additionally, the mutation rate of PHF14 in CRC patients accounts for a striking proportion of 18%. PHF14 is also implicated in the expression of several oncogenes. In vitro, PHF14 was significantly expressed in patient tissues and in various CRC cell lines, and its expression was closely associated with cell proliferation and growth. Knockdown of PHF14 mediated severe DNA damage and activation of the ATR-CHK1-H2A.X pathway, leading to apoptosis. Strikingly, PHF14 interacted with KIF4A and contributes to the formation of BRCA2/Rad51 foci, indicating that PHF14 is a newly discovered factor that may participate in the formation and recruitment of DNA damage response complexes. These impairments, however, could be alleviated by restoring PHF14 expression. Importantly, inhibiting PHF14 expression in CRC cells might reduce carcinogenesis in vivo. In conclusion, PHF14 is necessary for CRC cell proliferation and growth, and therefore, it might be used as a novel biomarker and therapeutic target for the disease.

Focal adhesion kinase (FAK) inhibitor-defactinib suppresses the malignant progression of human esophageal squamous cell carcinoma (ESCC) cells via effective blockade of PI3K/AKT axis and downstream molecular network.

The clinical therapeutic efficacy toward esophageal squamous cell carcinoma (ESCC) is undesirable, due to the lack of targeted agents. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in multiple fields of tumorigenesis, recently has been indicated as a promising therapeutic target in ESCC treatment. Here, we revealed that defactinib, a specific FAK inhibitor, effectively suppressed the malignancy of ESCC cells. Mechanistically, defactinib dose and time-dependently induced the dissociation of phosphoinositide-3-kinase (PI3K) from FAK, resultantly led to blockade of protein kinase B (AKT) signaling, and the expression of several oncogenes, such as SOX2, MYC, EGFR, MET, MDM2, or TGFBR2, identified by microarray and real-time polymerase chain reaction assay. Specifically, this FAK inhibition-mediated suppression of PI3K/AKT signaling and downstream ESCC specific biomarkers was maintained to 24 h in in vitro experiments to guarantee the treatment durability and efficacy. Importantly, defactinib suppressed tumor growth, metastatic ability, and increased overall survival of xenografted animals without producing significantly systematic toxicity. Our data suggest that FAK inhibition provides an excellent targeted therapy toward ESCC by effectively inhibiting PI3K/AKT pathway and downstream molecular network.

An Updated Focus on Quadruplex Structures as Potential Therapeutic Targets in Cancer.

Non-canonical, four-stranded nucleic acids secondary structures are present within regulatory regions in the human genome and transcriptome. To date, these quadruplex structures include both DNA and RNA G-quadruplexes, formed in guanine-rich sequences, and i-Motifs, found in cytosine-rich sequences, as their counterparts. Quadruplexes have been extensively associated with cancer, playing an important role in telomere maintenance and control of genetic expression of several oncogenes and tumor suppressors. Therefore, quadruplex structures are considered attractive molecular targets for cancer therapeutics with novel mechanisms of action. In this review, we provide a general overview about recent research on the implications of quadruplex structures in cancer, firstly gathering together DNA G-quadruplexes, RNA G-quadruplexes as well as DNA i-Motifs.

Anti-Angiogenic Effects of Phytochemicals on miRNA Regulating Breast Cancer Progression.

Several phytochemicals have been identified for their role in modifying miRNA regulating tumor progression. miRNAs modulate the expression of several oncogenes and tumor suppressor genes including the genes that regulate tumor angiogenesis. Hypoxia inducible factor-1 alpha (HIF-1α) signaling is a central axis that activates oncogenic signaling and acts as a metabolic switch in endothelial cell (EC) driven tumor angiogenesis. Tumor angiogenesis driven by metabolic reprogramming of EC is crucial for tumor progression and metastasis in many different cancers, including breast cancers, and has been linked to aberrant miRNA expression profiles. In the current article, we identify different miRNAs that regulate tumor angiogenesis in the context of oncogenic signaling and metabolic reprogramming in ECs and review how selected phytochemicals could modulate miRNA levels to induce an anti-angiogenic action in breast cancer. Studies involving genistein, epigallocatechin gallate (EGCG) and resveratrol demonstrate the regulation of miRNA-21, miRNA-221/222 and miRNA-27, which are prognostic markers in triple negative breast cancers (TNBCs). Modulating the metabolic pathway is a novel strategy for controlling tumor angiogenesis and tumor growth. Cardamonin, curcumin and resveratrol exhibit their anti-angiogenic property by targeting the miRNAs that regulate EC metabolism. Here we suggest that using phytochemicals to target miRNAs, which in turn suppresses tumor angiogenesis, should have the potential to inhibit tumor growth, progression, invasion and metastasis and may be developed into an effective therapeutic strategy for the treatment of many different cancers where tumor angiogenesis plays a significant role in tumor growth and progression.

The mRNA-binding Protein TTP/ZFP36 in Hepatocarcinogenesis and Hepatocellular Carcinoma.

Hepatic lipid deposition and inflammation represent risk factors for hepatocellular carcinoma (HCC). The mRNA-binding protein tristetraprolin (TTP, gene name ZFP36) has been suggested as a tumor suppressor in several malignancies, but it increases insulin resistance. The aim of this study was to elucidate the role of TTP in hepatocarcinogenesis and HCC progression. Employing liver-specific TTP-knockout (lsTtp-KO) mice in the diethylnitrosamine (DEN) hepatocarcinogenesis model, we observed a significantly reduced tumor burden compared to wild-type animals. Upon short-term DEN treatment, modelling early inflammatory processes in hepatocarcinogenesis, lsTtp-KO mice exhibited a reduced monocyte/macrophage ratio as compared to wild-type mice. While short-term DEN strongly induced an abundance of saturated and poly-unsaturated hepatic fatty acids, lsTtp-KO mice did not show these changes. These findings suggested anti-carcinogenic actions of TTP deletion due to effects on inflammation and metabolism. Interestingly, though, investigating effects of TTP on different hallmarks of cancer suggested tumor-suppressing actions: TTP inhibited proliferation, attenuated migration, and slightly increased chemosensitivity. In line with a tumor-suppressing activity, we observed a reduced expression of several oncogenes in TTP-overexpressing cells. Accordingly, ZFP36 expression was downregulated in tumor tissues in three large human data sets. Taken together, this study suggests that hepatocytic TTP promotes hepatocarcinogenesis, while it shows tumor-suppressive actions during hepatic tumor progression.

Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells

The transcriptional regulator BRD4 has been shown to be important for the expression of several oncogenes including MYC. Inhibiting of BRD4 has broad antiproliferative activity in different cancer cell types. The small molecule JQ1 blocks the interaction of BRD4 with acetylated histones leading to transcriptional modulation. Depleting BRD4 via engineered bifunctional small molecules named PROTACs (proteolysis targeting chimeras) represents the next-generation approach to JQ1-mediated BRD4 inhibition. PROTACs trigger BRD4 for proteasomale degradation by recruiting E3 ligases. The aim of this study was therefore to validate the importance of BRD4 as a relevant target in colorectal cancer (CRC) cells and to compare the efficacy of BRD4 inhibition with BRD4 degradation on downregulating MYC expression. JQ1 induced a downregulation of both MYC mRNA and MYC protein associated with an antiproliferative phenotype in CRC cells. dBET1 and MZ1 induced degradation of BRD4 followed by a reduction in MYC expression and CRC cell proliferation. In SW480 cells, where dBET1 failed, we found significantly lower levels of the E3 ligase cereblon, which is essential for dBET1-induced BRD4 degradation. To gain mechanistic insight into the unresponsiveness to dBET1, we generated dBET1-resistant LS174t cells and found a strong downregulation of cereblon protein. These findings suggest that inhibition of BRD4 by JQ1 and degradation of BRD4 by dBET1 and MZ1 are powerful tools for reducing MYC expression and CRC cell proliferation. In addition, downregulation of cereblon may be an important mechanism for developing dBET1 resistance, which can be evaded by incubating dBET1-resistant cells with JQ1 or MZ1.

2003-P: The Role of Re1-Silencing Transcription Factor (REST) in Adipocytes

We have reported that several neuronal genes, glutamate receptor 7 and synuclein γ, are expressed in adipocytes. Since the individual roles of these neuronal genes are different, it is unlikely that these genes carry out any common functions together. Numerous neuronal genes are negatively regulated by REST. Therefore, we generated the mice lacking REST in adipocytes to investigate the role of these genes. Rest2lox/2lox mice were mated with Adipoq-Cre mice, and the resultant Adipoq-Cre/Rest2lox/2lox mice were compared with Adipoq-Cre mice. Male Adipoq-Cre/Rest2lox/2lox mice exceeding 10 weeks of age showed increased body weight and fat mass. There was no difference in food consumption, glucose tolerance evaluated by intraperitoneal GTT (ip-GTT) and adipocyte size. However, glucose tolerance was deteriorated in Adipoq-Cre/Rest2lox/2lox mice after treated with high fat diet (HFD) for 8 weeks. The characteristics of gene expression in epididymal fat isolated from Adipoq-Cre/Rest2lox/2lox mice was examined with microarray with Adipoq-Cre/Rest2lox/2lox mice as a control. Significant increased mRNA levels were detected numerous neuronal genes such as Gdap1, Nrxn1, Nrsn1 and Syn1, in Adipoq-Cre/Rest2lox/2lox mice. In addition, expression of several oncogenes, including Srrm3, Lhfpl5, Stmn3 and Chga (a marker of neuro-endocrine tumor such as pheochromocytoma) were increased in these mice. On the other hand, brown adipose tissue (BAT) in Adipoq-Cre/Rest2lox/2lox mice showed white adipose tissue (WAT)-like appearance. These results suggested that REST might regulate adipocyte differentiation and function in both WAT and BAT. Disclosure M. Fuwa: None. K. Kajita: None. K. Taguchi: None. Y. Kitada: None. T. Ikeda: None. T. Ishizuka: None. H. Morita: None.

The homeobox transcription factor MEIS2 is a regulator of cancer cell survival and IMiDs activity in Multiple Myeloma: modulation by Bromodomain and Extra-Terminal (BET) protein inhibitors

The transcription factor Myeloid Ecotropic Insertion Site 2 (MEIS2) has been identified as a cellular substrate of the E3-ubiquitin ligase complex CRL4-cereblon (CRL4CRBN) in crystal structure and by biochemical screen. Emerging evidence suggests that IMiDs can block MEIS2 from binding to CRBN facilitating the subsequent activation of a CRL4CRBNIMiD-E3-ubiquitin ligase activity and proteasome-mediated degradation of critical substrates regulators of Multiple Myeloma (MM) cell survival and proliferation. Bromodomain and Extra-Terminal (BET) family of proteins are important epigenetic regulators involved in promoting gene expression of several oncogenes, and many studies have revealed important anticancer activities mediated by BET inhibitors (BETi) in hematologic malignancies including MM. Here, we investigated MEIS2 in MM, the role of this protein as a modulator of IMiDs activity and the ability of BETi to inhibit its expression. Our observations indicate that inhibition of MEIS2 in MM cells by RNA interference correlates with reduced growth, induction of apoptosis and enhanced efficacy of different anti-MM drugs. In addition, MEIS2 regulates the expression of Cyclin E/CCNE1 in MM and induction of apoptosis after treatment with the CDK inhibitor Seliciclib/Roscovitine. Interestingly, modulation of MEIS2 can regulate the expression of NKG2D and DNAM-1 NK cell-activating ligands and, importantly, the activity of IMiDs in MM cells. Finally, BETi have the ability to inhibit the expression of MEIS2 in MM, underscoring a novel anticancer activity mediated by these drugs. Our study provides evidence on the role of MEIS2 in MM cell survival and suggests therapeutic strategies targeting of MEIS2 to enhance IMiDs anti-myeloma activity.

The anti-cancer drugs curaxins target spatial genome organization

Recently we characterized a class of anti-cancer agents (curaxins) that disturbs DNA/histone interactions within nucleosomes. Here, using a combination of genomic and in vitro approaches, we demonstrate that curaxins strongly affect spatial genome organization and compromise enhancer-promoter communication, which is necessary for the expression of several oncogenes, including MYC. We further show that curaxins selectively inhibit enhancer-regulated transcription of chromatinized templates in cell-free conditions. Genomic studies also suggest that curaxins induce partial depletion of CTCF from its binding sites, which contributes to the observed changes in genome topology. Thus, curaxins can be classified as epigenetic drugs that target the 3D genome organization.

MiR-19a Overexpression in FTC-133 Cell Line Induces a More De-Differentiated and Aggressive Phenotype.

In recent years, microRNAs (miRNAs) have received increasing attention for their important role in tumor initiation and progression. MiRNAs are a class of endogenous small non-coding RNAs that negatively regulate the expression of several oncogenes or tumor suppressor genes. MiR-19a, a component of the oncogenic miR-17-92 cluster, has been reported to be highly expressed only in anaplastic thyroid cancer, the most undifferentiated, aggressive and lethal form of thyroid neoplasia. In this work, we evaluated the putative contribution of miR-19a in de-differentiation and aggressiveness of thyroid tumors. To this aim, we induced miR-19a expression in the well-differentiated follicular thyroid cancer cell line and evaluated proliferation, apoptosis and gene expression profile of cancer cells. Our results showed that miR-19a overexpression stimulates cell proliferation and alters the expression profile of genes related to thyroid cell differentiation and aggressiveness. These findings not only suggest that miR-19a has a possible involvement in de-differentiation and malignancy, but also that it could represent an important prognostic indicator and a good therapeutic target for the most aggressive thyroid cancer.

Abstract 4138: CK1δ-BRD4 pathway as novel therapeutic target for SHH subtype of medulloblastoma

Abstract Background: Medulloblastoma is the most common malignant pediatric brain tumor with variable prognosis due to its clinical and genomic heterogeneity. Despite decades of treatment advances, nearly 40% of children experience tumour recurrence, and 30% will die from this disease. Thus, new drugs and drug combinations need to be developed that effectively treat medulloblastoma. Casein kinase 1δ (CK1δ) is a serine/threonine kinase that controls cell cycle progression, signal transduction and neurogenesis, and we previously reported high levels of CK1δ in mouse models of medulloblastoma. BRD4 is an epigenetic reader protein that controls expression of several oncogenes. We wanted to determine the mechanism through which CK1δ regulates BRD4 activity in medulloblastoma and assess whether a combination of CK1δ and BRD4 inhibitors could be more effective in decreasing medulloblastoma progression in vivo. Methods: Granule cell progenitors (GCPs), medulloblastoma Ptch-/-P53-/- mouse cells and SUFU-/- mouse embryonic fibroblasts were incubated with the CK1δ inhibitor SR-1277 and the phosphorylation levels of BRD4 were determined by WB and its binding to chromatin by ChIP. In vitro phosphorylation studies with human BRD4 purified from E. coli were performed and whether genetic disruption of CK1δ reduces BRD4 phosphorylation was tested. We combined BRD4 and CK1δ inhibitors (JQ1 and SR-1277, respectively) and analysed Gli1 mRNA expression and EdU incorporation. We deleted CK1δ in GCPs in Ptch-/-;P53-/- mouse models of medulloblastoma by breeding these mice with a Tg(Atoh1-Cre);CK1δfl/fl strain. Furthermore, we intracranially transplanted human SHH medulloblastoma PDX cells expressing luciferase in mice, to validate the combination therapy. Results: CK1δ inhibitor treatment and CK1δ knockout reduced BRD4 phosphorylation, suggesting that CK1δ phosphorylates BRD4 and is required for BRD4 recruitment to chromatin. In vitro phosphorylation studies with purified human BRD4 and CK1δ confirmed CK1δ-mediated BRD4 phosphorylation and CK1δ is required for BRD4 phosphorylation at serine 492 and 494 in vivo. Using loss of function studies in GCPs, both CK1δ and BET inhibition reduced BRD4 association with Gli1 promoter, thereby reducing Gli1 mRNA levels. In addition, SR-1277 suppressed SHH signaling downstream of SUFU of medulloblastoma cells, in vitro and in vivo. We also found synergy by combining these compounds in vitro and in reducing tumor burden in the in vivo model with intracranial allografts. Conclusion: Together, our studies validate the CK1δ-BRD4 pathway as a novel target in medulloblastoma. The significance of our work is underscored by the possibility that simultaneous CK1δ-BRD4 inhibition could overcome the resistance observed with BRD4 inhibitors and enhance therapeutic benefit to patients. We are also the first group to demonstrate that CK1δ acts downstream of SHH signalling in SHH-driven medulloblastoma. Citation Format: Sze Kiat Tan, Clara Penas, Cheng-Ming Chiang, David Robbins, Nagi Ayad. CK1δ-BRD4 pathway as novel therapeutic target for SHH subtype of medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4138.

E6 Protein Expressed by High-Risk HPV Activates Super-Enhancers of the EGFR and c-MET Oncogenes by Destabilizing the Histone Demethylase KDM5C

The high-risk (HR) human papillomaviruses (HPV) are causative agents of anogenital tract dysplasia and cancers and a fraction of head and neck cancers. The HR HPV E6 oncoprotein possesses canonical oncogenic functions, such as p53 degradation and telomerase activation. It is also capable of stimulating expression of several oncogenes, but the molecular mechanism underlying these events is poorly understood. Here, we provide evidence that HPV16 E6 physically interacts with histone H3K4 demethylase KDM5C, resulting in its degradation in an E3 ligase E6AP- and proteasome-dependent manner. Moreover, we found that HPV16-positive cancer cell lines exhibited lower KDM5C protein levels than HPV-negative cancer cell lines. Restoration of KDM5C significantly suppressed the tumorigenicity of CaSki cells, an HPV16-positive cervical cancer cell line. Whole genome ChIP-seq and RNA-seq results revealed that CaSki cells contained super-enhancers in the proto-oncogenes EGFR and c-MET Ectopic KDM5C dampened these super-enhancers and reduced the expression of proto-oncogenes. This effect was likely mediated by modulating H3K4me3/H3K4me1 dynamics and decreasing bidirectional enhancer RNA transcription. Depletion of KDM5C or HPV16 E6 expression activated these two super-enhancers. These results illuminate a pivotal relationship between the oncogenic E6 proteins expressed by HR HPV isotypes and epigenetic activation of super-enhancers in the genome that drive expression of key oncogenes like EGFR and c-METSignificance: This study suggests a novel explanation for why infections with certain HPV isotypes are associated with elevated cancer risk by identifying an epigenetic mechanism through which E6 proteins expressed by those isotypes can drive expression of key oncogenes. Cancer Res; 78(6); 1418-30. ©2018 AACR.