Oncogenic Molecules Research Articles

Risk SNP-Mediated Enhancer-Promoter Interaction Drives Colorectal Cancer through Both FADS2 and AP002754.2.

Although genome-wide association studies (GWAS) have identified more than 100 colorectal cancer risk loci, most of the biological mechanisms associated with these loci remain unclear. Here we first performed a comprehensive expression quantitative trait loci analysis in colorectal cancer tissues adjusted for multiple confounders to test the determinants of germline variants in established GWAS susceptibility loci on mRNA and long noncoding RNA (lncRNA) expression. Combining integrative functional genomic/epigenomic analyses and a large-scale population study consisting of 6,024 cases and 10,022 controls, we then prioritized rs174575 with a C>G change as a potential causal candidate for colorectal cancer at 11q12.2, as its G allele was associated with an increased risk of colorectal cancer (OR = 1.26; 95% confidence interval = 1.17-1.36; P = 2.57 × 10-9). rs174575 acted as an allele-specific enhancer to distally facilitate expression of both FADS2 and lncRNA AP002754.2 via long-range enhancer-promoter interaction loops, which were mediated by E2F1. AP002754.2 further activated a transcriptional activator that upregulated FADS2 expression. FADS2, in turn, was overexpressed in colorectal cancer tumor tissues and functioned as a potential oncogene that facilitated colorectal cancer cell proliferation and xenograft growth in vitro and in vivo by increasing the metabolism of PGE2, an oncogenic molecule involved in colorectal cancer tumorigenesis. Our findings represent a novel mechanism by which a noncoding variant can facilitate long-range genome interactions to modulate the expression of multiple genes including not only mRNA, but also lncRNA, which provides new insights into the understanding of colorectal cancer etiology. SIGNIFICANCE: This study provides an oncogenic regulatory circuit among several oncogenes including E2F1, FADS2, and AP002754.2 underlying the association of rs174575 with colorectal cancer risk, which is driven by long-range enhancer-promoter interaction loops. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/9/1804/F1.large.jpg.

Colocalization of MID1IP1 and c-Myc is Critically Involved in Liver Cancer Growth via Regulation of Ribosomal Protein L5 and L11 and CNOT2.

Though midline1 interacting protein 1 (MID1IP1) was known as one of the glucose-responsive genes regulated by carbohydrate response element binding protein (ChREBP), the underlying mechanisms for its oncogenic role were never explored. Thus, in the present study, the underlying molecular mechanism of MID1P1 was elucidated mainly in HepG2 and Huh7 hepatocellular carcinoma cells (HCCs). MID1IP1 was highly expressed in HepG2, Huh7, SK-Hep1, PLC/PRF5, and immortalized hepatocyte LX-2 cells more than in normal hepatocyte AML-12 cells. MID1IP1 depletion reduced the viability and the number of colonies and also increased sub G1 population and the number of TUNEL-positive cells in HepG2 and Huh7 cells. Consistently, MID1IP1 depletion attenuated pro-poly (ADP-ribose) polymerase (pro-PARP), c-Myc and activated p21, while MID1IP1 overexpression activated c-Myc and reduced p21. Furthermore, MID1IP1 depletion synergistically attenuated c-Myc stability in HepG2 and Huh7 cells. Of note, MID1IP1 depletion upregulated the expression of ribosomal protein L5 or L11, while loss of L5 or L11 rescued c-Myc in MID1IP1 depleted HepG2 and Huh7 cells. Interestingly, tissue array showed that the overexpression of MID1IP1 was colocalized with c-Myc in human HCC tissues, which was verified in HepG2 and Huh7 cells by Immunofluorescence. Notably, depletion of CCR4-NOT2 (CNOT2) with adipogenic activity enhanced the antitumor effect of MID1IP1 depletion to reduce c-Myc, procaspase 3 and pro-PARP in HepG2, Huh7 and HCT116 cells. Overall, these findings provide novel insight that MID1IP1 promotes the growth of liver cancer via colocalization with c-Myc mediated by ribosomal proteins L5 and L11 and CNOT2 as a potent oncogenic molecule.

Targeted Protein Degradation: An Emerging Therapeutic Strategy in Cancer.

Drug discovery in the scope of cancer therapy has been focused on conventional agents that nonselectively induce DNA damage or selectively inhibit the activity of key oncogenic molecules without affecting their protein levels. An emerging therapeutic strategy that garnered attention in recent years is the induction of Targeted Protein Degradation (TPD) of cellular targets by hijacking the intracellular proteolysis machinery. This novel approach offers several advantages over conventional inhibitors and introduces a paradigm shift in several pharmacological aspects of drug therapy. While TPD has been found to be the major mode of action of clinically approved anticancer agents such as fulvestrant and thalidomide, recent years have witnessed systematic endeavors to expand the repertoire of proteins amenable to therapeutic ablation by TPD. Such endeavors have led to three major classes of agents that induce protein degradation, including molecular glues, Proteolysis Targeting Chimeras (PROTACs) and Hydrophobic Tag (HyT)-based degraders. Here, we briefly highlight agents in these classes and key advances made in the field with a focus on clinical translation in cancer therapy.

A Novel Model of Cancer Drug Resistance: Oncosomal Release of Cytotoxic and Antibody-Based Drugs.

Extracellular vesicles (EVs), such as exosomes or oncosomes, often carry oncogenic molecules derived from tumor cells. In addition, accumulating evidence indicates that tumor cells can eject anti-cancer drugs such as chemotherapeutics and targeted drugs within EVs, a novel mechanism of drug resistance. The EV-releasing drug resistance phenotype is often coupled with cellular dedifferentiation and transformation in cells undergoing epithelial-mesenchymal transition (EMT), and the adoption of a cancer stem cell phenotype. The release of EVs is also involved in immunosuppression. Herein, we address different aspects by which EVs modulate the tumor microenvironment to become resistant to anticancer and antibody-based drugs, as well as the concept of the resistance-associated secretory phenotype (RASP).

Abstract B98: IGFBP2 reprograms pancreatic cancer immune surveillance at epitranscriptome levels

Abstract In recent years, checkpoint blockade has been used successfully to treat many cancer types including melanoma, non-small cell lung, and colorectal cancers. In stark contrast, the response to these new immunotherapeutics has been poor in patients with pancreatic ductal adenocarcinoma (PDAC), the most common and lethal type of pancreatic cancer. Thus, PDAC is considered a cold tumor for immunotherapy and the mechanism underlying this resistance is poorly understood. Insulin-like growth factor binding protein-2 (IGFBP2), a secreted cytokine, is elevated in the serum, pancreatic juice, and PDAC tissues in many pancreatic cancer patients, and we demonstrated that high levels associate to reduced overall patient survival and promote the immunosuppressive M2 tumor-associated macrophage phenotype in the tumor microenvironment (TME). We recently identified IGFBP2 as a key oncogenic signaling molecule for PDAC metastasis and epithelial-to-mesenchymal transition. Our preliminary microarray analysis showed that major histocompatibility complex class II antigen (MHC class II) genes were significantly downregulated in AsPC-1 pancreatic cancer cells overexpressing IGFBP2. MHC class II molecules are constitutively expressed in antigen-presenting cells (APCs) such as dendritic and Langerhans cells, macrophages, and B lymphocytes. In PDAC, increased MHC class II expression correlates to a higher histologic grade of differentiation. Also, CD4+ T lymphocyte numbers in PDAC tissues positively correlate to MHC class II expression levels. We envision that high IGFBP2 represses MHC class II gene expression in the tumor-associated APCs, resulting in failure of recruitment and activation of CD4+ lymphocytes, augmenting the immunosuppressive TME. The mechanism whereby IGFBP2 regulates MHC class II gene expression, however, is unknown. IGFBP2 was reported to physically interact with Pim-1-associated protein-1 (PAP-1)-associated protin-1 (PAPA-1) in the nucleus. PAPA-1 (or INO80B) is a key component in the ATP-dependent INO80 chromatin-remodeling complex. A recent study shows that INO80 is required for super-enhancer-mediated oncogenic transcription and tumor growth in melanoma. Interestingly, the MHC class II genes HLA-DR and HLA-DQ were found to be regulated by a super-enhancer mediated mechanism in autoimmune disease. We hypothesized that tumor-secreted IGFBP2 directly regulates INO80 function and inhibits the MHC class II expression in APCs in the TME. Our preliminary data show that exogenous IGFBP2 translocates into the cell nucleus within hours, and co-immunoprecipitation confirmed that IGFBP2 interacts with INO80B in vitro. Furthermore, MHC Class II expression was downregulated by IGFBP2 in the murine DC 2.4 dendritic cell line and in mouse bone marrow-derived dendritic cells by RT-PCR and flow cytometry. Our data suggest that PDAC IGFBP2 levels might predict checkpoint blockade therapy readiness, and importantly, serve as a target mechanism for enabling immunotherapy effectiveness. Citation Format: Ping-Chieh Chou, Liang Liu, Elizabeth Forbes, Ashley Ballard, Tao Li, Wei Zhang. IGFBP2 reprograms pancreatic cancer immune surveillance at epitranscriptome levels [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B98.

Vascular mimicry: Triggers, molecular interactions and in vivo models.

Vascular mimicry is induced by a wide array of genes with functions related to cancer stemness, hypoxia, angiogenesis and autophagy. Vascular mimicry competent (VM-competent) cells that form de novo blood vessels are common in solid tumors facilitating tumor cell survival and metastasis. VM-competent cells display increased levels of vascular mimicry selecting for stem-like cells in an O2-gradient-dependent manner in deeply hypoxic tumor regions, while also aiding in maintaining tumor cell metabolism and stemness. Three of the principal drivers of vascular mimicry are EphA2, Nodal and HIF-1α, however, directly or indirectly many of these molecules affect VE-Cadherin (VE-Cad), which forms gap-junctions to bind angiogenic blood vessels together. During vascular mimicry, the endothelial-like functions of VM-competent cancer stem cells co-opt VE-Cad to bind cancer cells together to create cancer cell-derived blood conducting vessels. This process potentially compensates for the lack of access to blood and nutrient in avascular tumors, simultaneously providing nutrients and enhancing cancer invasion and metastasis. Current evidence also supports that vascular mimicry promotes cancer malignancy and metastasis due to the cooperation of oncogenic signaling molecules driving cancer stemness and autophagy. While a number of currently used cancer therapeutics are effective inhibitors of vascular mimicry, developing a new class of vascular mimicry specific inhibitors could allow for the treatment of angiogenesis-resistant tumors, inhibit cancer metastasis and improve patient survival. In this review, we describe the principal vascular mimicry pathways in addition to emphasizing the roles of hypoxia, autophagy and select proangiogenic oncogenes in this process.

The Role of Lysophosphatidic Acid Receptors in Ovarian Cancer: A Minireview

Lysophosphatidic acid (LPA) is a bioactive lipid component of ovarian cancer activating factor, which is present at a high concentration in the ascitic fluid and plasma of patients with ovarian cancer. A group of six lysophosphatidic acid receptors (LPARs), LPAR1 through LPAR6, which belong to the G protein-coupled receptor superfamily (GPCR), mediate cellular activities of LPA and activates a series of downstream molecules and cellular responses, including biological and pathological effects. LPARs are widely expressed in normal ovary, benign tumor, and ovarian cancer tissues and cancer cell lines with a broad range of levels. The LPA/LPAR axis is involved in tumorigenesis and development of ovarian cancer through mediating the cellular responses to LPA and influencing the expression and function of oncogenic molecules. In the present review, the roles of LPARs in ovarian cancer, including the expression, function, and downstream molecules, are summarized, and we discuss the implications for ovarian cancer treatment that targets LPARs.

MicroRNA-147b promotes lung adenocarcinoma cell aggressiveness through negatively regulating microfibril-associated glycoprotein 4 (MFAP4) and affects prognosis of lung adenocarcinoma patients

BackgroundLung adenocarcinoma (LUAD) is widely known as the leading cause of death in patients with lung cancer. Extensive evidence has determined that microRNAs (miRNAs) exert critical effects on various biological processes in tumorigenesis. microRNA-147b (miR-147b) has been reported to serve as an oncogenic molecule in colorectal cancer and hepatocellular carcinoma, however, its prognostic value and biological effect in LUAD remain rare. Materials and methodsmiR-147b and microfibril-associated glycoprotein 4 (MFAP4) data were collected from The Cancer Genome Atlas (TCGA) database to determine their expression levels in LUAD tissues. Kaplan-Meier method was used to plot the overall survival curves for the prognostic power of miR-147b and MFAP4 identification. Chi-square test was utilized to demonstrate the association between clinical characteristics and miR-147b or MFAP4 in LUAD. Luciferse reporter assay was implemented to identify the correlation between miR-147b and MFAP4. The mRNA and protein levels were detected by qRT-PCR and western blotting, respectively. To explore the effects of miR-147b and its potential mechanism in LUAD, cell counting kit 8 (CCK-8), colony formation and transwell assays were performed in LUAD cells with abnormal expression of miR-147b or/and MFAP4. ResultsOur results showed that miR-147b was up-regulated in LUAD tissues and cell lines, which induced poor outcome. Conversely, MFAP4, the putative target gene of miR-147b, was down-regulated in LUAD. The expression of MFAP4 in LUAD cells was negatively regulated by miR-147b. Results of experiments in vitro revealed that miR-147b could promote cell proliferation, colony formation, invasion and migration, while up-regulation of MFAP4 suppressed the impacts of miR-147b on cell malignant aggressiveness in A549 and Calu-3 cells. ConclusionIn conclusion, these findings determined that miR-147b contributed to the progression of LUAD via targeting MFAP4. Thus, understanding the potential mechanism of miR-147b/MFAP4 may improve the treatment of cancers, especially LUAD.

The Clinical Significance of a Novel microRNA Signature in Multiple Myeloma

Multiple myeloma (MM) is a hematologic malignancy characterized by infiltration of the bone marrow (BM) by malignant plasma cells. Multiple myeloma diagnosis is made by the presence of one or more of the CRAB criteria or one of the recently added biomarkers of malignancy. Smoldering MM (SMM) is a plasma cell dyscrasia preceding multiple myeloma, characterized by bone marrow infiltration of 10-60% and/or serum monoclonal protein ≥3g/dL or urinary monoclonal protein ≥500 mg per 24h, along with the absence of myeloma-defining events. MicroRNAs (miRNA) are single-stranded, small non-coding RNA molecules (~21 nt) that regulate protein-coding gene expression at the post-transcriptional level, mainly through interactions with the 3′-untranslated region of target mRNAs. The results of such interactions can be mRNA degradation and/or translational repression, depending on the complementarity of the miRNA seed sequence with the mRNAs 3′-untranslated region. They can function as oncogenes or tumor suppressors, possessing a vital role in several aspects of all stages of tumorigenesis and cancer progression. In the present study, we have investigated the clinical value of a molecular signature consisting of 10 cancer-related miRNAs in MM: miR-15a, miR-16, miR-21, miR-221, miR-222, miR-25, miR-125, miR-155, miR-223, and miR-181a. These molecules were selected due to their well-documented role and clinical significance in numerous human malignancies. More specifically, miR-15a and miR-16 expression levels have been associated with chronic lymphocytic leukemia. The deletion 13q14, the most prevalent alteration in CLL, leads to the deletion of these miRNAs, which act on cell proliferation and in the process of apoptosis. miR-221 and miR-222 form a cluster that has been correlated with tumorigenesis and unfavorable prognosis in human malignancies, while miR-155 is a pro-inflammatory, oncogenic molecule, with a potential role in chronic lymphocytic leukemia. Bone marrow aspiration samples were collected from 94 patients with MM and SMM, and CD138+ plasma cells were positively selected using magnetic beads coated with an anti-CD138 antibody. Total RNA was isolated using TRIZol. Thereafter, 200ng RNA of each sample were polyadenylated at the 3´ end and reversely transcribed. An in-house developed real-time quantitative PCR assay was conducted and the results were biostatistically analyzed. For the normalization of the expression levels of each miRNA, the mean expression of two small nucleolar RNAs (RNU43 and RNU48) was used as reference. Seventy six out of the 94 BM aspiration samples were derived from MM patients and 18 of them from SMM patients, at the time of diagnosis. The MM patients were classified, according to the R-ISS staging system, as follows: 15 patients had stage I disease, 42 patients had stage II, and 19 patients stage III MM. Forty nine myeloma patients presented with osteolytic lesions at diagnosis. The statistical analysis revealed significantly lower expression levels of miR-16 (p=0.036) and miR-155 (p=0.045) in CD138+ cells of MM patients, compared to those from SMM patients, highlighting their potential value to discriminate MM from SMM. Furthermore, miR-221 and miR-222 expression levels were negatively correlated with R-ISS; thus, miR-221 and miR-222 expression was significantly downregulated in MM patients with R-ISS stage III (p=0.004 and 0.034, respectively). This tendency reveals a potential favorable prognostic value of miR-221/222 cluster in MM. Next, miR-15a and miR-16 expression was shown to be associated with the presence of osteolytic lesions. In this regard, the expression levels of miR-15a (p=0.048) and miR-16 (p=0.047) were decreased in MM patients with bone disease, compared to those without bone disease. The observed decreased expression of these two miRNAs in symptomatic MM patients could constitute a predictive biomarker for the occurrence of bone disease and, hence, a putative predictive biomarker of SMM patients at high risk of evolution to symptomatic disease with bone lesions. We conclude that miR-221/222 correlate with more favorable R-ISS stage, while miR-15a and miR-16 correlate with the presence of osteolytic disease in MM. This ongoing study will further reveal the possible prognostic significance of this 10 miRNAs signature studied, when response to therapy, progression-free and overall survival is available. Disclosures Kastritis: Genesis: Honoraria; Prothena: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria. Gavriatopoulou:Takeda: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Amgen: Honoraria; Genesis: Honoraria, Other: Travel expenses. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Takeda: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding.

PDCT-16. A PHASE I STUDY OF SUBEROYLANILIDE HYDROXAMIC ACID (SAHA) WITH TEMSIROLIMUS IN CHILDREN WITH NEWLY DIAGNOSED OR PROGRESSIVE DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract Diffuse intrinsic pontine glioma (DIPG) in children remains essentially incurable with a median survival of 12 months. Radiation therapy is currently the standard treatment modality. To date chemotherapy or biologic agents have had no meaningful increase in survival. Data from biopsied DIPG showed the PI3K/AKT/mTOR pathway as a major oncogenic player. Thus targeting these pathways with mTOR inhibitors could hold promise. Genomic and epigenetic insights have shown the role of histone deacetylase (HDAC) in these tumors. Both temsirolimus (mTOR inhibitor) and SAHA (HDAC inhibitor) are well described in pediatric clinical trials with an established maximum tolerated dose (MTD) for phase II studies. This is the first ongoing phase I multi-targeted therapeutic study, in pediatric DIPG, using SAHA and temsirolimus in a combinatorial approach, targeting the key oncogenic pathway and molecules (NCT02420613 currently recruiting). Patients with a performance score of 50 or greater, with newly diagnosed (Stratum I), or progressive (stratum II) DIPG will be eligible. Biopsy is not mandatory. A standard 3 + 3 design is being used for this study. 1 dose escalation and 2 dose de-escalations are permitted beyond the starting dose level. In stratum I patients receive radiation therapy concurrently with vorinostat on the days of radiation, followed by adjuvant therapy with vorinostat and temsirolimus for 10 cycles if they do not have progressive disease. In stratum II patients receive vorinostat and temsirolimus for 12 cycles (28 day cycle), if they do not have progressive disease. Correlative studies evaluating histone acetylation, phosphorylated 70S6K and Akt are being performed. Currently 5 patients have been enrolled, and the study is at the dose escalation level with no dose limiting toxicity (DLT) seen so far. Safety, adverse effects, biological correlatives, and clinical outcomes will be reported once study is completed.

DJ-1 Contributes to Self-renewal of Stem Cells in the U87-MG Glioblastoma Cell Line.

DJ-1, an oncogenic molecule, helps to maintain somatic stem cells by reducing the intracellular level of reactive oxygen species (ROS). This study investigated the role of DJ-1 in glioma stem cells (GSCs). U87-MG (U87) and U251-MG (U251) glioblastoma cell lines that express wild-type and mutant p53, respectively, were used. These were cultured with DJ-1-targeting siRNA and subjected to a variety of in vitro experiments or intracranial transplantation into nude mice. Knockdown of DJ-1 reduced clonogenicity only in U87 cells, which was rescued by p53 depletion. ROS accumulated in DJ-1-depleted cells, although treatment with N-acetyl cysteine, which quenches ROS, did not affect exhaustion of CSCs among U87 cells by DJ-1 knockdown. In a serial transplantation study, DJ-1 knockdown prolonged the survival of mice in secondary transplantation. DJ-1 plays a pivotal role in maintenance of stem cell self-renewal in the U87 cell line.

Mushroom polysaccharides and jiaogulan saponins exert cancer preventive effects by shaping the gut microbiota and microenvironment in ApcMin/+ mice

The incidence of colorectal cancer (CRC) is alarming among younger peoples. While no effective chemopreventive drug available in the market, researchers have been searching for alternative strategies against CRC that are in demand. Therefore, we tested the cancer-preventive properties of Ganoderma lucidum (Lingzhi) polysaccharides (GLP), along with the saponins extracted from Gynostemma pentaphyllum (GpS), an herbal tea with prebiotic-like effects. Here, we report that saponins from Gynostemma pentaphyllum (GpS) and polysaccharides from Ganoderma lucidum (GLP together with GpS) profoundly improved the inflamed gut barrier of ApcMin/+ mice by reducing polyps, shifting colonic M1 to M2 macrophages, positively reverting E-cadherin/N-cadherin ratio, and downregulating oncogenic signaling molecules. The treatments also markedly promoted short-chain fatty acids (SCFAs)˗producing bacteria and abridged sulfate-reducing bacteria in a time-dependent manner. G-protein coupled-receptors were significantly stimulated in the treated mice, accompanied by the modulated expressions of histone deacetylases, anti-cancer gut hormone PYY, and PPAPγ. These findings suggest that some of the herbal medicinal foods could modulate the relationship between the host and the gut microbiota (GM) to exert their beneficial properties to the host. Our study also implicates that these dietary mushroom polysaccharides and the Gp saponins have the potential to be developed as new preventive medicines against CRC.

Abstract 2048: Stat3 downstream transthyretin stimulates tumor growth through regulation of tumor, immune and endothelial cells

Abstract Lung cancer is a very aggressive malignant form of cancer, and is one of the biggest public health challenges facing the United States and many other countries. Although incidence rates have been stabilized, an estimated 154,050 Americans are expected to die from lung & bronchus cancer in 2018, accounting for approximately 25.3 percent of all cancer deaths. Lung cancer is a difficult disease to detect in its early stages, with greater than 50% of patients diagnosed with lung cancer presenting with metastatic disease. Early detection of lung cancer is an important opportunity for decreasing mortality while it is still treatable and curable. The overall 5-year survival rate is ~15 percent. Thus, it is essential to better understand the mechanisms that initiate lung carcinogenesis and find easy-use biomarkers for more accurate lung cancer detection. Due to heterogeneity of lung cancers, a panel of biomarkers should be used for more accurate lung cancer detection and classification. Signal transducer and activator of transcription 3 (Stat3) is well known for its lung cancer-promoting activity. To assess the consequences of STAT3 persistent activation in the lung, a doxycycline-controlled CCSP-rtTA/(tetO)7-Stat3C bitransgenic mouse model was generated that over-expresses STAT3C (a constitutively active form of STAT3) in alveolar type II (AT II) epithelial cells. In sequential steps, Stat3C over-expression up-regulated pro-inflammatory molecules, increased inflammatory cell infiltration and caused adenocarcinomas in the lung. Using this lung tumor model, thirteen secretory proteins that are Stat3 downstream gene products were identified as a panel of biomarkers for lung cancer detection in human sera. This panel of biomarkers potentially differentiates different types of lung cancer for classification. Among them, the transthyretin (TTR) concentration was highly increased in human serum of lung cancer patients. TTR concentration was also induced in the serum, bronchoalveolar lavage fluid, alveolar type II epithelial cells and alveolar myeloid cells of the CCSP-rtTA/(tetO)7-Stat3C lung tumor mouse model. Recombinant TTR stimulated lung tumor cell proliferation and growth, which were mediated by activation of mitogenic and oncogenic molecules. TTR possesses cytokine functions to stimulate myeloid cell differentiation, which are known to play roles in tumor environment. TTR demonstrated a great influence on a wide spectrum of endothelial cell functions to control tumor and immune cell migration and infiltration. TTR-treated endothelial cells suppressed T cell proliferation. Taken together, Stat3 downstream inducible secretory protein biomarkers potentially can be used as clinical targets for lung cancer personalized treatment if their expression levels are increased in a given lung cancer patient in the blood. Citation Format: Xinchun Ding. Stat3 downstream transthyretin stimulates tumor growth through regulation of tumor, immune and endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2048.

Long noncoding RNA EMS connects c-Myc to cell cycle control and tumorigenesis

Deregulated expression of c-Myc is an important molecular hallmark of cancer. The oncogenic function of c-Myc has been largely attributed to its intrinsic nature as a master transcription factor. Here, we report the long noncoding RNA (lncRNA) E2F1 messenger RNA (mRNA) stabilizing factor (EMS) as a direct c-Myc transcriptional target. EMS functions as an oncogenic molecule by promoting G1/S cell cycle progression. Mechanistically, EMS cooperates with the RNA binding protein RALY to stabilize E2F1 mRNA, and thereby increases E2F1 expression. Furthermore, EMS is able to connect c-Myc to cell cycle control and tumorigenesis via modulating E2F1 mRNA stability. Together, these findings reveal a previously unappreciated mechanism through which c-Myc induces E2F1 expression and also implicate EMS as an important player in the regulation of c-Myc function.

Abstract 4394: Molecular interplay between Tank-binding kinase (TBK1) and Yes-associated protein (YAP1) in KRAS mutant NSCLC

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in the United States. Lung adenocarcinomas are highly correlated with smoking and are characterized by mutations in KRAS, EGFR, BRAF and other oncogenes. KRAS mutations are widespread in adenocarcinomas among smokers and known to be a key player in various downstream signaling pathways contributing to the tumorigenesis. Recently, a non-canonical IκB kinase, Tank Binding Kinase 1 (TBK1), has been found to contribute in KRAS mutant cancers. TBK1 has well documented functions in immune response, cell survival and in mitosis. While it has been suggested that TBK1-mediated regulation of Akt signaling might facilitate oncogenesis, the molecular mechanisms underlying TBK1 function downstream of KRAS is not fully elucidated. Yes associated protein 1 (YAP1) is an oncogenic component of the Hippo signaling cascade which could promote KRAS mediated oncogenesis and could substitute for the loss of Kras in mouse models of pancreatic cancer. In our present study, we demonstrate a unique and novel interplay between TBK1 and the oncogenic Hippo effector molecule, YAP1. YAP1 and its paralog, TAZ are known transcriptional co-activators that function to maintain organ size during development and is often activated in cancers. We find that TBK1 could physically interact with YAP1 and phosphorylate it at T110, T114, S128 and S131 residues in vitro. Knocking down (KD) or knock out (KO) of TBK1 resulted in a significant elevation of YAP1 expression at the protein level; surprisingly, without any effect at the mRNA level. Interestingly, the upregulation of YAP1 upon depletion of TBK1 was restricted to KRAS mutant NSCLC cell-lines and not in EGFR mutant cell lines. This elevation of YAP1 upon TBK1 KD was mainly observed in the nucleus; notably, there were only minimal changes in the levels of MST and LATS, raising the possibility that these changes occur independent of the classic hippo signaling pathway. Treatment with cycloheximide, an inhibitor of protein-translation, could not diminish the elevated level of YAP1 protein in the TBK1 depleted cells, indicating that the increased YAP1 level is due to enhanced protein stability, probably as a result of post-translational modification(s). Depletion of TBK1 also resulted in the induction of EMT-like features, promoting cell-migration in scratch assays and elevated the proportion of stem-like side-population cells, probably in a YAP1-dependent manner. An unbiased RNA-Seq analysis in A549 and H460 cells indicated that MAP kinase (MAPK) pathway is activated upon TBK1 KD, which might also be involved in the YAP1 protein regulation. Our recent experiments further support this argument. The in-depth molecular mechanism(s) by which TBK1 regulates YAP1 in KRAS mutant cells are under investigation, and we hypothesize that this regulatory event contributes to KRAS mediated oncogenesis in NSCLC. Citation Format: Biswarup Saha, Neha Jaiswal, Namrata Bora-Singhal, Srikumar Chellappan. Molecular interplay between Tank-binding kinase (TBK1) and Yes-associated protein (YAP1) in KRAS mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4394.

Abstract CT113: A Phase I study of Suberoylanilide Hydroxamic Acid (SAHA) with Temsirolimus in children with newly diagnosed or progressive diffuse intrinsic pontine glioma (DIPG)

Abstract Background/rationale: Diffuse intrinsic pontine glioma (DIPG) in children remains essentially incurable with a median survival of 12 months. Radiation therapy is currently the standard treatment modality. To date chemotherapy or biologic agents have had no meaningful increase in survival, making it imperative to profile novel therapeutic combinations. Data from biopsied DIPG showed the PI3K/AKT/mTOR pathway as a major oncogenic player. At the gene level high PDGFR amplification (most commonly seen) and overexpression of the PI3K-Akt-mTOR pathway were noted in these tumors. Focal amplifications of genes within the receptor tyrosine kinase-Ras-phosphoinositol 3-kinase signaling pathway were found in 47% of DIPG. Thus targeting this pathways with mTOR inhibitors could hold therapeutic promise. Elevated expression of DNA repair proteins in the NHEJ and HRR pathway are known to induce resistance to radiation. Preclinical data shows inhibition of these pathways potentiates the sensitivity of these tumors to radiation. SAHA a pan HDAC inhibitor, has been associated with decreased expression of key DNA repair proteins in both the NHEJ and HRR pathways. Both temsirolimus (an mTOR inhibitor) and SAHA are well described in pediatric clinical trials with an established maximum tolerated dose (MTD). The efficacy of the combination of temsirolimus and SAHA has been well-established in other malignancies including prostate cancer, mantle lymphoma, and renal carcinoma. This is the first multi-targeted therapeutic trial in pediatric DIPG, using SAHA and temsirolimus in a combinatorial approach, targeting the key oncogenic pathway and molecules along with enhancing the radiation sensitivity. Correlative study: Stratum I (chemoradiotherapy phase): Histone acetylation will be quantified from peripheral blood mononuclear cell (PBMC) collected prior to the first dose of vorinostat and radiation and at the end of radiation. Stratum I and II (maintenance phase): Evaluation of histone acetylation, phosphorylated 70S6K and Akt on day 1 and 8 (prior to chemotherapy with temsirolimus) during cycle 1 and cycle 2. Methods Patients with a performance score of 50 or greater with newly diagnosed (Stratum 1) or progressive DIPG (stratum II) as confirmed by gadolinium enhanced MRI are eligible. Biopsy is not mandatory. Stratum I: Patients will receive radiation therapy concurrently with vorinostat on the days of radiation at a dose of 230mg/m2/dose, followed by adjuvant therapy with vorinostat and temsirolimus for 10 cycles if patients do not have progressive disease. Stratum II: Patients will receive vorinostat and temsirolimus for 12 cycles (28 day cycle), if patient does not have progressive disease. SAHA will be given once daily at 230 mg/m2 orally from day 1 to day 8, along with temsirolimus at 25mg/m2 intravenously on day 1 and day 8. This will be the dose level 1 and 2 dose escalations and 1 dose de-escalation will be allowed. A standard 3+3 design will be used and a minimum of 9 patients and a maximum of 18 patients will be enrolled. Trial is ongoing (30% of planned patients enrolled), with patients now in the second dose level (enrollment of this cohort started on September 2018). No dose limiting toxicity (DLT) were noted at the first dose level. Trial: NCT02420613 (currently recruiting). Note: This abstract was not presented at the meeting. Citation Format: Soumen Khatua, Joya Chandra, Miriam M. Morrell, Heather B. Meador, David I. Sandberg, Jeffrey Weinberg, Greg Fuller, Leena Ketonen, Jason M. Johnson, Kenneth R. Hess, Wafik Zaky. A Phase I study of Suberoylanilide Hydroxamic Acid (SAHA) with Temsirolimus in children with newly diagnosed or progressive diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT113.

MicroRNA-191 targets CCAAT/enhanced binding protein β and functions as an oncogenic molecule in human non-small cell lung carcinoma cells.

The aberrant expression of microRNAs (miRs) may be involved in tumor growth and progression in human non-small cell lung carcinoma (NSCLC). The present study aimed to investigate the potential roles of miR-191 in NSCLC. Western blotting and reverse transcription-quantitative polymerase chain reaction were performed to assess protein and/or mRNA levels. Scratch wound healing and transwell assays were performed to determine the NSCLC cell migration and invasion. A luciferase demonstrated that CCAAT/enhanced binding protein β (C/EBPβ) was a target of miR-191. Previously, miR-191 has been reported to act as an oncogenic player in multiple human cancers. C/EBPβ has been identified as a target gene of miR-191; however, the roles and underlying mechanisms of miR-191 associated with the regulation of tumor invasion in NSCLC remain unknown. In the present study, it was demonstrated that miR-191 expression levels were higher in human NSCLC tumors compared with in normal adjacent tissue and elevated miR-191 expression levels were closely associated with tumor node metastasis stage in patients with NSCLC. Furthermore, transfection with miR-191 mimic inhibited C/EBPβ expression at the mRNA and protein levels and promoted A549 cell migration and invasion. C/EBPβ was reported to be the direct target gene of miR-191 using a dual luciferase reporter assay. Finally, C/EBPβ siRNA can mimic the effects of miR-191. These findings indicated that miR-191 may function as an oncogene in NSCLC, at least partially due to its negative regulatory on C/EBPβ.

Knockdown of lncRNA-UCA1 inhibits cell viability and migration of human glioma cells by miR-193a-mediated downregulation of CDK6.

Long noncoding RNA urothelial carcinoma-associated 1 (lncRNA-UCA1) is generally recognized as an oncogenic molecule in several human malignant tumors. However, the available evidence does not necessarily imply an unequivocal causal function of UCA1 in glioblastoma. The current study was aimed to probe the biological function of lncRNA-UCA1 in human glioblastoma cell lines. Besides, we further investigated the potential mechanisms. Cell viability, apoptosis, as well as migration and invasion were measured using a commercial cell counting kit-8, flow cytometry, and 24-Transwell assay, respectively. LncRNA-UCA1, microRNA-193a (miR-193a), and CDK6 at messenger RNA levels were evaluated by quantitative real-time polymerase chain reaction method. Protein level was examined by Western blot analysis. RNA immunoprecipitation was utilized to validate lncRNA-UCA1 associated with miR-193a. Luciferase activity assay was used to identify the miR-193a-targeted CDK6 3'-untranslated region. lncRNA-UCA1 knockdown weakened cell viability, augmented apoptosis progression, as well as suppressed migration and invasion behaviors in glioblastoma cells, whereas lncRNA-UCA1 silence exhibited the opposite functions. lncRNA-UCA1 functioned as an endogenous sponge of miR-193a. miR-193a silence reversed the biological function of lncRNA-UCA1 knockdown on U-118 MG cells. miR-193a negatively regulated the expression of CDK6, and it affected the U-118 MG cells through regulating CDK6 expression. CDK6 overexpression abrogated the blockage of PI3K/AKT, mitogen-activated protein kinase (MAPK), and Notch signaling pathways. Furthermore, lncRNA-UCA1 and miR-193a could affect these signaling cascades through regulating CDK6 expression. The regulatory mechanisms of lncRNA-UCA1 were further consolidated in clinical specimens. lncRNA-UCA1 silence reduced cell viability, promoted apoptosis progression, while impeding the migration and invasion of glioblastoma cells by miR-193a-mediated silence of CDK6, with blockage of PI3K/AKT, MAPK, and Notch pathways.

RON and RONΔ160 promote gastric cancer cell proliferation, migration, and adaption to hypoxia via interaction with β-catenin.

Aberrant accumulation of the receptor tyrosine kinase recepteur d’origine nantais (RON) has been verified in gastric adenocarcinoma. Upregulation of RON and its splice variant RONΔ160 contribute to the growth and migration in gastric cancer cells in vitro. However, the mechanisms of RON/RONΔ160-mediated gastric cancer growth and metastasis remain vague. We therefore examined the actions of RON, RONΔ160, and β-catenin in gastric cancer cells and tissue samples, and their effects on cell growth in vitro and in vivo. We found that in gastric cancer samples and cell lines, there was positive correlation between RON/RONΔ160 and β-catenin levels, and that they formed a RON/RONΔ160-β-catenin complex which was translocated to the nucleus. Hypoxia led the binding of hypoxia-inducible factor-1α to the RON/RONΔ160-β-catenin complex, which increased nuclear translocation and expression of downstream oncogenic signaling molecules. Overexpression of RON/RONΔ160 promoted the proliferation and migration of gastric cancer cells, which were also enhanced by hypoxia. Suppression of RON using siRNA or anti‑RON monoclonal antibody diminished gastric cancer cell and tumor growth in vitro and in vivo. These findings establish a link between the receptor tyrosine kinase RON and β-catenin and provide insight into the mechanism by which they contribute to gastric cancer progression.

The Role of AMP-Activated Protein Kinase as a Potential Target of Treatment of Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide with a very high recurrence rate and very dismal prognosis. Diagnosis and treatment in HCC remain difficult, and the identification of new therapeutic targets is necessary for a better outcome of HCC treatment. AMP-Activated Protein Kinase (AMPK) is an essential intracellular energy sensor that plays multiple roles in cellular physiology and the pathological development of chronic diseases. Recent studies have highlighted the important regulation of AMPK in HCC. This review aims to comprehensively and critically summarize the role of AMPK in HCC. Methods: Original studies were retrieved from NCBI database with keywords including AMPK and HCC, which were analyzed with extensive reading. Results: Dysregulation of the kinase activity and expression of AMPK was observed in HCC, which was correlated with survival of the patients. Loss of AMPK in HCC cells may proceed cell cycle progression, proliferation, survival, migration, and invasion through different oncogenic molecules and pathways. Conclusions: We identified several AMPK activators which may possess potential anti-HCC function, and discussed the clinical perspective on the use of AMPK activators for HCC therapy.