The long noncoding RNA (lncRNA) MEG3 is significantly downregulated in pancreatic neuroendocrine tumors (PNETs). MEG3 loss corresponds with aberrant upregulation of the oncogenic hepatocyte growth factor (HGF) receptor c-MET in PNETs. Meg3 overexpression in a mouse insulin-secreting PNET cell line, MIN6, downregulates c-Met expression. However, the molecular mechanism by which MEG3 regulates c-MET is not known. Using romatinsolation by NAurification and uencing (ChIRP-Seq), we identified Meg3 binding to unique genomic regions in and around the c-Met gene. In the absence of Meg3, these c-Met regions displayed distinctive enhancer-signature histone modifications. Furthermore, Meg3 relied on functional enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), to inhibit c-Met expression. Another mechanism of lncRNA-mediated regulation of gene expression utilized triplex-forming GA-GT rich sequences. Transfection of such motifs from Meg3 RNA, termed triplex-forming oligonucleotides (TFOs), in MIN6 cells suppressed c-Met expression and enhanced cell proliferation, perhaps by modulating other targets. This study comprehensively establishes epigenetic mechanisms underlying Meg3 control of c-Met and the oncogenic consequences of Meg3 loss or c-Met gain. These findings have clinical relevance for targeting c-MET in PNETs. There is also the potential for pancreatic islet β-cell expansion through c-MET regulation to ameliorate β-cell loss in diabetes.

Epigenetic regulation is emerging as a key feature in the molecular characteristics of various human diseases. Epigenetic aberrations can occur from mutations in genes associated with epigenetic regulation, improper deposition, removal or reading of histone modifications, DNA methylation/demethylation and impaired non-coding RNA interactions in chromatin. Menin, the protein product of the gene causative for the multiple endocrine neoplasia type 1 (MEN1) syndrome, interacts with chromatin-associated protein complexes and also regulates some non-coding RNAs, thus participating in epigenetic control mechanisms. Germline inactivating mutations in the gene that encodes menin predispose patients to develop endocrine tumors of the parathyroids, anterior pituitary and the duodenopancreatic neuroendocrine tissues. Therefore, functional loss of menin in the various MEN1-associated endocrine cell types can result in epigenetic changes that promote tumorigenesis. Because epigenetic changes are reversible, they can be targeted to develop therapeutics for restoring the tumor epigenome to the normal state. Irrespective of whether epigenetic alterations are the cause or consequence of the tumorigenesis process, targeting the endocrine tumor-associated epigenome offers opportunities for exploring therapeutic options. This review presents epigenetic control mechanisms relevant to the interactions and targets of menin, and the contribution of epigenetics in the tumorigenesis of endocrine cell types from menin loss.

Background: Pancreatic-islet ␤-cell tumors (insulinomas) that lack menin express the phospho-isoform of the differentiation factor HLXB9. Results: Phospho-HLXB9 interacts with the survival factor p54nrb/Nono and also activates the oncogenic c-Met pathway by down-regulating the c-Met inhibitor Cblb. Conclusion: Targeting the HLXB9-Nono interaction and c-Met in insulinomas can be therapeutic. Significance: ␤-Cell proliferation mechanisms propose tumor therapy and strategies to alleviate ␤-cell loss in diabetes.

Loss of tumor suppressors and gain of oncogenic properties is a hallmark of cancer. But the pathways leading to tumor formation are poorly understood. A good example is the multiple endocrine neoplasia type 1 (MEN1) syndrome, in which patients inherit germline mutations in the MEN1 gene, predisposing to tissue-specific loss of the encoded tumor suppressor protein menin. Consequently, tumors develop in multiple endocrine organs - the pituitary, the parathyroids, and the duodeno-pancreatic tissues, including pancreatic neuroendocrine tumors (PNETs). Insulinomas are the most common type of functioning PNETs that also occur in MEN1 patients. We investigated the molecular pathways associated with insulinomas. We previously showed that menin loss downregulated Maternally Expressed Gene 3 (MEG3), a long non-coding RNA (LncRNA), by eliciting promoter hypermethylation along with the loss of Histone H3 lysine 4 trimethylation (H3K4me3). Gene expression microarray analyses and RT-PCR, of mouse insulinoma MIN6 cells stably transfected with Meg3, showed a 5-fold decrease in the proto-oncogenic signaling receptor c-Met, suggesting a tumor suppressor function for Meg3. We also found MEG3 downregulation with concurrent upregulation of c-MET in PNETs. However, the molecular underpinnings of MEG3 governed c-MET repression remain elusive. To identify the mechanisms by which MEG3 inhibits oncogenic c-MET signaling to suppress tumorigenesis, we examined the effect of various Meg3 isoforms, explored Meg3 association with the epigenetic regulatory machinery, and RNA-DNA triplex formation. We provide direct evidence for the first time that ectopically expressed Meg3 isoforms could attenuate the highly abundant c-Met transcript in MIN6 insulinoma cells. Meg3 also interacted with components of the epigenetic machinery, such as the Polycomb Repressive Complex 2 (PRC2) protein conglomerate, to regulate c-Met RNA expression. Additionally, analyses of triplex forming oligos (TFOs) in MIN6 cells revealed that triple helix formation between Meg3 and dsDNA could potentially disrupt c-Met transcription. Combined, these data offer mechanistic insight into the dysregulation of c-MET repression in PNETs, and support the conclusion that MEG3 acts as an important determinant of oncogenic signaling in MEN1-associated endocrine tumor cells (insulinoma). These findings warrant further investigation into the tumorigenic pathways that may result from the loss of tumor suppressor MEG3 and gain of oncogenic c-MET signaling in other MEN1-associated endocrine tumors. Citation Format: Sucharitha Iyer, Sunita Agarwal. The lncRNA Meg3 acts as an epigenetic determinant of oncogenic signaling in multiple endocrine neoplasia type 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2544. doi:10.1158/1538-7445.AM2017-2544

BACKGROUND: The FDA’s modified risk authorization for IQOS® is contingent upon approved post-market surveillance studies. The IQOS® Cross-Sectional Post-Market Adult Consumer Study (hereinafter termed IQOS® CS PACS) and the IQOS® Longitudinal Cohort Post-Market Adult Consumer Study (hereinafter termed IQOS® LC PACS) are contiguous surveys designed to fulfill this proviso. OBJECTIVES: IQOS® CS PACS seeks to assess tobacco use patterns in IQOS® users, risk perceptions of IQOS®, and tobacco transition and cessation behaviors related to IQOS®. The IQOS® LC PACS aims to follow over time, and in comparison with cigarette users, these same parameters with additional emphasis on transitions and health outcomes. METHODS AND RESULTS: The IQOS® CS PACS is a repeated cross-sectional study to be conducted annually for four years. The IQOS® LC PACS is a longitudinal study, planned to follow a cohort of new IQOS® users for two years. Potential adult IQOS® consumers aged 21 and older will be recruited from an IQOS® consumer database. Both studies will use self-administered online screening and survey assessment. At least 250 adult ever established IQOS® users (current and former) constitute the target sample size for each administration of the IQOS® CS PACS. The target sample size for the IQOS® LC PACS is 2,100 adult IQOS® users and 1,600 adult cigarette smokers as control. Data analysis includes descriptive statistics for pre-defined outcomes and inferential statistics (e.g., generalized estimating equations and propensity scoring) to compare outcomes among IQOS® and cigarette smokers. The IQOS® CS PACS is designed to commence one year after IQOS® modified risk tobacco product authorization (MRTPA) and will recur annually over the course of four years. The IQOS® LC PACS will begin two years after issuance of the IQOS® modified risk order and has been designed to follow up with participants at 3-, 6-, 12-, 18-, and 24-months from initiation. Final reports will be generated and shared with the FDA when the studies are completed. CONCLUSIONS: Postmarket studies can help inform outcomes related to risk perceptions, tobacco use patterns, and health status related to IQOS® use in a real-world setting.