Kaveh Daneshvar

MUC1 induces drug resistance in pancreatic cancer cells via upregulation of multidrug resistance genes

MUC1 (CD227), a membrane tethered mucin glycoprotein, is overexpressed in >60% of human pancreatic cancers (PCs), and is associated with poor prognosis, enhanced metastasis and chemoresistance. The objective of this study was to delineate the mechanism by which MUC1 induces drug resistance in human (BxPC3 and Capan-1) and mouse (KCKO, KCM) PC cells. We report that PC cells that express high levels of MUC1 exhibit increased resistance to chemotherapeutic drugs (gemcitabine and etoposide) in comparison with cells that express low levels of MUC1. This chemo resistance was attributed to the enhanced expression of multidrug resistance (MDR) genes including ABCC1, ABCC3, ABCC5 and ABCB1. In particular, levels of MRP1 protein encoded by the ABCC1 gene were significantly higher in the MUC1-high PC cells. In BxPC3 and Capan-1 cells MUC1 upregulates MRP1 via an Akt-dependent pathway, whereas in KCM cells MUC1-mediated MRP1 upregulation is via an Akt-independent mechanism. In KCM, BxPC3 and Capan-1 cells, the cytoplasmic tail motif of MUC1 associates directly with the promoter region of the Abcc1/ABCC1 gene, indicating a possible role of MUC1 acting as a transcriptional regulator of this gene. This is the first report to show that MUC1 can directly regulate the expression of MDR genes in PC cells, and thus confer drug resistance.

MicroRNA miR-308 regulates dMyc through a negative feedback loop in Drosophila

Summary The abundance of Myc protein must be exquisitely controlled to avoid growth abnormalities caused by too much or too little Myc. An intriguing mode of regulation exists in which Myc protein itself leads to reduction in its abundance. We show here that dMyc binds to the miR-308 locus and increases its expression. Using our gain-of-function approach, we show that an increase in miR-308 causes a destabilization of dMyc mRNA and reduced dMyc protein levels. In vivo knockdown of miR-308 confirmed the regulation of dMyc levels in embryos. This regulatory loop is crucial for maintaining appropriate dMyc levels and normal development. Perturbation of the loop, either by elevated miR-308 or elevated dMyc, caused lethality. Combining elevated levels of both, therefore restoring balance between miR-308 and dMyc levels, resulted in lower apoptotic activity and suppression of lethality. These results reveal a sensitive feedback mechanism that is crucial to prevent the pathologies caused by abnormal levels of dMyc.

Myc Localizes to Histone Locus Bodies during Replication in Drosophila

Myc is an important protein at the center of multiple pathways required for growth and proliferation in animals. The absence of Myc is lethal in flies and mice, and its over-production is a potent inducer of over-proliferation and cancer. Myc protein is localized to the nucleus where it executes its many functions, however the specific sub-nuclear localization of Myc has rarely been reported. The work we describe here began with an observation of unexpected, punctate spots of Myc protein in certain regions of Drosophila embryos. We investigated the identity of these puncta and demonstrate that Myc is co-localized with coilin, a marker for sub-nuclear organelles known as Cajal Bodies (CBs), in embryos, larvae and ovaries. Using antibodies specific for U7 snRNP component Lsm11, we show that the majority of Myc and coilin co-localization occurs in Histone Locus Bodies (HLBs), the sites of histone mRNA transcription and processing. Furthermore, Myc localizes to HLBs only during replication in mitotic and endocycling cells, suggesting that its role there relates to replication-dependent canonical histone gene transcription. These results provide evidence that sub-nuclear localization of Myc is cell-cycle dependent and potentially important for histone mRNA production and processing.

DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC Expression to Control Definitive Endoderm Differentiation of Embryonic Stem Cells

Long noncoding RNAs (lncRNAs) exhibit diverse functions, including regulation of development. Here, we combine genome-wide mapping of SMAD3 occupancy with expression analysis to identify lncRNAs induced by activin signaling during endoderm differentiation of human embryonic stem cells (hESCs). We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. Disruption of the gene encoding DIGIT and depletion of the DIGIT transcript reveal that DIGIT is required for definitive endoderm differentiation. In addition, we identify the mouse ortholog of DIGIT and show that it is expressed during development and promotes definitive endoderm differentiation of mouse ESCs. DIGIT regulates GSC in trans, and activation of endogenous GSC expression is sufficient to rescue definitive endoderm differentiation in DIGIT-deficient hESCs. Our study defines DIGIT as a conserved noncoding developmental regulator of definitive endoderm.

Identification and characterization of m6A circular RNA epitranscriptomes

This study brings together the expanding fields of RNA modifications and circular (circ) RNAs. We find that cells express thousands of m6A methylated circRNAs, with cell-type specificity observed between human embryonic stem cells and HeLa cells. m6A-circRNAs were identified by RNA sequencing of total RNA following ribosome depletion and m6A immunoprecipitation. The presence of m6A-circRNAs is corroborated by the identification of complexes between circRNAs and YTHDF1 and YTHDF2, proteins that “read” m6A sites in mRNAs. Furthermore, m6A modifications on non-linear RNAs depend on METTL3 and METTL14, the known m6A methyltransferase “writer” complex components, suggesting that circRNAs are methylated by the same complexes responsible for m6A modification of linear RNAs. Despite sharing m6A readers and writers, m6A-circRNAs are frequently derived from exons not methylated in mRNAs. Nevertheless, m6A-mRNAs that are methylated on the same exons as those composing m6A-circRNAs exhibit less stability than other m6A-mRNA, and this circRNA-mRNA cross-talk is regulated by YTHDF2. Thus, our results expand the m6A regulatory code through identification of the first circRNA epitranscriptome.

Abstract LB-348: Chemoresistance in pancreatic cancer is conferred by MUC1 cytoplasmic tail binding to the promoter region of the ABCC1 (MRP-1) gene

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: The high mortality rate of pancreatic cancer (PC) is in part, due to their high propensity for metastasis and multidrug resistance. The three most common ways by which cancer cells acquire chemoresistance is via deregulation of apoptotic pathways, upregulation of survival pathways and upregulation of multi-drug resistance (MDR) genes. The MDR genes encode for ATP-binding cassette (ABC) membrane transporters, which extrudes chemotherapeutic drugs from cancer cells. However, the mechanisms behind the up-regulation of the MDR genes are not entirely understood. More than 90% of metastatic PC patients overexpress MUC1 (CD227), a membrane tethered mucin glycoprotein that has been linked to increased metastatic phenotype and drug resistance. The cytoplasmic tail (CT) of MUC1 has seven conserved tyrosine residues which are important for the signal transduction role of MUC1. The aim of this study was to understand the underlying mechanism by which MUC1 induces drug resistance in PC cells. Method: We used a panel of PC cells lines that endogenously express low or high levels of MUC1. The cells lines expressing low endogenous MUC1 were stably infected with the retroviral vector containing empty plasmid (Neo), or plasmid carrying full-length MUC1 (MUC1 WT), or full length MUC1 with the tyrosine residues of MUC1 CT mutated to phenylalanine (MUC1 Y0). The cells were treated with three commonly used chemotherapeutic/preventive drugs (Gemcitabine, Etoposide and Celecoxib). The level of dead cells was evaluated by H3 incorporation assay and Annexin V staining. We also checked for the activation of prosurvival pathways Erk1/2 and PI3K by Western Blot. The mRNA level of MDR genes were determined by RTPCR. CHIP (Chromatin Immunoprecipitation) was performed to determine the localization of MUC1 CT to the promoter region of ABCC1 gene (MRP1). Results: Our results indicate that PC cells expressing MUC1 WT or MUC1 Y0 have intrinsic resistance to chemotherapeutic, compared to cells expressing low levels of MUC1. We observed an enhanced activation of the prosurvival pathways Erk1/2 and PI3K in MUC1 high PC cells, compared to cells expressing low MUC1 or MUC1 Y0. Upon inhibition of the PI3K/Akt pathway, a partial loss in the chemoresistance was seen, indicating that this pathway is partially responsible for the chemoresistance. DNA microarray and RT PCR showed that the expression of MDR genes (ABCC1, ABCC3, ABCC5 and ABCB1) was significantly higher in PC cells expressing MUC1 WT or MUC1 Y0. Most importantly, for the first time, we showed that MUC1 CT localizes to the promoter region of a MDR gene, ABCC1. This suggests that MUC1 directly drives the transcription and expression of ABCC1 gene. The implications of the data are clinically significant as small molecule inhibitors can be designed to specifically block MUC1 CT signaling and transcriptional activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-348. doi:1538-7445.AM2012-LB-348