MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells

Pancreaticcancer (PC)has theworstprognosisamongallcancersdue to its late diagnosis and lack of effective therapies. Therefore,identiﬁcation of novel gene targets, which are differentially ex-pressed in PC and functionally involved in malignant phenotypes,is critical to achieve early diagnosis and development of effectivetherapeutic strategies. We have shown previously that MUC4,an aberrantly overexpressed transmembrane mucin, promotesgrowth, invasion and metastasis of PC cells, thus underscoringits potential as a clinical target. Here, we report a novel micro-RNA(miRNA)-mediatedmechanismunderlyingaberrantexpres-sion of MUC4 in PC. We demonstrate that the 3# untranslatedregion of MUC4 contains a highly conserved miRNA-150 (miR-150) binding motif and its direct interaction with miR-150 down-regulates endogenous MUC4 protein levels. We also show thatmiR-150-mediated MUC4 downregulation is associated witha concomitant decrease in human epidermal growth factor recep-tor 2 and its phosphorylated form, leading to reduced activationof downstream signaling. Furthermore, our ﬁndings demonstratethat miR-150 overexpression inhibits growth, clonogenicity, mi-gration and invasion and enhances intercellular adhesion in PCcells. Finally, our data reveal a downregulated expression of miR-150 in malignant pancreatic tissues, which is inversely associatedwith MUC4 protein levels. Altogether, these ﬁndings establishmiR-150 as a novel regulator of MUC4 and a tumor suppressormiRNA in PC.IntroductionPancreatic cancer (PC) is a highly lethal malignancy and has the worstprognosis among all cancers. Currently, it is the fourth leading causeof cancer-related deaths in the USA (1). The collective median sur-vival for all patients with PC is 2–8 months, and only 1–4% of allpatients survive 5 years after diagnosis (2). Such a grim prognosis ofPC is explained by the fact that at the time of diagnosis, majority ofpatients have already developed an aggressive form of the disease thuslimiting the potential for therapeutic intervention (3). Even smalladenocarcinoma of pancreas at diagnosis is genetically advancedand carries numerous genetic and epigenetic aberrations that co-operatively act to confer aggressive malignant phenotypes (4,5).Recent years have witnessed important advances in our understandingof the molecular progression of PC, and several important targets havebeen identiﬁed and experimentally tested for their functional participa-tion in the disease processes (4,6,7).MUC4 is a high-molecular weight glycoprotein that belongs to thefamily of membrane-bound mucins (8). It is overexpressed in pancre-atic adenocarcinomas and tumor cell lines while remains undetectablein the normal pancreas (9). Expression analysis of MUC4 in increas-ing grade pancreatic intraepithelial neoplasias and malignant lesionsdemonstrated a positive correlation of MUC4 with disease progres-sion (10). Importantly, in our earlier studies, we have shown a patho-genic role of MUC4 in promoting pancreatic tumor growth andmetastasis (11,12). Furthermore, aberrant MUC4 expression is alsoreported in other malignancies indicating its clinical relevance asa target for therapeutic intervention (8,13). However, there is still littleknown about the molecular mechanisms that regulate MUC4 expres-sion and whose perturbation ultimately leads to its aberrant expressionduring cancer initiation and progression (8,14).Recently, a novel class of endogenous small non-coding gene reg-ulatory RNAs, termed as microRNAs (miRNAs or miRs), has gainedsigniﬁcant attention (15). These small molecules exert their regulatoryeffects by base pairing with partially complementary messengerRNAs (mRNAs) and function by two mechanisms: degrading targetmRNA or inhibiting their translation (16,17). It is now well estab-lished that miRNAs play critical roles in the development of cancer byaltering the expression of oncogenes and tumor suppressor genes(15,16). In the present study, we have investigated the role of micro-RNA-150 (miR-150) in the regulation of MUC4 expression in PCcells. Our ﬁndings demonstrate that 3# untranslated region (UTR)of MUC4 contains putative binding site for miR-150, which is highlyconserved across several mammalian species. Furthermore, we exper-imentally show that miR-150 directly targets the 3# UTR of MUC4 tosuppress its expression. Downregulation of MUC4 by miR-150 alsoleads to a concomitant decrease in human epidermal growth factorreceptor 2 (HER2), an interacting partner of MUC4 (18), and itsphosphorylated form leading to reduced activation of downstreamsignaling molecules. Our ﬁndings also demonstrate that miR-150overexpression leads to reduced growth, clonogenicity, migrationand invasion in PC cells. Finally, our data reveal a discordant expres-sion of MUC4 at the transcript and protein levels, which is inverselyassociated with miR-150 expression in malignant clinical specimens.Altogether, our study characterizes a novel miRNA-mediated mech-anism of MUC4 regulation and suggests tumor suppressive actions ofmiR-150 in PC cells.Materials and methods