Tgfb- Mediated Control of RNA-Binding Proteins and Noncoding Rnas Harinarayanan Janakiraman, Reniqua P

Published OnlineFirst March 19, 2018; DOI: 10.1158/1541-7786.MCR-17-0547

Review Molecular Cancer Research The Long (lncRNA) and Short (miRNA) of It: TGFb- Mediated Control of RNA-Binding Proteins and Noncoding RNAs Harinarayanan Janakiraman, Reniqua P. House, Vamsi K. Gangaraju, J. Alan Diehl, Philip H. Howe, and Viswanathan Palanisamy

Abstract

RNA-binding proteins (RBP) and noncoding RNAs (ncRNA), importantly, transforming growth factor-beta (TGFb) signaling such as long noncoding RNAs (lncRNA) and microRNAs plays a signiﬁcant role in controlling gene expression patterns by (miRNA), control co- and posttranscriptional gene regulation targeting RBPs and ncRNAs. Because of TGFb signaling in cancer, (PTR). At the PTR level, RBPs and ncRNAs contribute to pre- RBP-RNA or RNA-RNA interactions are altered and cause mRNA processing, mRNA maturation, transport, localization, enhanced cell growth and tumor cell dissemination. This review turnover, and translation. Deregulation of RBPs and ncRNAs focuses on the emerging concepts of TGFb signaling on posttran- promotes the onset of cancer progression and metastasis. Both scriptional gene regulation and highlights the implications of RBPs and ncRNAs are altered by signaling cascades to cooperate or RBPs and ncRNAs in cancer progression and metastasis. Mol Cancer compete with each other to bind their nucleic acid targets. Most Res; 16(4); 1–13. 2018 AACR.

Introduction mRNA splicing and translation via RNA-binding proteins (RBP) and noncoding RNAs (ncRNA; refs. 6, 7). The transforming growth factor beta (TGFb) superfamily of RBPs are versatile regulators of gene expression. They control all secreted proteins are potent regulators of cellular development aspects of messenger RNA maturation, including pre-mRNA and differentiation. Once the TGFb ligands bind to their cognate splicing, mature mRNA export, localization, turnover, and trans- receptors, multitudes of downstream effector molecules are lation. Due to their ability to regulate the expression of hundreds recruited to orchestrate a collective cellular response. The TGFb of coding and ncRNAs, the loss of a single RBP can dramatically signaling cascade is a tightly controlled process, and aberrant alter cellular transcriptomic and proteomic landscapes, which induction of this pathway is the driving mechanism underlying could be detrimental to cellular development (8, 9). Therefore, several human disorders (1, 2). Therefore, a comprehensive signaling pathways, such as the TGFb pathway, capable of altering understanding of the molecular mediators controlling TGFb- RBP expression will have strong biological implications. induced cellular transformation may provide novel targets for The noncoding class of RNAs (ribosomal RNA, transfer RNA, therapeutic intervention. small nuclear RNA, piwi, micro, circular, and long noncoding) TGFb possesses tumor suppressive and tumor-promoting attri- constitutes >90% of the total pool of transcribed RNAs in mam- butes in multiple cancers. It is accepted that the strength, response, malian cells (10). Most extensively studied noncoding miRNAs and source of TGFb signaling in the context of cancer are highly have profound impact in cancer biology and also influenced by dependent on the number of accrued driving oncogenic muta- TGFb (11). The long noncoding RNAs (lncRNA) are more than tions in premalignant cells (3, 4). Traditionally, activation of the 200 nt in length, emerged as co- and posttranscriptional regula- TGFb signaling pathway is perceived as a transcriptional event, tors of gene expression. Typically, miRNAs and lncRNAs are lowly mediated through receptor phosphorylation and subsequent abundant in cells, and evidence supports their role as contributors activation of downstream transcription factors, to promote or to many disease including cancer. In this review, we summarize repress gene expression (3, 5). However, TGFb signaling can also the current field of ncRNA (miRNAs and lncRNAs) and RBP- control posttranscriptional gene regulatory processes such as mediated potentiation of TGFb signaling in cancer. In addition, we discuss the potential avenues of research for the burgeoning field of TGFb regulation of ncRNA expression in malignant cellular transformation. Department of Biochemistry and Molecular Biology, College of Medicine, Medical University of South Carolina, Charleston, South Carolina. TGFb Signaling Cascade H. Janakiraman and R.P. House contributed equally to this article. TGFb was originally identified and characterized as a factor Corresponding Author: Viswanathan Palanisamy, Department of Biochemistry central to the malignant transformation of fibroblasts (12). Since and Molecular Biology, College of Medicine, Charleston, SC 29425. Phone: 843- its discovery 40 years ago, TGFb has emerged as a critical regulator 792-5701. Fax: 843-792-8304; E-mail: [email protected] of multiple biological processes, including epithelial–mesenchy- doi: 10.1158/1541-7786.MCR-17-0547 mal transition (EMT; ref. 13), stem cell regeneration (14), cell 2018 American Association for Cancer Research. proliferation, and immune response (15). The TGFb family

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consists of 33 secreted signaling molecules identified by their signaling on tumorigenesis, it was observed that transgenic mice shared 3-dimensional cysteine knot motif (16). The 33 members with reduced TGFb signaling did not form spontaneous tumors. are broadly subdivided into five classes: TGFb, nodal, bone However, in the presence of carcinogenic stimuli, the loss of TGFb morphogenic proteins (BMP), activins/inhibins, and growth and signaling enhanced neoplastic lesions in the liver (24) and lung differentiation factors (GDF; ref. 17). TGFb is an obligate dimer (24) compared with WT mice. These studies support a tumor that binds to heteromeric type-II and I serine/threonine kinase suppressive role for TGFB signaling in cancer. In contrast, in a receptors and activates canonical or noncanonical downstream mammary carcinogenesis model, premalignant cells with an TGFb signaling molecules. In the following section, we briefly intact TGFb signaling pathway exhibited a marked reduction in describe the pathways of TGFb canonical signaling. proliferation rates compared with the dominant-negative type II TGFb receptor (DNR) expressing cells (25). However, TGFb Canonical TGFb Signaling Pathway signaling enhanced lung colonization in metastatic cells, and loss b There are seven type I (ALK1, ALK2, ALK3/BMPR1A, ALK4/ of TGF signaling in the DNR expressing cells yielded a 60% ACVR1B, ALK5/TGFBR1, ALK6/BMPR1B, and ALK7) and five type reduction in metastatic lung lesions (25). This study demonstrat- b fi II (TGFBR2, BMPR2, AMHR2, ACVR2, and ACVR2B) TGFb recep- ed that the TGF signaling axis modi es its cellular response tors (3). These receptors form heteromeric complexes upon ligand depending on the oncogenic mutational status of cells. Lastly, in a b binding, which induce type I signaling receptors undergo type II recent study, using a mouse model to track and manipulate TGF - fi b receptor-mediated phosphorylation, thus promoting intracellular responsive cells, Oshimori and colleagues identi ed that TGF adaptor protein binding. In canonical TGFb signaling, type I promotes tumor heterogeneity and drug resistance in squamous b receptors phosphorylate receptor regulated SMAD (R-SMADS) cell carcinoma (26). The report demonstrated that TGF -respon- transcription factors. SMADs 1, 2, 3, 5, and 8 are R-SMADs and sive cancer stem cells had reduced proliferative rates and altered b associate with the co-SMAD, SMAD4, to form a trimeric SMAD glutathione metabolism due to TGF -driven expression of pro- complex in the cytoplasm. The SMAD2/3/4 oligomers translocate teins such as p21 and NRF2 (26). Altogether, these studies b from the cytoplasm to the nucleus and with the aid of additional highlight the critical yet paradoxical role of TGF signaling in cofactors alter transcription. For example, phosphorylated R- cancer. SMADs 2/3 associate with SMAD4 to activate TGFb-responsive EMT is an important biological process in the initiation of p21WAF1/Cip1 or repress c-MYC transcription (18). Attenuation of tumor metastasis. It is a developmental process co-opted by activated TGFb signaling occurs through activation of inhibitory tumors, which helps tumor cell seeding and colonization of areas SMADs (I-SMADS) 6 and 7 (19). distant from the primary site of the tumor. EMT is characterized by SMAD6 solely functions in BMP signaling; however, SMAD7 the loss of epithelial cellular polarity, disruption of cell adhesion, can inhibit TGFb signaling by either competing with SMAD-2 and and acquisition of protease production capacity resulting in -3 for type-I receptor binding or regulating the turnover of the increased cellular motility (27). Loss of E-cadherin, a potent fi TGFb receptor through association with the ubiquitin ligase tumor suppressor, has also been identi ed as a characteristic Smurf (20). Depending on the cellular context, the transcriptional feature in the process of epithelial cellular EMT (28). The process events activated by canonical TGFb signaling can enhance cell of EMT is also governed by genetic alterations in tumor cells and growth, promote programmed cell death, or facilitate malignant their microenvironment. Also, cytokines, chemokines, extracel- transformation. In addition to the canonical mode of activation, lular matrix (ECM), and growth factors, which are altered by TGFb can also stimulate ancillary intracellular signaling pathways hypoxic conditions, are crucial for the development of EMT (29). b via SMAD-dependent or independent mechanisms. The extent of Among these, members of the TGF family of cytokines are vital, b TGFb stimulation of the MAPK, PI3K/AKT, Wnt, and RhoGTPase because TGF signaling initiates EMT through the activation of signaling pathways and others is highly context dependent EMT-inducing transcription factors (EMT-TF), including Snail/ fi (21, 22). The TGFb-mediated crosstalk between these noncanon- Slug, Twist, and zinc- nger E-box-binding homeobox 1/2 (ZEB1/ ical pathways is a complex, intricate network of signaling mole- 2; ref. 28). EMT is also marked by the repression of E-cadherin by cules whose concerted efforts promote epithelial-to-mesenchy- TFs including Snail, Slug, Twist, ZEB1, and ZEB2 via binding of its mal transition (EMT), cell proliferation, or apoptosis. For detailed promoter (28). discussions of noncanonical TGFb signaling, we direct the readers Posttranscriptional mechanisms that regulate E-cadherin to the following reviews (22, 23). Although TGFb control of expression and EMT have gained much attention in recent years. transcriptional events is important, there are several intermediate The precise coordination between transcriptional, posttranscrip- factors that are pivotal in the regulation of gene expression such as tional, and posttranslational events is crucial for the regulation of RBPs and ncRNAs. Below we discuss how TGFb regulated RBPs, protein expression and function. RBPs are one of the well-known miRNAs, and lncRNAs activate or repress TGFb-induced EMT and posttranscriptional regulators and have been reported to regulate act as central signaling nodes for TGFb-mediated malignant EMT through utilization of posttranscriptional gene control mea- transformation in cancer. sures such as alternative splicing and translation (30, 31). In addition, the ncRNAs such as miRNAs and long noncoding RNAs TGFb-mediated Posttranscriptional Gene (lncRNA) were also found to modulate gene expression at posttranscriptional levels. Speci fically, ncRNA-mediated regulation of Regulation in EMT and Cancer EMT was found to be mediated through EMT-TFs and EMT- Since its discovery in the late 1970s as a factor capable of associated signaling. In the following sections, we focus on RBPs, transforming normal fibroblasts in vitro, TGFb has emerged as an miRNAs, and lncRNAs involved both in the regulation of EMT and integral player in the development of cancer (12). Interestingly, cancer progression, and we discuss the posttranscriptional TGFb exhibits both protumorigenic and antitumorigenic activi- mechanisms that contribute to regulation of cancer-associated ties. In early mouse models, to determine the effect of TGFb EMT and tumorigenesis (Tables 1 and 2 and Figs. 1 and 2).

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TGFb, RBPs, and EMT treatment increased the expression of EMT-inducing dEF1 family d hnRNP E1 proteins, EF1 and SIP1, which bound to the promoter regions of both ESRP1 and 2 and suppressed their transcription in response Heterogeneous nuclear ribonucleoproteins (hnRNP) belong to to TGFb treatment. The authors also demonstrated that TGFb a class of RBPs involved in mRNA processing such as alternative induced downregulation of ESRPs in EpRas cells resulted in the splicing, mRNA stability, and translational regulation. Recently, upregulation of FGFR1IIIc, which is the mesenchymal isoform of hnRNPE1 has been shown to repress EMT promoting diabled-2 FGFR1 and downregulation of FGFR2IIIb, which is the epithelial (Dab2) and interleukin-like EMT inducer (ILEI), by binding the isoform of FGFR2, and both of these isoforms are the splicing structural, 33 nucleotide long TGFb-activated translation (BAT) targets of ESRP proteins. Importantly, ESRP 1 and 2 proteins have element present in their 30-UTRs (30). In addition, TGFb-medi- been reported to bind to the GU-rich, auxiliary cis-element ISE/ ated phosphorylation of hnRNP E1 at Ser43 by protein kinase Bb/ ISS-3 of the FGFR2 gene to modulate its splicing (33). Further- Akt2 was found to enhance the translational activation of Dab2 more, in breast cancer JygMC(A) cells, which autonomously and ILE1, by inducing the release of hnRNPE1 from the BAT secrete TGFb, the expression of ESRP1 and 2 was shown to be element of these mRNAs (30). In addition to Dab2 and ILEI, increased upon treatment with a TGFb type I receptor (TbR-I) several hnRNP E1 target mRNAs translationally regulated by inhibitor, SB431542. In addition, overexpression of ESRP 1 and 2 hnRNPE1 were identified as mediators of EMT under TGFb in human breast cancer MDA-MB-231 cells was found to upre- treatment (32). Specifically, a panel of 36 genes that possess BAT gulate E-cadherin expression thereby resulting in the attenuation element in the 30-UTR (termed BAT genes) including EMT-asso- of EMT (6). Therefore, the authors speculated that the alternative ciated ZEB2, Eukaryotic initiation factor 5A2, Moesin, Egfr, and splicing events mediated by ESRP 1 and 2 may regulate uniden- inhibin beta A was identified. Thus, TGFb-inducible hnRNP E1 tified E-cadherin inducers or epithelial regulators, causing a posttranscriptional regulon controls EMT process during devel- transition from mesenchymal-to-epithelial phenotype via opment and metastatic progression of tumors (32). increase in E-cadherin expression. ESRP 1 and 2 Epithelial splicing regulatory proteins (ESRP) 1 and 2 are RBPs RBFOX family that promote an epithelial phenotype by facilitating splicing of The RNA-binding Fox (RBFOX) family of proteins is known to transcripts such as fibroblast growth factor receptor 2 (FGFR2) be a crucial player in the regulation of alternative splicing (AS) of and ENAH, which have been shown to possess well-documented pre-mRNA (34). The RBFOX family of proteins has been reported and essential roles in EMT (31). Recently, Horiguchi and collea- to regulate AS by specifically recognizing the (U)GCAUG gues demonstrated that TGFb drives EMT via downregulation of sequence in regulated exons or flanking introns, and either ESRP1 and 2 (6). Specifically, in normal mammary gland, epi- promotes or represses the expression of the target exons (34). thelial cell line NMuMG and EpRas cancer cell lines, TGFb Recently, Rbfox2 (RNA binding Fox family protein 2) was found

Table 1. RBPs, miRNAs, and lncRNAs involved in TGFb-induced EMT Symbol Name Function References RNA binding proteins hnRNP E1 Heterogeneous ribonucleoprotein E1 Promotes EMT in breast cancer via translational repression of (30) Dab2 and ILEI mRNAs ESRP 1 and 2 Epithelial splicing regulatory proteins 1 and 2 Upregulate E-cadherin expression and cause attenuation of EMT (6) in breast cancer RBFOX2 RNA-binding Fox protein 2 Rbfox2 promoted EMT associated tissue invasiveness through (35) splicing events RBFOX3 RNA-binding Fox protein 3 Plays an important role in TGFb-induced EMT through (36) posttranscriptional regulation of a subset of EMT-related genes microRNAs miR-200 family microRNA 200 family Inhibits EMT in breast cancer (40, 42) miR-203 microRNA 203 Inhibits cancer invasion via upregulation of the transcription of (43, 44) SNAI2 miR-181a microRNA 181a Promotes cell migration and invasion through abrogation of (45, 46) proper tight junction formation miR-10b microRNA 10a Promotes TGFb1-induced EMT in breast cancer cells (47, 48) Long noncoding RNAs lncRNA Hotair Long intergenic noncoding RNA Hotair Promotes EMT via upregulation of EMT associated genes such as (60) ZEB1, SNAI1, and TWIST lncRNA MALAT1 Long noncoding RNA metastasis associated lung Promotes EMT via repression of E-cadherin expression by (63) adenocarcinoma transcript 1 associating with suz12 lncRNA ATB Long noncoding RNA activated by TGFb Induces EMT in breast cancer, colorectal cancer and gastric cancer (64, 65) through the TGFb/miR-200s/ZEB axis lncRNA HULC Long noncoding RNA highly upregulated in promotes TGFb-induced EMT in HCC via reduction of E-Cad, and (66) cancer upregulation of N-Cadherin, Snail, and ZEB1 LINC01186 Long intergenic noncoding RNA 01186 Is regulated by TGFb/SMAD3 and inhibits migration and invasion (67) through EMT in lung cancer lncRNA PE Long noncoding RNA PE promotes invasion and EMT in HCC through the miR-200a/b-ZEB1 (69) pathway

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Table 2. TGFb-regulated noncoding RNAs in cancer Symbol Name Function References miRNAs miR-21 microRNA-21 Inhibited the expression of MutS homolog 2 (MSH2) and (7, 49) contribute to chemoresistance in cancer miR-494 microRNA-494 Reduce cell proliferation, migration, and invasion in pancreatic (50) ductal adenocarcinomas miR-34a microRNA-34a Induces tumor suppression in HBV positive HCC (52) miR-584 microRNA-584 Inhibits cell migration in breast cancer (54) miR-182 microRNA-182 Promotes NF-kB signaling pathway and causes aggressive (55) phenotype in glioma lncRNA lncRNA ROR Long noncoding RNA-ROR lncRNA ROR was upregulated by TGFb (70) lncRNA Smad7 lncRNA Smad7 anti-apoptotic effect upon stimulation by TGFb in mouse breast (71) cancer cell line JygMC(A) HOXD-AS1 HOXD antisense growth-associated HOXD-AS1 is activated by the PI3K/Akt pathway and is involved in (72) long noncoding RNA cell differentiation retinoid acid treatment induced cell differentiation in neuroblastoma

to regulate EMT-associated AS and mediate cellular invasion in miR-200 a murine breast cancer cell line, PY2T, and its expression was The miR200 family consists of five members—miR-200a, miR- shown to be increased by TGFb treatment–induced EMT (35). 200b, miR200c, miR-141, and miR-429—and are one of the well- Further, it was demonstrated that in PY2T cells, Rbfox2 regu- known regulators of EMT. Interestingly, all the five miRNAs of the lated the splicing of its targets Cortactin (Cttn), Pard3 and miR200 family have been reported to be markedly downregulated dynamin 2 (Dnm2), which have been shown to control molec- in cells that undergo TGFb-induced EMT. Conversely, enforced ular events of EMT such as actin polymerization and regulation expression of either miR-200b–200a–429 cluster or the miR- of cell polarity (35). Very recently, RBFOX3 (RNA binding Fox 200c–141 cluster via a lentivirus system driven by cytomegalo- family protein 3), another well-known regulator of AS, was virus (CMV) promoter prevented TGFb-induced EMT. The reported to be transcriptionally downregulated by TGFb1treat- authors also demonstrated that a cooperative regulation of E- ment in A549 lung cancer cells, and CRISPR-Cas9 mediated cadherin transcriptional repressors ZEB1 and SIP1, by these five knockdown of RBFOX3 promoted EMT via the downregulation miRNAs, inhibited EMT in breast cancer (40). Although the of both E-cadherin and Claudin1 (36). Although a reduction of authors of the above study did not show how TGFb regulates RBFOX3 mRNA was observed upon treatment with TGFb1, the the expression of miR-200, a later study by Gregory and colleagues authors did not identify the specific transcriptional regulator of demonstrated that prolonged exposure to TGFb leads to DNA RBFOX3; instead, they speculated that one of the Smad proteins hypermethylation of the promoters of miR-200b and miR-200c, might be involved in the TGFb-mediated inhibition of which resulted in the long-term repression of miR-200 expression RBFOX3. Furthermore, the authors only demonstrated that (41). Recently, it was found that TGFb-mediated downregulation expression of RBFOX3 depleted lung cancer cells; however, the of miR-200 induced the migration of triple-negative breast cancer authors did not investigate the mechanism by which RBFOX3 (TNBC) cells through increasing the expression of ZEB2 (42). controls E-cadherin and/or claudin (36). Taken together, data from the above studies suggest that several RBPs are either miR-203 activated or repressed by TGFb promote EMT in multiple MicroRNA-203 (miR-203) is located on chromosome cancers via some of the well-known PTR mechanisms as illus- 14q32-33, and in breast cancer, it has recently been shown to trated in Table 1 and Fig. 1. inhibit cancer invasion via transcriptional downregulation of snail homolog 2 (SNAI2 or SLUG; ref. 43). Furthermore, the b authors demonstrated that miR-203 is downregulated in met- TGF , miRNAs, and EMT astatic breast cancer cells and promotes cell growth and inva- MicroRNAs are a group of small (22 nucleotides) ncRNAs, sion. The authors also indicated that miR-203 downregulation which bind to complementary sequences within mRNA mole- was mediated via methylation of its promoter, as observed in cules and control mRNA translation. MicroRNAs influence several all the three metastatic breast cancer cell lines used in the study physiological conditions and their deregulation cause a myriad of (43). Interestingly, a recent observation by Ding and colleagues diseases including cancer (37). TGFb is well known for its dual demonstrated that TGFb-induced SNAI2 repressed miR-203 by role in cancer: In the early stages of carcinogenesis, TGFb functions directly binding to its promoter, and SNAI2 expression pro- as a tumor suppressor, whereas in the advanced stages, it switches moted EMT in a group of both lowly invasive (MCF7, MDA- to promotion of cellular metastasis through the process EMT (38). MB-468, BT474, and T47D) and highly invasive (MDA-MB- The interplay between miRNAs, EMT and TGFb signaling, has 231, BT549, Hs578T, and SUM159) metastatic breast cancer been studied extensively in the recent past, and studies have also cell lines (44). Conversely, miR-203 was also shown to directly reported that miRNA maturation can either be enhanced or target SNAI2 and inhibit metastasis in breast cancer cells. Thus, inhibited by the TGFb pathway (39). In the section below, we a negative feedback loop between miR-203 and SNAI2 was discuss how TGFb-regulated miRNAs control cancer-associated found to control EMT and tumor invasive growth and metas- EMT (Table 1 and Fig. 1). tasisinamodelofbreastcancer.

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Figure 1. Regulation of TGFb induced EMT by RBPs. RBPs such as hnRNPE1 and RB fox protein 2 (RBFOX2) are upregulated by TGFb and promote EMT through posttranscriptional mechanisms such as translational regulation and splicing of EMT- associated target mRNAs. On the other hand, RBPs such as ESRP1 and ESRP2, RBFOX3, which have been reported to be repressed by TGFb, repress the EMT process via the upregulation of E-cadherin. RBPs are denoted by dark blue rectangles, and RBP-mediated modulations of EMT are denoted by black arrows and lines.

miR-181a leagues reported that TGFb treatment signiﬁcantly upregulated miR-181a belongs to the miR-181 family, which consists of miR-181a in nontransformed PH5CH8 hepatocyte cell line and three other members, namely, miRs 181b, 181c, and 181d. In induced EMT. Importantly, miR-181a was shown to induce the several of the metastatic breast tumor cell lines, miR-181a was expression of EMT-associated genes BMP1, MMP2/MMP9, and signiﬁcantly upregulated by TGFb treatment, possibly via Smad4- Snail in TGFb-treated PH5CH8 cells. Moreover, the authors also independent processing of pre-miR-181a transcripts, and this observed increased levels of miR-181a in human HCC tissues upregulation enhanced the motility and invasion of breast cancer compared with normal liver. Given the recent literature evidence cells (45). Conversely, inhibition of miR-181a activity was shown for the involvement of TGFb-mediated upregulation of miR-181a to abrogate TGFb-induced EMT, migration, invasion, and meta- in the promotion of EMT in breast cancer, and in nontransformed static outgrowth of TNBC cells. Recently, Brockhausen and col- PH5CH8 hepatocyte cell line in this study, it is reasonable to

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Figure 2. Regulation of TGFb induced EMT by microRNAs and lncRNAs. TGFb upregulates miRNAs miR-10b, miR-181a, and miR-200 family, which in turn promote the EMT, process via repression of E-cadherin in various human cancers. In contrast, miR-203, which inhibits repressors of E-cadherin, is repressed by TGFb to promote EMT. lncRNAs ATB, HULC, LINC01186, MALAT-1, and PE, which promote EMT either via repression of E-cadherin or through an E-cadherin–independent mechanism, are upregulated by TGFb in order to promote EMT. However, lncRNAs Hotair, although upregulated by TGFb, inhibits EMT via inhibition of one of the E-cadherin repressors such as ZEB1/2 or ZNF217. miRNAs are denoted by blue ovals and miRNA-mediated regulations of EMT are denoted by blue arrow and lines and lncRNAs are denoted red rectangles and lncRNA-mediated control of the EMT process is denoted by red arrows and lines. Transcription factors are denoted by dark yellow rounded rectangles and canonical TGFb pathway members such as Smads and noncanonical TGFb members such as PI3/Akt are denoted by light blue or yellow rectangles and dark green round single corner rectangles, respectively.

speculate that TGFb-induced miR-181a upregulation might pro- while decreasing the expression of vimentin, with a concomitant mote HCC metastasis via EMT (46). decrease in the invasive potential of breast cancer cells (47). Furthermore, the expression of miR-10b was also found to be miR-10b abundant in breast cancer in contrast to adjacent nontumor Han and colleagues identiﬁed miR-10b as a target gene of tissues and miR-10b expression was found to be closely correlated TGFb1 and found that expression of miR-10b was increased with breast cancer aggressiveness. These data suggested the pos- during TGFb1-induced EMT in breast cancer cells. Conversely, sibility that miR-10b upregulation coupled with TGFb1-induced inhibition of miR-10b increased the expression of E-cadherin EMT might be responsible for the promotion of invasion and

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metastasis in breast cancer cells. Unfortunately, the authors did growth of HCC cells (52). Conversely, the authors showed that not discuss the molecular mechanism by which TGFb1 upregu- ectopic expression of miR-34a reduced the mRNA and protein lated miR-10b expression. Interestingly, TGFb1-induced upregu- production of its predominant direct target CCL22, and sup- lation of miR-10b has been shown to be involved in the regulation pressed HCC tumor growth, whereas overexpression of a non- of glioblastoma (GBM) cell proliferation, migration, and EMT. targetable form of CCL22 largely eliminated miR-34a–induced Specifically, TGFb1-induced miR-10b directly targeted E-cad- tumor suppression in HBV-positive HCC (52). herin, apoptotic protease activating factor 1 (Apaf-1), and phosphatase and tensin homolog (PTEN) genes (48). miR-584 miR-584 is located at chromosomal region 5q32, and it was TGFb, miRNAs, and Cancer found to be tumor suppressive in clear cell renal carcinoma miR-21 (ccRCC), via direct inhibition of the oncogene ROCK-1 mRNA by binding to its 30-UTR (53). Recently, an investigation by Fils- The miR-21 has been suggested to be a prognostic factor in Aime and colleagues identified miR-584 as a novel target of TGFb cancer patients, based on the correlation between high miR-21 in breast cancer and found that inhibition of miR-584 expression levels and poor overall survival in various carcinomas. Interest- by TGFb is required for cell migration. Importantly, overexpres- ingly, a study by Yu and colleagues identified that TGFb-induced sion of ectopic miR-584 was reported to reverse TGFb-induced cell miR-21 inhibited the expression of MutS homolog 2 (MSH2), by 0 migration via inhibition of mRNA and protein expression of targeting its 3 -UTR region in HER2-transformed MCF10A mam- protein phosphatase and actin regulator 1 (PHACTR1; ref. 54). mary epithelial cells and in breast cancer cells (7). MSH2 is a central component of the DNA mismatch repair (MMR) system miR-182 that recognizes chemotherapy drug-induced DNA adducts and triggers MMR at the damaged sites and causes cell-cycle arrest and miR-182 is located on chromosome 7q32.1 and is often ampli- fi apoptosis. The authors speculated that miRNA-mediated post- ed in clinical gliomas. Recently, it was reported that in gliomas, fl TGFb treatment markedly increased the expression of miR-182 transcriptional inhibition of MSH2 might in uence genomic 0 instability and thereby contribute to chemoresistance in cancer and miR-182 directly targeted the 3 -UTR and suppressed the (7). In human gliomas, TGFb1-mediated upregulation of miR-21 expression of multiple genes, including cylindromatosis (CYLD), k was found to be inhibited by antitumor agent ursolic acid (UA), to which function as negative regulators of the NF- B signaling k promote apoptosis and attenuate proliferation of glioma cells. pathway. Consequently, NF- B hyperactivation mediated by b Specifically, UA was reported to suppress the attenuation of TGF -induced miR-182 resulted in enhanced aggressiveness of b programmed cell death 4 (PDCD4) protein expression mediated gliomas (55). Interestingly, in normal cells, TGF has been shown k k by TGFb1-induced miR-21, consequently promoting apoptosis of to repress NF- B activity, whereas in cancer cells, NF- B has been b k U251 glioma cells (49). reported to be activated by TGF , suggesting that NF- B acts as an oncogenic mediator of TGFb signaling in tumors. As demonstrated in this study, NF-kB is indeed activated by TGFb via miR-182, miR-494 leading to tumor aggressiveness in human gliomas (55). The miR-494 is located in the Dlk1-Dio3–imprinted locus on human chromosome 14q32, a region containing 54 miRNAs, and miRNAs Regulate the TGFb Signaling is supposedly one of the largest miRNA clusters in the human genome. Myeloid-derived suppressor cells (MDSC) mediated Pathway suppression of antitumor immune responses favors tumor angio- Several miRNAs have been shown to target the components of genesis and metastasis. In a quest to identify the regulatory net- the TGFb signaling pathway, including TGFb1 ligands and pro- works that govern the accumulation of tumor-expanded MDSCs, teins in multiple cancers (56). For example, a recent study dem- Liu and colleagues found that TGFb-induced upregulation of onstrated that miR-744 binds directly to the 30-UTR of TGFb1 and miR-494 via the Smad3-dependent pathway inhibited the protein posttranscriptionally inhibits the endogenous expression of expression of phosphatase and tensin homolog (PTEN). The TGFb1 in human renal proximal tubule epithelial cells (reviewed inhibition of PTEN was found to be essential for both the in ref. 56). In multiple cancers, miRNAs have been shown to accumulation and activity of MDSCs (50). It was recently reported posttranscriptionally target SMAD proteins and regulate the TGFb that TGFb-mediated upregulation of miR-494, and miR-494- pathway, resulting in either tumor promotion or suppression. For mediated negative regulation of FOXM1 protein expression could example, in HCC, miR-148a was reported to attenuate the cancer reduce cell proliferation, migration, and invasion in pancreatic stem cell properties of HepG2, Huh-7, and MHCC97H cell lines ductal adenocarcinomas (PDAC). Precisely, the authors demon- by targeting the 30-UTR of SMAD2 protein and decreasing its strated that in PDAC, FOXM1 is a direct target of TGFb-induced expression and function (56). In gastric cancer, miR-424-5p was miR-494, and miR-494 affects FOXM1 expression through a direct found to target and downregulate SMAD3 and promote the 0 interaction with FOXM1 3 -UTR (51). proliferation of gastric cancer cells (56). In addition to the SMAD proteins, miRNAs regulate the TGFb pathway via direct targeting miR-34a and controlling the expression of either TGFb type I (TGFb RI) or The miR-34a belongs to the miR-34 family, which consists of type II (TGFb RII) receptors in cancers such as anaplastic thyroid miR-34a, miR-34b, and miR-34c. In hepatitis B virus (HBV)– carcinoma (ATC), glioblastoma, and non–small cell lung carci- positive HCC, a study by Yang and colleagues demonstrated that noma (NSCLC). For detailed discussions on miRNA-mediated TGFb-mediated suppression of miR-34a, possibly via the auto- regulation of the TGFb pathway via targeting either, one of the crine activity of the TGFb produced by HepG2 cells, enhanced SMAD proteins or TGFb receptors in cancer, we direct the readers production of the chemokine CCL22 and promoted tumor to the review in ref. 56.

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TGFb, LncRNAs and EMT (64). More precisely, based on the expression level of n ¼ LncRNAs are ncRNA molecules greater than 200 nucleotides in lncRNA-ATB, patients ( 183) were divided into high lncRNA n ¼ n ¼ length and can control gene expression via transcriptional and ATB group ( 105) and low lncRNA ATB group ( 83). The posttranscriptional regulatory mechanisms (57). Recent evidence expression level of miR-200c and ZEB1 was elevated in the high suggests that lncRNAs play a crucial role in embryogenesis, lncRNA ATB group compared with the low lncRNA ATB group, b cellular development, tumorigenesis, and EMT (58, 59). As a and the treatment of gastric cancer cell lines with TGF resulted in major inducer of EMT in several systems, the TGFb pathway has the upregulation of lncRNA ATB and ZEB1, and downregulation also been shown to regulate several lncRNAs involved in EMT of miR-200c and CDH1. More recently, Yue and colleagues (Table 1 and Fig. 2). reported that lncRNA ATB was upregulated both in colon cancer and metastatic colon cancer tissues. In addition, relatively higher Hotair levels of lncRNA ATB and concurrent low levels of E-cadherin were Hotair is a member of the subclass of lncRNA called large also observed recently in three highly invasive colon cancer cell intergenic noncoding RNAs (lincRNA) and primarily is known for lines (65). Importantly, reduction of lncRNA ATB expression its ability to regulate epigenetic states through the recruitment of increased the expression of epithelial markers E-cadherin and chromatin-modifying complexes to specific target sequences (60). ZO-1 and decreased expression of mesenchymal markers ZEB1 Hotair expression is associated with breast cancer metastasis and and N-cadherin (N-cad) in these colon cancer cell lines (65). poor outcomes in several neoplasia. Padua and colleagues dem- HULC onstrated that TGFb treatment increased the expression of Hotair in both colon and breast cancer cells, required for EMT. The Highly upregulated in liver cancer (HULC), is a 500nt long authors observed that overexpression of Hotair in cancer cells lncRNA located on chromosome 6p24.3. Very recently, HULC was b upregulated EMT-associated genes such as ZEB1, SNAI1, TWIST, reported to promote TGF -induced EMT in HCC via reduction of CTNNA1 (b-catenin), including the mesenchymal markers E-Cadherin expression, and increased expressions of N-Cadherin, fi vimentin (VIM) and fibronectin (FN1; ref. 61). Although the Snail, and ZEB1 in SMMC-7721 cancer cells (66). Speci cally, authors demonstrated a positive correlation between TGFb treat- HULC-mediated promotion of EMT occurred through the seques- tration of miR-200a-3p, which positively regulates E-Cadherin ment and upregulation of Hotair, and induction of EMT, the 0 mechanism involved in the TGFb mediated upregulation of and negatively regulates ZEB1 by binding to their 3 -UTR. Hotair was not addressed in the study. Although the authors demonstrated that HULC functioned as ceRNA to upregulate ZEB1 by sequestering miR-200a-3p, the MALAT1 molecular mechanism by which TGFb affected the expression Metastasis-associated lung adenocarcinoma transcript 1 level of HULC was not discussed in the study. (MALAT1) is a lncRNA that was recently reported to be involved in bladder cancer cell migration, and its expression was found to Linc01186 be significantly increased in primary tumors that became subse- LINC01186 was originally identified in lung cancer tissues, as quently metastatic compared with those that did not (62). one of the 291 lncRNAs differentially expressed between lung Furthermore, downregulation of MALAT-1 resulted in impaired cancer tissues and adjacent normal tissues (67). Interestingly, bladder cancer cell migration and inhibited the EMT process, LINC01186 expression was found to be significantly downregu- which was associated with a decrease in ZEB1, ZEB2, and Slug lated in TGFb1-treated A549 lung cancer cells. The authors dem- expression levels (62). Importantly, a recent report by Fan onstrated that overexpression of LINC01186 in A549 cells, inhib- and colleagues demonstrated the underlying mechanisms of ited migration, and the invasive capacity of lung cancer cells (67). TGFb-induced MALAT1-mediated regulation of cancer metastasis The study also reported that SMAD3 repressed LINC01186, in bladder cancer (63). The authors showed that TGFb treatment indicating that LINC01186 is a downstream target of SMAD3 in induced MALAT1 expression by a yet unidentified mechanism, A549 lung cancer cells. Thus, the authors established a critical role which was followed by reduction of E-cadherin mRNA and for LINC01186 (a previously uncharacterized lncRNA) in TGFb- protein levels in RT4 bladder cancer cells. Of note, the authors induced EMT, in a model of lung cancer. also reported a significant negative correlation between the mRNA levels of E-cadherin and malat1 expression levels in vivo. On the LincRNA PNUTS other hand, mRNA and protein levels of mesenchymal markers We recently reported that a novel lncRNA PNUTS controlled by N-cadherin and fibronectin were also increased by TGFb treat- RBP hnRNP E1 is overexpressed in breast cancer tissues (68). In ment, but suppressed by malat1 silencing, indicating the induc- this report, Dr. Howe and colleagues demonstrate that in response tion of EMT by TGFb and involvement of malat1 in TGFb-induced to TGFb, hnRNP E1 promotes alternative splicing and generates EMT in bladder cancer. Interestingly, the authors also revealed the noncoding lncRNA PNUTS. The lncRNA-PNUTS serves as a that MALAT1 represses E-cadherin expression by associating with competitive sponge for miR-205 during EMT and appears to be suz12, a member of the polycomb repressive complex 2 (PRC2). tightly regulated by hnRNP E1 and tumor context. Thus, TGFb Also, the authors used RNA immuneprecipitation (RIP) assay cooperatively controls both RBP and lncRNA to modulate EMT. with antibodies against EZH2 and SUZ12 and demonstrated that lncRNA MALAT1 association with SUZ12 but not EZH2. LncRNA-PE In a screen for lncRNAs that might promote EMT and HCC LncRNA ATB progression, Shen and colleagues identified a 1454-bp long Long noncoding RNA activated by TGFb (lncRNA ATB) was lncRNA called lncRNA-PE (originally named as BC013423) in found to promote invasion and metastasis in gastric cancer HCC cells (69). In addition, TGFb treatment upregulated the through the TGFb/miR-200s/ZEB axis leading to poor prognosis expression of lncRNA-PE and lncRNA-PE in turn induced EMT in

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Figure 3. TGFb modulated miRNAs and lncRNAs in cancer. TGFb-upregulated miR-21 inhibits cancer cell apoptosis via posttranscriptional repression of proteins such as MSH2 and PDCD4, which are known to induce apoptosis, whereas miR-494 upregulated by TGFb promotes proliferation of cancer cells. TGFb-downregulated miR-34a also promotes cancer cell proliferation. TGFb-upregulated lncRNAs have been shown to play opposing roles in cancer cell apoptosis. For example, lnc-ROR promotes apoptosis whereas lncRNA- Smad7 inhibits apoptosis of cancer cells. LncRNA-HOX-D AS1 upregulated by TGFb-activated PI3/Akt promotes angiogenesis. miRNAs are denoted by blue ovals, black arrow and lines denote miRNA-mediated regulations of target genes, and lncRNAs are denoted by red rectangles, and red arrows and lines denote lncRNA-mediated control of the EMT process. Transcription factors are denoted by yellow rounded rectangles and canonical TGFb pathway members such as Smads and noncanonical TGFb members such as PI3/Akt, JNK, and ERK1/2 are denoted by light blue or yellow rectangles and light green, respectively.

SK-Hep-1 HCC cells. More precisely, siRNA-mediated knock- reported that lncRNA-ROR, a stress-responsive and TGFb-induced down of lncRNA-PE in SK-Hep1 cells reduced the expression of lncRNA, promoted chemo resistance of HCC cancer cells via þ N-cadherin and vimentin, whereas enhanced the protein levels of CD133 tumor-initiating cells (70). Importantly, TGFb was E-cadherin and ZO-1, consequently inhibiting HCC cell migra- found to reduce the chemosensitivity of HCC cells to the drugs tion and invasion (69). In search of a mechanistic insight into the sorafenib or doxorubicin and increased the expression of lnc- role of lncRNA-PE in the regulation of HCC associated EMT, the ROR, consequently reducing the chemotherapy induced cell authors demonstrated that lncRNA-PE enhanced ZEB1 expression death of HCC cells. In this study, although the authors have not via downregulation of miR-200a/b. Importantly, an analysis of demonstrated a direct association of lncRNA-ROR with its the distribution of lncRNA-PE in SK-Hep-1 cells revealed that it target genes, they showed that siRNA mediated knockdown of was localized both in the nucleus and the cytoplasm, indicating lncRNA-ROR in HepG2 cells signiﬁcantly increased the expres- that lncRNA-PE might function as a miRNA sponge. Thus, sion of apoptosis associated genes caspase 8 and p53. Thus, the lncRNA-PE might play a crucial role in the development of HCC authors speculated that the effects of lncRNA-ROR in HCC could via the miR-200a/b-ZEB1 pathway. be mediated through p53 signaling (70).

TGFb, LncRNAs, and Cancer LncRNA-Smad7 LncRNA-ROR In epithelial cells, TGFb has been shown to exhibit both The lncRNA-ROR is a 2.6-kb long and is one of the most proapoptotic and antiapoptotic effects. To study the down- signiﬁcantly upregulated lncRNA in HCC. Recently, it was stream regulatory mechanisms governing the TGFb-mediated

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Figure 4. LncRNAs can compete with or act as decoys for RBPs and RNAs. ia–ic, Long noncoding RNAs can be generated from (ia) intragenic, (ib) intergenic, (ic) or naturally occurring antisense sequences. In the nucleus, (ii) lncRNAs can bind to transcription factors, TFs, preventing speciﬁc gene transcription. In the cytoplasm, (iiia) lncRNAs can compete with miRNAs for mRNA binding sites or (iiib) or act as miRNA decoys affecting target mRNA stability and translation. Additionally, (iiic) lncRNAs can compete with or act as decoys for RBP regulation of mRNA fate. Lastly, (iv) naturally occurring antisense lncRNAs can regulate the stability and translation of the coding mRNA transcripts from which it was derived.

antiapoptotic functions in breast cancer, Arase and colleagues apoptosis (71). The identity of lncRNA-SMAD7 antiapoptotic conducted RNA sequencing in NmuMG cells and identiﬁed the targets is currently unknown. antiapoptotic lncRNA-Smad7 as a target of TGFb (71). LncRNA- Smad7 is located adjacent to the mouse Smad7 gene, found to HOXD-AS1 be induced by TGFb, and displays antiapoptotic functions in The HOXD-AS1 is a lncRNA located in the HOXD cluster, NMuMG and mouse breast cancer cell line JygMC (A). Inter- between HOXD1 and HOXD3 genes, and it is evolutionarily estingly, silencing lncRNA-Smad7 did not alter TGFb-induced conserved among hominids (72). In a model of human metastatic EMT or expression of Smad7 gene, suggesting that lncRNA- neuroblastoma, HOXD-AS1 was activated by TGFb induction of Smad7-mediated TGFb functions may be restricted only to the PI3K/Akt pathway (72). Moreover, knockdown of HOXD-AS1

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decreased the expression of several protein-coding genes associ- addition, there is a significant knowledge gap in TGFb signaling ated with angiogenesis and inflammation, indicating that HOXD- mediated control of PTR in the tumor microenvironment and AS1 controls these processes through regulation of the expression associated tumorigenesis. of its target genes (ref. 72; please see Fig. 3). Additional major consideration is whether TGFb-initiated changes in gene expression patterns are coupled with co- and Outlook posttranscriptional changes during RNA processing. This interro- gation is crucial because TGFb promotes the expression of several The changes in gene expression and altered transcription under transcription factors (75), which could serve as modifiers of TGFb signaling are well established (73); however, TGFb signaling transcriptional changes enhance the expression of ncRNAs and mediated posttranscriptional mechanisms that control pre- coding RNAs. One could assume establishment of the noncoding mRNA splicing, export, turnover, and translation are poorly (intron or intergenic) transcript variant can act as a decoy or understood. Understanding TGFb-mediated posttranscriptional competitor for the same or different coding transcript (Fig. 4). alterations in cancer may provide avenues for better therapeutic Advances in genome sequencing, single-cell transcriptomics, and opportunities. Several studies have delineated the role of TGFb- bioinformatic tools for investigating RNA expression are needed mediated miRNAs in cancer and discovered the possible mechan- to dissect this pathway. isms underlying the interaction between TGFb and miRNAs. Finally, as TGFb signaling is strongly linked with metastasis, However, TGFB-mediated changes in lncRNAs and their contri- the RBPs, mRNAs, miRNAs, and lncRNAs associated with this bution to cancer progression and metastasis remains understu- process will allow us to identify several metastatic biomarkers. died. Therefore, by better understanding of TGFb-mediated With better knowledge of TGFb-driven posttranscriptional changes in lncRNAs may provide an opportunity to modulate changes that target tumor progression and metastasis, the RBPs the expression of target genes and serve as a strategy for the or RNA molecules that are involved in this process can be treatment of cancer. On the other hand, silencing ncRNAs may reengineered to fabricate novel drugs. A powerful search for have deleterious effects in cancer, which leads to overexpression of posttranscriptional level effects of TGFb on cancer cell progres- oncogenes or tumor-promoting effects. For such scenario, altering sion and metastasis is warranted, as cancer cells constantly the expression of these ncRNAs or downregulating the expression evolve for multidrug resistance and reoccurrence. Last but not of target genes may have beneficial effects. Thus, by understanding least, a successful development of targeting TGFb pathway the molecular and epigenetic mechanisms, underlying the rela- members and its contribution to the control of single or tionship between TGFb and lncRNAs in cancer and their role in multiple RBPs, miRNAs, and lncRNAs would be ideal to mech- gene expression may facilitate the development of new therapeu- anistically understand the PTR and cancer progression. tic strategies targeting the tumor. Using high-throughput transcriptomics and proteomics fl approach, one can identify novel TGFb signaling mediated RNA Disclosure of Potential Con icts of Interest fl transcripts and proteins altered at the posttranscriptional level. No potential con icts of interest were disclosed. How the RNA transcripts are selectively regulated by TGFb signaling is largely unknown, except HNRNP E1, which has been Acknowledgments reported to utilize mRNA translation machinery (74). Although This study was supported by the grants from NIHDE022776 and several attempts have been made to address TGFb signaling DE025920 to V. Palanisamy, CA154664 and CA555536 to P.H. Howe, b CA11360 to J.A. Diehl, and EY023427 to V. Gangaraju.Supported in part mechanism, more in-depth understanding of TGF signaling by pilot research funding, Hollings Cancer Center's Cancer Center Support mediated pre-mRNA processing is needed. For example, identi- Grant P30 CA138313 at the Medical University of South Carolina. fying a regulatory loop involving TGFb, RBPs and ncRNAs will We sincerely apologize to colleagues whose work has been overlooked from uncover innovative tools to target TGFb-mediated cellular transi- this review owing to space limitations. tions from normal to benign and cancer. Likewise, the TGFb signaling mediated posttranslational modifications of RBPs and Received September 27, 2017; revised October 31, 2017; accepted December their impact on gene expression patterns are largely unknown. In 18, 2017; published first March 19, 2018.

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The Long (lncRNA) and Short (miRNA) of It: TGFβ-Mediated Control of RNA-Binding Proteins and Noncoding RNAs

Harinarayanan Janakiraman, Reniqua P. House, Vamsi K. Gangaraju, et al.

Mol Cancer Res Published OnlineFirst March 19, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-17-0547

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