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LIN28B Fosters Colon Cancer Migration, Invasion and Transformation Through Let-7-Dependent and -Independent Mechanisms

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LIN28B Fosters Colon Cancer Migration, Invasion and Transformation Through Let-7-Dependent and -Independent Mechanisms Oncogene (2011) 30, 4185–4193 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE LIN28B fosters colon cancer migration, invasion and transformation through let-7-dependent and -independent mechanisms CE King1,3,LWang1,RWinograd1,BBMadison1,3,PSMongroo1,3, CN Johnstone1 and AK Rustgi1,2,3 1Gastroenterology Division and Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 2Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA and 3Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Lin28b is an RNA-binding protein that inhibits biogenesis as inhibition of let-7 microRNA biogenesis is a cardinal of let-7 microRNAs. LIN28B is overexpressed in diverse feature of their functions. cancers, yet a specific role in the molecular pathogenesis The let-7 family of microRNAs comprises isoforms of colon cancer has to be elucidated. We have determined with highly conserved sequences that exhibit functional that human colon tumors exhibit decreased levels of redundancy (Pasquinelli et al., 2000; Zhao et al., 2010). mature let-7 isoforms and increased expression of Let-7 biogenesis resembles that of most microRNAs, in LIN28B. To determine LIN28B’s mechanistic role in which the various isoforms are transcribed initially by colon cancer, we expressed LIN28B in immortalized RNA polymerase II as pri-microRNAs, and processed colonic epithelial cells and human colon cancer cell lines. by Drosha and DGCR8 into pre-microRNAs that are We found that LIN28B promotes cell migration, invasion subsequently exported from the nucleus (Lee et al., and transforms immortalized colonic epithelial cells. 2002, 2003; Gregory et al., 2004; Han et al., 2004). In addition, constitutive LIN28B expression increases The hairpin loops of pre-microRNAs are cleaved by expression of intestinal stem cell markers LGR5 and dicer in the cytoplasm to yield microRNA:microRNA PROM1 in the presence of let-7 restoration. This may duplexes that are disassociated to release mature let-7 occur as a result of Lin28b protein binding LGR5 and (Lee et al., 2002, 2003). MicroRNAs are incorpo- PROM1 mRNA, suggesting that a subset of LIN28B rated into the RNA-induced silencing complex and functions is independent of its ability to repress let-7. Our bind the 30 untranslated region (UTR) of target tran- findings establish a new role for LIN28B in human colon scripts to provide post-transcriptional gene regulation cancer pathogenesis, and suggest LIN28B post-transcrip- by mRNA sequestration or cleavage (Esquela-Kerscher tionally regulates LGR5 and PROM1 through a let-7- and Slack, 2006). independent mechanism. Several established let-7 mRNA targets have tumor- Oncogene (2011) 30, 4185–4193; doi:10.1038/onc.2011.131; promoting properties, including the canonical target published online 30 May 2011 HMGA2 (Lee and Dutta, 2007; Mayr et al., 2007; Park et al., 2007) and the classic oncogenes KRAS and Keywords: LIN28B; LIN28; Let-7; colon cancer; LGR5; c-MYC (Johnson et al., 2005, 2007; Akao et al., 2006). PROM1 In addition, let-7 microRNAs have been described as tumor suppressors and are implicated as prognostic factors in a variety of divergent cancers (Takamizawa Introduction et al., 2004; Akao et al., 2006; Shell et al., 2007). Importantly, Lin28 and Lin28b may relieve let-7 target LIN28B is a homolog of LIN28 (also called LIN28A) suppression by binding to immature let-7 molecules and (Guo et al., 2006), which induces pluripotency in blocking further processing (Heo et al., 2008, 2009; somatic cells when expressed in concert with KLF4, Hagan et al., 2009). SOX2 and NANOG (Yu et al., 2007). A high degree of In approximately two-thirds of colon cancers evalu- homology exists between LIN28B, LIN28 and the ated, we find that let-7 microRNA levels are decreased heterochronic gene lin-28 in C. elegans (Moss and Tang, when compared with adjacent normal colonic mucosa 2003). Human LIN28 and LIN28B each contain a (unpublished observations). Interestingly, pri-let-7 levels cold-shock domain and CCHC zinc fingers that confer are maintained in colon cancer cells that display reduced RNA binding ability (Moss and Tang, 2003). The ability mature let-7 levels, suggesting a post-transcriptional- to bind RNA is critical to both Lin28 and Lin28b, mediated mechanism of let-7 downregulation. The regulator of let-7 biogenesis LIN28B, also a let-7 target Correspondence: Dr AK Rustgi, Division of Gastroenterology, (Boyerinas et al., 2008), is specifically implicated in this Department of Medicine, Department of Genetics, Abramson Cancer process because it is transactivated by c-myc (Chang Center, University of Pennsylvania, 415 Curie Boulevard, 600 CRB, et al., 2009). Nearly 70% of colorectal tumors harbor Philadelphia, PA 19104, USA. E-mail: [email protected] elevated levels of c-myc (Erisman et al., 1985); upregula- Received 16 August 2010; revised 14 March 2011; accepted 16 March tion occurs in the early stages of colon carcinoma as a 2011; published online 30 May 2011 consequence of Wnt pathway deregulation and b-catenin LIN28B fosters colon cancer migration through let-7 CE King et al 4186 stabilization (Stewart et al., 1986; Sikora et al., 1987; colon adenocarcinomas paired with adjacent normal Powell et al., 1992; Rubinfeld et al., 1993; He et al., colonic mucosa by measuring levels of mature let-7a and 1998; Clevers, 2006). let-7b. Of the 22 pairs assayed, 10 tumors displayed We hypothesized that LIN28B promotes colon reduced levels (greater than 60% reduction) of let-7a tumorigenesis via suppression of let-7. To test this and let-7b compared with their normal colonic epithe- hypothesis, we constitutively expressed LIN28B in lium counterpart (Figure 2a; Table 1). Initially, we immortalized colonic epithelial cells and human colon surmised this variation in levels of mature let-7 was cancer cell lines. We found that LIN28B expression attributable to differences in expression levels. Yet, increases cell migration, invasion and soft-agar colony qPCR for the let-7a-3-b cluster pri-microRNA sequence formation. The ability of constitutive LIN28B expres- revealed similar expression in tumors (data not shown). sion to promote migration and invasion is partially Alternatively, post-transcriptional regulatory mechan- reversed by concomitant let-7 expression, suggesting isms may account for let-7a and let-7b downregulation that these phenotypes are dependent on Lin28b’s ability in tumors. As LIN28B may be transactivated by c-myc, to repress let-7 biogenesis. Notably, we also found that which is frequently upregulated in colon cancer, we the intestinal/colonic stem cell markers LGR5 and hypothesized that LIN28B overexpression occurs in PROM1 are upregulated with constitutive LIN28B colon tumors results in inhibition of mature let-7 expression. LGR5 and PROM1 do not contain putative biogenesis. Consequently, we examined LIN28B expres- let-7 binding sites in their 30 UTR, and are not predicted sion in colon tumors, and found that LIN28B transcript let-7 targets. LGR5 and PROM1 remain upregulated levels are increased in a subset of tumors when even in the presence of let-7 restoration in LIN28B- compared with adjacent normal colonic epithelium expressing cells. Mechanistically, LGR5 and PROM1 (Figure 2b; Table 1). Furthermore, LIN28B expression transcripts are enriched in Lin28b mRNA-binding negatively correlates with levels of mature let-7a assays. Furthermore, Lin28b induces LGR5 and (r ¼À0.47, P ¼ 0.0297) and let-7b (r ¼À0.41, P ¼ 0.0637) PROM1 30 UTR sequences in luciferase reporter in colon tumors. assays. These data suggest that Lin28b modulates these genes in a let-7 independent manner, which is a novel LIN28B transforms immortalized colonic epithelial cells finding. Taken together, our data demonstrate that and promotes tumorigenesis Lin28b promotes migration, invasion and transforma- To assess the potential oncogenic functions of Lin28b in tion, while upregulating stem cell genes through mRNA the colon, we constitutively expressed human LIN28B in binding. immortalized intestinal epithelial (IEC-6) via retroviral transduction (Figure 3a). Human Lin28b expression in IEC-6 cells reduces levels of mature let-7 isoforms Results (Figure 3b). However, cellular proliferation rates were not affected (data not shown). We further examined cell Reduced levels of mature let-7a and let-7b microRNAs behavior using in vitro transwell migration and invasion and increased LIN28B expression in colon tumors assays, and found that Lin28b promoted both invasion We measured levels of mature let-7 microRNA isoforms and migration of IEC-6 cells in culture (Figure 3c). in four samples of human normal colonic epithelia via Additionally, IEC-6 cells, which do not form colonies in qPCR. We found that let-7a and let-7b are the soft agar, do so in the presence of constitutive LIN28B predominant let-7 isoforms present in the normal colon expression (Figure 3d). These observations also extend (Figure 1). Subsequently, we interrogated 22 human to human colon cancer cell lines. Constitutive expression of LIN28B in DLD-1 (data not shown) and LoVo colon cancer cells reduces mature let-7a and let-7b levels (Figures 4a and b). Furthermore, Lin28b increases migration, invasion (Figure 4c) and soft-agar colony formation in colon cancer cell lines (Figure 4d). Let-7 expression partially abrogates phenotype induced by LIN28B constitutive expression We sought to determine whether the increase in migration and invasion induced by constitutive LIN28B expression in colon cancer cell lines was let-7 dependent. To that end, we co-expressed a decoy let-7a hairpin molecule in cells constitutively expressing LIN28B. The loop structure of this miRNA is derived from mir-30, Figure 1 Expression of let-7 isoforms in isolated human colonic thereby eliminating the Lin28b binding site and allowing epithelia. Levels of mature let-7 microRNA family members were these let-7 precursors to evade Lin28b-mediated inhibi- measured in four isolated normal human colonic epithelia via qPCR. Levels of mature microRNAs are depicted relative to let-7a.
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