Human tumors display a global reduction in miRNA expression^1,2. Inhibition of the miRNA processing pathway via knockdown of miRNA processing enzymes promotes cellular transformation and tumorigenesis; this effect can be attenuated by ectopic expression of let-7g, suggesting that inhibition of let-7 processing is likely to be central to promoting the transformed state³. Low levels of let-7 correlate with increased transformation capacity in vitro³, and have been observed in various malignancies, including lung cancer,^9,10 hepatocellular carcinoma¹¹, melanoma¹², and ovarian cancer¹³. Over-expression of let-7 represses cellular proliferation pathways, inhibits cell growth, and impairs tumor development in both xenograft models and in an autochthonous model of non-small cell lung cancer^3,14,15,16. Copy number loss^3,17–19 and epigenetic silencing²⁰ of individual let-7 family members have been reported in human cancers, and such events have been suggested to contribute to the disease state. However, in tissues where let-7 family members are co-expressed, inactivation of individual let-7 miRNAs would be expected to only modestly decrease let-7 dosage, given the functional redundancy among family members. Therefore, we hypothesized that activation of LIN28/LIN28B could serve as a physiological mechanism by which levels of all let-7 family miRNAs are coordinately repressed in the context of human malignancy.

We initially sought to determine whether over-expression of Lin28 could promote cellular transformation in experimental settings. We observed that over-expression of Lin28 in NIH/3T3 cells potently depletes levels of multiple mature let-7 family member miRNAs (Fig. 1a), leading to a concomitant increase in protein levels of c-Myc, a let-7 target (Fig. 1b). NIH/3T3 cells overexpressing Lin28 formed colonies in soft agar (Fig 1c–d) at a frequency comparable to BCR-ABL (Fig. S1) and slightly lower than that reported for c-Myc (1–2%), both of which are weakly transforming in this assay²¹. Expression of Lin28 together with BCR-ABL resulted in an increase in both colony size and number as compared with either Lin28 or BCR-ABL alone (Fig. S1). NIH/3T3 cells expressing either Lin28 or LIN28B formed tumors in nude mice (Fig. S1b and Table S1). Although the tumors were small and of delayed latency, they displayed evidence of local invasion (Fig. S1b), similar to what has been observed upon global inhibition of miRNA processing³, and consistent with a recent report that Lin28 enhances metastasis²². Co-expression of Lin28 and 7S21L (a let-7 loop mutant capable of being processed to mature let-7 in the presence of Lin28²³) abrogated the ability to form colonies in semisolid medium (Fig 1c–d); thus, Lin28 transforms NIH/3T3 cells via the de-repression of let-7 target genes. We also observed that Lin28 over-expression enhanced transformation of a mouse lung adenocarcinoma cell line in which impairment of miRNA processing had been previously reported to be transforming³ (Fig. S2).

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Figure 1

Lin28 Transforms NIH/3T3 cells

(A) Levels of mature miR species in representative infection determined by quantitative PCR in NIH/3T3 cells infected with pMSCV.Neo or pMSCV.Neo.Lin28, and selected on G418. (B) Western blot analysis of extracts from NIH/3T3 cells infected with MSCV.Neo/pBabe.Puro, pMSCV.Neo.Lin28/pBabe.Puro, or pMSCV.Neo.Lin28/pBabe.Puro.7S21L, selected with Puromycin and G418. (C) Levels of mature let-7g in NIH/3T3 cells infected with MSCV.Neo/pBabe.Puro,pMSCV.Neo.Lin28/pBabe.Puro, or pMSCV.Neo.Lin28/pBabe.Puro.7S21L, selected with Puromycin and G418. (D) Colony numbers in semisolid media for 25,000 NIH/3T3 cells infected with pMSCV.Neo/pBabe.Puro, pMSCV.Neo.Lin28/pBabe.Puro, or pMSCV.Neo.Lin28/pBabe.Puro.7S21L, selected with Puromycin and G418. Colonies were counted after 4 weeks with five random fields counted per well. Results are plotted as average colony number +/− S.E.M., N=3. (E) Western blot analysis on cell extracts from Ba/F3 cells infected with pMSCV.Neo and pMSCV.Neo.Lin28, selected on G418. (F) Levels of mature miR species in representative infection determined by quantitative PCR in Ba/F3 cells infected with pMSCV.Neo or pMSCV.Neo.Lin28, and selected on G418. (G) Western blot analysis on cell extracts from Ba/F3 cells infected with pEYK.Puro.SrcY530F and either pMSCV.Neo or pMSCV.Neo.Lin28 and selected on puromycin and G418 (H) Ba/F3 lines established in (C) were plated in media without IL-3 at 10,000 cells per well. Cells were stained with Trypan blue and viable cells were counted 6d after plating. (I) Colony formation of Ba/F3 cells expressing Src/Y530F or Src/Y530F + Lin28 in semisolid medium, plated at 50,000 cells per well in medium without IL-3. Quantitation of colony number from soft agar assay after 21d of growth. Results are plotted as average colony number per well +/− S.E.M., N=3. (J) Survival of immunocompromised mice injected with 10⁷ Ba/F3 cells expressing Src-Y530F or Src-Y530F+Lin28. Average survival time 51.4d vs. 33.2d vs. respectively, p=0.00276 (K) Western blot analysis of extracts from Ba/F3 cells infected with pBabe.Puro or pBabe.Puro.Lin-28, selected on Puromycin. (L) Ba/F3 lines established in (C) were plated in media without IL-3 at 10,000 cells per well. Cells were stained with Trypan blue and counted 3d after plating. (M) Quantitation of colony number from soft agar assay. Results are plotted as average colony number per well +/− S.E.M., N=3. *, p<0.05; **, p<0.01.

We next tested Lin28 for its ability to transform cytokine-dependent BaF/3 cells to factor independence. Over-expression of Lin28 in BaF/3 cells potently depleted mature let-7 family members and led to the upregulation of the let-7 targets K-Ras and c-Myc (Fig. 1e–f). Co-expression of Lin28 and a weakly transforming variant of c-SRC (SRC/Y530F) conferred factor independence upon Ba/F3 cells (Fig. 1g–h) and strongly promoted growth in semisolid medium (Fig. 1i). Mice injected with Ba/F3 cells co-expressing Lin28 and Src/Y530F succumbed to leukemic disease more rapidly than mice injected with Ba/F3 cells expressing Src/Y530F alone (Fig. 1j). Lin28 cooperated similarly with a weakly transforming variant of c-Abl (Abl/G2A) to transform Ba/F3 cells (Fig. 1k–m). Notably, BCR-ABL, v-abl, and v-src have all been suggested to transform lymphoid cells via a similar pathway²⁴. Together, these experiments demonstrate that over-expression of Lin28 facilitates cellular transformation in multiple in vitro assays and accelerates disease progression in a murine model.

To determine whether LIN28 and LIN28B are aberrantly expressed in the setting of human cancer, we examined LIN28 expression by microarray analysis of a large panel of human cancer cell lines; we observed expression in 3.2% of cell lines (17/527; Fig. S3a), including lines derived from germ cell tumors, Wilms’ tumor (WT), and hepatocellular carcinomas (HCC). Analysis of microarray data available for the National Cancer Institute panel of 60 cell lines (NCI-60) revealed LIN28 expression in 5% (3/60) and LIN28B expression in 10% (6/60) of the lines (Fig. S3b and Table S2). We also performed quantitative PCR analysis on a select panel of cancer cell lines and found several that express LIN28B, including multiple cell lines derived from patients in the blast crisis phase of chronic myeloid leukemia (CML) (Table S2). Examination of LIN28 expression in a range of primary tumor types by immunohistochemistry on a multi-tumor tissue microarray revealed that LIN28 was highly overexpressed in diverse tumor types including colon cancer, breast cancer, lung cancer, and cervical cancer (Fig. S4 and Table S3). Together, these data show that LIN28/LIN28B are aberrantly expressed in ~15% of surveyed cancer samples.

We surmised that activation of LIN28/LIN28B could represent a physiologic means by which let-7 levels are coordinately repressed in a subset of human cancers, and that over-expression of these factors might modify disease severity. Given that we observed over-expression in cell lines from patients with CML, we examined levels of LIN28 and LIN28B in peripheral blood from patients in either chronic phase (CP) or blast crisis (BC) of CML. LIN28 expression was detected more frequently in patients with BC-CML (42.8%, 6/14) or accelerated phase CML (40%, 4/10) than in patients with CP-CML (9.1%, 1/11) (Fig. 2a). Furthermore, analysis of 13 paired CP-BC samples showed activation of LIN28B upon progression to BC in three patients (23%, 3/13) (Table S4). Therefore LIN28/LIN28B expression is associated with disease progression in a subset of CML patients. Interestingly, we observed that expression of HMGA2, a let-7 target gene, shows a similar relationship with disease progression in CML, as has also been reported elsewhere²⁵ (Fig. S5). This supports the notion that LIN28/LIN28B activation drives progression in CML via let-7 pathways. To determine whether cell lines derived from patients with BC-CML might be dependent on LIN28/LIN28B for growth, we inhibited LIN28B expression in K562 cells by RNA interference. Infection of K562 cells with an shRNA against LIN28B restored levels of mature let-7 miRNAs (Fig. 2b–c), decreased levels of c-MYC (Fig. 2d), impaired proliferative capacity and the ability to form colonies in soft agar (Fig. 2e–f), and resulted in morphological changes suggestive of differentiation (Fig. 2g)²⁶. Similar results were obtained in a second CML-BC cell line and in a lung adenocarcinoma cell line (Fig. S6–Fig. S7), both of which express LIN28B.

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Figure 2

LIN28B Knockdown Impairs Growth and Triggers Differentiation of K562 Cells

(A) Lin28 expression as determined by quantitative PCR in peripheral blood from patients in either chronic phase, accelerated phase, or myeloid blast crisis. (B) Levels of LIN28B expression determined by quantitative PCR in K562 cells infected with pLKO.controlshRNA or pLKO.LIN28BshRNA, and selected on Puromycin. (C) Levels of mature miR species determined by quantitative PCR in K562 cells were infected with pLKO.controlshRNA or pLKO.LIN28BshRNA, and selected on Puromycin. (D) Western blot for K-Ras and c-Myc on cell extracts from K562 cells infected with pLKO.controlshRNA or pLKO.LIN28BshRNA and selected on Puromycin. (E) Cell proliferation of K562 cells infected with pLKO.controlshRNA or pLKO.LIN28BshRNA, selected on Puromycin, and seeded at 10000 cells per well. Results are plotted as average cell number per well +/− S.E.M. N=3. (F) Colony formation for K562 cells infected with pLKO.controlshRNA or pLKO.LIN28BshRNA and plated in semisolid medium. 5000 cells were plated per well and colonies were counted after 21d. (G) Wright-Giemsa stain on cytospin preparation of K562 cells infected with pLKO.controlshRNA or pLKO.LIN28BshRNA, selected on Puromycin. *, p<0.05; **, p<0.01.

We next sought to determine whether LIN28/LIN28B might be similarly associated with advanced disease in HCC. We performed genome-wide mRNA and microRNA profiling on 89 HCC samples. LIN28B was highly expressed in a subset of HCCs, and these tumors displayed coordinate repression of all let-7 family miRNAs (Fig. 3a). Gene set enrichment analysis revealed that expression of let-7 target genes and myc target genes were significantly enriched in LIN28B-expressing tumors (Fig. 3b), suggesting that LIN28B represses let-7 and functionally activates let-7 targets in the setting of HCC. Tumors with high LIN28B expression accumulated in a molecular subclass defined by a meta-analysis of published transcriptome datasets (YH and TRG, data not shown), which is characterized by high serum alpha-fetoprotein level, higher tumor grade, and c-MYC activation (p=0.006, Fisher’s exact test). Consistently, LIN28B expression was observed in 66% (4/6) of tumors from patients with serum alpha-fetoprotein (AFP) > 10,000 ng/ml as compared to 16.3% (15/92) (p=0.00981, Fisher’s Exact Test). LIN28B-expressing tumors also fell predominantly within a subclass characterized by a proliferative nature as defined by unsupervised clustering²⁷ (p=0.004), and most LIN28B-expressing tumors were of advanced stage (Fig. 3d). Moreover, LIN28B expression was associated with a significantly increased incidence of early recurrence (within 2 years of resection, p=0.02, log-rank test), but was not associated with late recurrence (Fig. 3c). Early recurrence is an indicator of malignant disease while late recurrence is thought to occur as a de novo event from the carcinogenetic microenvironment of the cirrhotic liver²⁸. Knockdown of LIN28B in HepG2 cells (an HCC cell line expressing LIN28B) led to a marked decrease in the number of colonies formed in semisolid medium (Fig. 3e). Together, these results demonstrate that LIN28B is associated with advanced disease and poor clinical outcome in HCC.

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Figure 3

LIN28B is associated with poor outcome in hepatocellular carcinoma

(A) Expression of let-7 family miRNAs measured as part of genome-wide microRNA profile in 89 human HCC Tumor samples were ordered according by decreasing expression level of LIN28B as measured on an mRNA expression microarray on the same samples. (B) Induction of experimentally-defined MYC target gene signatures (left and middle) and let-7 target gene expression (right) was evaluated in LIN28B-high tumors using Gene Set Enrichment Analysis (C) LIN28B-high tumors showed significantly higher incidence of early recurrence (within 2 years after surgical treatment, p=0.02, log-rank test), but not late recurrence (p=0.22), as compared with LIN28B-low tumors. (D) The percentage of tumors expressing LIN28B is higher in BCLC-C stage HCC compared to BCLC-A or BCLC-B stage HCC. [Barcelona Clinic Liver Cancer (BCLC) staging system: A:early, B : intermediate C: advanced, D: end –stage⁵³] (E) Serum AFP values were plotted against log2-normalized LIN28B expression values for patients with log2N signal > 6.00. (F) Colony formation for HepG2 cells infected with pLKO.controlshRNA or pLKO.LIN28BshRNA and plated in semisolid medium. 50,000 cells were plated per well and colonies were counted after 14d. *, p<0.05; **, p<0.01.

To determine whether LIN28/LIN28B are associated with advanced malignancy in other settings, we interrogated published microarray data from multiple tumor types and found high levels of LIN28 and/or LIN28B expression in a discrete subset of human tumors including WT (Fig. S8), ovarian carcinoma (Fig. S9), and germ cell tumors (West et al., submitted). Strikingly, LIN28/LIN28B expression was specifically associated with advanced disease in all of these tumor types. In WT, LIN28/LIN28B expression was only observed in Stage 3 or Stage 4 tumors (p= 0.00361, Fisher’s exact test), but not in lower stage tumors (Fig. S8). Among ovarian carcinomas, LIN28/LIN28B expression was observed predominantly in tumors of histological Grade 2 or 3, rather than Grade 1 (Fig. S9).

Various mechanisms may explain LIN28/LIN28B expression in human cancers, including genomic amplification, aberrant hypomethylation, and expression in a rare cell of origin that acquires additional malignant lesions. Amplification of regions containing the LIN28 (1p36) ²⁹ and LIN28B (6q21) ^30,31 loci in human cancers have been reported. We surveyed SNP array data from 3330 primary tumors and cell lines to check for focal copy number changes at the LIN28 and LIN28B loci. Although we observed no appreciable copy number changes at the LIN28 locus, a small subset of cancers showed focal copy number increases of LIN28B (1.7% overall) (Table S5 and Figure S10). However, most amplification events were rare and typically broad, involving both LIN28B and numerous neighboring genes. Analysis of SNP array data in 31 CML-BC samples (22 myeloid, 9 lymphoid) revealed no focal alterations at either the LIN28 or LIN28B loci (data not shown). Thus, while genomic amplification may explain LIN28B expression in a small number of tumors, it is unlikely to be a dominant mechanism of activation. We also assessed methylation status at a CpG island located within the LIN28B locus, and observed relative hypomethylation in LIN28B-expressing cell lines (Fig. S11), suggesting that aberrant hypomethylation may poise the LIN28B locus for transcriptional activation in some tumors. Interestingly, this region of aberrant hypomethylation overlaps with a recently defined c-Myc binding site within the LIN28B locus³². This, together with our observation that LIN28B-expressing tumors cluster in a high-Myc expression subclass, suggests that Myc may transcriptionally activate LIN28B in some contexts.

Notably, there have been several reported cases of WT with a translocation involving 6q21^33–37, the band on which LIN28B is located. To determine whether these translocations may be associated with altered expression of LIN28B, we analyzed a case of sporadic WT with a previously described chromosomal translocation t(6;15)(q21;q21)^33,38. Previous mapping by FISH analysis had narrowed the 6q21 breakpoint in this case to a region intergenic to two 6q21 genes, HACE1 and LIN28B, and had demonstrated that loss of HACE1 expression in a cohort of WT cases³⁹ was associated with altered methylation of several CpG islands in the region, including CpG-29 directly adjacent to the LIN28B gene (Fig S12a). To further map the 6q21 breakpoint in this case, we performed Southern blot assay using probes from the intergenic region. A probe covering CpG-29 and flanking sequences lying ~3–4 kb upstream of the LIN28B transcriptional start site demonstrated rearrangement using PstI-digested genomic DNA from the index WT case compared to matching normal kidney and other controls (Figure S12a–c). Moreover, quantitative RT-PCR showed dramatically increased expression of LIN28B in the index case compared to patient-matched normal kidney (Figure S12d). A second WT cause carrying a t(6;15) (Fig. S12e) also displayed dramatically increased expression of LIN28B in tumor sample compared with normal kidney (Fig. S12f). Therefore, LIN28B may be activated by translocation, although the frequency of such events remains to be determined.

Lin28 and Lin28b are expressed in various stem cell populations^8,40,41, although there is scarce expression in most terminally differentiated somatic tissues⁴². Although our data suggest that altered gene expression of LIN28 or LIN28B can promote tumorigenesis and is associated with advanced malignancy, we cannot rule out the possibility that in some settings, LIN28 or LIN28B may be endogenously expressed in a rare cell of origin that develops into an aggressive tumor. We have observed relatively strong LIN28 expression within cells of the normal ovarian surface epithelium, thought to be the cells of origin of epithelial ovarian cancer (Fig. S9)⁴³. Some poor-prognosis HCCs have also been suggested to arise from a primitive cell of origin that shares gene expression patterns with fetal hepatoblasts⁴⁴.

Recently, LIN28 was used together with OCT4, NANOG, and SOX2 to reprogram human somatic fibroblasts to pluripotency⁴⁵. Over-expression of other stem cell factors, such as BMI1⁴⁶ and OCT4^47,48, has been reported to promote oncogenesis by driving self-renewal and proliferation. Moreover, poorly differentiated, aggressive human tumors have recently been shown to have an embryonic stem cell-like gene expression signature⁴⁹; other embryonic factors have also been reported to have roles in tumor progression^50,51. Our data add LIN28/LIN28B to a growing list of embryonic genes involved in oncogenesis, and suggest a mechanism whereby repression of let-7 by LIN28/LIN28B may help to confer self-renewal capacity on cancer stem cells⁵². Antagonizing LIN28 and/or LIN28B function in tumors that overexpress these genes may provide a means to reactivate the expression of let-7 family tumor suppressors, and may thus be therapeutically beneficial.