Let-7 Levels Research Articles

Abstract PR04: LIN28B promotes growth and tumorigenesis of the intestinal epithelium primarily via Let-7 repression

Abstract The RNA-binding proteins LIN28A and LIN28B have diverse functions in embryonic stem cells, cellular reprogramming, growth, and oncogenesis. Many of these effects occur via direct inhibition of Let-7 miRNAs, although Let-7 independent effects have been surmised. Our past studies have shown that LIN28B expression in colorectal cancers is associated with poor prognosis and cancer recurrence, and that LIN28B promotes migration, invasion, and metastasis of CRC cell lines in mouse xenograft models. Considering these studies, and the observation that LIN28B is more frequently up-regulated in a variety of other cancers, we sought to elucidate the molecular mechanisms of LIN28B function in intestinal homeostasis and colorectal cancer. We report that intestine-targeted expression of Lin28b in mice causes intestinal hypertrophy, crypt expansion, and Paneth cell loss. Lgr5-positive crypt base columnar cells were not expanded, nor compromised by the depletion of Paneth cells. However, Lin28b fostered intestinal polyp and adenocarcinoma formation. We observed that higher Lin28b levels also correlated with increased incidence of adenocarcinoma. This is consistent with human epidemiological data illustrating that increased LIN28B levels are observed at early stages of CRC, with high expression at both stages I and II disease correlating inversely with patient survival and higher probability of recurrence. To examine potential Let-7-independent functions of LIN28B, we pursued ribonucleoprotein cross-linking, immunoprecipitation, and high-throughput sequencing (CLIP-Seq) to identify direct RNA targets. This revealed that LIN28B protein binds a substantial number of mRNAs, many of which are specific to epithelial cells. LIN28B modestly augmented protein levels of these target mRNAs, in vivo. Conversely, Let-7 had a profound effect; modulation of Let-7 levels via deletion of the mirLet7c2/mirLet7b genes recapitulated effects of Lin28b overexpression. Furthermore, intestine-specific Let-7 expression could reverse hypertrophy and Paneth cell depletion caused by Lin28b. Previous reports have linked Let-7 to the regulation of the PI3K-Akt-mTOR signaling pathway via regulation of Insr, Igf1r, and Irs2 mRNAs, yet we found that Lin28b did not have a significant impact on these mRNAs or insulin-PI3K-mTOR signaling. Kras and Myc mRNAs, which are also reported to be Let-7 targets, were likewise unaffected. Instead, we found profound up-regulation of Hmga2, Igf2bp1, Igf2bp2, and E2f5 in the context of Let-7 depletion or Lin28b over-expression. Our study reveals that Let-7 miRNAs are critical for repressing tissue growth and promoting Paneth cell differentiation. These results highlight the critical nature of LIN28B and Let-7 dosage, where a stepwise reduction of Let-7 levels has a direct effect on target mRNAs and adenocarcinoma risk. These Let-7 dependent effects of LIN28B appear to supersede any Let-7 independent effects. In sum, LIN28B can definitively act as an oncogene in the absence of canonical genetic alteration in intestinal carcinogenesis. This abstract is also presented as poster A42. Citation Format: Blair Madison, Qi Liu, Xue Zhong, Christopher M. Hahn, Nan Lin, Matthew Emmet, Ben Z. Stanger, Anil K. Rustgi. LIN28B promotes growth and tumorigenesis of the intestinal epithelium primarily via Let-7 repression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR04.

Cloning, expression and target gene prediction of let-7 in Paralichthys olivaceus metamorphosis

The let-7 gene is a critical regulator of developmental timing events of the larval-to-adult transition.Here,Paralichthys olivaceus let-7 was identified by a stem-loop RT-PCR.The precursor of let-7 was cloned and was proven to be highly conserved among different species.Quantitative analysis revealed that the expression levels of let-7 were generally variably enhanced during the first five metamorphic stages and peaked during the late metamorphic stages(36 dph).At the end of P.olivaceus metamorphosis(41 dph),expression of let-7 was re-duced.To study the effects of triiodothyronine(T3) on the expression of let-7,six different concentrations(0 nmol?L?1,50 nmol?L?1,75 nmol?L?1,100 nmol?L?1,200 nmol?L?1) were used to treat P.olivaceus kidney cells in vitro.Quantitative PCR showed that let-7 levels increased after T3 treatment,especially at 75 nmol?L?1,indicating that T3 regulated the expression of let-7.Further investigation of target genes demonstrated that the 3′-untranslated regions(3?-UTRs) of 29 mRNAs were possible target sites matched with let-7.These mRNA targets played key roles in diverse biological processes: development,cell proliferation,signal transduction,virus immune response,and signaling pathways.These re sults suggested that let-7 played an essential role in regulating the developmental timing of P.olivaceus metamorphosis.

LIN28Expression in Malignant Germ Cell Tumors Downregulateslet-7and Increases Oncogene Levels

Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n = 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed "GAGGUA" was enriched in the 3' untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention.

Novel MicroRNA Reporter Uncovers Repression of Let-7 by GSK-3β

Several members of the let-7 microRNA family are downregulated in ovarian and other cancers. They are thought to act as tumor suppressors by lowering growth-promoting and anti-apoptotic proteins. In order to measure cellular let-7 levels systematically, we have developed a highly sensitive let-7 reporter assay system based on the expression of a chimeric mRNA that contains the luciferase coding region and a 3′-untranslated region (UTR) bearing two let-7-binding sites. In cells expressing the reporter construct, termed pmirGLO-let7, luciferase activity was high when let-7 was absent, while luciferase activity was low when let-7 levels were elevated. The ovarian cancer cell lines BG-1 and UCI-101 were transfected with the let-7 reporter and surveyed with a library of kinase inhibitors in order to identify pathways affecting let-7 activity. Among the inhibitors causing changes in endogenous let-7 abundance, the lowering of glycogen synthase kinase 3 (GSK-3)β function specifically increased let-7 levels and lowered luciferase activity. Similarly, silencing GSK-3β increased both mature and primary-let-7 levels in BG-1 cells, and decreased BG-1 cell survival. Further studies identified p53 as a downstream effector of the GSK-3β-mediated repression of let-7 biosynthesis. Our studies highlight GSK-3β as a novel therapeutic target in ovarian tumorigenesis.

MicroRNA profiles in nasal mucosa of patients with allergic and nonallergic rhinitis and asthma

Rhinitis and asthma commonly coexist and are often regarded as "unified airways disease." Evidence exists that microRNAs are important in controlling inflammatory processes, but little is known about their role in airway inflammation. The present study evaluated the inflammatory profiles of patients with allergic rhinitis (AR), with and without concomitant asthma, and of patients with nonallergic rhinitis (NAR). We analyzed inflammatory cells, cytokines, and microRNAs from nasal biopsies and measured nasal nitric oxide (nNO) levels in 159 young adult subjects subdivided into 4 groups: (1) AR; (2) AR+asthma; (3) NAR; and (4) controls. We observed the upregulation of T-helper 2 (Th2) cytokines and the trend of elevation of nNO levels in AR patients compared to controls. Subjects with current AR symptoms had increased levels of miR-155, miR-205, and miR-498, but reduced levels of let-7e. In addition, patients with positive skin prick test (SPT) reactions exhibited increased miR-155 and miR-205 expression and a decreased level of let-7e, compared to subjects with negative SPT findings. Concomitant asthma had little effect on the inflammatory profile of AR. No significant changes in inflammatory markers were found in NAR patients compared to healthy controls. Our results suggest that microRNAs miR-155, miR-205, miR-498, and let-7e may be important in the allergic inflammation present in nasal mucosa. Regarding NAR, our findings support the view that mechanisms other than inflammation are pivotal.

MYCN/LIN28B/Let-7/HMGA2 pathway implicated by meta-analysis of GWAS in suppression of post-natal proliferation thereby potentially contributing to aging

Mammalian organ and body growth slows and finally terminates because of a progressive suppression of cell proliferation, however little is known about the genetic regulatory mechanisms responsible. A meta-analysis of genome-wide association studies using growth and development-related traits revealed that two genes, HMGA2 and LIN28B, had multiple associations. Altered HMGA2 expression has been shown to result in both overgrowth and pygmy phenotypes in mice and overgrowth in humans. These genes are members of the MYCN/LIN28B/Let-7/HMGA2 pathway and homologs of LIN28B and let-7 are known to regulate developmental timing in Caenorhabditis elegans. Strikingly, expression levels of let-7 and Hmga2 in murine stem cells continue to increase and decrease, respectively, after growth terminates, suggesting that this pathway may contribute to regulating the pace of both development and age-related degenerative phenotypes.

Tumor suppressor p53 plays a key role in induction of both tristetraprolin and let-7 in human cancer cells

Tristetraprolin (TTP) and let-7 microRNA exhibit suppressive effects on cell growth through down-regulation of oncogenes. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. However, the precise mechanism of this repression is unknown. We here demonstrate that p53 stimulated by the DNA-damaging agent doxorubicin (DOX) induced the expression of TTP in cancer cells. TTP in turn increased let-7 levels through down-regulation of Lin28a. Correspondingly, cancer cells with mutations or inhibition of p53 failed to induce the expression of both TTP and let-7 on treatment with DOX. Down-regulation of TTP by small interfering RNAs attenuated the inhibitory effect of DOX on let-7 expression and cell growth. Therefore, TTP provides an important link between p53 activation induced by DNA damage and let-7 biogenesis. These novel findings provide a mechanism for the widespread decrease in TTP and let-7 and chemoresistance observed in human cancers.

Highly lymphatic metastatic pancreatic cancer cells possess stem cell-like properties

Cancer stem cells are thought to be the origin of tumor metastasis. However, evidence of cancer stem cells as the source of lymphatic metastasis in pancreatic cancer is not clear. In this study, we examined the stem cell-like properties of the highly lymphatic metastatic pancreatic cancer cells BxPC-3-LN. Compared with the parental BxPC-3 cells, the BxPC-3-LN cells showed stem cell-like properties, including high lymphatic metastasis potential, self-renewal ability and chemoresistance. In addition, the BxPC-3-LN cells also expressed higher levels of sonic hedgehog and migrating cancer stem cell surface markers (CD133 and CXCR4) compared to the parental BxPC-3 cells. The growth of BxPC-3-LN cells was significantly inhibited by gemcitabine combined with the sonic hedgehog inhibitor cyclopamine. The BxPC-3-LN cells expressed lower levels of let-7, miR-34, miR-107, miR-125, miR-128, miR-130, miR-132 and miR-141 than the parental BxPC-3 cells detected by microRNA PCR array, which were reported to have close relation to stem cell factors. This study provides evidence that cancer stem cells are the major sources of pancreatic cancer lymphatic metastasis, and microRNAs may regulate lymphatic metastasis in pancreatic cancer through modulating cancer stem cells.

Stat3-coordinated Lin-28–let-7–HMGA2 and miR-200–ZEB1 circuits initiate and maintain oncostatin M-driven epithelial–mesenchymal transition

Inflammation can act as a crucial mediator of epithelial-to-mesenchymal transition (EMT). In this study, we show that oncostatin M (OSM) is expressed in an autocrine/paracrine fashion in invasive breast carcinoma. OSM stimulation promotes spontaneous lung metastasis of MCF-7 xenografts in nude mice. A conspicuous epigenetic transition was induced by OSM stimulation not only in breast cancer cell lines but also in MCF-7 xenografts in nude mice. The expression of miR-200 and let-7 family members in response to OSM stimulation was downregulated in a signal transducer and activator of transcription factor 3 (Stat3)-dependent manner, resulting in comprehensive alterations of the transcription factors and oncoproteins targeted by these microRNAs. Inhibition of Stat3 activation or the ectopic expression of let-7 and miR-200 effectively reversed the mesenchymal phenotype of breast cancer cells. Stat3 promotes the transcription of Lin-28 by directly binding to the Lin-28 promoter, resulting in the repression of let-7 expression and concomitant upregulation of the let-7 target, high-mobility group A protein 2 (HMGA2). Knock down of HMGA2 significantly impairs OSM-driven EMT. Our data indicate that downregulation of let-7 and miR-200 levels initiates and maintains OSM-induced EMT phenotypes, and HMGA2 acts as a master switch of OSM-induced EMT. These findings highlight the importance of Stat3-coordinated Lin-28B-let-7-HMGA2 and miR-200-ZEB1 circuits in the cytokine-mediated phenotypic reprogramming of breast cancer cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Mono-Uridylation of Pre-MicroRNA as a Key Step in the Biogenesis of Group II let-7 MicroRNAs

RNase III Drosha initiates microRNA (miRNA) maturation by cleaving a primary miRNA transcript and releasing a pre-miRNA with a 2 nt 3' overhang. Dicer recognizes the 2 nt 3' overhang structure to selectively process pre-miRNAs. Here, we find that, unlike prototypic pre-miRNAs (group I), group II pre-miRNAs acquire a shorter (1 nt) 3' overhang from Drosha processing and therefore requirea 3'-end mono-uridylation for Dicer processing. The majority of let-7 and miR-105 belong to group II. We identify TUT7/ZCCHC6, TUT4/ZCCHC11, and TUT2/PAPD4/GLD2 as the terminal uridylyl transferases responsible for pre-miRNA mono-uridylation. The TUTs actspecifically on dsRNAs with a 1 nt 3' overhang, thereby creating a 2 nt 3' overhang. Depletion of TUTs reduces let-7 levels and disrupts let-7 function. Although the let-7 suppressor, Lin28, induces inhibitory oligo-uridylation in embryonic stem cells, mono-uridylation occurs in somatic cells lacking Lin28 to promote let-7 biogenesis. Our study reveals functional duality of uridylation and introduces TUT7/4/2 as components of the miRNA biogenesis pathway.

LIN28 Binds Messenger RNAs at GGAGA Motifs and Regulates Splicing Factor Abundance

LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.

O026 Negative regulation of toll like receptor signaling by IL-10 dependent microRNAs

Introduction MicroRNAs (miRs) have emerged as important controllers of Toll-like-Receptor (TLR) but their functional role in the inflammatory response remains incompletely understood. Methods Cells. Human monocytes obtained from healthy donor buffycoats and THP-1 monocytic cell lines were stimulated with 100 ng/ml LPS or Pam3CSK4. Constructs. 3′UTR of potential target genes were cloned in psiCHECK vector for the evaluation of miR activity by luciferase assay. For the transduction of THP-1 cell line we generated miRNA/lentiviral-based expression vectors (pRRL-cluster) and cytokines levels were evaluated by ELISA assay. Bioinformatics and statistical analysis. The IPA software has been used for the study of pathways associated to predicted miR-125a∼99b∼let-7e targets. Statistical evaluation was determined using the Student t -test or the One-way ANOVA. Results To identify candidate miRs potentially involved in the response of human monocytes to stimuli of bacterial origin, we previously analyzed miR expression profile of monocytes stimulated for 8 h with LPS [1] . Among the miRs strongly induced by LPS stimulation we identified the cluster of miR-125a, miR-99b and let-7e. We observed a higher expression of miR-125a, miR-99b and let-7e levels at later time points. As IL-10 is a cytokine late-induced during monocyte activation and exert its anti-inflammatory activity through modulation of gene expression program triggered by LPS, we measured the expression levels of these miRs in monocytes stimulated with IL-10, in the presence or not of LPS, showing a potentiating effect of IL-10 on LPS-induced cluster miR-125a∼99b∼let-7e expression. We identified a putative STAT3 binding site and demonstrated the binding of STAT3 to this site in the condition of IL-10 stimulation. These data were also consistent with the reduction of cluster miR-125a∼99b∼let-7e expression levels in the presence of blocking antibody against IL-10R and JAK-STAT inhibitors. An in silico analysis was performed to assess the potential role of cluster miR-125a∼99b∼let-7e in the context of inflammation and strikingly we found that the TLR pathway was potentially regulated by these miRs at multiple steps of the signaling cascade. We validated both the TLR4 and CD14 receptors as direct targets, as well as key adaptor/signalling proteins, in particular MyD88 and IRAK1 proteins and also most of the pro-inflammatory cytokines and chemokines expressed in THP-1 monocytic cells. Furthermore, the enforced expression of cluster miR-125a∼99b∼let-7e in LPS stimulated human monocytes led to an extensive down-regulation of pro-inflammatory cytokines. Conclusion Altogether, our results identify a class of IL-10-responsive miRs with anti-inflammatory activity in monocytes based on multiple targeting of components of the TLR4 pathway. We provide a mechanistic insight into the role of these miRs in the suppression of the inflammatory role and candidate them as new feedback modulators of LPS response involved in the resolution of inflammation. A broader and deeper understanding of these issues may well lead to novel therapeutic approaches to those diseases shown to be marked by dysregulated inflammatory responses.

DMP1 is a target of let-7 in dental pulp cells

Members of the let-7 family have been shown to play a critical role in cell differentiation and tumorigenesis. However, potential targets of let-7 are still unclear. In the current study, we used bioinformatic analysis combined with DNA sequence analysis to identify potential let-7 targets. We discovered that dentin matrix protein 1 (DMP1), which is a non-collagenous protein essential in the mineralization of dentin and bone, has a let-7 binding site in its 3'-untranslated region. Furthermore, reporter assays demonstrated that the DMP1 3'-untranslated region can be regulated directly by the members of let-7. Gene expression levels of let-7 and DMP1 were validated by qRT-PCR of dental pulp cells cultured in a mineralizing medium. Our results suggest that DMP1 is regulated post-transcriptionally by let-7 during odontoblast differentiation.

Abstract LB-385: The role of tumor suppressing microRNA let-7 in undifferentiated nasopharyngeal carcinoma

Abstract AIMS:This study aimed at evaluating the potential anti-proliferative effects of the microRNA let-7 family in nasopharyngeal carcinoma (NPC) cells. In addition, the association between let-7 suppression and DNA hypermethylation is examined. MATERIALS AND METHODS:Levels of mature let-7 family members (-a, -b, -d, -e, -g, and -i) in normal nasopharyngeal cells (NP69 and NP460) and nasopharyngeal carcinoma cells (HK1 and HONE1) were measured by real-time quantitative PCR. Cell-proliferation assay and c-Myc immunohistochemical staining were performed on NPC cells transfected with let-7 precursor molecules. In addition, expression changes in let-7 family members in response to demethylating agents (5-azacytidine and zebularine) were also examined. RESULTS:In comparison with the normal nasopharyngeal cells, let-7 (-a, -b, -d, -e, -g, and -i) levels were reduced in nasopharyngeal carcinoma cells. Ectopic expression of the let-7 family in nasopharyngeal carcinoma cells resulted in inhibition of cell proliferation through downregulation of c-Myc expression. Demethylation treatment of nasopharyngeal carcinoma cells caused activation of let-7 expression in poorly differentiated nasopharyngeal carcinoma cells only. CONCLUSION:Our results suggested that miRNA let-7 might play a role in the proliferation of NPC. DNA methylation is a potential regulatory pathway, which is affected when let-7 is suppressed in NPC cells. However, the extent of DNA hypermethylation/hypomethylation in regulating let-7 expression requires further elucidation. 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-385. doi:1538-7445.AM2012-LB-385

Circulating MicroRNA Profiles as Potential Biomarkers for Diagnosis of Papillary Thyroid Carcinoma

There are no known effective and reliable biomarkers to distinguish benign thyroid nodules from papillary thyroid carcinomas (PTC). Previous studies have indicated that serum microRNA (miRNA) profiles may be diagnostic and/or prognostic markers for numerous other cancers. We studied circulating miRNA profiles in patients with PTC or benign nodules and healthy controls to identify serum miRNA that may be useful as markers for PTC. Genome-wide serum miRNA expression profiles were determined using Solexa sequencing followed by extensive quantitative RT-PCR validation in 245 subjects (106 patients with PTC, 95 patients with benign nodules, and 44 healthy controls). A panel of miRNA was used to assess the expression of specific miRNA in the sera and thyroid tissues of patients with PTC or benign nodules. The expression of serum let-7e, miR-151-5p, and miR-222 was significantly increased in PTC cases relative to benign cases and healthy controls. Receiver operating characteristic curve analyses indicated that use of these three miRNA had a high diagnostic sensitivity and specificity for PTC. Serum let-7e, miR-151-5p, and miR-222 levels were found to be well correlated with certain clinicopathological variables, such as nodal status, tumor size, multifocal lesion status, and Tumor-Node-Metastasis stage. Expression of serum miR-151-5p and miR-222 in a subset of PTC patients decreased significantly after tumor excision. Increased expression of miR-151-5p and miR-222 was also found in the tissue of PTC patients. Our study demonstrates that serum miRNA profiles may be used as novel and minimally invasive diagnostic markers for PTC.

FSH stimulates expression of the embryonic gene HMGA2 by downregulating let-7 in normal fimbrial epithelial cells of ovarian high-grade serous carcinomas

FSH may increase the risk of ovarian malignancy and play a key role in ovarian carcinogenesis, although the mechanism(s) are undefined. HMGA2 overexpression has been observed to be an early genetic event in tumorigenesis. The present study was designed to investigate the effect of FSH on let-7, HMGA2 and p53 expression in normal fimbrial epithelial cells of ovarian high-grade serous carcinomas (HGSCs). A primary human Fallopian tube (FT) fimbrial epithelium ex vivo culture system of low-grade serous carcinomas (LGSCs) and HGSCs was established. The levels of HMGA2, let-7, p53 and FSHR were evaluated by western blotting and reverse transcription (RT)-PCR. Treatment with FSH significantly increased HMGA2 expression in a time-dependent manner and the let-7 expression levels decreased gradually over time in the normal fimbrial epithelial cells of HGSCs. However, we did not observe similar results in LGSCs. In addition, knockdown of let-7 suppressed HMGA2 expression. p53 was not detected in the normal fimbrial epithelial cells before or after FSH administration. Our results indicate that FSH increases the expression of HMGA2 by downregulating the expression of let-7 in normal fimbrial epithelial cells of HGSCs, but no occurrence of p53 mutation. The susceptibility of fimbria to FSH in HGSCs compared with those in LGSCs is different.

P2-07-02: Germline Genetic Variants Disturbing the Let-7/LIN28 Double-Negative Feedback Loop Alter Breast Cancer Susceptibility.

Abstract Previous studies have shown that let-7 can repress the post-transcriptional translation of LIN28, and LIN28, in turn, could block the maturation of let-7, forming a double-negative feedback loop. In this study, we investigated the effect of germline genetic variants on regulation of the homeostasis of the let-7/LIN28 loop and breast cancer risk. We initially demonstrated that the T/C variants of rs3811463, a single nucleotide polymorphism (SNP) located near the let-7 binding site in LIN28, could lead to differential regulation of LIN28 by let-7. Specifically, the C allele of rs3811463 weakened let-7-induced repression of LIN28 mRNA, resulting in increased production of LIN28 protein, which could in turn downregulate the level of mature let-7. This effect was then validated at the tissue level in that the normal breast tissue of individuals with the rs3811463-TC genotype expressed significantly lower levels of let-7 and higher levels of LIN28 protein than those individuals with the rs3811463-TT genotype. Because previous in vitro and ex vivo experiments have consistently suggested that LIN28 could promote cellular transformation, we then systematically evaluated the relationship between rs3811463 as well as other common LIN28 SNPs and the risk of breast cancer in a stepwise manner. The first hospital-based association study (n = 2,300) demonstrated that two SNPs were significantly associated with breast cancer risk, one of which was rs3811463, while the other was rs6697410. The C allele of the rs3811463 SNP corresponded to an increased risk of breast cancer with an odds ratio (OR) of 1.25 (P = 0.0091), which was successfully replicated in a second independent study (n = 1,156) with community-based controls. The combined P-value of the two studies was 8.0 × 10−5. Taken together, our study demonstrates that host genetic variants could disturb the regulation of the let-7/LIN28 double-negative feedback loop and alter breast cancer risk. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-07-02.

Metformin may antagonize Lin28 and/or Lin28B activity, thereby boosting let-7 levels and antagonizing cancer progression

Cancer cells with stem cell characteristics are harbored by most tumors, and are characterized by epithelial–mesenchymal transition (EMT) – which promotes invasive growth and metastasis – chemoresistance, and the capacity to reconstitute new tumors. Hence, the control or destruction of cancer stem cells should be a major goal of cancer management. The let-7 family of microRNAs has cancer suppressor activity, and recent evidence suggests that markedly reduced levels of let-7 are not only a typical feature of cancer stem cells, but may be largely responsible for cancer stemness. It is therefore particularly intriguing that metformin, a diabetes drug thought to have potential in the prevention and treatment of cancer, has recently been found to oppose cancer cell stemness, to markedly potentiate chemotherapeutic control of cancer in mouse xenograft models, and to notably boost let-7a levels in cancer stem cells. It is proposed that this latter effect of metformin may reflect AMPK-mediated inhibition of the expression or activity of Lin28/Lin28A, proteins which act post-transcriptionally to decrease the levels of all let-7 family members. The transcription of Lin28B is promoted by NF-kappaB and by Myc; hence, practical measures which antagonize NF-kappaB or Myc activity may complement the utility of metformin for boosting let-7 expression and controlling cancer stemness; salsalate, antioxidants, tyrosine kinase and cox-2 inhibitors, ribavirin, vitamin D, gamma-secretase inhibitors (when available), and parenteral curcumin may have some utility in this regard. Although the impact of histone deacetylase inhibitors on let-7 expression has not been assessed, there is reason to suspect that these drugs might complement let-7’s impact on chemoresistance, EMT, and stemness. Multifocal strategies centering on metformin may have considerable potential for reversing cancer stemness and rendering advanced cancers more susceptible to long term control.

Germline Genetic Variants Disturbing the Let-7/LIN28 Double-Negative Feedback Loop Alter Breast Cancer Susceptibility

Previous studies have shown that let-7 can repress the post-transcriptional translation of LIN28, and LIN28 in turn could block the maturation of let-7, forming a double-negative feedback loop. In this study, we investigated the effect of germline genetic variants on regulation of the homeostasis of the let-7/LIN28 loop and breast cancer risk. We initially demonstrated that the T/C variants of rs3811463, a single nucleotide polymorphism (SNP) located near the let-7 binding site in LIN28, could lead to differential regulation of LIN28 by let-7. Specifically, the C allele of rs3811463 weakened let-7–induced repression of LIN28 mRNA, resulting in increased production of LIN28 protein, which could in turn down-regulate the level of mature let-7. This effect was then validated at the tissue level in that the normal breast tissue of individuals with the rs3811463-TC genotype expressed significantly lower levels of let-7 and higher levels of LIN28 protein than those individuals with the rs3811463-TT genotype. Because previous in vitro and ex vivo experiments have consistently suggested that LIN28 could promote cellular transformation, we then systematically evaluated the relationship between rs3811463 as well as other common LIN28 SNPs and the risk of breast cancer in a stepwise manner. The first hospital-based association study (n = 2,300) demonstrated that two SNPs were significantly associated with breast cancer risk, one of which was rs3811463, while the other was rs6697410. The C allele of the rs3811463 SNP corresponded to an increased risk of breast cancer with an odds ratio (OR) of 1.25 (P = 0.0091), which was successfully replicated in a second independent study (n = 1,156) with community-based controls. The combined P-value of the two studies was 8.0×10−5. Taken together, our study demonstrates that host genetic variants could disturb the regulation of the let-7/LIN28 double-negative feedback loop and alter breast cancer risk.