Expression Of Lin28 Research Articles

Abstract 1244: An in vitro biological assay to identify small molecule upregulators of let-7 miRNA in LIN28- positive ovarian cancer cells

Abstract Onco-fetal LIN28, present in two isoforms A and B and expressed in cancer cells, promotes tumorigenesis by suppressing the biogenesis of the tumor suppressor microRNA, let-7, thereby activating an array of oncogenes including RAS, MYC, HMGA2 and Cyclin D1. Studies have demonstrated a positive correlation between LIN28 A/B expression and advanced epithelial ovarian cancers (Histological grade 2 or 3). Correlation of LIN28 expression to a poor progression-free survival and resistance to platinum-based therapies makes LIN28-let-7 pathway an attractive target for ovarian cancer chemotherapy. The overall goal of this study was to identify small molecules that upregulate let-7 levels in LIN28A or LIN28B positive ovarian cancer cells. We hypothesize that induction of let-7 levels would potentially improve prognosis for ovarian cancer patients bearing LIN28 positive tumors. To test this hypothesis, we developed a dual luciferase let-7 reporter assay in which let-7 binding sites found in the 3’ UTR of the HMGA2 gene were cloned downstream of the nanoluciferase reporter gene. In this assay, elevated let-7 levels would result in repression of the nanoluciferase signal. Data obtained was normalized to Firefly luciferase activity. We tested efficacy of the assay system using let-7 mimics and inhibitors in LIN28A (OVK-18) and LIN28B (TOV-112D) positive ovarian cancer cell lines stably expressing the reporter cassette that were generated in house. Transfection of the let-7 mimic resulted in a 0.5 fold decrease in both cell lines relative to the negative controls, whereas transfection of the let-7 inhibitor resulted in a two-fold increase in relative luminescence. We miniaturized the assay to 384-well format for small molecule screening. The Z prime (Z’) scores for both cell lines was 0.58 and 0.84 respectively, indicating that the assay was robust to carry out the screens. We performed a primary screen using the Prestwick and LOPAC1280 libraries for each cell line in duplicate as well as a counter screen using a reporter cassette that lacked let-7 binding sites. A compound was identified as a hit if it ranked within the top 100 compounds in the primary screen and outside the top 250 compounds in the counter screen. The hit rate for LIN28A positive cell line was 80 % for the Prestwick and 74 % for the LOPAC1280 libraries. For the LIN28 B positive cell line, the hit rate was 73 % and 53 % for the Prestwick and LOPAC libraries. The hits were validated by assaying for let-7 levels using qRTPCR. The hits identified included compounds that inhibited PI3K-mTOR, nFkB, c-MYC, Cyclin D1, BET1 as well as aurora kinase. These targets have been reported in the literature to downregulate let-7 levels. In conclusion, our assay-system is the first to utilize a biological assay of let-7 levels for small molecule library screening thus serving as a valuable tool for cancer drug discovery. Citation Format: Miriam-Rose Menezes, Goeun Bae, Yong Sung Park, Julien Balzeau, Clifford C. Stephan, John P. Hagan. An in vitro biological assay to identify small molecule upregulators of let-7 miRNA in LIN28- positive ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1244. doi:10.1158/1538-7445.AM2017-1244

The Bile Acid Nuclear Receptor FXRα Is a Critical Regulator of Mouse Germ Cell Fate.

SummarySpermatogenesis is the process by which spermatozoa are generated from spermatogonia. This cell population is heterogeneous, with self-renewing spermatogonial stem cells (SSCs) and progenitor spermatogonia that will continue on a path of differentiation. Only SSCs have the ability to regenerate and sustain spermatogenesis. This makes the testis a good model to investigate stem cell biology. The Farnesoid X Receptor alpha (FXRα) was recently shown to be expressed in the testis. However, its global impact on germ cell homeostasis has not yet been studied. Here, using a phenotyping approach in Fxrα−/− mice, we describe unexpected roles of FXRα on germ cell physiology independent of its effects on somatic cells. FXRα helps establish and maintain an undifferentiated germ cell pool and in turn influences male fertility. FXRα regulates the expression of several pluripotency factors. Among these, in vitro approaches show that FXRα controls the expression of the pluripotency marker Lin28 in the germ cells.

RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts.

Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts). Activation potential was monitored using a luciferase reporter system and we found that both CRISPRa and SAM can efficiently activate the proximal promoter of all five genes. We also observed that the guide RNA (gRNA) target sites and number of gRNAs have a major effect on gRNA-guided activation efficiency. Furthermore, increased activation efficiency (>3-folds) could be achieved by the SAM system compared to CRISPRa. In addition, we discovered that only the SAM system could efficiently activate LIN28, OCT4, and SOX2 expression (up to 100-folds compared to coexpression with a scrambled gRNA) in primary human fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular reprogramming using ATFs.

Abstract TMEM-024: CHARACTERIZATION OF STEM CELLS IN HIGH GRADE SEROUS OVARIAN CARCINOMA CELLS

Abstract PURPOSE: The objectives of this study are to understand how transcription factors and miRNAs impact stemness in epithelial ovarian cancer (EOC). Our goal is to target EOC cancer stem cells (CSC) in order to arrest disease progression. Ovarian Cancer is the most fatal gynecological diagnosis for women. It is typically treated with surgery and chemotherapy, which is initially successful in that tumors shrink; however, most patients experience relapse. Cancer Stem Cells (CSCs) express a similar set of stem cell markers as pluripotent cells, and are thought to contribute to chemoresistance and relapse. Chemotherapy is effective on differentiated cells of the tumor, however cancer stem cells survive, and have the ability to repair and renew the tumor. Cancer cells can be re-sensitized to chemotherapy if stem cells are targeted. Several stem cell markers are expressed in advanced ovarian malignancies. We study the effect of the epithelial to mesenchymal transition (EMT) on stemness. During EMT, cells acquire mesenchymal characteristics and lose epithelial ones. EMT is caused by SNAI1 and other transcription factors that repress epithelial and adhesion molecules such as E-cadherin. EMT results in a stem cell-like phenotype in breast cancer. There is evidence that the EMT factor SNAI1 represses the miRNA let-7, which we hypothesize results in a higher proportion of CSC in EOC cells. Let-7's major function is to promote terminal differentiation and tumor suppression by targeting oncogenes and pluripotency factors. EXPERIMENTAL PROCEDURES: We performed flow cytometry for CD133, CD44, CD117, and aldehyde dehydrogenase to determine proportion of positive cells using published markers. q-RT-PCR was used to determine expression of pluripotency markers Oct4, Lin28 and Nanog. We used immunofluorescence microscopy to identify CSCs. Using a panel of biomarkers specific to CSCs we labeled for SNAI1, NANOG, LIN28A, OCT4, SSEA4, ALDH, TRA1-60, CD133, E-CAD, and N-CAD followed by fluorescent secondary antibodies and nuclear labeling with DAPI. We characterized three epithelial ovarian cell (EOC) lines (OVCAR8, OVSAHO, and COV 318) and patient-derived cells, with and without Snai1 manipulation. RESULTS: Cells were positive for stem cell markers to varying degrees. OVCAR-8 was positive for LIN28A, NANOG, CD44, and ALDH; OVSAHO for Lin 28a and ALDH; and COV318 for NANOG and ALDH. OVSAHO displayed most epithelial (E) characteristics, COV318 and patient samples were most mesenchymal (M), and OVCAR8 appeared to be a M/E hybrid. There was imperfect correlation between mRNA and protein expression of pluripotency markers. CONCLUSIONS: Higher expression of Snai1 correlates with a stem cell phenotype on a population level. On a single cell level, most Nanog-positive cells are Snail positive. However, no ovarian cancer cell perfectly mirrors the pluripotent marker expression seen in NCCIT, our positive control. SNAI1 is a potential target for therapy targeting ovarian cancer stem cells. Citation Format: Maricela Gallardo, Alyse Huisken-Hill, Hanmin Wang, Christine Castanon, Juli J. Unternaehrer. CHARACTERIZATION OF STEM CELLS IN HIGH GRADE SEROUS OVARIAN CARCINOMA CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-024.

Inhibition of neurotensin receptor 1 induces intrinsic apoptosis via let-7a-3p/Bcl-w axis in glioblastoma

Backgroud:Glioblastoma is a kind of highly malignant and aggressive tumours in the central nervous system. Previously, we found that neurotensin (NTS) and its high-affinity receptor 1 (NTSR1) had essential roles in cell proliferation and invasiveness of glioblastoma. Unexpectedly, cell death also appeared by inhibition of NTSR1 except for cell cycle arrest. However, the mechanisms were remained to be further explored.Methods:Cells treated with SR48692, a selective antagonist of NTSR1, or NTSR1 shRNA were stained with Annexin V-FITC/PI and the apoptosis was assessed by flow cytometry. Cytochrome c release was detected by using immunofluorescence. Mitochondrial membrane potential (MMP, ΔΨm) loss was stained by JC-1 and detected by immunofluorescence or flow cytometry. Apoptosis antibody array and microRNA microarray were performed to seek the potential regulators of NTSR1 inhibition-induced apoptosis. Interaction between let-7a-3p and Bcl-w 3′UTR was evaluated by using luciferase assay.Results:SR48692 induced massive apoptosis, which was related to mitochondrial cytochrome c release and MMP loss. Knockdown of NTSR1 induced slight apoptosis and significant MMP loss. In addition, NTSR1 inhibition sensitised glioblastoma cells to actinomycin D or doxorubicin-induced apoptosis. Consistently, NTSR1 inhibition-induced mitochondrial apoptosis was accompanied by downregulation of Bcl-w and Bcl-2. Restoration of Bcl-w partly rescued NTSR1 deficiency-induced apoptosis. In addition, NTSR1 deficiency promoted higher let-7a-3p expression and inhibition let-7a-3p partly rescued NTSR1 inhibition-induced apoptosis. In addition, let-7a-3p inhibition promoted 3′UTR activities of Bcl-w and the expression of c-Myc and LIN28, which were the upstream of let-7a-3p, decreased after NTSR1 inhibition.Conclusions:NTSR1 had an important role in protecting glioblastoma from intrinsic apoptosis via c-Myc/LIN28/let-7a-3p/Bcl-w axis.

The LIN28/let-7 Pathway in Cancer.

Among all tumor suppressor microRNAs, reduced let-7 expression occurs most frequently in cancer and typically correlates with poor prognosis. Activation of either LIN28A or LIN28B, two highly related RNA binding proteins (RBPs) and proto-oncogenes, is responsible for the global post-transcriptional downregulation of the let-7 microRNA family observed in many cancers. Specifically, LIN28A binds the terminal loop of precursor let-7 and recruits the Terminal Uridylyl Transferase (TUTase) ZCCHC11 that polyuridylates pre-let-7, thereby blocking microRNA biogenesis and tumor suppressor function. For LIN28B, the precise mechanism responsible for let-7 inhibition remains controversial. Functionally, the decrease in let-7 microRNAs leads to overexpression of their oncogenic targets such as MYC, RAS, HMGA2, BLIMP1, among others. Furthermore, mouse models demonstrate that ectopic LIN28 expression is sufficient to drive and/or accelerate tumorigenesis via a let-7 dependent mechanism. In this review, the LIN28/let-7 pathway is discussed, emphasizing its role in tumorigenesis, cancer stem cell biology, metabolomics, metastasis, and resistance to ionizing radiation and several chemotherapies. Also, emerging evidence will be presented suggesting that molecular targeting of this pathway may provide therapeutic benefit in cancer.

Disturbance of the let-7/LIN28 double-negative feedback loop is associated with radio- and chemo-resistance in non-small cell lung cancer.

Radio- and chemo-resistance represent major obstacles in the therapy of non-small-cell lung cancer (NSCLC) and the underlying molecular mechanisms are not known. In the present study, during induction of radio- or chemo-resistance in NSCLC cells, dynamic analyses revealed that decreased expression of let-7 induced by irradiation or cisplatin resulted in increased expression of its target gene LIN28, and increased expression of LIN28 then contributed to further decreased expression of let-7 by inhibiting its maturation and biogenesis. Moreover, we showed that down-regulation of let-7 and up-regulation of LIN28 expression promoted resistance to irradiation or cisplatin by regulating the single-cell proliferative capability of NSCLC cells. Consequently, in NSCLC cells, let-7 and LIN28 can form a double-negative feedback loop through mutual inhibition, and disturbance of the let-7/LIN28 double-negative feedback loop induced by irradiation or chemotherapeutic drugs can result in radio- and chemo-resistance. In addition, low expression of let-7 and high expression of LIN28 in NSCLC patients was associated significantly with resistance to radiotherapy or chemotherapy. Therefore, our study demonstrated that disturbance of the let-7/LIN28 double-negative feedback loop is involved in the regulation of radio- and chemo-resistance, and that let-7 and LIN28 could be employed as predictive biomarkers of response to radiotherapy or chemotherapy in NSCLC patients.

H19/let-7/LIN28 reciprocal negative regulatory circuit promotes breast cancer stem cell maintenance

Long noncoding RNA-H19 (H19), an imprinted oncofetal gene, has a central role in carcinogenesis. Hitherto, the mechanism by which H19 regulates cancer stem cells, remains elusive. Here we show that breast cancer stem cells (BCSCs) express high levels of H19, and ectopic overexpression of H19 significantly promotes breast cancer cell clonogenicity, migration and mammosphere-forming ability. Conversely, silencing of H19 represses these BCSC properties. In concordance, knockdown of H19 markedly inhibits tumor growth and suppresses tumorigenesis in nude mice. Mechanistically, we found that H19 functions as a competing endogenous RNA to sponge miRNA let-7, leading to an increase in expression of a let-7 target, the core pluripotency factor LIN28, which is enriched in BCSC populations and breast patient samples. Intriguingly, this gain of LIN28 expression can also feedback to reverse the H19 loss-mediated suppression of BCSC properties. Our data also reveal that LIN28 blocks mature let-7 production and, thereby, de-represses H19 expression in breast cancer cells. Appropriately, H19 and LIN28 expression exhibits strong correlations in primary breast carcinomas. Collectively, these findings reveal that lncRNA H19, miRNA let-7 and transcriptional factor LIN28 form a double-negative feedback loop, which has a critical role in the maintenance of BCSCs. Consequently, disrupting this pathway provides a novel therapeutic strategy for breast cancer.

Stage-Specific Timing of the microRNA Regulation of lin-28 by the Heterochronic Gene lin-14 in Caenorhabditis elegans.

In normal development, the order and synchrony of diverse developmental events must be explicitly controlled. In the nematode Caenorhabditis elegans, the timing of larval events is regulated by hierarchy of proteins and microRNAs (miRNAs) known as the heterochronic pathway. These regulators are organized in feedforward and feedback interactions to form a robust mechanism for specifying the timing and execution of cell fates at successive stages. One member of this pathway is the RNA binding protein LIN-28, which promotes pluripotency and cell fate decisions in successive stages. Two genetic circuits control LIN-28 abundance: it is negatively regulated by the miRNA lin-4, and positively regulated by the transcription factor LIN-14 through a mechanism that was previously unknown. In this report, we used animals that lack lin-4 to elucidate LIN-14's activity in this circuit. We demonstrate that three let-7 family miRNAs-miR-48, miR-84, and miR-241-inhibit lin-28 expression. Furthermore, we show genetically that these miRNAs act between lin-14 and lin-28, and that they comprise the pathway by which lin-14 positively regulates lin-28 We also show that the lin-4 family member mir-237, also regulates early cell fates. Finally, we show that the expression of these miRNAs is directly inhibited by lin-14 activity, making them the first known targets of lin-14 that act in the heterochronic pathway.

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells

Objective: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. Methods: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. Results: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. Conclusion: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

Abstract B31: Sulindac inhibition of tumor cell transformation

Abstract Cell transformation is the process in which normal cells acquire the properties of cancer. Sulindac Sulfide (SS) is one of non-steroidal anti-inflammatory drugs (NSAIDs), and its anticancer activity in prevention of tumor occurrence and progression has been documented in various human cancers. In this study, we observed the anti-transformative activity of SS in NIH/3T3 cells by using a two-stage transformation assay induced by the carcinogens, 3-methylcholanthrene (MCA) and 2-O-tetradecanoylphorbol-13-acetate (TPA) in which foci formation is used to characterize the transformed cells. We found that SS could inhibit foci formation by attenuation of TPA's promotive effect. K-Ras was upregulated in the transformed cells, while SS treatment could selectively inhibit the growth of transformed cell in which K-Ras was suppressed significantly. Loss-of-function study demonstrated that knockdown of K-Ras in NIH/3T3 cells could significantly reduce foci formation, which support that K-Ras might be one of the key targets involved in SS inhibiting tumor cell transformation. When studying the mechanism by which SS regulates K-Ras, we triggered two tumor suppressor miRNAs, let-7b and let-7g, which were previously reported to target K-Ras and repress its expression through the post-transcriptional or translational modulation. We found that let-7b and let-7g could be upregulated in the transformed cells by SS treatment. However, the inhibitory effect of SS on foci formation was significantly attenuated when let-7b and let-7g were knocked down by CRISPR/cas9. In parallel, K-Ras maintained static expression in the transformed cells, regardless of SS treatment. These results suggest that upregulation of let-7b and let-7g is the key step for SS to inhibit tumor transformation. In addition, we demonstrated that SS could suppress the expression of Lin28 that interacts with let-7 through a documented feedback loop. Given our previous studies reporting that SS was able to inhibit the transcriptional activity of NF-kappaB that is involved in regulation of Lin28 expression, our study demonstrates that the NF-kappaB/Lin28/Let-7/K-Ras axis addresses a new mechanism responsible for anti-transformative activity of SS. These results will provide novel insights into understanding the mechanisms involved in tumorigenesis and benefiting identification of new targets for drug development in prevention of tumor progression. This study is supported by the NIH/NCI R01 Grant (1R01CA192395) and the American Cancer Society Research Scholar Grant (RSG-13-265-01-RMC). Citation Format: Zhipin Liang, Xiangling Feng, Hong Chang, Bin Yi, Ruixia Ma, Yaguang Xi. Sulindac inhibition of tumor cell transformation. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B31.

The Lin28 Expression in Stallion Testes.

The molecular markers for specific germ cell stages can be utilized for identifying, monitoring, and separating a particular stage of germ cells. The RNA-binding protein Lin28 is expressed in gonocytes of human fetal testes. The Lin28 expression is restricted to a very small population of spermatogonial cells in human, mice, and monkey. The main objective of this study was to investigate the expression pattern of Lin28 in stallion testes at different reproductive stages. Based on the presence or absence of full spermatogenesis and lumina in seminiferous tubules, the testicular samples were categorized into two reproductive stages pre-pubertal and post-pubertal. We performed a reverse transcription polymerase chain reaction to confirm the presence of Lin28 mRNA in the testicular tissues and a western blot analysis to verify the cross-reactivity of rabbit Lin28 antibody with horse testicular tissue. For immunohistochemistry, Lin28 (rabbit anti-human), GATA4 (goat anti-human) or DAZL (goat anti-human) antibodies were used. The results of RT-PCR confirmed the expression of Lin28 mRNA in the stallion testes. The western blot analysis showed that the expression of 28 kDa Lin28 protein was localized in the cytoplasm of spermatogonia at both reproductive stages. The numbers of Lin28-positive germ cells per 1000 Sertoli cells in pre- and post-pubertal stages were 253 ± 8.66 and 29.67 ± 2.18, respectively. At both reproductive stages, all Lin28 positive cells showed no co-stained with GATA4 antibody, whereas only some of the Lin28-positive germ cells showed co-staining with DAZL antibody. The results from whole-mount staining showed that the Lin28 expression was limited to Asingle (As) and Apaired (Apr) spermatogonia. In conclusion, Lin28 might be utilized as a molecular marker for undifferentiated spermatogonial stem cells when used with DAZL antibody.

Back to basics: Refined nuclear reprogramming techniques yield higher-quality stem cells.

Back to basics: Refined nuclear reprogramming techniques yield higher-quality stem cells.

Polycomb Group Protein Pcgf6 Acts as a Master Regulator to Maintain Embryonic Stem Cell Identity.

The polycomb repressive complex 1 (PRC1) is a multi-subunit complex that plays critical roles in the epigenetic modulation of gene expression. Here, we show that the PRC1 component polycomb group ring finger 6 (Pcgf6) is required to maintain embryonic stem cell (ESC) identity. In contrast to canonical PRC1, Pcgf6 acts as a positive regulator of transcription and binds predominantly to promoters bearing active chromatin marks. Pcgf6 is expressed at high levels in ESCs, and knockdown reduces the expression of the core ESC regulators Oct4, Sox2, and Nanog. Conversely, Pcgf6 overexpression prevents downregulation of these factors and impairs differentiation. In addition, Pcgf6 enhanced reprogramming in both mouse and human somatic cells. The genomic binding profile of Pcgf6 is highly similar to that of trithorax group proteins, but not of PRC1 or PRC2 complexes, suggesting that Pcgf6 functions atypically in ESCs. Our data reveal novel roles for Pcgf6 in directly regulating Oct4, Nanog, Sox2, and Lin28 expression to maintain ESC identity.

Treatment Outcome and Prognostic Molecular Markers of Supratentorial Primitive Neuroectodermal Tumors.

BackgroundTo identify prognostic factors and define the optimal management of patients with supratentorial primitive neuroectodermal tumors (sPNETs), we investigated treatment outcomes and explored the prognostic value of specific molecular markers.MethodsA total of 47 consecutive patients with pathologically confirmed sPNETs between May 1985 and June 2012 were included. Immunohistochemical analysis of LIN28, OLIG2, and Rad51 expression was performed and correlated with clinical outcome.ResultsWith a median follow-up of 70 months, 5-year overall survival (OS) and progression-free survival (PFS) was 55.5% and 40%, respectively, for all patients. Age, surgical extent, and radiotherapy were significant prognostic factors for OS and PFS. Patients who received initially planned multimodal treatment without interruption (i.e., radiotherapy and surgery (≥subtotal resection), with or without chemotherapy) showed significantly higher 5-year OS (71.2%) and PFS (63.1%). In 29 patients with available tumor specimens, tumors with high expression of either LIN28 or OLIG2 or elevated level of Rad51 were significantly associated with poorer prognosis.ConclusionsWe found that multimodal treatment improved outcomes for sPNET patients, especially when radiotherapy and ≥subtotal resection were part of the treatment regimen. Furthermore, we confirmed the prognostic significance of LIN28 and OLIG2 and revealed the potential role of Rad51 in sPNETs.

Long noncoding RNA MALAT1 promotes malignant development of esophageal squamous cell carcinoma by targeting β-cateninviaEzh2

Evidences have shown that lncRNAs involve in the initiation and progression of various cancers including esophageal squamous cell carcinoma (ESCC). The aberrant expression of lncRNA MALAT1 was investigated in 106 paired ESCC tissues and adjacent non-cancerous tissues by qRT-PCR. Down-regulated MALAT1 and Ezh2 over-expression plasmid were constructed respectively to analyze the expression of β-catenin, Lin28 and Ezh2 genes. We found that the MALAT1 expression level was higher in human ESCC tissues (P=0.0011), which was closely correlated with WHO grade (P=0.0395, P=0.0331), lymph node metastasis (P=0.0213) and prognosis (P=0.0294). Silencing of MALAT1 expression inhibited cell proliferation, migration and tumor sphere formation, while increasing cell apoptosis of esophageal cancer in vitro. Down-regulation of MALAT1 decreased the expression of β-catenin, Lin28 and Ezh2 genes, while over-expressed Ezh2 combined with MALAT1 down-regulation completely reversed the si-MALAT1-mediated repression of β-catenin and Lin28 in esophageal cancer cells. Animal experiments showed that knockdown of MALAT1 decreased tumor formation and improved survival. MALAT1 promotes the initiation and progression of ESCC, suggesting that inhibition of MALAT1 might be a potential target for treatment of ESCC.

LIN28: A Stem Cell Factor with a Key Role in Pediatric Tumor Formation.

Differentiation and development are normally unidirectional processes in which progenitor/stem cells differentiate into more mature cells. Transformation of adult cells into cancer cells is accompanied in many cases by dedifferentiation of the adult cell, while differentiation failure of progenitor cells can result in the formation of unique type of cancers called pediatric cancer. LIN28A and its paralog LIN28B are pluripotent genes that are expressed mainly in stem/progenitor cells. Since the first identification of LIN28 in mammals, numerous studies demonstrated the general oncogenic features of these genes. In this review, we emphasize the unique role of LIN28 in pediatric tumor formation. We show, based on comprehensive literature screen and analysis of published microarray data, that LIN28 expression in pediatric tumors is even more common than in adult tumors, and discuss the possibility that in the case of pediatric cancers, LIN28 acts by preventing normal development/differentiation rather than by transformation of mature cells into cancer cells. Overall, this review highlights the role of LIN28 as a bridge point between embryonic development, stem cell biology, and cancer.

Let-7a mimic attenuates CCL18 induced breast cancer cell metastasis through Lin 28 pathway.

MicroRNAs are believed to influence breast cancer cell tumorgenicity by interacting with the production of tumor associated macrophages. At this stage, this hypothesis lacks sufficient empirical evidence. Our study is an investigation of the effects of let-7a on the function of human breast cancer cell lines that had undergone chemokine ligand 18 (CCL18) stimulation. Two breast cancer cell lines MDA-MB-231 and MCF-7 were transfected with let-7a mimics with or without CCL18 simulation. The expression level of let-7a was evaluated with qRT-PCR. Our study examined cell proliferation, migration and cell cycles following let-7a treatment. The predicted target of let-7a was identified and confirmed in vitro by a dual luciferase reporter system. The associations between let-7a, CCL18 and target gene expression were evaluated using RT-PCR and the Western blotting method. The downregulated expression level of let-7a was observed in both breast cancer cell lines. When compared to the control and CCL18 stimulation groups, cell proliferation and migration in MDA-MB-231 and MCF-7 cells were significantly inhibited by let-7a. Furthermore, the cell cycle was dramatically blocked at the G2/M phase. The luciferase reporter identified Lin28 as the direct binding target of let-7a in both breast cancer cell lines. Upregulation of let-7a carries the potential to reverse CCL18 induced cell proliferation and migration alteration in breast cancer cells by regulating Lin28 expression. Our results provided evidence which suggests the use of let-7a as a therapeutic agent in the treatment of breast cancer.

Lin28: an emerging important oncogene connecting several aspects of cancer.

RNA-binding protein Lin28 was originally found as a heterochronic gene which played a significant role in the development of Caenorhabditis elegans. The tumor suppressor let-7 is a downstream target of Lin28, which has a wide variety of target genes which are involved in many aspects of cellular activities. By inhibition of let-7 and directly binding the target RNAs, Lin28 plays an important role in different biological and pathological processes including differentiation, metabolism, proliferation, pluripotency, and tumorigenesis. Overexpression of Lin28 has been reported in several kinds of cancers and is correlated with poor outcomes. It has been shown that Lin28 could affect the progression of cancers in several ways, such as promoting proliferation, increasing glucose metabolism, and inducing epithelial-mesenchymal transition (EMT) and cancer stem cells. Decrease of Lin28 expression or reactivation of let-7 in cancer cells could induce a reverse effect, indicating their therapeutic values in developing novel strategies for cancer treatment. Here, we will overview the regulatory mechanisms and functions of Lin28 in cancers.

Lin28 induces resistance to anti-androgens via promotion of AR splice variant generation.

Prostate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR). Sensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA. We show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells. Our findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa progression.