Expression Of Lin28 Research Articles

Episomal plasmid-based generation of induced pluripotent stem cells from fetal femur-derived human mesenchymal stromal cells

Human bone mesenchymal stromal cells derived from fetal femur 55days post-conception were reprogrammed to induced pluripotent stem cells using episomal plasmid-based expression of OCT4, SOX2, NANOG, LIN28, SV40LT, KLF4 and c-MYC and supplemented with the following pathway inhibitors — TGFβ receptor inhibitor (A-83-01), MEK inhibitor (PD325901), GSK3β inhibitor (CHIR99021) and ROCK inhibitor (HA-100). Successful induction of pluripotency in two iPS-cell lines was demonstrated in vitro and by the Pluritest.

Overexpression of Lin28 Decreases the Chemosensitivity of Gastric Cancer Cells to Oxaliplatin, Paclitaxel, Doxorubicin, and Fluorouracil in Part via microRNA-107.

Higher Lin28 expression is associated with worse pathologic tumor responses in locally advanced gastric cancer patients undergoing neoadjuvant chemotherapy. However, the characteristics of Lin28 and its mechanism of action in chemotherapy resistance is still unclear. In this study, we found that transfection of Lin28 into gastric cancer cells (MKN45 and MKN28) increased their resistance to the chemo-drugs oxaliplatin (OXA), paclitaxel (PTX), doxorubicin (ADM), and fluorouracil (5-Fu) compared with gastric cancer cells transfected with a control vector. When knockdown Lin28 expression by Lin28 small interfering RNA (siRNA) was evaluated in vitro, we found that the resistance to chemo-drugs was reduced. Furthermore, we found that Lin28 up-regulates C-myc and P-gp and down-regulates Cylin D1. Finally, we found that miR-107 is a target microRNA of Lin28 and that it participates in the mechanism of chemotherapy resistance. Our results suggest that the Lin28/miR-107 pathway could be one of many signaling pathways regulated by Lin28 and associated with gastric cancer chemo-resistance.

Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are leading candidate cells as raw materials for cell therapy products, because of their capacity for pluripotent differentiation and unlimited self-renewal. hPSC-derived products have already entered the scope of clinical application. However, the assessment and control of their tumorigenicity remains to be a critical challenge. Sensitive detection of the pluripotent cellular impurities is necessary for the safety and quality control of the hPSC-derived products. In the present study, we established a sensitive assay for detection of the residual undifferentiated hiPSCs in cardiomyocytes, using droplet digital PCR (ddPCR). The ddPCR method with a probe and primers for LIN28 significantly detected as low as 0.001% undifferentiated hiPSCs in primary cardiomyocytes, which is equivalent to the ratio of a single hiPSC to 1 × 105 cardiomyocytes. The ddPCR also showed that LIN28 expression is extremely low in human tissues including liver, heart, pancreas, kidney, spinal cord, corneal epithelium and lung. These results suggest that the ddPCR method targeting LIN28 transcripts is highly sensitive and useful for the quality assessment of various cell therapy products derived from hPSCs.

Tumorigenic hybrids between mesenchymal stem cells and gastric cancer cells enhanced cancer proliferation, migration and stemness.

BackgroundEmerging evidence indicates that inappropriate cell-cell fusion might contribute to cancer progression. Similarly, mesenchymal stem cells (MSCs) can also fuse with other cells spontaneously and capable of adopting the phenotype of other cells. The aim of our study was to investigate the role of MSCs participated cell fusion in the tumorigenesis of gastric cancer.MethodsWe fused human umbilical cord mesenchymal stem cells (hucMSCs) with gastric cancer cells in vitro by polyethylene glycol (PEG), the hybrid cells were sorted by flow cytometer. The growth and migration of hybrids were assessed by cell counting、cell colony formation and transwell assays. The proteins and genes related to epithelial- mesenchymal transition and stemness were tested by western blot、immunocytochemistry and real-time RT-PCR. The expression of CD44 and CD133 was examined by immunocytochemistry and flow cytometry. The xenograft assay was used to evaluation the tumorigenesis of the hybrids.ResultsThe obtained hybrids exhibited epithelial- mesenchymal transition (EMT) change with down-regulation of E-cadherin and up-regulation of Vimentin, N-cadherin, α-smooth muscle actin (α-SMA), and fibroblast activation protein (FAP). The hybrids also increased expression of stemness factors Oct4, Nanog, Sox2 and Lin28. The expression of CD44 and CD133 on hybrid cells was stronger than parental gastric cancer cells. Moreover, the migration and proliferation of heterotypic hybrids were enhanced. In addition, the heterotypic hybrids promoted the growth abilities of gastric xenograft tumor in vivo.ConclusionsTaken together, our results suggest that cell fusion between hucMSCs and gastric cancer cells could contribute to tumorigenic hybrids with EMT and stem cell-like properties, which may provide a flexible tool for investigating the roles of MSCs in gastric cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1780-1) contains supplementary material, which is available to authorized users.

LIN28A Modulates Splicing and Gene Expression Programs in Breast Cancer Cells.

LIN28 is an evolutionarily conserved RNA-binding protein with critical functions in developmental timing and cancer. However, the molecular mechanisms underlying LIN28's oncogenic properties are yet to be described. RNA-protein immunoprecipitation coupled with genome-wide sequencing (RIP-Seq) analysis revealed significant LIN28 binding within 843 mRNAs in breast cancer cells. Many of the LIN28-bound mRNAs are implicated in the regulation of RNA and cell metabolism. We identify heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein with multiple roles in mRNA metabolism, as a LIN28-interacting partner. Subsequently, we used a custom computational method to identify differentially spliced gene isoforms in LIN28 and hnRNP A1 small interfering RNA (siRNA)-treated cells. The results reveal that these proteins regulate alternative splicing and steady-state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of the ENAH exon 11a isoform. The expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. Intriguingly, analysis of publicly available array data from the Cancer Genome Atlas (TCGA) reveals that LIN28 expression in the HER2 subtype is significantly different from that in other breast cancer subtypes. Collectively, our data suggest that LIN28 may regulate splicing and gene expression programs that drive breast cancer subtype phenotypes.

Abstract 4215: Small molecule compounds that destabilize stem cell factors

Abstract Cancer stem cells (CSCs) are thought to be present in malignant tumors at various proportions, and their phenotype can drift over time. Based on the current view, this elusive population of cancer cells is likely responsible for distant metastases, drug resistance and recurrence. However, the process by which such cell population is generated and maintained in a tumor mass has been unclear. Based on our previous study (PNAS Vol. 110: 6097-102 2013) and others, this process may involve the elevated expression of stem cell factors (SOX2, OCT4, NANOG, LIN28, KLF4, MYC, MYCN and those with equivalent functions). If so, it is conceivable that destabilization of these proteins would facilitate the eradication of CSC compartments in tumor mass. We have previously identified several small molecule compounds that confer the anti-growth effect on the stem cell-like neuroblastoma cells via screening of the Prestwick Chemical Library®, containing 1200 FDA-approved small molecules and of other selected experimental small molecules by using MTS assays. These compounds were structurally and biologically diverse molecules, including Alexidine (Alex), Benzethonium (Benz), Ketoconazole (Keto), Flubendazole (Flu), Auranofin (Aura), Nifuroxazide (Nif), Itraconazole (Itac) and JQ1. In this study, we investigated the effects of these selected small molecules on the expression of stem cell factors using the human teratocarcinoma cell lines NT2 and NCCIT as model systems, because these cells retain the expression of the majority of stem cell factors. The teratocarcinoma cell lines were treated with the small molecules at 0.5 to 10 μM for 24 hours and subjected to Western blot analysis. The expression of stem cell factors was down-regulated in NT2 cells by the several small molecules tested. The expression of SOX2 was down-regulated by Benz (5 μM), Flu (10 μM), Nif (10 μM), Aura (1 μM) and JQ1 (5 μM); the expression of OCT4 was down-regulated by Benz, Flu, Nif, Alex and JQ1; the NANOG expression was down-regulated by the all compounds tested; the expression of LIN28 was down-regulated by Benz, Flu and Aura; the expression of MYC/MYCN was down-regulated by Benz, Flu, Nif, Alex, Keto and JQ1. Interestingly, Auranofin treatment augmented the expression of MYC/MYCN, although the compound was growth suppressive to NT2 cells. Unlike the stem cell factors, the expression of LIN28B, which is the homologue of LIN28, was not affected by any of the compounds tested. A similar observation was made for NCCIT cells, but the effect of the compounds on the expression of stem cell factors appeared to be weaker. Collectively, the data suggest that small molecules identified via the process described could become useful lead chemicals for developing drug-like compounds that can target the most malignant stem cell-like cancer cells. Citation Format: Naohiko Ikegaki, Sarah Lomahan, Xao Tang. Small molecule compounds that destabilize stem cell factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4215. doi:10.1158/1538-7445.AM2015-4215

Abstract 2300: LIN28 cooperates with Wnt signaling to drive invasive intestinal and colorectal adenocarcinoma in mouse and human

Abstract Despite extensive investigation into the molecular basis of tumor development, colorectal cancer (CRC) remains a major contributor to cancer-related mortality. LIN28A and LIN28B are highly related RNA-binding proteins that influence development, tissue regeneration and oncogenesis. Here we demonstrate that overexpression of LIN28 drives invasive intestinal adenocarcinoma and cooperates with the Wnt pathway to promote tumor initiation and progression in murine models. Importantly, conditional withdrawal of LIN28 reduced tumor volume and increased tumor differentiation, indicating that LIN28 contributes to tumor maintenance. Furthermore, co-expression of a LIN28-resistant form of the let-7 microRNA reduced tumor burden. We detected aberrant expression of LIN28 in 38% of a large series of tumor samples from human CRC, and LIN28 expression levels were associated with invasive tumor growth pattern. Our robust late-stage CRC murine model demonstrates strong roles for LIN28 in promoting CRC initiation, growth and progression, and suggests the therapeutic potential in targeting the LIN28/let-7 pathway. Citation Format: Ho-Chou Tu, Sarah Schwitalla, Zhirong Qian, Grace LaPier, Alena Yermalovich, Yuan-Chieh Ku, Shann-Ching Chen, Srinivas R. Viswanathan, Hao Zhu, Reiko Nishihara, Kentaro Inamura, Sun A. Kim, Reppi Morikawa, Kosuke Mima, Yasutaka sukawa, Juhong Yang, Gavin Meredith, Charles S. Fuchs, Shuji Ogino, George Q. Daley. LIN28 cooperates with Wnt signaling to drive invasive intestinal and colorectal adenocarcinoma in mouse and human. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2300. doi:10.1158/1538-7445.AM2015-2300

Abstract 4267: Integrated genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups

Abstract Hepatoblastoma (HB), the most common primary liver cancer in children, is a clinically heterogeneous embryonal malignancy. Previous studies of HB have revealed a paucity of genetic alterations in these tumors, other than mutations activating the WNT signaling pathway, and have not provided an adequate biological explanation for the diverse clinical outcomes of children with HB. To identify critical genes and pathways in the pathogenesis of HB and provide insight into the biologic basis and clinical heterogeneity of these tumors, we performed an integrated genomic analysis of a large cohort of clinically-annotated tumor-normal pairs utilizing whole exome sequencing (n = 35), mRNA and miRNA expression arrays (n = 51; n = 58), and high-resolution copy number arrays (n = 47). Somatic mutations/deletions of CTNNB1 or rare germline mutations of APC were identified in >90% of cases, confirming the central role of the WNT pathway in HB pathogenesis. The only other gene targeted by recurrent mutations in our cohort was the transcription factor NFE2L2 (NRF2), which was found to harbor hotspot mutations at a similar frequency (5-10%) to that reported for hepatocellular carcinoma. Expression profiling revealed near-universal WNT-pathway activation: 7 of the 20 most highly expressed genes in HB were WNT-pathway related genes, and the WNT-pathway marker DKK1 was expressed hundreds of standard deviations above its levels in normal liver. Unsupervised hierarchical clustering identified three distinct molecular HB clusters that were characterized by differential activation of hepatic progenitor cell and metabolic pathways. Analysis of the expression and/or inferred-activity of transcription factors and highly expressed genes identified prognostic biomarkers, including the significantly predictive regulons of NFE2L2, LIN28b, HNF1A, and NOTCH1. These were used to identify clinical groups that overlapped with HB expression clusters and were associated with patient survival. Tumors in the high-risk group were characterized by high NFE2L2, low NOTCH1 activity, and high LIN28b expression and activity (corresponding to low let-7b expression), as well as high expression of the onco-fetal proteins AFP and GPC3 and stem cell markers EPCAM, DLK1, and SALL4. In contrast, the low-risk group was characterized by high HNF1a and NOTCH1 activities and low LIN28 expression and activity. Rare chromosomal gains and losses were characteristic of the low risk group of tumors, while gains of 20q13 (SALL4) and 1q chromosomal regions were primarily found in the high and intermediate risk groups. Immunohistochemistry for LIN28b, NFE2L2, HNF1alpha, PTEN, and EPCAM is being performed on a validation set of tumors to assess their reliability to classify formalin-fixed tumor specimens. Prospective studies will test the prognostic utility of this method for children with HB. Citation Format: Dolores H. Lopez-Terrada, Pavel Sumazin, Yidong Chen, Lisa Trevino, Stephen Sarabia, Oliver Hampton, Kayuri Patel, Toni-Ann Mistretta, Barry Zorman, Sarah Comerford, David Wheeler, Murali Chintagumpala, Rebecka M. Meyers, Milton J. Finegold, Gail Tomlinson, Donald W. Parsons. Integrated genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4267. doi:10.1158/1538-7445.AM2015-4267

Generation of induced pluripotent stem cells from domestic goats.

The creation of genetically modified goats provides a powerful approach for improving animal health, enhancing production traits, animal pharming, and for ensuring food safety all of which are high-priority goals for animal agriculture. The availability of goat embryonic stem cells (ESCs) that are characteristically immortal in culture would be of enormous benefit for developing genetically modified animals. As an alternative to long-sought goat ESCs, we generated induced pluripotent stem cells (iPSC) by forced expression of bovine POU5F1, SOX2, MYC, KLF4, LIN-28, and NANOG reprogramming factors in combination with a MIR302/367 cluster, delivered by lentiviral vectors. In order to minimize integrations, the reprogramming factor coding sequences were assembled with porcine teschovirus-1 2A (P2A) self-cleaving peptides that allowed for tri-cistronic expression from each vector. The lentiviral-transduced cells were cultured on irradiated mouse feeder cells in a semi-defined, serum-free medium containing fibroblast growth factor (FGF) and/or leukemia inhibitory factor (LIF). The resulting goat iPSC exhibit cell and colony morphology typical of human and mouse ESCs-that is, well-defined borders, a high nuclear-to-cytoplasmic ratio, a short cell-cycle interval, alkaline phosphatase expression, and the ability to generate teratomas in vivo. Additionally, these goat iPSC demonstrated the ability to differentiate into directed lineages in vitro. These results constitute the first steps in establishing integration and footprint-free iPSC from ruminants. Mol. Reprod. Dev. 82: 709-721, 2015. © 2015 Wiley Periodicals, Inc.

Identification of small molecule inhibitors of Zcchc11 TUTase activity

The RNA-binding protein Lin28 regulates the expression of the let-7 family of microRNAs (miRNAs) during early embryonic development. Lin28 recruits the 3′ terminal uridylyl transferase (TUTase) Zcchc11 (TUT4) and/or Zcchc6 (TUT7) to precursor let-7 RNA (pre-let-7) to selectively block let-7 biogenesis. Uridylated pre-let-7 is targeted for decay by the downstream exonuclease Dis3l2 thereby preventing processing to mature let-7. Activation of this oncogenic pathway via up-regulation of Lin28 expression promotes cellular transformation, drives tumorigenesis in mouse models, and is frequently observed in a wide variety of cancer. Recent proof-of-principle experiments showed that Zcchc11 knockdown inhibits the tumorigenicity of Lin28-expressing human cancer cells and established this enzyme as a possible new therapeutic target for human malignancies. However, there are currently no known pharmacological agents capable of targeting this novel enzyme. In this study we developed and applied a sensitive biochemical assay that monitors Zcchc11 activity. Using this assay we performed an automated high-throughput screen of ∼15,000 chemicals to identify putative TUTase inhibitors. Several of these small molecules were validated as specific inhibitors of Zcchc11 activity. Our results demonstrate the feasibility of screening for TUTase inhibitors and present a relatively simple platform that can be exploited for future drug discovery efforts aimed at restoring let-7 expression in cancer.

Paclitaxel-sensitization enhanced by curcumin involves down-regulation of nuclear factor-κB and Lin28 in Hep3B cells

Although paclitaxel is an effective chemotherapeutic drug used in the treatment of many tumors, hepatoma cells, in particular, are known to be highly resistant to it. Previously, we discovered that Lin28 was closely associated with resistance to paclitaxel in Hep3B cells. The nuclear factor-kappa B (NF-κB) transcription factor, which plays an important role in tumor survival, directly activates Lin28 expression through a binding site on the first intron. Curcumin, a non-toxic anti-inflammatory agent, inhibits NF-κB activity in vitro. In this study, we reported that a combination of curcumin and paclitaxel exhibited synergistic anti-proliferative and pro-apoptosis effects on Hep3B cells, and curcumin down-regulated paclitaxel-induced enhanced expression of Lin28 and NF-κB activation. Furthermore, our results revealed that curcumin reduced Lin28 levels via mechanisms directly mediated by inhibition of NF-κB activity. These mechanism-based observations evidence that curcumin enhances the sensitivity of hepatoma cells to paclitaxe, and strongly support the notion that paclitaxel in combination with curcumin may provide a superior therapeutic index for HCC chemotherapy.

LIN28 cooperates with WNT signaling to drive invasive intestinal and colorectal adenocarcinoma in mice and humans.

Colorectal cancer (CRC) remains a major contributor to cancer-related mortality. LIN28A and LIN28B are highly related RNA-binding protein paralogs that regulate biogenesis of let-7 microRNAs and influence development, metabolism, tissue regeneration, and oncogenesis. Here we demonstrate that overexpression of either LIN28 paralog cooperates with the Wnt pathway to promote invasive intestinal adenocarcinoma in murine models. When LIN28 alone is induced genetically, half of the resulting tumors harbor Ctnnb1 (β-catenin) mutation. When overexpressed in Apc(Min/+) mice, LIN28 accelerates tumor formation and enhances proliferation and invasiveness. In conditional genetic models, enforced expression of a LIN28-resistant form of the let-7 microRNA reduces LIN28-induced tumor burden, while silencing of LIN28 expression reduces tumor volume and increases tumor differentiation, indicating that LIN28 contributes to tumor maintenance. We detected aberrant expression of LIN28A and/or LIN28B in 38% of a large series of human CRC samples (n = 595), where LIN28 expression levels were associated with invasive tumor growth. Our late-stage CRC murine models and analysis of primary human tumors demonstrate prominent roles for both LIN28 paralogs in promoting CRC growth and progression and implicate the LIN28/let-7 pathway as a therapeutic target.

Immunohistochemical expression of stem cell markers in pheochromocytomas/paragangliomas is associated with SDHx mutations.

Pheochromocytomas (PCCs) are neuroendocrine tumors that occur in the adrenal medulla, whereas paragangliomas (PGLs) arise from paraganglia in the head, neck, thorax, or abdomen. In a variety of tumors, cancer cells with stem cell-like properties seem to form the basis of tumor initiation because of their ability to self-renew and proliferate. Specifically targeting this small cell population may lay the foundation for more effective therapeutic approaches. In the present study, we intended to identify stem cells in PCCs/PGLs. We examined the immunohistochemical expression of 11 stem cell markers (SOX2, LIN28, NGFR, THY1, PREF1, SOX17, NESTIN, CD117, OCT3/4, NANOG, and CD133) on tissue microarrays containing 208 PCCs/PGLs with different genetic backgrounds from five European centers. SOX2, LIN28, NGFR, and THY1 were expressed in more than 10% of tumors, and PREF1, SOX17, NESTIN, and CD117 were expressed in <10% of the samples. OCT3/4, NANOG, and CD133 were not detectable at all. Double staining for chromogranin A/SOX2 and S100/SOX2 demonstrated SOX2 immunopositivity in both tumor and adjacent sustentacular cells. The expression of SOX2, SOX17, NGFR, LIN28, PREF1, and THY1 was significantly associated with mutations in one of the succinate dehydrogenase (SDH) genes. In addition, NGFR expression was significantly correlated with metastatic disease. Immunohistochemical expression of stem cell markers was found in a subset of PCCs/PGLs. Further studies are required to validate whether some stem cell-associated markers, such as SOX2, could serve as targets for therapeutic approaches and whether NGFR expression could be utilized as a predictor of malignancy.

AT-05 * TARGETING mTOR DOWNSTREAM OF LIN28 IN ATYPICAL TERATOID RHABDOID TUMORS PROMOTES APOPTOSIS AND SUPPRESSES TUMORIGENICITY

LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. One of the canonical downstream targets of LIN28 is the mTOR pathway. We have previously shown that overexpression of LIN28 in Atypical Teratoid Rhabdoid Tumors (AT/RT) contributes to the aggressive nature of the tumor. We hypothesized that AT/RT would have increased expression of mTOR signaling due to high-level expression of LIN28. We found that primary human AT/RT samples do have high levels of P-S6 and P-AKT expression by immunohistochemistry staining (mean H-score for P-S6 36.3; p-AKT 150.4). The dual TORC1/2 inhibitor MLN0128 has good brain penetration and is currently being tested in phase I clinical trials in children. Treatment of AT/RT cell lines with MLN0128 inhibits TORC1/2 targets in vitro and suppresses cell proliferation at 10nM concentration (MTS assay for BT12 p = 0.0034 vs DMSO control; BT37 p = 0.0064 vs DMSO control; and CHLA-06 p = 0.002 vs DMSO control by t-test). MLN0128 also slows cell proliferation via BrdU assay (BT-37 p = 0.0241 10nM vs DMSO control; CHLA-06 p = 0.045 10nM vs DMSO control by t-test) and induces apoptosis measured by Western blot for PARP and cleaved caspase 3 assay (BT-37 p = 0.001 100nM vs DMSO; CHLA-06 p = 0.007 10nM vs DMSO by t-test). MLN0128 treatment of AT/RT xenograft mouse models leads to rapid reduction in tumor volume compared to controls treated with vector alone (61.9% average reduction in tumor volume with MLN0128 treatment vs 272.1% average increase in tumor volume in vector control; p = 0.006 by t-test). Targeting the overexpressed mTOR pathway with the TORC1/TORC2 inhibitor MLN0128 leads to potent in vitro and in vivo activity against AT/RT. MLN0128 may be a candidate for future clinical trials to treat this aggressive tumor.

The role of Lin28b in myeloid and mast cell differentiation and mast cell malignancy.

Mast cells (MCs) are critical components of the innate immune system and important for host defense, allergy, autoimmunity, tissue regeneration and tumor progression. Dysregulated MC development leads to systemic mastocytosis (SM), a clinically variable but often devastating family of hematologic disorders. Here we report that induced expression of Lin28, a heterochronic gene and pluripotency factor implicated in driving a fetal hematopoietic program, caused MC accumulation in adult mice in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in LIN28B-expressing hematopoietic progenitors, with increased levels of LIN28B in common myeloid and basophil-MC progenitors altering gene expression patterns to favor cell fate choices that enhanced MC specification. In addition, LIN28B-induced MCs appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of MC terminal differentiation in the context of LIN28B upregulation. Finally, interrogation of human MC leukemia samples revealed upregulation of LIN28B in abnormal MCs from patients with SM. This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in MC disease.

Over-expression of cofilin-1 suppressed growth and invasion of cancer cells is associated with up-regulation of let-7 microRNA

Cofilin-1, a non-muscle isoform of actin regulatory protein that belongs to the actin-depolymerizing factor (ADF)/cofilin family is known to affect cancer development. Previously, we found that over-expression of cofilin-1 suppressed the growth and invasion of human non-small cell lung cancer (NSCLC) cells in vitro. In this study, we further investigated whether over-expression of cofilin-1 can suppress tumor growth in vivo, and performed a microRNA array analysis to better understand whether specific microRNA would be involved in this event. The results showed that over-expression of cofilin-1 suppressed NSCLC tumor growth using the xenograft tumor model with the non-invasive reporter gene imaging modalities. Additionally, cell motility and invasion were significantly suppressed by over-expressed cofilin-1, and down-regulation of matrix metalloproteinase (MMPs) -1 and -3 was concomitantly detected. According to the microRNA array analysis, the let-7 family, particularly let-7b and let-7e, were apparently up-regulated among 248 microRNAs that were affected after over-expression of cofilin-1 up to 7days. Knockdown of let-7b or let-7e using chemical locked nucleic acid (LNA) could recover the growth rate and the invasion of cofilin-1 over-expressing cells. Next, the expression of c-myc, LIN28 and Twist-1 proteins known to regulate let-7 were analyzed in cofilin-1 over-expressing cells, and Twist-1 was significantly suppressed under this condition. Up-regulation of let-7 microRNA by over-expressed cofilin-1 could be eliminated by co-transfected Twist-1 cDNA. Taken together, current data suggest that let-7 microRNA would be involved in over-expression of cofilin-1 mediated tumor suppression in vitro and in vivo.

Isolation of adipose and bone marrow mesenchymal stem cells using CD29 and CD90 modifies their capacity for osteogenic and adipogenic differentiation

Mesenchymal stem cells isolated from rats are frequently used for tissue engineering research. However, considerable differences have been identified between rat mesenchymal stem cells and those derived from humans, and no defined panel of markers currently exists for the isolation of these cells. The aim of this study was to examine the effects of cell sorting for CD29+/CD90+ cells from rat adipose and bone marrow tissues on their differentiation and expression of stem cell–associated genes. Flow cytometry showed 66% and 78% CD29+/CD90+ positivity within passage 1 of adipose and bone marrow cultures, respectively. CD29+/CD90+ cells showed a reduction in both osteogenic and adipogenic differentiation when compared with unsorted cells, as determined by alizarin red and Oil Red-O staining, respectively. These findings could not entirely be explained by fluorescence-activated cell sorting–induced cell injury as sort recovery was only modestly affected in adipose-derived cells. Maintaining cells in fluorescence-activated cell sorting buffer did not affect adipose-derived cell viability, but a significant (p < 0.05) reduction was found in bone marrow–derived cell viability. Additionally, CD29+/CD90+ selection was associated with a significant decrease in the expression of Lin28, Sox2, Nanog and CD73 in adipose-derived cell cultures, whereas differences in stem cell–associated gene expression were not observed in sorted bone marrow–derived cell cultures. In summary, this study demonstrated that fluorescence-activated cell sorting had differential effects on adipose-derived cells and bone marrow–derived cells, and both CD29+/CD90+ cells displayed a significantly reduced capacity for osteogenic/adipogenic differentiation. In conclusion, we identify that maintaining heterogeneity within the mesenchymal stem cell population may be important for optimal differentiation.

Overexpression of Lin28 inhibits the proliferation, migration and cell cycle progression and induces apoptosis of BGC-823 gastric cancer cells.

Lin28 plays important roles in the development, maintenance of pluripotency and progression of various types of cancers. Lin28 represses the biogenesis of let-7 microRNAs and is implicated in both development and tumorigenesis. Oncogenic regulation of let-7 microRNAs has been demonstrated in several human malignancies, yet their correlation with Lin28 has not yet been studied in gastric cancer. Therefore, in the present study, we explored the possible mechanisms involved in the effects by Lin28 on the proliferation, migration, cell cycle arrest and apoptosis in gastric cancer cells via alteration of let-7 miRNA. The expression levels of Lin28 and let-7 were detected by real-time PCR in gastric cancer cell lines in vitro. Lin28 was overexpressed in the BGC-823 cells via lentiviral transfection, and let-7 expression was assessed. Cell proliferation and migration capabilities were investigated by MTT and Transwell assays, while cell cycle distribution and the apoptosis rate were detected using flow cytometry. The expression of Lin28 was moderately expressed in the GES cells while underexpressed in the BGC-823, SGC-7901 and HGC-27 cells. Let-7a miRNA was highly expressed in the GES, BGC-823, SGC-7901 and HGC-27 cells. Overexpression of Lin28 was inversely correlated with the downregulated expression of let-7a, and markedly suppressed the proliferation, migration, cell cycle progression and induced apoptosis in the BGC-823 cells. These findings demonstrated that overexpression of Lin28 can suppress the biological behavior of gastric cancer in vitro, and let-7 miRNA may play an important role in the process. We suggest that Lin28 may be a candidate predictor or an anticancer therapeutic target for gastric cancer patients.

Ectopic Expression of Lin28 Biases Myeloid Differentiation to Promote an Immature Mast Cell State and Is Implicated in Aggressive Mastocytosis

Dysregulated mast cell development leads to systemic mastocytosis, a clinically variable but often devastating family of hematologic disorders. Lin28 is a heterochronic gene and pluripotency factor implicated in many types of malignancy, and prior studies suggest that Lin28 expression can restore a fetal hematopoietic program in adult mice. However, the role of Lin28 in hematologic malignancy remains controversial. In our study, we induced expression of Lin28 in adult mice using a doxycycline-responsive transgenic system. Lin28 induction caused marked mast cell accumulation in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in Lin28-expressing hematopoietic progenitors, with increased levels of Lin28 in common myeloid progenitors and basophil-mast cell progenitors altering gene expression patterns to favor cell fate choices that enhance mast cell specification. In addition, Lin28-induced mast cells appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of mast cell terminal differentiation in the context of Lin28 upregulation. Finally, interrogation of human mast cell leukemia samples revealed upregulation of LIN28 in abnormal mast cells from patients with aggressive systemic mastocytosis (ASM). This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in mast cell disease. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Let-7 Maturation By Lin28b Controls Timing of Embryonic and Adult Myeloid Progenitor Phenotypes during Development

Within the mammalian fetal liver (FL) or adult bone marrow (ABM), terminally differentiated neutrophils, monocytes, and red blood cells emerge from a pool of multipotent common myeloid progenitors (CMPs). At steady state, the output of these cells is finally balanced but highly adaptable to the pathophysiologic requirements of the host. In the FL, erythroid output predominates to generate adequate oxygen transport for the metabolic demands of rapid organism growth in a hypoxic intrauterine environment; while granulocytic production comprises a larger balance of myelopoietic output in the ABM to provide innate immunity. The genetic regulators that specify timing of developmental stage-specific granulocytic/erythrocytic output from the myeloid progenitor pool have not been described.Initially defined as regulators of developmental timing in C. elegans, the mammalian Lin28a and Lin28b genes encode RNA binding-proteins capable of contributing to pluripotency induction in fully differentiated cells. Lin28 proteins exert their effects in part through inhibition of the maturation of the let-7 family of microRNAs - of which there are eight forms in mammals - that collectively drive cell terminal differentiation. Therefore, Lin28a/b expression is largely confined to embryonic tissues and is associated with the undifferentiated pluripotent or multipotent state in vivo. Given this role in regulation of multipotent cell function, we assessed the relative roles of theLin28/let-7 axis in developmental timing of myeloid progenitor phenotypes.We first analyzed patterns of let-7 expression during FL and ABM myelopoiesis. We found that several mature let-7 forms accumulated in CMPs during the transition from FL to ABM hematopoiesis (up to 30-fold induction as observed in let-7b in ABM compared to FL), coincident with 5-fold downregulation of the let-7 target and fetal-specific hematopoietic regulator Hmga2, indicating that the presence of mature let-7species may specify ABM-like myelopoiesis.To investigate this concept, we utilized a model of ectopic expression of LIN28B to block maturation of all let-7 species in ABM. Mice with activated LIN28B expression had a FL-like erythroid-dominant hematopoietic phenotype compared to controls (megakaryocyte-erythroid progenitor [MEP]:granulocyte-macrophage progenitor [GMP] ratio 3.9 ± 0.9 vs 0.8 ± 0.1, P = 0.005), along with blunted output of mature neutrophils in the ABM (P = 0.0001). Expression of LIN28B decreased formation of mature let-7 species in CMPs. Taken together, these results suggest that LIN28B expression reprograms ABM CMPs to a FL-like phenotype.To further define the Lin28-let-7 interaction in myelopoiesis, we analyzed the expression patterns of Lin28a and Lin28b as well as precursor let-7 microRNAs and their host genes during myeloid development. We observed up to 30-fold activation of precursor let-7 microRNAs and 4-fold activation of the polycistron encoding let-7b and let-7c2 coincident with 5-fold downregulation of Lin28b in CMPs during the transition from FL to ABM myelopoiesis. These results are consistent with mechanisms regulating levels of mature let-7 microRNAs at both the transcriptional and post-transcriptional level.We next characterized the effects of dysregulation of the Lin28b-let-7 interaction in the FL. Enforced expression of let-7g in mouse embryos drove increased granulocytic output in FL myeloid progenitors in culture (P = 0.001). While ectopic expression of Lin28 has been shown to confer fetal hematopoietic programs in other systems, the impact of loss of Lin28 genes on fetal hematopoiesis has not been reported. Fetal loss of Lin28b skewed the myeloid progenitor pool toward an ABM-like phenotype compared to wild-type animals (MEP:GMP 2.8 ± 0.5 vs. 4± 0.4, P = 0.1), though this phenotype is blunted due to the relative absence of let-7 precursors in the FL compared to the ABM, resulting in deregulated maturation of only a subset of mature let-7 forms. However, Lin28b-null FLs contained increased numbers of mature neutrophils (P = 0.005). Together, these data identify a Lin28/let-7 genetic switch with multiple levels of regulation that controls timing of the developmental myelopoietic phenotype. DisclosuresNo relevant conflicts of interest to declare.