HMGA2 Research Articles

CD271 determines migratory properties of melanoma cells

Melanoma cell expression of the nerve growth factor receptor CD271 is associated with stem-like properties. However, the contributing role of the receptor in melanoma cell migration is elusive. Here, we explored extracranial (skin, soft tissue, lymph node and liver, n = 13) and matched brain metastases (BM, n = 12) and observed a heterogeneous distribution of phenotypically distinct subsets of CD271+ cells. In addition, we observed that CD271 expression gradually rises along with melanoma progression and metastasis by exploration of publicly available expression data of nevi, primary melanoma (n = 31) and melanoma metastases (n = 54). Furthermore, we observed highest levels of CD271 in BM. Sub-clustering identified 99 genes differentially expressed among CD271high and CD271low (p < 0.05) BM-subgroups. Comparative analysis of subsets revealed increased ( ≥ 1.5fold, log2) expression of migration-associated genes and enrichment of CD271-responsible genes involved in DNA-repair and stemness. Live cell-imaging based scratch-wound assays of melanoma cells with stable knock-down of CD271 revealed a significantly reduced cell migration (3.9fold, p = 1.2E-04) and a reduced expression of FGF13, CSPG4, HMGA2 and AKT3 major candidate regulatory genes of melanoma cell migration. In summary, we provide new insights in melanoma cell migration and suggest that CD271 serves as a candidate regulator, sufficient to determine cellular properties of melanoma brain metastatic cells.

MiR-let-7a regulates anti-citrullinated protein antibody-induced macrophage activation and correlates with the development of experimental rheumatoid arthritis

Anti-citrullinated protein antibodies (ACPAs) are a key serological marker of rheumatoid arthritis (RA). To investigate whether miR-let7a downregulated ACPA-induced macrophage activation and arthritis, miR-let7a levels were assessed in the synovial fluid from patients with RA or osteoarthritis (OA). In addition, expression of the pro-inflammatory genes HMGA2, PI3K, and IRF5 was examined in ACPA-induced macrophages and a collagen antibody-induced mouse model of arthritis. As expected, miR-let7a expression in synovial fluid macrophages was substantially lower in patients with RA than in those with OA. Moreover, ACPAs treatment (160IU/mL) suppressed miR-let7a expression in macrophages isolated from patients with RA. Mechanistic studies revealed that miR-let7a directly targets HMGA2 to suppress ACPA-induced IRF5 expression through PI3K in macrophages. Further, miR-let7a expression was markedly decreased in swollen ankle tissue and splenocytes isolated from arthritic mice, whereas HMGA2, PI3K, and IRF5 expression positively correlated with disease severity. However, injection miR-let7a agomir was unable to mitigate the development of experimental arthritis in model mice. Collectively, these data demonstrated that miR-let7a directly targets HMGA2 to downregulate ACPAs-induced macrophage activation, and correlated with experimental RA severity.

A rare case of pediatric lipoma with t(9;12)(p22;q14) and evidence of HMGA2-NFIB gene fusion

Lipoma is a benign tumor, typically of adulthood, with characteristic cytogenetic findings, including rearrangement of 12q13-15; these rearrangements often lead to the fusion of the HMGA2 gene at this locus to the transcriptional regulatory domain of its fusion partner, resulting in neomorphic activity that presumably facilitates the neoplastic process. Herein, we report a rare case of pediatric lipoma with t(9;12)(p22;q14) and evidence of HMGA2-NFIB gene fusion in a 9 year-old boy. This case provides further evidence of the link between NFIB rearrangement and early-onset, deep-seated lipomatous tumors.

Let-7 Contributes to Diabetic Retinopathy but Represses Pathological Ocular Angiogenesis.

The in vivo function of microRNAs (miRs) in diabetic retinopathy (DR) and age-related macular degeneration (AMD) remains unclear. We report here that let-7 family members are expressed in retinal and choroidal endothelial cells (ECs). In ECs, overexpression of let-7 by adenovirus represses EC proliferation, migration, and networking in vitro, whereas inhibition of the let-7 family with a locked nucleic acid (LNA)-anti-miR has the opposite effect. Mechanistically, silencing of the let-7 target HMGA2 gene mimics the phenotype of let-7 overexpression in ECs. let-7 transgenic (let-7-Tg) mice show features of nonproliferative DR, including tortuous retinal vessels and defective pericyte coverage. However, these mice develop significantly less choroidal neovascularization (CNV) compared to wild-type controls after laser injury. Consistently, silencing of let-7 in the eye increased laser-induced CNV in wild-type mice. Together, our data establish a causative role of let-7 in nonproliferative diabetic retinopathy and a repressive function of let-7 in pathological angiogenesis, suggesting distinct implications of let-7 in the pathogenesis of DR and AMD.

Lin28B facilitates the progression and metastasis of pancreatic ductal adenocarcinoma.

Lin28B, a Lin28 homologue, represses the biogenesis of let-7 microRNAs (miRNAs), has a role in tumorigenesis, and is considered a potential therapeutic target for various human malignancies. However, the associations between Lin28B and the clinical features and outcomes of patients with pancreatic ductal adenocarcinoma (PDAC) remain unclear. In this study, we explored the clinical significance of Lin28B in PDAC and its association with metastasis by examining tissues from patients with PDAC and elucidated the molecular mechanisms using PDAC cell lines. In patients, high Lin28B expression was significantly correlated with high levels of lymphatic metastasis, distant metastasis and a poor prognosis. Furthermore, the multivariate analysis identified Lin28B expression as an independent prognostic factor in patients. In cell lines, stable silencing of Lin28B inhibited cell proliferation, cell cycle transition, migration and the epithelial-mesenchymal transition (EMT). It also increased the expression of the c-MYC, HMGA2 and KRAS genes, which are targeted by the cancer-suppressor miRNA let-7. Lin28B overexpression in the PDAC cell lines had the opposite effect. In human PDAC samples, high Lin28B expression was associated with decreased let-7 expression and increased c-MYC, HMGA2 and KRAS expression. Thus, Lin28B is a novel marker for predicting the prognosis of patients with PDAC and might be a potential therapeutic target for PDAC.

RNA related markers of thyroid cancer

In recent years, there is still an unbridgeable area in the early diagnosis of thyroid carcinoma. Many scholars are committed to the research of molecular markers, and find that RNA markers associated with thyroid carcinoma are microRNAs, human telomerase reverse transcripase, trefail factor 3 and HMGA2 genes. Different genes are closely related to the occurrence, development of different types of thyroid cancers, and some genes may even affect the transfer of thyroid carcinoma. Key words: Thyroid neoplasms; MicroRNAs

Abstract 5019: HMGA1 amplifies Wnt signaling and expands the intestinal stem cell compartment to drive premalignant polyposis in transgenic mice

Abstract Emerging evidence suggests that cancer cells undergo chromatin remodeling and epigenetic reprogramming to co-opt stem cell properties and drive tumor progression. The HMGA1 chromatin remodeling protein is an architectural transcription factor that binds to DNA at AT-rich sequences where it “opens” chromatin, recruits transcriptional complexes, and modulates gene expression. The HMGA1 gene is highly expressed during embryogenesis and in adult stem cells, but silenced postnatally in differentiated tissues. HMGA1 becomes re-expressed in most high-grade cancers and high levels portend adverse clinical outcomes. In colon cancer, HMGA1 is among the genes most highly overexpressed compared to normal intestinal epithelium. We previously reported that HMGA1 drives tumor progression in colon cancer by inducing stem cell genes involved in an epithelial-mesenchymal transition. We also discovered that Hmga1 transgenic mice develop marked proliferative changes and pre-malignant polyposis in the intestinal epithelium. To determine how Hmga1 functions in the intestines during tissue homeostasis and carcinogenesis, we examined in transgenic mice and organoid models. Here, we uncover a novel role for Hmga1 in maintaining the intestinal stem cell (ISC) pool and Paneth cell niche. Hmga1 is required by ISCs to organize into three-dimensional organoids in vitro; silencing Hmga1 disrupts organoid formation and bud development. Conversely, overexpression of Hmga1 increases organoid formation, bud development, and replating efficiency, suggesting that Hmga1 enhances ISC function and/or number. We therefore crossed the Hmga1 transgenic mice onto the Lgr5-EGFP background to enumerate ISCs and found that Hmga1 expands the ISC compartment. To determine how this occurs, we performed in vivo imaging and discovered that Hmga1 enhances self-renewal of ISCs. Mechanistically, we found that Hmga1 amplifies Wnt/β-catenin signaling by inducing genes encoding both Wnt agonist receptors and downstream Wnt target genes. Surprisingly, Hmga1 also expands the Paneth cell niche, which is comprised of terminally differentiated crypt cells that secrete Wnt to support ISCs. Because Paneth cells require Sox9 for development, we determined whether Hmga1 regulates its expression. Hmga1 binds directly to the Sox9 promoter at 2 AT-rich sites to activate its expression. In human colonic epithelium, HMGA1 and SOX9 are positively correlated, and both become markedly up-regulated in colon carcinogenesis. This work not only provides new insights into the role of Hmga1 in intestinal homeostasis by maintaining both the stem cell pool and epithelial niche compartment, but also suggests that deregulated Hmga1 perturbs this equilibrium during polyposis and carcinogenesis. Our results also highlight the HMGA1-WNT-SOX9 pathway as rational therapeutic target in colon carcinogenesis. Citation Format: Lingling Xian, Dan Georgess, Li Luo, Lionel Chia, Qihua Gu, Tait Huso, Amy Belton, David Huso, Andrew Ewald, Linda M.S. Resar. HMGA1 amplifies Wnt signaling and expands the intestinal stem cell compartment to drive premalignant polyposis in transgenic mice [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 5019. doi:10.1158/1538-7445.AM2017-5019

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

Scattered genomic amplification in dedifferentiated liposarcoma

BackgroundAtypical lipomatous tumor (ALT), well differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) are cytogenetically characterized by near-diploid karyotypes with no or few other aberrations than supernumerary ring or giant marker chromosomes, although DDLS tend to have somewhat more complex rearrangements. In contrast, pleomorphic liposarcomas (PLS) have highly aberrant and heterogeneous karyotypes. The ring and giant marker chromosomes contain discontinuous amplicons, in particular including multiple copies of the target genes CDK4, HMGA2 and MDM2 from 12q, but often also sequences from other chromosomes.ResultsThe present study presents a DDLS with an atypical hypertriploid karyotype without any ring or giant marker chromosomes. SNP array analyses revealed amplification of almost the entire 5p and discontinuous amplicons of 12q including the classical target genes, in particular CDK4. In addition, amplicons from 1q, 3q, 7p, 9p, 11q and 20q, covering from 2 to 14 Mb, were present. FISH analyses showed that sequences from 5p and 12q were scattered, separately or together, over more than 10 chromosomes of varying size. At RNA sequencing, significantly elevated expression, compared to myxoid liposarcomas, was seen for TRIO and AMACR in 5p and of CDK4, HMGA2 and MDM2 in 12q.ConclusionsThe observed pattern of scattered amplification does not show the characteristics of chromothripsis, but is novel and differs from the well known cytogenetic manifestations of amplification, i.e., double minutes, homogeneously staining regions and ring chromosomes. Possible explanations for this unusual distribution of amplified sequences might be the mechanism of alternative lengthening of telomeres that is frequently active in DDLS and events associated with telomere crisis.

Clinical and molecular characterization of a second family with the 12q14 microdeletion syndrome and review of the literature.

The 12q14 microdeletion syndrome is a rare condition characterized by low birth weight, failure to thrive, short stature, learning disabilities, and osteopoikilosis. To date, 20 cases of 12q14 deletion have been reported in the literature, displaying both phenotypic than genetic variability. We report on three familial cases, a mother and two brothers, with severe short stature. The mother and elder brother presented with osteopoikilosis while the younger brother had severe short stature and developmental delay. SNP array analysis revealed a 1.9 Mb heterozygous 12q14.2q14.3 deletion in all three patients encompassing 14 genes and 3 miRNAs. In addition, the younger brother carried a paternal 11q13.4 duplication including the SHANK2 gene. This latter patient was investigated for developmental delay and did not show osteopoikilosis, confirming the role of age in the clinical presentation of this condition. To the best of our knowledge, this is the second family described with the syndrome. Comparing the clinical and molecular data of our patients with those previously reported we performed a detailed genotype-phenotype correlation confirming the association between growth retardation and osteopoikilosis when the rearrangement includes both LEMD3 and HMGA2 genes. In addition, we suggest the XPOT, TBK1, WIF1 genes as candidates for the clinical features observed in our patients and discuss for the first time the possible involvement of some microRNAs, when deleted, in the etiology of the phenotypes in 12q14 microdeletion syndrome patients. We expect the interpretation of our findings to be useful both from a molecular point of view and for genetic counseling.

Alteration of the exDNA profile in blood serum of LLC-bearing mice under the decrease of tumour invasion potential by bovine pancreatic DNase I treatment.

Taking into account recently obtained data indicating the participation of circulating extracellular DNA (exDNA) in tumorigenesis, enzymes with deoxyribonucleic activity have again been considered as potential antitumour and antimetastatic drugs. Previously, using murine Lewis lung carcinoma and hepatocellular carcinoma A1 tumour models, we have shown the antimetastatic activity of bovine DNase I, which correlates with an increase of DNase activity and a decrease of exDNA concentration in the blood serum of tumour-bearing mice. In this work, using next-generation sequencing on the ABS SOLiD™ 5.500 platform, we performed a search for molecular targets of DNase I by comparing the exDNA profiles of healthy animals, untreated animals with Lewis lung carcinoma (LLC) and those with LLC treated with DNase I. We found that upon DNase I treatment of LLC-bearing mice, together with inhibition of metastasis, a number of strong alterations in the patterns of exDNA were observed. The major differences in exDNA profiles between groups were: i) the level of GC-poor sequences increased during tumour development was reduced to that of healthy mice; ii) levels of sequences corresponding to tumour-associated genes Hmga2, Myc and Jun were reduced in the DNase I-treated group in comparison with non-treated mice; iii) 224 types of tandem repeat over-presented in untreated LLC-bearing mice were significantly reduced after DNase I treatment. The most important result obtained in the work is that DNase I decreased the level of B-subfamily repeats having homology to human ALU repeats, known as markers of carcinogenesis, to the level of healthy animals. Thus, the obtained data lead us to suppose that circulating exDNA plays a role in tumour dissemination, and alteration of multiple molecular targets in the bloodstream by DNase I reduces the invasive potential of tumours.

Dwarfism and Altered Craniofacial Development in Rabbits Is Caused by a 12.1 kb Deletion at the HMGA2 Locus.

The dwarf phenotype characterizes the smallest of rabbit breeds and is governed largely by the effects of a single dwarfing allele with an incompletely dominant effect on growth. Dwarf rabbits typically weigh under 1 kg and have altered craniofacial morphology. The dwarf allele is recessive lethal and dwarf homozygotes die within a few days of birth. The dwarf phenotype is expressed in heterozygous individuals and rabbits from dwarf breeds homozygous for the wild-type allele are normal, although smaller when compared to other breeds. Here, we show that the dwarf allele constitutes a ∼12.1 kb deletion overlapping the promoter region and first three exons of the HMGA2 gene leading to inactivation of this gene. HMGA2 has been frequently associated with variation in body size across species. Homozygotes for null alleles are viable in mice but not in rabbits and probably not in humans. RNA-sequencing analysis of rabbit embryos showed that very few genes (4-29 genes) were differentially expressed among the three HMGA2/dwarf genotypes, suggesting that dwarfism and inviability in rabbits are caused by modest changes in gene expression. Our results show that HMGA2 is critical for normal expression of IGF2BP2, which encodes an RNA-binding protein. Finally, we report a catalog of regions of elevated genetic differentiation between dwarf and normal-size rabbits, including LCORL-NCAPG, STC2, HOXD cluster, and IGF2BP2 Levels and patterns of genetic diversity at the LCORL-NCAPG locus further suggest that small size in dwarf breeds was enhanced by crosses with wild rabbits. Overall, our results imply that small size in dwarf rabbits results from a large effect, loss-of-function (LOF) mutation in HMGA2 combined with polygenic selection.

Myolipoma of Soft Tissue: Clinicopathologic Analysis of 34 Cases.

Myolipoma of soft tissue, which was first described by Meis and Enzinger (1991), is a rare benign neoplasm characterized by the admixture of mature adipocytes and well-differentiated smooth muscle cells. Recently, cytogenetic alteration of the HMGA2 gene has been reported in 2 myolipomas. We present the clinicopathologic features of 34 cases of myolipoma of soft tissue, study immunoreactivity for HMGA2, and review the previous literature. In our series, there were 32 women and 2 men, with age at presentation ranging from 35 to 94 years (median, 55 y). The most frequently affected site was retroperitoneum (47%), followed by pelvis (15%), abdominal wall (12%), and intra-abdominal sites (9%). Follow-up information was available for 17 patients (50%), ranging from 1 to 202 months (mean, 41 mo). None has developed local recurrence or metastasis. Grossly, tumors were well circumscribed, and the cut surface showed an admixture of yellowish adipose tissue and tan-whitish nodules. The size ranged from 2.4 to 60 cm (median 10.5 cm). Histologically, the tumors were composed of an intimate admixture of mature fat cells and bland spindle-shaped cells with brightly eosinophilic cytoplasm, arranged in fascicles. Some cases showed the following unusual features focally: hypercellular fascicular pattern (N=2), degenerative nuclear atypia (N=1), round cell morphology (N=1), hemosiderin deposition (N=1), metaplastic cartilage (N=1), metaplastic bone (N=1), and eosinophil infiltrates (N=1). Immunohistochemically, spindle cells showed strong and diffuse positivity for desmin (26/26 cases), SMA (20/21), and ER (13/15). Nuclear positivity for HMGA2 was identified in 15 of 25 cases (60%). MDM2 and CDK4 were usually negative (14/15, 8/9, respectively). In summary, myolipoma of soft tissue is a distinctive benign tumor composed of mature fat cells and smooth muscle cells and arises most commonly in deep-seated locations of middle-aged women. In our study, 60% of cases showed nuclear staining for HMGA2 by immunohistochemistry, which supports the possibility that these tumors harbor aberration of the HMGA2 gene, as seen in lipomas and leiomyomas elsewhere.

Features of miRNA binding with mRNA of candidate genes of breast cancer subtypes

To determine associations of miRNA and mRNA of their target genes, binding characteristics of miRNA and mRNA of candidate genes of four subtypes of the breast cancer have been studied. Half of candidate genes of the triple-negative subtype had binding sites for several miRNAs. mRNA of ATM gene had seven binding sites for miR-5095, miR-619-5p, miR-5096, miR-5585-3p, miR-1273a, miR-1273g-3p, all of which bind in the 3'UTR. mRNA of AXL gene, the tyrosine kinase receptor, had binding sites for five miRNAs that are localized in 3'UTR, CDS, and 5'UTR. From five miRNA, miR-1908-3p may the most effective regulated the expression of protooncogene CBL. mRNA of СЕАСАМ5 gene contained binding sites of miR-5095, miR-619-5p, miR-5585-3p with a high degree of complementarity. mRNA of F2RL1, IAPP genes have binding sites predominantly for miR-5095, miR-619-5p, miR-5585-3p, miR-5096. Based on the obtained data, it is necessary to control the expression of candidate genes of the triple-negative subtype with miR-5095, miR-619-5p, miR-5585-3p, miR-5096 and miR-1273a, miR-1273e, miR-1273g-3p. A high free binding energy was detected for pairs of miR-6089 and triple-negative subtype RUNX1 and SFN candidate genes mRNA. mRNA of IL11, MAGEA10 and STMN1 genes had binding sites of miR-619-5p and miR-1273a, miR-1273d, miR-1273e, miR-1273f. &nbsp;mRNA of the subtype her2 candidate genes ADAM17, AURKA and BRCA2 strongly bind miR-619-5p. mRNA of BRIP1 gene has sites for miR-1285-5p, miR-5095, miR-619-5p, miR-5585-3p, miR-1273a, miR-1273g-3p. mRNA of CDK6 gene has binding sites for miR-548 family and multiple sites for miR-466. The presence of such binding sites in mRNA of CDK6 gene several times increases the probability of its interaction with these miRNAs. The key candidate gene ERBB3 of the her2 subtype interacts with miR-619-5p with high complementarity. 12 miRNAs can bind to mRNA of MAZ gene, binding sites are located in 5'UTR and CDS. mRNA of candidate genes of the subtype luminal A, B can bind: HMGA2 gene - five miRNA, MAPT gene - six miRNA, SMAD3 gene - four miRNA, TGFB1 gene - six miRNA. mRNA of TGFB1 and SMAD3 genes had four and three effective miR-6089 binding sites, respectively. A special feature of candidate genes of the subtype luminal A, B is the absence in their mRNA binding sites of the unique miRNA family miR-1273 and group miR-5095, miR-619-5p, miR-5585-3p, miR-5096, miR-1285-5p.

ANALYSIS OF RELATIVE EXPRESSION OF THE HMGA2 GENE AND ONCOGENIC MICRORNA-221 IN CYTOLOGICAL SLIDES OBTAINED BY A FINE-NEEDLE ASPIRATION BIOPSY OF THE THYROID NODULES

Background. Fine-needle aspiration biopsy is recognized as the “gold standard” in the preoperative diagnosis of thyroid cancer. However, this method does not always allow reliable differentiation between benign and malignant thyroid nodules. Thus, the cytological report of a “follicular neoplasm or suspicious for a follicular neoplasm” suggests surgical lobectomy. However, in most cases, this conclusion does not point to follicular cancer but to benign neoplasm, follicular adenoma, where the surgical intervention is excessive. This implies the great relevance of finding biological markers capable of enhancing the specificity of detecting malignant neoplasms. Such markers may include an increase in the level of expression of oncogenes or a change in the expression of microRNA, since it is known that the content of a number of microRNAs changes significantly during the development of thyroid tumors. Materials and methods. The expression level of the HMGA2 gene, normally active at the embryonic stage, and oncogenic miRNA-221 microRNA was analyzed using real-time reverse transcription polymerase chain reaction in 713 cytological preparations of the thyroid gland (stained material dried on glasses) obtained by standard fine-needle aspiration biopsy. The sample included preparations corresponding to different cytological diagnoses: benign (n = 375), follicular neoplasm or suspicious for a follicular neoplasm (n = 143), medullary carcinoma (n = 7), papillary carcinoma (n = 186) and anaplastic carcinoma (n = 2). Results. The content of messenger RNA (mRNA) HMGA2 (P = 1.6 × 10–66, area under curve ROC 0.946) and miR-221 (P = 6.3 × 10 –61 , area under curve ROC 0.927) proved to be significantly elevated in papillary carcinoma compared to benign tumors. Follicular neoplasms showed significant heterogeneity in the quantity of both molecular markers. At the same time, an elevated level of miR-221 (3 times on average) was also characteristic of samples from this group with an increased level of expression of the HMGA2 gene (an average of 85 times). With regard to the quantity of these markers, the group of follicular neoplasms, in which no increase in the HMGA2 mRNA level was detected, did not differ from the group of benign nodules. Conclusions. The obtained results show that assessing the expression of HMGA2 mRNA and oncogenic miR-221 makes it possible to differentiate the papillary thyroid carcinoma from goiter at the preoperative stage, and based on the content of the HMGA2 mRNA, the group of follicular neoplasms can be divided into subgroups presumably differing in the risk of malignancy.

Abstract B16: Loss of SIRT6 reactivates the RNA-binding protein Lin28b to drive pancreatic cancer

Abstract Chromatin remodeling proteins are frequently dysregulated in human cancer; however, little is known about how they control tumorigenesis. The NAD+ dependent histone deacetylase Sirtuin 6 (SIRT6) is a nutrient sensor that reprograms the epigenome in response to nutrient stress and shows reduced expression in human pancreatic ductal adenocarcinoma (PDAC) relative to normal tissue. Thus, we aimed to determine the role of SIRT6 in PDAC and to determine how the loss of this chromatin modifier could influence PDAC formation, progression and metastasis. Here, we uncover an epigenetic program mediated by SIRT6 that is critical for the suppression of PDAC. We demonstrate that SIRT6 inactivation cooperates with oncogenic KRAS to greatly accelerate the development of lethal pancreatic tumors regardless of p53 status. In addition to developing PDAC and high-grade pancreatic intraepithelial neoplasia (PanIN) at an earlier age, SIRT6-deficient tumors had a greater propensity to metastasize to the lung, compared to their SIRT6 wild-type (WT) counterparts. Through genome-wide profiling of SIRT6 WT and knock-out (KO) PDAC cell lines, we identified the oncofetal RNA-binding protein Lin28b as a target of Sirt6-mediated transcriptional repression. Loss of SIRT6 in both murine and human PDAC cell lines resulted in hyperacetylation of histone 3 lysine 9 and lysine 56 at the Lin28b promoter, MYC recruitment, and pronounced induction of Lin28b expression. Knocking down Lin28b resulted in potent suppression of cell proliferation and tumor sphere formation in Sirt6null/low murine and human cell lines, while murine and human Sirt6WT/high PDAC lines were completely insensitive to the same treatment. Functionally, Lin28b binds to and induces the degradation of the let-7 family of microRNA, thus resulting in increased expression of let-7 target genes such as the IGF2BPs and HMGA2, which have been correlated with increased agressiveness and metastasis in pancreatic tumors. Furthermore, Gene Set Enrichment Analysis (GSEA) comparing PDAC tumors and cell lines with high versus low expression of LIN28B revealed that LIN28Bhigh tumors were strongly enriched for the expression of let-7 targets. Finally, either loss of SIRT6 or the reactivation of Lin28b or the let-7 target genes IGF2BP3 and HMGA2 correlated with a worse overall survival in human PDAC patients. Thus, our critical findings define SIRT6 as a tumor suppressor in PDAC, working as a transcriptional repressor of the oncofetal RNA-binding protein Lin28b. We demonstrate that loss of Sirt6 creates a permissive hyperacetylated chromatin state, allowing for MYC-dependent transactivation of Lin28b. This work not only provides new insights into the epigenetic mechanisms governing the reactivation of oncofetal proteins in cancer but also demonstrates that this pathway is well-conserved in human PDAC, where reduced SIRT6 expression defines a specific subset of tumors which may benefit from novel therapeutic approaches aimed at targeting components of the LIN28B/let-7 pathway. This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section PR09 of the Conference Proceedings. Citation Format: Sita Kugel, Carlos Sebastián, Julien Fitamant, Kenneth N. Ross, Supriya K. Saha, Esha Jain, Adrianne Gladden, Kshitij S. Arora, Yasutaka Kato, Miguel N. Rivera, Sridhar Ramaswamy, Ruslan I. Sadreyev, Alon Goren, Vikram Deshpande, Nabeel Bardeesy, Raul Mostoslavsky.{Authors}. Loss of SIRT6 reactivates the RNA-binding protein Lin28b to drive pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B16.

Abstract PR09: Loss of SIRT6 reactivates the RNA-binding protein lin28b to drive pancreatic cancer

Abstract Chromatin remodeling proteins are frequently dysregulated in human cancer; however, little is known about how they control tumorigenesis. The NAD+ dependent histone deacetylase Sirtuin 6 (SIRT6) is a nutrient sensor that reprograms the epigenome in response to nutrient stress and shows reduced expression in human pancreatic ductal adenocarcinoma (PDAC) relative to normal tissue. Thus, we aimed to determine the role of SIRT6 in PDAC and to determine how the loss of this chromatin modifier could influence PDAC formation, progression and metastasis. Here, we uncover an epigenetic program mediated by SIRT6 that is critical for the suppression of PDAC. We demonstrate that SIRT6 inactivation cooperates with oncogenic KRAS to greatly accelerate the development of lethal pancreatic tumors regardless of p53 status. In addition to developing PDAC and high-grade pancreatic intraepithelial neoplasia (PanIN) at an earlier age, SIRT6-deficient tumors had a greater propensity to metastasize to the lung, compared to their SIRT6 wild-type (WT) counterparts. Through genome-wide profiling of SIRT6 WT and knock-out (KO) PDAC cell lines, we identified the oncofetal RNA-binding protein Lin28b as a target of Sirt6-mediated transcriptional repression. Loss of SIRT6 in both murine and human PDAC cell lines resulted in hyperacetylation of histone 3 lysine 9 and lysine 56 at the Lin28b promoter, MYC recruitment, and pronounced induction of Lin28b expression. Knocking down Lin28b resulted in potent suppression of cell proliferation and tumor sphere formation in Sirt6null/low murine and human cell lines, while murine and human Sirt6WT/high PDAC lines were completely insensitive to the same treatment. Functionally, Lin28b binds to and induces the degradation of the let-7 family of microRNA, thus resulting in increased expression of let-7 target genes such as the IGF2BPs and HMGA2, which have been correlated with increased aggressiveness and metastasis in pancreatic tumors. Furthermore, Gene Set Enrichment Analysis (GSEA) comparing PDAC tumors and cell lines with high versus low expression of LIN28B revealed that LIN28Bhigh tumors were strongly enriched for the expression of let-7 targets. Finally, either loss of SIRT6 or the reactivation of Lin28b or the let-7 target genes IGF2BP3 and HMGA2 correlated with a worse overall survival in human PDAC patients. Thus, our critical findings define SIRT6 as a tumor suppressor in PDAC, working as a transcriptional repressor of the oncofetal RNA-binding protein Lin28b. We demonstrate that loss of Sirt6 creates a permissive hyperacetylated chromatin state, allowing for MYC-dependent transactivation of Lin28b. This work not only provides new insights into the epigenetic mechanisms governing the reactivation of oncofetal proteins in cancer but also demonstrates that this pathway is well-conserved in human PDAC, where reduced SIRT6 expression defines a specific subset of tumors which may benefit from novel therapeutic approaches aimed at targeting components of the LIN28B/let-7 pathway. This abstract is also being presented as Poster B16 Citation Format: Sita Kugel, Carlos Sebastián, Julien Fitamant, Kenneth N. Ross, Supriya K. Saha, Esha Jain, Adrianne Gladden, Kshitij S. Arora, Yasutaka Kato, Miguel N. Rivera, Sridhar Ramaswamy, Ruslan I. Sadreyev, Alon Goren, Vikram Deshpande, Nabeel Bardeesy, Raul Mostoslavsky.{Authors}. Loss of SIRT6 reactivates the RNA-binding protein lin28b to drive pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr PR09.

Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice

It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing. We performed a genome-wide CRISPR/Cas9-based knockout screen of P53-null mouse embryonic liver progenitor cells that overexpressed MYC. We infected p53-/-;Myc;Cas9 hepatocytes with the mGeCKOa lentiviral library of 67,000 single-guide RNAs (sgRNAs), targeting 20,611 mouse genes, and transplanted the transduced cells subcutaneously into nude mice. Within 1 month, all the mice that received the sgRNA library developed subcutaneous tumors. We performed high-throughput sequencing of tumor DNA and identified sgRNAs increased at least 8-fold compared to the initial cell pool. To validate the top 10 candidate tumor suppressors from this screen, we collected data from patients with hepatocellular carcinoma (HCC) using the Cancer Genome Atlas and COSMIC databases. We used CRISPR to inactivate candidate tumor suppressor genes in p53-/-;Myc;Cas9 cells and transplanted them subcutaneously into nude mice; tumor formation was monitored and tumors were analyzed by histology and immunohistochemistry. Mice with liver-specific disruption of p53 were given hydrodynamic tail-vein injections of plasmids encoding Myc and sgRNA/Cas9 designed to disrupt candidate tumor suppressors; growth of tumors and metastases was monitored. We compared gene expression profiles of liver cells with vs without tumor suppressor gene disrupted by sgRNA/Cas9. Genes found to be up-regulated after tumor suppressor loss were examined in liver cancer cell lines; their expression was knocked down using small hairpin RNAs, and tumor growth was examined in nude mice. Effects of the MEK inhibitors AZD6244, U0126, and trametinib, or the multi-kinase inhibitor sorafenib, were examined in human and mouse HCC cell lines. We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation. Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.

131 EFFECT OF PROLONGED PROGESTERONE EXPOSURE OF BEEF COWS ON THE EXPRESSION OF OOCYTE DEVELOPMENTAL COMPETENCE-ASSOCIATED GENES

Progesterone is a key hormone in reproduction and plays a crucial regulatory role during the oestrous cycle and pregnancy. The effect of progesterone on the developmental competence of bovine oocytes is yet to be fully understood. Molecular tools might provide further information on the role of progesterone on expression of developmentally important genes. The objective of this study was to evaluate the effect of the prolonged exposure of beef cows (days = 32) to different progesterone levels on the expression of genes associated with oocyte developmental competence. Fifteen cows were used in the experiment and were divided in 3 treatment groups (group n = 5). Treatment 1 consisted of no progesterone supplementation, treatment 2 consisted of supplementation by insertion of one controlled internal drug release device (CIDR) per animal, and treatment 3 consisted of supplementation with the insertion of 2 CIDR per animal. All cows were subjected to follicle ablation 4 days before initiation of the experiment. At Day 0, prostaglandin F2α (PGF2α, 25 mg of dinoprost tromethamine, i.m., Lutalyze®) was administered and CIDR devices were inserted in cows designated to treatments 2 and 3. Cumulus-oocyte complexes were collected through ovum pickup where follicles &gt;2 mm were aspirated. Samples evaluated were obtained at Day 0, 8, 16, 24, and 32. The oocyte developmental competence-associated genes METAP2, FOXM1, HMGA1 were analysed in the study. Polyadenylate polymerase was used as normalization gene. Relative gene expression was calculated utilising a comparative CT method (2−ΔΔCT) where data were calculated as fold increase (&gt;1) or decrease (&lt;1) from a calibrator included in each PCR reaction. Mean values of the fold increase/decrease were compared between treatments and were analysed by repeated-measures ANOVA. No statistical difference was detected between treatments at Day 0, 8, 16, 24, or 32 when analysing independent relative expression of oocyte developmental competence-associated genes METAP2, FOXM1, or HMGA1 (P &gt; 0.05). Similarly, no significant differences between treatments was detected in relative expression of METAP2, FOXM1, or HMGA1 across time (P &gt; 0.05). Prolonged exposure of progesterone at different levels does not affect the expression of METAP2, FOXM1, and HMGA1 genes in bovine cumulus-oocyte complexes as indicated by the results of this experiment.

HMGA1P7-pseudogene regulates H19 and Igf2 expression by a competitive endogenous RNA mechanism.

Recent studies have revealed that pseudogene transcripts can function as competing endogenous RNAs, and thereby can also contribute to cancer when dysregulated. We have recently identified two pseudogenes, HMGA1P6 and HMGA1P7 for the HMGA1 gene whose overexpression has a critical role in cancer progression. These pseudogenes work as competitive endogenous RNA decoys for HMGA1 and other cancer related genes suggesting their role in carcinogenesis. Looking for new HMGA1 pseudogene ceRNAs, we performed RNA sequencing technology on mouse embryonic fibroblasts deriving from transgenic mice overexpressing HMGA1P7. Here, we report that HMGA1P7 mRNA sustains the H19 and Igf2 overexpression by acting as miRNA decoy. Lastly, the expression of HMGA1P7 was significantly correlated with H19 and IGF2 levels in human breast cancer thereby suggesting a role for HMGA1P7 deregulation in this neoplasia.