Krystian Jażdżewski

Polymorphic mature microRNAs from passenger strand of pre-miR-146a contribute to thyroid cancer

Prior work has shown that heterozygosity G/C of single nucleotide polymorphism (SNP rs2910164) within the precursor of microRNA-146a predisposes to PTC (odds ratio = 1.62, P = 0.000007) although the mechanism was unclear. Here, we show that GC heterozygotes differ from both GG and CC homozygotes by producing 3 mature microRNAs: 1 from the leading strand (miR-146a), and 2 from the passenger strand (miR-146a*G and miR-146a*C), each with its distinct set of target genes. TaqMan analysis of paired tumor/normal samples revealed 1.5- to 2.6-fold overexpression of polymorphic miR-146a* in 7 of 8 tumors compared with the unaffected part of the same gland. The microarray data showed that widely different transcriptomes occurred in the tumors and in unaffected parts of the thyroid from GC and GG patients. The modulated genes are mainly involved in regulation of apoptosis leading to exaggerated DNA-damage response in heterozygotes potentially explaining the predisposition to cancer. We propose that contrary to previously held views transcripts from the passenger strand of miRs can profoundly affect the downstream effects. Heterozygosity for polymorphisms within the premiR sequence can cause epistasis through the production of additional mature miRs. We propose that mature miRs from the passenger strand may regulate many genetic processes.

Next generation sequencing reveals microRNA isoforms in liver cirrhosis and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents the major histological subtype of liver cancer. Tumorigenic changes in hepatic cells potentially result from aberrant expression of microRNAs (miRNAs). Individual microRNA gene may give rise to miRNAs of different length, named isomiRNAs that proved to be functionally relevant. Since microRNA length heterogeneity in hepatic tissue has not been described before, we employed next-generation sequencing to comprehensively analyze microRNA transcriptome in HCC tumors (n=24) and unaffected tissue adjacent to tumors (n=24), including samples with (n=15) and without cirrhosis (n=9). We detected 374 microRNAs expressed in liver, including miR-122-5p that constituted over 39% of the hepatic miRnome. Among the liver expressed miRs, the levels of 64 significantly differed between tumor and control samples (FDR<0.05, fold change>2). Top deregulated miRNAs included miR-1269a (T/N=22.95), miR-3144-3p (T/N=5.24), miR-183-5p (T/N=4.63), miR-10b-5p (T/N=3.87), miR-490-3p (T/N=0.13), miR-199a-5p (T/N=0.17), miR-199a-3p/miR-199b-3p (T/N=0.19), miR-214-5p (T/N=0.20) and miR-214-3p (T/N=0.21). Almost all miRNA genes produced several mature molecules differing in length (isomiRNAs). The reference sequence was not the most prevalent in 38.6% and completely absent in 10.5% of isomiRNAs. Over 26.1% of miRNAs produced isoforms carrying≥2 alternative seed regions, of which 35.5% constituted novel, previously unknown seeds. This fact sheds new light on the percentage of the human genome regulated by microRNAs and their variants. Among the most deregulated miRNAs, miR-199a-3p/miR-199b-3p (T/N fold change=0.18, FDR=0.005) was expressed in 9 isoforms with 3 different seeds, concertedly leading to upregulation of TGF-beta signaling pathway (OR=1.99; p=0.004). In conclusion, the study reveals the comprehensive miRNome of hepatic tissue and provides new tools for investigation of microRNA-dependent pathways in cirrhotic liver and hepatocellular carcinoma. This article is part of a Directed Issue entitled: Rare Cancers.

SRGAP1 Is a Candidate Gene for Papillary Thyroid Carcinoma Susceptibility

BACKGROUND Papillary thyroid carcinoma (PTC) shows high heritability, yet efforts to find predisposing genes have been largely negative. OBJECTIVES The objective of this study was to identify susceptibility genes for PTC. METHODS A genome-wide linkage analysis was performed in 38 families. Targeted association study and screening were performed in 2 large cohorts of PTC patients and controls. Candidate DNA variants were tested in functional studies. RESULTS Linkage analysis and association studies identified the Slit-Robo Rho GTPase activating protein 1 gene (SRGAP1) in the linkage peak as a candidate gene. Two missense variants, Q149H and A275T, localized in the Fes/CIP4 homology domain segregated with the disease in 1 family each. One missense variant, R617C, located in the RhoGAP domain occurred in 1 family. Biochemical assays demonstrated that the ability to inactivate CDC42, a key function of SRGAP1, was severely impaired by the Q149H and R617C variants. CONCLUSIONS Our findings suggest that SRGAP1 is a candidate gene in PTC susceptibility. SRGAP1 is likely a low-penetrant gene, possibly of a modifier type.

Genomic sequence matters: a SNP in microRNA-146a can turn anti-apoptotic.

Transcription factor NFκB is a key regulator of immune response. It also is constitutively active in many cancers as a major activator of anti-apoptotic gene expression that prolongs cancer cell survival by enhancing such features as proliferation, angiogenesis and metastasis.1,2 NFκB is activated in numerous inflammation-driven cancers, e.g., colitis-associated colorectal cancer, hepatocellular carcinoma and lymphoma of mucosal-associated lymphoid tissue. Moreover, it also has a role in tumors such as thyroid carcinoma in which association with immune response is not obvious. NFκB is activated in all subtypes of human thyroid neoplasms. The activity of NFκB is particularly strong in poorly differentiated carcinoma. In thyroid cancer cell lines it correlates with resistance to drug-induced apoptosis.3 In undifferentiated thyroid carcinoma, activation of NFκB by tumor necrosis factor alpha (TNF-α) led to cytomorphologic differentiation of cancerous cells, and induction of thyroid-specific secretion of thyroglobulin.4 The influence of microRNAs, especially miR-155 and miR-146, on the immune response has been extensively studied.5 That miR-146a might be up-regulated by NFκB was recently shown.6 NFκB induces the expression of miR-146a upon ligation of toll-like receptors (TLR2, TLR4 or TLR5) as well as stimulation by TNF-α or interleukin 1 β.7–9 It is suggested that miR-146a serves as an important negative regulator of the TLR and NFκB signaling pathway as the IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 genes (TRAF6) were proved to be direct targets of miR-146a.6 Thus, microRNA-146a seems to function as a pro-apoptotic molecule by inhibiting the NFκB pathway and blocking its impact on cell proliferation, tumor-progression and cancer cell survival. When expressed in the highly metastatic human breast cancer cell line MDA-MB-231 microRNA-146a leads to impaired NFκB activity. Overexpression of miR-146 through inhibition of the NFκB pathway led to impaired invasion and migration in vitro and suppressed experimental lung metastasis in mice.10 MiR-146 has been reported to be highly expressed in breast, pancreatic, ovarian, stomach and thyroid cancer. In papillary thyroid carcinoma (PTC) miR-146a is up-regulated 19 fold11 and its expression is dependent on a sequence variation.12 A single nucleotide polymorphism (SNP) within pre-miR-146a (rs2910146; G/C) seems to disrupt the pro-apoptotic function of the miR by decreasing the total amount of mature miR-146a with consequent reduced inhibition of miR target genes such as IRAK1 and TRAF6.12,13 In an association study of 608 PTC patients and 901 controls, the GC heterozygous state was associated with an increased risk of acquiring PTC (odds ratio = 1.62, p = 0.000007), whereas both homozygous states (GC and CC) were protective with odds ratio = 0.42 for the CC genotype (p = 0.003) and odds ratio = 0.69 for the GG genotype (p = 0.0006). Moreover, 4.7% of informative tumors had undergone somatic mutations of the SNP sequence probably as a step in the clonal selection during carcinogenesis.12 Heterozygosity as a genetic risk rather than either homozygosity is a rare phenomenon (referred to as overdominance) and should be critically evaluated. This led to the observation that the passenger strand of the pre-miR produces mature miR. Thus, in GC heterozygotes totally 3 mature miRs were produced (main strand; passenger strand G, passenger strand C), each with distinct target genes.14 To begin to unravel the functional consequences on target genes a microarray expression analysis was performed on thyroid tumors from 16 PTC patients who were either GG or GC, revealing significantly different transcriptomes. In particular, the genes involved in regulation of apoptosis were differentially transcribed in heterozygotes compared to GG homozygotes (44 genes, p=0.0001). Moreover, among genes differentially regulated in heterozygotes 12 genes were designated “positive regulators of NFκB” (p=0.009).14 In unstimulated cells, NFκB is retained in the cytoplasm by specific inhibitors, the IκB proteins. Stimulation with the proinflammatory cytokines, TNF-α or IL-1, induces IκB phosphorylation and ubiquitin-dependent proteasomal degradation resulting in nuclear entry of NFκB dimers to initiate target gene transcription. IκB phosphorylation is catalyzed by the IκB kinase (IKK) complex.15 In the study described above,14 numerous genes leading to activation of NFκB were up-regulated in heterozygotes, as were many genes targeted by NFκB including the proinflammatory chemokines IL8 and CFLAR (Table 1). Caspase 4 and caspase 8 were also up-regulated; however in the presence of induced CFLAR the latter was most likely inactive.14 Table 1 Impact of the miR-146a polymorphism (GC vs. GG) on the expression of genes involved in the NF-κB pathway and in the regulation of apoptosis Thus, these results suggest significantly higher activation of the NFκB pathway in GC heterozygotes of miR-146a as compared to GG homozygotes. We speculate that this phenomenon might promote tumor progression and be responsible for the increased risk of PTC in heterozygotes. A possible stimulus for an induction of the NFκB pathway in thyroid might be ionizing radiation, the strongest environmental risk factor for thyroid cancer. Ionizing radiation is also used for the treatment of thyroid cancer. It induces DNA damage, e.g. double strand DNA breaks, and leads to significant activation of p53 and apoptosis. NFκB is rapidly activated in the course of an immediate early response of thyroid cancer cells to ionizing radiation leading to increased thyroid cell survival. In an undifferentiated thyroid cancer cell line inhibition of NFκB increased radiosensitivity and enhanced ionizing radiation-induced apoptosis.16 Cells that do not undergo apoptosis after ionizing radiation are prone to genetic instability, increased rate of mutation, and accelerated cancer evolution. We suggest that thyroid cells heterozygous at miR-146a present higher activity of NFκB and lower potential of inhibition of this pathway and therefore are more likely to survive after ionizing radiation.

MicroRNA-related sequence variations in human cancers

Abstract MicroRNAs are emerging as a most promising field in basic and translational research, explaining the pathogenesis of numerous human diseases and providing excellent tools for their management. This review considers the effects of microRNA sequence variations and their implication in pathogenesis and predisposition to human cancers. Although the role of microRNAs still remains to be elucidated, functional, and populational studies indicate that microRNA variants are important factors underlying the process of carcinogenesis. Further understanding of the cellular and molecular basis of microRNA action will lead to the identification of their new target genes and microRNA-regulated pathways. As a consequence, novel models of cancer pathogenesis can be proposed, and serve as a basis for elucidation of new prognostic and diagnostic tools for human cancers.

Variants in the ATM-CHEK2-BRCA1 Axis Determine Genetic Predisposition and Clinical Presentation of Papillary Thyroid Carcinoma

The risk of developing papillary thyroid carcinoma (PTC), the most frequent form of thyroid malignancy, is elevated up to 8.6‐fold in first‐degree relatives of PTC patients. The familial risk could be explained by high‐penetrance mutations in yet unidentified genes, or polygenic action of low‐penetrance alleles. Since the DNA‐damaging exposure to ionizing radiation is a known risk factor for thyroid cancer, polymorphisms in DNA repair genes are likely to affect this risk. In a search for low‐penetrance susceptibility alleles we employed Sequenom technology to genotype deleterious polymorphisms in ATM, CHEK2, and BRCA1 in 1,781 PTC patients and 2,081 healthy controls. As a result of the study, we identified CHEK2 rs17879961 (OR = 2.2, P = 2.37e‐10) and BRCA1 rs16941 (odds ratio [OR] = 1.16, P = 0.005) as risk alleles for PTC. The ATM rs1801516 variant modifies the risk associated with the BRCA1 variant by 0.78 (P = 0.02). Both the ATM and BRCA1 variants modify the impact of male gender on clinical variables: T status (P = 0.007), N status (P = 0.05), and stage (P = 0.035). Our findings implicate an important role of variants in the ATM‐ CHEK2‐ BRCA1 axis in modification of the genetic predisposition to PTC and its clinical manifestations.

Family of microRNA-146 Regulates RARβ in Papillary Thyroid Carcinoma

Retinoic acid is a promising tool in adjuvant cancer therapies, including refractory thyroid cancer, and its biological role is mediated by the retinoic acid receptor beta (RARβ). However, expression of RARβ is lowered in papillary thyroid carcinoma (PTC), contributing to promotion of tumor growth and inefficiency of retinoic acid and radioactive iodine treatment. The causes of aberrant RARB expression are largely unknown. We hypothesized that the culpable mechanisms include the action of microRNAs from the miR-146 family, previously identified as significantly upregulated in PTC tumors. To test this hypothesis, we assessed the expression of RARB as well as miR-146a-5p and miR-146b-5p in 48 PTC tumor/normal tissue pairs by Taqman assay to reveal that the expression of RARB was 3.28-fold decreased, and miR-146b-5p was 28.9-fold increased in PTC tumors. Direct interaction between miRs and RARB was determined in the luciferase assay and further confirmed in cell lines, where overexpression of miR-146a-5p and miR-146b-5p caused a 31% and 33% decrease in endogenous RARB mRNA levels. Inhibition of miR-146a and miR-146b resulted in 62.5% and 45.4% increase of RARB, respectively, and a concomitant decrease in proliferation rates of thyroid cancer cell lines, analyzed in xCELLigence system.We showed that two microRNAs of the miR-146 family directly regulate RARB. Inhibition of miRs resulted in restoration of RARB expression and decreased rates of proliferation of thyroid cancer cells. By restoring RARB levels, microRNA inhibitors may become part of an adjuvant therapy in thyroid cancer patients.

Association between GWAS-Derived rs966423 Genetic Variant and Overall Mortality in Patients with Differentiated Thyroid Cancer.

Purpose: Five germline genetic variants (rs116909374, rs965513, rs944289, rs966423, and rs2439302) have been associated in genome-wide association studies (GWAS) with increased risk of differentiated thyroid cancer (DTC), but their role in mortality of patients has not been established. Also, no preoperative marker of the clinical outcome of thyroid cancer had yet been identified. The aim of the study was to investigate the relationship between the variants and overall mortality in patients with DTC. Experimental Design: Retrospective study of 1,836 patients (1,643 women, 193 men) with median age at diagnosis of 49 years and overall median follow-up time of 8.7 years after initial treatment at a single comprehensive cancer center between 1990 and 2013. Results: Among 5 variants, rs966423 was associated with increased mortality, which was 6.4% (33 of 518) versus 3.7% (47 of 1,259) in TT carriers versus CC/CT carriers (P = 0.017). The HR of TT versus TC/CC carriers was 1.6 [95% confidence interval (CI), 1.02–2.49; P = 0.038] after adjustment for age at diagnosis and sex. Importantly, the association of rs966423 with mortality remained valid when clinicopathologic risk factors were included in the model (HR, 1.89; 95% CI, 1.14–3.13; P = 0.014). Higher rs966423–associated patient mortality of TT versus CC/CT carriers was also observed in interaction with angioinvasion (adjusted HR, 3.48; 95% CI, 1.67–7.22; P < 0.001), lymph node metastasis (adjusted HR, 3.47; 95% CI, 1.16–10.4; P = 0.018), extrathyroidal invasion (adjusted HR, 2.07; 95% CI, 1.15–3.73; P = 0.013). Conclusions: The presence of the rs966423-TT genotype was associated with a significant increase in overall mortality of patients with DTC. Contrary to BRAF mutation and other somatic changes, the status of germline rs966423 is known before the treatment and might be used in the management of mortality risk by means of modification of therapy. Clin Cancer Res; 22(5); 1111–9. ©2015 AACR.

Next-generation sequencing reveals microRNA markers of adrenocortical tumors malignancy

Background Adrenocortical carcinoma is a rare finding among common adrenocortical tumors, but it is highly aggressive and requires early detection and treatment. Still, the differential diagnosis between benign and malignant lesions is difficult even for experienced pathologists and there is a significant need for novel diagnostic methods. In this study we aimed to reveal a complete set of microRNAs expressed in the adrenal gland and to identify easily detectable, stable and objective biomarkers of adrenocortical malignancy. Methods We employed next-generation sequencing to analyze microRNA profiles in a unique set of 51 samples, assigned to either a learning dataset including 7 adrenocortical carcinomas (ACCs), 8 adrenocortical adenomas (AAs) and 8 control samples (NAs), or a validation dataset including 8 ACCs, 10 AAs and 10 NAs. The results were validated in real-time Q-PCR. Results We detected 411 miRNAs expressed in 1763 length isoforms in the examined samples. Fifteen miRNAs differentiate between malignant (ACC) and non-malignant (AA + NA) tissue in the test set of independent samples. Expression levels of 6 microRNAs, miR-503-5p, miR-483-3p, miR-450a-5p, miR-210, miR-483-5p, miR-421, predict sample status (malignancy/non-malignancy) with at least 95% accuracy in both datasets. The best single-gene malignancy marker, miR-483-3p, has been validated by real-time RT PCR. Conclusions As a result of the study we propose clinically valid and easily detectable biomarkers of adrenocortical malignancy that may significantly facilitate morphological examination. Since microRNAs can be detected in blood, the study brings tools for development of non-invasive diagnostics of adrenocortical carcinomas.

MECHANISMS IN ENDOCRINOLOGY: MicroRNA in diagnostics and therapy of thyroid cancer

MicroRNAs, short non-coding regulators of the gene expression, are subjects of numerous investigations assessing their potential use in the diagnostics and management of human diseases. In this review, we focus on studies that analyze the utility of microRNAs as novel diagnostic and therapeutic tools in follicular cell-derived thyroid carcinomas. This very interesting and promising field brings new insight into future strategies for personalized medicine.