Glycosylation and Glycoproteins in Thyroid Cancer: A Potential Role for Diagnostics

Rozdział książki Updates in the Understanding and Management of Thyroid Cancer Edited by Thomas J. Fahe

Ekspresja genów kodujących enzymy związane z O-GlcNAcylacją w rakach błony śluzowej trzonu macicy: korelacja z parametrami kliniczno-patologicznymi

Objectives: O-GlcNAcylation is an abundant modification of cellular proteins which consist of single N-acetylglucosamine residues attached by O-linkage to serine or threonine residues. Abnormal O-GlcNAcylation seems to be a feature of malignant cancer cells. The aim of the present study was to determine the relationship between the expression of genes encoding O-GlcNAc cycling enzymes (OGT and MGEA5) and clinicopathological parameters of endometrial carcinomas. Materials and methods: The mRNA expression levels of O-GlcNAc cycling enzymes in series of 76 samples of endometrial carcinoma were studied by real time RT-PCR method. Results: The OGT and MGEA5 mRNA expression was significantly higher in tumors of higher histological grade than in well-differentiated tumors. Statistically significant association was found between OGT and MGEA5 mRNA expression and depth of myometrial invasion. Both OGT and MGEA5 expression profiles showed no significant association with the clinical stage of endometrial cancer. Conclusion: O-GlcNAcylation may be an important regulatory modification involved in endometrial cancer pathogenesis but the actual significance of this modification for endometrial cancer progression needs to be investigated further.Cel pracy: O-GlcNAcylacja jest powszechną modyfikacją białek komórkowych polegającą na przyłączeniu wiązaniem O-glikozydowym pojedynczych reszt N-acetyloglukozoaminy do reszt seryny i treoniny. Zaburzenia O-GlcNAcylacji wydają się być istotną cechą związaną z agresywnością komórek nowotworowych. Celem prezentowanej pracy było określenie zależności pomiędzy ekspresją genów kodujących enzymy związane z O-GlcNAcylacją białek a kliniczno-patologicznymi parametrami raka błony śluzowej trzonu macicy. Materiał i metody: Poziom ekspresji mRNA enzymów analizowano techniką real time RT-PCR w 76 preparatach raków błony śluzowej trzonu macicy. Wyniki: Nowotwory o wyższym stopniu złośliwości histologicznej wykazywały wyższą ekspresję mRNA dla OGT i MGEA5 w porównaniu z rakami dobrze zróżnicowanymi. Stwierdzono również istotną statystycznie zależność pomiędzy ekspresją badanych genów a głębokością naciekania mięśniówki macicy. Nie stwierdzono natomiast zależności pomiędzy ekspresją mRNA OGT i MGEA5 a stopniem klinicznego zaawansowania nowotworu. Wniosek: Wydaje się, że O-GlcNAcylacja może być ważną regulatorową modyfikacją włączoną w patogenezę raka błony śluzowej trzonu macicy ale dokładne określenie jej roli w progresji tego nowotworu wymaga dalszych badań

Metallothionein 2A genetic polymorphisms and risk of ductal breast cancer

Metallothioneins (MTs) are a family of metal binding proteins that play an important role in cellular processes such as proliferation and apoptosis. Metallothionein 2A is the most expressed MT isoform in the breast cells. A number of studies have demonstrated increased MT2A expression in various human tumors, including breast cancer. We carried out an association study to examine whether MT2A gene polymorphisms are associated with risk of breast cancer. Information on lifestyle risk factors was collected via a self-administered questionnaire. Genotyping was conducted using polymerase chain reaction–restriction fragment length polymorphism technique. Three single nucleotide polymorphisms (SNP) rs28366003, rs1610216 and rs10636 were genotyped in 534 breast cancer cases and 556 population controls. One SNP in MT2A (rs28366003) showed a positive association with breast cancer. Compared with homozygous common allele carriers, heterozygous for the G variant [odds ratio (OR) = 1.92, 95 % confidence interval (CI):1.28–2.81, p trend <0.01; the OR assuming a dominant model 1.93 (95 % CI: 1.29–2.89, p dominant <0.02) after adjustment for age, family history, smoking status, BMI, menarche, parity, menopausal status and use of contraceptive and menopausal hormones] had a significantly increased risk of breast cancer in Polish population, as well as women with haplotypes, including variant allele of rs28366003 SNP (OR = 1.58, CI: 0.41–6.33, p global = 0.03). Our data suggest that the rs28366003 SNP in MT2A is associated with risk of breast cancer in Polish population.This work was supported, in part, by the statutory fund for the Department of Cytobiochemistry, University of Łód

Wpływ metforminy na przeżywalność komórek raka jajnika linii SKOV-3 oraz ekspresję genów kodujących enzymy związane z O-GlcNAcylacją

Objectives: The aim of the study was to evaluate the cytotoxic effect of metformin on the ovarian cancer cells SKOV-3 and analyze the impact of this compound on the expression of genes coding for O-GlcNAc cycling enzymes, i.e. O-GlcNAc transferase (OGT) and β-N-acetylglucosaminidase (OGA). Materials and methods: Viability and proliferation of control cells and cells treated with metformin were evaluated by MTT test and trypan blue staining. OGT and OGA mRNA expressions analysis was performed using real-time PCR method. Results: A metformin concentration-dependent decrease of SKOV-3 cell viability was observed. The IC50 parameter for metformin cytotoxicity was 14 mM. The SKOV-3 cell doubling time was 45 hours. The cell population treated with 10 mM metformin did not double even after 72 hours. There was no significant difference in mRNA level of OGA between control cells and cells treated with metformin. The OGT mRNA level was significantly higher in cells treated with metformin for 24 hours as compared to the control cells. The increase of OGT mRNA was dependent on time of incubation. Cells treated with metformin for 48 hour showed higher expression of OGT than cells treated for 24 hours. Conclusion: Antiproliferative activity of metformin suggests that this compound may be considered as a candidate for potential chemotherapeutic agent. However, taking into account its impact on the expression of O-GlcNAc transferase, further studies on the molecular mechanism of metformin action are necessary.Cel pracy: Celem pracy była analiza wpływu metforminy na proliferację komórek raka jajnika linii SKOV-3 oraz ekspresję genów, kodujących białka zaangażowane w proces O-GlcNAcylacji – O-GlcNAc transferazy (OGT) i β-N-acetylo-D-glukozaminidazy (OGA). Materiał i metody: Przeżywalność komórek po działaniu metforminy oznaczano metodą spektrofotometryczną z użyciem bromku 3-(4,5-dimetylo-2-tiazolilo)-2,5-difenylo-2H-tetrazoliowego (MTT). Żywotność komórek oceniano stosując metodę barwienia błękitem trypanu. Analizę ekspresji genów OGT i OGA na poziomie mRNA przeprowadzono przy użyciu techniki real-time PCR. Wyniki: Zaobserwowano obniżenie przeżywalności komórek poddanych działaniu metforminy wraz ze wzrostem stosowanych stężeń. Cytotoksyczność metforminy została oszacowana na podstawie parametru IC50 wynoszącego 14mM, w odniesieniu do komórek linii SKOV-3. Czas potrzebny do podwojenia komórek linii SKOV-3 wynosił 45 godzin, natomiast w przypadku prób traktowanych 10 mM metforminą nie uzyskano efektu podwojenia liczby komórek nawet po 72 godzinach. Wniosek: Metformina z uwagi na jej antyproliferacyjne działanie może być rozpatrywana jako potencjalny chemioterapeutyk, niemniej z uwagi na fakt, iż zmienia ona ekspresję jednego z dwóch enzymów odpowiedzialnych za proces O-glikozylacji białek - O-GlcNAc transferazy, należy dogłębnie zbadać ten molekularny mechanizm

Topoisomerase IIβ Binding Protein 1 c.*229C>T (rs115160714) Gene Polymorphism and Endometrial Cancer Risk

TopBP1 (topoisomerase IIβ binding protein 1) protein is involved in DNA replication, DNA damage checkpoint response and transcriptional regulation. In this study we investigated whether alterations in the TopBP1 gene can influence the risk of endometrial cancer. We examined the association between five single nucleotide polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 3′UTR region of the TopBP1 gene and endometrial cancer risk as well as allele-specific gene expression. One hundred twenty-one endometrial cancer patients were genotyped for these SNPs. Allele-specific TopBP1 mRNA and protein expressions were determined by real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with endometrial cancer. Compared to homozygous common allele carriers, heterozygous for the T variant had significantly increased risk of endometrial cancer [adjusted odds ratio (OR) = 5.59, 95 % confidence interval (CI): 1.96–15.91, p = 0.0003]. Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT genotype. There was a significant association between the rs115160714 and tumor grade and FIGO classification. Most carriers of minor allele had a high grade tumors (G3) classified as FIGO III/IV. The results of our study raise a possibility that a genetic variation of TopBP1 may be implicated in the etiology of endometrial cancer

Expression, Localization, and Phosphorylation of Akt1 in Benign and Malignant Thyroid Lesions

The serine/threonine protein kinase Akt is a key molecule in the phosphatidyl inositol 3-kinase pathway that is often overactivated in human cancers. Three Akt isoforms (Akt1, Akt2, Akt3) have been identified in human cells and they show different distribution and have non-redundant functions. The aim of this study was to determine whether the expression, phosphorylation, and localization of Akt1 isoform in human thyroid malignant lesions are different from those in benign lesions. Nuclear and cytoplasmic fractions were isolated from tissue samples and Western blot method was used to detect Akt1 presence in both cellular fractions. Akt1 expression was also assessed by ELISA method. To estimate Akt1 phosphorylation, kinase was immunoprecipitated from cell lysates and tested with anti-phospho-Akt antibodies. The Akt1 expression in majority of thyroid cancer samples was significantly higher than in benign lesions (p < 0.05). Akt1 both in differentiated cancers (follicular and papillary) and benign lesions was localized mainly in cytoplasmic fraction. In two of three anaplastic cancer samples Akt1 was predominantly localized in nucleus. The ratio of phosphorylated Akt1 to total Akt1 was lower in cancers than in non-neoplastic lesions and adenomas. Thus, although Akt1 seems to be overexpressed in thyroid neoplasms, its high phosphorylation is not characteristic for thyroid cancers

Association between the c.*229C>T polymorphism of the topoisomerase IIb binding protein 1 (TopBP1) gene and breast cancer

Topoisomerase IIb binding protein 1 (TopBP1) is involved in cell survival, DNA replication, DNA damage repair and cell cycle checkpoint control. The biological function of TopBP1 and its close relation with BRCA1 prompted us to investigate whether alterations in the TopBP1 gene can influence the risk of breast cancer. The aim of this study was to examine the association between five polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 30UTR region of the TopBP1 gene and breast cancer risk as well as allele-specific gene expression. Five hundred thirty-four breast cancer patients and 556 population controls were genotyped for these SNPs. Allele-specific Top- BP1 mRNA and protein expressions were determined by using real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with breast cancer. Compared to homozygous common allele carriers, heterozygous and homozygous for the T variant had significantly increased risk of breast cancer (adjusted odds ratio = 3.81, 95 % confidence interval: 1.63–8.34, p = 0.001). Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT or TT genotype. There was a significant association between the rs115160714 and tumor grade and stage. Most carriers of minor allele had a high grade (G3) tumors classified as T2-T4N1M0. Our study raises a possibility that a genetic variation of TopBP1 may be implicated in the etiology of breast cancer

Gene and protein expression of glucose transporter 1 and glucose transporter 3 in human laryngeal cancer—the relationship with regulatory hypoxia-inducible factor-1α expression, tumor invasiveness, and patient prognosis

Increased glucose uptake mediated by glucose transporters and reliance on glycolysis are common features of malignant cells. Hypoxia-inducible factor-1α supports the adaptation of hypoxic cells by inducing genes related to glucose metabolism. The contribution of glucose transporter (GLUT) and hypoxia-inducible factor-1α (HIF-1α) activity to tumor behavior and their prognostic value in head and neck cancers remains unclear. The aim of this study was to examine the predictive value of GLUT1, GLUT3, and HIF-1α messenger RNA (mRNA)/protein expression as markers of tumor aggressiveness and prognosis in laryngeal cancer. The level of hypoxia/metabolic marker genes was determined in 106 squamous cell laryngeal cancer (SCC) and 73 noncancerous matched mucosa (NCM) controls using quantitative realtime PCR. The related protein levels were analyzed by Western blot. Positive expression of SLC2A1, SLC2A3, and HIF-1α genes was noted in 83.9, 82.1, and 71.7 % of SCC specimens and in 34.4, 59.4, and 62.5 % of laryngeal cancer samples. Higher levels of mRNA/protein for GLUT1 and HIF-1α were noted in SCC compared to NCM (p<0.05). SLC2A1 was found to have a positive relationship with grade, tumor front grading (TFG) score, and depth and mode of invasion (p<0.05). SLC2A3 was related to grade and invasion type (p<0.05). There were also relationships of HIF-1α with pTNM, TFG scale, invasion depth and mode, tumor recurrences, and overall survival (p<0.05). In addition, more advanced tumors were found to be more likely to demonstrate positive expression of these proteins. In conclusion, the hypoxia/metabolic markers studied could be used as molecular markers of tumor invasiveness in laryngeal cancer.This work was supported, in part, by the statutory fund of the Department of Cytobiochemistry, University of Łódź, Poland (506/811), and by grant fromtheNational Science Council, Poland (N403 043 32/2326)

Gene expression of O-GlcNAc cycling enzymes in human breast cancers

O-GlcNAcylation is an abundant, dynamic, and inducible posttranslational modification in which single β-N-acetylglucosamine residues are attached by O-glycosidic linkage to serine or treonine residues. It is suggested that abnormally regulated O-GlcNAcylation may contribute to the pathology of cancer. Cycling of O-GlcNAc residues on intracellular proteins is controlled by two enzymes, O-GlcNAc transferease (OGT), which catalyses the addition of O-GlcNAc residues and nucleocytoplasmic β-N-acetylglucosaminidase (O-GlcNAcase; encoded by MGEA5 gene), an enzyme involved in the removal of O-GlcNAc. In this study, relationship between the mRNA expressions of genes coding O-GlcNAc cycling enzymes in breast ductal carcinomas and clinicopathological parameters were analyzed. The results showed that poorly differentiated tumors (grade II and III) had significantly higher OGT expression than grade I tumors. Contrary, MGEA5 transcript levels were significantly lower in grade II and III in comparison with grade I tumors. The Spearman rank correlation showed the expressions of OGT and MGEA5 in breast cancer was negatively correlated (r = −0.430, P = 0.0002). Lymph node metastasis status was significantly associated with decreased MGEA5 mRNA expression. This result suggests that elevation in O-GlcNAc modification of proteins may be implicated in breast tumor progression and metastasis