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Signalling pathways in renal cell carcinoma with a focus on telomerase regulation
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Telomerase is a ribonucleoprotein complex that catalyses telomeric repeat addition at the ends of chromosomes. The catalytic subunit, hTERT, acts as a key determinant for telomerase activity control; the induction of hTERT expression is required for telomerase activity. hTERT participates in cellular immortalization and is elevated in certain malignant tissues. Several tumours exhibit telomerase activity, which contributes to the infinite proliferation capacity that promotes tumour progression.

Renal cell carcinoma (RCC) represents 2% of all adult malignancies and has a high mortality rate. The WHO classifies RCC into several sub-types based on cytogenetic aberrations and morphological features; the most prevalent sub-types are clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC). The aims of this thesis were to study the expression patterns of various signalling molecules, to elucidate the functional links among them, and to define the roles of these signalling molecules in the regulation of hTERT gene expression and telomerase activity in RCC. The first paper included in this thesis revealed mRNA overexpression of DJ-1 (a PTEN inhibitor), cMyc, and hTERT in clinical ccRCC samples compared to tumour-free kidney cortex tissues. Significant, positive correlations were detected for DJ-1, cMyc, and hTERT mRNA levels in ccRCC, but not in pRCC. In vitro knockdown of DJ-1 by siRNA in ccRCC cells induced downregulation of p-Akt, cMyc, hTERT, and telomerase activity. Forced overexpression of DJ-1 in an ovarian carcinoma cell line was followed by increased hTERT promoter activity, which appeared to be dependent on cMYC binding to the promoter. Collectively, the in vitro studies verified a functional link among DJ-1, cMyc, and hTERT as implied in the clinical ccRCC samples. The second paper included in this thesis demonstrated overexpression of NBS1 mRNA levels in ccRCC compared to the kidney cortex. NBS1 mRNA levels exhibited significant, positive correlations with DJ-1, cMyc, and S phase, but not with hTERT. In vitro experiments suggested that DJ-1 could regulate NBS1 gene expression. The role of the hTERT transcriptional repressor WT1 in RCC was evaluated in the third paper included in this thesis. ccRCC samples displayed low WT1 mRNA levels compared to kidney cortex samples. Interestingly, WT1 expression was negatively associated with hTERT and cMyc both of which were elevated in ccRCC. Forced overexpression of WT1 isoforms in a ccRCC cell line increased the expression of several negative transcriptional regulators of hTERT and diminished the expression of hTERT positive regulators. In consequence, hTERT mRNA levels and telomerase activity were reduced. Chromatin immunoprecipitation verified direct binding of WT1 to the cMyc, Smad3, and hTERT promoters. Taken together, these data suggested that in ccRCC, WT1 affects hTERT at the transcriptional level via a combined effect on both positive and negative regulators. In conclusion, DJ-1 can regulate hTERT and telomerase activity through the PI3K pathway encompassing PTEN, NBS1, p-Akt, and cMyc in ccRCC, but not in pRCC. WT1 negatively regulates hTERT and telomerase activity directly and indirectly through multiple pathways in ccRCC.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå University , 2010. , s. 74
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1391
Nyckelord [en]
Renal cell carcinoma, DJ-1, NBS1, PTEN, WT1, cMyc, hTERT, telomerase
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
patologi
Identifikatorer
URN: urn:nbn:se:umu:diva-38121ISBN: 978-91-7459-111-8 (tryckt)OAI: oai:DiVA.org:umu-38121DiVA, id: diva2:372486
Disputation
2010-12-17, Hörsal Betula, byggnad 6M, NUS, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2010-12-06 Skapad: 2010-11-25 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
Delarbeten
1. The PTEN regulator DJ-1 is associated with hTERT expression in clear cell renal cell carcinoma
Öppna denna publikation i ny flik eller fönster >>The PTEN regulator DJ-1 is associated with hTERT expression in clear cell renal cell carcinoma
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2009 (Engelska)Ingår i: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 125, nr 4, s. 783-790Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

DJ-1 is as a novel regulator of the tumor suppressor PTEN with stimulatory effects on PI3K-AKT/PKB signaling, one possible target of which is cMyc. The catalytic unit of the telomerase complex, hTERT, can be activated at different levels, including transcriptionally by cMyc and through phosphorylation by AKT/PKB. The aim of the study was to analyze the putative signaling pathway encompassing DJ-1, cMyc and hTERT in a series of 176 renal cell carcinomas (RCC) and experimentally in cell lines. DJ-1 mRNA expression was significantly elevated in clear cell RCC (ccRCC) compared with in papillary RCC (pRCC; p = 0.005) and kidney cortex tissue (p < 0.001). ccRCC and pRCC demonstrated higher cMyc RNA levels than in kidney cortex (p < 0.001 for both) as well as increased levels of hTERT RNA (p < 0.001 and p = 0.011, respectively). DJ-1 was positively correlated to cMyc and hTERT in ccRCC (p < 0.001 and p = 0.019, respectively), but not in pRCC, indicating that this pathway could have a functional significance in ccRCC. siRNA knock down of DJ-1 induced downregulation of cMyc and hTERT mRNA associated with decreased expression of pAKT and cMyc protein levels. hTERT promoter activity was upregulated after DJ-1 transfection and this upregulation was inhibited after mutation of the cMyc binding sites. These experimental data support the functional link among DJ-1, cMyc and hTERT expression as indicated in the tumor material. Neither DJ-1, cMyc nor hTERT mRNA levels were associated with proliferation (S-phase fraction), telomere length or prognosis in ccRCC.

Nyckelord
renal cell carcinoma; DJ-1; cMyc; hTERT; telomere length; proliferation; prognosis
Identifikatorer
urn:nbn:se:umu:diva-26484 (URN)10.1002/ijc.24335 (DOI)19384955 (PubMedID)2-s2.0-67650095406 (Scopus ID)
Tillgänglig från: 2009-10-12 Skapad: 2009-10-12 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
2. The NBS1 gene is overexpressed and regulated by DJ-1 in clear cell renal cell carcinoma
Öppna denna publikation i ny flik eller fönster >>The NBS1 gene is overexpressed and regulated by DJ-1 in clear cell renal cell carcinoma
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nyckelord
Renal cell carcinoma, ␣NBS1, cMYC, DJ 1, hTERT
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
patologi
Identifikatorer
urn:nbn:se:umu:diva-38142 (URN)
Tillgänglig från: 2010-11-25 Skapad: 2010-11-25 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
3. Wilms' tumour 1 can suppress hTERT gene expression and telomerase activity in clear cell renal cell carcinoma via multiple pathways
Öppna denna publikation i ny flik eller fönster >>Wilms' tumour 1 can suppress hTERT gene expression and telomerase activity in clear cell renal cell carcinoma via multiple pathways
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2010 (Engelska)Ingår i: British Journal of Cancer, ISSN 0007-0920, E-ISSN 1532-1827, Vol. 103, nr 8, s. 1255-1262Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

BACKGROUND: Wilms' tumour 1 (WT1) gene was discovered as a tumour suppressor gene. Later findings have suggested that WT1 also can be oncogenic. This complexity is partly explained by the fact that WT1 has a number of target genes.

METHOD: WT1 and its target gene human telomerase reverse transcriptase (hTERT) were analysed in clear cell renal cell carcinoma (ccRCC). In vitro experiments were performed to examine the functional link between WT1 and hTERT by overexpression of WT1 isoforms in the ccRCC cell line, TK-10.

RESULTS: WT1 demonstrated lower RNA expression in ccRCC compared with renal cortical tissue, whereas hTERT was increased, showing a negative correlation between WT1 and hTERT (P=0.005). These findings were experimentally confirmed in vitro. The WT1 generated effect on hTERT promoter activity seemed complex, as several negative regulators of hTERT transcription, such as SMAD3, JUN (AP-1) and ETS1, were activated by WT1 overexpression. Downregulation of potential positive hTERT regulators, such as cMyc, AP-2α, AP-2γ, IRF1, NFX1 and GM-CSF, were also observed. Chromatin immunoprecipitation analysis verified WT1 binding to the hTERT, cMyc and SMAD3 promoters.

CONCLUSION: The collected data strongly indicate multiple pathways for hTERT regulation by WT1 in ccRCC.

Nyckelord
renal cell carcinoma; WT1; hTERT; telomerase activity and pathways
Identifikatorer
urn:nbn:se:umu:diva-38095 (URN)10.1038/sj.bjc.6605878 (DOI)000283542900016 ()20842112 (PubMedID)2-s2.0-77957988663 (Scopus ID)
Tillgänglig från: 2010-11-25 Skapad: 2010-11-24 Senast uppdaterad: 2023-03-24Bibliografiskt granskad

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