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The role of transforming growth factor‐β signaling and hypoxia‐inducible factors in renal cell carcinoma
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology. Umeå university. (Maréne Landström)ORCID iD: 0000-0001-5022-1782
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Renal cell carcinoma (RCC) is the cancer of the kidneys; about 1100 patients with RCC are diagnosed in Sweden each year. RCC can be classified into several subtypes, clear cell renal cell carcinoma (ccRCC) is most common accounting to about 70% of all RCCs, and also the most lethal; papillary renal cell carcinoma (pRCC) accounts to about 10%‐15%, while chromophobe renal cell carcinoma (chRCC) accounts to about 5% of all RCCs. There is a need to study the distinguishing features of RCC subtypes to design treatment. Von Hippel‐Lindau tumor suppressor gene (VHL) is often inactivated in ccRCC, unlike in pRCC or chRCC. Transforming growth factor‐β (TGF‐β) is a cytokine involved in various biological processes such as differentiation, proliferation, apoptosis, migration, andepithelial‐mesenchymal transition. TGF‐β exerts its functions through canonical (Smad‐dependent) and non‐canonical (Smadindependent) signaling pathways. In the first study, we have shown that both canonical and non‐canonical TGF‐β signaling pathways are associated with ccRCC tumor progression. VHL is known to have a dampening effect on TGF‐β signaling in RCC. However, the effects of pVHL status on the TGF‐β signaling pathway in ccRCC and non-ccRCC has not yet been studied in detail. In the second study, we have investigated the effects of the TGF‐β signaling pathway in the presence or absence of pVHL in ccRCC and non‐ccRCC. We show that, in ccRCC, VHL has an inhibiting effect exclusively on canonical TGF‐β signaling, and has no effect on non‐canonical TGF‐β signaling via ALK5‐ICD. In non‐ccRCC, TGF‐β signaling did not have an effect on tumor progression. Further, we demonstrate that VHL, through its ubiquitin ligases activity ubiquitinates ALK5 in a K48 dependent manner and subjects it to proteasomal degradation. During the normoxic conditions, VHL is implicated in ubiquitination and proteasomal degradation of Hypoxia‐inducible factors (HIFs). In hypoxic conditions or when the loss of VHL occurs, HIFs accumulates in the cytoplasm and enters the nucleus to initiate angiogenesis, cell proliferation, and tumor progression. In the third study, we have explored a potential synergistic cross‐talk between TGF‐β signaling and hypoxia in ccRCC. We demonstrate a correlation between TGF‐β signaling components and HIF‐1α/2α in ccRCC. We have also shown that TGF‐β signaling enhances the expression of HIF‐1α/2α and their target genes even under normoxic conditions, dependent on the kinase activity of ALK5 and dictated by the status of VHL. We present novel data that the synergistic crosstalk between hypoxia and TGF‐β is orchestrated through interactions between ALK5 and HIF‐1α/2α. HIF‐3α is only limited studied, compared with HIF‐1α and HIF‐2α. In the fourth study, we have analyzed the roles of HIF‐3α in ccRCC and pRCC and show that HIF‐3α is associated with advanced stage and metastasized tumors. We also found that HIF‐3α is associated with TRAF6, a crucial component of non‐canonical TGF‐β signaling.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2019. , p. 84
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2061
Keywords [en]
TGF-β, Hypoxia, Renal cell carcinoma, ccRCC, non-ccRCC, transforming growth factor-β, ALK5, pVHL, HIF-α, SNAIL1
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-165401ISBN: 9789178551583 (print)OAI: oai:DiVA.org:umu-165401DiVA, id: diva2:1372462
Public defence
2019-12-18, E04, Byggnad 6E, Norrlands universitetssjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2019-11-27 Created: 2019-11-23 Last updated: 2019-11-25Bibliographically approved
List of papers
1. Transforming growth factor-β promotes aggressiveness and invasion of clear cell renal cell carcinoma
Open this publication in new window or tab >>Transforming growth factor-β promotes aggressiveness and invasion of clear cell renal cell carcinoma
2016 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 24, p. 35917-35931Article in journal (Refereed) Published
Abstract [en]

The molecular mechanisms whereby transforming growth factor-β (TGF-β) promotes clear cell renal cell carcinoma (ccRCC) progression is elusive. The cell membrane bound TGF-β type I receptor (ALK5), was recently found to undergo proteolytic cleavage in aggressive prostate cancer cells, resulting in liberation and subsequent nuclear translocation of its intracellular domain (ICD), suggesting that ALK5-ICD might be a useful cancer biomarker. Herein, the possible correlation between ALK5 full length (ALK5-FL) and ALK5-ICD protein, phosphorylated Smad2/3 (pSmad2/3), and expression of TGF-β target gene PAI-1, was investigated in a clinical ccRCC material, in relation to tumor grade, stage, size and cancer specific survival. Expression of ALK5-FL, ALK5-ICD, pSmad2/3 and PAI-1 protein levels were significantly higher in higher stage and associated with adverse survival. ALK5-ICD, pSmad2/3 and PAI-1 correlated with higher grade, and ALK5-FL, pSmad2/3 and PAI-1 protein levels were significantly correlated with larger tumor size. Moreover, the functional role of the TGF-β - ALK5-ICD pathway were investigated in two ccRCC cell lines by treatment with ADAM/MMP2 inhibitor TAPI-2, which prevented TGF-β-induced ALK5-ICD generation, nuclear translocation, as well as cell invasion. The present study demonstrated that canonical TGF-β Smad2/3 pathway and generation of ALK5-ICD correlates with poor survival and invasion of ccRCC in vitro.

Keywords
ccRCC, ALK5, pSmad2/3, PAI-1, TGF-β signaling pathway
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-120254 (URN)10.18632/oncotarget.9177 (DOI)000377756800034 ()27166254 (PubMedID)
Available from: 2016-05-12 Created: 2016-05-12 Last updated: 2019-11-23Bibliographically approved
2. VHL status regulates transforming growth factor-β signaling pathways in renal cell carcinoma
Open this publication in new window or tab >>VHL status regulates transforming growth factor-β signaling pathways in renal cell carcinoma
2018 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 9, no 23, p. 16297-16310Article in journal (Refereed) Published
Abstract [en]

To evaluate the role of pVHL in the regulation of TGF-β signaling pathways in clear cell renal cell carcinoma (ccRCC) as well as in non-ccRCC; the expression of pVHL, and the TGF-β pathway components and their association with clinicopathological parameters and patient’s survival were explored. Tissue samples from 143 ccRCC and 58 non-ccRCC patients were examined by immunoblot. ccRCC cell lines were utilized for mechanistic in-vitro studies. Expression levels of pVHL were significantly lower in ccRCC compared with non-ccRCC. Non-ccRCC and ccRCC pVHL-High expressed similar levels of pVHL. Expression of the TGF-β type I receptor (ALK5) and intra-cellular domain were significantly higher in ccRCC compared with non-ccRCC. In non-ccRCC, expressions of ALK5-FL, ALK5-ICD, pSMAD2/3, and PAI-1 had no association with clinicopathological parameters and survival. In ccRCC pVHL-Low, ALK5-FL, ALK5-ICD, pSMAD2/3, and PAI-1 were significantly related with tumor stage, size, and survival. In ccRCC pVHL-High, the expression of PAI-1 was associated with stage and survival. In-vitro studies revealed that pVHL interacted with ALK5 to downregulate its expression through K48-linked poly-ubiquitination and proteasomal degradation, thus negatively controlling TGF-β induced cancer cell invasiveness. The pVHL status controls the ALK5 and can thereby regulate the TGF-β pathway, aggressiveness of tumors, and survival of the ccRCC and non-ccRCC patients.

Place, publisher, year, edition, pages
Impact Journals, LLC, 2018
Keywords
ccRCC, non-ccRCC, ALK5, pVHL, TGF-β signaling
National Category
Cancer and Oncology Cell and Molecular Biology
Research subject
molecular cell biology; Cancer Epidemiology
Identifiers
urn:nbn:se:umu:diva-146504 (URN)10.18632/oncotarget.24631 (DOI)2-s2.0-85044474424 (Scopus ID)
Available from: 2018-04-11 Created: 2018-04-11 Last updated: 2019-11-23Bibliographically approved
3. Interactions between TGF-β type I receptor and hypoxia-inducible factor-alpha mediates a synergistic crosstalk leading to poor prognosis for patients with clear cell renal cell carcinoma
Open this publication in new window or tab >>Interactions between TGF-β type I receptor and hypoxia-inducible factor-alpha mediates a synergistic crosstalk leading to poor prognosis for patients with clear cell renal cell carcinoma
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2019 (English)In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 18, no 17, p. 2141-2156Article in journal (Refereed) Published
Abstract [en]

To investigate the significance of expression of HIF-1 alpha, HIF-2 alpha, and SNAIL1 proteins; and TGF-beta signaling pathway proteins in ccRCC, their relation with clinicopathological parameters and patient's survival were examined. We also investigated potential crosstalk between HIF-alpha and TGF-beta signaling pathway, including the TGF-beta type 1 receptor (ALK5-FL) and the intracellular domain of ALK5 (ALK5-ICD). Tissue samples from 154 ccRCC patients and comparable adjacent kidney cortex samples from 38 patients were analyzed for HIF-1 alpha/2 alpha, TGF-beta signaling components, and SNAIL1 proteins by immunoblot. Protein expression of HIF-1 alpha and HIF-2 alpha were significantly higher, while SNAIL1 had similar expression levels in ccRCC compared with the kidney cortex. HIF-2 alpha associated with poor cancer-specific survival, while HIF-1 alpha and SNAIL1 did not associate with survival. Moreover, HIF-2 alpha positively correlated with ALK5-ICD, pSMAD2/3, and PAI-1; HIF-1 alpha positively correlated with pSMAD2/3; SNAIL1 positively correlated with ALK5-FL, ALK5-ICD, pSMAD2/3, PAI-1, and HIF-2 alpha. Intriguingly, in vitro experiments performed under normoxic conditions revealed that ALK5 interacts with HIF-1 alpha and HIF-2 alpha, and promotes their expression and the expression of their target genes GLUT1 and CA9, in a VHL dependent manner. We found that ALK5 induces expression of HIF-1 alpha and HIF-2 alpha, through its kinase activity. Under hypoxic conditions, HIF-alpha proteins correlated with the activated TGF-beta signaling pathway. In conclusion, we reveal that ALK5 plays a pivotal role in synergistic crosstalk between TGF-beta signaling and hypoxia pathway, and that the interaction between ALK5 and HIF-alpha contributes to tumor progression.

Place, publisher, year, edition, pages
Taylor & Francis, 2019
Keywords
ALK5, clear cell renal cell carcinoma, HIF-α, SNAIL1, transforming growth factor-β
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-162654 (URN)10.1080/15384101.2019.1642069 (DOI)000478075700011 ()31339433 (PubMedID)
Available from: 2019-09-05 Created: 2019-09-05 Last updated: 2019-11-23Bibliographically approved
4. Expression and association of HIF-3α with hypoxic and TGF-β signalling components in renal cell carcinoma
Open this publication in new window or tab >>Expression and association of HIF-3α with hypoxic and TGF-β signalling components in renal cell carcinoma
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Gastroenterology and Hepatology
Research subject
Molecular Biology; biology
Identifiers
urn:nbn:se:umu:diva-164847 (URN)
Available from: 2019-11-04 Created: 2019-11-04 Last updated: 2019-11-25

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Mallikarjuna, Pramod

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