| 1. |
- Aripaka, Karthik, 1986-
(author)
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Studies on the biological functions of interaction between components in Wnt, TGF-β and HIF pathways for cancer progression
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Cancer is a disease that involves aggressive changes in the genome and aberrant signals between the living cells. Signalling pathways such as TGF-β (Transforming growth factor-β), Wnt, EGF (epidermal growth factor) and HIF (Hypoxia-inducible factor) evolved to regulate growth and development in mammals. These factors are also implicated for tumorigenesis due to failure or aberrant expression of components in these pathways. Cancer progression is a multistep process, and these steps reflect genetic alterations driving the progressive transformation of healthy human cells into highly malignant derivatives. Many types of cancers are diagnosed in the human population, such as head & neck, cervical, brain, liver, colon, prostate, uterine, breast, and renal cell cancer.Prostate cancer is the second most common cancer and one of the foremost leading cancer-related deaths in men in the world. Aberrant Wnt3a signals promote cancer progression through the accumulation of β-Catenin. In the first paper, we have elucidated intriguing functions for Tumour necrosis factor receptor-associated factor 6 (TRAF6) as a coregulatory factor for the expression of Wnt-target genes which was confirmed in vivo by using CRISPR/Cas9 genomic editing, in zebrafish. Our data suggest that Wnt3a promotes TRAF6 interaction with Wnt components, and TRAF6 is required for gene expression of β-Catenin as well as for the Wnt-ligand co-receptor LRP5. From the in vivo studies, we elucidated positive regulation of TRAF6, which is crucial for survival and development of zebrafish. This study identifies TRAF6 as an evolutionary conserved co-regulatory protein in the Wnt pathway that also promotes the progression of prostate and colorectal cancer due to its positive effects on Wnt3a signalling.Hypoxia is a condition due to O2 deprivation, and Hypoxia-inducible factors (HIF) transcription factors are responsible for the maintenance of oxygen homeostasis in living cells. Irregularities in these HIF transcription factors trigger pathological cellular responses for initiation and progression of malignant cancers. Renal cell carcinoma, malignant cancer arising in renal parenchyma and renal pelvis and, hypoxia plays a vital role in its progression. In the second paper, we have investigated the clinicopathological relevance of several hypoxic and TGF-β component proteins such as HIF-1α/2α/3α, TGF-β type 1 receptor (ALK5-FL) and the intracellular domain of ALK5 (ALK5-ICD), SNAI1 and PAI-1 with patient survival in clear cell renal cell carcinoma (ccRCC). We showed that HIF-2α associated with low cancer-specific survival. HIF-2α and SNAI1 positively correlated with ALK5-ICD, pSMAD2/3, PAI-1 and SNAI1 with HIF-2α; HIF-1α positively correlated with pSMAD2/3. Further, under normoxic conditions, our data suggest that ALK5 interacts with HIF-1α and HIF-2α, and promotes their expression and target genes such as GLUT1 and CA9, in a VHL dependent manner through its kinase activity. These findings shed light on the critical aspect of cross-talk between TGF-β signalling and hypoxia pathway, and also the novel finding of an interaction between ALK5 and HIF-α might provide a more in-depth understanding of mechanisms behind tumour progressionIn the third paper, an ongoing study, we investigated the role of HIF-3α in the progression of Renal cell carcinoma and its association with the components of TGF-β and HIF pathways. We have observed increased levels of HIF-3α in ccRCC and pRCC (papillary renal cell carcinoma) which are associated with advanced tumour stage, metastasis and larger tumours. Also, we found HIF-3α show a significant positive association with pro-invasive gene SNAI1, which is a crucial regulator of epithelial to mesenchymal transition. TRAF6 an E3 ligase known to be a prognostic marker in RCC and we observed HIF-3α associates with TRAF6.
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| 2. |
- Sundar, Reshma, 1983-
(author)
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TRAF6, a key regulator of TGFβ-induced oncogenesis in prostate cancer
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Prostate cancer is the most common cancer in men, with the incidence rapidly increasing in Europe over the past two decades. Reliable biomarkers for prostate cancer are currently unavailable. Thus, there is an urgent need for improved biomarkers to diagnose prostate cancer at an early stage and to determine the best treatment options. Higher expression of transforming growth factor-β (TGFβ) has been reported in patients with aggressive cancer.TGFβ is a multifunctional cytokine that acts as a tumor suppressor during early tumor development, and as a tumor promoter at later stages of cancer. TGFβ signals through the canonical Smad or non-Smad cascade via TGFβ type II and type I receptors. The TGFβ signaling cascade is regulated by various post-translational modifications of its key components. The present investigation aimed to identify a potential function of TRAF6 in TGFβ-induced responses in prostate cancer.The first two articles of this thesis unveil the proteolytic cleavage of TGFβ type I receptor (TβRI), and the biological importance of the liberated TβRI intracellular domain (TβRI-ICD) in the nucleus. We found that tumor necrosis factor receptor-associated factor 6 (TRAF6) polyubiquitinates TβRI, which leads to cleavage of TβRI by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta (PKCζ)-dependent manner. Following ectodomain shedding, TβRI undergoes a second cleavage by presenilin 1 (PS1), which liberates TβRI-ICD. TβRI-ICD translocates to the nucleus, where it regulates its own expression as well as expression of the pro-invasive gene Snail1, thereby promoting invasion. We further found that TβRI-ICD associates with Notch intracellular domain (NICD) to drive expression of the pro-invasive gene Snail1, as well as Notch1 ligand Jag1.The third article provides evidence that TRAF6 promotes Lys63-linked polyubiquitination of TβRI at Lys178 in a TGFβ-dependent manner. TβRI polyubiquitination was found to be a prerequisite for TβRI nuclear translocation, and thus for regulation of the genes involved in cell cycle, differentiation, and invasion of prostate cancer cells.In the fourth article we investigated the role of the pro-invasive gene Snail1 in TGFβ-induced epithelial-to-mesenchymal transition (EMT) in prostate cancer cells.
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| 3. |
- Mallikarjuna, Pramod, 1987-
(author)
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The role of transforming growth factor‐β signaling and hypoxia‐inducible factors in renal cell carcinoma
- 2019
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Doctoral thesis (other academic/artistic)abstract
- 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.
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| 4. |
- Song, Jie, 1984-
(author)
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Non-canonical TGFb signaling pathways in prostate cancer
- 2016
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Doctoral thesis (other academic/artistic)abstract
- Prostate cancer is the second leading cause of cancer-related death in men in the Western world. Deregulation of transforming growth factor β (TGFβ) signaling pathway is frequently detected in prostate cancer and contributes to tumor growth, migration, and invasion. In normal tissue and the early stages of cancer, TGFβ acts as a tumor suppressor by regulating proliferation, differentiation, and apoptosis. In later stages of cancer, TGFβ acts as a tumor promoter by inducing angiogenesis, tumor invasion, and migration. Thus, it is important to investigate the molecular mechanisms behind the tumor-promoting effects of TGFβ, which is the topic of this thesis. The tumor necrosis factor receptor–associated factor 6 (TRAF6) controls non-canonical TGFβ signals due to its enzymatic activity, causing polyubiquitination of the cell membrane–bound, serine/threonine kinase TGFβ type I receptor (TβRI) and its subsequent cleavage in the extracellular domain by tumor necrosis factor a–converting enzyme (TACE) in a protein kinase C ζ (PKCζ)-dependent manner. TRAF6 also recruits the active g-secretase complex to the TβRI, resulting in a second cleavage in the transmembrane region and the liberation of the TβRI intracellular domain (TβRI-ICD), which enters the nucleus, where it associates with the transcriptional co-regulator p300. In Paper I, the aim was to elucidate by which mechanisms TβRI-ICD enters the nucleus. We found that the endocytic adaptor protein APPL1 interacts with TβRI and PKCζ. APPL proteins are required for TβRI translocation from endosomes to the nucleus via microtubules in a TRAF6-dependent manner. Moreover, APPL proteins are important for TGFβ-induced cell invasion, and high levels of APPL1 are detected by immunohistochemistry in prostate cancer. Finally, we demonstrated that the APPL1–TβRI complex visualized with the in situ proximity ligation assay (PLA) correlates with Gleason score, indicating that it might be a novel prognostic marker for aggressive prostate cancer. In Paper II, the aim was to explore by which mechanisms TGFβ causes activation of the AKT pathway, which regulates migration and therapy resistance of cancer cells. We found that the E3 ligase activity of TRAF6 induces Lys63-linked polyubiquitination of p85α upon TGFβ stimulation, resulting in plasma membrane recruitment, Lys63-linked polyubiquitination, and subsequent activation of AKT. Moreover, the TRAF6 and PI3K/AKT pathway were found to be crucial for the TGFβ-induced migration. Importantly, we demonstrated, by PLA, a correlation between Lys63-linked polyubiquitination of p85α and aggressive prostate cancer in tissue sections from patients with prostate cancer. In Paper III, the aim was to investigate the mechanisms for TGFβ-induced activation of PKCζ and the role of PKCζ in tumor regression. We found that TRAF6 caused Lys63-linked polyubiquitination of PKCζ. By using two novel chemical compounds that inhibit PKCζ, we demonstrated that PKCζ is crucial for prostate cancer cell survival and invasion. In Paper IV, the aim was to investigate further the target genes for the nuclear TβRI-ICD-APPL1 complex identified in Paper I. We provide evidence that APPL proteins and the TGFβ signaling pathway are important for cell proliferation. In summary, the results reported in this thesis suggest the potential usefulness of the identified signaling components of the tumor-promoting effects of TGFβ as drug targets and biomarkers for aggressive prostate cancer.
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