| 1. |
- Aripaka, Karthik, 1986-
(author)
-
Studies on the biological functions of interaction between components in Wnt, TGF-β and HIF pathways for cancer progression
- 2019
-
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.
|
|
| 2. |
- Herdenberg, Carl, 1982-
(author)
-
Molecular and physiological functions of LRIG proteins and netrin-1 in health and disease
- 2021
-
Doctoral thesis (other academic/artistic)abstract
- The leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family has three members, LRIG1, LRIG2, and LRIG3, that encode three structurally similar transmembrane proteins. LRIG1 is a receptor tyrosine kinase regulator, tumor suppressor, and stem cell marker in the skin, intestine, and brain. LRIG2 and LRIG3 have been less studied but shown to interact with LRIG1. The different roles and mechanisms of action of LRIG proteins have not yet been fully elucidated. In Caenorhabditis elegans (C. elegans), the LRIG homolog SMA-10 regulates bone morphogenetic protein (BMP) signaling; however, this function has not been demonstrated for mammalian LRIG proteins. In mice, the gene encoding the neurodevelopmental guidance cue netrin-1, Ntn1, interacts with Lrig3 in inner ear development. The physical interactions between LRIG proteins and other proteins are mostly unknown. Here, we describe an LRIG1-centered protein interaction network that regulates growth factor receptor levels. The LRIG1 interactome comprised LRIG2 and LRIG3 as well as many unanticipated proteins. An unbiased pathological examination of female mice with different Lrig3 genotypes (homozygous, heterozygous, or knockout) revealed a reduced incidence of spontaneous fatty liver and lymphocytic hyperplasia of the spleen in Lrig3-null mice. Female Lrig3-null mice also had a lower incidence of microvesicular cytoplasm in the liver after eight weeks on a high-fat diet. To further explore the molecular and physiological functions of LRIG proteins, we generated Lrig-null (Lrig1-/-;Lrig2-/-;Lrig3-/-) mouse embryonic fibroblasts (MEFs), which displayed a deficiency in adipogenesis caused by impaired BMP signaling. LRIG1 and LRIG3, but not LRIG2, sensitized cells to BMP and rescued the adipogenesis deficiency in Lrig-null MEFs. In C. elegans, the LRIG homolog sma-10 was needed for proper lipid accumulation. By analyzing data from the UK Biobank and GENiAL cohort, we found that certain LRIG1 gene variants were associated with a higher body mass index (BMI) yet protected against type 2 diabetes. This effect was probably mediated by altered adipocyte morphology. CRISPR/Cas9-mediated ablation of Ntn1 revealed that the BMP-promoting function of LRIG1 and LRIG3 was opposed by netrin-1, which functioned as an inhibitor of BMP signaling via its receptor neogenin.In summary, the present thesis describes a novel LRIG protein interaction network, the regulation of BMP signaling by LRIG proteins and netrin-1, and an important function of LRIG proteins in regulating fat metabolism with implications for human metabolic health.
|
|
| 3. |
- Wang, Tianyan, 1983-
(author)
-
Utility of novel drug targets for treatment of metastatic cancer
- 2022
-
Doctoral thesis (other academic/artistic)abstract
- Metastasis is the leading cause of cancer death because of a lack of early diagnosis tools and efficient treatment drugs. The lipid kinase phosphatidylinositol 4-phosphate 5 kinase (PIP5K1α) has been shown to play a vital role in the PI3K/AKT and KRAS signaling pathways. My PhD work, therefore, aims: (i) to study the role of PIP5K1α as a potential target for cancer treatment and the utility of its inhibitor ISA-2011B for the treatment of castration-resistant prostate cancer (CRPC) and pancreatic cancer, (ii) to establish genetically engineered mouse models and murine syngeneic models to recapitulate pancreatic cancer progression and test targeted anticancer drugs, (iii) to utilize the state-of-the-art molecularly imprinting technique for cancer biomarker detection.My thesis work has shown a clear inhibitory effect of ISA-2011B on human CRPC cell lines C4-2, DU145, and PC-3. The siRNA-mediated downregulation of PIP5K1α and ISA-2011B treatments both showed inhibition of the in vitro growth in all three cell lines. The PC-3 cell and its xenograft tumor can be inhibited by tamoxifen or ISA-2011B treatment alone, and a combination treatment from both compounds can selectively block the ERα and PIP5K1α/AKT network. The results, therefore, suggest that it is possible to treat CRPC by targeting PIP5K1α/AKT and ERα pathways.We established the KPC [Krastm4Tyj Trp53tm1Brn Tg(Pdx1-Cre/Esr1*)] mouse model, in which spontaneous pancreatic ductal adenocarcinoma (PDAC) develops under tamoxifen induction. Three PDAC cell lines bearing KRASG12D and P53 mutations from spontaneous tumors were established and characterized. ISA-2011B in vitro treatment on those cell lines showed that KRASG12D and pErk were significantly decreased in at least one of the cell lines. It suggests that PIP5K1α is a potential target, and its inhibitor ISA-2011B is a promising drug for treating KRAS-mutated PDAC. The syngeneic PDAC model was also prepared by subcutaneous injection of the three cell lines back into the KPC mice, which will be used as an in vivo model to study the function of PIP5K1α in PDAC further.We developed molecularly imprinted polymers (MIPs) for the potential biomarker Neu5Acα2-6GalNAcα-O-Ser/Thr (STn), as well as the non-imprinted polymers(NIPs) as a control. We identified human PDAC cell lines CFPAC-1 and BxPC-3 are STn-positive and -negative cells, respectively. Although STn-MIPs have a higher affinity than NIPs to both cancer cell lines, STn-MIPs cannot differentiate the STn-positive CFPAC-1 cells from the STn-negative BxPC-3 cells. It remains challenging to apply MIPs to detect biological molecules.Our data provide a novel therapeutic strategy to treat advanced cancers such as CRPC and KRAS-mutated PDAC by targeting PIP5K1α-associate PI3K/AKT and/or KRAS signaling pathways.
|
|
| 4. |
- Sundar, Reshma, 1983-
(author)
-
TRAF6, a key regulator of TGFβ-induced oncogenesis in prostate cancer
- 2015
-
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.
|
|
| 5. |
- Ekman, Maria, 1979-
(author)
-
The role of Smad7 and TRAF6 in Prostate Cancer Cell Invasion, Migration and Survival
- 2011
-
Doctoral thesis (other academic/artistic)abstract
- Transforming growth factor (TGF) β is a tumor suppressor during early tumor development, by inhibiting proliferation and inducing apoptosis. At later stages of cancer, it becomes a tumor promoter, and promotes tumor cell migration and invasion. TGFβ signals via its type II and type I receptors to several downstream signaling pathways. In the present work we have focused on the TRAF6 (tumor necrosis factor receptor-associated factor 6)/ TAK1 (TGFβ activated kinase 1) signaling pathway and the Smad7-dependent activation of p38 in prostate carcinoma cells (PC3U). We found that TGFβ-induced activation of the ubiquitin ligase TRAF6 was needed for cell invasion, by a mechanism that involves activation of the metalloproteinase TNFα converting enzyme (TACE), via protein kinase Cζ (PKCζ). TACE cleaves the TβRI, whereafter the intracellular domain (ICD) translocates to the nucleus, where it binds to the transcriptional co-activator p300 and regulates gene expression, promoting invasion. Interestingly, the translocation of the TβRI ICD was observed in several cancer cell lines and in sections of primary tumors, but not in primary prostate epithelial cells. We also found that Smad7 and adenomatous polyposis coli (APC) are important for TGFβ- and epidermal growth factor (EGF)-induced cell migration in PC3U cells. TGFβ induces the formation of a complex consisting of Smad7, p38, glycogene synthase kinase 3β (GSK-3β), APC and β-catenin, which localizes to the membrane ruffles in the leading edge of migrating cells. The complex links the TβRI to the microtubule system and promotes membrane ruffling and microtubule polarization, which are known to be important for cell migration. In the EGF signaling pathway, Smad7 was found to be important for phosphorylation of the EGF receptor at Tyr1068, for the activation of p38 and JNK, and for induction of membrane ruffles. Smad7 is required for TGFβ-induced activation of p38 and apoptosis. We found that Smad7 forms a complex with p38 and ataxia telangiectasia mutated (ATM), which is important for activation of p53 mediated apoptosis. Many tumor cells including the PC3U cells lack a functional p53, which is one of the reasons to why cancer cells can avoid the tumor suppressor effects of TGFβ.
|
|
| 6. |
- Gudey, Shyam Kumar, 1982-
(author)
-
TRAF6 stimulates TGFβ-induced oncogenic signal transduction in cancer cells
- 2014
-
Doctoral thesis (other academic/artistic)abstract
- Prostate cancer is one of the leading causes of cancer-related deaths in men worldwide, with 10,000 new cases/year diagnosed in Sweden. In this context, there is an urgent need to identify new biomarkers to detect prostate cancer at an initial stage for earlier treatment intervention. Although how prostate cancer develops has not been fully established, the male sex hormone testosterone is a known prerequisite for prostate cancer development. High levels of transforming growth factor-β (TGFβ) are prognostically unfavorable in prostate cancer patients.TGFβ is a multifunctional cytokine that regulates a broad range of cellular responses. TGFβ signals through either the canonical Smad or the non-Smad signaling cascade. Cancerous cells develop different strategies to evade defense mechanisms and metastasize to different parts of the body. This thesis unveils one such novel mechanism related to TGFβ signaling.The first two articles provide evidence that TGFβ receptor type I (TβRI) is ubiquitinated by tumor necrosis factor receptor-associated factor 6 (TRAF6) and is cleaved at the ectodomain region by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta type-dependent manner. After TβRI is shed from the ectodomain, it undergoes a second cleavage by presenilin 1 (PS1), a γ-secretase catalytic subunit, which liberates the TβRI intracellular domain (TβRI-ICD) from the cell membrane. TRAF6 promotes TGFβ-dependent Lys63-linked polyubiquitination and recruitment of PS1 to the TβRI complex, and facilitates the cleavage of TβRI by PS1 to generate a TβRI-ICD. The TβRI-ICD then translocates to the nucleus, where it binds with the transcriptional co-activator p300 and regulates the transcription of pro-invasive target genes such as Snail1. Moreover, the nuclear translocated TβRI-ICD cooperates with the Notch intracellular domain (NICD), a core component in the Notch signaling pathway, to drive the expression of invasive genes. Interestingly, treatment with g-secretase inhibitors was able to inhibit cleavage of TβRI and inhibit the TGFβ-induced oncogenic pathway in an in vivo prostate cancer xenograft model.In the third article, we identified that Lysine 178 is the acceptor lysine in TβRI that is ubiquitinated by TRAF6. The TβRI K178R mutant was neither ubiquitinated nor translocated to the nucleus, and prevented transcriptional regulation of invasive genes in a dominant negative manner.In the fourth article, we show that TGFβ utilizes the E3-ligase TRAF6 and the p38 mitogen-activated protein kinase to phosphorylate c-Jun. In turn, the phosphorylated c-Jun activates p21 and Snail1 in a non-canonical Smad-independent pathway, and thereby promotes invasion in cancerous cells.In summary, we elucidate a new mechanism of TGFβ-induced oncogenic signal transduction in cancer cells in which TRAF6 plays a fundamental role. This opens a new avenue in the field of TGFβ signaling.
|
|
| 7. |
- Kermpatsou, Despoina
(author)
-
Method development for the analysis of protein interactions
- 2023
-
Doctoral thesis (other academic/artistic)abstract
- Biological processes take place through interactions between macromolecules, such as nucleic acids and proteins. It is, therefore, fundamental to understand the functions of proteins and how they form complexes in order to carry out their role. Importantly, including spatial information in the analyses of protein complexes allows us to account for cell or tissue heterogeneity, highlighting the importance of in situ studies in biologically and clinically relevant material.To that end, methods for the analysis of protein complexes in situ have been developed, such as in situ proximity ligation assay (PLA) and proximity-dependent initiation of hybridisation chain reaction (proxHCR). Both methods depend on antibodies for target recognition and utilise oligonucleotide systems in order to generate reporter signals with fluorescence readout. While in situ PLA employs rolling circle amplification, proxHCR is an enzyme-free method that takes advantage of DNA hybridisation properties. Both methods, however, yield a polymerised reporter signal of protein complex formation.To further the use of proxHCR, we optimised the design of the oligonucleotide system as well as the experimental procedure, so as to increase the robustness and versatility of the assay. In addition, we developed a novel method, MolBoolean, that simultaneously reports the levels of free proteins as well as protein complexes. In this way, we address limitations of earlier methods and provide the opportunity to obtain a more comprehensive picture of biological processes. Our methods provide a means to circumvent the resolution limits of light microscopy by utilising molecular tricks so that protein binding events, that occur below the resolving power of conventional instrumentation, are made visible. The methods presented in the present doctoral thesis provide powerful tools in the analysis of protein interactions and have applications in cell biology studies as well as in diagnostics. Part of this thesis was the examination of the function of 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) receptor, potentially linked to vitamin D3. We investigated the expression and subcellular localisation of 1,25D3-MARRS in an array of cell lines and employed siRNA-mediated depletion to examine effects on cellular processes in androgen-independent prostate cancer cell models. Our data suggest that 1,25D3-MARRS supports cell proliferation and might have a role in cell migration. Additionally, we observe an effect on the regulation of intracellular vitamin D3 levels. With this study, we contribute to the understanding of the role of 1,25D3-MARRS in prostate cancer cells, that could potentially prove of value in the adaptation of therapeutic strategy for prostate carcinoma.
|
|
| 8. |
- Mallikarjuna, Pramod, 1987-
(author)
-
The role of transforming growth factor‐β signaling and hypoxia‐inducible factors in renal cell carcinoma
- 2019
-
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.
|
|
| 9. |
- Song, Jie, 1984-
(author)
-
Non-canonical TGFb signaling pathways in prostate cancer
- 2016
-
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.
|
|
| 10. |
- Davoodpour, Padideh, 1966-
(author)
-
2-ME-Induced Apoptotic Signalling in Prostate Cancer PC3 Cells
- 2005
-
Doctoral thesis (other academic/artistic)abstract
- Prostate cancer is common in the Western society and current treatments are often associated with side effects, therefore improved therapeutic strategies are desired. 2-methoxyestradiol (2-ME), an endogenous metabolite of estradiol-17β inhibits tumor growth in vivo as it prevents angiogenesis. 2-ME has also direct cytotoxic effects on tumor cells. In this study, we have investigated the potential use of PET to record effects 2-ME on prostate cancer cell (PC3) aggregates. The anti-proliferative and pro-apoptotic effects of 2-ME on PC3 cell aggregates in vitro were correlated with the uptake of deoxy-D-glucose, FMAU and choline labeled with 18F, 11C or 3H. 2-ME clearly reduced growth of PC3 aggregates and induced apoptosis in a dose-dependent manner. However, the PET tracers failed to record the cytotoxicity of 2-ME on PC3 aggregates. Further, the signaling events responsible for 2-ME induced prostate cancer cell death were investigated. We found that Smad7, previously implicated in TGF-β-induced responses, is required for 2-ME-induced p38 MAPK activation and subsequent apoptosis in PC-3U cells, as shown by the use of antisense or siRNA techniques and a specific inhibitor of p38 MAPK (SB203580). Interestingly, Smad7 also regulated the expression of the pro-apoptotic Bim protein. Shb is a Src Homology 2 domain adapter protein with pro-apoptotic effects. PC3 clones overexpressing Shb exhibited increased rates of apoptosis, both in the presence or absence of 2-ME, as they failed to activate survival mechanisms through ERK and Akt in response to 2-ME. Notably, Shb cells displayed increased activity of the pro-apoptotic kinase c-Abl. Pre-treatment with SB203580 or c-Abl (STI-571) inhibitors completely blocked the apoptotic response to 2-ME. In conclusion, Smad7 and Shb appear to be crucial for 2-ME-induced PC3 cell apoptosis via their activation of p38 MAPK and c-Abl. Future therapies exploring these pathways can be envisaged as treatment of prostate cancer.
|
|
