p73 shares many structural and functional similarities with its homologue, the p53 tumour suppressor. Many p73 protein isoforms result from both alternative splicing and differential promoter utilization. Transcriptionally active (TA) isoforms are pro-apoptotic, while N-terminally truncated (dN) variants are anti-apoptotic and act as dominant-negative inhibitors of TAp73 and p53. p73 has been shown to activate the transcription of genes involved in cell cycle arrest and apoptosis in response to DNA damaging agents, including chemotherapies. We hypothesize that the activity of p73, like many transcription factors, may be modulated by binding to regulatory proteins. A novel p73-binding protein that we have identified is special AT-rich sequence binding protein 2 (SATB2), which is a nuclear matrix attachment region (MAR)-binding protein. It has important functions in the regulation of gene transcription, neuronal and osteoblast (OB) differentiation, and craniofacial patterning. The SATB2 family member, SATB1, has been implicated in breast tumour growth and metastasis. Here, I describe the initial characterization of the p73-SATB2 interaction. SATB2 binds to and stabilizes TAp73a, leading to the enhancement of TAp73a-mediated induction of p53-upregulated modulator of apoptosis (PUMA; apoptotic gene) and p21 (cell cycle gene). Conversely, SATB2 does not bind to but decreases TAp73b levels, resulting in inhibition of TAp73b-mediated transcription. Thus, our findings reveal a role for SATB2 in the regulation of p73 stability and function. Moreover, we demonstrate that SATB2 is overexpressed in osteosarcoma (OS), while it is undetectable in other sarcomas, thereby suggesting that SATB2 may be a potential biomarker that can distinguish OS from other sarcomas. Loss of SATB2 also inhibits the migration and invasion capabilities of OS cells, and suggests that SATB2 promotes both migration and invasion in OS. Therefore, I present work showing that SATB2 is a novel p73-binding partner that differentially regulates the stability and function of the C-terminal isoforms of p73, as well as the novel involvement of SATB2 in OS invasion.

p73 shares many structural and functional similarities with its homologue, the p53 tumour suppressor. Many p73 protein isoforms result from both alternative splicing and differential promoter utilization. Transcriptionally active (TA) isoforms are pro-apoptotic, while N-terminally truncated (dN) variants are anti-apoptotic and act as dominant-negative inhibitors of TAp73 and p53. p73 has been shown to activate the transcription of genes involved in cell cycle arrest and apoptosis in response to DNA damaging agents, including chemotherapies. We hypothesize that the activity of p73, like many transcription factors, may be modulated by binding to regulatory proteins. A novel p73-binding protein that we have identified is special AT-rich sequence binding protein 2 (SATB2), which is a nuclear matrix attachment region (MAR)-binding protein. It has important functions in the regulation of gene transcription, neuronal and osteoblast (OB) differentiation, and craniofacial patterning. The SATB2 family member, SATB1, has been implicated in breast tumour growth and metastasis. Here, I describe the initial characterization of the p73-SATB2 interaction. SATB2 binds to and stabilizes TAp73a, leading to the enhancement of TAp73a-mediated induction of p53-upregulated modulator of apoptosis (PUMA; apoptotic gene) and p21 (cell cycle gene). Conversely, SATB2 does not bind to but decreases TAp73b levels, resulting in inhibition of TAp73b-mediated transcription. Thus, our findings reveal a role for SATB2 in the regulation of p73 stability and function. Moreover, we demonstrate that SATB2 is overexpressed in osteosarcoma (OS), while it is undetectable in other sarcomas, thereby suggesting that SATB2 may be a potential biomarker that can distinguish OS from other sarcomas. Loss of SATB2 also inhibits the migration and invasion capabilities of OS cells, and suggests that SATB2 promotes both migration and invasion in OS. Therefore, I present work showing that SATB2 is a novel p73-binding partner that differentially regulates the stability and function of the C-terminal isoforms of p73, as well as the novel involvement of SATB2 in OS invasion.

Effector caspase activation is a critical regulatory step in apoptosis, as well as an essential inductive cut in numerous non-death related processes that occur within complex cell systems. Here we report two novel studies detailing mechanisms by which effector caspase activation advances muscle cell differentiation. In the first study, we demonstrate that caspase 3 triggered DNA damage leads to rapid formation of XRCC1 repair foci within differentiating myonuclei, which dissipates as the maturation program proceeds. Skeletal myoblast deletion of XRCC1 does not impact cell growth, yet leads to perinatal lethality, with sustained DNA damage and impaired myofiber development. These observations demonstrate that the temporal deployment of the XRCC1-related DNA repair mechanisms are effector caspase mediated, and essential for muscle cell differentiation. In the second study, we sought to investigate whether effector caspase enzymes altered chromatin structure to promote the early differentiation of muscle progenitor cells. Past research has shown that Matrix Attachment region proteins known as Special AT-rich binding proteins are expressed abundantly in stem and progenitor cells, showing rapid decrease in expression as the cell advances into its mature phenotype. Here we demonstrate that effector caspase-7 is responsible for cleavage of Satb2, rather than caspase 3. Satb2 degradation alters the expressed genetic profile leading to acceleration of the muscle differentiation program. Our cumulative work adds novel roles in which effector caspases are vital in the development of cells.

Kolorektalcancer (CRC) är en av de vanligaste förekommande cancerformerna i världen och är även den näst dödligaste typen. Vid immunohistokemiska (IHC) analyser använder man sig av markörer som cytokeratin 20 (CK20) och caudal-typ homeobox 2 (CDX2) för diagnostisering av CRC. En ytterligare markör som kan användas är special AT-rikt sekvensbindningsprotein 2 (SATB2). SATB2 är ett DNA-bindande protein som finns mellan 70-97% hos CRC-fall. Ett automatiserat färgningsinstrument, BenchMark Ultra använder IHC, där analysprincipen går ut på att den inmärkta sekundära antikroppen med pepparrotsperoxidas (HRP) binder till den primära antikroppen och dess antigen. I närvaro av 3’3-diaminobenzindine (DAB) bildas en brun färgprodukt tillsammans med väteperoxidaset (H2O2) i HRP. Syftet med arbetet var att framställa ett färgningsprotokoll för SATB2 och se hur detta protokoll fungerar vid riktiga patientfall. Två klossar med två tarmvävnader på varje och en testkontroll snittades, färgades med BenchMark Ultra och monterades för mikroskopi. Samma procedur utfördes med patientfallen. Resultatet visade att preparaten med SATB2-antikroppsspädningen 1:200 fungerade bäst med värmebehandling vid både ultraView respektive OptiView DAB. Proteasbehandling gav ospecifik infärgning. Det valda optimala protokollet med SATB2-antikroppsspädning på 1:200, värmebehandling 36 min i ultraView DAB kördes på patientfallen. Resultatet jämfördes med tidigare genomförda IHC-analyser med positivitet för CK20 och CDX2. SATB2 utföll positiv för båda patientfallen, vilket indikera på att protokollet fungerar. Slutsatsen kom blev att optimala protokollet för SATB2 är spädning på 1:200 och värmebehandling 36 min i ultraView DAB. Med detta kan SATB2 införas som ett tillägg till CK20 och CDX2 vid diagnostisering av CRC i rutinverksamheten. / Colorectal cancer (CRC) is a common and the second most deadly cancer form in the world. With immunohistochemistry (IHC) analyses, biomarkers as cytokeratine 20 (CK20) and caudal type homeobox 2 (CDX2) are used for diagnosing CRC. Another biomarker that can be used is special AT-rich sequence binding protein 2 (SATB2). SATB2 is a DNA-binding protein that is found in 70-90% of CRC cases. BenchMark Ultra is an automatic staining instrument which uses IHC. The principle is that a marked secondary antibody with horse radish peroxidase (HRP) binds to the primary antibody and it’s antigen. Peroxidase (H2O2) in HRP produces a brown color with 3’3-diaminobenzindine (DAB). The aim of this study was to create a protocol for SATB2 and see how it can be used in real cases. Two embedded colon samples and a test control were cut, stained and mounted on slides for microscopy. Same process was carried out with the patient cases. Results showed samples with SATB2-antibody dilution 1:200 worked best with heat treatment in both detection kits. The protease treatment gave non-specific staining. The optimal protocol with SATB2-antibody dilution 1:200 and heat treatment for 36 min with ultraView DAB were tested on patient cases. The results of the patient cases were compared with previous conducted staining with CK20 and CDX2, SATB2 was positive for both cases. In conclusion, the protocol with dilution 1:200 and heat treatment for 36 min with ultraView DAB suited best for SATB2. SATB2 will be introduced as an additional antibody for diagnosis of CRC.

No description available.

Le cortex cérébral des mammifères est divisé en plusieurs domaines tangentiels appelés des aires fonctionnelles, députées à l'élaboration des afférences motrice et sensorielles, la mise en oeuvre des plans moteurs. Chaque aire fonctionnelle est constituée par six couches de neurones de projections avec différentes morphologies, connectivité et codes moléculaires. Plusieurs facteurs de transcription spécifiant différentes sous-Classes de neurones ont été identifiés à ce jour. Fezf2 et Ctip2 induisent la spécification des neurones sous-Cérébraux, alors que Satb2 induit l'identité calleuse, essentiellement en réprimant la transcription de Ctip2 par le recrutement du complexe NURD au locus de Ctip2. Dans cette étude, je montre que la population de cellules co-Exprimant des marqueurs moléculaires des neurones calleux et sous-Cérébraux, Satb2 et Ctip2, sont d’abord spécifié dans les couches V et VI du néocortex rostro-Médiale chez la souris au stades périnataux, et augmente progressivement entre P0 et P21. En absence de COUP-TFI, un facteur de transcription impliqué dans l'aréalisation, le nombre de cellules co-Exprimant Satb2 et Ctip2 augmente fortement dans la couche V de l’aire somatosensorielle primaire éventuelle. J'ai démontré que l’expression ectopique et prématurée de LMO4 dans la lignée mutante pour COUP-TFI, de-Réprime Ctip2 dans les cellules Satb2 positives en perturbant l’assemblage du complexe NURD au locus de Ctip2. En plus, par l'utilisation d'une lignée transgénique exprimant la GFP dans les neurones de la couche V et de colorants vitaux, j'ai analysé la morphologie, la connectivité et Les propriétés électrophysiologiques de cette classe hybride de neurones. / The mammalian cerebral cortex is subdivided into several tangential domains calledfunctional areas, deputed to the elaboration of motor and sensory inputs and implementation of motor plans. Each functional area is constituted by six layers of projection neurons with different morphologies, connectivity and molecular codes.Several transcription factors specifying different subclasses of neurons have been identified so far. Fezf2 and Ctip2 promote the specification of subcerebralPNs, whereas Satb2 promotes callosal identity, mainly by repressing Ctip2transcription through the recruitment of the NURD complex to the Ctip2 locus.However, little is known about the mechanisms specifying their features in a timeandareal-Specific manner. In this study I show that a population of cells co-Expressing molecular markers of CPN and SCPN neurons, Satb2 and Ctip2, becomes first specified in layers V and VI of rostro-Medial mouse neocortex at perinatal stages and progressively increases between P0 and P21 in somatosensory areas. I found that in neocortices lacking COUP-TFI, a transcription factor involved in arealization, the number of Satb2/Ctip2 co-Expressing cells increases abnormally in layer V of the prospective somatosensory area. I demonstrated that LMO4 ectopic and premature expression of LMO4 in COUP-TFI mutant transgenic line derepresses Ctip2 in Satb2 positive cells by disturbing the assembly of the NURD complex at Ctip2 locus. Moreover, by the use of a transgenic line expressing GFP in layer V neurons and of vital dyes, I analysed morphology, connectivity and electrophysiological activity of this hybrid class of neurons.development. Toget

p63(alpha) belongs to the p53-family of proteins and has full-length (TA) as well as truncated ((delta)N) p63(alpha) isoforms. Previous studies have shown that TA and (delta)Np63(alpha) play multiple roles in cancer and development. In cancer, (delta)Np63(alpha)-mediated transcriptional repression promotes oncogenesis while transactivation by TAp63(alpha) is critical during development. Despite their importance, little is known regarding how TA or (delta)Np63(alpha) is regulated and factors influencing the function of p63(alpha) have yet to be identified. Here, I identify Special AT-rich Binding Protein 2 (SATB2) as a protein that forms a complex with and modulates the function of p63(alpha). SATB2 is detected in multiple head and neck squamous cell carcinoma (HNSCC) cell lines that also show overexpression of (delta)Np63(alpha). Histological analysis on tumor specimens revealed that SATB2 is predominantly expressed in advanced-stage HNSCC cancers. SATB2 increases DNA-binding capabilities of (delta)Np63(alpha), augmenting (delta)Np63(alpha) repression of apoptotic gene expression. Knockdown of SATB2 in HNSCC cells sensitizes cancer cells towards chemotherapy- and radiation-induced apoptosis. These results indicate that SATB2 functions as a co-factor and promotes the transrepression function of (delta)Np63(alpha) in HNSCC. In addition to examining the role of SATB2 in HNSCC, I also investigated the effect of SATB2 on the ability of TAp63(alpha) to induce gene expression. In particular, perp has been shown to be a critical downstream target of p63 during development. ChIP analysis revealed that while SATB2 increases TAp63(alpha)-binding to apoptotic gene promoters, SATB2 decreases TAp63(alpha) localization on the perp promoter and inhibits p63(alpha)-mediated perp induction. SATB2 more readily interacts with human disease-associated p63(alpha) mutations that are found in the SAM domain, further inhibiting transcriptional properties of these mutants. Together, my results suggest that SATB2 is an important modulator of p63(alpha) in cancer and development.

p63(alpha) belongs to the p53-family of proteins and has full-length (TA) as well as truncated ((delta)N) p63(alpha) isoforms. Previous studies have shown that TA and (delta)Np63(alpha) play multiple roles in cancer and development. In cancer, (delta)Np63(alpha)-mediated transcriptional repression promotes oncogenesis while transactivation by TAp63(alpha) is critical during development. Despite their importance, little is known regarding how TA or (delta)Np63(alpha) is regulated and factors influencing the function of p63(alpha) have yet to be identified. Here, I identify Special AT-rich Binding Protein 2 (SATB2) as a protein that forms a complex with and modulates the function of p63(alpha). SATB2 is detected in multiple head and neck squamous cell carcinoma (HNSCC) cell lines that also show overexpression of (delta)Np63(alpha). Histological analysis on tumor specimens revealed that SATB2 is predominantly expressed in advanced-stage HNSCC cancers. SATB2 increases DNA-binding capabilities of (delta)Np63(alpha), augmenting (delta)Np63(alpha) repression of apoptotic gene expression. Knockdown of SATB2 in HNSCC cells sensitizes cancer cells towards chemotherapy- and radiation-induced apoptosis. These results indicate that SATB2 functions as a co-factor and promotes the transrepression function of (delta)Np63(alpha) in HNSCC. In addition to examining the role of SATB2 in HNSCC, I also investigated the effect of SATB2 on the ability of TAp63(alpha) to induce gene expression. In particular, perp has been shown to be a critical downstream target of p63 during development. ChIP analysis revealed that while SATB2 increases TAp63(alpha)-binding to apoptotic gene promoters, SATB2 decreases TAp63(alpha) localization on the perp promoter and inhibits p63(alpha)-mediated perp induction. SATB2 more readily interacts with human disease-associated p63(alpha) mutations that are found in the SAM domain, further inhibiting transcriptional properties of these mutants. Together, my results suggest that SATB2 is an important modulator of p63(alpha) in cancer and development.

The Human Protein Atlas project is a Swedish research initiative that uses antibody-based proteomics for large scale protein profiling in human tissues and cells. Affinity-purified antibodies are produced within the project and used for immunohistochemical staining on tissue micro arrays (TMAs) in order to map the human proteome and publish the result in a protein atlas (www.proteinatlas.org). In this thesis, TMAs were used for analysis of protein expression patterns in order to identify and explore potential biomarkers of clinical relevance. In Paper I, protein expression of SATB2 was studied in colorectal cancer. The results show that SATB2 is a sensitive and specific biomarker for colorectal cancer, staining 85% of all investigated tumors. Moreover, SATB2 in combination with CK20 showed positivity in 97% of all colorectal carcinomas and is therefore suitable as a complementary tool in clinical differential diagnostics of cancer. In Paper II, ANLN was explored as a prognostic biomarker for breast cancer. A high nuclear fraction of ANLN in breast cancer was significantly correlated to large tumor size, high histological grade, hormone receptor negative tumors, high proliferation rate and poor prognosis. Furthermore, ANLN depletion in breast cancer cell lines resulted in cell cycle arrest and cellular senescence with altered cell morphology. In Paper III, young age at breast cancer diagnosis was investigated as an independent risk factor for poor prognosis. TMAs were produced from a selection of patients from a previously defined register-based cohort. The analysis shows that young women with luminal B tumors have a 2.2-fold higher risk of dying of breast cancer compared to older women. In Paper IV, vascular expression of CD93 was explored by image analysis of the tissue-based breast cancer cohort produced in Paper III. The analysis shows that young women with breast cancer display a significantly higher CD93-positive vessel area in their tumors. High CD93-positive vessel area was significantly associated with hormone receptor negative tumors, grade, Ki-67, EGFR and a poor prognosis. In conclusion, this thesis shows that protein expression profiling using TMAs is an important tool for identifying and exploring potential novel biomarkers for cancer.

No description available.