Alterations to the tumour microenvironment is a common feature of many cancers, including breast cancer, and there is increasing evidence that alterations to the microenvironment, including; increased integrin expression, ECM deposition and protease activity, promote cancer progression. Most invasive breast cancers arise from a preinvasive stage, ductal carcinoma in situ (DCIS). Previous work in our laboratory has shown the microenvironment of DCIS is altered, such that myoepithelial cells (MECs) switch to a tumour-promoting phenotype, associated with upregulation of integrin αvβ6 and fibronectin (FN) expression. Mechanisms by which integrin αvβ6 and FN expression are regulated is unclear. We show DCIS progression into invasion is accompanied by an increase in MEC expression of integrin αvβ6 and periductal FN deposition, and their expression were associated in DCIS. These findings were modelled in isolated primary DCIS-MECs, primary normal MECs and MEC lines, with and without integrin αvβ6 expression, where integrin αvβ6-positive MECs upregulating FN expression. We identified integrin αvβ6-positive DCIS ducts were larger than integrin αvβ6-negative DCIS ducts, and mechanical stretching of primary normal MECs and a normal MEC line led to upregulation of integrin αvβ6 expression and FN deposition in a TGFβ-dependent manner. We further show upregulation of integrin αvβ6 and FN by MECs mediate TGFβ-dependent upregulation of MMP13 which promotes breast cancer cell invasion in vitro. These data show altered tissue mechanics in DCIS and MEC expression of integrin αvβ6 and FN deposition are linked, and implicate TGFβ in their activation. These findings suggest integrin αvβ6 and FN may be used as markers to stratify DCIS patients.

Squamous cell carcinoma of the head and neck (SCCHN), the 6th most common cancer worldwide, has a low 5-year survival. Disease as well as treatment often causes patients severe functional and aesthetic problems. In order to improve treatment and diagnosis at earlier stages of tumour development it is important to learn more about the molecular mechanisms behind the disease. p63, an important regulator of epithelial formation, has been suggested to play a role in the development of SCCHN. Six different isoforms of p63 have been found and shown to have various functions. The aim of the studies in this thesis was to learn more about the role of p63 and proteins connected to p63 in SCCHN. Expression of p63, Cox-2, EGFR, beta-catenin, PP2A and p53 isoforms was mapped in tumours and normal tumour adjacent tissue from patients with SCCHN using western blot or RT-PCR. Results showed no significant difference between tumours and normal tumour adjacent tissue concerning expression of EGFR and beta-catenin. Cox-2 and PP2A showed significantly higher expression in tumours while p63 was more expressed in normal tumour adjacent tissue. However, expression of all these proteins in normal tumour adjacent tissue differed from tissue from disease-free non-smoking individuals. Smoking in itself did not affect expression of these proteins. The p53 isoforms p53, p53beta, p53gamma, ∆133p53, ∆133p53beta and ∆133p53gamma were expressed at RNA level in samples both from tumours and normal tumour adjacent tissue, though most of them at fairly low levels. The functional properties of the different p63 isoforms have not been fully mapped. By establishing stable cell lines over-expressing the different p63 isoforms we investigated their specific effect on tumour cells from SCCHN. Only the ∆Np63 isoforms could be stably over-expressed, whereas no clones over-expressing TAp63 could be established. Using microarray technique, cell lines stably expressing the ∆Np63 isoforms were studied and CD44, Keratins 4, 6, 14, 19 and Cox-2 were found to be regulated by p63. In conclusion, the present project adds new data to the field of p63 and SCCHN. For example, we have shown that clinically normal tumour adjacent tissue is altered compared to normal oral mucosa in non tumour patients, and that smoking does not change expression of p63, Cox-2, EGFR, beta-catenin or PP2A in oral mucosa. Novel p53 isoforms are expressed in SCCHN, and even though levels are very low they should not be overlooked. Furthermore, CD44, keratins 4, 6, 14, 19 and Cox-2 were identified as p63 targets in SCCHN.

Fibrosis is a sequel of chronic inflammation and is defined as an excessive deposition of collagen that ultimately leads to organ dysfunction. To date there are no effective treatments for fibrosis. The main cell type involved in collagen deposition and organization is the myofibroblast. In the first study we examined how myofibroblasts differentiate in human fibrotic conditions and in experimental animal models. Human tissues were stained with antibodies that recognize integrin receptors and in addition we also stained for α-SMA, a myofibroblast marker. We found a co-localization between these two markers in stromal cells and hypothesized that integrin α1 is important for the acquisition of the myofibroblast phenotype. To tests this hypothesis we used knockout animals for this integrin subunit. These animals showed a reduction of α-SMA positive fibroblasts, indicating that the α1 integrin subunit is required for proper myofibroblast differentiation. In the second study we used a neuroblastoma tumor model to study tumour growth when a drug targeting the synthesis of cellular NAD was administered. In treated animals an expansion of the nonvascular stroma was observed compared to controls. Normalization of the vasculature was observed in treated tumors together with a decrease in hypoxia. Moreover, this was followed by a decrease in stromal PDGF-B and VEGF expression, suggesting a deactivation of the stroma. In the third study the effects of over-expression of the two pro-fibrotic growth factors TGF-β and PDGF-B in skin was evaluated. We observed that both growth factors induced fibrosis. Over time, a decrease in blood vessel density was observed in both treatment groups. Both factors also stimulated an expansion of the connective tissue cell population originating from the microvascular pericyte, but the phenotype of these cells differed in the different treatments with regards to expression of markers. Furthermore, in tissue over-expressing PDGF-B but not TGF-β, the fibrotic process was partially reversible.

In addition to malignant cells, solid tumors comprise supporting stromal tissue that consists of extra cellular matrix (ECM), connective tissue cells, inflammatory cells and blood vessels. The stromal compartment and the malignant cells together shape the tumor microenvironment that in turn determines tumor progression and efficacy of anti-tumor treatments. In this thesis, studies that investigate the roles of different kinds of interactions between tumor cells and stromal cells were undertaken. Further, growth factors that have important roles in interactions between tumor cells and stromal cells were investigated in a non-tumor environment. Tumor cells were found to modulate the response to the platelet derived growth factor  (PDGF) by microvascular pericytes, a cell type found in the vasculature of solid tumors. The importance of this growth factor in biology of tumors has earlier been shown, but here it was shown that PDGF also modulate the ECM phenotype of solid tumors. The ECM of tumors treated with an inhibitor of PDGF receptor (PDGFR) signaling induced a less fibrotic collagen scaffold, which could explain how PDGFR inhibition in earlier reports lowered tumor interstitial fluid pressure (IFP). Lowering the normally high IFP in tumors increases efficacy of chemotherapy. The integrin αVβ3 is activated downstream of PDGF-B in acute inflammations, and this integrin is important for raising IFP in loose connective tissue in such conditions. However, in tumors we found that lack of the β3 subunit lead to an increased IFP, which were attributed to a more fibrotic ECM phenotype. In addition to PDGF-B, transforming growth factor β (TGFβ) is an important growth factor in the biology of tumors. These two growth factors were separately overexpressed in mouse skin and they both induced an inflammatory response. Expressed in a tumor free context, they evoked a response that was in many ways reminiscent of what can be observed in the tumor microenvironment. This thesis contributes further understanding of how the complex tumor microenvironment affects the phenotype of solid tumors.

Malignant glioma is a highly lethal and destructive disease with no proper cure. We have investigated some of the hallmarks of cancer in connection to glioma and found ways to disrupt these and prevent tumor growth. The work is done within the context of a glioma subtype distinguished by activation of PDGF signaling termed the proneural subtype. In two of the studies we have investigated mechanisms regulating the glioma cells themselves, and in the other two we have focused on the tumor stroma. In the first study, glioma-initiating cells were isolated in defined serum free culture medium from PDGF-B driven murine glioma and shown to be independent of EGF and FGF2 for self-renewal and proliferation. When cultured in serum the GICs displayed an aberrant differentiation pattern that was reversible. Specific depletion of the transduced PDGF-B caused a loss of self-renewal and tumorigenicity and induced oligodendrocyte differentiation. The transcription factor S-SOX5 has previously been shown to have a tumor suppressive effect on PDGF-B induced murine glioma, and to induce cellular senescence in PDGF-B stimulated cells in vitro. We found that S-SOX5 had a negative effect on proliferation of newly established human glioma cells cultured under stem cell conditions. We also revealed a connection between alterations causing up-regulation of SOX5 with the proneural subgroup and a tendency towards co-occurrence with PDGFRA alterations. Angiogenesis, the formation of new blood vessels from existing ones, is an important hallmark for glioma malignancy. We found that the anti-angiogenic protein HRG had a negative effect on glioma progression in PDGF-B induced experimental tumors and that HRG was able to completely prevent formation of glioblastomas. Subsequently it was shown that HRG could skew pro-tumorigenic tumor associated macrophages into an anti-tumorigenic phenotype. Stromal cells had not previously been fully investigated in gliomas. We observed a correlation between tumor malignancy and increased numbers of tumor-associated macrophages as well as pericytes in PDGF-B induced gliomas. There was also a correlation between tumor grade and vessel functionality that had not previously been shown. Our results offer further understanding of gliomagenesis and present possible future therapies.

Neurofibromatosis type 2 (NF2) is an inherited, multiple tumour disease caused by loss of the tumour suppressor protein, Merlin. There are several tumours associated with NF2 including; ependymomas, meningiomas and schwannomas. Merlin loss can also occur sporadically in all of these tumours and is associated with upregulation of various growth factor receptors and their relevant signalling pathways. At present the only treatment options for NF2 are surgery or radiosurgery, both of which incur serious morbidity and are unable to prevent recurrence of tumours. Either new drug treatments, or re-profiling of other drugs already commercially available, are urgently needed to improve outcome for NF2 patients. Cellular prion protein (PrPC), encoded by PRNP gene, is involved in tumour development by altering proliferation, adhesion, and survival in some cancers via focal adhesion kinase (FAK) /Src/ NFκB, cyclin D1 and p53 -proteins. Our group previously showed a strong elevation of PRNP gene activity in schwannoma. I hypothesise that PrPC may contribute to schwannoma development. To study the role of PrPC in schwannoma development I have used the well-established in vitro model of schwannoma that comprises primary human Schwann and schwannoma cells. I show that PrPC is upregulated in schwannoma as well as in Merlin-deficient meningiomas and human malignant mesotheliomas. In schwannoma PrPC is released both via exosomes and by α-cleavage which forms biologically active N- and C-terminal portions of the protein. PrPC contributes to pathological proliferation, adhesion and survival of schwannoma cells by activating ERK1/2, PI3K/AKT, cyclin D1, FAK, p53 pathways via the 37/67kDa non-integrin laminin receptor (LR/37/67kDa) and CD44. Furthermore, schwannoma cells appear to be intrinsically drug-resistant due to upregulation of MDR1 protein p-glycoprotein (p-gp) expression. P-gp expression is dependent on PrPC thus, inhibiting PrPC may be a good potential new therapeutic option for schwannoma patients, either alone or in combination with Sorafenib and p-gp inhibitor Valspodar (PSC833). An inhibitor of LR/37/67kDa/PrP interaction, NSC47924, or Bortezomib, a proteasome/NFκB inhibitor which has been approved for the treatment of multiple myeloma, could also be of beneficial therapeutic effect and is something to investigate in future work. I conclude that PrPC is an interesting new therapeutic target through its involvement with schwannoma patholgenesis and resistance to drug treatments PrPC may prove to be a good therapeutic target in other NF2-related tumours like meningiomas and schwannomas.

<p>Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the <i>MEN1</i> and <i>VHL</i> tumour suppressor genes, is still elusive. The protein product of the <i>MEN1</i> gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.</p><p>Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous <i>Men1</i> mice. For comparison, normal and <i>MEN1</i> non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between <i>Men1</i> expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in <i>Men1</i> mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and <i>Men1</i> mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of <i>Men1</i> and wt mice were observed.</p><p>In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.</p><p>Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.</p>

<p>Varje år diagnostiseras över 50 000 individer med cancer i Sverige. Ett cancerbesked väcker blandade känslor och associeras ofta med lidande och död. Ett svårt besked kan leda till en förändrad livssituation och kan även ses som början på en lång och mödosam resa. En vetenskaplig litteraturstudie baserad på 15 originalartiklar genomfördes med syftet att belysa patientens upplevelse av ett cancerbesked och därmed öka sjuksköterskans förståelse för patientens situation samt fördjupa kunskaperna inom ämnet. Genom litteraturgranskningen identifierades tre teman: information, emotionella reaktioner samt psykosocialt stöd. Patienten upplever att det är viktigt att informationen ges på ett öppet och ärligt sätt. Vidare framkom det betydelsefullt att uppmärksamma patientens emotionella reaktioner i samband med beskedet. Eftersom upplevelsen av ett cancerbesked påverkar patientens fortsatta upplevelse av sin sjukdom, har sjuksköterskan en viktig roll att fylla genom att erbjuda psykosocialt stöd i de olika tänkbara situationer som kan uppkomma i samband med ett livsavgörande besked. Fortsatt forskning behövs för att jämföra hur de rekommendationer som finns angående delgivandet av ett svårt besked överensstämmer med patienternas egna upplevelser och önskemål.</p> / <p>Every year, over 50 000 individuals in Sweden are diagnosed with cancer. The disclosure of the cancer diagnosis arouses emotions and is often associated with suffering and death. Receiving bad news may lead to changes in life and can also be seen as the beginning of a long and difficult journey. A scientific study based on 15 original articles was carried out with the purpose to identify the patient’s experience of receiving a cancer diagnosis and therefore increase the nurse’s understanding for the patient’s situation and deepen the knowledge of the subject. When examining the articles three themes were identified: information, emotional reactions and psychosocial support. The patient experience that it is important that the information is given in an open and honest manner. Patients also find it important that their emotional reaction is being observed as receiving the diagnosis. As the disclosure of the cancer diagnosis affects the patient’s further perception of the disease the nurse has an important role providing psychosocial support in various situations that may arise in connection with the disclosure. Continued research is needed to compare how guidelines for giving bad news to a patient correspond with the patient’s own experiences and preferences.</p>

Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the MEN1 and VHL tumour suppressor genes, is still elusive. The protein product of the MEN1 gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling. Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous Men1 mice. For comparison, normal and MEN1 non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between Men1 expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in Men1 mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and Men1 mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of Men1 and wt mice were observed. In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression. Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.

The incidence of cutaneous malignant melanoma has increased dramatically in Caucasians the last few decades, an increase that is partly explained by altered sun exposure habits. For the individual patient, with a localized disease, the tumor thickness of the excised lesion is the most important prognostic factor. However, there is a need to identify characteristics that can place patients in certain risk groups. In this study, the protein expression of multiple proteins in malignant melanoma tumors was studied, with the aim of identifying potential new candidate biomarkers. Representative samples from melanoma tissues were assembled in a tissue microarray format and protein expression was detected using immunohistochemistry. Multiple cohorts were used and for a subset of proteins the expression was also analyzed in melanocytes in normal skin and in benign nevi. The immunohistochemical staining was evaluated manually and for part of the proteins also with an automated algorithm. The protein expression of STX7 was described for the first time in tumors of the melanocytic lineage. Stronger expression of STX7 and SOX10 was seen in superficial spreading melanomas compared with nodular malignant melanomas. An inverse relationship between STX7 expression and T-stage was seen and between SOX10 expression and T-stage and Ki-67, respectively. In a population-based cohort the expression of MITF was analyzed and found to be associated with prognosis. Twenty-one potential biomarkers were analyzed using bioinformatics tools and a protein signature was identified which had a prognostic value independent of T-stage. The protein driving this signature was RBM3, a protein not previously described in malignant melanoma. Other markers included in the signature were MITF, SOX10 and Ki-67. In conclusion, the protein expression of numerous potential biomarkers was extensively studied and a new prognostic protein panel was identified which can be of value for risk stratification.