The epidermal growth factor receptor (EGFR) family consists of four (EGFR, ErbB2, Erbb3, and ErbB4) receptor tyrosine kinases (RTK) whose signalling is important for physiological and malignant cellular functions such as proliferation, survival, migration, and differentiation. EGFR and ErbB2 in particular are established oncogenes in many solid tumours and are targets for anti-cancer treatment. LRIG1 (leucine-rich repeats and immunoglobulin-like domains-1) is a protein that negatively regulates the EGFR-family, and other RTKs and is a proposed tumour suppressor. This thesis examines the expression of the EGFR-family members and LRIG1 in renal cell carcinoma (RCC) and in prostate cancer (PC). In RCC, up-regulation of EGFR was shown for all RCC types analysed: clear cell (ccRCC), papillary (pRCC), and chromophobe (chRCC). ErbB2 was down-regulated in ccRCC. ErbB3 expression was low in non-neoplastic kidney and not significantly altered in RCC. ErbB4 was strongly down-regulated in the vast majority of RCCs of all types. LRIG1 was down-regulated in ccRCC. No prognostic value was found for any of these factors in RCC. In prostate cancer cells, LRIG1 was shown to be up-regulated by androgen stimulation and suppressed the growth of prostate cancer cells. In prostate cancer, the expression and prognostic value of LRIG1 was investigated in two patient series, one with untreated patients and one with patients who had undergone prostatectomy. In the untreated patient series, LRIG1 correlated with malignancy grade (Gleason score) and poor outcome for patients (both cancer specific and overall survival), being an independent prognostic factor. In contrast, in the series of patients who had undergone prostatectomy, LRIG1 expression correlated with a good outcome (overall survival). Thus in RCC, there were alterations in gene-expression of the EGFR-family members and LRIG1 between kidney cortex and RCC and between the RCC types. Despite few associations with clinical factors, these alterations are likely to be of biological importance. In prostate cancer LRIG1 was up-regulated by androgen stimulation and inhibited cell proliferation. LRIG1 expression had prognostic value in prostate cancer, maybe as a secondary marker of androgen receptor activation or because of growth inhibition of prostate cancer cells. Contradicting findings in untreated patients and patients treated with prostatectomy poses the question of whether the prognostic value of LRIG1 and other markers vary depending on the specific biological and clinical circumstances in the materials studied.

Telomerase is a ribonucleoprotein complex that catalyses telomeric repeat addition at the ends of chromosomes. The catalytic subunit, hTERT, acts as a key determinant for telomerase activity control; the induction of hTERT expression is required for telomerase activity. hTERT participates in cellular immortalization and is elevated in certain malignant tissues. Several tumours exhibit telomerase activity, which contributes to the infinite proliferation capacity that promotes tumour progression. Renal cell carcinoma (RCC) represents 2% of all adult malignancies and has a high mortality rate. The WHO classifies RCC into several sub-types based on cytogenetic aberrations and morphological features; the most prevalent sub-types are clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC). The aims of this thesis were to study the expression patterns of various signalling molecules, to elucidate the functional links among them, and to define the roles of these signalling molecules in the regulation of hTERT gene expression and telomerase activity in RCC. The first paper included in this thesis revealed mRNA overexpression of DJ-1 (a PTEN inhibitor), cMyc, and hTERT in clinical ccRCC samples compared to tumour-free kidney cortex tissues. Significant, positive correlations were detected for DJ-1, cMyc, and hTERT mRNA levels in ccRCC, but not in pRCC. In vitro knockdown of DJ-1 by siRNA in ccRCC cells induced downregulation of p-Akt, cMyc, hTERT, and telomerase activity. Forced overexpression of DJ-1 in an ovarian carcinoma cell line was followed by increased hTERT promoter activity, which appeared to be dependent on cMYC binding to the promoter. Collectively, the in vitro studies verified a functional link among DJ-1, cMyc, and hTERT as implied in the clinical ccRCC samples. The second paper included in this thesis demonstrated overexpression of NBS1 mRNA levels in ccRCC compared to the kidney cortex. NBS1 mRNA levels exhibited significant, positive correlations with DJ-1, cMyc, and S phase, but not with hTERT. In vitro experiments suggested that DJ-1 could regulate NBS1 gene expression. The role of the hTERT transcriptional repressor WT1 in RCC was evaluated in the third paper included in this thesis. ccRCC samples displayed low WT1 mRNA levels compared to kidney cortex samples. Interestingly, WT1 expression was negatively associated with hTERT and cMyc both of which were elevated in ccRCC. Forced overexpression of WT1 isoforms in a ccRCC cell line increased the expression of several negative transcriptional regulators of hTERT and diminished the expression of hTERT positive regulators. In consequence, hTERT mRNA levels and telomerase activity were reduced. Chromatin immunoprecipitation verified direct binding of WT1 to the cMyc, Smad3, and hTERT promoters. Taken together, these data suggested that in ccRCC, WT1 affects hTERT at the transcriptional level via a combined effect on both positive and negative regulators. In conclusion, DJ-1 can regulate hTERT and telomerase activity through the PI3K pathway encompassing PTEN, NBS1, p-Akt, and cMyc in ccRCC, but not in pRCC. WT1 negatively regulates hTERT and telomerase activity directly and indirectly through multiple pathways in ccRCC.

Antibody-based targeting techniques play an increasingly important role in cancer research. By targeting a structure that is abundant in tumour cells, but rare in healthy tissues, an antibody can mediate the delivery of radioactivity specifically to tumour cells in the body. This idea is particularly appealing for head and neck squamous cell carcinoma (HNSCC), as the advanced stages have a large fraction of spread disease that is difficult to treat with procedures available today. In this thesis, we have investigated possible radioimmunotargeting structures for HNSCC, and found that CD44v6 is a suitable target for antibody-based radiotherapy and diagnostics in this patient group. We have identified radiohalogens as attractive nuclides for such use, and have investigated the possibility of radiohalogenating the anti CD44v6 chimeric monoclonal antibody (cMAb) U36. Several feasible labelling methods were identified, using both direct and indirect labelling. The cMAb U36 was then successfully labelled with 211At and 131I, and preclinically evaluated for therapeutic use. Results proved the astatinated conjugate to be most efficient in this context, demonstrating a specific and dose-dependent cytotoxicity. The cMAb U36 was then evaluated for diagnostic use in thyroid anaplastic carcinoma, using 124I as the diagnostic nuclide. Results in tumour-bearing mice were promising, with all of the tumours identified in micro-PET studies. These results demonstrate how antibody-based radionuclide targeting can provide more sensitive and specific methods for identifying and treating head and neck cancer, and hopefully help improve long-term survival rates for this patient group in the future.

A prognostic factor is a marker or a feature that can be used to estimate the risk of recurrence of disease, metastatic spread and clinical outcome. Despite intensive search for more sophisticated markers in renal cell carcinoma (RCC), few have added prognostic information to earlier described factors like stage of disease, nuclear grade, tumour type, and in metastatic disease; performance status, anaemia, hypercalcaemia and increased erythrocyte sedimentation. In the dominating tumour type, clear cell renal RCC (cRCC), hypoxia is common, leading to an up-regulation of hypoxia inducible factor (HIF). The majority of cRCC have a mutation in the von Hippel Lindau gene (VHL-gene), which regulates HIF and in turn leads to up-regulation of a number of target genes for potential growth factors. The aim of the study was to evaluate the possible prognostic information of a few factors associated to pVHL/HIF, anemia and/or hypercalcaemia in RCC; erythropoietin (EPO) and it´s receptor (EPO-R), carbonic anhydrase IX (CA IX), parathyroid hormone-related protein (PTHrP) and osteopontin (OPN). Patients diagnosed with RCC between 1982-2007 were included in the studies. The tumour tissue expressions of EPO, EPO-R and PTHrP were assessed using immunohistochemistry. Serum/plasma levels of EPO, CA IX, PTHrP and OPN were also analyzed using immunometric methods. Our study demonstrated that the expression of EPO and EPO-R were related, and the expressions differed significantly between RCC types. The serum EPO levels did not associate to the tumour expression of EPO or EPO-R, indicating that circulating EPO derives from other sources than tumour cells. Erythropietin receptor expression was more frequent in advanced stages of disease, but neither EPO, nor EPO-R, were independent prognostic factors for survival. Serum CA IX levels were higher in cRCC compared to papillary RCC (pRCC). In cRCC, the CA IX serum levels correlated positively to TNM stage, but serum CA IX did not add independent prognostic information. Parathyroid hormone-related protein is a cause of hypercalcaemia in malignancy, and we observed that circulating PTHrP related to hypercalcaemia in RCC. The tumour expression of PTHrP associated positively to serum PTHrP, but not to serum calcium. We found an association between PTHrP and OPN in plasma, and both plasma PTHrP and OPN were positively associated to TNM stage.  Neither serum/plasma PTHrP nor tumour expression of PTHrP were independent prognostic factors for survival. The serum OPN levels were higher in pRCC but no impact on survival was observed in this RCC type. In contrast, plasma/serum OPN was an independent prognostic factor for disease-specific survival in cRCC. Our results support a role for these factors in RCC. The expressions vary between tumour types, which can be explained by different gene aberrations. Some of the factors have a close relation to para-malignant symptoms like hypercalcaemia. Most of the factors correlate positively to TNM-stage, reflecting a relation to advanced disease. Although expression of EPO, EPO-R, PTHrP and CA IX did not add independent prognostic information, the results might contribute to greater understanding of important mechanisms and associations in RCC. Osteopontin is a strong independent prognostic factor in cRCC, and should be further evaluated as a tool in the clinic when treating RCC patients.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Activity of the hypoxia inducible HIF-alpha transcription factors drive the hypoxic response, resulting in enhancement of angiogenesis, tumour growth, invasion and metastasis. Seeking to uncover a role for Tid1 in control of HIF2-alpha, we used lentiviral shRNA to knock-down Tid1 in 786-0 RCC cells with and without pVHL. In 786-0 cells stably expressing pVHL30, Tid1 knock-down resulted in a dramatic reduction in HIF2-alpha levels relative to controls. Adenoviral-mediated overexpression of Tid1S rescued this decline in HIF2-alpha levels, while overexpression of Tid1L enhanced this decline. A protective role of Tid1S for HIF2-alpha was reproduced in a HEK293 cell model. Immunoprecipitations in HEK293 cells revealed a lack of direct binding between HIF2-alpha and Tid1 in vivo, while adenoviral-mediated overexpression of Tid1 in this model failed to alter in vitro binding between HIF2-alpha and pVHL30. We present a model in which Tid1 regulates HIF2-alpha stability through regulation of pVHL30 nuclear import.

Clear cell renal cell carcinoma (ccRCC) is an aggressive malignancy characterized by inactivation of the von Hippel-Lindau tumour suppressor gene, the protein product of which mediates degradation of the transcription factor hypoxia-inducible factor (HIF). GAL3ST1 is a sulfotransferase which catalyzes the production of sulfatide, a plasma membrane sulfolipid previously implicated in metastasis. We observed GAL3ST1 overexpression in primary ccRCC tumours relative to matched-normal tissue and subsequently asked if GAL3ST1 was a HIF-responsive gene that facilitates ccRCC metastasis. GAL3ST1 expression was suppressed in ccRCC cells by stable reconstitution of wild-type VHL and also siRNA-mediated knockdown of HIF1alpha and HIF2alpha. Dual luciferase assays and chromatin immunoprecipitation revealed a hypoxia-response element in the GAL3ST1 5’-UTR that appeared to be crucial for HIF-mediated upregulation. Finally, stable knockdown of GAL3ST1 significantly impeded ccRCC cell invasion through an in vitro basement membrane mimic. These results suggest GAL3ST1 is a HIF-responsive gene that promotes tumour cell invasion.

Clear cell renal cell carcinoma (ccRCC) is an aggressive malignancy characterized by inactivation of the von Hippel-Lindau tumour suppressor gene, the protein product of which mediates degradation of the transcription factor hypoxia-inducible factor (HIF). GAL3ST1 is a sulfotransferase which catalyzes the production of sulfatide, a plasma membrane sulfolipid previously implicated in metastasis. We observed GAL3ST1 overexpression in primary ccRCC tumours relative to matched-normal tissue and subsequently asked if GAL3ST1 was a HIF-responsive gene that facilitates ccRCC metastasis. GAL3ST1 expression was suppressed in ccRCC cells by stable reconstitution of wild-type VHL and also siRNA-mediated knockdown of HIF1alpha and HIF2alpha. Dual luciferase assays and chromatin immunoprecipitation revealed a hypoxia-response element in the GAL3ST1 5’-UTR that appeared to be crucial for HIF-mediated upregulation. Finally, stable knockdown of GAL3ST1 significantly impeded ccRCC cell invasion through an in vitro basement membrane mimic. These results suggest GAL3ST1 is a HIF-responsive gene that promotes tumour cell invasion.

Activity of the hypoxia inducible HIF-alpha transcription factors drive the hypoxic response, resulting in enhancement of angiogenesis, tumour growth, invasion and metastasis. Seeking to uncover a role for Tid1 in control of HIF2-alpha, we used lentiviral shRNA to knock-down Tid1 in 786-0 RCC cells with and without pVHL. In 786-0 cells stably expressing pVHL30, Tid1 knock-down resulted in a dramatic reduction in HIF2-alpha levels relative to controls. Adenoviral-mediated overexpression of Tid1S rescued this decline in HIF2-alpha levels, while overexpression of Tid1L enhanced this decline. A protective role of Tid1S for HIF2-alpha was reproduced in a HEK293 cell model. Immunoprecipitations in HEK293 cells revealed a lack of direct binding between HIF2-alpha and Tid1 in vivo, while adenoviral-mediated overexpression of Tid1 in this model failed to alter in vitro binding between HIF2-alpha and pVHL30. We present a model in which Tid1 regulates HIF2-alpha stability through regulation of pVHL30 nuclear import.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.