Orthotopic PC-3 Tumor Xenografts in Studies on Prostate Cancer Growth and Metastasis

Prostate cancer is generally a slowly developing disease. However, some cancers develop into an aggressive, metastasic and consequently life-threatening state. The mechanisms of prostate cancer spread are still mainly unidentified but hormones and growth factors are known to been involved. The forming of new blood vessels i.e. angiogenesis is crucial for tumor growth. Blood vessels and lymphatic vessels are also prominent routes for metastasis. Both angiogenic and lymphangiogenic factors are overexpressed in prostate cancer. We established an in vivo model to study the factors effecting human prostate cancer growth and metastasis. Tumors were produced by the orthotopic inoculation of PC-3 prostate cancer cells into the prostates of immunodeficient mice. Like human prostate tumors, these tumors metastasized to prostate-draining lymph nodes. Treatment of the mice with the bisphosphonate alendronate known to decrease prostate cancer cell invasion in vitro inhibited metastasis and decreased tumor growth. Decreased tumor growth was associated with decreased angiogenesis and increased apoptosis of tumor cells. To elucidate the role of angiogenesis in prostate cancer progression, we studied the growth of orthotopic PC-3 tumors overexpressing fibroblast growth factor b (FGF8b) known to be expressed in human prostate cancer. FGF8b increased tumor growth and angiogenesis, which were both associated with a characteristic gene expression pattern. To study the role of lymphangiogenesis, we produced orthotopic PC-3 tumors overexpressing vascular endothelial growth factor C (VEGF-C). Blocking of VEGF-C receptor (VEGFR3) completely inhibited lymph node metastasis whereas overexpression of VEGF-C increased tumor growth and angiogenesis. VEGF-C also increased lung metastases but, surprisingly, decreased spread to lymph nodes. This suggests that the expanded vascular network was primarily used as a route for tumor spreading. Finally, the functionality of the capillary network in subcutaneous FGF8b-overexpressing PC-3 tumors was compared to that of tumors overexpressing VEGF. Both tumors showed angiogenic morphology and grew faster than control tumors. However, FGF8b tumors were hypoxic and their perfusion and oxygenation was poor compared with VEGF tumors. This suggests that the growth advantage of FGF8b tumors is more likely due to stimulated proliferation than effective angiogenesis. In conclusion, these results show that orthotopic prostate tumors provide a useful model to explore the mechanisms of prostate cancer growth and metastasis.

The Role of Integrins in Enterovirus Infections and in Metastasis of Cancer

Integrins are a family of transmembrane glycoproteins, composed of two different subunits (alpha and beta). Altered expression of integrins in tumor cells contributes to metastasis tendency by influencing on the cells‟ attachment to adjacent cells and their migration. Viral pathogens, including certain enteroviruses, use integrins as receptors. Enteroviruses have also been suggested to be involved in the etiopathogenesis of type 1 diabetes. The study focuses on the role of integrins in the pathogenesis of metastasis to cortical bone and on type 1 diabetes (T1D) and echovirus 1 infection. In the first part of the thesis, the role of different integrins in the initial attachment of MDA-MD-231 breast cancer cells to bovine cortical bone disks was studied. A close correlation between alpha2beta1 and alpha3beta1 integrin receptor expression and the capability of the tumor to attach to bone were observed. In the second part, a possible correlation between susceptibility to enterovirus infections in diabetic children and differences in enterovirus receptor genes, including certain integrins, was investigated. In parallel, virus-specific neutralizing antibodies and diabetic risk alleles were studied. In the diabetic group, an amino acid change was detected in the polio virus receptor and the neutralizing antibody titers against echovirus 30 were lower. However, to obtain statistically sustainable results, a larger number of individuals should be analyzed. Echovirus 1 (EV1) enters cells by attaching to the alpha2I domain of the alpha2beta1 integrin. In the third part EV1 was shown to attach to a chimeric receptor construct of the transferrin receptor and the alpha2I domain and to enter cells through clathrin-mediated endocytosis that is normally not used by the virus. The chimeric receptor was recycled to the plasma membrane, whereas the virus remained in intracellular vesicles. The virus replication cycle was initiated in these cells, suggesting that evolution pressure could possibly cause the virus to evolve to use a different entry mechanism. Moreover, a cDNA microarray analysis of host gene expression during EV1 replication showed that 0.53% of the total genes, including several immediate early genes, were differently expressed.

Dissecting the molecular mechanisms of breast cancer bone metastasis for therapeutic targeting

Breast cancer that has metastasized to bone is currently an incurable disease, causing significant morbidity and mortality. The aim of this thesis work was to elucidate molecular mechanisms of bone metastasis and thereby gain insights into novel therapeutic approaches. First, we found that L‐serine biosynthesis genes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1) and phosphoserine phosphatase (PSPH), were up‐regulated in highly bone metastatic MDA‐MB‐231(SA) cells as compared with the parental breast cancer cell line. Knockdown of serine biosynthesis inhibited proliferation of MDA‐MB‐231(SA) cells, and L‐serine was essential for the formation of bone resorbing osteoclasts. Clinical data demonstrated that high expression of PHGDH and PSAT1 was associated with decreased relapse‐free and overall survival and with features typical of poor outcome in breast cancer. Second, RNA interference screening pointed out heparan sulfate 6‐O‐sulfotransferase 2 (HS6ST2) as a critical gene for transforming growth factor β (TGF‐β)‐induced interleukin 11 (IL‐11) production in MDA‐MB‐231(SA) cells. Exogenous heparan sulfate glycosaminoglycans heparin and K5‐NSOS also inhibited TGF‐β‐induced IL‐11 production in MDA‐MB‐231(SA) cells. Furthermore, K5‐NSOS decreased osteolytic lesion area and tumor burden in bone in mice. Third, we discovered that the microRNAs miR‐204, ‐211 and ‐379 inhibited IL‐11 expression in MDA‐MB‐231(SA) cells through direct targeting of the IL‐11 mRNA. MiR‐379 also inhibited Smad‐mediated signaling. Gene expression profiling of miR‐204 and ‐379 transfected cells indicated that these microRNAs down‐regulate several bone metastasis‐relevant genes, including prostaglandin‐endoperoxide synthase 2 (PTGS2). Taken together, this study identified three potential treatment strategies for bone metastatic breast cancer: inhibition of serine biosynthesis, heparan sulfate glycosaminoglycans and restoration of miR‐204/‐211/‐379.

Generation and Characterization of Knockout Mice and Analysis of Patient Material Demonstrates a Role for Tissue Inhibitor of Metalloproteinases 4 (Timp4) in the Cardiovascular System and Tumor Metastasis

This thesis focuses on tissue inhibitor of metalloproteinases 4 (TIMP4) which is the newest member of a small gene and protein family of four closely related endogenous inhibitors of extracellular matrix (ECM) degrading enzymes. Existing data on TIMP4 suggested that it exhibits a more restricted expression pattern than the other TIMPs with high expression levels in heart, brain, ovary and skeletal muscle. These observations and the fact that the ECM is of special importance to provide the cardiovascular system with structural strength combined with elasticity and distensibility, prompted the present molecular biologic investigation on TIMP4. In the first part of the study the murine Timp4 gene was cloned and characterized in detail. The structure of murine Timp4 genomic locus resembles that in other species and of the other Timps. The highest Timp4 expression was detected in heart, ovary and brain. As the expression pattern of Timp4 gives only limited information about its role in physiology and pathology, Timp4 knockout mice were generated next. The analysis of Timp4 knockout mice revealed that Timp4 deficiency has no obvious effect on the development, growth or fertility of mice. Therefore, Timp4 deficient mice were challenged using available cardiovascular models, i.e. experimental cardiac pressure overload and myocardial infarction. In the former model, Timp4 deficiency was found to be compensated by Timp2 overexpression, whereas in the myocardial infarct model, Timp4 deficiency resulted in increased mortality due to increased susceptibility for cardiac rupture. In the wound healing model, Timp4 deficiency was shown to result in transient retardation of re-epithelialization of cutaneous wounds. Melanoma tumor growth was similar in Timp4 deficient and control mice. Despite of this, lung metastasis of melanoma cells was significantly increased in Timp4 null mice. In an attempt to translate the current findings to patient material, TIMP4 expression was studied in human specimens representing different inflammatory cardiovascular pathologies, i.e. giant cell arteritis, atherosclerotic coronary arteries and heart allografts exhibiting signs of chronic rejection. The results showed that cardiovascular expression of TIMP4 is elevated particularly in areas exhibiting inflammation. The results of the present studies suggest that TIMP4 has a special role in the regulation of tissue repair processes in the heart, and also in healing wounds and metastases. Furthermore, evidence is provided suggesting the usefulness of TIMP4 as a novel systemic marker for vascular inflammation.