Identification of novel regulators for tumor progression in breast cancer

Breast cancer is the most frequent solid tumor among women and the leading cause of cancer related death in women worldwide. The prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor. In this thesis, the aim was to identify novel regulators of tumor progression in breast cancer as well as to get insights into the molecular mechanisms of breast cancer progression and metastasis. First, the role of phospholipid remodeling genes and enzymes important for breast cancer progression was studied in breast cancer samples as well as in cultured breast cancer cells. Tumor samples displayed increased de novo synthesized fatty acids especially in aggressive breast cancer. Furthermore, RNAi mediated cell based assays implicated several target genes critical for breast cancer cell proliferation and survival. Second, the role of arachidonic acid pathway members 15-hydroxyprostaglandin dehydrogenase (HPGD) and phospholipase A2 group VII (PLA2G7) in tumorigenesis associated processes was explored in metastatic breast cancer cells. Both targets were found to contribute to epithelial-mesenchymal transition related processes. Third, a high-throughput RNAi lysate microarray screen was utilized to identify novel vimentin expression regulating genes. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was found to promote cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion. Taken together, this study identified several putative targets for breast cancer therapy. In addition, these results provide novel information about the mechanisms and factors underlying breast cancer progression.

VAP-1 in Leukocyte Trafficking

The extravasation of leukocytes from the blood stream into the tissues is a prerequisite for adequate immune surveillance and immune reaction. The leukocyte movement from the bloodstream into the tissues is mediated by molecular bonds. The bonds are formed between adhesion molecules on endothelial cells and their counterparts expressed on leukocytes. Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule mediating leukocyte interactions with endothelium. It is also an enzyme having semicarbazide sensitive amine oxidase (SSAO) activity. The SSAOactivity catalyses deamination of primary amines into corresponding aldehyde and during the enzymatic reaction hydrogen peroxide and ammonia are produced. The aim of this study was to investigate the relationship between the adhesive and enzymatic activities of VAP-1. The role of VAP-1 in leukocyte traffic was studied in vivo under normal and pathological conditions in VAP-1 deficient mice. The results from in vitro flow-based assays indicated that VAP-1 uses both SSAOactivity and its adhesive epitope to bind leukocytes, and both are perquisites for VAP-1 mediated adhesion. Furthermore, in vivo results demonstrated that leukocyte trafficking was impaired in vivo by deleting VAP-1 or inhibiting SSAO-activity. There was impairment in lymphocyte recirculation as well as leukocyte accumulation into the inflamed area. Moreover, the VAP-1 deficient mice did not show generalized defects in antimicrobial responses, whereas significant reduction in tumor progression and neovascularization was observed. These results indicate that VAP-1 could be used as a target in anti-adhesive therapies either by blocking its adhesive epitope with antibodies or by inhibiting its SSAO-activity using inhibitors. Moreover, targeting of VAP-1 may provide a new way of inhibiting neovascularization in tumors.

Complement system in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) consists 20% of keratinocytederived non-melanoma skin cancers (NMSC), the incidence of which is increasing globally. cSCC is the most common metastatic skin cancer and it causes approximately 20% of skin cancer-related deaths. At present, there are no molecular markers for predicting which cSCC lesions are aggressive or metastasize rapidly. UV radiation is the most important risk factor for cSCC. During the development of cSCC, normal epidermal keratinocytes are transformed and form actinic keratosis (AK), which progresses to cSCC in situ (cSCCIS, Bowen’s disease) and finally to invasive and metastatic cSCC. Inflammatory factors and cells are a part of cancer microenvironment and cSCC can develop in the chronically irritated skin or in the context of chronic inflammation. The complement system is a central part of innate immunity and it regulates normal immunological and inflammatory processes. In this study, the role of complement system components and inhibitors were studied in the progression of cSCC in culture and in vivo. Elevated expression of complement factor H (CFH), complement factor I (CFI), complement component C3 and complement factor B (CFB) was noted in cSCC cells in culture. The analysis with immunohistochemistry (IHC) revealed that the expression of CFH, CFI, C3 and CFB was specifically noted in tumor cells in vivo. The staining intensity of CFH, CFI, C3 and CFB was also stronger in invasive cSCC than in AK or cSCCIS samples. The knockdown of CFH, CFI and CFB with specific siRNAs decreased cSCC cell viability and migration, whereas the knockdown of C3 reduced only cSCC cell migration. Moreover, the knockdown of CFI, C3 and CFB inhibited growth of cSCC xenograft tumors established in SCID mice in vivo. In these tumors, CFI, C3 and CFB knockdown decreased the number of proliferating cells. Moreover, the knockdown of CFI increased local inflammation and complement activation. This study provides evidence for the roles of CFH, CFI, C3 and CFB in the tumor progression indicating these as molecular biomarkers and putative therapeutic targets of cSCC.

Filopodia and stromal extracellular matrix as regulators of cancer cell invasion and growth

Bidirectional exchange of information between the cancer cells and their environment is essential for cancer to evolve. Cancer cells lose the ability to regulate their growth, gain the ability to detach from neighboring cells and finally some of the cells disseminate from the primary tumor and invade to the adjacent tissue. During cancer progression, cells acquire features that promote cancer motility and proliferation one of them being increased filopodia number. Filopodia are dynamic actin-rich structures extending from the leading edge of migrating cells and the main function of these structures is to serve as environmental sensors. It is nowadays widely appreciated, that not only the cancer cells, but also the surrounding of the tumor – the tumor microenvironment- contribute to cancer cell dissemination and tumor growth. Activated stromal fibroblasts, also known as cancer-associated fibroblasts (CAFs) actively participate on tumor progression. CAFs are the most abundant cell type surrounding the cancer cells and they are the main cell type producing the extracellular matrix (ECM) within tumor stroma. CAFs secrete growth factors to promote tumor growth, direct cancer cell invasion as well as modify the stromal ECM architecture. The aim of this thesis was to investigate the function of filopodia, particularly the role of filopodia-inducing protein Myosin-X (Myo10), in breast cancer cell invasion and metastasis. We found that Myo10 is an important regulator of basal type breast cancer spreading downstream of mutant p53. In addition, I investigated the role of CAFs and their secreted matrix on tumor growth. According to the results, CAF-derived matrix has altered organization and stiffness which induces the carcinoma cell proliferation via epigenetic mechanisms. I identified histone demethylase enzyme JMJD1a to be regulated by the stiffness and to participate in stiffness induced growth control.

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.

Molecular markers for progression of squamous cell carcinoma of the skin

Incidence of nonmelanoma skin cancer (NMSC) is increasing. Ultraviolet (UV) –light is a major risk factor for the development of cutaneous SCC. Cutaneous SCCs that develop to chronic ulcers are known to progress and metastasize more easily than UV-induced SCCs. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes which are suggested to have a role in cancer growth and invasion. The molecular background for progression of cutaneous SCC was examined by immunohistochemistry (IHC) using tissue samples of recessive dystrophic epidermolysis bullosa (RDEB) –associated SCC, sporadic UV-induced SCC, and SCC precursors. IHC studies using tissue microarray (TMA) technique revealed overexpression of MMP-7 and MMP-13 in SCC tumor cells. MMP-7 expression was enhanced especially in the SCC tumor cells of the RDEB –associated SCCs. Studies with SCC cell lines showed that tumor cell derived MMP-7 activated heparin binding epidermal growth factor –like growth factor (HB-EGF) which enhanced the growth of SCC tumor cells. Further, it was shown that type VII collagen (COL7) is expressed in sporadic SCC tumor cells. Interestingly, it was shown that SCC –associated MMP-13 is capable of cleaving COL7 in vitro. COL7 cleavage may have a role in the progression of cutaneous SCC. Studies on serine proteinase inhibitor gene family using SCC tumor cell gene array, quantitative real-time PCR, SCC cell lines, normal human epidermal keratinocytes and IHC of TMA samples showed that serine proteinase inhibitor clade A, member 1 (serpinA1, alpha-1-antitrypsin) is expressed and produced by human SCC tumor cells but not by normal keratinocytes. Moreover, serpinA1 expression was shown to correlate with the progression of cutaneous SCC using transformed HaCaT-cell lines and mouse chemically induced skin SCC model. SerpinA1 may serve as a novel biomarker for the progression of cutaneous SCC. This study elucidated putative mechanisms of the progression of cutaneous SCC and revealed novel biomarker candidates for the progression of SCC of the skin.

Roles of novel biomarkers in progression of cutaneous squamous cell carcinoma

Roles of novel biomarkers was studied in progression of cutaneous squamous cell carcinoma (cSCC) as the most common metastatic skin cancer. The incidence of cSCC is increasing worldwide due to lifestyle changes such as recreational exposure to sunlight and the aging of the population. Because of an emerging need for molecular markers for the progression of cSCC, we set our goal to characterize three distinct novel markers overexpressed in cSCC cells. Our results identified overexpression of serpin peptidase inhibitor clade A member 1 (SerpinA1), EphB2 and absent in melanoma 2 (AIM2) in cSCC cell lines compared with normal human epidermal keratinocytes (NHEKs). Immunohistochemical analysis of SerpinA1, EphB2 and AIM2 revealed abundant tumor cell-specific expression of cytoplasmic SerpinA1 and AIM2 and cytoplasmic and membranous EphB2 in cSCC tumors in vivo. The staining intensity of SerpinA1, EphB2 and AIM2 was significantly stronger in cSCC as compared with carcinoma in situ (cSCCIS) and actinic keratosis (AK). Tumor cell-associated SerpinA1 and EphB2 was noted in chemically induced mouse skin SCC, and the staining intensity was stronger in mouse cSCCs than in untreated skin. AIM2 staining intensity was significantly more abundant in cSCC of organ transplant recipients (OTR) than in sporadic cSCC in vivo. EphB2 knockdown resulted in inhibition of migration in cSCC cells. In addition, knockdown of EphB2 and AIM2 was found to inhibit the proliferation and invasion of cSCC cells and to delay the growth and vascularization of cSCC xenografts in vivo. Altogether, these findings identify SerpinA1 as a novel biomarker for cSCC. In addition, characterization of the roles of EphB2 and AIM2 in the progression of cSCC was implicated them as possible therapeutic targets for the treatment of cSCC particularly in unresectable and metastatic tumors.