Jani Seppänen

Expression of fibroblast growth factor (FGF)-8 isoforms and FGF receptors in human ovarian tumors

FGF‐8 is a mitogenic growth factor, which is widely expressed during embryonic development but only at a very low level in adult tissues. Alternative splicing of the human FGF‐8 gene potentially allows coding for 4 protein isoforms (a, b, e, f), which differ in their transforming capacity. The FGF‐8 isoforms preferentially activate the receptors FGFR1IIIc, FGFR2IIIc, FGFR3IIIc and FGFR4. FGF‐8 is over‐expressed in human breast and prostate cancers. Expression has also been found in RT‐PCR studies of human ovarian and testicular cancers. The present study was undertaken to examine which FGF‐8 isoforms are expressed in ovarian cancer and whether FGF‐8 receptors are also expressed. Specimens from 5 normal human ovaries and 51 ovarian tumors (1 benign tumor, 8 borderline malignancies, 42 malignant tumors of different histopathological types) were studied by RT‐PCR and immunohistochemistry. FGF‐8 isoform b was expressed in all ovarian tumors and in all 7 ovarian‐cancer cell lines studied. Isoform a was co‐expressed in 9 malignant ovarian tumors. FGF‐8 mRNA was not detected by RT‐PCR of 3 normal ovary samples. Immunohistochemical staining localized FGF‐8 protein to cancer cells. In general, the increased intensity of FGF‐8 staining was associated with loss of differentiation within the tumors (Bowkers test, p = 0.37). FGF‐8 staining of surface epithelium observed on 2 normal ovaries was very faint. RT‐PCR showed that FGFR1IIIc, FGFR2IIIc and FGFR4 were the FGF‐8 receptors expressed in normal ovaries and in ovarian tumors. FGF‐8 receptor immunoreactivity was preferentially found in normal ovary surface epithelium and tumor cells but also in some stromal cells. Collectively, our results show that ovarian cancers of a wide variety of histological types expressing receptors for FGF‐8 have acquired the capacity of expressing FGF‐8. This suggests that FGF‐8 has an important role in ovarian tumorigenesis. Int. J. Cancer 88:718–725, 2000.

FGF-8b increases angiogenic capacity and tumor growth of androgen-regulated S115 breast cancer cells

Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has transforming potential. Alternative splicing of the mouse Fgf-8 gene potentially codes for eight protein isoforms (a–h) which differ in their transforming capacity in transfected cells. S115 mouse mammary tumor cells express a transformed phenotype and secrete FGF-8 in an androgen-dependent manner. In order to study the role of FGF-8 isoforms in the induction of transformed phenotype of breast cancer cells, we over-expressed FGF-8 isoforms a, b and e in S115 cells. Over-expression of FGF-8b, but not FGF-8a or FGF-8e, induced androgen and anchorage independent growth of S115 cells. FGF-8b-transfected S115 cells formed rapidly growing tumors with increased vascularization when injected s.c. into nude mice. FGF-8a also slightly increased tumor growth and probably tumor vascularization but FGF-8e was not found to have any effects. The angiogenic activity of FGF-8b and heparin-binding growth factor fraction (HBGF) of S115 cell conditioned media was tested in in vitro and in vivo models for angiogenesis using immortomouse brain capillary endothelial cells (IBEC) and chorion allantoic membrane (CAM) assays. Recombinant FGF-8b protein was able to stimulate proliferation, migration, and vessel-like tube formation of IBECs. In addition, stimulatory effect of S115-HBGF on IBE cell proliferation was evident. A positive angiogenic response to FGF-8b was also seen in CAM assay. The results demonstrate that the expression of Fgf-8b is able to promote vessel formation. Angiogenic capacity probably markedly contributes to the ability of FGF-8b to increase tumor growth of androgen-regulated S115 mouse breast cancer cells.

Estrogen and the selective estrogen receptor modulator (SERM) protection against cell death in estrogen receptor alpha and beta expressing U2OS cells

In the current work, we compared the ability of 17beta-estradiol (E2) and the selective estrogen receptor modulators (SERMs), tamoxifen (Tam), raloxifene (Ral) and ospemifene (Osp) to promote the survival of osteoblast-derived cells against etoposide-induced apoptosis. In order to compare the roles of the two estrogen receptor (ER) isotypes, we created a U2OS human osteosarcoma cell line stably expressing either ERalpha (ERalpha) or ERbeta (ERbeta). Transfection with either of the ERs was able to render the U2OS cells sensitive to E2. We show that E2 opposed etoposide-induced apoptosis and that the effect was mediated via both ER isotypes. The ER isotype selective agonists propyl-pyrazole-triol (PPT) and diarylpropionitrile (DPN) had the same effect in U2OS/ERalpha and U2OS/ERbeta cells, respectively. Osp also opposed apoptosis at least in U2OS/ERalpha cells. Tam and Ral were not able to protect against etoposide-induced cell death. In order to evaluate the protective effects of E2 and Osp upon etoposide challenge, we studied the expression of two E2-regulated, osteoblast-produced cytokines, IL-6 and OPG in E2 and SERM-treated U2OS/ERalpha and U2OS/ERbeta cells. Etoposide strongly increased expression of IL-6 and decreased that of OPG. E2 opposed IL-6 increase only in U2OS/ERalpha cells and OPG decrease primarily in ERbeta cells. Osp opposed the effect of etoposide on OPG primarily in U2OS/ERbeta cells but interestingly, it had little effect on IL-6 expression. E2, PPT, DNP and Osp also inhibited etoposide-induced death and cytokine changes in SAOS-2 osteosarcoma cells expressing endogenous ERalpha and ERbeta. Collectively, our results suggest that the osteoblast protective anti-apoptotic effects of E2 are mediated by both ERalpha and ERbeta but those of Osp primarily by ERalpha. In addition, E2 and Osp opposed the etoposide-induced increase of IL-6 and decrease of OPG which changes would increase osteoclastic activity. These anti-resorptive effects of E2 and Osp upon etoposide challenge differed from each other and they seemed to be differentially mediated in ERalpha and ERbeta expressing osteoblast-derived U2OS cells.

Overexpression of vascular endothelial growth factor C increases growth and alters the metastatic pattern of orthotopic PC-3 prostate tumors

BackgroundProstate cancer metastasizes to regional lymph nodes and distant sites but the roles of lymphatic and hematogenous pathways in metastasis are not fully understood.MethodsWe studied the roles of VEGF-C and VEGFR3 in prostate cancer metastasis by blocking VEGFR3 using intravenous adenovirus-delivered VEGFR3-Ig fusion protein (VEGFR3-Ig) and by ectopic expression of VEGF-C in PC-3 prostate tumors in nude mice.ResultsVEGFR3-Ig decreased the density of lymphatic capillaries in orthotopic PC-3 tumors (p < 0.05) and inhibited metastasis to iliac and sacral lymph nodes. In addition, tumor volumes were smaller in the VEGFR3-Ig-treated group compared with the control group (p < 0.05). Transfection of PC-3 cells with the VEGF-C gene led to a high level of 29/31 kD VEGF-C expression in PC-3 cells. The size of orthotopic and subcutaneous PC-3/VEGF-C tumors was significantly greater than that of PC-3/mock tumors (both p < 0.001). Interestingly, while most orthotopic PC-3 and PC-3/mock tumors grown for 4 weeks metastasized to prostate-draining lymph nodes, orthotopic PC-3/VEGF-C tumors primarily metastasized to the lungs. PC-3/VEGF-C tumors showed highly angiogenic morphology with an increased density of blood capillaries compared with PC-3/mock tumors (p < 0.001).ConclusionThe data suggest that even though VEGF-C/VEGFR3 pathway is primarily required for lymphangiogenesis and lymphatic metastasis, an increased level of VEGF-C can also stimulate angiogenesis, which is associated with growth of orthotopic prostate tumors and a switch from a primary pattern of lymph node metastasis to an increased proportion of metastases at distant sites.

Androgen and fibroblast growth factor 8 (FGF8) downregulation of thrombospondin 1 (TSP1) in mouse breast cancer cells

In the search for androgen target genes responsible for malignant growth in S115 mouse mammary tumor cells we found that thrombospondin 1 (TSP1) expression was strongly downregulated by testosterone (Te). Experiments with cycloheximide suggested that Te repression of TSP1 was dependent on de novo protein synthesis. TSP1 repression by Te was preceded by the induction of fibroblast growth factor 8 (FGF8) expression. FGF8 has previously been shown to mediate androgen effects on proliferation of S115 cells by autocrine/paracrine mechanisms. It has also been shown to increase breast cancer cell growth as tumors in nude mice and to stimulate tumor angiogenesis. We studied here the possibility that FGF8 belonged to the Te-induced de novo synthesized proteins that mediate the effect of Te on TSP1 expression in these cells. We found that addition of FGF8b to in vitro cultures or ectopic expression of FGF8b in S115 cells repressed TSP1 expression at mRNA and protein levels even in the absence of Te. FGF2, another angiogenic member of FGF family, also downregulated TSP1 mRNA level in the in vitro cultures of S115 cells. The antisense oligonucleotides for FGF8 did not, however, prevent Te-repression of TSP1 mRNA expression and a neutralizing anti-FGF8b antibody only partially opposed Te induced downregulation of TSP1. These results suggest that both androgen and FGF8 inhibit TSP1 expression independently. They also suggest that opposite to many other androgen-induced responses in S115 cells, the effect of Te on the expression TSP1 is not mediated by FGF8.

Fast growth associated with aberrant vasculature and hypoxia in fibroblast growth factor 8b (FGF8b) over-expressing PC-3 prostate tumour xenografts

BackgroundProstate tumours are commonly poorly oxygenated which is associated with tumour progression and development of resistance to chemotherapeutic drugs and radiotherapy. Fibroblast growth factor 8b (FGF8b) is a mitogenic and angiogenic factor, which is expressed at an increased level in human prostate tumours and is associated with a poor prognosis. We studied the effect of FGF8b on tumour oxygenation and growth parameters in xenografts in comparison with vascular endothelial growth factor (VEGF)-expressing xenografts, representing another fast growing and angiogenic tumour model.MethodsSubcutaneous tumours of PC-3 cells transfected with FGF8b, VEGF or empty (mock) vectors were produced and studied for vascularity, cell proliferation, glucose metabolism and oxygenation. Tumours were evaluated by immunohistochemistry (IHC), flow cytometry, use of radiolabelled markers of energy metabolism ([18F]FDG) and hypoxia ([18F]EF5), and intratumoral polarographic measurements of pO2.ResultsBoth FGF8b and VEGF tumours grew rapidly in nude mice and showed highly vascularised morphology. Perfusion studies, pO2 measurements, [18F]EF5 and [18F]FDG uptake as well as IHC staining for glucose transport protein (GLUT1) and hypoxia inducible factor (HIF) 1 showed that VEGF xenografts were well-perfused and oxygenised, as expected, whereas FGF8b tumours were as hypoxic as mock tumours. These results suggest that FGF8b-induced tumour capillaries are defective. Nevertheless, the growth rate of hypoxic FGF8b tumours was highly increased, as that of well-oxygenised VEGF tumours, when compared with hypoxic mock tumour controls.ConclusionFGF8b is able to induce fast growth in strongly hypoxic tumour microenvironment whereas VEGF-stimulated growth advantage is associated with improved perfusion and oxygenation of prostate tumour xenografts.

Abstract 2680: Improved imaging of orthotopic BxPC3 pancreatic adenocarcinoma xenograft using animal PET/CT.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Treatment of pancreatic adenocarcinoma is difficult because the location of the lesion is often unknown. 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]FDOPA) is used for imaging of neuroendocrine tumours, which take up and decarboxylate amine precursors. [18F]FDOPA use in diagnostic pancreatic imaging has increased, since the exocrine pancreas synthesises dopamine. We studied the uptake of [18F]FDOPA in healthy and diseased pancreas. Mice bearing orthotopic BxPC3 pancreatic adenocarcinoma and sham-operated controls were pre-treated with enzyme inhibitors of aromatic amino acid decarboxylase (AADC), catechol-O-methyl transferase (COMT), monoamine oxidase A (MAO-A) or combinations of these inhibitors. Mice were injected with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) or [18F]FDOPA and scanned via positron emission tomography/computed tomography. The absolute [18F]FDOPA uptake was determined from selected tissues using a well counter. The intratumoural biodistribution pattern of [18F]FDOPA was recorded autoradiographically. The main FDOPA metabolites present in pancreatic samples were determined with high-performance liquid chromatography. [18F]FDG uptake was high in pancreatic tumours, while [18F]FDOPA uptake was highest in healthy pancreas and significantly lower in tumour-bearing pancreas, independent of pre-treatment. [18F]FDOPA uptake in pancreas was lowest with vehicle pre-treatment and highest with pre-treatment with the inhibitor of AADC. When mice received COMT+MAO-A inhibitors, uptake was high in healthy pancreas but low in tumour-bearing pancreas. Unequal uptake may be due to blockade of metabolism and increased availability of [18F]fluorodopamine due to the employed enzyme inhibitors. Combined use of [18F]FDG and [18F]FDOPA is suitable for imaging pancreatic tumours. Pre-treatment with COMT+MAO-A inhibitors improved the differentiation of pancreas from surrounding tissue and healthy pancreas from tumour. Citation Format: Johanna M. Tuomela, Jani Seppanen, Laura Haavisto, Sarita Fosrback, Tero Vahlberg, Olof Solin, Merja Haaparanta. Improved imaging of orthotopic BxPC3 pancreatic adenocarcinoma xenograft using animal PET/CT. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2680. doi:10.1158/1538-7445.AM2013-2680

Abstract 5272: A novel organotypic 3D invasion platform to advance anticancer drug development

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tumor cell invasion is traditionally studied in three-dimensional (3D) organotypic models composed of type I collagen and fibroblasts, where various cell types and the extracellular matrix in the tumor microenvironment (TME) affects tumor cell behavior. As both molecular and cellular components of TME are cardinal participants in tumor progression, special attention should be addressed in the growth environment. As there is urgent need for more predictive models to be utilized in drug discovery and development, organotypic 3D in vitro assay provides an applicable platform. We characterized a novel organotypic model based on human uterine leiomyoma tissue to be used in drug development. To evaluate the general applicability of the myoma model, human tongue squamous cell carcinoma cells (HSC-3) and human triple negative breast cancer cells (MDA-MB-231(SA)-GFP) were cultured on top of human uterine leiomyoma tissue. Organotypic sections were examined by immunohistochemical stainings against cytokeratin and GFP, where invasion area and depth were measured. The organotypic myoma model also gives opportunity for ELISA, RIA and Western blotting analysis of the culture media. The used cell lines were found to invade in highly distinct patterns. This organotypic 3D myoma model provides a potent tool for analyzing the behavior of tumor cells. Moreover, we conclude that established organotypic 3D in vitro platform gives remarkable advantages to screen potential therapeutic options by producing totally new commercially usable models and innovative treatment approaches that target both the cancer cells and the TME components. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5272. doi:1538-7445.AM2012-5272