Publications by authors named "Pirkko L Härkönen"

27 Publications

  • Page 1 of 1

Dovitinib dilactic acid reduces tumor growth and tumor-induced bone changes in an experimental breast cancer bone growth model.

J Bone Oncol 2019 Jun 19;16:100232. Epub 2019 Mar 19.

University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland.

Advanced breast cancer has a high incidence of bone metastases. In bone, breast cancer cells induce osteolytic or mixed bone lesions by inducing an imbalance in bone formation and resorption. Activated fibroblast growth factor receptors (FGFRs) are important in regulation of tumor growth and bone remodeling. In this study we used FGFR1 and FGFR2 gene amplifications containing human MFM223 breast cancer cells in an experimental xenograft model of breast cancer bone growth using intratibial inoculation technique. This model mimics bone metastases in breast cancer patients. The effects of an FGFR inhibitor, dovitinib dilactic acid (TKI258) on tumor growth and tumor-induced bone changes were evaluated. Cancer-induced bone lesions were smaller in dovitinib-treated mice as evaluated by X-ray imaging. Peripheral quantitative computed tomography imaging showed higher total and cortical bone mineral content and cortical bone mineral density in dovitinib-treated mice, suggesting better preserved bone mass. CatWalk gait analysis indicated that dovitinib-treated mice experienced less cancer-induced bone pain in the tumor-bearing leg. A trend towards decreased tumor growth and metabolic activity was observed in dovitinib-treated mice quantified by positron emission tomography imaging with 2-[F]fluoro-2-deoxy-D-glucose at the endpoint. We conclude that dovitinib treatment decreased tumor burden, cancer-induced changes in bone, and bone pain. The results suggest that targeting FGFRs could be beneficial in breast cancer patients with bone metastases.
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http://dx.doi.org/10.1016/j.jbo.2019.100232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434100PMC
June 2019

Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells.

Oncotarget 2018 Aug 24;9(66):32593-32608. Epub 2018 Aug 24.

University of Turku, Institute of Biomedicine, FI-20520 Turku, Finland.

Bisphosphonates are used for prevention of osteoporosis and metastatic bone diseases. Anti-invasive effects on various cancer cells have also been reported, but the mechanisms involved are not well-understood. We investigated the effects of the nitrogen-containing bisphosphonate alendronate (ALN) on the regulation of actin cytoskeleton in PC-3 cells. We analyzed the ALN effect on the organization and the dynamics of actin, and on the cytoskeleton-related regulatory proteins cofilin, p21-associated kinase 2 (PAK2), paxillin and focal adhesion kinase. Immunostainings of cofilin in ALN-treated PC-3 cells and xenografts were performed, and the role of cofilin in ALN-regulated F-actin organization and migration/invasion in PC-3 cells was analyzed using cofilin knockdown and transfection. We demonstrate that disrupted F-actin organization and decreased cell motility in ALN-treated PC-3 cells were associated with decreased levels of total and phosphorylated cofilin. PAK2 levels were also lowered but adhesion-related proteins were not altered. The knockdown of cofilin similarly impaired F-actin organization and decreased invasion of PC-3 cells, whereas in the cells transfected with a cofilin expressing vector, ALN treatment did not decrease cellular cofilin levels and migration as in mock transfected cells. ALN also reduced immunohistochemical staining of cofilin in PC-3 xenografts. Our results suggest that reduction of cofilin has an important role in ALN-induced disruption of the actin cytoskeleton and inhibition of the PC-3 cell motility and invasion. These data also support the idea that the nitrogen-containing bisphosphonates could be efficacious in inhibition of prostate cancer invasion and metastasis, if delivered in a pharmacological formulation accessible to the tumors.
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http://dx.doi.org/10.18632/oncotarget.25961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135693PMC
August 2018

Adenosine inhibits tumor cell invasion via receptor-independent mechanisms.

Mol Cancer Res 2014 Dec 30;12(12):1863-74. Epub 2014 Jul 30.

Department of Medical Microbiology, University of Turku, Turku, Finland.

Unlabelled: Extracellular adenosine mediates diverse anti-inflammatory, angiogenic, and other signaling effects via binding to adenosine receptors, and it also regulates cell proliferation and death via activation of the intrinsic signaling pathways. Given the emerging role of adenosine and other purines in tumor growth and metastasis, this study evaluated the effects of adenosine on the invasion of metastatic prostate and breast cancer cells. Treatment with low micromolar concentrations of adenosine, but not other nucleosides or adenosine receptor agonists, inhibited subsequent cell invasion and migration through Matrigel- and laminin-coated inserts. These inhibitory effects occurred via intrinsic receptor-independent mechanisms, despite the abundant expression of A2B adenosine receptors (ADORA2B). Extracellular nucleotides and adenosine were shown to be rapidly metabolized on tumor cell surfaces via sequential ecto-5'-nucleotidase (CD73/NT5E) and adenosine deaminase reactions with subsequent cellular uptake of nucleoside metabolites and their intracellular interconversion into ADP/ATP. This was accompanied by concurrent inhibition of AMP-activated protein kinase and other signaling pathways. No differences in the proliferation rates, cytoskeleton assembly, expression of major adhesion molecules [integrin-1β (ITGB1), CD44, focal adhesion kinase], and secretion of matrix metalloproteinases were detected between the control and treated cells, thus excluding the contribution of these components of invasion cascade to the inhibitory effects of adenosine. These data provide a novel insight into the ability of adenosine to dampen immune responses and prevent tumor invasion via two different, adenosine receptor-dependent and -independent mechanisms.

Implications: This study suggests that the combined targeting of adenosine receptors and modulation of intracellular purine levels can affect tumor growth and metastasis phenotypes.
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http://dx.doi.org/10.1158/1541-7786.MCR-14-0302-TDOI Listing
December 2014

Novel interaction of Rab13 and Rab8 with endospanins.

FEBS Open Bio 2013 22;3:83-8. Epub 2013 Jan 22.

Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland.

Rab GTPases regulate vesicular traffic in eukaryotic cells by cycling between the active GTP-bound and inactive GDP-bound states. Their functions are modulated by the diverse selection of effector proteins that bind to specific Rabs in their activated state. We previously described the expression of Rab13 in bone cells. To search for novel Rab13 interaction partners, we screened a newborn rat bone marrow cDNA library for Rab13 effectors with a bacterial two-hybrid system. We found that Rab13 binds to the C-terminus of Endospanin-2, a small transmembrane protein. In addition to Rab13 also Rab8 bound to Endospanin-2, while no binding of Rab7, Rab10, Rab11 or Rab32 was observed. Rab13 and Rab8 also interacted with Endospanin-1, a close homolog of Endospanin-2. Rab13 and Endospanin-2 colocalised in perinuclear vesicular structures in Cos1 cells suggesting direct binding also in vivo. Endospanin-2 is implicated in the regulation of the cell surface growth hormone receptor (GHR), but the inhibition of Rab13 expression did not affect GHR cell surface expression. This suggests that the Rab13-Endospanin-2 interaction may have functions other than GHR regulation. In conclusion, we have identified a novel interaction for Rab13 and Rab8 with Endospanin-2 and Endospanin-1. The role of this interaction in cell physiology, however, remains to be elucidated.
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http://dx.doi.org/10.1016/j.fob.2013.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668521PMC
June 2013

Differential roles of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in the regulation of S115 breast cancer cell growth.

PLoS One 2012 21;7(11):e49970. Epub 2012 Nov 21.

Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Turku, Finland.

Fibroblast growth factors (FGFs) regulate the growth and progression of breast cancer. FGF signaling is transduced through FGF receptors 1-4, which have oncogenic or anti-oncogenic roles depending on the ligand and the cellular context. Our aim was to clarify the roles of FGFR1-3 in breast cancer cell growth in vitro and in vivo. Pools of S115 mouse breast cancer cells expressing shRNA against FGFR1, 2 and 3 were created by lentiviral gene transfer, resulting in cells with downregulated expression of FGFR1, FGFR2 or FGFR3 (shR1, shR2 and shR3 cells, respectively) and shLacZ controls. FGFR1-silenced shR1 cells formed small, poorly vascularized tumors in nude mice. Silencing of FGFR2 in shR2 cells was associated with strong upregulation of FGFR1 expression and the formation of large, highly vascularized tumors compared to the control tumors. Silencing FGFR3 did not affect cell survival or tumor growth. Overexpressing FGFR2 in control cells did not affect FGFR1 expression, suggesting that high FGFR1 expression in shR2 cells and tumors was associated with FGFR2 silencing by indirect mechanisms. The expression of FGFR1 was, however, increased by the addition of FGF-8 to starved shLacZ or MCF-7 cells and decreased by the FGFR inhibitor PD173074 in shR2 cells with an elevated FGFR1 level. In conclusion, our results demonstrate that FGFR1 is crucial for S115 breast cancer cell proliferation and tumor growth and angiogenesis, whereas FGFR2 and FGFR3 are less critical for the growth of these cells. The results also suggest that the expression of FGFR1 itself is regulated by FGF-8 and FGF signaling, which may be of importance in breast tumors expressing FGFs at a high level.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049970PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503871PMC
May 2013

Rab13 is upregulated during osteoclast differentiation and associates with small vesicles revealing polarized distribution in resorbing cells.

J Histochem Cytochem 2012 Jul 4;60(7):537-49. Epub 2012 May 4.

Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland.

Osteoclasts are bone-resorbing multinucleated cells that undergo drastic changes in their polarization due to heavy vesicular trafficking during the resorption cycle. These events require the precise orchestration of membrane traffic in order to maintain the unique characteristics of the different membrane domains in osteoclasts. Rab proteins are small GTPases involved in regulation of most, if not all, steps of vesicle trafficking. The investigators studied RAB genes in human osteoclasts and found that at least 26 RABs were expressed in osteoclasts. Out of these, RAB13 gene expression was highly upregulated during differentiation of human peripheral blood monocytic cells into osteoclasts. To study its possible function in osteoclasts, the investigators performed immunolocalization studies for Rab13 and various known markers of osteoclast vesicular trafficking. Rab13 localized to small vesicular structures at the superior parts of the osteoclast between the trans-Golgi network and basolateral membrane domain. Rab13 localization suggests that it is not involved in endocytosis or transcytosis of bone degradation products. In addition, Rab13 did not associate with early endosomes or recycling endosomes labeled with EEA1 or TRITC-conjugated transferrin, respectively. Its involvement in glucose transporter traffic was excluded as well. It is suggested that Rab13 is associated with a putative secretory function in osteoclasts.
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http://dx.doi.org/10.1369/0022155412448069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460351PMC
July 2012

Stromal activation associated with development of prostate cancer in prostate-targeted fibroblast growth factor 8b transgenic mice.

Neoplasia 2010 Nov;12(11):915-27

Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland.

Expression of fibroblast growth factor 8 (FGF-8) is commonly increased in prostate cancer. Experimental studies have provided evidence that it plays a role in prostate tumorigenesis and tumor progression. To study how increased FGF-8 affects the prostate, we generated and analyzed transgenic (TG) mice expressing FGF-8b under the probasin promoter that targets expression to prostate epithelium. Prostates of the TG mice showed an increased size and changes in stromal and epithelial morphology progressing from atypia and prostatic intraepithelial neoplasia (mouse PIN, mPIN) lesions to tumors with highly variable phenotype bearing features of adenocarcinoma, carcinosarcoma, and sarcoma. The development of mPIN lesions was preceded by formation of activated stroma containing increased proportion of fibroblastic cells, rich vasculature, and inflammation. The association between advancing stromal and epithelial alterations was statistically significant. Microarray analysis and validation with quantitative polymerase chain reaction revealed that expression of osteopontin and connective tissue growth factor was markedly upregulated in TG mouse prostates compared with wild type prostates. Androgen receptor staining was decreased in transformed epithelium and in hypercellular stroma but strongly increased in the sarcoma-like lesions. In conclusion, our data demonstrate that disruption of FGF signaling pathways by increased epithelial production of FGF-8b leads to strongly activated and atypical stroma, which precedes development of mPIN lesions and prostate cancer with mixed features of adenocarcinoma and sarcoma in the prostates of TG mice. The results suggest that increased FGF-8 in human prostate may also contribute to prostate tumorigenesis by stromal activation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978914PMC
http://dx.doi.org/10.1593/neo.10776DOI Listing
November 2010

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

BMC Cancer 2010 Oct 30;10:596. Epub 2010 Oct 30.

Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Turku, Finland.

Background: Prostate 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.

Methods: Subcutaneous 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.

Results: Both 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.

Conclusion: FGF8b 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.
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http://dx.doi.org/10.1186/1471-2407-10-596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2984431PMC
October 2010

Inhibition of GGTase-I and FTase disrupts cytoskeletal organization of human PC-3 prostate cancer cells.

Cell Biol Int 2010 Aug;34(8):815-26

Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, FI20520 Turku, Finland.

The mevalonate synthesis pathway produces intermediates for isoprenylation of small GTPases, which are involved in the regulation of actin cytoskeleton and cell motility. Here, we investigated the role of the prenylation transferases in the regulation of the cytoskeletal organization and motility of PC-3 prostate cancer cells. This was done by using FTI-277, GGTI-298 or NE-10790, the specific inhibitors of FTase (farnesyltransferase), GGTase (geranylgeranyltransferase)-I and -II, respectively. Treatment of PC-3 cells with GGTI-298 and FTI-277 inhibited migration and invasion in a time- and dose-dependent manner. This was associated with disruption of F-actin organization and decreased recovery of GFP-actin. Immunoblot analysis of various cytoskeleton-associated proteins showed that the most striking change in GGTI-298- and FTI-277-treated cells was a markedly decreased level of total and phosphorylated cofilin, whereas the level of cofilin mRNA was not decreased. The treatment of PC-3 cells with GGTI-298 also affected the dynamics of GFP-paxillin and decreased the levels of total and phosphorylated paxillin. The levels of phosphorylated FAK (focal adhesion kinase) and PAK (p-21-associated kinase)-2 were also lowered by GGTI-298, but levels of paxillin or FAK mRNAs were not affected. In addition, GGTI-298 had a minor effect on the activity of MMP-9. RNAi knockdown of GGTase-Ibeta inhibited invasion, disrupted F-actin organization and decreased the level of cofilin in PC-3 cells. NE-10790 did not have any effect on PC-3 prostate cancer cell motility or on the organization of the cytoskeleton. In conclusion, our results demonstrate the involvement of GGTase-I- and FTase-catalysed prenylation reactions in the regulation of cytoskeletal integrity and motility of prostate cancer cells and suggest them as interesting drug targets for development of inhibitors of prostate cancer metastasis.
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http://dx.doi.org/10.1042/CBI20090288DOI Listing
August 2010

Fibroblast growth factor 8 increases breast cancer cell growth by promoting cell cycle progression and by protecting against cell death.

Exp Cell Res 2010 Mar 4;316(5):800-12. Epub 2009 Dec 4.

Department of Laboratory Medicine, Tumour Biology, Lund University, CRC, Building 91, Plan 10, Entrance 72, UMAS, 20502 Malmö, Sweden.

Fibroblast growth factor 8 (FGF-8) is expressed in a large proportion of breast cancers, whereas its level in normal mammary gland epithelium is low. Previous studies have shown that FGF-8b stimulates breast cancer cell growth in vitro and in vivo. To explore the mechanisms by which FGF-8b promotes growth, we studied its effects on cell cycle regulatory proteins and signalling pathways in mouse S115 and human MCF-7 breast cancer cells. We also studied the effect of FGF-8b on cell survival. FGF-8b induced cell cycle progression and up-regulated particularly cyclin D1 mRNA and protein in S115 cells. Silencing cyclin D1 with siRNA inhibited most but not all FGF-8b-induced proliferation. Inhibition of the FGF-8b-activated ERK/MAPK pathway decreased FGF-8b-stimulated proliferation. Blocking the constitutively active PI3K/Akt and p38 MAPK pathways also lowered FGF-8b-induced cyclin D1 expression and proliferation. Corresponding results were obtained in MCF-7 cells. In S115 and MCF-7 mouse tumours, FGF-8b increased cyclin D1 and Ki67 levels. Moreover, FGF-8b opposed staurosporine-induced S115 cell death which effect was blocked by inhibiting the PI3K/Akt pathway but not the ERK/MAPK pathway. In conclusion, our results suggest that FGF-8b increases breast cancer cell growth both by stimulating cell cycle progression and by protecting against cell death.
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http://dx.doi.org/10.1016/j.yexcr.2009.11.019DOI Listing
March 2010

FGF-8b induces growth and rich vascularization in an orthotopic PC-3 model of prostate cancer.

J Cell Biochem 2009 Jul;107(4):769-84

Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Finland.

Fibroblast growth factor 8 (FGF-8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF-8b on proliferation of PC-3 prostate cancer cells and growth of PC-3 tumors, and to identify FGF-8b-associated molecular targets. Expression of ectopic FGF-8b in PC-3 cells caused a 1.5-fold increase in cell proliferation in vitro and a four- to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF-8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT-qPCR of FGF-8b mRNA. Microarray analyses revealed significantly altered, up- and downregulated, genes in PC-3 cell cultures (169 genes) and in orthotopic PC-3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell-to-cell signaling and energy production, and the downregulated genes associated with differentiation (DKK-1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT-qPCR. In conclusion, our results demonstrate that FGF-8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF-8b actions on prostate cancer progression and metastasis.
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http://dx.doi.org/10.1002/jcb.22175DOI Listing
July 2009

Alendronate decreases orthotopic PC-3 prostate tumor growth and metastasis to prostate-draining lymph nodes in nude mice.

BMC Cancer 2008 Mar 28;8:81. Epub 2008 Mar 28.

Institute of Biomedicine, Department of Anatomy, University of Turku, Finland.

Background: Metastatic prostate cancer is associated with a high morbidity and mortality but the spreading mechanisms are still poorly understood. The aminobisphosphonate alendronate, used to reduce bone loss, has also been shown to inhibit the invasion and migration of prostate cancer cells in vitro. We used a modified orthotopic PC-3 nude mouse tumor model of human prostate cancer to study whether alendronate affects prostate tumor growth and metastasis.

Methods: PC-3 cells (5 x 10(5)) were implanted in the prostates of nude mice and the mice were treated with alendronate (0.5 mg/kg/day in PBS, s.c.) or vehicle for 4 weeks. After sacrifice, the sizes of tumor-bearing prostates were measured and the tumors and prostate-draining regional iliac and sacral lymph nodes were excised for studies on markers of proliferation, apoptosis, angiogenesis and lymphangiogenesis, using histomorphometry and immunohistochemistry.

Results: Tumor occurrence in the prostate was 73% in the alendronate-treated group and 81% in the control group. Mean tumor size (218 mm3, range: 96-485 mm3, n = 11) in the alendronate-treated mice was 41% of that in the control mice (513 mm3, range: 209-1350 mm3, n = 13) (p < 0.05). In the iliac and sacral lymph nodes of alendronate-treated mice, the proportion of metastatic area was only about 10% of that in control mice (p < 0.001). Immunohistochemical staining of tumor sections showed that alendronate treatment caused a marked decrease in the number of CD34-positive endothelial cells in tumors (p < 0.001) and an increase in that of ISEL positive apoptotic cells in tumors as well as in lymph node metastases (p < 0.05) compared with those in the vehicle-treated mice. The density of m-LYVE-1-stained lymphatic capillaries was not changed.

Conclusion: Our results demonstrate that alendronate treatment opposes growth of orthotopic PC-3 tumors and decreases tumor metastasis to prostate-draining lymph nodes. This effect could be at least partly explained by decreased angiogenesis and increased apoptosis. The results suggest that bisphosphonates have anti-tumoral and anti-invasive effects on primary prostate cancer.
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http://dx.doi.org/10.1186/1471-2407-8-81DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2294135PMC
March 2008

Role of fibroblast growth factor 8 in growth and progression of hormonal cancer.

Cytokine Growth Factor Rev 2007 Jun-Aug;18(3-4):257-66. Epub 2007 May 23.

Institute of Biomedicine, Department of Anatomy, University of Turku, Turku, Finland.

Hormonal cancers such as breast and prostate cancer arise from steroid hormone-regulated tissues. In addition to breast and prostate cancer hormonal regulation has also a role in endometrial, ovarian, testis and thyroid carcinomas. The effects of estrogens, androgens and progestagens on tumor growth are largely mediated by paracrine and autocrine target molecules which include growth factors and growth factor receptors. During cancer progression the hormonal growth regulation is often lost or overcome by an inappropriate activation of growth factor signaling cascades. One of the growth factors which have been associated with the regulation of growth and progression of hormonal cancer is fibroblast growth factor 8 (FGF8) which has also been recognized as an oncogene. FGF8 is widely expressed during embryonic development. It has been shown to mediate embryonic epithelial-mesenchymal transition and to have a crucial role in gastrulation and early organization and differentiation of midbrain/hindbrain, pharyngeal, cardiac, urogenital and limb structures. During adulthood FGF8 expression is much more restricted but in hormonal cancers it becomes frequently activated. High level of FGF8 expression in tumors is associated with a poor prognosis at least in prostate cancer. In experimental models FGF8 induces and facilitates prostate tumorigenesis and increases growth and angiogenesis of tumors. Several lines of evidence for autocrine and paracrine loops in the growth regulation of breast, prostate and ovarian cancer by FGF8 have been suggested.
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http://dx.doi.org/10.1016/j.cytogfr.2007.04.010DOI Listing
August 2007

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

Luminescence 2007 May-Jun;22(3):163-70

Laboratory of Biophysics, Department of Anatomy, and Medicity Research Laboratories, Institute of Biomedicine, University of Turku, Turku, Finland.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.
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http://dx.doi.org/10.1002/bio.943DOI Listing
July 2007

Monocyte-macrophage system as a target for estrogen and selective estrogen receptor modulators.

Ann N Y Acad Sci 2006 Nov;1089:218-27

Lund University, Department of Laboratory Medicine, Tumor Biology, Malmö University Hospital, CRC Entrance 72, 205 02 Malmö, Sweden.

Postmenopausal decline of estrogen production is associated with development of several degenerative disorders such as osteoporosis, neuroinflammatory diseases and vascular wall degeneration. These are associated with the activation of the cells of the monocyte-macrophage system in a context-dependent manner. Estrogen regulates differentiation, maturation and function of many cell types in this system directly or indirectly via other cells by autocrine/paracrine mechanisms. Estrogen effects on the monocyte-macrophage system are primarily repressive. Most of these effects are mediated by repression of expression of genes for cytokines or modulation of other inflammatory mediators by the estrogen receptor (ER)-dependent or nongenomic pathways. The ER-dependent mechanisms mostly involve modulation of the nuclear factor kappa B (NF-kappaB) pathway for transcriptional regulation of cytokine or other mediator genes. In the context of hormone-regulated cancer, estrogen can influence production of cytokines or other inflammatory mediators by both tumor cells and tumor-invading macrophages. The interactions of breast and prostate cancer cells with tumor-associated macrophages (TAMs) may play an important role in tumor progression and even in the development of resistance to hormonal treatment. Regulation of the monocyte-macrophage system by estrogen and cross-talk between the ER and cytokine-mediated pathways provides multiple novel targets for development of selective ER modulator (SERM) molecules for prevention and treatment of postmenopausal degenerative and neoplastic diseases.
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http://dx.doi.org/10.1196/annals.1386.045DOI Listing
November 2006

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

Mol Cell Endocrinol 2006 Jul 24;253(1-2):36-43. Epub 2006 May 24.

Institute of Biomedicine, Department of Anatomy, University of Turku, 20520 Turku, Finland.

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.
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http://dx.doi.org/10.1016/j.mce.2006.04.007DOI Listing
July 2006

Regulation of osteoblast differentiation: a novel function for fibroblast growth factor 8.

Endocrinology 2006 May 26;147(5):2171-82. Epub 2006 Jan 26.

Institute of Biomedicine, Department of Anatomy, University of Turku, Finland.

Several members of the fibroblast growth factor (FGF) family have an important role in the development of skeletal tissues. FGF-8 is widely expressed in the developing skeleton, but its function there has remained unknown. We asked in this study whether FGF-8 could have a role in the differentiation of mesenchymal stem cells to an osteoblastic lineage. Addition of FGF-8 to mouse bone marrow cultures effectively increased initial cell proliferation as well as subsequent osteoblast-specific alkaline phosphatase production, bone nodule formation, and calcium accumulation if it was added to the cultures at an early stage of osteoblastic differentiation. Exogenous FGF-8 also stimulated the proliferation of MG63 osteosarcoma cells, which was blocked by a neutralizing antibody to FGF-8b. In addition, the heparin-binding growth factor fraction of Shionogi 115 (S115) mouse breast cancer cells, which express and secrete FGF-8 at a very high level, had an effect in bone marrow cultures similar to that of exogenous FGF-8. Interestingly, experimental nude mouse tumors of S115 cells present ectopic bone and cartilage formation as demonstrated by typical histology and expression of markers specific for cartilage (type II and IX collagen) and bone (osteocalcin). These results demonstrate that FGF-8 effectively predetermines bone marrow cells to differentiate to osteoblasts and increases bone formation in vitro. It is possible that FGF-8 also stimulates bone formation in vivo. The results suggest that FGF-8, which is expressed by a great proportion of malignant breast and prostate tumors, may, among other factors, also be involved in the formation of osteosclerotic bone metastases.
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http://dx.doi.org/10.1210/en.2005-1502DOI Listing
May 2006

Estrogen and testosterone use different cellular pathways to inhibit osteoclastogenesis and bone resorption.

J Bone Miner Res 2005 Dec 1;20(12):2224-32. Epub 2005 Aug 1.

Department of Anatomy, Institute of Biomedicine, University of Turku, Finland.

Unlabelled: Using human peripheral blood CD14(+) osteoclast precursors, we show that testosterone directly inhibits osteoclast formation and bone resorption at physiological concentrations. Instead, estrogen has no direct effects, whereas its action seems to be mediated through osteoblasts by producing osteoprotegerin. Both estrogen and testosterone acts through their cognate receptors.

Introduction: Estrogen (E2) deficiency is associated with both the development of postmenopausal and senile form of osteoporosis in elderly women. Testosterone (Te) deficiency, on the other hand, may cause osteoporosis in men. In both sexes, osteoporosis is associated with disturbed bone turnover, including increased bone resorption caused by enhanced osteoclast formation and increased osteoclast activity. However, the mechanisms by which E2 or Te act on bone are not fully understood, and one of the central questions is whether these hormones act directly on osteoclast precursors or whether their action is mediated through osteoblastic cells.

Materials And Methods: We cultured human peripheral blood CD14(+) osteoclast precursors in the presence of RANKL, macrophage-colony stimulating factor (M-CSF), TNF-alpha, and dexamethasone to induce them to differentiate into osteoclasts. To study the possible osteoblast-mediated effects, osteoclast precursors were also co-cultured either with human MG-63 or SaOS-2 osteoblast-derived osteosarcoma cells. These cultures were treated with 10(-8)-10(-12) M of E2 or Te for 7 days.

Results: E2 did not have any direct effect on osteoclast formation, whereas testosterone inhibited osteoclast formation and bone resorption in a dose-dependent manner. In co-cultures, where MG-63 or SaOS-2 cells were present, E2 and Te inhibited osteoclast formation in a dose-dependent manner. At the same time, E2 and Te treatment in MG-63 or SaOS-2 cell-containing cultures stimulated significantly the formation of osteoprotegerin (OPG) compared with untreated cultures measured by ELISA assay from the culture medium. The effects of E2 and Te on osteoclast formation and bone resorption were completely antagonized by an E2 receptor (ER) antagonist, ICI 182,780, and an androgen receptor (AR) antagonist, flutamide, suggesting ER- and AR-mediated mechanisms, respectively, in these cultures.

Conclusions: Te is likely to have direct and indirect inhibitory effects on human osteoclast formation and bone resorption, whereas the effect of E2 on osteoclast precursors and osteoclasts seems to be mediated by osteoblastic cells. Inhibitory effect of E2 is associated with the stimulated secretion of OPG by osteoblast-derived osteosarcoma cells. Mechanism of action of E2 and Te is mediated by ER and AR, respectively.
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http://dx.doi.org/10.1359/JBMR.050803DOI Listing
December 2005

Two-photon excitation in fluorescence polarization receptor-ligand binding assay.

J Biomol Screen 2005 Jun;10(4):314-9

Laboratory of Biophysics, Institute of Biomedicine, University of Turku, Finland.

Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.
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http://dx.doi.org/10.1177/1087057104273334DOI Listing
June 2005

Role of estrogens in development of prostate cancer.

J Steroid Biochem Mol Biol 2004 Nov 19;92(4):297-305. Epub 2004 Dec 19.

Department of Laboratory Medicine, Tumor Biology, Lund University, 20502 Malmö, Sweden.

Estrogens have previously been extensively used in prostate cancer treatment. Serious side effects, primarily in cardiovascular system have, however, limited their use. The therapeutic effect of estrogen in preventing prostate cancer growth was mainly obtained indirectly by feedback inhibition of the hypothalamic release of LRH leading to lowered serum androgen levels and castration like effects. Prostate tissue is also most probably a target for direct regulation by estrogens. Prostate contains estrogen receptor alpha (ERalpha) and beta (ERbeta), which are localized characteristically in stroma and epithelium, respectively. The physiological function of these receptors is not known but there is evidence of the role of estrogens in prostatic carcinogenesis. Developing prostate seems particularly sensitive to increased level of endogenous and/or exogenous estrogens. Perinatal or neonatal exposure of rats and mice to estrogens leads to "imprinting" of prostate associated with increased proliferation, inflammation and dysplastic epithelial changes later in life. Prolonged treatment of adult rodents with estrogens along with androgens also leads to epithelial metaplasia, PIN-like lesions and even adenocarcinoma of prostate speaking for the role of estrogen in prostate cancer development. Recent results concerning antiestrogen inhibition of prostate cancer development beyond PIN-type lesions in transgenic mouse models further suggests a role for estrogens in prostate cancer progression. These results also suggest that direct inhibition of estrogen action at the level of prostate tissue may provide an important novel principle of development of prostate cancer therapies.
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http://dx.doi.org/10.1016/j.jsbmb.2004.10.016DOI Listing
November 2004

The in vitro cytotoxic and apoptotic activity of Triphala--an Indian herbal drug.

J Ethnopharmacol 2005 Feb 25;97(1):15-20. Epub 2004 Dec 25.

Department of Botanical Sciences, Guru Nanak Dev University, Amritsar 143005, India.

A study on cytotoxic effect of acetone extract of "Triphala" whose antimutagenicity has already been tested. The in vitro antimutagenic activity of Triphala--an Indian herbal drug. Food Chemistry and Toxicology 40, 47-54) was extended to test its cytotoxic effects on cancer cell-lines using Shionogi 115 (S115) and MCF-7 breast cancer cells and PC-3 and DU-145 prostate cancer cells as models. The results revealed that acetone extract of "Triphala" showed a significant cytotoxic effect on these cancer cell-lines and the effect was similar on all cancer cell lines used in this study. The major phenolic compounds in the most potent acetone extracts were isolated and purified. Structural analysis was conducted using spectroscopic techniques including mass spectroscopy, nuclear magnetic resonance (NMR) and infrared (IR) which showed gallic acid as the major component. The suppression of the growth of cancer cells in cytotoxic assays may be due to the gallic acid-a major polyphenol observed in "Triphala".
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http://dx.doi.org/10.1016/j.jep.2004.09.050DOI Listing
February 2005

Comparative study of the short-term effects of a novel selective estrogen receptor modulator, ospemifene, and raloxifene and tamoxifen on rat uterus.

J Steroid Biochem Mol Biol 2004 Feb;88(2):143-56

Medicity Research Laboratory, Department of Anatomy, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland.

To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1-24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.
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http://dx.doi.org/10.1016/j.jsbmb.2003.11.009DOI Listing
February 2004

Studies on correlation of antimutagenic and antiproliferative activities of Juglans regia L.

J Environ Pathol Toxicol Oncol 2003 ;22(1):59-67

Department of Botanical Sciences, Guru Nanak Dev University, Amritsar, India.

We investigated the effect of water and acetone extract of Juglans regia L. to evaluate its antimutagenic and antiproliferative activities. The antimutagenic study using TA98 and TA100 tester strains of Salmonella revealed the water and acetone extracts to be more effective than the benzene and chloroform extracts in inhibiting the revertants induced by 2-aminoflourene (2AF) in TA100 tester strains. The most effective extracts in the Ames assay were further evaluated using the Lucifer luciferase assay and in time course studies for antiproliferative activities using the Hoechst staining to observe apoptotic cell deaths. The acetone extract showed a correlation of antimutagenic activities in the Ames assay with its antiproliferative effect in different cell lines, while the water extract exerted its effect distinctly in each cell line. Further studies are still needed to evaluate the cytotoxicity in experiments carried out in vivo.
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April 2003

The effects of tamoxifen and toremifene on bone cells involve changes in plasma membrane ion conductance.

J Bone Miner Res 2003 Mar;18(3):473-81

Department of Surgery and Anatomy, University of Oulu, Oulu, Finland.

Selective estrogen receptor modulators (SERMs), tamoxifen (Tam) and toremifene (Tor), are widely used in the treatment of breast cancer. In addition, they have been demonstrated to prevent estrogen deficiency-induced bone loss in postmenopausal women. These effects are thought to be caused by the interaction of the SERMs with the estrogen receptor, although SERMs have also been shown to conduct non-receptor-mediated effects such as rapid changes in membrane functions. We compared the effects of Tam, Tor, and 17beta-estradiol (E2) on the viability of rat osteoclasts and osteoblasts. Both Tam and Tor were found to cause osteoclast apoptosis in in vitro cultures, which was reversed by E2. In addition, at higher concentration (10 microM), both SERMs had an estrogen receptor-independent effect, which involved interaction with the plasma membrane as demonstrated with UMR-108 osteosarcoma cells by Tam and Tor, but not E2. A leak of protons leading to changes in intracellular pH was shown both in medullary bone derived membrane vesicles and in intact cells. These effects were followed by a rapid loss of cell viability and subsequent cell lysis. Our results show that both Tam and Tor have an ionophoric effect on the plasma membranes of bone cells and that these SERMs differed in this ability: Tor induced rapid membrane depolarization only in the presence of high concentration of potassium. These non-receptor-mediated effects may be involved in therapeutic responses and explain some clinical side effects associated with the treatment of patients with these SERMs.
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http://dx.doi.org/10.1359/jbmr.2003.18.3.473DOI Listing
March 2003

Alendronate inhibits invasion of PC-3 prostate cancer cells by affecting the mevalonate pathway.

Cancer Res 2002 May;62(9):2708-14

Institute of Biomedicine, Department of Anatomy and Medicity Research Laboratory, University of Turku, FIN-20520 Turku, Finland.

Breast and prostate cancer preferentially metastasize in the skeleton, inducing locally increased bone resorption by osteoclasts. Bisphosphonates (BPs), potent inhibitors of osteoclasts and bone resorption, are able to reduce metastatic bone lesions, but the metastasis-related cellular target molecules for BPs have not yet been identified. In osteoclasts, nitrogen-containing BPs inhibit the function of the mevalonate pathway, impairing the prenylation and activation of small GTPases. In addition, direct effects of BPs on cancer cells have been suggested. In the present study, the effects of two clinically used BPs, the amino-BP alendronate and clodronate, on adhesion, invasion, and migration of human PC-3 prostate cancer cells were examined in vitro. We also studied the possible role of the mevalonate pathway in invasion and migration of PC-3 cells using the beta-hydroxy-beta-methylglutaryl-CoA reductase inhibitor mevastatin and the mevalonate pathway intermediates mevalonate (mevalonic acid lactone), geranylgeraniol, and trans-trans-farnesol. The results demonstrate that alendronate pretreatment very effectively inhibited in vitro invasion of prostate cancer cells in a dose-dependent manner, with an IC50 as low as approximately 1 pM. The inhibition was similar to that of mevastatin. Clodronate also inhibited invasion, but the IC50 was 0.1 microM. Importantly, geranylgeraniol and trans-trans-farnesol reversed the inhibitory effect of alendronate and mevastatin but not the clodronate-induced inhibition of invasion. Alendronate pretreatment also inhibited migration, which was partially reversed by geranylgeraniol and trans-trans-farnesol. Adhesion of PC-3 cells to various matrices was reduced, and their F-actin organization was changed. Alendronate pretreatment also inhibited invasion of human Du-145 prostate and MDA-MB-231 breast cancer cells. As a conclusion, the results demonstrate that the mevalonate pathway leading to protein prenylation is important for cancer cell invasion and migration in vitro. They further suggest that interference with this pathway is involved in inhibition of invasion and migration of prostate cancer cells by the amino-BP alendronate but that the mechanism of clodronate inhibition is different. It is possible that BPs have therapeutic potential in preventing the spread of prostate cancer.
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May 2002

VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors.

Int J Cancer 2002 Apr;98(6):946-51

Department of Anatomy, Institute of Biomedicine and MediCity Research Laboratory, University of Turku, Turku, Finland.

The spread of cancer cells to regional lymph nodes through the lymphatic system is the first step in the dissemination of breast cancer. In several human cancers including those of the breast and prostate, the expression of vascular endothelial growth factor C (VEGF-C) is associated with lymph node metastasis. Our study was undertaken to evaluate the effect of VEGF-C on metastasis of poorly invasive, estrogen dependent human MCF-7 breast cancer cells. MCF-7 breast cancer cells transfected with VEGF-C (MCF-7-VEGF-C) were grown as tumors in the mammary fat pads of nude mice implanted with subcutaneous estrogen pellets. Tumor lymphangiogenesis and lymph node metastasis were studied immunohistochemically using antibodies against lymphatic vessel hyaluronan receptor -1 (LYVE-1), VEGF receptor-3 (VEGFR-3), PECAM-1, pan-cytokeratin and estrogen dependent pS2 protein. Overexpression of VEGF-C in transfected MCF-7 cells stimulated in vivo tumor growth in xenotransplanted mice without affecting estrogen responsiveness. The resulting tumors metastasized to the regional lymph nodes in 75% (in 6 mice out of 8, Experiment I) and in 62% (in 5 mice out of 8, Experiment II) of mice bearing orthotopic tumors formed by MCF-7-VEGF-C cells whereas no metastases were observed in mice bearing tumors of control vector-transfected MCF-7 cells (MCF-7-Mock). The density of intratumoral and peritumoral lymphatic vessels was increased in tumors derived from MCF-7-VEGF-C cells but not MCF-7-Mock cells. Taken together, our results show that VEGF-C overexpression stimulates tumor lymphangiogenesis and induces normally poorly metastatic estrogen-dependent MCF-7 tumors to disseminate to local lymph nodes. These data suggest that VEGF-C has an important role in lymph node metastasis of breast cancer even at its hormone-dependent early stage.
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http://dx.doi.org/10.1002/ijc.10283DOI Listing
April 2002

PRL signal transduction in the epithelial compartment of rat prostate maintained as long-term organ cultures in vitro.

Endocrinology 2002 Jan;143(1):228-38

United States Military Cancer Institute, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA.

Using long-term organ cultures of rat prostate tissue explants, we previously demonstrated that PRL both stimulates proliferation and acts as an androgen-independent suppressor of apoptosis in prostate epithelial cells, leading to epithelial hyperplasia. In this work we delineate intracellular signaling molecules activated by PRL in prostate tissue to identify candidate signaling proteins that are responsible for maintaining survival and proliferation of prostate epithelium in androgen-deprived growth environment. We now show that signal transducer and activator of transcription-5a (Stat5a) and Stat5b become tyrosine phosphorylated in response to PRL stimulation in rat prostate using prostate organ culture as an experimental model. Stat5 was translocated to the nuclei of epithelial cells of prostate tissue as demonstrated by immunohistochemistry. Furthermore, EMSA showed PRL-inducible binding of Stat5a homodimers and Stat5a/5b heterodimers to the PRL response element of the beta-casein gene promoter. Signaling molecules Stat3, Stat1, MAPK, or protein kinase B, which can be activated by PRL in other target cells, were not activated by PRL in prostate tissue. Furthermore, we show that Stat5a and Stat5b are continuously phosphorylated in rat prostate in vivo, although they are expressed to varying degree in separate lobes of rat prostate. Collectively, our results suggest that PRL signaling in rat prostate tissue is primarily transduced via Stat5a and Stat5b. The Stat5 pathway represents one candidate signaling mechanism, used by PRL and possibly other growth factors and cytokines, that supports the viability of prostate epithelial cells during long-term androgen deprivation.
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http://dx.doi.org/10.1210/endo.143.1.8576DOI Listing
January 2002