As solid tumor types, breast and prostate cancer are rivalled only by lung cancer in their propensity to metastasize to bone in the later stages of disease. At advanced stages of disease, approximately 80% of breast and 90% of prostate cancer patients will present with bone metastases. Bone metastases are often a painful conclusion to the lives of these patients, resulting in bone pain, hypercalcemia, pathological fractures and spinal cord compression. The culmination of these comorbidities considerably reduces a patient’s quality of life and prolonged survival. Hormone depletion is used as a first line of treatment in the majority of cases, negatively regulating bone health due to increased bone resorption by osteoclasts and decreased bone formation by osteoblasts. Not only is bone integrity undermined, but this action of increased bone turnover is beneficial for the colonization of metastasizing cells which co-opt and enhance the same mechanisms to establish and maintain their own growth. This is termed ‘the vicious cycle’ of osteolytic bone metastasis. Current research approaches aim to identify bone-targeted therapies which not only inhibit tumor growth but concurrently protect bone. In this study, Dickkopf-1 (DKK-1), mechanistic target of rapamycin (mTOR) and p38 mitogen-activated kinases (p38 MAPK) are presented as novel targets. Pro-tumor roles have been described for all and clinical trials are currently investigating their efficacy in different cancer types. In normal bone biology DKK-1 is an inhibitor of the canonical Wnt signaling pathway which promotes osteoblastogenesis while mTOR signaling is a promoter of osteoclastogenesis. P38 MAPK inhibitors have been shown to regulate DKK-1 expression and bone destruction in preclinical models of multiple myeloma. The aims of this current study were to 1) investigate the role of DKK-1 in the biology of osteotropic breast cancer, 2) to assess the potential bone protective effects of mTOR inhibition by everolimus in the context of osteotropic cancers and 3) to test the hypothesis that p38 MAPK is a regulator of DKK-1 expression in prostate cancer, potentially supporting an osteolytic phenotype by impairing osteoblastogenesis. In aim 1, analysis of a breast cancer tissue microarray demonstrated that DKK-1 expression was elevated in advanced and invasive tumor stages. Strikingly, positive DKK-1 expression correlated with a significantly reduced survival rate only in estrogen receptor-negative (ER-) breast cancer patients compared to patients with tumors which were negative for DKK-1 expression. In MDA-MB-231 breast cancer xenograft models, neutralization of secreted DKK-1 by treating mice with the monoclonal DKK-1 antibody BHQ880 or knocking out the expression of DKK-1 in MDA-MB-231 cells using CRISPR-Cas9 mediated gene editing, resulted in reduced tumor growth and burden by ≥ 50% (p < 0.05). In aim 2, the mTOR inhibitor everolimus is presented as an anti-tumor and bone-protective agent. The anti-tumor effects of everolimus were confirmed in two subcutaneous tumor models and a model of breast cancer bone metastasis, were tumor burden in the bone was reduced by 45.4% (p < 0.01). Bone loss induced by a hormone-deprived environment in ovariectomized mice was prevented with everolimus treatment as was bone destruction in the metastasis model. In more detail, it could be shown that everolimus maintained osteoblast function while specifically inhibiting osteoclast function. In aim 3, p38 MAPK is presented as a regulator of DKK-1 in prostate cancer. While the activation of p38 MAPK upregulated DKK-1, inhibition of p38 MAPK using small molecule inhibitors and siRNAs inhibited DKK-1 expression. Furthermore, assessment of different p38 MAPK isoforms revealed MAPK11 as the most effective regulator of DKK-1 and inhibition of DKK-1 by interfering with p38 MAPK signaling was sufficient to prevent the inhibitory effects of prostate cancer-derived DKK-1 on osteoblastogenesis in vitro. This study has assessed multiple targets and their concurrent roles in cancer and bone cell biology. Specifically, DKK-1 has been proven to be a tumor promoter in ER- breast cancer and can be targeted therapeutically to inhibit tumor growth. MTOR inhibition by everolimus has been shown to be an effective mono-therapy in ER- breast cancer, inhibiting the growth of subcutaneous tumor and bone metastases and preventing bone loss induced by estrogen ablation. This further supports its use in postmenopausal women with breast cancer who are predisposed to developing osteoporosis and bone metastases. It also supports the use of everolimus in hormone receptor-negative or triple receptor-negative breast cancer, for which it has not yet been approved. A clear link has been made between p38 MAPK signaling and DKK-1 expression in prostate cancer and its consequent regulation of osteoblastogenesis. A future focus on the inhibition of a specific MAPK isoform, MAPK11 in particular, may help in translating these encouraging in vitro results into promising pre-clinical trials in vivo. As a whole, these investigations provide a foundation for further research and could be valuable for the design of future clinical trials, leading to improvements in the treatment and prognosis of osteolytic bone metastases.

As solid tumor types, breast and prostate cancer are rivalled only by lung cancer in their propensity to metastasize to bone in the later stages of disease. At advanced stages of disease, approximately 80% of breast and 90% of prostate cancer patients will present with bone metastases. Bone metastases are often a painful conclusion to the lives of these patients, resulting in bone pain, hypercalcemia, pathological fractures and spinal cord compression. The culmination of these comorbidities considerably reduces a patient’s quality of life and prolonged survival. Hormone depletion is used as a first line of treatment in the majority of cases, negatively regulating bone health due to increased bone resorption by osteoclasts and decreased bone formation by osteoblasts. Not only is bone integrity undermined, but this action of increased bone turnover is beneficial for the colonization of metastasizing cells which co-opt and enhance the same mechanisms to establish and maintain their own growth. This is termed ‘the vicious cycle’ of osteolytic bone metastasis. Current research approaches aim to identify bone-targeted therapies which not only inhibit tumor growth but concurrently protect bone. In this study, Dickkopf-1 (DKK-1), mechanistic target of rapamycin (mTOR) and p38 mitogen-activated kinases (p38 MAPK) are presented as novel targets. Pro-tumor roles have been described for all and clinical trials are currently investigating their efficacy in different cancer types. In normal bone biology DKK-1 is an inhibitor of the canonical Wnt signaling pathway which promotes osteoblastogenesis while mTOR signaling is a promoter of osteoclastogenesis. P38 MAPK inhibitors have been shown to regulate DKK-1 expression and bone destruction in preclinical models of multiple myeloma. The aims of this current study were to 1) investigate the role of DKK-1 in the biology of osteotropic breast cancer, 2) to assess the potential bone protective effects of mTOR inhibition by everolimus in the context of osteotropic cancers and 3) to test the hypothesis that p38 MAPK is a regulator of DKK-1 expression in prostate cancer, potentially supporting an osteolytic phenotype by impairing osteoblastogenesis. In aim 1, analysis of a breast cancer tissue microarray demonstrated that DKK-1 expression was elevated in advanced and invasive tumor stages. Strikingly, positive DKK-1 expression correlated with a significantly reduced survival rate only in estrogen receptor-negative (ER-) breast cancer patients compared to patients with tumors which were negative for DKK-1 expression. In MDA-MB-231 breast cancer xenograft models, neutralization of secreted DKK-1 by treating mice with the monoclonal DKK-1 antibody BHQ880 or knocking out the expression of DKK-1 in MDA-MB-231 cells using CRISPR-Cas9 mediated gene editing, resulted in reduced tumor growth and burden by ≥ 50% (p < 0.05). In aim 2, the mTOR inhibitor everolimus is presented as an anti-tumor and bone-protective agent. The anti-tumor effects of everolimus were confirmed in two subcutaneous tumor models and a model of breast cancer bone metastasis, were tumor burden in the bone was reduced by 45.4% (p < 0.01). Bone loss induced by a hormone-deprived environment in ovariectomized mice was prevented with everolimus treatment as was bone destruction in the metastasis model. In more detail, it could be shown that everolimus maintained osteoblast function while specifically inhibiting osteoclast function. In aim 3, p38 MAPK is presented as a regulator of DKK-1 in prostate cancer. While the activation of p38 MAPK upregulated DKK-1, inhibition of p38 MAPK using small molecule inhibitors and siRNAs inhibited DKK-1 expression. Furthermore, assessment of different p38 MAPK isoforms revealed MAPK11 as the most effective regulator of DKK-1 and inhibition of DKK-1 by interfering with p38 MAPK signaling was sufficient to prevent the inhibitory effects of prostate cancer-derived DKK-1 on osteoblastogenesis in vitro. This study has assessed multiple targets and their concurrent roles in cancer and bone cell biology. Specifically, DKK-1 has been proven to be a tumor promoter in ER- breast cancer and can be targeted therapeutically to inhibit tumor growth. MTOR inhibition by everolimus has been shown to be an effective mono-therapy in ER- breast cancer, inhibiting the growth of subcutaneous tumor and bone metastases and preventing bone loss induced by estrogen ablation. This further supports its use in postmenopausal women with breast cancer who are predisposed to developing osteoporosis and bone metastases. It also supports the use of everolimus in hormone receptor-negative or triple receptor-negative breast cancer, for which it has not yet been approved. A clear link has been made between p38 MAPK signaling and DKK-1 expression in prostate cancer and its consequent regulation of osteoblastogenesis. A future focus on the inhibition of a specific MAPK isoform, MAPK11 in particular, may help in translating these encouraging in vitro results into promising pre-clinical trials in vivo. As a whole, these investigations provide a foundation for further research and could be valuable for the design of future clinical trials, leading to improvements in the treatment and prognosis of osteolytic bone metastases.

No description available.

Die Interaktion zwischen Tumorzellen und Stromazellen spielt eine wichtige Rolle für die lokale Tumorprogression, die Invasivität und Metastasierung von soliden Tumoren wie dem Mammakarzinom. Es ist bekannt, dass die Kokultivierung von MCF-7 Mammakarzinomzellen mit humanen Makrophagen zu einer Wnt5a abhängigen Invasivitätssteigerung der Mammkarzinomzellen führt, welche durch den Wnt-Antagonisten Dkk-1 verhindert werden kann. Unbekannt war, ob sich primär hoch invasive Mammakarzinomzellen wie etwa die tripe-negative (TN) Mammakarzinomzelllinie MDA-MB-231 und die schwach invasive Zelllinie MCF-7 hinsichtlich ihrer Expression von Wnt- und Wnt-abhängigen Genen unterscheiden. So zeigten sich sowohl die nicht-kanonischen Wnt-Liganden Wnt5a und Wnt5b als auch die Wnt-assoziierten Gene VEGF-A und PLAU-R in der MDA-MB-231 Zelllinie als deutlich höher exprimiert im Vergleich zu MCF-7. Insbesondere die Expressionsunterschiede von Wnt5a und Wnt5b waren zuvor unbekannt und erweitern die molekulare Charakteristik dieser Zelllinien. In Kokulturexperimten von MCF-7 Mammakarzinomzellen und humanen Makrophagen zeigte sich in dieser Arbeit eine signifkant höhere Expression von Wnt5a, VEGF-A und TNF-α in MCF-7 nach 24h. Dies ist ein weiterer Aspekt für die molekularen Mechanismen, welche zu einer Invasivitätssteigerung solider Tumore durch Tumor-assoziierte Makrophagen (TAM) führen können. Interessanterweise blieb diese Regulation unter Zugabe von rh Dkk-1 aus, was eine wichtige Rolle von Dkk-1 möglicherweise auch aus therapeutischer Sicht nahelegt.

No description available.

S100A4 spielt eine zentrale Rolle für die Metastasierung des Dickdarmkrebses. Die Hemmung der S100A4 Expression stellt damit einen vielversprechenden therapeutischen Ansatz dar. Die vorliegende Arbeit präsentiert Niklosamid und Calcimycin als neue Inhibitoren der S100A4 Transkription. In Kolonkarzinomzellen, die mit einem der beiden Inhibitoren behandelt wurden, wurde die S100A4 Expression konzentrations- und zeitabhängig unterdrückt. Des Weiteren war die Zellmigration und -invasion in Abhängigkeit von S100A4 in behandelten Zellen vermindert. Niklosamid und Calcimycin Behandlung verhinderten die Zellproliferation und die Koloniebildung von Kolonkarzinomzellen. Beide Inhibitoren hemmten den konstitutiv aktiven Wnt Pathway von Kolonkarzinomzellen. Calcimycin Behandlung verminderte die Expression von beta-catenin. Niklosamid hemmte die Bildung des beta-catenin/TCF Komplexes und unterband damit die Expression von Wnt Pathway Genen, wie z.B. S100A4. Im Rahmen dieser Arbeit wurde ein in vivo Tiermodell entwickelt, mit dem die S100A4-induzierte Metastasierung mit Hilfe von nicht-invasivem Biolumineszenz Imaging visualisiert werden konnte. In diesem Model konnte gezeigt werden, dass Niklosamid signifikant die S100A4 Expression im Tumor vermindert und damit die Metastasierung hemmt. Des Weiteren zeigt diese Arbeit, dass S100A4 die Expression des Wnt Pathway Antagonisten DKK-1 in Kolonkarzinomzellen hemmt. DKK-1 selbst konnte als endogener Inhibitor der S100A4 Expression identifiziert werden. Zusammenfassend beschreibt die vorliegende Arbeit einen neuen regulativen Mechanismus im Wnt Pathway, der die S100A4 Expression im Kolonkarzinom fördert. Diese Beobachtung verdeutlicht die Notwendigkeit für wirksame S100A4 Inhibitoren, wie Niklosamid und Calcimycin, die das Potenzial haben, in einer klinischen Anwendung die Metastasierung von Kolonkarzinompatienten mit erhöhter S100A4 Expression zu hemmen und damit deren Überlebenschance wesentlich zu verbessern. / S100A4 promotes metastasis in colon cancer patients thereby reducing their five-year survival chances to less than 10%. Consequently, inhibition of S100A4 expression is a promising strategy for anti-metastatic treatment of colon cancer patients. The present study characterizes the small molecules niclosamide and calcimycin as transcriptional inhibitors of S100A4 which reduced S100A4 expression concentration- and time-dependently. Niclosamide and calcimycin treatment restricted cell migration, invasion and wound healing capabilities in a S100A4-specific manner, and inhibited cell proliferation and colony formation of colon cancer cells. Both small molecule inhibitors interfere with the constitutively active Wnt pathway. Targeting β-catenin expression by calcimycin or interfering with the β-catenin/TCF transcription activating complex by niclosamide resulted in reduced Wnt target gene transcription, among them S100A4. The study further presents a human colon cancer xenograft mouse model for monitoring S100A4-induced metastasis formation via non-invasive bioluminescence imaging. Treatment of xenograft mice with niclosamide resulted in a significant reduction of the S100A4 mRNA level in the tumor accompanied by inhibition of metastasis formation. Moreover, this study presents evidence that S100A4 is an inhibitor of DKK-1 expression. In colon cancer cells DKK-1 and S100A4 expression was negatively correlated. Ectopic S100A4 overexpression inhibited DKK-1 expression. Targeting S100A4 via shRNA recovered the repressed DKK-1 expression and vice versa. In summary, the study describes a novel positive feedback loop in the Wnt pathway regulation formed by S100A4 repressing its antagonist DKK-1. This novel mechanism further strengthens the need for S100A4 inhibitors such as niclosamide or calcimycin. Consequently, such small molecules provide immense potential for the treatment of colon cancer patients who are at high risk for S100A4-induced colon cancer metastasis.

No description available.