A Novel Lung Metastasis Signature Links Wnt Signaling with Cancer Cell Self-Renewal and Epithelial-Mesenchymal Transition in Basal-like Breast Cancer

The establishment of metastasis depends on the ability of cancer cells to acquire a migratory phenotype combined with their capacity to recreate a secondary tumor in a distant tissue. In epithelial cancers, such as those of the breast, the epithelial-mesenchymal transition (EMT) is associated with basal-like breast cancers, generates cells with stem-like properties, and enables cancer cell dissemination and metastasis. However, the molecular mechanism(s) that connects stem cell–like characteristics with EMT has yet to be defined. Using an orthotopic model of human breast cancer metastasis to lung, we identified a poor prognosis gene signature, in which several components of the wnt signaling pathway were overexpressed in early lung metastases. The wnt genes identified in this signature were strongly associated with human basal-like breast cancers. We found that inhibiting wnt signaling through LRP6 reduced the capacity of cancer cells to self-renew and seed tumors in vivo. Furthermore, inhibition of wnt signaling resulted in the reexpression of breast epithelial differentiation markers and repression of EMT transcription factors SLUG and TWIST. Collectively, these results provide a molecular link between self-renewal, EMT, and metastasis in basal-like breast cancers

Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment

INTRODUCTION: Polyamines affect proliferation, differentiation, migration and apoptosis of cells, indicating their potential as a target for cancer chemotherapy. Ornithine decarboxylase converts ornithine to putrescine and is the rate-limiting step in polyamine synthesis. α-Difluoromethylornithine (DFMO) irreversibly inhibits ornithine decarboxylase and MDA-MB-435 human breast cancer metastasis to the lung without blocking orthotopic tumor growth. This study tested the effects of DFMO on orthotopic tumor growth and lung colonization of another breast cancer cell line (MDA-MB-231) and the effects on bone metastasis of MDA-MB-435 cells. METHODS: MDA-MB-231 cells were injected into the mammary fat pad of athymic mice. DFMO treatment (2% per orally) began at the day of tumor cell injection or 21 days post injection. Tumor growth was measured weekly. MDA-MB-231 cells were injected into the tail vein of athymic mice. DFMO treatment began 7 days prior to injection, or 7 or 14 days post injection. The number and incidence of lung metastases were determined. Green fluorescent protein-tagged MDA-MB-435 cells were injected into the left cardiac ventricle in order to assess the incidence and extent of metastasis to the femur. DFMO treatment began 7 days prior to injection. RESULTS: DFMO treatment delayed MDA-MB-231 orthotopic tumor growth to a greater extent than growth of MDA-MB-435 tumors. The most substantial effect on lung colonization by MDA-MB-231 cells occurred when DFMO treatment began 7 days before intravenous injection of tumor cells (incidence decreased 28% and number of metastases per lung decreased 35–40%). When DFMO treatment began 7 days post injection, the incidence and number of metastases decreased less than 10%. Surprisingly, treatment initiated 14 days after tumor cell inoculation resulted in a nearly 50% reduction in the number of lung metastases without diminishing the incidence. After intracardiac injection, DFMO treatment decreased the incidence of bone metastases (55% vs 87%) and the area occupied by the tumor (1.66 mm(2 )vs 4.51 mm(2), P < 0.05). CONCLUSION: Taken together, these data demonstrate that DFMO exerts an anti-metastatic effect in more than one hormone-independent breast cancer, for which no standard form of biologically-based treatment exists. Importantly, the data show that DFMO is effective against metastasis to multiple sites and that treatment is generally more effective when administered early

Breast Cancer Metastasis Suppressor-1 Differentially Modulates Growth Factor Signaling*S⃞

That metastatic tumor cells grow in selective non-native environments suggests an ability to differentially respond to local microenvironments. BRMS1, like other metastasis suppressors, halts ectopic growth (metastasis) without blocking orthotopic tumor formation. BRMS1-expressing tumor cells reach secondary sites but do not colonize distant tissues, compelling the hypothesis that BRMS1 selectively restricts the ability of tumor cells to respond to exogenous regulators in different tissues. Here we report that BRMS1 expression in metastatic human breast cancer cells leads to a selective reduction in epidermal growth factor receptor expression and downstream (AKT) signaling. Signaling through another receptor tyrosine kinase, hepatocyte growth factor receptor (c-Met), remains unaltered despite reduced levels of the signaling intermediate phosphatidylinositol (4,5)-bisphosphate. Interestingly, reduced downstream calcium signaling is observed following treatment with platelet-derived growth factor, consistent with decreased phosphatidylinositol (4,5)-bisphosphate. However, platelet-derived growth factor receptor expression is unaltered. Thus, BRMS1 differentially attenuates cellular responses to mitogenic signals, not only dependent upon the specific signal received, but at varying steps within the same signaling cascade. Specific modulation of signaling responses received from the microenvironment may ultimately dictate which environments are permissive/restrictive for tumor cell growth and provide insights into the biology underlying metastasis

Α-Difluoromethylornithine (DFMO) treatment decreases lung metastasis of MDA-MB-231 cells

<p><b>Copyright information:</b></p><p>Taken from "Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment"</p><p>Breast Cancer Research 2005;7(5):R819-R827.</p><p>Published online 9 Aug 2005</p><p>PMCID:PMC1242150.</p><p>Copyright © 2005 Richert et al, licensee BioMed Central Ltd.</p> MDA-MB-231 cells were injected into the tail vein of athymic mice. DFMO treatment (2% per orally) began either 7 days prior to (DFMO-7), or 7 days (DFMO+7) or 14 days (DMFO+14) after, tumor cell injection. Mice were killed either 4 weeks (Experiment 1) or 6 weeks post injection (Experiment 2). Lungs were removed and fixed in Bouin's fixative diluted 1:5 in neutral buffered formalin. Each lung was examined for the presence of surface metastases. The incidence of mice with lung metastases was graphed as a percentage of the total number of mice (= 10 for each group). The average number of metastases per lung was graphed as a percentage of the control (= 10 for each group). Experiment 1 (black) and Experiment 2 (small hatched) are graphed separately as well as combined (large hatched)

Α-Difluoromethylornithine (DFMO) (2% per orally in drinking water) decreases polyamine levels in mammary fat pad tumors of MDA-MB-231 cells

<p><b>Copyright information:</b></p><p>Taken from "Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment"</p><p>Breast Cancer Research 2005;7(5):R819-R827.</p><p>Published online 9 Aug 2005</p><p>PMCID:PMC1242150.</p><p>Copyright © 2005 Richert et al, licensee BioMed Central Ltd.</p> Polyamine levels were determined in control subcutaneous (s.c.) and mammary fat pad (mfp) tumors as well as in DFMO-treated tumors grown in the mammary fat pad. DFMO treatment began at either 7 days prior to tumor cell injections (DFMO-7), on the day of injection (DFMO 0) or 21 days post injection (DFMO+21). Putrescine (black), spermidine (diagonal line) and spermine (cross-hatched) levels are graphed as nanomoles per milligram of tumor

Α-Difluoromethylornithine (DFMO) decreases the incidence and size of bone metastases from MDA-MB-435 cells

<p><b>Copyright information:</b></p><p>Taken from "Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment"</p><p>Breast Cancer Research 2005;7(5):R819-R827.</p><p>Published online 9 Aug 2005</p><p>PMCID:PMC1242150.</p><p>Copyright © 2005 Richert et al, licensee BioMed Central Ltd.</p> Green fluorescent protein-labeled MDA-MB-435 cells were injected intracardially into athymic mice. DFMO (2% per orally in drinking water ) treatment began 7 days prior to injection and continued for 6 weeks post injection. Bone metastases were visualized using a fluorescence microscope. Photographs of femurs from control (= 10) and DFMO-treated (= 10) mice show the presence and size of the bone metastases. Incidence of metastasis was determined by counting the number of mice with green fluorescence in the femur regardless of size of the mass. Image analysis was used to quantify fluorescence in each bone. The area of tumor in bone was quantified by comparing the total area of the femur with the area containing green fluorescence. The percentage of bone occupied by the tumor was also determined

Α-Difluoromethylornithine (DFMO) treatment affects both cell proliferation and cell death in MDA-MB-231 orthotopic tumors

<p><b>Copyright information:</b></p><p>Taken from "Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment"</p><p>Breast Cancer Research 2005;7(5):R819-R827.</p><p>Published online 9 Aug 2005</p><p>PMCID:PMC1242150.</p><p>Copyright © 2005 Richert et al, licensee BioMed Central Ltd.</p> TUNEL analysis and BrdU analysis of MDA-MB-231 orthotopic tumors from either control mice or mice treated with 2% DFMO in the drinking water beginning at the day of injection