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Supplementary Figure 5 from Cadherin-11 Increases Migration and Invasion of Prostate Cancer Cells and Enhances their Interaction with Osteoblasts

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Cell adhesion molecules have been implicated in the colonization of cancer cells to distant organs. Prostate cancer (PCa) has a propensity to metastasize to bone, and cadherin-11, which is an osteoblast cadherin aberrantly expressed in PCa cells derived from bone metastases, has been shown to play a role in the metastasis of PCa cells to bone. However, the mechanism by which cadherin-11 is involved in this process is not known. Here, we show that expression of cadherin-11 in cadherin-11–negative C4-2B4 cells increases their spreading and intercalation into an osteoblast layer and also stimulates C4-2B4 cell migration and invasiveness. The downregulation of cadherin-11 in cadherin-11–expressing metastatic PC3 cells decreases cell motility and invasiveness. Further, both the juxtamembrane (JMD) and β-catenin binding domains (CBS) in the cytoplasmic tail of cadherin-11 are required for cell migration and invasion, but not spreading. Gene array analyses showed that several invasion-related genes, including MMP-7 and MMP-15, are upregulated in cadherin-11–expressing, but not in cad11-ΔJMD–expressing or cad11-ΔCBS–expressing, C4-2B4 cells. These observations suggest that cadherin-11 not only provides a physical link between PCa cells and osteoblasts but also increases PCa cell motility and invasiveness that may facilitate the metastatic colonization of PCa cells in bone. Cancer Res; 70(11); 4580–9. ©2010 AACR.

Supplementary Figure 3 from Cadherin-11 Increases Migration and Invasion of Prostate Cancer Cells and Enhances their Interaction with Osteoblasts

Despite advances in our understanding of the molecular mechanisms underlying the aggressiveness of triple-negative breast cancer (TNBC), the contribution of tumor-associated macrophages (TAMs) to TNBC pathogenesis has not been therapeutically exploited. TAMs are the most abundant cell types in the tumor microenvironment (TME) and the key contributor to tumor progression and invasion. We have found that c-Jun NH2-terminal kinase (JNK), a member of the MAPK family and a major regulator of inflammation, contributes to TNBC tumorigenesis by promoting the cancer stem-like cell phenotype. However, whether the JNK pathway regulates TAMs and their cross-talk with tumor cells in TNBC remains unknown. Here, we tested the hypothesis that JNK signaling contributes to TNBC aggressiveness by promoting the tumor cell–TAM cross-talk that facilitates TNBC cell invasiveness. We found that, among 80 patients with primary inflammatory breast cancer (IBC), TNBC tumors (n=18) had 2-fold more TAMs than non-TNBC tumors (n=62, P=0.05) and that high TAM counts were correlated with shorter disease-free survival of patients with IBC (P=0.05). Both JNK1 and c-Jun were highly activated in TAMs, and JNK-IN-8, a pan-inhibitor of JNK, suppressed c-Jun activation. JNK-IN-8 also increased expression of M1 macrophage markers (CD80 and HLA-DR) but reduced expression of TAM markers (CD163 and CD206), suggesting that JNK suppresses M1 macrophage differentiation but promotes TAM differentiation. Co-culture with TAMs significantly enhanced migration and invasion of HCC70 and MDA-MB-468 human and 4T1 murine TNBC cells. Similarly, an enhancement in TNBC cells migration and invasion was observed following culture with TAM-conditioned medium, suggesting that TAMs enhance TNBC cellular activities through paracrine signaling. In addition, inhibition of JNK signaling in TNBC cells or in TAMs by JNK-IN-8 significantly suppressed TAM-promoted enhancement of TNBC cell migration and invasion. These studies strongly suggest that JNK regulates M1/TAM differentiation and TNBC cell–TAM cross-talk. Furthermore, cytokine/chemokine profiling analysis showed that, of the identified molecules, MCP-1 (secreted by TAMs) and VEGF (secreted by TNBC cells) had the highest expression levels and that their expression was dramatically reduced following JNK-IN-8 treatment. Stimulation with recombinant VEGF increased proliferation of MDA-MB-468 cells, and stimulation with recombinant MCP-1 enhanced migration of the cells. These findings suggest that VEGF and MCP-1 are involved in JNK-mediated TNBC cell–TAM cross-talk. Together, our results suggest that JNK signaling regulates tumor cell–TAM cross-talk through MCP-1– and/or VEGF-mediated paracrine signaling and that JNK is an important therapeutic target in TNBC. Further animal studies using JNK-knockout TNBC cells co-injected with TAMs are needed to confirm our in vitro findings. Citation Format: Xie X, Otsuka S, Chu K, Lu AY, Tripathy D, Dalby KN, Hittelman WN, Van Laere S, Bartholomeusz C, Ueno NT. JNK signaling regulates tumor cell–tumor-associated macrophage cross-talk in triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-03.

Real-time energy monitoring is a way of monitoring energy consumption and quality of power within a building or a network of buildings. It provides a more efficient approach to using energy by making informed decisions based on data that has been collected in the past and using it to predict future trends. In this project, we have developed a Real-Time Energy Monitoring and Reporting Tool for the Hal Marcus College of Science and Engineering building of the University of West Florida (UWF). This project is a culmination of work done by two Capstone Project teams, with one team working on the hardware configuration of the project for recording the data in a database and the other working on the software interface for viewing, monitoring and reporting the data. This project allows the University to track energy consumption and power quality in real time, review historical data, and find patterns that can be used to regulate energy usage and power quality. The visualization and reporting software has been written in Python, has a MYSQL database, and is used in conjunction with a separately developed hardware interface. The software is embedded in a hardware interface that uses IoT connected sensors to measure data from UWF Buildings. Data such as voltage, current, power, frequency, etc. is collected from the sensors and stored in a database. The software developed in this project is used to interpret the data and display it in user defined graphical and tabular outputs. The developed energy-monitoring and reporting software has been installed in the central data center located in the Department of Electrical and Computer Engineering.

// Adam R. Wolfe 1, 2 , Nicholaus J Trenton 5 , Bisrat G. Debeb 1, 2 , Richard Larson 1, 2 , Brian Ruffell 6 , Khoi Chu 4 , Walter Hittelman 4 , Michael Diehl 5 , Jim M Reuben 1 , Naoto T. Ueno 1, 3 , Wendy A. Woodward 1, 2 1 MD Anderson Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5 Department of Bioengineering, Rice University, Houston, TX, USA 6 Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Correspondence to: Wendy A. Woodward, email: wwoodward@mdanderson.org Keywords: inflammatory breast cancer, macrophages, mesenchymal stem cells, IL-6, statins Received: August 23, 2016 Accepted: October 06, 2016 Published: October 15, 2016 ABSTRACT Inflammatory breast cancer (IBC) is a unique and deadly disease with unknown drivers. We hypothesized the inflammatory environment contributes to the IBC phenotype. We used an in vitro co-culture system to investigate interactions between normal and polarized macrophages (RAW 264.7 cell line), bone-marrow derived mesenchymal stem cells (MSCs), and IBC cells (SUM 149 and MDA-IBC3). We used an in vivo model that reproduces the IBC phenotype by co-injecting IBC cells with MSCs into the mammary fat pad. Mice were then treated with a macrophage recruitment inhibitor, anti-CSF1. MSC and macrophages grown in co-culture produced higher levels of pro-tumor properties such as enhanced migration and elevated IL-6 secretion. IBC cells co-cultured with educated MSCs also displayed enhanced invasion and mammosphere formation and blocked by anti-IL-6 and statin treatment. The treatment of mice co-injected with IBC cells and MSCs with anti-CSF1 inhibited tumor associated macrophages and inhibited pSTAT3 expression in tumor cells. Anti-CSF1 treated mice also exhibited reduced tumor growth, skin invasion, and local recurrence. Herein we demonstrate reciprocal tumor interactions through IL-6 with cells found in the IBC microenvironment. Our results suggest IL-6 is a mediator of these tumor promoting influences and is important for the IBC induced migration of MSCs.

Purpose: Brain metastasis poses a major treatment challenge and remains an unmet clinical need. Finding novel therapies to prevent and treat brain metastases requires an understanding of the biology and molecular basis of the process, which currently is constrained by a dearth of experimental models and specific therapeutic targets. The purpose of this study was to develop preclinical models and identify molecular mediators of brain metastasis from breast cancer. Methods: We used MDA-MB-IBC3 (ER-/HER2+), SUM149 (ER-/HER2-), MCF7 (ER+/HER2-), SUM159 (ER-/HER2-) and MDA-MB-231 (ER-/HER2-) cell lines for this study. GFP-labeled cells were injected via tail vein into SCID/beige mice and metastatic colonization to the brain and lung evaluated by fluorescent stereomicroscope and histology 8-weeks after injection. miRNA microarray was performed with miRNA 3.0 Array. Stable knockdown of miR-141 was achieved with the lentiviral miRZip system. MiR-141 serum levels in 105 breast cancer patients were measured using quantitative PCR. Results: We developed novel brain metastasis models in which tail-vein injection of both triple-negative and a HER2-overexpressing inflammatory breast cancer lines led to a high rate of brain metastases (67%) in SCID/Beige mice (SUM149, 6 of 9 mice; MDA-MB-IBC3, 10 of 15 mice). Sub-lines derived from lung or brain metastases in these models were morphologically and molecularly distinct. The brain metastasis-derived sublines showed epithelial morphology and overexpressed epithelial markers and miR-141 while sublines from lung metastases showed mesenchymal morphology and overexpressed mesenchymal markers. Knockdown of miR-141 significantly inhibited metastatic colonization to the brain compared to controls (miR-141 knockdown vs. control: SUM149, 0 of 8 mice vs. 6 of 9 mice, p=0.009; MDA-MB-IBC3, 2 of 14 mice vs. 10 of 15 mice, p=0.007) but it did not affect colonization to the lung. Importantly, ectopic expression of miR-141 in non-expressing MDA-MB-231 significantly enhanced brain metastatic colonization (5 of 9 mice vs. 0 of 10 mice, P=0.02). On multivariate analyses high serum level of miR-141 was an independent predictor of progression free survival [HR 4.8 (95%CI, 2.6-8.7), P Conclusion: We demonstrated high rates of brain metastases from a heterogeneous group of cell lines that have not previously been associated with brain metastases, demonstrated miR-141 as a key regulator of brain metastasis and provided clinical evidence supporting the prognostic relevance of miR-141. We propose that miR-141 should be examined as a biomarker and potential target in the prevention and treatment of brain metastases from breast cancer. Citation Format: Bisrat G Debeb, Lara Lacerda, Simone Anfossi, Parmeswaran Diagaradjane, Khoi Chu, Lei Huo, Caimiao Wei, Richard A Larson, Adam R Wolfe, Wei Xu, Daniel L Smith, Li Li, Cristina Ivan, Pamela K Allen, Xiang H Zhang, George A Calin, Savitri Krishnamurthy, Naoto T Ueno, Thomas A Buchholz, James M Reuben, Wendy A Woodward. The microRNA miR-141 is a key regulator of brain metastasis from breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-16-01.

Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2cre/Osxf/f/SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osxf/f/SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa.

Introduction: Inflammatory breast cancer (IBC) is a distinct and aggressive subtype of locally advance breast cancer associated with increased aldehyde dehydrogenase 1 (ALDH1) positive cancer stem cells (CSCs). IBC is associated with a poor survival rate (40% 5-year survival), with few therapeutic strategies identified that effectively blocked the growth and metastasis of IBC. Our previous in vitro studies revealed that the pan-histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) effectively targets self-renewal of IBC tumor cells. Materials and Methods: The IBC cell line, SUM149 was tagged with the Luciferase gene (SUM149-LUC) and their in vivo growth in immune compromised mice tested in both orthotopic and metastatic settings. Results: SUM149-LUC rapidly develop primary tumors and metastatic lesions at a variety of locations including lung, brain, bone, liver, reproductive organs and adrenal glands. SAHA effectively blocked growth of SUM149 primary tumors and inhibited metastasis, which was synergistic with the microtubule stabilizer, Paclitaxel. The therapeutic efficacy of SAHA was associated with a significant decrease in ALDH1+ CSCs populations. Conclusions: Collectively, these results suggest that strategies of combining agents such as SAHA that target CSCs with Paclitaxel that targets the bulk of proliferating tumor cells warrant further investigation for their potential effectiveness in IBC patients, who have the lowest survival of breast cancer patients and have few therapeutic options. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-14.

// Khoi Chu 1 , Kimberley M. Boley 1 , Ricardo Moraes 1 , Sanford H Barsky 2 and Fredika M. Robertson 1 1 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 2 Department of Pathology, The University of Nevada School of Medicine, Reno, NV Correspondence: Fredika M. Robertson, email: // Keywords : E-Cadherin, Inflammatory Breast Cancer, Mesenchymal-Epithelial Transition (MET), Hypoxia-inducible 1α transcription factor (HIF-1α), Metabolism, Glycolysis. Received : March 15, 2013 Accepted : March 21, 2013 Published : March 21, 2013 Abstract E-Cadherin is a cell:cell adhesion molecule critical for appropriate embryonic and mammary development. In cancer, E-Cadherin has been primarily viewed as being lost during the process of epithelial-mesenchymal transition (EMT), which occurs with a switch from E-Cadherin expression to a gain of N-Cadherin and other mesenchymal markers. EMT has been shown to play a role in the metastatic process while the reverse process, mesenchymal-epithelial transition (MET), is important for metastatic colonization. Here we report an unexpected role of E-Cadherin in regulating tumorigenicity and hypoxia responses of breast tumors in vivo . Reduced expression of E-Cadherin led to a dramatic reduction of the in vivo growth capability of SUM149, Mary-X and 4T1 tumor cells. Furthermore, over-expression of ZEB1 , a known transcriptional repressor of E-Cadherin, led to reduced in vivo growth of SUM149 tumors. Gene set enrichment analysis identified the loss of hypoxia response genes as a major mechanism in mediating the lack of in vivo growth of SUM149 cells that lacked E-Cadherin or over-expressed ZEB1 . The in vivo growth defect of SUM149 E-Cadherin knockdown tumors was rescued by the hypoxia-inducible 1α transcription factor (HIF-1α). Given the importance of HIF-1α in cellular metabolism, we observed reduced glycolytic capacity in SUM149 and 4T1 cells that had E-Cadherin knocked down. Our observations shed light on the complex functions of E-Cadherin in retention of an epithelial phenotype and as a mediator of survival of aggressive breast cancer under hypoxic conditions in vivo . Furthermore, we find that patients with basal subtype breast cancer and high E-Cadherin expression in their tumors had a poor clinical outcome. Our data suggests a novel function for E-Cadherin as a bona fide signaling molecule required for the in vivo growth of aggressive breast cancer tumor cells, that retain E-Cadherin expression, in mediating their metabolic function.

Background: Genetic abnormalities in the anaplastic lymphoma kinase (ALK) gene result in activation of signaling pathways including Akt, mTor, and JAK/Stat3. ALK has been shown to be a primary oncogenic driver in a variety of human tumors, including both hematologic malignancies such as anaplastic large cell lymphoma, as well as solid tumors including neuroblastoma, non-small cell lung cancer, myofibroblastic tumors and most recently, high grade serous ovarian carcinoma. While only ∼3% of all breast cancers have been reported to have ALK genetic abnormalities, our studies revealed that inflammatory breast cancer (IBC), the most lethal variant of breast cancer, is characterized by prevalent ALK gene amplification with activated ALK signaling. The present studies investigated the role of ALK in breast cancer by expressing full-length wild type ALK in MCF-7 cells. Materials and Methods: Clones of MCF-7 breast cancer cells expressing wild type ALK or non-target vector were produced by lentivirus infection and selection of single cell clones. MCF-7 ALK clones were evaluated using live cell and phase contrast imaging, immunofluorescent staining with confocal imaging, gene profiling, phospho-protein array analysis, western blot and ELISA validation. In vivo studies were performed by injection of MCF-7 ALK clones into using NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice using IACUC approved animal protocols. Results: When cultured on plastic substrates, MCF-7 ALK clones formed tumor cell aggregates instead of monolayer cultures, and when cultured as tumor spheroids under non-adherent 3D conditions had a distinct cellular phenotype with significantly greater clonogenicity than either non-target vector MCF-7 clones or the parental cell line. Whole transcriptome analysis, with validation using protein arrays, western blots and ELISA analysis revealed that the presence of ALK up-regulated phospho-src. In vivo studies revealed that ALK expressing MCF-7 clones formed tumor emboli that were enwrapped by dermal lymphatic vessels, essentially recapitulating the phenotype of IBC tumor emboli that exhibit encircling lymphovasculogenesis. Enforced expression of wild type ALK in another breast cancer cell line resulted in similar formation of tumor emboli. Discussion: These studies provide first time evidence for the association between full length ALK and formation of highly invasive tumor emboli enwrapped by lymphatic vessels, which is a primary characteristic of IBC. These studies, taken together with discovery of the prevalence of ALK gene amplification in IBC patients, indicate that ALK represents an important therapeutic target for IBC, with the availability of new ALK targeted therapies to evaluate as single agents and in combinations with other agents that may effectively target IBC tumor emboli that we have now linked to ALK and which represent the metastatic lesion of this lethal variant of breast cancer. Funding by Susan G. Komen Organization Promise Grant KG081287 (FMR and MC). Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-04.

Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC. Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis. Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC. Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.

Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of locally advanced breast cancer. Pathologically, IBC is characterized by the presence of nests of cells, defined as tumor emboli, that undergo lymphovascular invasion into the skin and chest wall and represent the metastatic lesion of IBC. Due to the distinct presentation of IBC, it is often misdiagnosed due to a lack of defined biomarkers that are specific for tumor emboli in biopsy samples of IBC patients. To date, the only validated marker that has shown to consistently identify IBC tumor emboli is the surface glycoprotein E-cadherin. Materials and Methods: With the goal of identifying genes specifically expressed by IBC tumor emboli, the present studies performed whole transcriptome analysis of tumor emboli from skin punch biopsy samples from IBC patients and tumor emboli from the Mary-X pre-clinical model of IBC which were isolated using laser capture microdissection. Results: Secretory leukocyte peptidase inhibitor (SLPI) was a gene identified as being highly expressed by tumor emboli. This gene was previously identified as a metastasis related gene, has been reported to be associated with poor prognosis in serous ovarian carcinoma and is implicated in mediating resistance to paclitaxel. Immunofluorescence staining and confocal microscopy revealed that SLPI is a robust marker of tumor emboli in skin punch biopsies from IBC patient and in Mary-X tumor emboli. SLPI differentially delineates between IBC tumor emboli and hair follicles present in the skin, which also stain with E-cadherin. An additional marker, Cytokeratin 15 (CK15), can be used in combination with SLPI to differentially identify the components of the skin that are CK15 positive, and IBC tumor emboli that reside within the skin, which are CK15 negative. Use of podoplanin antibodies, which stain lymphovascular endothelium, demonstrate that IBC tumor emboli that express SLPI protein, are completely encircled within the dermal lymphovasculature. Conclusions: These studies are among the first to identify SLPI as a potential biomarker of tumor emboli in skin punch biopsy samples from IBC patients and in pre-clinical models of IBC. Studies are ongoing to establish the molecular function and role of SLPI in mediating the survival and invasion of IBC tumor emboli. Collectively, these studies are the first to identify SLPI as a potential biomarker of IBC tumor emboli in skin punch biopsy samples. In addition to serving as a potential biomarker of tumor emboli, SLPI may also serve as a therapeutic target for development of approaches to eradicate IBC tumor emboli. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-08.

Dasatinib, a dual Src family kinase and Abl inhibitor, is being tested clinically for the treatment of prostate cancer bone metastasis. Bidirectional interactions between osteoblasts and prostate cancer cells are critical in the progression of prostate cancer in bone, but the effect of dasatinib on osteoblasts is unknown. We found that dasatinib inhibited proliferation of primary mouse osteoblasts isolated from mouse calvaria and the immortalized MC3T3-E1 cell line. In calvarial osteoblasts from Col-luc transgenic mice carrying osteoblast-specific Col1alpha1 promoter reporter, luciferase activity was inhibited. Dasatinib also inhibited fibroblast growth factor-2-induced osteoblast proliferation, but strongly promoted osteoblast differentiation, as reflected by stimulation of alkaline phosphatase activity, osteocalcin secretion and osteoblast mineralization. To determine how dasatinib blocks proliferative signaling in osteoblasts, we analyzed the expression of a panel of tyrosine kinases, including Src, Lyn, Fyn, Yes and Abl, in osteoblasts. In the Src family kinases, only Src was activated at a high level. Abl was expressed at a low level in osteoblasts. Phosphorylation of Src-Y419 or Abl-Y245 was inhibited by dasatinib treatment. Knockdown of either Src or Abl by lenti-shRNA in osteoblasts enhances osteoblast differentiation, suggesting that dasatinib enhances osteoblast differentiation through inhibition of both Src and Abl.

Men with castration-resistant prostate cancer (PCa) frequently develop metastasis in bone. The reason for this association is unclear. We have previously shown that cadherin-11 (also known as OB-cadherin), a homophilic cell adhesion molecule that mediates osteoblast adhesion, plays a role in the metastasis of PCa to bone. Here, we report that androgen deprivation therapy upregulates cadherin-11 expression in PCa. In human PCa specimens, immunohistochemical staining showed that 22 of 26 (85%) primary PCa tumors from men with castration-resistant PCa expressed cadherin-11. In contrast, only 7 of 50 (14%) androgen-dependent PCa tumors expressed cadherin-11. In the MDA-PCa-2b xenograft animal model, cadherin-11 was expressed in the recurrent tumors following castration. In the PCa cell lines, there is an inverse correlation between expression of cadherin-11 and androgen receptor (AR), and cadherin-11 is expressed in very low levels or not expressed in AR positive cell lines, including LNCaP, C4-2B4, and VCaP cells. We showed that AR likely regulates cadherin-11 expression in PCa through an indirect mechanism. Although re-expression of AR in the AR-negative PC3 cells led to the inhibition of cadherin-11 expression, depletion of androgen from the culture medium or down regulation of AR by RNA interference in the C4-2B4 cells or VCaP cells only produce a modest increase of cadherin-11 expression. Promoter analysis indicated that cadherin-11 promoter does not contain a typical AR binding element, and AR elicits a modest inhibition of cadherin-11 promoter activity, suggesting that AR does not regulate cadherin-11 expression directly. Together, these results suggest that androgen deprivation upregulates cadherin-11 expression in prostate cancer and this may contribute to the metastasis of PCa to bone. Our study suggests that therapeutic strategies that block cadherin-11 expression or function may be considered when applying androgen ablation therapy.

Bone is the most common site of metastases from prostate cancer. The mechanism by which prostate cancer cells metastasize to bone is not fully understood, but interactions between prostate cancer cells and bone cells are thought to initiate the colonization of metastatic cells at that site. Here, we show that cadherin-11 (also known as osteoblast-cadherin) was highly expressed in prostate cancer cell line derived from bone metastases and had strong homophilic binding to recombinant cadherin-11 in vitro. Down-regulation of cadherin-11 in bone metastasis–derived PC3 cells with cadherin-11–specific short hairpin RNA (PC3-shCad-11) significantly decreased the adhesion of those cells to cadherin-11 in vitro. In a mouse model of metastasis, intracardiac injection of PC3 cells led to metastasis of those cells to bone. However, the incidence of PC3 metastasis to bone in this model was reduced greatly when the expression of cadherin-11 by those cells was silenced. The clinical relevance of cadherin-11 in prostate cancer metastases was further studied by examining the expression of cadherin-11 in human prostate cancer specimens. Cadherin-11 was not expressed by normal prostate epithelial cells but was detected in prostate cancer, with its expression increasing from primary to metastatic disease in lymph nodes and especially bone. Cadherin-11 expression was not detected in metastatic lesions that occur in other organs. Collectively, these findings suggest that cadherin-11 is involved in the metastasis of prostate cancer cells to bone. (Mol Cancer Res 2008;6(8):1259–67)

We previously reported using statins was correlated with improved metastasis-free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple-negative breast cancer (TNBC) cells. TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in eight public breast cancer gene expression datasets including 1479 patients. Simvastatin increased G1/S-phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pre-treatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted.

Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA binding domain (DBD) and represses the transcriptional activity of various nuclear receptors. In this study, we examined the novel cross talk between SHP and BETA2/NeuroD, a basic helix-loop-helix transcription factor. In vitro and in vivo protein interaction studies showed that SHP physically interacts with BETA2/NeuroD, but not its heterodimer partner E47. Moreover, confocal microscopic study and immunostaining results demonstrated that SHP colocalized with BETA2 in islets of mouse pancreas. SHP inhibited BETA2/NeuroD-dependent transactivation of an E-box reporter, whereas SHP was unable to repress the E47-mediated transactivation and the E-box mutant reporter activity. In addition, SHP repressed the BETA2-dependent activity of glucokinase and cyclin-dependent kinase inhibitor p21 gene promoters. Gel shift and in vitro protein competition assays indicated that SHP inhibits neither dimerization nor DNA binding of BETA2 and E47. Rather, SHP directly repressed BETA2 transcriptional activity and p300-enhanced BETA2/NeuroD transcriptional activity by inhibiting interaction between BETA2 and coactivator p300. We also showed that C-terminal repression domain within SHP is also required for BETA2 repression. However, inhibition of BETA2 activity was not observed by naturally occurring human SHP mutants that cannot interact with BETA2/NeuroD. Taken together, these results suggest that SHP acts as a novel corepressor for basic helix-loop-helix transcription factor BETA2/NeuroD by competing with coactivator p300 for binding to BETA2/NeuroD and by its direct transcriptional repression function.

NeuroD1(BETA2) and Tpit are cell-specific activators of pituitary proopiomelanocortin (POMC) gene transcription. Expression of both factors slightly precedes that of POMC at embryonic d 12.5 of mouse pituitary development. We now report that NeuroD1(BETA2) is required for early corticotroph differentiation. In agreement with the transcriptional synergism observed between Tpit and basic helix-loop-helix dimers containing NeuroD1(BETA2), POMC expression is delayed in NeuroD1-deficient mice. However, this differentiation defect does not reflect a change of corticotroph commitment as revealed by Tpit expression. The delay of corticotroph terminal differentiation is transient and coincides with the developmental window of NeuroD1 expression in corticotrophs. In contrast to their requirement in other NeuroD1-expressing cells, the neurogenin genes do not appear to be necessary for corticotroph differentiation. Taken together with a similar requirement of Tpit for corticotroph differentiation but not for commitment, the present data indicate that the POMC promoter is a point of convergence for independent corticotroph differentiating signals.

Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of this disease and is known to be enriched for cells with a cancer stem cell phenotype. IBC is characterized by the presence of cell aggregates, defined as tumor emboli, that metastasize into skin and chest wall. The only documented biomarker of tumor emboli is the surface glycoprotein, E-cadherin. We previously demonstrated that IBC tumor emboli express the alarm anti-protease, secretory leukocyte peptidase inhibitor (SLPI), a metastasis related gene highly expressed in IBC patient tumors. Since the function of SLPI in IBC is unknown, the present studies used zinc finger technology to knockout (KO) copies of SLPI in the SUM149 IBC cell line to define the role of SLPI in IBC. Materials and Methods: Using CompoZr zinc finger nuclease (ZFN) technology (Sigma-Aldrich), SLPI KO cell lines were generated by disrupting all alleles (3) in exon 1 of SUM149 cells derived from an IBC primary tumor with a high CD44+cell population. The target-specific ZFNs bound DNA at a sequence-specific location and created double strand breaks repaired by non-homologous end joining, resulting in deletions at the SLPI locus. Single cell SLPI KO clones were isolated and serially passaged to establish stable cell lines. Functional assays were used to assess proliferation, invasion, tube formation and clonogenicity. Global transcriptional profiling was performed to identify genes and signaling pathways directly regulated by SLPI. Results: SUM149 SLPI KO clones did not produce SLPI protein and had a significantly slower turn-over time of 75 hrs compared with 24 hrs in SUM149 wild type clones. Loss of SLPI blocked invasion by 50%, and completely inhibited formation of tube-like structures, an activity defined as vasculogenic mimicry, characteristic of IBC. The loss of only 1 SLPI allele resulted in the inability of SUM149 cells to grow as anchorage independent clones in soft agar, commonly used as a predictor of in vivo tumorigenicity. SLPI directly regulated expression of multiple genes within the embryonic stem cell pleuripotency canonical pathway, including WNT, Frizzled, PDK-1, platelet derived growth factor receptor and sphingosine-1-phosphate receptor. Studies are underway to determine the specific role of SLPI in IBC tumor growth and metastasis. Conclusions: Our previous studies demonstrated that SLPI is expressed by IBC tumor emboli and can be used as a biomarker of tumor emboli in IBC core and skin punch biopsies. SLPI was found to not only regulate critical functional activities of IBC tumor cells but also to directly regulate genes within pathways critically important to maintenance of pleuripotency of tumors with a cancer stem cell phenotype. Collectively, these studies demonstrate the power and utility of the zinc finger technology, which enables the interrogation of tumor cells to discern the direct function and role of specific genes of interest. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-03.

BETA2/NeuroD, a basic helix-loop-helix transcription factor, is expressed in pancreatic endocrine cells during development and regulates insulin gene expression. We demonstrated previously that the endocrine pancreas of BETA2/NeuroD-deficient mice undergoes massive apoptosis and, consequently, animals die of diabetes shortly after birth. Here we show that a significant fraction of BETA2-deficient mice in a new genetic background can survive diabetes and live to adulthood through the process of beta-cell neogenesis. Morphometric examination indicates that pancreatic beta-, but not alpha-cell mass, was restored to a level comparable to that of wild-type animals. However, the newly formed islet cells cannot form mature islets of Langerhans, indicating an indispensable role of BETA2 in morphogenesis of normal islet structure. Furthermore, immunohistochemical examinations revealed that newly formed beta-cells of BETA2/NeuroD-deficient mice come from two sources: either directly budding from the pancreatic ductal tree or from the preexisting beta-cells in the residual endocrine pancreas. Our results indicate that beta-cell neogenesis in our BETA2/NeuroD-deficient mice contributes to their survival, and these mice may provide a useful model for studying the mechanism of beta-cell regeneration.

Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. IBC has distinctive characteristics including invasion of tumor emboli into the skin and rapid disease progression. Given our previous studies suggesting that HDAC inhibitors have promise in targeting IBC, the present study revealed that the class I HDAC inhibitor Romidepsin (FK-288, Istodax; Celgene Corporation, Summit, NJ) potently induced destruction of IBC tumor emboli and lymphatic vascular architecture. associated with inhibition of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha, (HIF1alpha) proteins in the Mary-X pre-clinical model of IBC. Romidepsin treatment induced clinically relevant biomarkers in including induction of acetylated Histone 3 (Ac-H3) proteins, apoptosis, and increased p21WAF1/CIP1. Romidepsin, alone and synergistically when combined with Paclitaxel, effectively eliminated both primary tumors and metastatic lesions at multiple sites formed by the SUM149 IBC cell line. This is the first report of the ability of an HDAC inhibitor to eradicate IBC tumor emboli, to destroy the integrity of lymphatic vessel architecture and to target metastasis. Furthermore, Romidepsin, in combination with a taxane, warrants evaluation as a therapeutic strategy that may effectively target the skin involvement and rapid metastasis that are hallmarks of IBC.

The BETA2 (neuroD) gene is expressed in endocrine cells during pancreas development and is essential for proper islet morphogenesis. The objective of this study is to identify potential upstream regulators of the BETA2 gene during pancreas development. We demonstrated that the expression of neurogenin 3 (ngn3), an islet- and neuron-specific basic-helix-loop-helix transcription factor, partially overlaps that of BETA2 during early mouse development. More importantly, overexpression of ngn3 can induce the ectopic expression of BETA2 in Xenopus embryos and stimulate the endogenous RNA of BETA2 in endocrine cell lines. Furthermore, overexpression of ngn3 could cause a dose-dependent activation on the 1.0-kb BETA2 promoter in islet-derived cell lines. Deletion and mutation analyses revealed that two proximal E box sequences, E1 and E3, could bind to ngn3-E47 heterodimer and mediate ngn3 activation. Based on these results, we hypothesize that ngn3 is involved in activating the expression of BETA2 at an early stage of islet cell differentiation through the E boxes in the BETA2 promoter.

We have previously demonstrated that the oxytocin (OT) gene is expressed in the rat uterine epithelium and that its expression is upregulated in vivo and in vitro by estrogen. This hormonal regulation is mediated by a hormone response element (HRE) located in the OT gene promoter. Here we show that the same OT-HRE is also capable of interacting with two novel members of the orphan nuclear receptor family, rat COUP-TFII and Ear-2, and that this interaction antagonizes the estrogenic induction of the OT promoter. By Northern blot analysis and immunocytochemistry, using specific cDNA probes and antibodies, respectively, we demonstrate furthermore that both orphan receptors are expressed in uterine epithelial cells. Therefore, the present findings indicate that uterine OT gene expression is under stimulatory as well as inhibitory influences which are both mediated by the same HRE. More detailed analysis of the sequences necessary for estrogen receptor action and for orphan receptor action, using site-directed mutagenesis, revealed that the specific recognition sequences are overlapping but distinct: whereas the (imperfect) palindromic structure of the HRE constitutes the estrogen response element (ERE), orphan receptor action relies on an underlying direct TGACC repeat which forms part of the OT-HRE structure and overlaps with the estrogen response element.

Inflammatory breast cancer (IBC) is the most lethal variant of locally advanced breast cancer and carries with it a very low survival rate of 40 % at 5 years. IBC does not present as a lump but rather mimics characteristics of an inflammation that first appears as swelling of the breast, with edema, redness, and common lymph node involvement. The physical changes in the breast are associated with the presence of nests of aggregated tumor cells, defined as tumor emboli that are encircled by lymphatic vessels, effectively blocking lymphatic drainage. Little is understood about IBC, in part due to the lack of preclinical models that recapitulate its distinct characteristics. This chapter provides an overview of our studies that have profiled all available preclinical models of IBC, including two new models recently developed, to elucidate the molecular underpinnings of this lethal variant of breast cancer. Our studies demonstrate that IBC is enriched for cells that express CD44+ and CD133+ and have aldehyde dehydrogenase-1 (ALDH1) activity, supporting a cancer stem cell/tumor initiating phenotype, associated with a very high metastatic potential to multiple distant organ sites. IBC has a distinct gene signature including E-cadherin expression with associated loss of expression of ZEB1, a transcriptional repressor of E-cadherin. IBC is also characterized by loss of expression of genes within the transforming growth factor-beta (TGFβ) signaling pathway, which is permissive for cohesive invasion by IBC tumor emboli. Taken together, these studies suggest that IBC is a very distinct variant of breast cancer characterized by epithelial plasticity, enrichment of a stem cell phenotype, and cohesive invasion as an adaptive survival mechanism, consistent with the definition of IBC as the most metastatic variant of breast cancer.

BACKGROUND Brain metastasis poses a major treatment challenge and remains an unmet clinical need. Finding novel therapies to prevent and treat brain metastases requires an understanding of the biology and molecular basis of the process, which currently is constrained by a dearth of experimental models and specific therapeutic targets. METHODS Green Fluorescent Protein (GFP)-labeled breast cancer cells were injected via tail vein into SCID/Beige mice (n = 10-15 per group), and metastatic colonization to the brain and lung was evaluated eight weeks later. Knockdown and overexpression of miR-141 were achieved with lentiviral vectors. Serum levels of miR-141 were measured from breast cancer patients (n = 105), and the association with clinical outcome was determined by Kaplan-Meier method. All statistical tests were two-sided. RESULTS Novel brain metastasis mouse models were developed via tail vein injection of parental triple-negative and human epidermal growth factor receptor 2 (HER2)-overexpressing inflammatory breast cancer lines. Knockdown of miR-141 inhibited metastatic colonization to brain (miR-141 knockdown vs control: SUM149, 0/8 mice vs 6/9 mice,P= .009; MDA-IBC3, 2/14 mice vs 10/15 mice,P= .007). Ectopic expression of miR-141 in nonexpressing MDA-MB-231 enhanced brain metastatic colonization (5/9 mice vs 0/10 mice,P= .02). Furthermore, high miR-141 serum levels were associated with shorter brain metastasis-free survival (P= .04) and were an independent predictor of progression-free survival (hazard ratio [HR] = 4.77, 95% confidence interval [CI] = 2.61 to 8.71,P< .001) and overall survival (HR = 7.22, 95% CI = 3.46 to 15.06,P< .001). CONCLUSIONS Our study suggests miR-141 is a regulator of brain metastasis from breast cancer and should be examined as a biomarker and potential target to prevent and treat brain metastases.

Purpose: Brain metastasis poses a major treatment challenge and remains an unmet clinical need. Finding novel therapies to prevent and treat brain metastases requires an understanding of the biology and molecular basis of the process, which currently is constrained by a dearth of experimental models and specific therapeutic targets. The purpose of this study was to identify molecular mediators of brain metastasis from breast cancer using novel preclinical brain metastasis models we developed recently. Methods: GFP-labeled cells were injected via tail vein into SCID/beige mice and metastatic colonization to the brain and lung was evaluated by fluorescent stereomicroscope and histology 8-weeks after injection. miRNA microarray was performed with miRNA 3.0 Array. Stable knockdown and overexpression of miR-141 was achieved with lentiviral vectors. miR-141 serum levels in patients with metastatic breast cancer was measured using quantitative PCR and was associated with outcome data. Results: We developed novel brain metastasis models in which tail-vein injection of parental triple-negative and a HER2-overexpressing inflammatory breast cancer lines led to a high rate of brain metastases (67%) in SCID/Beige mice (SUM149, 6 of 9 mice; MDA-MB-IBC3, 10 of 15 mice). Sub-lines derived from brain metastases (BrMS) or lung metastases (LuMS) tissues were morphologically and molecularly distinct. The BrMS showed epithelial morphology and overexpressed epithelial markers and miR-141 while the LuMS showed mesenchymal morphology and overexpressed mesenchymal markers. Knockdown of miR-141 significantly inhibited metastatic colonization to the brain compared to parental controls in both cell lines (miR-141 knockdown vs. control: SUM149, 0 of 8 mice vs. 6 of 9 mice, p=0.009; MDA-MB-IBC3, 2 of 14 mice vs. 10 of 15 mice, p=0.007). Further, ectopic expression of miR-141 in non-expressing MDA-MB-231 significantly enhanced brain metastatic colonization via tail-vein injection (5 of 9 mice vs. 0 of 10 mice, P=0.02). On multivariate analyses high serum level of miR-141 was an independent predictor of progression free survival [HR 4.8 (95%CI, 2.6-8.7), P Conclusion: We have generated novel brain metastasis models that can be utilized to develop therapies. Further, we demonstrated miR-141 as a key regulator of brain metastasis and provided clinical evidence supporting the prognostic relevance of miR-141 in patients with brain metastasis. Our work could lead to novel targets for the prevention and treatment of brain metastasis that could improve patient survival in addition to fundamentally advancing the field of brain metastasis research. We propose that miR-141 should be examined as a biomarker and potential target to prevent or treat brain metastases. Citation Format: Bisrat G. Debeb, Lara Lacerda, Simone Anfossi, Parmeswaran Diagaradjane, Richard Larson, Khoi Chu, Lei Huo, Daniel Smith, Li Li, Cristina Ivan, Pamela Allen, Xiang Zhang, George Calin, Savitri Krishanmurthy, Thomas Buchholz, Naoto Ueno, James Reuben, Wendy A. Woodward. A novel mediator of brain metastasis from breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A55.

Study background: Inflammatory Breast Cancer (IBC) is the most metastatic variant of breast cancer. Although IBC is recognized as a distinct variant of breast cancer, the molecular basis for the rapid progression of IBC remains largely undefined, in part due to the lack of preclinical models that recapitulate the human disease as well as a lack of comprehensive analysis of the preclinical models of IBC that are available. Methods: All available 7 pre-clinical models of IBC, including 2 new models, FC-IBC01 and FC-IBC02 developed from pleural effusion, were used to identify genes and molecular pathways that are selectively altered compared to non IBC breast tumor models. Laser capture micro dissection of biopsy tissue from core biopsy and skin punch biopsies were also analyzed by whole transcriptome analysis. Results: Whole transcriptome analysis defined 7 pre-clinical models of IBC as being within either the triple negative or ErbB2/Her-2 expressing subtypes, similar to the prevalence of these subtypes of breast cancer observed in IBC patients. Comparative analysis of the FC-IBC01, FC-IBC02 and Mary-X models of IBC demonstrated that each of these recapitulate the formation of tumor emboli with encircling lymphovasculogenesis. The majority (6/7) of the pre-clinical models of IBC express CDH1, which encodes for E-cadherin, which was associated with a loss of ZEB1, a transcriptional repressor of E-cadherin. The lack of ZEB1 expression was validated in a limited set of 4 skin punch biopsy samples from IBC patients that were isolated by laser capture micro-dissection, demonstrating concordance with loss of ZEB1 in pre-clinical models of IBC. Expression of other transcription factors involved in acquisition of a cancer stem cell phenotype, including Snai1, which encodes for Snail, SNAI2, which encodes for Slug and TWIST1, was retained in pre-clinical models of IBC. Maintenance of E-cadherin in pre-clinical models of IBC was associated with a loss of genes within the transforming growth factor beta (TGFβ signaling pathway, with expression of SMAD6, a known repressor of TGFβ. This is similar to a recent study reporting the persistence of E-cadherin and loss of TGFβ signaling in IBC patient tumors based on gene profiling of 3 independent data sets. Conclusion: The present studies provide first time comparison of gene signatures of 7 pre-clinical models of IBC, including our 2 newly developed pre-clinical models, FC-IBC01 and FC-IBC02, that recapitulate formation of tumor emboli with encircling lymphatic vessels, similar to that observed in biopsy tissues of IBC patients. We demonstrate that E-cadherin expression was associated with both loss of ZEB1 and diminished expression of multiple genes within the TGFβ signaling pathway, with retention of expression of transcription factors and surface markers consistent with maintenance of a cancer stem cell phenotype, as has been reported to be a characteristic of IBC tumors. Collectively, these observations provide first time characterization of the molecular signatures of all available pre-clinical models of IBC, and suggest that IBC has a signature of epithelial plasticity, with characteristics of their ability to undergo the mesenchymal to epithelial reverting transition. The loss of genes within the TGFβ signaling is also consistent with the tight cell: cell aggregation of IBC tumor cells within tumor emboli that exhibit “cohesive invasion”. The new pre-clinical models of IBC that recapitulate the human disease will serve as useful tools to accelerate our understanding of the molecular underpinnings and therapeutic targets of IBC as the most lethal form of breast cancer.

The molecular signature of inflammatory breast cancer (IBC) includes activation of target genes of the nuclear factor-kappa B (NF-κB) transcription factor. These NF-κB target genes are differentially activated in IBC tumors and primarily produce pro-inflammatory mediators such as the chemokine interleukin-8 (IL-8), the lipid mediator prostaglandin E2, the chemokine receptor CXCR4 and its ligand partner CXCL12, and the axis defined by IL-6/Janus kinases and signal tranducer and activator of transcription 3 (STAT3). While these genes are known to regulate innate immune responses, they also are critically important to survival of tumor cells and to metastatic progression. Ongoing research is defining the roles of these inflammatory mediators and associated signaling pathways in breast cancer, in general, and in IBC. Some of these studies have evaluated pharmacological and biological agents that effectively target these pro-inflammatory mediators and have led to development of new therapeutics that may effectively abrogate IBC growth and metastasis. In summary, this chapter reviews the inflammatory mediators that have been identified as part of the molecular fingerprint of IBC and describes new evidence for the potential for inhibitors of these mediators to target specific populations of cells within IBC tumors that contribute to tumor initiation and metastatic progression.

Katanin p60 is a microtubule-severing protein and is involved in microtubule cytoskeleton organization in both mitotic and non-mitotic processes. Its role in cancer metastasis is unknown.Differential protein profiles of bone marrow aspirates were analyzed by chromatography, electrophoresis, and mass spectrometry. Expression of katanin p60 in primary and metastatic prostate cancer was examined by immunohistochemistry. Cellular function of katanin p60 was further examined in prostate cell lines.In a proteomic profiling of bone marrow aspirates from men with prostate cancer, we found that katanin p60 was one of the proteins differentially expressed in bone metastasis samples. Immunohistochemical staining showed that katanin p60 was expressed in the basal cells in normal human prostate glands. In prostatic adenocarcinomas, in which the basal cells were absent, katanin p60 was expressed in the prostate cancer cells. In the specimens from bone metastasis, katanin p60 was detectable in the metastatic cancer cells. Strikingly, some of the metastatic cancer cells also co-expressed basal cell biomarkers including the tumor suppressor p53-homologous protein p63 and the high molecular weight cytokeratins, suggesting that the metastatic prostate cancer cells may have a basal cell-like phenotype. Moreover, overexpression of katanin p60 inhibited prostate cancer cell proliferation but enhanced cell migration activity.Katanin p60 was aberrantly expressed during prostate cancer progression. Its expression in the metastatic cells in bone was associated with the re-emergence of a basal cell-like phenotype. The elevated katanin p60 expression may contribute to cancer cell metastasis via a stimulatory effect on cell motility.

Tumors that display a highly metastatic phenotype contain subpopulations of cells that display characteristics similar to embryonic stem cells. These cells exhibit the ability to undergo self-renewal; slowly replicate to retain a nucleoside analog label, leading to their definition as "label-retaining cells"; express specific surface markers such as CD44(+)/CD24(-/low) and CD133; and can give rise to cells of different lineages (i.e., they exhibit multipotency). Based on these characteristics, as well as their demonstrated ability to give rise to tumors in vivo, these cells have been defined as tumor-initiating cells (TICs), tumor-propagating cells, or cancer stem cells (CSCs). These cells are highly resistant to chemotherapeutic agents and radiation and are believed to be responsible for the development of both primary tumors and metastatic lesions at sites distant from the primary tumor. Established cancer cell lines contain CSCs, which can be propagated in vitro using defined conditions, to form 3D tumor spheroids. Because the vast majority of studies to identify cancer-associated genes and therapeutic targets use adherent cells grown in 2 dimensions on a plastic substrate, the multicellular composition of these 3D tumor spheroids presents both challenges and opportunities for their imaging and characterization. The authors describe approaches to image and analyze the properties of CSCs within 3D tumor spheroids, which can serve as the basis for defining the gene and protein signatures of CSCs and to develop therapeutic strategies that will effectively target this critically important population of cells that may be responsible for tumor progression.

Cell adhesion molecules have been implicated in the colonization of cancer cells to distant organs. Prostate cancer (PCa) has a propensity to metastasize to bone, and cadherin-11, which is an osteoblast cadherin aberrantly expressed in PCa cells derived from bone metastases, has been shown to play a role in the metastasis of PCa cells to bone. However, the mechanism by which cadherin-11 is involved in this process is not known. Here, we show that expression of cadherin-11 in cadherin-11–negative C4-2B4 cells increases their spreading and intercalation into an osteoblast layer and also stimulates C4-2B4 cell migration and invasiveness. The downregulation of cadherin-11 in cadherin-11–expressing metastatic PC3 cells decreases cell motility and invasiveness. Further, both the juxtamembrane (JMD) and β-catenin binding domains (CBS) in the cytoplasmic tail of cadherin-11 are required for cell migration and invasion, but not spreading. Gene array analyses showed that several invasion-related genes, including MMP-7 and MMP-15, are upregulated in cadherin-11–expressing, but not in cad11-ΔJMD–expressing or cad11-ΔCBS–expressing, C4-2B4 cells. These observations suggest that cadherin-11 not only provides a physical link between PCa cells and osteoblasts but also increases PCa cell motility and invasiveness that may facilitate the metastatic colonization of PCa cells in bone. Cancer Res; 70(11); 4580–9. ©2010 AACR.

Osteoblast cadherin (OB-cadherin, also known as cadherin-11) is a Ca 2+ -dependent homophilic cell adhesion molecule that is expressed mainly in osteoblasts. OB-cadherin is expressed in prostate cancer and may be involved in the homing of metastatic prostate cancer cells to bone. The extracellular domain of OB-cadherin may be used to inhibit the adhesion between prostate cancer cells and osteoblasts. In this report, we describe the expression of the extracellular domain of OB-cadherin as an Fc fusion protein (OB-CAD-Fc) in human embryonic kidney 293FT cells using a bicistronic retroviral vector. Coexpression of GFP and OB-CAD-Fc through the bicistronic vector permitted enrichment of OB-CAD-Fc-expressing cells by fluorescence-activated cell sorting. Recombinant OB-CAD-Fc proteins were secreted into cell medium, and about 0.85 mg of purified OB-CAD-Fc protein was purified from 1 l of the conditioned medium using immobilized protein A-affinity chromatography. The purified OB-CAD-Fc was biologically active because it supported the adhesion of PC3 cells and L cells transduced with OB-cadherin. The availability of OB-CAD-Fc offers opportunities to test whether OB-CAD-Fc can be used to inhibit OB-cadherin-mediated prostate cancer bone metastasis in vivo or to generate antibodies for inhibiting the adhesion between prostate cancer cells and osteoblasts.

The propensity for prostate cancer to metastasize to bone led us and others to propose that bidirectional interactions between prostate cancer cells and bone are critical for the preferential metastasis of prostate cancer to bone. We identified previously a secreted isoform of ErbB3 (p45-sErbB3) in bone marrow supernatant samples from men with prostate cancer and bone metastasis and showed by immunohistochemical analysis of human tissue specimens that p45-sErbB3 was highly expressed in metastatic prostate cancer cells in bone. Here, we show that p45-sErbB3 stimulated mouse calvaria to secrete factors that increased the invasiveness of prostate cancer cells in a Boyden chamber invasion assay. Using gene array analysis to identify p45-sErbB3–responsive genes, we found that p45-sErbB3 up-regulated the expression of osteonectin/SPARC, biglycan, and type I collagen in calvaria. We further show that recombinant osteonectin increased the invasiveness of PC-3 cells, whereas osteonectin-neutralizing antibodies blocked this p45-sErbB3–induced invasiveness. These results indicate that p45-sErbB3 enhances the invasiveness of PC-3 cells in part by stimulating the secretion of osteonectin by bone. Thus, p45-sErbB3 may mediate the bidirectional interactions between prostate cancer cells and bone via osteonectin. [Cancer Res 2007;67(14):6544–8]

In diabetes, the death of insulin-producing β-cells by apoptosis leads to insulin deficiency. The lower prevalence of diabetes in females suggests that female sex steroids protect from β-cell injury. Consistent with this hypothesis, 17β-estradiol (estradiol) manifests antidiabetic actions in humans and rodents. In addition, estradiol has antiapoptotic actions in cells that are mediated by the estrogen receptor-a (ERα), raising the prospect that estradiol antidiabetic function may be due, in part, to a protection of β-cell apoptosis via ERα. To address this question, we have used mice that were rendered estradiol-deficient or estradiol-resistant by targeted disruption of aromatase (ArKO) or ERα (αERKO) respectively. We show here that in both genders, ArKO −/− mice are vulnerable to β-cell apoptosis and prone to insulin-deficient diabetes after exposure to acute oxidative stress with streptozotocin. In these mice, estradiol treatment rescues streptozotocin-induced β-cell apoptosis, helps sustain insulin production, and prevents diabetes. In vitro , in mouse pancreatic islets and β-cells exposed to oxidative stress, estradiol prevents apoptosis and protects insulin secretion. Estradiol protection is partially lost in β-cells and islets treated with an ERα antagonist and in αERKO islets. Accordingly, αERKO mice are no longer protected by estradiol and display a gender nonspecific susceptibility to oxidative injury, precipitating β-cell apoptosis and insulin-deficient diabetes. Finally, the predisposition to insulin deficiency can be mimicked in WT mice by pharmacological inhibition of ERα by using the antagonist tamoxifen. This study demonstrates that estradiol, acting, at least in part, through ERα, protects β-cells from oxidative injury and prevents diabetes in mice of both genders.

BETA2 (NeuroD1) is a member of the basic helix-loop-helix transcription factor family. BETA2 plays an important role in the development of the pancreas and the nervous system. Using microarray technology, we identified neuronatin (Nnat) as differentially expressed between wild-type (WT) and knockout (KO) pancreatic RNA from embryonic day 14 (e14.5). NNAT is a member of the proteolipid family of amphipathic polypeptides and is believed to be involved in ion channel transport or channel modulation. Northern blot and in situ hybridization analysis of WT and KO samples confirmed the downregulation of Nnat in pancreas of mutant BETA2 embryos. Chromatin immunoprecipitation and gel shift assays were performed and demonstrated the presence of BETA2 on the Nnat promoter, thus confirming the direct transcriptional regulation of Nnat by BETA2. To assess NNAT potential function, we performed knockdown studies by siRNA in NIT cells and observed a reduction in the ability of the NIT cells to respond to glucose. These results suggest for the first time an important role for NNAT in insulin secretion and for proper β-cell function.

Background: Inflammatory breast cancer (IBC) is a rare but very aggressive form of breast cancer. IBC is characterized by nests of tightly aggregated cells, defined as tumor emboli, that exhibit characteristics of cancer stem cells (CSCs). IBC tumor emboli express E-cadherin which is required to maintain their integrity and our recent evidence demonstrates that expression of E-cadherin by tumor emboli is associated with lack of ZEB1 expression, a transcriptional repressor of E-cadherin. This is at odds with the current hypothesis that metastatic progression is associated with the process of epithelial mesenchymal transition (EMT), with loss of E-cadherin and gain of transcription factors including ZEB1, acquisition of CSC characteristics and enhanced invasive capabilities. Materials and Methods: shRNA knockdown and over-expression methods, real time PCR arrays, western blotting, and in vitro assays to evaluate proliferation, invasion, growth in soft agar and clonogenicity and in vivo animal studies were used. Results: Expression of E-cadherin was reduced by shRNA and ZEB1 was expressed in SUM149 IBC tumor cells. Numerous EMT-related genes were upregulated with loss of E-cadherin and gain of ZEB1, including N-cadherin and vimentin. However, there were marginal differences in the in vitro parameters of proliferation, Matrigel invasion and anchorage independent growth in soft agar between SUM149-shECad or SUM149-ZEB1 clones and their respective vector control cells. The loss of E-cadherin and gain of ZEB1 altered the morphology of SUM149 cells when cultured under low adherence conditions permissive for the enrichment of CSC, exhibiting a reversion in grape-like morphology to more well defined spheres, which was accompanied by increased clonogenicity in both SUM149-shECad and SUM149-ZEB1 cells. The loss of E-cadherin and the gain of ZEB1 significantly inhibited tumor growth of cells injected in the mammary fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumor volume at 56 days for E-cadherin vector control cells was 771.9 mm3 +/− 185.6 compared to shECadherin tumors, which was 13.6 mm3 +/− 7.2. Tumor volume of ZEB1 vector control tumors was 346.1 mm3 +/− 96 compared to volume of ZEB1 expressing tumors, which was 21.5 mm3 +/− 7.2.Conclusions: E-cadherin with lack of ZEB1 expression in IBC is consistent with a mesenchymal-epithelial transition (MET), consistent with the retention of the epithelial phenotype while maintaining a program of rapid metastasis and colonization of lymph nodes and distant organ sites. Furthermore, we demonstrate that the E-cadherin-ZEB1 axis is critical for the in vivo growth of IBC tumor cells. Although SUM149 cells are fully capable of undergoing an EMT process, which is under negative regulation by E-cadherin, the process of EMT does not drive in vivo tumor growth in IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-03.

Background: Inflammatory breast cancer (IBC) is the most aggressive and deadly form of breast cancer and is phenotypically distinct from other forms of locally advanced breast cancer. The 5-year survival for IBC patients (40%) has not improved even with the implementation of multi-disciplinary care and targeted therapies (e.g. anti-HER2 or anti-estrogen therapies). Lymph node involvement is common at first diagnosis and the accelerated metastasis program that characterizes IBC account for a disproportionate number of the 40,000 women who will die from breast cancer. The most common molecular marker for IBC is the cell adhesion surface glycoprotein, E-Cadherin. Classically, E-Cadherin is thought to be a tumor suppressor and its loss is associated with the epithelial-mesenchymal transition (EMT) and acquisition of an invasive phenotype. The importance of EMT in metastasis is well accepted in numerous tumor models although the presence of EMT in patient samples is rarely demonstrated. Given the propensity of IBC to metastasize, one would expect a strong EMT signature in IBC cell lines, however the persistent robust expression of E-Cadherin in IBC patient tumors and in IBC cell lines, with a lack of expression of ZEB1, a transcriptional repressor of E-cadherin and diminished expression of genes within the transforming growth factor beta signaling pathway, suggests otherwise. Methods: To further define the role of E-Cadherin in IBC, we used the SUM149 IBC cell line to manipulate E-Cadherin via shRNA knockdown and by overexpression of ZEB1 a known transcriptional repressor of E-cadherin. Results: While the lack of E-Cadherin or ZEB1 overexpression in SUM149 cells led to an EMT phenotype in vitro, E-Cadherin was required for in vivo growth of SUM149 tumor cells. Gene profiling identified novel E-Cadherin signaling pathways including an hypoxia gene signature through HIF1a as well as cytokine responsive pathways. The requirement of HIF-1a for in vivo growth of SUM149 cells was confirmed by shRNA knockdown of HIF-1a. Conclusions: The absolute requirement for E-Cadherin and maintenance of the mesenchymal-epithelial transition in IBC tumor growth is demonstrated. Furthermore, a novel functional link between E-Cadherin and HIF-1a and in their critical role in survival of IBC tumors is identified for the first time and warrants further investigation. Supported in part by The Susan G Komen Organization Promise Grant KG081287 (FMR and MC) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-05.

We and others have previously identified functional estrogen (E) and retinoic acid (RA) response elements in the human and rat oxytocin (OT) gene promoters. Whereas there is no direct evidence for a significant role of E or RA in the regulation of rat hypothalamic OT gene expression, we have recently demonstrated that in vivo adminstration of E strongly stimulates uterine OT gene expression. Here, we show that in vivo administration of RA similarly induces a significant increase in uterine OT gene expression. Moreover, we report that the E and RA effects are reproducible in vitro. Using short-term uterine organ explant cultures derived from 18-day pregnant rats, we found that E (50 nM) and RA (0.4 nM) increased OT mRNA levels 5.2- and 3-fold, respectively, suggesting a direct action of these agents on uterine OT gene expression. Finally, we analyzed uterine E and RA receptor gene expression during pregnancy. Using semi-quantitative Northern blot analysis, we found that mRNAs encoding the E receptor, the RA receptor α and RA receptor β are present in rat uterus and that their levels rise by 3.7-, 3.6- and 5.8-fold, respectively, between day 14 of gestation and term. Taken together, the data suggest that, at term, the rat uterus has an increased capacity to respond to E and RA, and that both agents may be involved in mediating the dramatic increase of OT mRNA accumulation observed in the uterus at term.

Publisher Summary The posterior pituitary contains a strong uterotonic activity known as the nonapeptide oxytocin (OT). It has been determined that uterus itself represents a major site of OT production. Therefore, the activation of uterine contractions by circulating OT is only one aspect of OT action. A significant portion of OT activity originates from within the uterus and, therfore, acts via autocrine and paracrine mechanisms. This chapter provides an overview of the role of circulating oxytocin and its receptors. The uterus contains its own intrinsic OT system. The ligand and its cognate receptor are both expressed in the same organ and, to some extent, in the same cells. The experiments described in the chapter showed that the genes for the receptor and its cognate ligand were subject to strict and very dramatic regulation during gestation and also, to a lesser extent, during the estrous cycle.

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast can[[Unsupported Character - Codename ­]]cer and it is associated with a poor prognosis in spite of appropriate multidisciplinary treatments. The disease is characterized by peculiar molecular and clinical features and usually affects younger patients, mostly under the age of 50 years at diagnosis. IBC has shown the capacity to spread early, primarily through lymphatic channels and secondarily through blood vessels causing the inflammatory signs and development of early metastasis. The high incidence of metastatic disease at presentation, the persistence of residual disease after induction chemotherapy associated with significant risk of disease recurrence immediately surgery strongly support our hypothesis that these patients develop micro metastatic disease early in the disease course (Cristofanilli et al., Cancer 2007;110:1436-44). Moreover, the lower survival rate of IBC patients may be due to the early activation of metastatic process associated with a peculiar phenotype of cancer stem cells (CSCs) (Cristofanilli et al., Oncologist 2003;8:141-8). Although IBC research has been ongoing for over 15 years, very few molecular alterations have been associated specifically with IBC and few preclinical models are currently available to evaluate the peculiar biology of IBC. The majority of IBC studies have been performed using the cell lines SUM149 and SUM 190 which were developed from the primary tumor and, KPL-4 isolated from the pleural effusion of IBC patients. Although both the SUM149 and KPL-4 injected into immuno- compromised mice form primary tumors, there are currently only two in vivo xenograft models of IBC, the Mary-X and the WIBC-9 models that recapitulate the tumor emboli that are the signature of IBC in humans. We have developed another IBC model, FC-IBC-02, derivate from the pleural effusion of a 49 years old IBC triple negative patient. Tumor cells from the pleural effusion were ER(-) Pgr(-) and Her2(-) and strongly positive for tetraspanin CD151 and E-cadherin. Cells from the pleural effusion of this patient were growth under non-adherent conditions in serum-free mammary epithelial growth medium (MEGM). These tumor spheroids have Aldhehyde Dehydrogenase 1 (ALDH-1) activity and characteristics of cancer stem cells (CSC) and they rapidly developed tumors when they were injected in the mammary fat pad of SCID mice. These tumors were poorly differentiated carcinomas ER-negative PgR-negative Her2-negative and the mice developed micro metastasis in the lungs. In contrast, cells isolated under adherent conditions were unable to produce tumor in SCID mice. In summary, our studies demonstrated that IBC is a disease enriched for cells with a stem cell phenotype and these cells are highly tumorigenic. IBC may represent an ideal model to evaluate stem cell targeting therapeutic modalities. 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 5342. doi:1538-7445.AM2012-5342

Background: Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer. It is associated with a poor survival rate (40% 5-year survival) despite appropriate multidisciplinary care. For such an aggressive type of cancer, IBC has been understudied, in part due to the lack of adequate numbers of cell lines and mouse models that recapitulate the human disease. To expand our understanding of IBC, we have obtained all of the previously developed and characterized IBC cell lines and models including Mary-X, SUM149, SUM190, KPL-4, MDA-IBC-3 and have developed two new IBC models, designated as FC-IBC01 and FC-IBC02, using tumor cells derived from pleural effusion of IBC patients. Materials and Methods: Each of these IBC cell lines has been luciferase (LUC)-tagged, allowing the growth of orthotopic injection or subcutaneous implantation to be evaluated by bioluminescent imaging (BLI). Alternatively, the LUC-tagged IBC cells can by injected via either intra-cardiac or intravenous route of delivery, which promotes rapid tumor colonization, resulting in both visceral and skeletal metastasis. Growth of IBC tumors can then be monitored immediately using BLI, thus eliminating the lag time needed for the physical detection of palpable tumors. BLI imaging also allows for monitoring of the kinetics and location of development of metastatic lesions. Whole transcriptome analysis was performed on IBC cell lines and xenograft tissues to define the heterogeneity of IBC as a distinct variant of breast cancer Results: These models have allowed us to identify micro-metastatic foci in multiple sites distant from the IBC primary tumor in each of these models of IBC and allow the quantitation of anti-tumor and anti-metastatic effects of targeted therapeutics as single agents as well as the potential synergy of combinations of agents. As an example, injection of LUC-tagged IBC cell lines such as SUM149-Luc, into the left ventricle of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice allows the metastatic tumor burden to be monitored longitudinally by whole animal BLI, which can be validated at necropsy and by immunohistochemical analysis. Whole transcriptome analysis of pre-clinical models of IBC reflect the molecular subtypes observed in IBC patients, with the majority of IBC models being of the basal like, luminal B and Her2 amplified. Discussion: First time analysis of known and newly developed pre-clinical models of IBC allows a more complete analysis of IBC as a distinct variant of breast cancer. Furthermore, these approaches allow rapid evaluation of the promising targeted therapeutics identified based on whole transcriptome analysis of both IBC patient tumors and pre-clinical models developed from IBC patients. We believe that this extensive collection of LUC-tagged IBC cell lines is an invaluable tool for IBC research since the cell lines encompass the broad spectrum of IBC heterogeneity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-06.