[Show abstract][Hide abstract] ABSTRACT: We previously identified dermicidin (DCD), which encodes a growth and survival factor, as a gene amplified and overexpressed in a subset of breast tumors. Patients with DCD-positive breast cancer have worse prognostic features. We therefore searched for specific molecular signatures in DCD-positive breast carcinomas from patients and representative cell lines.
DCD expression was evaluated by qRT-PCR, immunohistochemical and immunoblot assays in normal and neoplastic tissues and cell lines. To investigate the role of DCD in breast tumorigenesis, we analyzed the consequences of its downregulation in human breast cancer cell lines using three specific shRNA lentiviral vectors. Genes up- and down-regulated by DCD were identified using Affymetrix microarray and analyzed by MetaCore Platform.
We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines. DCD expression in breast tumors from patients with clinical follow up data correlated with high histological grade, HER2 amplification and luminal subtype. We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice. Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands.
These findings imply that DCD promotes breast tumorigenesis via modulation of ERBB signaling pathways. As ERBB signaling is also important for neural survival, HER2+ breast tumors may highjack DCD's neural survival-promoting functions to promote tumorigenesis.
[Show abstract][Hide abstract] ABSTRACT: Introduction:
There are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized.
Microarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers.
Analysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification.
BCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.
Full-text · Article · Sep 2015 · Breast cancer research: BCR
[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) is an incurable neoplasm caused by proliferation of malignant plasma cells in the bone marrow (BM). MM is characterized frequently by a complete or partial deletion of chromosome 13q14, seen in more than 50% of patients at diagnosis. Within this deleted region the tripartite motif containing 13 (TRIM13, also termed RFP2) gene product has been proposed to be a tumour suppressor gene (TSG). Here, we show that low expression levels of TRIM13 in MM are associated with chromosome 13q deletion and poor clinical outcome. We present a functional analysis of TRIM13 using a loss-of-function approach, and demonstrate that TRIM13 downregulation decreases tumour cell survival as well as cell cycle progression and proliferation of MM cells. In addition, we provide evidence for the involvement of TRIM13 downregulation in inhibiting the NF kappa B pathway and the activity of the 20S proteasome. Although this data does not support a role of TRIM13 as a TSG, it substantiates important roles of TRIM13 in MM tumour survival and proliferation, underscoring its potential role as a novel target for therapeutic intervention.
No preview · Article · May 2013 · British Journal of Haematology
[Show abstract][Hide abstract] ABSTRACT: Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116). Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.
[Show abstract][Hide abstract] ABSTRACT: Deregulated Wnt/β-catenin signaling underlies the pathogenesis of a broad range of human cancers, yet the development of targeted therapies to disrupt the resulting aberrant transcription has proved difficult because the pathway comprises large protein interaction surfaces and regulates many homeostatic functions. Therefore, we have directed our efforts toward blocking the interaction of β-catenin with B cell lymphoma 9 (BCL9), a co-activator for β-catenin-mediated transcription that is highly expressed in tumors but not in the cells of origin. BCL9 drives β-catenin signaling through direct binding mediated by its α-helical homology domain 2. We developed a stabilized α helix of BCL9 (SAH-BCL9), which we show targets β-catenin, dissociates native β-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and exhibits mechanism-based antitumor effects. SAH-BCL9 also suppresses tumor growth, angiogenesis, invasion, and metastasis in mouse xenograft models of Colo320 colorectal carcinoma and INA-6 multiple myeloma. By inhibiting the BCL9-β-catenin interaction and selectively suppressing oncogenic Wnt transcription, SAH-BCL9 may serve as a prototype therapeutic agent for cancers driven by deregulated Wnt signaling.
Full-text · Article · Aug 2012 · Science translational medicine
[Show abstract][Hide abstract] ABSTRACT: Bruton tyrosine kinase (Btk) has a well-defined role in B-cell development, whereas its expression in osteoclasts (OCs) further suggests a role in osteoclastogenesis. Here we investigated effects of PCI-32765, an oral and selective Btk inhibitor, on osteoclastogenesis as well as on multiple myeloma (MM) growth within the BM microenvironment. PCI-32765 blocked RANKL/M-CSF-induced phosphorylation of Btk and downstream PLC-γ2 in OCs, resulting in diminished TRAP5b (ED50 = 17 nM) and bone resorption activity. PCI-32765 also inhibited secretion of multiple cytokines and chemokines from OC and BM stromal cell cultures from both normal donors (ED50 = 0.5 nM) and MM patients. It decreased SDF-1-induced migration of MM cells, and down-regulated MIP1-α/CCL3 in MM cells. It also blocked MM cell growth and survival triggered by IL-6 or coculture with BM stromal cells or OCs in vitro. Importantly, PCI-32765 treatment significantly inhibits in vivo MM cell growth (P < .03) and MM cell-induced osteolysis of implanted human bone chips in SCID mice. Moreover, PCI-32765 prevents in vitro colony formation by stem-like cells from MM patients. Together, these results delineate functional sequelae of Btk activation mediating osteolysis and growth of MM cells, supporting evaluation of PCI-32765 as a novel therapeutic in MM.
[Show abstract][Hide abstract] ABSTRACT: Cytotoxic chemotherapy targets elements common to all nucleated human cells, such as DNA and microtubules, yet it selectively
kills tumor cells. Here we show that clinical response to these drugs correlates with, and may be partially governed by, the
pretreatment proximity of tumor cell mitochondria to the apoptotic threshold, a property called mitochondrial priming. We
used BH3 profiling to measure priming in tumor cells from patients with multiple myeloma, acute myelogenous and lymphoblastic
leukemia, and ovarian cancer. This assay measures mitochondrial response to peptides derived from proapoptotic BH3 domains
of proteins critical for death signaling to mitochondria. Patients with highly primed cancers exhibited superior clinical
response to chemotherapy. In contrast, chemoresistant cancers and normal tissues were poorly primed. Manipulation of mitochondrial
priming might enhance the efficacy of cytotoxic agents.
[Show abstract][Hide abstract] ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
Full-text · Article · Nov 2011 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma is characterized by frequent chromosomal alterations. Deletion of chr 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. Here, we functionally validate the role of the microRNAs-15a/16-1 cluster, centered at the deleted region, as TSGs and delineate their downstream target genes in MM. Using "sponge" lentiviral vectors to competitive stably inhibit mature microRNAs in vitro and in vivo, we have documented enhanced proliferative and invasive capacity of cells with stably inhibition of miR-16. Importantly, miR-16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load and host angiogenesis. Expression profiling analysis of miR-16-deficient cells identified a large number of downstream target genes including FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. We validated designated genes showing binding sites within the conserved 3'-UTR and also within the mRNA coding region as direct miR-16 targets, thus indicating that the miRNAs may have many more targets than anticipated by conventional prediction methods. This loss-of-function system, which mimics the 13q chromosomal deletion, provides a valuable tool to investigate their function in MM pathogenesis and their potential use as therapeutic targets.
[Show abstract][Hide abstract] ABSTRACT: The PIK3 signaling pathway has been identified as one of the most important and most frequently mutated pathways in breast cancer. Somatic mutations in the catalytic subunit of PIK3CA have been found in a significant fraction of breast carcinomas, and it has been proposed that mutant PIK3CA plays a role in tumor initiation. However, the majority of primary human tumors analyzed for genetic alterations in PIK3CA have been invasive breast carcinomas and the frequency of PIK3CA mutations in preinvasive lesions has not been explored. To investigate this, we sequenced exons 9 and 20 of PIK3CA in pure ductal carcinoma in situ (DCIS), DCIS adjacent to invasive carcinoma, and invasive ductal breast carcinomas. In a subset of cases, both in situ and invasive areas were analyzed from the same tumor. We found that the frequency of PIK3CA mutations was essentially the same ( approximately 30%) in all three histologic groups. In some cases, in situ and invasive areas of the same tumor were discordant for PIK3CA status, and in two cases in which multiple invasive and adjacent in situ areas within the same tumor were analyzed independently, we detected intratumor heterogeneity for PIK3CA mutations. Our results suggest that mutation of PIK3CA is an early event in breast cancer that is more likely to play a role in breast tumor initiation than in invasive progression, although a potential role for exon 9 mutations in the progression of a subset of DCIS cases cannot be excluded.
[Show abstract][Hide abstract] ABSTRACT: Constitutive B-cell lymphoma 6 (Bcl-6) expression was undetectable in multiple myeloma (MM) cell lines, except U266 cells. However, it was up-regulated by coculture with bone marrow (BM) stromal cell-culture supernatant (SCCS). Bcl-6 expression in patient MM cells in the BM was positive. Anti-interleukin-6 (IL-6)-neutralizing antibody significantly blocked SCCS-induced Bcl-6 in MM cells. Indeed, IL-6 strongly triggered Bcl-6 expression in MM cells, whereas Janus kinase inhibitor and STAT3 siRNA down-regulated Bcl-6. Tumor necrosis factor-alpha (TNF-alpha) also triggered Bcl-6, but independently of STAT3, whereas IkappaB kinasebeta inhibitor down-regulated TNF-alpha-induced Bcl-6, indicating that the canonical nuclear factor-kappaB pathway mediates TNF-alpha-induced Bcl-6 expression. Importantly, down-regulation of Bcl-6 by shRNA significantly inhibited MM cell growth in the presence of SCCS. Our results therefore suggest that Bcl-6 expression in MM cells is modulated, at least in part, via Janus kinase/STAT3 and canonical nuclear factor-kappaB pathways and that targeting Bcl-6, either directly or via these cascades, inhibits MM cell growth in the BM milieu.
[Show abstract][Hide abstract] ABSTRACT: Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as in vitro and in vivo functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances beta-catenin-mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling.
[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) is a cancer of plasma cells with complex molecular characteristics that evolves from monoclonal gammopathy of undetermined significance, a highly prevalent premalignant condition. MM is the second most frequent hematologic cancer in the United States, and it remains incurable, thereby highlighting the need for new therapeutic approaches, particularly those targeting common molecular pathways involved in disease progression and maintenance, shared across different MM subtypes. Here we report that Wnt/beta-catenin is one such pathway. We document the involvement of beta-catenin in cell-cycle regulation, proliferation, and invasion contributing to enhanced proliferative and metastatic properties of MM. The pleiotropic effects of beta-catenin in MM correlate with its transcriptional function, and we demonstrate regulation of a novel target gene, Aurora kinase A, implicating beta-catenin in G2/M regulation. beta-catenin and Aurora kinase A are present in most MM but not in normal plasma cells and are expressed in a pattern that parallels progression from monoclonal gammopathy of undetermined significance to MM. Our data provide evidence for a novel functional link between beta-catenin and Aurora kinase A, underscoring a critical role of these pathways in MM disease progression.
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of malignant melanoma involves the interplay of tumor cells with normal host elements, but the underlying mechanisms are incompletely understood. Here, we show that milk fat globule EGF-8 (MFG-E8), a secreted protein expressed at high levels in the vertical growth phase of melanoma, promotes disease progression through coordinated alpha(v)beta(3) integrin signaling in the tumor microenvironment. In a murine model of melanoma, MFG-E8 enhanced tumorigenicity and metastatic capacity through Akt-dependent and Twist-dependent pathways. MFG-E8 augmented melanoma cell resistance to apoptosis, triggered an epithelial-to-mesenchymal transition (EMT), and stimulated invasion and immune suppression. In human melanoma cells, MFG-E8 knockdown attenuated Akt and Twist signaling and thereby compromised tumor cell survival, EMT, and invasive ability. MFG-E8-deficient human melanoma cells also showed increased sensitivity to small molecule inhibitors of insulin-like growth factor I receptor and c-Met. Together, these findings delineate pleiotropic roles for MFG-E8 in the tumor microenvironment and raise the possibility that systemic MFG-E8 blockade might prove therapeutic for melanoma patients.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the expression of cathepsin K (CTSK) and CXCL14 in stromal and epithelial cells in human breast tumor progression.
We did immunohistochemical analyses of CTSK and CXCL14 expression in normal breast tissue, biopsy sites, benign lesions, ductal carcinoma in situ, and invasive breast tumors of different stages. Expression patterns were related to histopathologic characteristics of the tumors and clinical outcome. The effect of CTSK+ breast stromal fibroblasts on CTSK- breast cancer cells was assessed in coculture.
Epithelial expression of CTSK was rarely detected in any of the tissue samples analyzed, whereas CXCL14-positive epithelial cells were found in all tissue types. The expression of CXCL14 was not associated with any tumor or patient characteristics analyzed. Stromal CTSK expression was significantly higher in invasive compared with in situ carcinomas, and in one of the two data sets analyzed, it correlated with higher tumor stage. Among all samples examined, the highest stromal CTSK levels were detected in biopsy sites. Neither epithelial nor stromal expression of CTSK was significantly associated with recurrence-free or overall survival. Coculture of CTSK+ fibroblasts enhanced the invasion of CTSK- breast tumor epithelial cells and this was blocked by CTSK inhibitors.
CTSK may function as a paracrine factor in breast tumorigenesis. CTSK+ fibroblasts may play a role in tumor progression by promoting the invasiveness of tumor epithelial cells. The possibility that CTSK inhibitors may have a clinical role in decreasing the risk of tumor progression merits further investigation.
Full-text · Article · Oct 2008 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a poorly understood key event in breast tumor progression. Here, we analyzed the role of myoepithelial cells and fibroblasts in the progression of in situ carcinomas using a model of human DCIS and primary breast tumors. Progression to invasion was promoted by fibroblasts and inhibited by normal myoepithelial cells. Molecular profiles of isolated luminal epithelial and myoepithelial cells identified an intricate interaction network involving TGFbeta, Hedgehog, cell adhesion, and p63 required for myoepithelial cell differentiation, the elimination of which resulted in loss of myoepithelial cells and progression to invasion.