Life Sciences Commons^™

Brca1 And Brca2 5′ Noncoding Region Variants Identified In Breast Cancer Patients Alter Promoter Activity And Protein Binding, Leslie J. Burke, Jan Sevcik, Gaetana Gambino, Emma Tudini, Eliseos J. Mucaki, Ben C. Shirley, Phillip Whiley, Michael T. Parsons, Kim De Leeneer, Sara Gutiérrez-Enríquez, Marta Santamariña, Sandrine M. Caputo, Elizabeth Santana Dos Santos, Jana Soukupova, Marketa Janatova, Petra Zemankova, Klara Lhotova, Lenka Stolarova, Mariana Borecka, Alejandro Moles-Fernández, Siranoush Manoukian, Bernardo Bonanni, Stacey L. Edwards, Marinus J. Blok, Thomas Van Overeem Hansen, Maria Rossing, Orland Diez, Ana Vega, Kathleen B.M. Claes, David E. Goldgar, Etienne Rouleau

Biochemistry Publications

© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc. The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5′ noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C>T and PAX5 binding to BRCA2:c.-296C …

Lung-Derived Selectins Interact With Cd44 And Enhance The Migration Of Breast Cancer Cells, Sami U. Khan

Electronic Thesis and Dissertation Repository

The lung is among the deadliest sites of breast cancer metastasis. Previous work from our laboratory demonstrated that aggressive CD44-expressing breast cancer cells preferentially metastasize to the lung in vivo, and observed the presence of multiple CD44-interacting proteins in the lung including E-, L- and P-selectin. We hypothesized that lung-derived selectins promote breast cancer migration and/or growth via interactions with CD44. Using an ex vivo model of the soluble lung-microenvironment, we demonstrate that lung-derived selectins enhance in vitro migration but not proliferation of breast cancer cells. Co-immunoprecipitation experiments reveal that CD44 expressed by breast cancer cells in vitro interacts …

Proteomic Characterization Of Ovarian And Breast Cancer Microenvironments For Improved Diagnostics And Therapeutic Targeting, Dylan Z. Dieters-Castator

Electronic Thesis and Dissertation Repository

Cancers exist within complex microenvironments formed by heterogeneous cell types. This diversity creates significant challenges for detection, diagnosis and treatment. Mass spectrometry-based proteomics is a powerful approach capable of characterizing complex biological systems which are characteristic of cancer biology. In this thesis, proteomics was utilized to answer several questions related to ovarian cancer diagnosis and detection, and the effects of NODAL, an embryonic morphogen, on the breast cancer secretome and stromal cell recruitment. First, I compared multiple sample preparation techniques and found high-pH/low-pH fractionation to yield the greatest proteome coverage over commonly used approaches. Second, I compared the proteomes from …

Influence Of The Metastatic Microenvironment On Stem-Like Human Breast Cancer Cells, Ashkan Sadri

Electronic Thesis and Dissertation Repository

Ninety percent of breast cancer-related mortalities result from metastasis. We have previously demonstrated that stem-like ALDHhiCD44+ breast cancer cells are critical for metastasis, and preferentially target the lung and bone marrow (BM). We hypothesize organ tropism occurs through promotion of the ALDHhiCD44+ phenotype. Using a 2D ex vivo model, lung and BM conditioned media (CM) were utilized to assess their influence on stem-like phenotype and behavior. Exposure of human breast cancer cells to lung-CM significantly decreased the proportion of cells with a stem-like ALDHhiCD44+ phenotype, decreased expression of cancer stem cell (CSC)-related genes, and increased gene expression related to migration …

Predicting Outcomes Of Hormone And Chemotherapy In The Molecular Taxonomy Of Breast Cancer International Consortium (Metabric) Study By Biochemically-Inspired Machine Learning, Peter Rogan, Eliseos J. Mucaki, Katherina Baranova, Huy Q. Pham, Iman Rezaeian, Dimo Angelov, Alioune Ngom, Luis Rueda

Biochemistry Publications

Genomic aberrations and gene expression-defined subtypes in the large METABRIC patient cohort have been used to stratify and predict survival. The present study used normalized gene expression signatures of paclitaxel drug response to predict outcome for different survival times in METABRIC patients receiving hormone (HT) and, in some cases, chemotherapy (CT) agents. This machine learning method, which distinguishes sensitivity vs. resistance in breast cancer cell lines and validates predictions in patients; was also used to derive gene signatures of other HT (tamoxifen) and CT agents (methotrexate, epirubicin, doxorubicin, and 5-fluorouracil) used in METABRIC. Paclitaxel gene signatures exhibited the best performance, …

First-Order Statistical Speckle Models Improve Robustness And Reproducibility Of Contrast-Enhanced Ultrasound Perfusion Estimates, Matthew R. Lowerison

Electronic Thesis and Dissertation Repository

Contrast-enhanced ultrasound (CEUS) permits the quantification and monitoring of adaptive tumor responses in the face of anti-angiogenic treatment, with the goal of informing targeted therapy. However, conventional CEUS image analysis relies on mean signal intensity as an estimate of tracer concentration in indicator-dilution modeling. This discounts additional information that may be available from the first-order speckle statistics in a CEUS image. Heterogeneous vascular networks, typical of tumor-induced angiogenesis, lead to heterogeneous contrast enhancement of the imaged tumor cross-section.

To address this, a linear (B-mode) processing approach was developed to quantify the change in the first-order speckle statistics of B-mode cine …

Mt1-Mmp Mediates The Migratory And Tumourigenic Potential Of Breast Cancer Cells Via Non-Proteolytic Mechanisms, Mario Cepeda

Electronic Thesis and Dissertation Repository

Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease that affects cell function via proteolytic and non-proteolytic mechanisms such as promoting degradation of the extracellular matrix (ECM) or augmentation of cell migration and viability, respectively. MT1-MMP has been implicated in metastatic progression ostensibly due to its ability to degrade ECM components and to allow migration of cells through the basement membrane. Despite in vitro studies demonstrating this principle, this knowledge has not translated into the use of MMP inhibitors (MMPi) that inhibit substrate catalysis as effective cancer therapeutics, or been corroborated by evidence of in vivo ECM degradation mediated by …

Generation Of Organ-Conditioned Media And Applications For Studying Organ-Specific Influences On Breast Cancer Metastatic Behavior, Matthew M. Piaseczny, Graciella M. Pio, Jenny E. Chu, Ying Xia, Kim Nguyen, David Goodale, Alison Allan

Anatomy and Cell Biology Publications

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. …

The Role Of Connexins And Pannexins In Mammary Gland Development And Tumorigenesis, Michael K. G. Stewart

Electronic Thesis and Dissertation Repository

The identification of key regulators of breast cancer onset and progression is critical for the development of targeted therapies. Connexins and pannexins are characterized by their ability to form large-pore channels and are frequently dysregulated in cancer. However, their role in breast cancer progression remains poorly understood due to a lack of in vivo models capable of assessing the proposed and opposing roles of connexins and pannexins as both tumor suppressors and/or facilitators in multiple stages of the disease. Using 2 previously uncharacterized genetically-modified mice, connexin43 (Cx43) and connexin26 (Cx26) were evaluated for their role in normal mammary gland development …

The Lung Microenvironment Influences The Metastatic Behaviour Of Breast Cancer Cells In An Innovative 3d Ex Vivo Pulmonary Metastasis Model, Matt M. Piaseczny

Electronic Thesis and Dissertation Repository

Lung metastasis remains a leading cause of death in breast cancer patients. This study established an innovative 3D ex vivo pulmonary metastasis assay (PuMA) to test the hypothesis that the lung microenvironment promotes metastatic behaviour of whole population and stem-like ALDH^hiCD44⁺ breast cancer cells. Following in vivo delivery of breast cancer cells to mice, lungs were excised, maintained in culture and imaged to observe breast cancer growth over time. We observed metastatic progression of breast cancer cells in the PuMA, most notably of ALDH^hiCD44⁺ cells which progressed rapidly from single cells to multicellular colonies …

The Role Of Mir-526b In Cox-2 Mediated Human Breast Cancer Progression And Induction Of Stem-Like Phenotype Via Ep4 Receptor Signaling, Erin O. Landman

Electronic Thesis and Dissertation Repository

Our laboratory previously established that aberrant expression of cyclo-oxygenase (COX)-2 promotes breast cancer progression and metastasis via multiple mechanisms, including stem-like cell (SLC) induction, owing to activation of the prostaglandin E2 receptor EP4. COX-2 expression was linked to up-regulation of miRNA-526b. We hypothesized that miR-526b is regulated by EP4 activity, and that miR-526b supports breast cancer progression and induction of SLCs. Using stably miR-526b transfected MCF-7 and SKBR-3 cells in functional assays, including tumorsphere formation in vitro and lung colony formation in vivo, we observed enhanced migration, invasion, proliferation, tumorsphere formation, and in vivo tumorigenecity compared to controls. EP4 …

The Synthesis And Evaluation Of Peptide-Based Probes For The Imaging Of Rhamm Expressing Carcinoma And Glp-1r On Pancreatic Beta Cells, Ashley Esarik

Electronic Thesis and Dissertation Repository

Peptide-based probes are developed into imaging agents to target two different receptors: receptor for HA mediated motility (RHAMM) and glucagon-like peptide 1 receptor (GLP-1R).

In the first project, peptides are derived from tubulin and mimic HA (hyaluronan), the natural ligand of RHAMM. In order to develop the lead candidate peptides into imaging agents, a DOTA chelator which can coordinate a radiometal was coupled onto the lead peptides. Gallium labelling studies were performed and the labelled probes were used in in vivo mouse studies. Various analogues of two of the candidate peptides were also synthesized using bioisosteric, unnatural as well as …

Kiss1r Induces Human Mammary Epithelial Cell Invasiveness And Promotes Breast Cancer Cell Migration And Invasion Via Iqgap1, Donna Cvetkovic

Electronic Thesis and Dissertation Repository

Kisspeptins (KP), peptide products of the kisspeptin-1 (KISS1) gene are the endogenous ligands for a G protein-coupled receptor (KISS1R). KISS1 acts as a metastasis suppressor in numerous human cancers. However, recent studies have demonstrated that an increase in KISS1 and KISS1R expression in human breast tumors correlates with higher tumor grade and metastatic potential. We have previously shown that KP-10, the most potent KP, stimulates invasion of estrogen receptor (ER)-negative breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR). Here, I report that KP-10 treatment of the ER-negative non-malignant mammary epithelial MCF10A cells, or stable …

The Embryonic Protein Nodal Supports Metastatic Phenotypes In Breast Cancer, Daniela F. Quail

Electronic Thesis and Dissertation Repository

Metastasis is the process by which tumour cells disseminate to distant organ sites. Aberrant expression of stem cell-associated proteins within tumours is associated with metastasis and poor patient prognosis. One example of a stem cell factor that is associated with cancer progression is Nodal, a member of the TGF-β superfamily. Nodal is normally limited to pluripotent stem cells during embryonic development, and to specialized dynamic adult tissue (such as the cycling endometrium), but is aberrantly re-expressed in multiple cancer types, including melanoma, glioma, prostate cancer, and pancreatic cancer. The central objective of this thesis is to determine the role of …

High Aldehyde Dehydrogenase And Expression Of Cancer Stem Cell Markers Selects For Breast Cancer Cells With Enhanced Malignant And Metastatic Ability, Alysha K. Croker, David Goodale, Jenny Chu, Carl Postenka, Benjamin D. Hedley, David A. Hess, Alison L. Allan

Anatomy and Cell Biology Publications

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however, the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre-clinical models of metastasis. Therefore, the objective of this study was to develop suitable pre-clinical model systems for studying stem-like cells in breast cancer metastasis, and to test the hypothesis that stem-like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA-MB-435, MDA-MB-231, MDA-MB-468, MCF-7) …