Summer Student Research Program
Project Description
FACULTY SPONSOR’S NAME AND DEGREE: Pranela Rameshwar, PhD
PHONE: (973) 972 - 0625
DEPARTMENT AND INTERNAL MAILING ADDRESS: MSB, Room E-579
E-MAIL: rameshwa@umdnj.edu
PROJECT TITLE (200 Characters max):
miRNA is central to breast cancer quiescence in bone marrow
HYPOTHESIS:
The overarching hypothesis states that breast cancer cells accept specific microRNA (miRNAs)
from bone marrow stroma via gap junctional communication to facilitate their integration close
to the endosteum. The specific hypothesis states that miRNAs-222, -223, -127, -31 and -197
maintain cell cycle checkpoint at G1/S, and reduce the production of membrane and soluble
CXCL12 (SDF-1), reported to facilitate breast cancer cell quiescence in bone marrow (1). The
G2/M phase is achieved by miR27A and survival by decreases in the apoptotic-linked miR-21,15 and -16 (2).
PROJECT DESCRIPTION (Include design, methodology, data collection, techniques, data analysis to be
employed and evaluation and interpretation methodology)
Innovation: This study challenges the paradigm of an absolute direct bone invasion by breast
cancer cells. The study proposes to focus on the biology of BC, prior to bone invasion. The longterm goal is to understand why breast cancer cells, close to the endosteum of bone marrow are
highly resistant to current therapies. The studies are relevant to the early phase of breast cancer,
and also during remission. At both times, we propose that the cancer cells are `hidden’ as noncycling cells within regions close to the endosteum.
The immediate objective is to understand how BCCs adapt a quiescence phenotype in BM by in
vitro methods. The long-term goals will identify methods to `flush’ BCCs from BM for targeting.
Methods: Feasibility: Breast cancer cells and bone marrow marrow stroma form functional gap
junctions (1), which promote the passage of miRNAs (feasibility studies are supported by arrays
of 365 targets). Analyses with miRanda olgarithm, combined with published reports have
selected the proposed miRNAs (2-4).
Experimental Model: An established co-culture method of primary human BM stroma and low
aggressive T47D or MCF7 will serve as the experimental model (1).
Experiment 1: I. What are the changes in the proposed miRNA before and after co-culture? II. Are the
changes due to passage from stroma?
I. At 16 h, the gap junctions will be inhibited with 5 mM 18-glycyrrhetinic acid (GA). Cells
will be de-adhered with acutase and each subset separated with anti-cytokeratin conjugated
magnetic beads. Control cultures will subject the cancer cells to magnetic beads. Total RNA
from each subset will be quantitated for the proposed miRNA by qPCR (5). II. Determine if the
changes in miRNA are due to passage from stroma by repeating the co-culture. At 16 h, gap
junction functions will be inhibited with 5 mM GA, thereby preventing further passage of
miRNA from stroma. Vehicle will be added to controls. At 12-h intervals up to 72 h, miRNA
levels will be quantiated in each cell subset by qPCR.
Experiment 2: Are CXCL12-specific miRNAs passed from stroma to breast cancer cells?
Summer Student Research Program
Project Description
miRanda olgarithym indicate potential binding of the proposed miRNAs to the 3′UTR of
CXCL12. Those increased (Expt 1) will be knockdown with specific anti-miRs in stroma before
co-culture. At 16 h, co-cultured cancer cells will be studied for CXCL12 mRNA by qPCR and
protein by ELISA. For ELISA, the isolated cancer cells will be replated in media with 2% sera.
After 16 h, the culture media will be analyzed by ELISA. We will also determine if the antimiRs prevent gap junction formation by immunocytochemistry and western blots (with
membrane extracts). The proliferative state of breast cancer cells, and their viability (apoptosis
and necrosis) will also be studied.
LITERATURE CITED
Moharita AL, Taborga M, Corcoran KE, Bryan M, Patel PS, Rameshwar P. SDF-1
regulation in breast cancer cells contacting bone marrow stroma is critical for normal
hematopoiesis. Blood 2006;108:3245.
Mertens-Talcott S, Chintharlapalli S, Li X, Safe S. The oncogenic microRNA-27a targets
genes that regulate specificity protein transcription factors and the G2-M checkpoint in
MDA-MB-231 breast cancer cells. Cancer Res 2007;67:11001.
miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA
2005;13944.
MicroRNA-21 is an antiapoptotis factor in human glioblastoma cells. Cancer Res
2005;65:6029.
Greco SJ, Rameshwar P. miRNAs regulate synthesis of the neurotransmitter substance P in
human mesenchymal stem cell-derived neuronal cells. Proc Natl Acad Sci USA
2007;104:15484.
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SPONSOR’S MOST RECENT PUBLICATIONS RELEVANT TO THIS RESEARCH:
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Corcoran KE, Trzaska KA, Fernandes H, Bryan M, Taborga M, Srinivas V, Packman K,
Patel PS, Rameshwar P. (2008) Mesenchymal stem cells in early entry of breast cancer
into bone marrow. Plos ONE 3:e2563.
IS THIS PROJECT SUPPORTED BY EXTRAMURAL FUNDS?
Yes
or
No
(IF YES, PLEASE SUPPLY THE GRANTING AGENCY’S NAME)
Department of Defense
THIS PROJECT IS:
Clinical
Laboratory
Behavioral
Other
THIS PROJECT EMPLOYS RADIOISOTOPES
THIS PROJECT INVOLVES THE USE OF ANIMALS
PENDING
APPROVED
IACUC PROTOCOL #
THIS PROJECT INVOLVES THE USE OF HUMAN SUBJECTS
PENDING
APPROVED
IRB PROTOCOL # M
WHAT WILL THE STUDENT LEARN FROM THIS EXPERIENCE?
The student will learn cellular and laboratory cultures; qPCR; data analyses; reading of literature