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

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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-1a), 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 long-term 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 non-cycling 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 18a-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?

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 anti-miRs 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

1.  Moharita AL, Taborga M, Corcoran KE, Bryan M, Patel PS, Rameshwar P. SDF-1a regulation in breast cancer cells contacting bone marrow stroma is critical for normal hematopoiesis. Blood 2006;108:3245.

2.  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.

3.  miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA 2005;13944.

4.  MicroRNA-21 is an antiapoptotis factor in human glioblastoma cells. Cancer Res 2005;65:6029.

5.  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.

SPONSOR’S MOST RECENT PUBLICATIONS RELEVANT TO THIS RESEARCH:

-  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