Graduation Year

2010

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Pathology and Cell Biology

Major Professor

Jin Q. Cheng, M.D., Ph.D.

Co-Major Professor

Santo V. Nicosia, M.D.

Committee Member

Patricia Kruk, Ph.D.

Committee Member

Domenico Coppola, M.D.

Committee Member

Jerry Wu, Ph.D.

Keywords

MicroRNA, EMT, Apoptosis, TGF-β, Post-transcriptional Regulation

Abstract

Recent statistics reveal breast cancer as the most common cancer among women

and accounts for approximately 41,000 mortalities per year. In diagnosis, features such

as stage, grade, lymph node metastasis are important prognostic indicators that help guide

physicians and oncologist towards optimal patient care. Presence of established

pathological markers such as ER, PR, and Her2/neu status would indicate ideal adjuvant

therapy situation. Although treatment of these types of breast cancer is well established,

cancer that lack all three receptors, “triple negatives” or “basal like” do not respond to

adjuvant therapy and are considered more aggressive in that patients tend to recur early

and experience visceral metastasis. Although scientists have uncovered numerous

molecular biology mechanisms in search of an understanding in cancer, leading to

development of fields such as apoptosis or growth pathways; cell cycle; angiogenesis;

metastasis; and more recently cancer stem cells, much work remains as cancer is still not

eradicated.

MicroRNAs (miRNAs) are post transcriptional regulators of gene expression.

Their discovery and functional understanding have only been uncovered in the past ten

years. Long pri-miRNAs are transcribed from the genome and processed into premiRNAs

by Dicer; and then into short single stranded mature miRNAs complexed with

Argonaute proteins to inhibit protein translation. The first link of miRNAs to cancer was

made only relatively recently, but the field has expanded exponentially since.

TGF-

β induced Epithelial to Mesenchymal Transition model in Normal Mouse

Mammary Gland Epithelia Cells (NMuMG) is a commonly used model to dissect the

molecular processes of breast cancer metastasis. Using miRNA microarray, we

demonstrated miR-155 was upregulated along with alterations of other miRNAs. This

observation was validated with Northern and qRT-PCR analysis. Promoter and ChIP

analysis revealed TGF-

β activated the Smad4 transcriptional complex to induce the

expression of miR-155. The reduction of RhoA protein levels by ubiquitination has been

described to be a critical step during EMT, and we showed miR-155 down regulates

RhoA proteins without degrading its mRNA levels; therefore, preventing de novo

synthesis of RhoA proteins in the course of EMT. The interaction between miR-155 and

RhoA’s 3’UTR was confirmed by reporter assays. In summary, we reported the

importance of miR-155 during TGF

β induced EMT in NMuMG cells.

FOXO3a is a well studied tumor suppressor transcriptional factor and resides in

the nucleus to transcribe pro-apoptotic genes such as Bim, or p27 in the active state.

During conditions when cells are signaled to grow and divide, it is phosphorylated by

oncogenes such as AKT or IKK β, becomes inactivated and translocates into the

cytoplasm. We have shown for the first time that FOXO3a activity is also regulated by

miRNAs, specifically miR-155. Western and Northern analysis revealed a correlation

between FOXO3a protein and mature miR-155 RNA levels in breast cancer cell lines

along with breast tumor and normal tissues. Specifically, miR-155 expression is low in

BT474 and high in HS578T, and inversely correlates with endogenous FOXO3a protein

levels. Overexpression of miR-155 decreased endogenous FOXO3a protein and

knockdown of miR-155 HS578T rescued its expression. Reporter assay experiments

validated the interaction between miR-155 and FOXO3a 3’UTR. More importantly,

overexpression of miR-155 in BT474 protected the cells from apoptosis induced by drugs

while knockdown of miR-155 in HS578T initiated cell death even in the absence of drugs.

In summary, we have shown the importance of miR-155 in chemosensitivity by targeting

FOXO3a in breast cancer.

MiR-155 has been previously shown up-regulated in multiple types of

malignancies, including breast cancer. In addition, miR-155 expression was reported to

correlate very strongly to survival in lung and pancreatic cancer. We validated by qRTPCR

and Northern analysis that miR-155 expression is detected only in breast tumors and

not normal breast tissue. In situ hybridization of breast cancer tissue microarrays

revealed similar results. In light of previous studies that showed a correlation between

miR-155 and survival in lung and pancreatic cancers, we performed an X-tile analysis to

determine an optimal cut point for miR-155 level in our breast cancer sample population

that would correlate to ten years overall survival. Verification using Kaplan-Meier

validated a cut point at 90.14 to significantly correlate to overall survival (P=0.007). In

addition, Chi-square analysis revealed miR-155 expression to correlate with high tumor

stage, grade and lymph node metastasis. However, miR-155 expression did not

correspond to ER, PR, or HER2/neu status, but this is hardly surprising since

computational analysis does not predict miR-155 to target these genes. In summary, we

have shown deviant expression of miR-155 in breast cancer. Due to its correlation with

overall survival; higher grade and stage; lymph node metastasis, and triple negative

subtype, miR-155 may prove to be a valuable prognostic marker and therapeutic target

for breast cancer intervention.

Share

COinS