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.

Committee Member

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. viii 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 TGFβ 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 IKKβ, 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 ix 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 x 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.

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