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.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-4802 |
| Date | 20 July 2010 |
| Creators | Kong, William |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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