Year: 2010
Type: dissertation
Abstract: Recent statistics reveal breast cancer as the most common cancer among women\nand accounts for approximately 41,000 mortalities per year. In diagnosis, features such\nas stage, grade, lymph node metastasis are important prognostic indicators that help guide\nphysicians and oncologist towards optimal patient care. Presence of established\npathological markers such as ER, PR, and Her2/neu status would indicate ideal adjuvant\ntherapy situation. Although treatment of these types of breast cancer is well established,\ncancer that lack all three receptors, “triple negatives” or “basal like” do not respond to\nadjuvant therapy and are considered more aggressive in that patients tend to recur early\nand experience visceral metastasis. Although scientists have uncovered numerous\nmolecular biology mechanisms in search of an understanding in cancer, leading to\ndevelopment of fields such as apoptosis or growth pathways; cell cycle; angiogenesis;\nmetastasis; and more recently cancer stem cells, much work remains as cancer is still not\neradicated.\nMicroRNAs (miRNAs) are post transcriptional regulators of gene expression.\nTheir discovery and functional understanding have only been uncovered in the past ten\nyears. Long pri-miRNAs are transcribed from the genome and processed into premiRNAs\nby Dicer; and then into short single stranded mature miRNAs complexed with\nArgonaute proteins to inhibit protein translation. The first link of miRNAs to cancer was\nmade only relatively recently, but the field has expanded exponentially since.\nTGF-\nβ induced Epithelial to Mesenchymal Transition model in Normal Mouse\nMammary Gland Epithelia Cells (NMuMG) is a commonly used model to dissect the\nmolecular processes of breast cancer metastasis. Using miRNA microarray, we\ndemonstrated miR-155 was upregulated along with alterations of other miRNAs. This\nobservation was validated with Northern and qRT-PCR analysis. Promoter and ChIP\nanalysis revealed TGF-\nβ activated the Smad4 transcriptional complex to induce the\nexpression of miR-155. The reduction of RhoA protein levels by ubiquitination has been\ndescribed to be a critical step during EMT, and we showed miR-155 down regulates\nRhoA proteins without degrading its mRNA levels; therefore, preventing de novo\nsynthesis of RhoA proteins in the course of EMT. The interaction between miR-155 and\nRhoA’s 3’UTR was confirmed by reporter assays. In summary, we reported the\nimportance of miR-155 during TGF\nβ induced EMT in NMuMG cells.\nFOXO3a is a well studied tumor suppressor transcriptional factor and resides in\nthe nucleus to transcribe pro-apoptotic genes such as Bim, or p27 in the active state.\nDuring conditions when cells are signaled to grow and divide, it is phosphorylated by\noncogenes such as AKT or IKK β, becomes inactivated and translocates into the\ncytoplasm. We have shown for the first time that FOXO3a activity is also regulated by\nmiRNAs, specifically miR-155. Western and Northern analysis revealed a correlation\nbetween FOXO3a protein and mature miR-155 RNA levels in breast cancer cell lines\nalong with breast tumor and normal tissues. Specifically, miR-155 expression is low in\nBT474 and high in HS578T, and inversely correlates with endogenous FOXO3a protein\nlevels. Overexpression of miR-155 decreased endogenous FOXO3a protein and\nknockdown of miR-155 HS578T rescued its expression. Reporter assay experiments\nvalidated the interaction between miR-155 and FOXO3a 3’UTR. More importantly,\noverexpression of miR-155 in BT474 protected the cells from apoptosis induced by drugs\nwhile knockdown of miR-155 in HS578T initiated cell death even in the absence of drugs.\nIn summary, we have shown the importance of miR-155 in chemosensitivity by targeting\nFOXO3a in breast cancer.\nMiR-155 has been previously shown up-regulated in multiple types of\nmalignancies, including breast cancer. In addition, miR-155 expression was reported to\ncorrelate very strongly to survival in lung and pancreatic cancer. We validated by qRTPCR\nand Northern analysis that miR-155 expression is detected only in breast tumors and\nnot normal breast tissue. In situ hybridization of breast cancer tissue microarrays\nrevealed similar results. In light of previous studies that showed a correlation between\nmiR-155 and survival in lung and pancreatic cancers, we performed an X-tile analysis to\ndetermine an optimal cut point for miR-155 level in our breast cancer sample population\nthat would correlate to ten years overall survival. Verification using Kaplan-Meier\nvalidated a cut point at 90.14 to significantly correlate to overall survival (P=0.007). In\naddition, Chi-square analysis revealed miR-155 expression to correlate with high tumor\nstage, grade and lymph node metastasis. However, miR-155 expression did not\ncorrespond to ER, PR, or HER2/neu status, but this is hardly surprising since\ncomputational analysis does not predict miR-155 to target these genes. In summary, we\nhave shown deviant expression of miR-155 in breast cancer. Due to its correlation with\noverall survival; higher grade and stage; lymph node metastasis, and triple negative\nsubtype, miR-155 may prove to be a valuable prognostic marker and therapeutic target\nfor breast cancer intervention.
Author: William Kong
Institution: University of South Florida
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1577336727