Date of Award

6-2013

Document Type

Thesis

Degree Name

Master of Science in Medical Sciences (MSMS)

Department

Medical Education

First Advisor

Dr.Samir Attoub

Second Advisor

Prof. Sherif Karam

Third Advisor

Dr. Ahmed AI Marzouqi

Abstract

Breast cancer is the most common cancer seen in women worldwide accounting for more than 1.3 million cases and 458 000 deaths a year worldwide. Breast cancer patients are at high risk of recurrence in the form of metastatic disease. Genes associated with invasion and metastasis provides an aggressive edge in proliferation and growth during colonization of the metastatic site. The SMARCAD1 (SWI/SNF-related, Matrix-associated, Actin-dependent Regulator of Chromatin, containing DEAD/H box 1) protein is the human homolog of yeast Fun30 (Function Unknown Now 30) proteins. These proteins are members of the Snf2 helicase motif-containing protein families, and are known to act as ATP-dependent DNA translocators. Previous studies have shown that yeast Fun30 deletions are resistant to ultraviolet (UV) radiation. The over-expression of Fun30 has been shown to affect chromosome stability, integrity, and segregation. Fun30 has also been shown to be a potential cyclin-dependent kinase (Cdk1)/Cdc28 substrate. The SMARCAD1 is a DEAD/H box containing helicase which includes proteins essential for replication, repair, and transcription. In addition to two DEAD/H box and an ATP-binding motifs within SMARCAD1, it has a putative nuclear localization signal and several regions that may mediate protein-protein interactions. Expression analysis indicates that SMARCAD1 transcript are ubiquitous, with particularly high levels in endocrine tissue. The gene for SMARCAD1 has been mapped to the chromosome 4q22-q23m, a region rich in break points and deletion mutants of genes involved in several human diseases, notably soft tissue leiomyosarcoma, hepatocellular carcinoma, and hematologic malignancies. It has been recently reported that the binding sites of endogenous SMARCAD1/KIAA1122 are frequently found in the vicinity of transcriptional start sites. Moreover, human SMARCAD1 overexpression was observed in E1A-expressing cell line with increased capacity for gene reactivation events by genomic rearrangements suggesting that human SMARCAD1 may play a role in genetic instability. Previous studies in our laboratory showed that the breast cancer cells estrogen receptors (ER)-negative MDA-MB -231 and MDA-MB-231-1833 as well as ER-positive MCF-7 and T47D express a high level of SMARCAD1 in comparison with the normal breast epithelial cells NHME.

In my master research project, we investigated the impact of specific silencing SMARCAD1 on human breast cancer cell proliferation, colony growth, morphology, migration and invasion using human breast cancer cells estrogen receptors (ER)-negative MDA-MB-231.

In this respect, the cells were stably transfected with two different designs of SMARTvector 2.0 Lentiviral shRNA particles targeting SMARCAD1. Control cells were transfected with SMARTvector 2.0 Non-Targeting control particles. The positive clones (10 to 12 from each design) were selected under puromycin and the GFP positive clones were analyzed using western-blot to confirm specific SMARCAD1 silencing. The two different design of shRNA targeting SMARCAD1 induced 99% decreased in the SMARCAD1 protein level (SMARCAD1-shRNA1, and SMARCAD1-shRNA3). The selectivity of this silencing was confirmed by the fact that no impact on SMARCAD1 protein was observed in the cells transfected with shRNA-control particles (control-shRNA). We demonstrated for the first time that silencing of SMARCAD1 resulted in a significant inhibition of cellular proliferation and colonies formation in soft agar, as well as cellular migration, and invasiveness. All together, these results strongly suggest that SMARCAD1 may play an important role in breast cancer growth and metastasis.

Identification of new molecular effectors and signaling pathways involved in breast cancer growth, invasion, and metastasis could lead to effective targeted approaches in breast cancer therapy.

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