Identification of new negative regulators of ciliogenesis in breast cancer cells through high-throughput siRNA screening
Recorded On: 02/05/2018
Breast cancer is a major cause of death in women in the world. The basal subtypes, also recognized as triple negative breast cancers (TBNC), are the most aggressive type and account for the highest mortality rate in patients. Currently, there are no FDA approved targeted therapies for TNBC, and innovative approaches are necessary to develop new therapeutic options. The primary cilium is a membrane-bound, cell surface projection assembled from centrosomes and singularly expressed in the majority of cells in the human body, serving as a cellular 'antenna' in the recognition and transduction of extra-cellular stimuli, such as growth factors. This organelle forms during cellular quiescence and disassembles when cells enter the cell cycle and proliferate. Interestingly, primary cilia are frequently lost in malignant tumors, such as breast tumors. Thus primary cilia may play a repressive role in regulating cell proliferation and could lower breast cancer development. In order to identify negative regulators of ciliogenesis that could represent target for new drugs, we performed a high content screen using an arrayed library containing pooled siRNAs targeting 23,000 human genes in triplicate on Hs578T cells, a basal B breast cancer cell line which forms cilia at low frequency. Detecting cilia by automated immunofluorescence staining and imaging, we identified 350 candidate genes (~1-2%) that increased the number of ciliated cells. Candidate genes were retested in secondary screens in additional cell lines to distinguish the genes involved in cilia formation common to all cell lines and the ones specific to the (sub)types of (breast) cancer. There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers. In order to test the candidate genes from the 2D cell culture experiments in a tertiary screen to see their effect on tumor growth, migration and invasion, we grew Hs578T cells in ultra-low attachment (ULA) 96-well roundbottomed plates, where tumor cell suspensions formed a three-dimensional structure within 24 h. Three-dimensional spheroid assays are considered valid models to recapitulate features of tumors and, combined with new technologies of automated imaging and analysis, will contribute to a better understanding of ciliogenesis and breast cancer and to an important step in anticancer drug research.
NYU Pelmutter Cancer Institute
Since my Pharmacy studies, I wanted to work in basic research. I did an internship in the Neuropharmacology Center of the Pharmacy University of Milan where I learned basic proteomic research (Mallei A, et al., 2014). During my Master’s degree, I was in charge of the validation of a small scale siRNA screen on ciliogenesis. During my Ph.D., I focused on the characterization of two new Nephronophthisis candidate genes (Failler M et al., 2014). I used high resolution imaging (SIM and STED microscopy) and participated in setting up this imaging platform at our institution (Alby C et al., 2015). I now wish to continue understanding the role of ciliary dysfunction in cancer. Under the supervision of my mentor, I performed high-throughput siRNA screen in a breast cancer cell lines and identified candidate genes that allow cilia growth in these cells.