The RNA-binding Protein G3BP2 Regulates The Breast Tumor-initiating Cell State

BACKGROUND Tumors contain tumorigenic cancer cells,termed tumor-initiating cells(TICs),which are capable of replenishing both themselves and populations of non-tumorigenic cancer cells(non-TIC).Furthermore,it seems that TICs might be resistant to standard cancer therapy such as radiation,chemotherapy and biological therapy-This might explain the limitations of these therapies in curing cancer patients and the reason of relapse.Currently,little is known about the mechanisms that maintain these cells and regulate its switch.METHOD To identify genes potentially involved in the regulation of the breast TIC state,we developed a novel chemical approach for dissection of the tumor-initiating cell and drug resistance programs.We used high-throughput drug screen for anti-TIC compounds and explored the tructure of selected compounds.Then we identified the molecular target-G3BP2 for selected small molecules.The ALDEFLUOR assay was applied to identify the ALDEFLUOR+population in MDA-MB-453 cells with G3BP3 overexpression(pBabe-G3BP2)and pBabe as a control.Expression of CD44 and CD24 in MDA-MB-231 control and G3BP2 knock down cells analyzed by flow cytometry.When silencing G3BP2,western blot analysis of CD24 in MDA-MB-231,MDA-MB-453 and BT-474 cell lysates was performed.The clonogenic efficiency of control cancer cells and cancer cells with G3BP2 knock down were determined based on number of spheres that originate from cells plated at clonal density.Extreme limiting dilution assay(ELDA)for tumor forming frequency of G3BP2 shRNA MDA-MB-453 cells and scr-control shRNA MDA-MB-453 cells in NOD-SCID mice.Co-IP was used to determine the physical interaction of G3BP2 and SART3.Western blot,immunocytochemistry and fluorescent immunocytochemical staining were performed to determine the expression of SART3 in G3BP2 depletion cells and control cells.Western blot analysis was performed to detect OCT4 and Nanog expression in SART3 or G3BP2 knock down breast cancer cell lines.Representative images of mammosphere formation were observed in SART3 silenced BT-474 and MDA-MB-453 cells.SART3 mRNA was assessed by qPCR in cells with downregulation of G3BP2(red)in three different breast cancer cell lines.RNA immunoprecipitation(RIP)assay with BT-474 and MDA-MB-453 cell lysates was performed with downregulation of G3BP2.SART3 and GAPDH mRNAs were quantified using qPCR following pulldown with anti-G3BP2 Ab or IgG,and were represented as fold enrichment compared with control IgG for RIP assay.Retroviral constructs with full-length G3BP2,deletion of RNA binding morif of the G3BP2 gene(△1)(RB),and retrovirus alone were infected into MCF-7 cells.qPCR was performed to detect SART3 mRNA changes.Western blot analysis of SART3 protein in MCF-7 cells with different retroviral constructs.Representative images show correspondence between SART3 and G3BP2 staining patterns in 54 clinical samples.RESULT Of the 60,000 compounds screened,we selected 117 for the second screening step.We studied their efficacy at five different concentrations(0.12 mol/L,0.37 mol/L,1.11 mol/L,3.33 mol/L and 10 mol/L).We then selected 78 of these 117 compounds that showed dose dependent repression of cancer cell growth and that were non-toxic to normal.human umbilical vein endothelial cells(HUVEC).Flow cytometric plots depicting the Aldefluor-positive population in BT474 cells treated with control,C108,or C108 and paclitaxel.G3BP2 protein binds to C108 compound which was analyzed by mass spectroscopy.G3BP2 increases the Aldefluor positive population in breast cancer cells.Silencing G3BP2 results in increased CD24 protein levels.Knock down of G3BP2 inhibits mammosphere formation in BT-474 and MDA-MB-453 cells.Knocking down G3BP2 in BT-474 and MDA-MB-231 cells results in significantly fewer and smaller spheres.Tumor-initiating frequency of stable control and G3BP2 silenced cell lines BT-474 and MDA-MB-453 in NOD/SCID mice.SART3 was identified to interact physically with G3BP2.G3BP2 regulate embryonic stem cell transcriptional factors SART3,OCT4 and NANOG.Representative images of mammosphere formation observed in SART3 silenced BT-474 and MDA-MB-453 cells.SART3 mRNA was decreased in shRNA-G3BP2 cells.Total mRNAs interacting with G3BP2 were pulled down and SART3 mRNA was detected significantly decreased when silencing G3BP2.Moreover,SART3 mRNA and protein were significantly reduced in cells with deleted RRM.G3BP2 is highly expressed in invasive breast cancer clinical samples.Staining patterns for G3BP2 and SART3 were significantly correlated.CONCLUSION In this study,we undertook an unbiased screen to identify molecules involved in TIC maintenance,and have identified one RNA binding protein,G3BP2,as a potential regulator of breast cancer sternness through the intersection of its activity with the embryonic stem cell transcriptional factors Oct-4 and Nanog..