Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - CFS modelling (Chromosomal Common Fragile Sites: Unravelling their biological functions and the basis of their instability)
Teaser
In this project we aim to characterize the physiological functions and the mechanisms that regulate chromosomal Common Fragile Sites (CFSs) stability. CFSs are unusual genomic regions conserved in all the individuals that are prone to break specially under conditions of...
Summary
In this project we aim to characterize the physiological functions and the mechanisms that regulate chromosomal Common Fragile Sites (CFSs) stability. CFSs are unusual genomic regions conserved in all the individuals that are prone to break specially under conditions of replication stress (RS). Critically, CFSs are hotspots for chromosomal translocations and rearrangements in cancer. In other words, CFS instability drives tumorigenesis. Therefore, the study of the regulation of CFSs may unmask novel therapeutic strategies for cancer treatment. In addition, we expect that the knowledge generated in this project will contribute to open new directions in cancer research that may have an important impact in society.
Work performed
-We have generated human cell lines harbouring deletions of the 3 most frequently expressed CFSs (FRA7H, FRA3B and FRA16D). Remarkably, these delta-CFS cell lines are viable and proliferate at a similar rate than WT cells. So far, we have not detected significant signs of genomic instability, checkpoint defects or cell cycle abnormalities.
-In order to isolate and identity novel proteins at CFSs, we have implemented chromatin pull downs using specific FANCD2 antibodies. Using this protocol we have been able to obtain a reliable proteomics analysis of protein located at CFSs.
We have validated and characterized several novel factors identified in our proteomic screen. Of note, we have found that the chromatin remodeler ATRX is an important factor to maintain CFS stability. Given that ATRX is a tumor suppressor in glioblastoma, we believe that the CFS stability caused by ATRX mutations may drive tumorigenesis in glioblastoma. Our initial work on the characterization of the CFS proteome and the function of ATRX at regulating CFS stability is under review in Nucleic Acid Research.
-We have generated cohorts of WT, SuperChk1, SuperRRM2 and ATR-hypomorph mice to investigate the influence of these modifications on CFS stability, aging and cancer development.
In an initial work published at the beginning of this grant (Lopez-Contreras et al, Genes & Dev., 2015) we showed that CFSs are more stable in SuperRRM2 B cells, indicating that the supply of dNTPS is relevant for the maintenance of CFS stability. Interestingly, our latest data show that SuperRRM2 mice are more prone to develop tumors –mainly lymphomas- than WT mice. We will perform similar analyses in a novel mouse model generated deficient for PICH (Albers et al, Cell Reports, 2018).
Final results
In view of our initial results, new publications and discussions with colleagues at our Center and meetings, we have initiated new research directions relevant for this project that will contribute to a better understanding of CFS biology and its impact on cancer.
We have already characterized the relevance of ATRX on CFS stability (under review in Nucleic Acid Research). We expect to characterize several novel factors involved in CFS stability, and which may have implications in cancer development (similarly to ATRX). For the most interesting candidates identified, we aim to investigate their relevance in vivo, using available mouse models, or generating novel models. Ultimately, we expect to identify among them potential novel therapeutic targets for cancer treatment.
In regards to deletions of CFSs, we have identified that the role of FRA3B locus could be regulating FHIT expression throughout the cell cycle and in response to replication stress. Given the relevance of FHIT in various cancers, we are interested in characterizing in detail this regulation and the molecular functions of FHIT.
On the other hand, we have generated a PICH KO mouse model and published its initial characterization during embryonic development (Albers et al, Cell Reports, 2018). PICH is a protein involved in the resolution of chromosomal ultra fine anaphase bridges (UFBs) that are formed at CFSs. Thus, PICH deficiency may lead to increased CFS fragility. We have observed that PICH KO cells indeed accumulate chromosomal instability and we are currently characterizing its connection to CFS instability. We believe that the PICH deficient model will be a very valuable tool to study CFS fragility in vivo. Furthermore, our initial data and other recently published data (Yin et al, Cancer Cell Int, 2018; Zhong et al, Front Genet, 2018) point to a relevant role of PICH in relation to cancer. As for other factors identified in the CFS proteomics screen, we aim to establish whether targeting PICH could be a potential therapeutic strategy for some cancer types. For this, our PICH deficient mouse model will be crossed with cancer prone mouse models.
References:
Albers E, Sbroggiò M, Pladevall-Morera D, Bizard AH, Avram A, Gonzalez P, Martin-Gonzalez J, Hickson ID, Lopez-Contreras AJ. Loss of PICH Results in Chromosomal Instability, p53 Activation, and Embryonic Lethality. Cell Rep. 2018, 24: 3274-3284.
Yin X, Wang J, Zhang J. Identification of biomarkers of chromophobe renal cell carcinoma by weighted gene co-expression network analysis. Cancer Cell Int. 2018, 18: 206.
Zhong X, Liu Y, Liu H, Zhang Y, Wang L, Zhang H. Identification of Potential Prognostic Genes for Neuroblastoma. Front Genet. 2018, 9:589.