Live-cell imaging shows uneven segregation of extrachromosomal DNA elements and transcriptionally active extrachromosomal DNA hubs in cancer

Cancer + MetastasisCheng LabCRISPR/Cas + TALENImagingRepresentative
Eunhee Yi, Amit D Gujar, Molly Guthrie, Hoon Kim, Dacheng Zhao, Kevin C. Johnson, Samirkumar B Amin, Megan L Costa, Qianru Yu, Sunit Das, Nathaniel Jillette, Patricia A Clow, Albert W Cheng#, Roel GW Verhaak# (co-corresponding)
Cancer Discovery, doi: 10.1158/2159-8290.CD-21-1376
Publication year: 2021

Oncogenic extrachromosomal DNA elements (ecDNAs) play an important role in tumor evolution, but our understanding of ecDNA biology is limited. We determined the distribution of single-cell ecDNA copy number across patient tissues and cell line models and observed how cell-to-cell ecDNA frequency greatly varies. The exceptional intratumoral heterogeneity of ecDNA suggested ecDNA-specific replication and propagation mechanisms. To evaluate the transfer of ecDNA genetic material from parental to offspring cells during mitosis, we established the CRISPR-based ecTag method. EcTag leverages ecDNA-specific breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying ecTag during mitosis revealed disjointed ecDNA inheritance patterns, enabling rapid ecDNA accumulation in individual cells. Post-mitosis, ecDNAs clustered into ecDNA hubs, and ecDNA hubs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new light on mechanisms through which ecDNAs contribute to oncogenesis.

Activation of lytic genes in cancer cells

Cancer + MetastasisCheng LabCRISPR/Cas + TALENPatents
Albert Cheng, Kwok Wai Lo, Pol Man Tom Hau, Man Wu
WO2021173977A1
Publication year: 2021

Poison exon splicing regulates a coordinated network of SR protein expression during differentiation and tumorigenesis

Cancer + MetastasisCheng LabCRISPR/Cas + TALENRNA Splicing + RBPs
Leclair NK, Brugiolo M, Urbanski L, Lawson SC, Thakar K, Yurieva M, George J, Hinson JT, Cheng A, Graveley BR, Anczuków O
Molecular Cell 80(4):468-665.e9 doi: 10.1016/j.molcel.2020.10.019.
Publication year: 2020

C11orf95-RELA reprograms 3D epigenome in supratentorial ependymoma

Cancer + MetastasisCheng LabEpigenetics
Jacqueline Jufen Zhu, Nathaniel Jillette, Xiao-Nan Li, Albert Wu Cheng# & Ching C. Lau# (co-corresponding)
Acta Neuropathol (2020). https://doi.org/10.1007/s00401-020-02225-8
Publication year: 2020

Supratentorial ependymoma (ST-EPN) is a type of malignant brain tumor mainly seen in children. Since 2014, it has been known that an intrachromosomal fusion C11orf95-RELA is an oncogenic driver in ST-EPN [Parker et al. Nature 506:451–455 (2014); Pietsch et al. Acta Neuropathol 127:609–611 (2014)] but the molecular mechanisms of oncogenesis are unclear. Here we show that the C11orf95 component of the fusion protein dictates DNA binding activity while the RELA component is required for driving the expression of ependymoma-associated genes. Epigenomic characterizations using ChIP-seq and HiChIP approaches reveal that C11orf95-RELA modulates chromatin states and mediates chromatin interactions, leading to transcriptional reprogramming in ependymoma cells. Our findings provide important characterization of the molecular underpinning of C11orf95-RELA fusion and shed light on potential therapeutic targets for C11orf95-RELA subtype ependymoma.

Musashi proteins are post-transcriptional regulators of the epithelial-luminal cell state

Cancer + MetastasisPhDEraRNA Splicing + RBPs
Katz Y, Li F, Lambert NJ, Sokol ES, Tam WL, Cheng AW, Airoldi EM, Lengner CJ, Gupta PB, Yu Z, Jaenisch R, Burge CB
Elife 3:e03915
Publication year: 2014

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types.

Alternatively spliced mRNA isoforms as prognostic indicators for metastatic cancer

Cancer + MetastasisPatentsPhDEraRepresentativeRNA Splicing + RBPs
Christopher Boyce BURGE, Wu Albert CHENG, John S. Condeelis, Frank B. Gertler, Maja H. OKTAY, Irina M. SHAPIRO
WO2012116248 A1
Publication year: 2012

The present invention provides a method for identifying a tumor as likely to metastasize, or likely to have metastasized, comprising obtaining a sample of the tumor and quantitating alternatively spliced mRNA isoforms of a cell motility gene, a cell adhesion gene and /or an actin cytoskeletal remodeling gene in the sample, or any specified genes or the level of RNA binding proteins compared to a predetermined non-metastasizing control.

An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

Cancer + MetastasisPhDEraRepresentativeRNA Splicing + RBPs
Irina M. Shapiro*, Albert W. Cheng*, Nicholas C. Flytzanis, Michele Balsamo, John S. Condeelis, Maja H. Oktay, Christopher B. Burge , Frank B. Gertler
PLoS Genet 7(8): e1002218
Publication year: 2010

Abstract

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.