Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells.
BACKGROUND
Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival.


RESULTS
A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway.


CONCLUSIONS
We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.
Gene Silencing Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA.
Amanda B
AB
Hummon
Jason J
JJ
Pitt
Jordi
J
Camps
Georg
G
Emons
Susan B
SB
Skube
Konrad
K
Huppi
Tamara L
TL
Jones
Tim
T
Beissbarth
Frank
F
Kramer
Marian
M
Grade
Michael J
MJ
Difilippantonio
Thomas
T
Ried
Natasha J
NJ
Caplen
D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D016000:Cluster Analysis; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D004261:DNA Replication; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006657:Histones / Q000235:genetics*; D006801:Humans; D016900:Neurofilament Proteins / Q000235:genetics; D034622:RNA Interference; D014158:Transcription, Genetic; D059467:Transcriptome
D051017:Apoptosis Regulatory Proteins; C485604:CASP8AP2 protein, human; D002135:Calcium-Binding Proteins; D006657:Histones; D016900:Neurofilament Proteins; C051774:neurofilament protein H