Transforming Growth Factor β Signaling in Colorectal Cancer Cells With Microsatellite Instability Despite Biallelic Mutations in TGFBR2.
OBJECTIVE
Most colorectal cancer (CRC) cells with high levels of microsatellite instability (MSI-H) accumulate mutations at a microsatellite sequence in the gene encoding transforming growth factor β receptor II (TGFBR2). TGFβ signaling therefore is believed to be defective in these tumors, although CRC cells with TGFBR2 mutations have been reported to remain sensitive to TGFβ. We investigated how TGFβ signaling might continue in MSI-H CRC cells.


METHODS
We sequenced the 10-adenines microsatellite sequence in the TGFBR2 gene of 32 MSI-H colon cancer tissues and 6 cell lines (HCT116, LS180, LS411N, RKO, SW48, and SW837). Activation of TGFβ signaling was detected by SMAD2 phosphorylation and through use of a TGFβ-responsive reporter construct in all CRC cell lines. Transcripts of TGFBR2 were knocked-down in CRC cells using short hairpin RNA. Full-length and mutant forms of TGFBR2 were expressed in LS411N cells, which do not respond to TGFβ, and their activities were measured.


RESULTS
SMAD2 was phosphorylated in most MSI-H CRC tissues (strong detection in 44% and weak detection in 34% of MSI-H tumors). Phosphorylation of SMAD2 in MSI-H cells required TGFBR2—even the form encoding a frameshift mutation. Transcription and translation of TGFBR2 with a 1-nucleotide deletion at its microsatellite sequence still produced a full-length TGFBR2 protein. However, protein expression required preservation of the TGFBR2 microsatellite sequence; cells in which this sequence was replaced with a synonymous nonmicrosatellite sequence did not produce functional TGFBR2 protein.


CONCLUSIONS
TGFβ signaling remains active in some MSI-H CRC cells despite the presence of frameshift mutations in the TGFBR2 gene because the mutated gene still expresses a functional protein. Strategies to reactivate TGFβ signaling in colorectal tumors might not be warranted, and the functional effects of mutations at other regions of microsatellite instability should be evaluated.
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Noel F C C
NF
de Miranda
Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Leiden University Medical Center, Leiden, The Netherlands.
Maarten
M
van Dinther
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Brendy E W M
BE
van den Akker
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Tom
T
van Wezel
Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Leiden University Medical Center, Leiden, The Netherlands; Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. Electronic address: P.ten_Dijke@lumc.nl.
Peter
P
ten Dijke
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: j.morreau@lumc.nl.
Hans
H
Morreau
D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016368:Frameshift Mutation; D015972:Gene Expression Regulation, Neoplastic; D020022:Genetic Predisposition to Disease; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D053842:Microsatellite Instability; D010641:Phenotype; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000819:agonists* / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II
Colon Cancer; RNA Editing; Signal Transduction; Tumor Suppressor