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Transforming Growth Factor β Signaling in Colorectal Cancer Cells With Microsatellite Instability Despite Biallelic
Noel F C C de Miranda1, Maarten van Dinther2, Brendy E W M van den Akker1
1Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Background & Aims:
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.
Conclusion:
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.
Insights
Transforming growth factor β receptor II (TGFBR2) mutations in microsatellite instability-high colorectal cancer cells can still produce functional TGFBR2 protein, maintaining TGFβ signaling. This suggests that reactivating TGFβ signaling may not be effective for all colorectal tumors.
Area of Science:
- Oncology
- Molecular Biology
- Gastroenterology
Background:
- Microsatellite instability-high (MSI-H) colorectal cancer (CRC) often features mutations in the transforming growth factor β receptor II (TGFBR2) gene.
- TGFβ signaling is typically considered defective in these tumors, yet some MSI-H CRC cells remain TGFβ-sensitive despite TGFBR2 mutations.
Purpose of the Study:
- To investigate the mechanisms by which TGFβ signaling persists in MSI-H CRC cells.
- To determine if mutated TGFBR2 can still produce functional protein and mediate TGFβ signaling.
Main Methods:
- Sequencing of the TGFBR2 microsatellite sequence in MSI-H colon cancer tissues and cell lines.
- Assessment of TGFβ signaling activation via SMAD2 phosphorylation and reporter constructs.
- TGFBR2 knockdown and expression of full-length and mutant TGFBR2 in CRC cells.
Main Results:
- SMAD2 phosphorylation, indicative of TGFβ signaling, was detected in most MSI-H CRC tissues and cell lines.
- This phosphorylation required TGFBR2, even a mutated form with a frameshift mutation.
- A 1-nucleotide deletion in the TGFBR2 microsatellite sequence still allowed production of functional full-length TGFBR2 protein.
Conclusions:
- TGFβ signaling remains active in some MSI-H CRC cells due to the expression of functional TGFBR2 protein from mutated genes.
- Therapeutic strategies aimed at reactivating TGFβ signaling in colorectal tumors may not be universally effective.
- Further evaluation of functional consequences of mutations in other microsatellite instability regions is warranted.
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