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Rac1 in human breast cancer: overexpression, mutation analysis, and characterization of a new isoform, Rac1b
A Schnelzer1, D Prechtel, U Knaus
1Department of Obstetrics and Gynecology, Technische Universität München, Klinikum rechts der Isar, Ismaninger Str. 22, 0-81675 Munich, Germany.
Abstract:
Rac1 is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) that act as molecular switches to control cytoskeletal rearrangements and cell growth. Analogous to Ras, constitutively activating point mutations of Rac1 cause tumorigenic transformation of cell lines. However, there is no information about whether Rac1 is also mutated in vivo. After RT - PCR of Rac1, several clones of seven benign and 10 malignant breast cancer tissues as well as eight breast cancer cell lines were sequenced. Only single-nucleotide polymorphisms of Rac1 could be detected, and none of these corresponded to constitutively activating point mutations that have been used in cell lines for transformation. While sequencing Rac1 in breast tissues, a new Rac1 isoform with an insertion of 19 codons within the reading frame of Rac1 close to switch region II was identified and named Rac1b. The Rac1b protein acts like a fast cycling GTPase in GTP binding and hydrolysis assays. In Northern and Western blot experiments both Rac1 RNA and Rac1 protein had a significantly higher expression in breast cancer tissues compared to normal breast tissue samples. Immunohistochemical staining of Rac1 showed weak Rac1 expression in benign breast disease but high expression level in ductal carcinoma-in-situ, primary breast cancer, and lymph node metastases. In addition, breast tumor cells from patients with recurrent disease had Rac1 expression at the plasma membrane, suggesting activation of Rac1, in patients with aggressive breast cancer. Oncogene (2000).
Insights
Constitutively activating Rac1 mutations were not found in breast cancer tissues. However, a new Rac1 isoform, Rac1b, and increased Rac1 expression were identified in breast tumors, suggesting Rac1
Area of Science:
- Molecular biology
- Cancer research
- Cell signaling
Background:
- Rac1, a small GTPase, regulates cell growth and cytoskeletal dynamics.
- Constitutively activating Rac1 mutations transform cell lines but their in vivo relevance is unknown.
- Investigating Rac1 mutations in breast cancer is crucial for understanding tumorigenesis.
Purpose of the Study:
- To investigate in vivo mutations of Rac1 in breast cancer tissues.
- To identify novel Rac1 isoforms or alterations in breast cancer.
- To correlate Rac1 expression and localization with breast cancer progression and recurrence.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of Rac1 in breast cancer tissues and cell lines.
- Identification and characterization of Rac1 isoforms using molecular assays.
- Northern and Western blot analyses for Rac1 RNA and protein expression.
- Immunohistochemical staining to assess Rac1 localization and expression in tissue samples.
Main Results:
- No constitutively activating Rac1 point mutations were detected in vivo; only single-nucleotide polymorphisms were found.
- A novel Rac1 isoform, Rac1b, with a 19-codon insertion near switch region II, was identified.
- Rac1 RNA and protein expression were significantly elevated in breast cancer tissues compared to normal tissues.
- High Rac1 expression was observed in ductal carcinoma-in-situ, primary tumors, and metastases.
- Rac1 plasma membrane localization in recurrent breast cancer suggests activation in aggressive disease.
Conclusions:
- Rac1 activation, rather than specific activating mutations, may drive breast cancer progression.
- The novel Rac1b isoform warrants further investigation for its role in cancer.
- Elevated Rac1 expression and specific localization are potential biomarkers for aggressive breast cancer.