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KLF5 is frequently deleted and down-regulated but rarely mutated in prostate cancer
Ceshi Chen1, Hina V Bhalala, Robert L Vessella
1Department of Pathology, University of Virginia Health System, Charlottesville, Virginia, USA.
Background:
Previous studies mapped a region at the q21 band of chromosome 13 (13q21), which is frequently deleted in various human cancers including prostate cancer, suggesting the existence of a tumor suppressor gene at 13q21. The target gene of deletion in prostate cancer, however, has not been identified at present.
Methods:
We examined four non-neoplastic and 18 neoplastic prostatic cell lines or xenografts. Homozygous/hemizygous deletion was detected by assays of duplex PCR and real-time PCR. Expression levels of genes were determined by the methods of RT-PCR, real time PCR, and northern blot analysis. Mutations of KLF5 were detected by the approaches of single strand conformational polymorphism (SSCP) and direct sequencing. For the detection of promoter methylation, Southern blotting of genomic DNA and restriction digestion or SSCP analysis of methylation specific PCR products were used. Finally, an expression plasmid of KLF5 was introduced into prostate cancer cell lines with reduced KLF5 expression to investigate colony formation for cell growth.
Results:
A 2-Mb region of homozygous deletion at 13q21 was detected in the LUCaP70 xenograft of prostate cancer. This region of deletion was further narrowed to 142 Kb by a hemizygous deletion in the NCI-H660 cell line. KLF5 was identified as the only complete gene in the smallest region of deletion. Quantitative deletion of KLF5 genome occurred in six of the 18 (33%) prostate cancer xenografts/cell lines. Each of the six samples with deletion also showed loss of expression for KLF5, suggesting that hemizygous deletion is one mechanism for loss of KLF5 expression. In total, 16 of the 18 cases (89%) showed loss of KLF5 expression at different degrees. In contrast, mutations and promoter methylations were not detected in any of the samples. Functionally, restoration of KLF5 in DU 145 and 22Rv1 cell lines significantly inhibited their growth in vitro.
Conclusions:
Frequent genomic deletion and loss of expression as well as cell growth suppression indicate that KLF5 is a reasonable candidate for the tumor suppressor gene at 13q21 in prostate cancer. Mutation and promoter methylation are not common mechanisms for the inactivation of KLF5 in prostate cancer.
Insights
Krüppel-like factor 5 (KLF5) is identified as a tumor suppressor gene at chromosome 13q21 in prostate cancer. Frequent deletion and loss of KLF5 expression suppress tumor cell growth, suggesting KLF5 as a therapeutic target.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- A specific region on chromosome 13q21 is frequently deleted in various cancers, including prostate cancer.
- This deletion suggests the presence of a tumor suppressor gene, but it remained unidentified in prostate cancer.
- Previous research has not pinpointed the specific gene responsible for tumor suppression at this locus in prostate cancer.
Purpose of the Study:
- To identify the tumor suppressor gene located at the 13q21 region frequently deleted in prostate cancer.
- To investigate the role of Krüppel-like factor 5 (KLF5) in prostate cancer development and progression.
- To determine the mechanisms of KLF5 inactivation in prostate cancer cells.
Main Methods:
- Examined 18 prostate cancer cell lines/xenografts and four non-neoplastic controls.
- Utilized duplex PCR and real-time PCR to detect homozygous/hemizygous deletions.
- Assessed KLF5 gene expression via RT-PCR, real-time PCR, and northern blot analysis.
- Investigated KLF5 mutations using single-strand conformational polymorphism (SSCP) and direct sequencing.
- Analyzed promoter methylation through Southern blotting and methylation-specific PCR.
- Restored KLF5 expression in prostate cancer cell lines to evaluate its effect on cell growth.
Main Results:
- Identified KLF5 as the sole complete gene within the smallest deleted region at 13q21 (142 Kb).
- Found quantitative deletion of KLF5 in 33% of prostate cancer samples, correlating with loss of expression.
- Observed loss of KLF5 expression in 89% of cases, primarily due to genomic deletion rather than mutation or promoter methylation.
- Demonstrated that KLF5 restoration significantly inhibited prostate cancer cell growth in vitro.
Conclusions:
- KLF5 is a strong candidate tumor suppressor gene at 13q21 in prostate cancer, evidenced by frequent deletion, loss of expression, and growth suppression.
- Genomic deletion is the primary mechanism for KLF5 inactivation in prostate cancer.
- Mutation and promoter methylation are not common mechanisms for KLF5 inactivation in this cancer type.