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A Candidate Breast and Ovarian Tumor Suppressor Gene on Chromosome 17p13.3
Chromosome 17p13.3 has the highest rate of loss of heterozygosity (LOH) in breast cancer, with 50% of tumors having LOH at 17p13.3, compared with an all-chromosome-arms average LOH rate of 15%. LOH at 17p13.3 loci has been significantly correlated with poor survival in breast cancer, and with larger size, nodal metastasis, negative hormone receptors, and high proliferation. LOH at 17p13.3 is independent of LOH at TP53. There are two common regions of deletion, a subtelomeric one, and a more proximal one 1,753 - 2,650 kb from the telomere. Ovarian carcinoma also has an 80% LOH rate at 17p13.3, and its common region of deletion (1,884 - 2,251 kb) is included within the proximal breast cancer common region of deletion. Ovarian cancer 17p13.3 LOH also is independent of TP53 deletion.
Our hypothesis is that chromosome 17p13.3 contains one or more tumor suppressor genes involved in breast and ovarian carcinogenesis, and that the gene is located in the overlap of the regions of deletion. Eight genes are located in this overlap: DPH2L1, OVCA2, HIC1, KIAA0732, SRR, KIAA1401, KIAA0397, MNT. None of the three genes screened so far (DPH2L1, HIC1, MNT) have shown mutations in breast or ovarian cancer. Some functional information is available for HIC1, MNT, SRR, and KIAA0732, but little is known about the remaining genes. We have shown that FLAG-tagged KIAA1401 localizes to the nucleus. Transfection of constitutively expressed KIAA1401 into cell lines shows strong suppression of stable clonal outgrowth in ovarian cancer line ES2, moderate suppression in estrogen receptor (ER)-negative, HER2-amplified breast cancer line SKBR3, and no effect in ER-positive breast cancer line MCF7. Since 50% of breast cancers have 17p13.3 LOH, lack of effect in some cell lines is expected.
Two independent approaches will be used to examine the role of KIAA1401 in breast cancer:
Aim 1: Evaluate larger panel of breast and ovarian cancer cell lines for suppression of clonal outgrowth following transfection with constitutively promoted N-FLAG-KIAA1401, and correlate with native and exogenous mRNA and protein expression. Evaluate purified populations of cells transiently expressing KIAA1401 for growth rate, cell cycle population distribution by flow cytometry, apoptosis by annexin V and DNA ladder assays.
Aim 2: Screen for KIAA1401 mutations in a panel of 30 primary breast tumors and the breast and ovarian cancer cell lines examined in Aim 1, using denaturing HPLC and sequencing. Type the same panel for LOH/homozygosity at several SNP and microsatellite loci within or near the KIAA1401 gene.
Tumor suppressor genes are normally present in two copies, and in order to deactivate the gene's growth suppression function, both copies need to be lost or defective. 50% of invasive breast cancers have lost a section of one of the two copies of chromosome 17, band p13.3. Loss of this region correlates with decreased survival and decreased disease-free survival. Ovarian carcinoma also has a smaller region of 17p13.3 deletion that overlaps the breast cancer deletion. Our hypothesis is that there is a breast and ovarian cancer tumor suppressor gene in this region of chromosome 17p13.3, and that its loss is associated with more aggressive breast tumors.
Eight genes are located in the overlapping breast cancer and ovarian cancer regions of deletion. None of the three genes screened so far have shown mutations in breast or ovarian cancer. We have found that introduction of an intact copy of the KIAA1401 gene slows in vitro growth in an estrogen receptor-negative, HER2-amplified breast cancer cell line and an ovarian cancer line, but not in an estrogen receptor-positive breast cancer cell line. An effect seen in a poorly differentiated but not in a well differentiated breast cancer cell line might reflect the association of 17p13.3 deletion with poor prognosis. KIAA1401 protein is found in the nucleus, suggesting a possible role in RNA transcription or processing, cell cycle regulation, or apoptosis (programmed cell death). The gene does not have a strong structural similarity to genes of known function. Based on the growth suppression in the above assay, we wish to characterize the KIAA1401 gene in breast cancer.
Aim 1 will be to evaluate additional breast and ovarian cancer lines for in vitro growth suppression. Cell lines susceptible to growth suppression by KIAA1401 will be used to identify the general mechanism of the growth suppression (slow but continuous growth, cell cycle arrest, or apoptosis). Aim 2 will be to search for KIAA1401 mutations in a panel of 30 primary breast tumors and the breast and ovarian cancer cell lines examined in Aim 1, and to characterize the same tumor panel for loss of the normal copy of the gene.
The two benefits of identifying the breast cancer 17p13.3 tumor suppressor gene would be (1) a better understanding of breast cancer pathogenesis, and (2) development of a test for identifying poor-prognosis subgroups of patients that should receive adjuvant therapy. Should there prove to be a significant mutation rate in the KIAA1401 gene, the test developed in Aim 2 may be a useful tool for a larger prognostic study.