ID4: a Prognostic Factor of Breast Cancer Metastasis

Institution: John Wayne Cancer Institute
Investigator(s): David Hoon, MSc, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11IB-0166 Award: $283,200
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2005)
Breast cancer metastasis to regional lymph nodes is one of the most important factors in predicting disease outcome in early stage breast cancer. Our institute developed the surgical procedure to identify the first tumor-draining lymph node, the sentinel lymph node (SLN), likely to have micrometastasis. This has improved disease staging and reduced unnecessary surgery. Furthermore, we discovered a novel gene referred to as ID4 in breast cancer and found that its inactivation correlated with metastasis to the SLN. We will validate whether the inactivation of ID4 independently, or in combination with other prognostic factors leads to primary tumor spread.

The major aim is to identify if the genetic inactivation of ID4 is a predictive factor for tumor metastasis to SLN. The study will involve assessment of archival breast tissue specimens with known histopathology diagnosis. Breast patients who underwent the SLN procedure at our institute over the last 10 years will be assessed. First, we will assess ID4 DNA in primary tumors by molecular techniques and determine if its inactivation is predictive of metastasis to the SLN. Also, we will assess patients who have tumor in their SLN and determine if we can predict if adjacent nodes are negative. The next aim is to utilize our quantitative molecular assay to assess "circulating" ID4 DNA in blood as a marker for early disease recurrence and tumor spread. In a final aim, we will investigate the functional role of ID4 gene in breast cancer by inhibiting and enhancing its expression in breast cancer cell lines.

The major innovation of this study is the assessment of ID4 inactivation as a predictor of SLN metastasis in early stage breast cancer patients to determine who needs SLN surgery. The other goal of the study is to identify among patients who have tumors in their SLN, those whom will benefit from having additional node dissection. The study will utilize novel and innovative molecular techniques to determine the inactivation of ID4 by assessing very small tissue sections (8 micron) and to detect the presence of ID4 gene (DNA) in blood to identify breast cancer recurrence without invasive surgery. The assay and approach are innovative in addressing an issue not dealt with previously and may lead to improvement of early disease staging and prediction of disease outcome.

Final Report (2007)
Development of new molecular prognostic assays may improve survival and disease management. Metastasis of breast cancer to regional lymph nodes is one of the most important factors in predicting disease outcome. We identified a novel gene referred to as ID4 in breast cancer and found that its inactivation correlated with metastasis to the sentinel lymph node (SLN). This project was designed to validate whether the inactivation of ID4 gene relates to tumor progression and breast tumor spread. Our hypothesis was that the ID4 gene expression is epigenetically regulated (i.e., do not involve changes in the underlying DNA sequence) and controls other genes in breast cancer spreading. We examined both mRNA expression and methylation (i.e., a type of epigenetic gene silencing that involves the addition of a methyl group to DNA, especially cytosine) of the promoter region of ID4 gene.

This project involved: (1) the analysis of human breast cancer patient samples for expression levels and “epigenetic silencing” of ID4 for a correlation with cancer progress, and (2) cell-based studies to study the underlying mechanisms of ID4 functions. During this grant period we have accrued all the primary breast tissues and tumor-draining lymph nodes (SLN) for both DNA and RNA analysis of ID4. The accrual of patients material has been very successful and currently we analyzing the specimens and data. ID4 mRNA expression analysis by PCR in primary tumors and lymph node metastasis have been evaluated. We will continue to evaluate the data and hopefully publish the work even though the funding of the grant is terminated. The studies took longer than anticipated due to patient accrual logistics. The novel findings of this proposal indicate that the epigenetic state of the ID4 has potential as a prognostic marker of breast cancer. In the breast cancer cell-based studies, we have cloned ID4 and inserted the full length gene clone into a Lentivirus vector. This vector has been successfully introduced into several cell lines and shown to express the ID4 protein by Western blot analysis. This will allow us to determine the functional role of ID4 protein in breast cancer. Current studies are underway to delineate the mechanism of ID4 protein in breast cancer progression. We have identified two breast cancer cell lines that are "triple-negative" (ER and PR hormone receptor deficient and Her-2 negative) with ID4 expression approximately 3-4 folds lower than that in normal breast cell line (HBL-100). These cell lines are being assessed because of their resistant to current hormone therapies. ID4 differences in expression are an important observation. To assess the role of ID4 expression we also performed “gene knockdown” experiments. To determine the most efficient HuSH shRNA (29-mer) for ID4 knockdown we have stably and separately introduced four plasmid constructs carrying ID4 specific shRNA into breast cancer cells to determine the effect of downregulation of ID4 in normal and tumor cell lines. These cell studies have been highly successful and have reached the goals we anticipated with this grant.

The resources created with this CBCRP funding are very valuable will allow us to continue investigating ID4 mechanism in breast cancer tumor progression.