Molecular studies of metastasis facilitated by a rapid autopsy program

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA SY32-01 Considerable progress has been made in recent decades in the treatment of breast cancer. However, achieving a permanent cure by eradicating metastatic breast cancer has remained elusive. As a first step to devising suitable therapies, identification of alterations in expression levels of genes/proteins that are typical of metastatic breast cancer and its distant metastasis is necessary. It is also clear that communication between tumor cells and other cells in the microenvironment, such as the vascular endothelial cells (ECs), the stromal fibroblasts, and immune cells, is critical for tumor growth. These studies have been facilitated through a Rapid Autopsy Program (RAP) at Johns Hopkins. Since its inception two years ago, we have succeeded in banking hundreds of metastatic lesions from varied sites from 14 patients. These tissues, characterized for several markers and methylation profiles, some grown as xenografts, are a rich resource for research in metastasis. We have performed SAGE and microarray analysis to identify several epithelial molecular markers overexpressed in breast cancers. Considerable progress has been made in understanding the biology and function of two transcription factors, HOXB7 and HEYL. We found that these markers were also overexpressed in breast cancer that had metastasized to multiple organs. colelcted through the RAP. Biological and functional analysis of HOXB7 adn HEYL was performed at the biochemical and molecular level, and using multiple imaging modalities, Experimental evidence is mounting that HEYL may act as an oncogene by antagonizing the cytostatic effects of the TGF-beta pathway. HEYL binds SMAD 3 resulting in the repression of TGF-b pathway activity. HEYL is able to repress pAI and p15 mRNA expression, both of which are downstream targets of TGF-beta. HEYL IHC has been developed and TMAs of tumors with outcome information have been tested. HEYL overexpression correlates with poorer disease outcome. In an independent investigation, this antibody has been given to Dr. Nicole Urban to test its utility in ELISA assays for early detection of breast cancer in serum. HEYL can transform NIH3T3 to tumorigenicity. Compound AEE (Novartis) was partially effective in HEYL cells with maximum inhibition of 40% at doses from 0.1μM-1 mM. HOXB7 is overexpressed in approximately 30% of primary breast cancers and to greater extent in bone metastases. By 1H NMR imaging studies, altered choline phospholipids and increased vascular permeability were seen in HOXB7-expressing MCF-7 cells. MCF-7-HOXB7 cells express increased levels of tyrosine kinase receptors (e.g. EGFR and HER-2) as well as increased levels of ERα and appeared to be similar in this regard to MCF-7 cells that are resistant to letrozole, a model of endocrine resistant breast cancer. A search for compounds that target HOXB7 and inhibit cell growth in vitro and also tumor growth in vivo revealed that HOXB7-overexpressing MCF7 cells respond to nM levels of VN/14-1, a newly synthesized atypical RAMBA (retinoic acid metabolism blocking agent)/aromatase inhibitor active against both letrozole- and tamoxifen-resistant breast cancer cells. Consistent with this finding, the aromatase inhibitors letrozole and anastrozole are effective in suppressing proliferation of MCF-7-HOXB7 cells in vitro at IC50 concentrations 1-2 nM. This suggests that patients whose breast tumors overexpress HOXB7 and are resistant to treatment with tamoxifen may be responsive to sequential treatment with the aromatase inhibitors. In addition, the tyrosine kinase inhibitor gefitinib and AEE (Novartis) inhibited MCF-7-HOXB7 cell proliferation. The m-Tor inhibitor RAD-001 (Novartis) was also an effective inhibitor of HOXB-7 cells at low nM concentrations. These results compliment our findings in letrozole resistant cells and indicate that crosstalk is occurring between estrogen signaling and tyrosine kinase receptor pathways. VN/14-1 is poised to enter clinical trials. HOXB7 confers tamoxifen resistance and metastasis; both aspects were replicated in a newly developed model of mammary tumors arising in double transgenic HOXB7/neu mice. From this knowledge, we will finally devise combinations of immune and drug therapy that target the tumor and its microenvironment to prevent and treat metastatic breast cancer and its micro- and macrometastasis most effectively.