My laboratory is exploring the effects of intracellular calcium
homeostasis on cancer growth and dissemination. This interest directs our
current working hypotheses that the modulation of calcium and calcium-driven
signaling events alters cellular activity and gene expression. This work may
point to new directions for cancer therapy.
Our current line of research began when we observed that changes in cellular signaling, including blocking an increase in intracellular calcium, could abrogate the usual migration of tumor cells in response to growth factors and cytokines. Through a screening program consisting of motility assays and calcium-influx experiments, we identified a carboxyamido-triazole compound, which we call CAI, that inhibits calcium influx and calcium-influx-dependent signaling events. CAI has proven to be a useful tool in our investigation of the modulation of calcium concentrations and calcium-linked mechanisms both in vitro and in vivo.
Our studies have demonstrated that calcium homeostasis plays an important role in the process of angiogenesis, which is a form of physiologic invasion of new blood vessels into tissue that occurs during wound healing, pregnancy, and tumor growth. CAI disrupts the normal function of the cytoskeleton of endothelial cells, reduces expression of proteolytic enzymes, and decreases neovascular potential in vitro--all of which are key steps in angiogenesis, suggesting that CAI may be a useful agent in the treatment of cancer. We have also observed a marked anti-angiogenic effect of CAI in vivo, in chicken chorioallantoic membrane (CAM) assays. We are now investigating the immediate signaling effects of altered calcium balance in the endothelial cells as part of the hypothesis that calcium homeostasis is important in physiologic, as well as malignant, invasion.
We are also studying the regulation of gene expression as a function of calcium modulation, or signaling balance. We developed a human melanoma cell subline that is resistant to constant exposure to CAI and observed a phenotypic difference between resistant cells and nonresistant cells. Unexpectedly, the resistant cells displayed reduced tumorigenic potential as measured by reduced density-independent growth and reduced tumorigenesis in xenografts of human tumors in nude mice. This led to a molecular investigation comparing resistant and nonresistant cells that led to the discovery of several genes that are currently being cloned.
Early studies found that CAI treatment reversibly inhibited the proliferation and invasive capacity of more than 25 types of tumor cells. The oral administration of CAI to human xenograft-bearing mice resulted in a reduction in total tumor burden and metastatic dissemination without marked toxicity to normal tissues. Our in vitro and animal observations have led to phase I clinical trials of CAI in solid-tumor patients with advanced and refractory cancer. Since the trial was initiated in 1992, more than 60 patients have received CAI. So far, CAI has been well tolerated and has resulted in disease stabilization, as characterized by a reduction in both the size and number of tumors and by improved symptoms. Both the CAI study and a trial of CAI in combination with Taxol are ongoing and open to patient entry.
The major effort of my laboratory is currently focused on understanding
the molecular pathogenesis of human leukemias and lymphomas caused by nuclear
oncogenes. This effort often coincides with the lab's secondary interest--the
molecular regulation of B-lymphocyte development. Our early work defined a
novel lymphoid-restricted transcription factor, Oct-2, which was a founding
member of the POU domain class of homeobox transcription factors. Now we are
studying two lymphoid malignancies: diffuse large-cell lymphoma caused by the
BCL-6 oncogene and t(4;11) acute lymphoblastic leukemia caused by a
fusion oncoprotein involving the MLLandAF-4 genes.
Diffuse large-cell lymphoma, which is a malignancy of mature B lymphocytes,
accounts for 40% of all cases of non-Hodgkin's lymphoma. In 40% of diffuse
large cell lymphomas, theBCL-6 gene is rearranged by translocations
that leave the BCL-6 coding region intact but that substitute its
promoter region with regulatory regions from other genes. The BCL-6 gene
encodes a zinc-finger transcription factor that shares a
121-residue
amino-terminal homology domain, the POZ domain, with a subset of other
zinc-finger proteins.
In studies involving normal lymphocytes, we have shown that BCL-6 mRNA is highly expressed in mature B cells but not in terminally differentiated, antibody-producing plasma cells, and that activation of lymphocytes downregulates BCL-6 mRNA. The BCL-6 protein is phosphorylated and is expressed highly in the germinal center, the site where memory B cells and plasma cells are generated. These findings have led to our working hypothesis that BCL-6 expression must be downregulated for terminal B cell differentiation to occur and that such regulation is absent in diffuse large cell lymphoma. BCL-6 presumably transforms B lymphocytes by regulating the transcription of key target genes. We have identified high-affinity binding sites through which BCL-6 functions as a potent transcriptional repressor, and we have shown that its POZ domain is necessary and sufficient for repression. Currently, we are trying to identify the mechanism underlying this transcriptional repression and the natural targets of BCL-6 repression.
Our second major project is aimed at understanding the molecular pathogenesis of t(4;11) pro-B-cell acute lymphoblastic leukemia in which theAF-4 gene is fused to the MLL gene. This translocation is found in 60% of acute lymphoblastic leukemia cases in children under 12 months old. The MLL gene, which is a homologue of the Drosophila melanogaster regulatory protein, trithorax, is translocated to several different chromosomal loci in a variety of acute leukemias. Each translocation generates an in-frame fusion protein between the amino terminus of MLL and the carboxy terminus of the fusion partner. Our interest in this leukemia stems from our cloning of a lymphoid-restricted homologue of the AF-4 gene, termed LAF-4. Neither LAF-4 nor AF-4 show significant homology to previously cloned transcription factors. We have shown that LAF-4 is a nuclear protein and have found that both LAF-4 and AF-4 have potent transcriptional activation domains. Thus, LAF-4 and AF-4 are the founding members of a new family of nuclear transactivator proteins. Intriguingly, the AF-4 activation domain is retained in the MLL-AF-4 fusion oncoprotein, suggesting that this domain may contribute to the oncoprotein's transforming properties.