Professor
Department of Biochemistry

Cancer resistance to chemotherapy; genetic models of innate resistance or susceptibility to disease

Francesco Bellini Life Sciences Building
3649 promenade Sir-William-Osler
Office: Rm 366; Lab: Rm 370
Montreal, Quebec H3G 0B1
Tel: 514-398-7291; Lab: 514-398-2542
Fax: 514-398-2603
Email

1983 - PhD, McGill University

Distinguished Investigator of the CIHR.
Fellow of the Royal Society of Canada.

Research Interests

P-glycoprotein and MRP

The emergence of multidrug resistant tumor cells is a major limitation in the successful chemotherapeutic treatment of many types of human tumors. Multidrug resistance in cultured cells in vitro and in certain tumors in vivo is associated with the overexpression of P-glycoprotein (P-gp), or the Multidrug resistance protein 1 (MRP1), both integral membrane proteins members of the ABC superfamily of transporters, that efflux a large number of structurally unrelated chemotherapeutic drugs in an ATP-dependent fashion. The precise mechanism by which P-gp and MRP1 recognize and transport structurally dissimilar substrates, and the mechanism by which ATP hydrolysis at one or both of its nucleotide binding (NB) sites effects transport remain obscure and need to be better understood for the rational design of efficient inhibitors for clinical use. Three aspects of Pgp and MRP1 function are being studied:

1. ATP binding and hydrolysis by the two NB sites;
2. drug binding determinants located in the TM domains;
3. protein segments responsible for signaling between drug binding TM domains and ATP hydrolyzing NB sites.

Independently, we are attempting the identification of new genes and proteins possibly involved in cellular response and resistance to chemotherapeutic drugs in tumor cells. For this, a high throughput approach based on microarray technology is being used to establish cellular transcriptional profiles for different human cultured cells before and after short- and long-term exposures to different classes of chemotherapeutic drugs (different doses and exposure regimens). We are looking for genes up-regulated in response to different or specific types of drugs; the relevance of these up-regulated genes and proteins in protection against, and resistance to chemotherapy will be systematically assessed in transfection experiments. Parallel experiments using RNA specimens derived from primary human cells will also be carried out to test the possible relevance of these genes/proteins in resistance to chemotherapy in vivo in human tumors.

Loop-tail (Ltap)

The loop-tail mutation was one of the first mouse mutant ever described. Lp/+ heterozygotes have a "looped" tail, while homozygotes have a very severe form of neural tube defect (NTD) called craniorachischisis, characterized by a completely open tube and mid-stage embryonic death. Although described some 60 years ago, this mutant was only recently cloned in my lab. The protein encoded by this gene is particularly exciting since it is an integral membrane protein that is extremely conserved throughout evolution from insects to humans. The Ltap protein is believed to be essential for assembling a Wnt signaling complex at the cell membrane of proliferating epithelial cells, where it regulates the complex process of convergent extention during embryonic development.

During the coming years, we will be trying to characterize this membrane complex and understand how it provides spatial and polarity information to the cells. We also plan to study the intracellular signaling network regulated by this complex, and ultimately identify interacting proteins and transcriptional targets of their action. We have also recently cloned a close homolog of Ltap, named Stbm, which also shows embryonic expression in the developing neural tube. In the future, we will be working aggressively to determine if Ltap and Stbm are indeed involved in the etiology of spina bifida and anencephaly in humans.

Nramp1 and Nramp2

Several years ago, my laboratory identified by positional cloning the Nramp1 gene in which mutations cause susceptibility to a variety of infections including Mycobacterium, Salmonella and Leishmania in mice. Later, parallel genetic studies in humans established that polymorphic variants in the human NRAMP1 gene are associated with susceptibility to tuberculosis, leprosy and also to certain inflammatory conditions such as inflammatory bowel disease, and juvenile diabetes. We have shown that the Nramp1 protein plays a pivotal role in macrophages and neutrophils, where it functions as a metal efflux pump at the membrane of pathogen-containing phagosomes. The bacteriostatic/bactericidal activity of Nramp1 is complex and relies on deprivation of divalent metals in the phagosomal space, such metals being essential to the different pathogens for expression of their respective intracellular survival strategies. Many aspects of the molecular structure and biochemical mechanism of transport by this protein are being studied.

One particularly exciting area of future work will be the use of transcriptional profiling with bacterial genomes microarrays to identify bacterial genes that are differentially regulated in the phagosome in presence or absence of Nramp1. We believe that the pursuit and identification of such genes will provide significant insight into bacterial and mammalian genes playing a key role at the interface of host:parasite interaction. Such bacterial proteins could represent novel and exciting targets for drug development and intervention in such important diseases such as TB, diahrreal diseases and others. The close homolog Nramp2 is also a metal transporter and we recently shown that it is the major iron acquisition system of the body at the intestinal brush border, but also functions in the membrane of recycling endosomes in all cells to transport transferrin-delivered iron into the cytoplasm. Consequently, mouse mutants with inactivating mutations at Nramp2 develop severe microcytic anemia, and impaired iron metabolism in peripheral tissues.

We are interested in Nramp2 as a target for therapeutic intervention at two sites. First, at the duodenum brush border where its therapeutic inactivation by small molecules in situ could lead to new treatment opportunities for iron overload diseases such as hemochromatosis. Also, we have detected Nramp2 expression at the brush border of the kidney proximal tubule where it may act as a previously unsuspected iron re-uptake system. We will elucidate the physiological role of Nramp2 at that site. Should these experiments be positive, we believe that Nramp2 in the kidney would be a great target for drug development for disease such as hemochromatosis, through systemic inactivation of such a re-uptake system.

Lgn1 (Legionella pneumophila)

The chromosome 13 locus Lgn1 controls intracellular growth of Legionella pneumophila in mouse macrophages. The gene maps in a complex duplicated region of the genome that contains between 5 and 8 copies of the Naip gene (Naip1-Naip8), a gene previously shown to affect apoptosis in motor neurons, and found to be associated with spinal muscular atrophy in humans.

Despite having excellent genetic, and physical map of the locus, including a recently completed nucleotide sequence (475kb), positional cloning remained elusive until recently. We were able to implement a new cloning strategy, that of functional cloning in vivo in transgenic mice using bacterial artificial chromosomes overlapping large discrete fragments of the region. This approach identified Naip5 as the only Naip copy present in 2 BAC clones showing phenotypic rescue in vivo. Having shown that Naip protein expression is robust in macrophages, and further stimulated by phagocytosis of pathogens, including Legionella, our research focus in the next few years will be to understand the role of Naip protein in pathogenesis or normal response of macrophage to infections.

In particular, we will investigate how Naip proteins may regulate or antagonize microbial-induced apoptosis in phagocytes, the exclusive ecological niche of Legionella in mammals. These studies will include testing a possible implication of Naip in different cellular pathways known to be involved in apoptosis, and also try to determine how such pathway may antagonize the intracellular survival strategy of Legionella, including seclusion in a replicative vacuole, and inhibition of maturation to phagolysosome.

Complex Traits

Tuberculosis and malaria are 2 of the top 4 causes of death by infectious diseases worldwide, with a combined estimated 4 million death per year. In both cases genetic factors are known to influence response, but the genetic control is complex and difficult to study in humans.

Susceptibility to pulmonary tuberculosis is a complex trait under multigenic control in humans and mice. DBA/2J (D2) mice are extremely susceptible, with rapid bacillar replication in the lung, intense inflammatory response at that site, and they succumb rapidly to infection compared to C57Bl/6J (B6). Recently, we have used quantitative trait locus (QTL) mapping and whole genome scanning to localize four mouse loci (Trl-1, Trl-2, Trl-3, Trl-4) that control pulmonary replication and overall survival time following aerosol infection with M. tuberculosis, and that account for ~75% of the variance in a D2 X B6 cross.

In the coming years, the role of these loci, including contribution to individual aspects of host response and pathogenesis, will be studied after their isolation by breeding experiments in congenic strains. Examination of genomic DNA sequence near the mapped loci, together with transcriptional profiling studies in target tissues and cell types from these congenic lines will be used to select candidate genes. These candidates will be validated in gene targeting experiments, and will be used as entry points in parallel studies in humans to establish the possible role of these genes as pre-disposing factors for tuberculosis. Parallel studies will be conducted to understand better the molecular mechanism of action and role of these genes and proteins in pathogenesis of TB.

Using an experimental infection model with Plasmodium chabaudi, we have shown that susceptibility to Plasmodium infection (malaria) is also under complex genetic control in the mouse. So far, we and others have mapped loci on chromosomes 8 (char2) and 9 (char1) that independently control blood parasitemia and survival to infection, respectively. Novel additional loci regulating response to P. chabaudi infection were investigated using an alternative strategy based on a newly derived set of AcB/BcA recombinant congenic strains (RCS) bred from malaria-susceptible A/J (A), and resistant C57BL/6J (B6). Individual RCS contain a small amount (12.5%) of DNA from one parent fixed as a set of discrete congenic segments, on the background (87.5%) of DNA from the other parent. Therefore, individual resistance/susceptibility loci may independently segregate in individual RCS. The relatively small size of the congenic segments fixed in individual RCS also facilitates the search and testing of candidate genes. Using these strains, we have so far mapped a novel malaria resistance locus on Chr. 3 designated Char4.

In the coming years, we will characterize the physiological pathway regulated by Char4, and will elucidate its molecular mechanism of action, which so far appears to involve regulation of erythropoiesis. We have also identified another RCS which shows a unique resistance phenotype unrelated to the action of Char1, 2 and 4, and will pursue the genetic analysis of this strain as well. In these studies, we will again take advantage of the small size of the congenic segment, as well as a global transcriptional profiling and mapping strategy to identify the gene responsible.

Publications - Philippe Gros