Marialuisa Lavitrano - Associate Professor
Department and institution: School of Medicine and Surgery, University of Milano Bicocca
tel. +39 02 6448 8336 - 8050
institutional profile page: UNIMIB
Research Area(s): Target therapy, Xenotransplantation, Atherosclerosis, Plaque
Identification of novel molecular therapy targets to overcome drug resistance of p53 null (or mutated) cancer cells
Cancer is a multistep process during which the mutation of four to six genes has to occur for the cell to become fully oncogenic. Genes affected by such mutations encode for regulators of the cell cycle, signalling molecules which transduce external stimuli into the cell (leading it to division, differentiation or apoptosis), molecules belonging to the pathways that allow the repair of genetic damage or that control the apoptotic process. Despite basic knowledge of cancer mechanisms has been constantly increasing in the last decades, translation of this knowledge into rational and effective therapy - such as molecular therapy, targeting single molecules specifically crucial for the survival of the tumor cell - has been a difficult process. Many of the available drugs, even those affecting specific molecules, act aspecifically via induction of DNA damage, to which the cell respond (when the pathways sensing the DNA damage are intact) by induction of apoptosis. Beside aspecificity at the molecular level, another major limitation in cancer therapy is drug resistance, very often occuring even after few cycles of therapy. On the whole, in order to find rational targets to better tailor cancer therapy, is necessary to identify novel genes involved in drug resistance.
Several studies have been already published aimed to the identification of cancer specific alterations in gene expression by micro-array technology. Although providing important informations, this technology has severe limitations in that it provides only lists of cancer-associated changes in gene expression (molecular profile), without shedding light on the functional implications of these changes.
To achieve the goal of identifying novel, and relevant, genes involved in drug resistance, we recently started, in collaboration with Kristian Helin (BRIC, DK) a project based on a genome-wide loss-of-function screen (RNAi library screen) having a specific cellular phenotype as readout. The Bernards laboratory (NKI, NL) has recently generated a set of 25,000 RNAi retroviral vectors that target 8300 human transcripts.
Introduction of such vectors into mammalian cells results in the creation of a tagged knock-down cell carrying a permanent gene-specific identifier, easily isolated by PCR amplification using vector-derived PCR primers flanking the hairpin-encoding DNA sequence.
Using this RNAi library in a colon cell line p53-null, and therefore resistant to 5FU-induced apoptosis, we have been able to identify 49 genes whose silencing revert the resistant phenotype, although to a variable extent, both in short-term and long-term assays. RNAi of 34 of them suppressed completely or almost completely (80%) colony growth upon 5FU. RNAi of 10 putative targets reduced colony growth from 30 to 60% and the knock out of the last 5 only slightly reduced the resistance to 5FU. Moreover, > 30% of the targets were validated not only in the cell line in which the screen has been originally performed but also in two other p53-mutated and 5FU-resistant colon cell lines, with different mutational background. We are currently studying two targets in more detail, trying to characterize the apoptotic pathways triggered by 5FU in absence of the abovementioned proteins. Future directions include: in vivo validation (colon cell lines in which the target has been silenced will be grafted in nude mice, and tumor regression will be evaluated after FU treatment); ex vivo analysis (expression of the targets will be investigated in human tumor samples, by means of tissue micro-arrays and when possible, expression analysis will be paired to clinical data; primary cells from tumor samples will be used to study the apoptotic response to 5FU in the absence of the target proteins). Moreover, other targets are being evaluated to be studied in further detail: we are focusing on those that, after preliminary experiments, appear to be involved in mediating drug resistance also in other types of epithelial tumors (ovary and lung).
Molecular Medicine and Animal Biotechnologies for Successful Organ
Transplantation: Study of Hyperacute and Chronic Rejection in Xenotransplantation
Transplantation has become one of the most successful stories of the recent years turning hopeless fight with end stage organ failure into almost full recovery of health and quality of life. This has been achieved mainly thanks to the development of new immuno-suppressive drugs and mastering surgical techniques.
Transplantation is today the therapeutic option of choice in case of end stage organ failure, in particular for kidney, liver, pancreas, hearth and lung. However, major limitation has become shortage of organs resulting in extensive waiting lists. There are also two other major limitations of successful organ transplantation: the damage done by reperfusion of an organ after ischemia and the chronic rejection; both these clinical conditions are not affected by the immuno-suppressive treatment.
The objectives of the research project is to address the current problems related to organ transplantation: 1) organ shortage by generating an alternative source for organ transplantation and 2) long-term function and survival of xenotransplanted organs. The availability of pig organs and cells to treat patients in terminal organ failure would represent a revolution in human medicine and solve the problem of the organ shortage. Pigs are considered as the optimal source of organs for human transplantation. The biggest hurdle to xenotransplantation has been that of hyperacute and acute vascular xenograft rejection. It is our objective to identify and overexpress genes that encodes proteins blocking rejection factors. To accomplish this objective, we shall prevent the inflammatory process, thrombosis (platelet aggregation and coagulation) and apoptosis in xenotransplanted organs, and the first set of selected genes are the following: This specific knowledge would allow generation of a multi-gene transgenic source animal from which organs or cells could be taken and transplanted successfully to humans. Such a program could benefit hundreds of thousands of patients per year.