Research Grants Awarded
translational control of metastases
Tumor Cell Biology I
Cancer gene therapy is at the forefront of medicine, but is currently subject to three major constraints: 1) method of delivery, 2) specific gene expression, and 3) efficacy vs. toxicity. One promising strategy is based on HTK/ganciclovir (GCV) suicide system. Most of the previous work has been directed at targeting primary tumors, whereas treatment of metastasis remains largely uncharted. We propose a novel approach targeting a characteristic that distinguishes cancer from normal cells, i.e., elevated translation initiation factor eIF4E, thus allowing selective killing. eIF4E is a component of the helicase that unwinds excess structure in the 5’UTR of mRNAs. Elevated eIF4E specifically facilitates the translation of mRNAs with a long and structured 5’UTR. With this in mind, the expression of HTK was selectively regulated by placing the 5’UTR of FGF2, previously found to be translationally regulated, upstream of the HTK open reading frame. The idea behind this construct is to obtain a more selective target to GCV killing by limiting the expression of HTK to cancer cells, not normal cells which are unable to translate this mRNA. This is important, because in addition to killing cancer cells at the primary tumor site, the ability to selectively attack metastasis could be possible. Preliminary results show that this was feasible in one cell model. Experiments are proposed with a panel of normal and cancer cell lines to determine the general applicability of the system: pattern of HTK expression, differential sensitivity to GCV, and enzymatic activity. Additional constructs with synthetic hairpins at 5’UTR will also be tested. Various construct- transfection reagent complexes will be also injected in mice to determine efficacy (i.e., regression of tumors) vs. toxicity to distal organs. The capacity to reduce or eliminate lung metastases and to extend the survival of mice will be determined. All these effects will be correlated with the distribution of the vectors in tumors and normal tissues by real-time PCR and IHC analysis. The pattern of expression of HTK will also be monitored by a variety of methods, including an in vivo imaging (PET) system that takes advantage of the incorporation of [18F]GCV by cells expressing HTK. This allows for direct monitoring of the pattern of HTK expression in tumors and normal organs without killing the animals, which can be subsequently treated with GCV to eliminate the tumor nodules and verify organs toxicity.
Breast cancer affects over 200,000 women yearly in the US, with a mortality of ~25%. One of the major problems in cancer treatment today is the failure of standard therapy to treat cancer recurrence and metastasis. Specifically, patients succumb to outgrowths of resistant metastatic cancer, which do not respond to customary radiation and chemotherapeutic options without fatal toxicity. The dynamic cellular make-up of tumors, as well as their widespread dispersion to various organs, often prevents effective treatment. Advances in molecular biology have elucidated many cellular functions involved in tumor growth and spread. These tumor cell-specific properties are being exploited in a number of novel therapies in which the cancerous cells are targeted in ways that circumvent their resistance to standard treatment. Suicide gene therapy offers the hope of being able to deliver a “magic bullet” to cancer cells to obliterate primary tumors and metastases. Specific delivery to metastasis is a very complex problem and, to date, no delivery system developed was specific enough to guarantee that normal tissues will not be targeted, thus preventing lethal toxicity. To obviate this problem, our work is aimed at targeting a specific gene expression property of cancer cells, i.e., the ability to translate mRNAs with a complex structure upstream of the protein coding information (5’UTR), due to high level of a translation factor (eIF4E). Therefore, even if normal tissues and organs were transfected by the suicide gene/vector they would not be able to express the suicide product, which is an enzyme that converts a non-toxic drug into a toxin. Thus far, we have tested a natural 5’UTR of FGF-2 which would be efficiently translated in cancer, and not normal cells. We now want to optimize and fine-tune the molecular design of the suicide vector and the mode of delivery thereby, completing the preliminary development of this novel approach, which would set the stage for its use in clinical trials in the near future. Experiments are proposed to eliminate metastatic breast cancer in mice with a targeted suicide gene therapy approach.