Esther Chang, PhD - US grants

The studies of the molecular mechanism of inherited susceptibility to cancer are significantly aided by the identification and analysis of predisposing genes from familial cancer syndromes such as retinoblastoma, Wilms'Tumors, Neurofibromatosis Type I, and Familial Polyposis Coli. The tumor suppressor gene p53 has been shown to frequently altered in a wide variety of sporatic tumors. In these instances, one allele of p53 has been lost and the remaining allele has acquired a somatic mutation. The identification of a germ-line p53 mutation in cancer-prone families with Li-Fraumeni syndrome (LFS) has introduced a new role for p53, i.e., a role in genetic susceptibility to cancer. Our recent studies show that the normal skin fibroblast (NSFs) cells derived from individuals in the cancer- prone family express low levels of both wild-type (wt) and mutant (mt) p 53 protein at levels comparable to that of the wt p53 detected in normal cells. Furthermore, tumors arising from two germ-layers in four different individuals in this family exhibited the loss of the wt p53 allele and the retention of the mt allele. We have also known that many of the mutations in p53 found in LFS families appear to have a transdominant effect over the wt p53 in in vitro assays. To understand the role of germ-line p53 mutations in genetic predisposition and tumorigenesis, we propose to analyze the biological and biochemical characteristics of mt p53 found in this cancer-prone family with LFS. Therefore, we will examine the SV40 T Ag, MDM-2 and sequence specific DNA binding properties of p53 in these NSFs, and evaluate the effect of this wt/mt phenotype on cell cycle progression and apoptosis. Such analysis will provide information on the effects of mt p53 on the functions of wt p53 in a heterozygous situation. We will extend these studies to other LFS families in order to understand the broad implications of these findings for inherited p53 mutations. Since the p53 protein exhibits the properties of a transcription factor and binds to specific DNA sequences in vitro, the family NSFs with endogenous expression of both mt and wt p53 provide a good experimental system with which to identify target genes modulated by mutation in p53. The status of such genes will be evaluated by the analysis of either the expression of previously identified cell growth associated genes or the expression of genes which are differentially regulated in family NSFs (wt/mt). Thus, the experiments proposed in this application should serve not only to increase our understanding of the functional implications of mt p53 in predisposition, tumor formation and progression in cancer-prone families with LFS but should also yield information relevant to the function of p53 in tumorigenesis in general.

Squamous cell carcinoma of the head and neck (SCCHN), including that of the oral cavity, has a significant level of mortality and a high rate of recurrence. A significant portion of these clinical failures result from tumor cell radiation resistance (RR). Thus, the development of an effective method for sensitizing head and neck tumors to radiotherapy would have a profound effect on the treatment of this disease. Wild-type (wt) p53 plays a crucial role in apoptotic pathways leading to tumor cell death. The lack of functional p53 in many SCCHN tumor cells is thought to be responsible for their RR. The restoration of wtp53 function may restore the p53-mediated apoptotic pathway resulting in more efficient treatment. A combination of wtp53 gene therapy and radiation will be used to restore radiation sensitivity to RR SCCHN. A long-standing goal in gene therapy for cancer is a systemic delivery system that selectively targets tumor cells, including metastases. This application proposes to optimize a folate-linked liposome systemic delivery system for wtp53 to improve the efficacy of conventional radiotherapy. Preliminary in vivo results have proved in principle that restoration of wtp53 function enhances radiation induced apoptosis, leading to long term total tumor regression. In collaboration with a pharmaceutical partner, we will obtain GMP grade reagent, perform toxicology and pharmacokinetic studies to obtain IRB, IBC and FDA approval. Once approvals are obtained we will use this combination therapy in a Phase I clinical trial in an effort to translate this new and potentially more effective treatment modality into the clinic for head and neck cancer.

[unreadable] DESCRIPTION (provided by applicant): The low rate of cure of certain cancers such as lung cancer, the most common causes of cancer death, is an important health problem. Early detection appears currently to be the only way of improving the high mortality rate, but is quite difficult because of the lack of symptoms in early disease. Moreover, lung cancer is mimicked in its in vivo image appearance by benign lesions and processes that lower the specificity of detection. Current imaging methods include chest x-ray, CT, and MRI. While these current methods are able to identify curable lung cancer they also result in many false positives. They are also limited in the size of the lung nodules they can detect. Using available criteria, sensitivity for lung cancer detection is high, but specificity and positive predictive value are only moderate. Thus there is a need for enhanced sensitivity and specificity for cancer cells. Our Anti-transferrin Receptor scFv-antibody fragment (TfRscFv) immunoliposome complex (scL) is a nanoconstruct (~100 nm) for delivery of gene therapy to tumors. It has been shown to target various types of human tumor cells in vivo when implanted as xenografts in mice and is now in Phase I clinical trials for delivery of wtp53. What we are proposing in this application is a quantum jump in diagnostic accuracy, an approach specific to cancer and best for small cancers such as lung cancer. The method we are developing is a nano- sized immunoliposome complex delivering superparamagnetic iron oxide particles (SPIO). Iron Oxide particles are both paramagnetic and super-paramagnetic, giving a biphasic response with both T1 and T2* features. This complex targets cancer cells with high selectivity. Thus the efficient delivery of SPIO directly into the tumor cells by the scL-SPIO complex of this application can increase the conspicuity of the lung tumor cells. Moreover, based on previous studies, the nanocomplex delivered contrast agent which is the focus of this application should accumulate within the cancer cells themselves remaining for an extended period (hours) allowing the contrast in non-cancer areas to wash out, further enhancing cancer conspicuity. In preliminary studies using an as yet unoptimized scL-SPIO complex, we demonstrated tumor cell specificity as compared to free SPIO, and enhanced image intensity. More importantly, in earlier studies with scL complexed with another imaging agent, gadopentate dimeglumine (gad-d), in a lung tumor model, the scL-gad-d (and not free gad-d) was able to enhance and identify lung tumors as small as 1-4 pixels (0.1-0.4mm), a size smaller than possible with current technology. No toxicity was found with this complex. In this application we will optimize the scL- SPIO complex and fully characterize its capabilities for use in early detection of lung cancer in a mouse model of human lung cancer and extend our studies to a mouse model of primary lung cancer. In collaboration with investigators at NIST and NCI we will also asses the magnetic properties of the complex and determine it sub cellular localization and trafficking through the cell. Our goal is to perform the majority of the studies necessary for filing an IND as we aim to move rapidly towards clinical trials. If cancer is detected early (e.g. Stage I), it can in many instances be cured (lower mortality). The challenge is to be able to find and positively identify the cancer at this early stage, particularly lung cancer. While the current methods of detection are good, they can only detect tumors of a certain size. Moreover, lung cancer is often mimicked during imaging by non-cancerous lesions, resulting in uncertainty and many false positives, which are often resolved only by following growth of the tumor. Thus there is a pressing need for imaging agents that increase sensitivity and specificity. Our tumor-specific nano complex delivery of an MR imaging agent, e.g. gad-d and iron oxide, has shown great promise in our preliminary studies in this regard, demonstrating that its high affinity for cancer cells in the lung can result in improved sensitivity in detecting tumors and in overall specificity. The development of an imaging agent that can lead to earlier detection is a high priority in the war on cancer and could lead to increased survival. [unreadable] [unreadable] [unreadable]