A Novel HTP Assay for Identification of Compounds that Reduce PNC Prevalence

Northwestern University At Chicago, Evanston IL

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Abstract

[unreadable] DESCRIPTION (provided by applicant): Although extensive changes have been known to take place during transformation, the mechanism and the biological bases that promotes and maintains the malignant phenotype remains unclear due to the complexity of metastasis. Our lab has shown that the perinucleolar compartment (PNC), a multicomponent subnuclear structure, is unique to cancer cells derived from multiple solid tissues, both in vitro and in vivo (Huang et al., 1997; Kamath et al., 2005, Norton et al, 2008). We have shown that PNC prevalence (percentage of cells with at least one PNC) increases in parallel with breast cancer progression and reaches nearly 100% in distant metastases. In addition, high PNC prevalence in primary tumors correlates with poor patient prognosis (Kamath et al., 2005). Preliminary investigations of 4 other types of epithelial carcinomas; colon, ovarian, prostate, and cervix, show similar results. These findings suggest that PNC formation reflects advanced cellular transformation and is a surrogate biomarker of cellular metastatic capabilities. Importantly, the PNC is absent in normal cells in vivo, including human and mouse embryonic stem cells. Although the precise function of the PNC remains to be identified, the close association of the PNC with the metastatic phenotype makes it a simple morphological marker that reflects the complex trait of cellular malignancy. We hypothesize that PNC elimination/reduction can be used as a phenotypic marker for of a positive response to intervention with bioactive small molecules, thus, allowing for the discovery of novel compounds that selectively target malignant behaviors. The rationale is that compounds that eliminate the PNC should shift cell behavior toward benign by inhibiting processes that convey metastatic capability as reflected by the presence of the PNC. In other words, molecules that eliminate/reduce the PNC, although may not be directly targeting the PNC structure itself, may induce desired changes in cellular behavior upon treatment since the PNC reflects cellular malignancy. Our preliminary screening of NCI small compound libraries and others identified a group of compounds that effectively disassemble the PNC. Chemical-biology studies using representative compounds reveal that the PNC is directly associated with DNA and the nucleation of the PNC is dependent upon the integrity of the DNA structure. These results encourage further exploration of compounds with novel mode of action to help understand the function of PNCs. We have now developed a one-step cell based assay amenable for HTS. Our goal of this proposal is to identify PNC disrupting compounds that are selectively against malignant cells and malignant behaviors with novel modes of action, and to use these compounds to investigate the function of the PNC and its contribution to the malignant behavior of cancer cells. These studies will help understand cancer biology from a different angle and will potentially provide cellular targets and compounds for cancer treatment. PUBLIC HEALTH RELEVANCE: Cancer is a complex metastastic disease with high fatality. Although extensive changes that take place during transformation, the mechanism and the biological bases that promotes and maintains the malignant phenotype remains unclear. Our lab focus on a unique cellular structure that is selectively associated with metastastic phenotype of the cancer cells. We propose to use this marker to identify compounds that selectively target malignant cells, which will help understand the role of the structure in malignant behaviors of cancer cells and will also generate potential novel and selective anti-cancer agents. [unreadable] [unreadable] [unreadable]