Abstract 4448: Disruption of nedd4 and connexin 46 interaction by gap junction activator in breast cancer cells

Abstract

Abstract Breast cancer is the second most common cancer among women; it is also the second leading cause of cancer death among women in the United States. Progress in understanding the etiology of this disease and developing therapies has been slow due to multiple deregulations of various genes. Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). Most normal cells have functional gap junctional intercellular communication (GJIC), while most, if not all, tumors cells have dysfunctional GJIC. GJIC consists of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, cell signaling and previous studies have been suggested their participation in tumor formation and progression. It is believed that restoring GJIC is linked to drug sensitivity and reduction of tumorigenicity. Thus, increasing gap junction activity or enhancing GJIC in tumor cells provides the targets to enhance anti-neoplastic therapies. Several GJIC enhancers have been reported; however, an effective clinical drug targeting gap junction is not available at this time. Recently, we synthesized a new class of substituted quinolines (code name: PQ) and found that they possess potent inhibitory activities against T47D breast cancer cells (IC50 value of PQ1 is 119 nM) through the enhancement of GJIC. Since the mechanism of action of GJIC enhancement caused by PQ is unknown, the current study is to determine the target proteins that PQ interacts with, which may lead to the mechanism of enhancement of GJIC and inhibition of cancer cell growth. Using surface plasmon resonance (SPR) spectroscopy, we have found that PQ1 has a strong binding with Nedd4, E3 ubiquitin ligase which previously been shown to involve in the degradation of gap junction. T47D breast cancer cells were treated with various concentrations of PQ1 for 24 hours. Whole cell extract was isolated and subjected to immunoprecipitation assay, followed by western blot analysis. The results show that 100 nM PQ1 can cause a 2-fold decrease in the interaction of Nedd4 and connexin 46 compared to control without treatment or solvent. Since gap junction turnover has been shown to be facilitated by lysosomal and proteasomal degradation, disruption of Nedd4 and connexin 46 suggests that PQ1 may inhibit the lysosomal and proteasomal degradation of connexin 46 in breast cancer cells. Future study will focus on whether PQ causes an increase in gap junction activity via inhibition of Nedd4-ubiquination of connexin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4448.