This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Breast cancer is the most common cancer in women worldwide and mortality from breast cancer is consistent due to tumor metastasis. Breast cancer patients initially respond to estrogen ablation therapy, but estrogen-independent cells almost always aggressively emerge. The disease eventually progresses to estrogen-independent breast cancer. Tumor is no longer responsive to estrogen ablation therapy and unrestrained progression of the disease is inevitable. Progress in understanding the etiology of this disease and developing therapies has been slow due to multiple deregulations of various genes. The additive effects against mammary tumor cells might be achieved by combining antitumor agents directed against one or more altered mechanisms in cancer. Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). Cell communication is important in the cooperation between neighboring cells. It is mediated through extracellular signals such as hormones (i.e. estrogen) and growth factors (i.e. IGF, EGF, and FGF), or through cell to cell interaction between adjacent cells. One type of cell communication is gap junctional intercellular communication (GJIC), considered to be of fundamental importance. Gap junction (GJ) channels allow the transfer of small molecules, which involve in the regulation of cell growth, differentiation, and function. Gap junctions are the only communicating junctions found in animal tissues, in all species, which are responsible for the direct traffic of ions and molecules with molecular weights less than 1,200 Daltons. These traffic ways are formed by the interaction between two hemichannels on the surface of opposing cells. These hemichannels are formed by the association of six proteins, the connexins. Because of the importance of intercellular junctions in the maintenance of the cellular homeostasis, the modulation of intercellular junctions and expression of connexin is involved in carcinogenesis. Most normal cells have functional GJIC, while most, if not all, tumors cells have dysfunctional GJIC. 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 PQ7 is 16 nM and PQ1 is 119 nM) through the enhancement of GJIC. Our data showed that PQ1 significantly increases gap junction activity and inhibits cell viability and colony growth of T47D breast cancer cells. Moreover, PQ1 and PQ7 decrease 71% and 99%, respectively, of xenograft breast tumors bearing mice. Interestingly, PQ-treated animals have a normal histological pathology. Furthermore, PQ1 and PQ7 have no effect on normal primary epithelial mammary cells. We have found that PQ1 has a very strong binding with Nedd4 (from surface plasmon resonance studies), an E3 ubiquitin ligase, and decreases the interaction of Nedd4 and connexin 43 (Cx43) and the phosphorylation of Cx43, which lead to the enhancement of GJIC. Thus, the principle hypothesis of this proposal is that PQs can 1) increase gap junction activities, 2) inhibit the interaction of Nedd4 and Cx43 and phosphorylation of Cx43, and 3) attenuate tumor growth. The goal of this application is to identify the molecular target(s) of this novel class of molecules through Si RNA high-throughput screening and study the morphological changes of cells treated with PQs through a collaboration with Dr. Rathnam Chaguturu at the KU High-Throughput Screening Laboratory.
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