Abstract
Background. Triple-negative breast cancers (TNBC) are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. Due to the absence these molecular targets and to their intrinsic biological heterogeneity, treatment options for TNBC patients are limited to conventional chemotherapy. Unfortunately, standard chemotherapy is less effective in women with TNBC, indicating that new therapeutic approaches are urgently needed. Our previous findings demonstrated that TNBC cell lines are particularly sensitive to the purine-scafold Hsp90 inhibitor PU-H71. Moreover, PU-H71 exhibited a potent and durable anti-tumor activity in TNBC xenografts inducing complete responses without toxicity to the host (Abstract 5637, AACR-Annual Meeting 2008). Study design. In order to determine the mechanisms underlying the effect of PU-H71 in TNBC, we isolated Hsp90 complexes containing co-chaperons, adaptors and client proteins using PU-H71 immobilized on beads, followed by LC/MS/MS. Findings were independently confirmed by western blots. Three different TNBC cell lines were used (MDA-468, MDA-231 and Hcc1806) in triplicate experiments in vitro, and some of the results confirmed in tumor xenografts in vivo. Results. Several oncogenic pathways responsible for cell proliferation and migration are activated in TNBC including those driven by EGFR, IGF-IR, HER3 and c-Kit. These proteins activate the core unit composed by Ras/Raf/MEK/ERK that ultimate regulates the expression of critical genes for TNBC. We found that in TNBC cells, EGFR, IGF-IR, HER3, c-Kit and Raf-1, are in a complex with Hsp90 and are preferentially targeted for degradation by PU-H71. We also identified new Raf/MEK/ERK pathway components bound to Hsp90, such as ERK2 and p90RSK. Recent reports suggested that Akt activation plays a role in therapy resistance in breast cancer. We found that the Ser473 phosphorylated (active) Akt as well as its activator, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), are in complex with Hsp90 in TNBC cells and are sensitive targets of PU-H71 both in vitro and in vivo. PU-H71 was able to inactivate Akt in TNBC cells inducing apoptosis to a degree similar to specific Akt inhibitors, suggesting that activated Akt is an important apoptotic target of PU-H71. In addition, we found that PU-H71-dependent inhibition of NF-kB, Akt, ERK2, Tyk2 and PKC is involved in the reduction of the invasive potential of these tumors. Conclusion. Our findings indicate that a multitude of TNBC driving proteins, including those involved in cell cycle progression, proliferation, cell survival and invasion, exist in complex with the Hsp90 complexes and are recognized by PU-H71. In conclusion, the pluripotent effect of PU-H71 in TNBC cells is highly connected to its ability to inactivate these complexes in a parallel fashion, leading to downregulation of a large number of cancer activating molecules.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-7.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO
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