Alterations in tumor necrosis factor signaling pathways are associated with cytotoxicity and resistance to taxanes: a study in isogenic resistant tumor cells

INTRODUCTION: The taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance. METHODS: MCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR). RESULTS: MCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways. CONCLUSIONS: We report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

Background: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. Methods: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. Results: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. Conclusions: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer

Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance

<p>Abstract</p> <p>Background</p> <p>Anthracyclines and taxanes are commonly used in the treatment of breast cancer. However, tumor resistance to these drugs often develops, possibly due to overexpression of drug transporters. It remains unclear whether drug resistance <it>in vitro </it>occurs at clinically relevant doses of chemotherapy drugs and whether both the onset and magnitude of drug resistance can be temporally and causally correlated with the enhanced expression and activity of specific drug transporters. To address these issues, MCF-7 cells were selected for survival in increasing concentrations of doxorubicin (MCF-7_DOX-2), epirubicin (MCF-7_EPI), paclitaxel (MCF-7_TAX-2), or docetaxel (MCF-7_TXT). During selection cells were assessed for drug sensitivity, drug uptake, and the expression of various drug transporters.</p> <p>Results</p> <p>In all cases, resistance was only achieved when selection reached a specific threshold dose, which was well within the clinical range. A reduction in drug uptake was temporally correlated with the acquisition of drug resistance for all cell lines, but further increases in drug resistance at doses above threshold were unrelated to changes in cellular drug uptake. Elevated expression of one or more drug transporters was seen at or above the threshold dose, but the identity, number, and temporal pattern of drug transporter induction varied with the drug used as selection agent. The pan drug transporter inhibitor cyclosporin A was able to partially or completely restore drug accumulation in the drug-resistant cell lines, but had only partial to no effect on drug sensitivity. The inability of cyclosporin A to restore drug sensitivity suggests the presence of additional mechanisms of drug resistance.</p> <p>Conclusion</p> <p>This study indicates that drug resistance is achieved in breast tumour cells only upon exposure to concentrations of drug at or above a specific selection dose. While changes in drug accumulation and the expression of drug transporters does occur at the threshold dose, the magnitude of resistance cannot be attributed solely to changes in drug accumulation or the activity of drug transporters. The identities of these additional drug-transporter-independent mechanisms are discussed, including their likely clinical relevance.</p

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

Background: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells.Methods: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies.Results: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model.Conclusions: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.Peer reviewedBiochemistry and Molecular Biolog

Loss of Transforming Growth Factor-β Signaling in Mammary Fibroblasts Enhances CCL2 Secretion to Promote Mammary Tumor Progression through Macrophage-Dependent and -Independent Mechanisms

Whereas the accumulation of fibroblasts and macrophages in breast cancer is a well-documented phenomenon and correlates with metastatic disease, the functional contributions of these stromal cells on breast cancer progression still remain largely unclear. Previous studies have uncovered a potentially important role for CCL2 inflammatory chemokine signaling in regulating metastatic disease through a macrophage-dependent mechanism. In these studies, we demonstrate a significant regulatory mechanism for CCL2 expression in fibroblasts in mediating mammary tumor progression and characterize multiple functions for CCL2 in regulating stromal-epithelial interactions. Targeted ablation of the transforming growth factor-β (TGF-β) type 2 receptor in fibroblasts (Tgfbr2FspKO) results in a high level of secretion of CCL2, and cografts of Tgfbr2FspKO fibroblasts with 4T1 mammary carcinoma cells enhanced tumor progression associated with recruitment of tumor-associated macrophages (TAMs). Antibody neutralization of CCL2 in tumor-bearing mice inhibits primary tumor growth and liver metastases as evidenced by reduced cell proliferation, survival, and TAM recruitment. Both high and low stable expressions of small interfering RNA to CCL2 in Tgfbr2FspKO fibroblasts significantly reduce liver metastases without significantly affecting primary tumor growth, cell proliferation, or TAM recruitment. High but not low knockdown of CCL2 enhances tumor cell apoptosis. These data indicate that CCL2 enhances primary tumor growth, survival, and metastases in a dose-dependent manner, through TAM-dependent and -independent mechanisms, with important implications on the potential effects of targeting CCL2 chemokine signaling in the metastatic disease