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

OATP1B2 deficiency protects against paclitaxel-induced neurotoxicity

Paclitaxel is among the most widely used anticancer drugs and is known to cause a dose-limiting peripheral neurotoxicity, the initiating mechanisms of which remain unknown. Here, we identified the murine solute carrier organic anion–transporting polypeptide B2 (OATP1B2) as a mediator of paclitaxel-induced neurotoxicity. Additionally, using established tests to assess acute and chronic paclitaxel-induced neurotoxicity, we found that genetic or pharmacologic knockout of OATP1B2 protected mice from mechanically induced allodynia, thermal hyperalgesia, and changes in digital maximal action potential amplitudes. The function of this transport system was inhibited by the tyrosine kinase inhibitor nilotinib through a noncompetitive mechanism, without compromising the anticancer properties of paclitaxel. Collectively, our findings reveal a pathway that explains the fundamental basis of paclitaxel-induced neurotoxicity, with potential implications for its therapeutic management

Abstract 3550: Role of TNFα in the cytotoxicity of docetaxel and in docetaxel resistance in MCF-7 cells

Abstract Taxanes such as paclitaxel and docetaxel are widely used in cancer chemotherapy regimens due to their ability to stabilize microtubules and to arrest cell division in mitosis (Chazard, 1994). While the events leading to mitotic arrest are well understood, the mechanism by which the drug induces cytotoxicity is unclear. Paclitaxel has previously been shown to promote secretion of TNFα in macrophages; however this has not been observed in other cell lines (Bogdan, 1992). TNFα induces cell death in many cell lines through interaction with its receptor TNFR1 (Hsu, 1996). TNFα also binds to a second receptor, TNFR2, which does not possess a death domain. TNFR2 does however retain the ability to induce expression of NFκB-dependent survival genes. Our laboratory group recently selected MCF-7 cells for survival in increasing concentrations (doses) of paclitaxel or docetaxel (MCF-7TAX and MCF-7TXT, respectively). Resistance was only achieved at or above selection dose 9 (3.33nM docetaxel) (Hembruff et al., 2008). MCF-7 cells were also “selected” in the absence of these drugs to control for changes simply associated with extended cell propagation. The ABC transporter inhibitor cyclosporin A either had no effect on docetaxel sensitivity at selection dose 9, or partially restored sensitivity at selection dose 12 (Hembruff, 2008). These observations suggest there are likely additional mechanisms of docetaxel resistance at selection doses 9 through 12. Interestingly, we observed that docetaxel concentrations between 1.11nM and 3.33nM induced a &amp;gt;100-fold increase in TNFα secretion from MCF-7 cells, which likely contributes to cytotoxicity. Consistent with this view, a neutralizing TNFR1 antibody decreased cytotoxicity of docetaxel in MCF-7 cells. We also discovered that MCF-7TXT and MCF-7TAX cells at or above selection dose 9 were resistant to TNFα-induced cell death. Moreover, MCF-7TXT cells displayed downregulation of TNFR1 upon the acquisition of resistance to taxanes. Increased levels of TNFα were observed within the media of MCF-7TXT cells selected to dose levels 9 and 10, but not dose levels 11 or 12. We further provide evidence that the increased levels of TNFα in the medium of MCF-7TXT cells contributes to increased cell survivability through a TNFR2-dependent activation of NFκB and its resulting increase in the expression of cell survival genes. 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 3550.</jats:p