Alterations in tumor necrosis factor signaling pathways are associated with cytotoxicity and resistance to taxanes: a study in isogenic resistant tumor cells.
BACKGROUND
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.
Regional Cancer Program, Sudbury Regional Hospital, 41 Ramsey Lake Road, Sudbury ON P3E 5J1, Canada.
Jason A
JA
Sprowl
Kerry
K
Reed
Stephen R
SR
Armstrong
Carita
C
Lanner
Baoqing
B
Guo
Irina
I
Kalatskaya
Lincoln
L
Stein
Stacey L
SL
Hembruff
Adam
A
Tam
Amadeo M
AM
Parissenti
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms; D002470:Cell Survival / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D000077143:Docetaxel; D019008:Drug Resistance, Neoplasm; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D006801:Humans; D061986:MCF-7 Cells; D016328:NF-kappa B / Q000378:metabolism; D010051:Ovarian Neoplasms; D017239:Paclitaxel / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D059748:Proteolysis; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D043823:Taxoids / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D016328:NF-kappa B; D011500:Protein Synthesis Inhibitors; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D043823:Taxoids; D014409:Tumor Necrosis Factor-alpha; D000077143:Docetaxel; D003513:Cycloheximide; D017239:Paclitaxel