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

Taxanes are a family of chemotherapy drugs used to treat various human cancer types [1‐6]. The most common family members include paclitaxel and docetaxel, which block microtubule depolymerization, inducing cell-cycle arrest at mitosis and multinucleation of tumor cells [7, 8]. Taxanes also reduce tumor angiogenesis and cell migration, while stimulating the immune system against neoplasms [9‐11].

Another mechanism for taxane cytotoxicity may involve tumor-necrosis factor (TNF)-α production, because paclitaxel has been shown to augment TNF-α levels in murine macrophages [12]. TNF-α is a membrane-integrated cytokine (mTNF-α) generally produced in activated macrophages and monocytes [13], which can be released from cells in a soluble form (sTNF-α) by the action of the metalloproteinase ADAM-17 [14]. The release of sTNF-α from cells can then induce cell death or a cell-survival response, depending on the receptor to which it binds: TNFR1 or TNFR2, respectively (reviewed in [15]. Although mTNF-α binds with equal affinity to both receptors, sTNF-α preferentially binds to TNFR1, which has a death-effector domain that induces caspase-8 cleavage and apoptosis [16, 17]. Unlike TNFR1, TNFR2 does not contain a death domain. Limited reports suggest that TNFR2 activation promotes cell death, although the mechanism for this is poorly understood and may require the presence of TNFR1 [18, 19]. Nevertheless, TNFR2 has been shown to induce NF-κB activity and cell survival [20].

Many mechanisms associated with resistance to taxanes have been identified in vitro, such as overexpression of the drug-efflux pump Abcb1, β-tubulin gene mutations, or overexpression of β-tubulin (type III); however, their clinical relevance remains unclear [21, 22]. Multiple mechanisms of taxane resistance likely occur simultaneously in cells [23]. To restore tumor sensitivity to taxanes appreciably in cancer patients, all clinically relevant mechanisms of docetaxel resistance must be identified.

To better understand the various pathways associated with taxane resistance, our laboratory selected MCF-7 breast tumor cells for survival in increasing concentrations (doses) of paclitaxel (MCF-7_TAX-1 cells) [24] or docetaxel (MCF-7_TXT cells) [23]. Increased Abcb1 expression and decreased taxane uptake occurred on acquisition of taxane resistance in these cells, but a pan-ABC transporter inhibitor that restored taxane uptake had no or only a partial effect on drug sensitivity in these cells [23]. Therefore, additional mechanisms must contribute to taxane resistance, and these cell lines may serve as an attractive tool for assessing the possible role of TNF-α and other pathways in taxane cytotoxicity or resistance.

Although taxanes are known to inhibit cell division by preventing microtubule depolymerization and inducing multinucleation [8, 36], it is unclear whether these are their sole mechanisms of tumor cell growth arrest/death in vitro and in vivo. Paclitaxel has been shown to increase sTNF-α release from murine macrophages [12, 37], although the levels used in those studies would be unachievable in patients, and docetaxel had no effect on TNF-α expression in the same study. In our study, we showed for the first time that docetaxel (at concentrations between 3 and 45 nM) can stimulate TNF-α production and sTNF-α release from both breast and ovarian tumor cells. Such concentrations are clearly in the range of plasma levels of docetaxel in breast cancer patients after docetaxel infusion (10 to 75 nM) [38] and are likely sufficiently high to induce TNF expression in even poorly vascularized tumors. This newly identified TNF-dependent mechanism of docetaxel action may also account for its reported immunomodulatory activity [11, 39]. In addition, we show in this article that paclitaxel treatment (at 5 and 15 nM concentrations) can dramatically increase sTNF-α release from ovarian tumor cells.

Our study also illustrates that the acquisition of docetaxel resistance in breast tumor cells temporally correlates with increased production and release of sTNF-α from cells, despite the ability of sTNF-α to be cytotoxic to cells [40]. However, the onset of docetaxel resistance in MCF-7 cells (at docetaxel selection doses ≥ 3.33 nM) also correlated with strongly reduced levels of TNFR1, which would block the ability of TNF-α to induce cell death. Although the mechanism responsible for TNFR1 reduction remains undefined, neither changes in TNFR1 transcript levels nor increased levels of sTNFR1 in the media were found, suggesting that the receptor was not shed from cells by the ADAM-17 protease [14]. In fact, MCF-7_TXT9 and MCF-7_TXT10 cells exhibited decreased levels of sTNFR1 in the medium in which it was grown. It is possible that increased levels of sTNFα produced by these cells bound to sTNFR1 in the medium, preventing its detection by the TNFR1 antibody. Taken together, our findings suggest that downregulation of TNFR1 occurs posttranscriptionally, because of either reduced translation of the TNFR1 transcript or increased TNFR1 proteolysis.

A recent study [41] found that TNF-α or paclitaxel induced NF-κB activity in C2C12 myotubes. However, paclitaxel did not induce increased TNF-α production, and inhibition of TNFR1 blocked TNF-α-induced NF-κB activation but did not abolish paclitaxel-induced NF-κB activity [41]. It is important to note that, in these studies, TNF-α levels were assessed only 4 hours after treatment with paclitaxel (10 nM to 10 μM).

Whereas docetaxel selection doses between 3 and 5 nM resulted in highly elevated sTNF-α production, higher selection doses (≥ 15 nM) did not. This was despite the ability of the drug to induce TNF-α production in wild-type cells over a large concentration range (Figure 1). This may be explained by the increased expression of the Abcb1 drug transporter and reduced docetaxel uptake that we observed in MCF-7_TXT11 and MCF-7_TXT12 cells. Expression was maximal at the highest selection doses (≥ 15 nM) [42]. We propose that docetaxel accumulates at sufficient concentrations to induce production of sTNF-α in MCF-7_TXT9 and MCF-7_TXT10 cells. However, at or above 15 nM docetaxel, MCF-7_TXT cells exhibit reduced drug uptake, such that docetaxel accumulation is insufficient to stimulate TNF-α production.

The mechanism for resistance to taxanes and TNF-α in MCF-7_TAX-1 cells appears to differ from that of MCF-7_TXT cells. TNFR1 levels were equivalent in MCF-7_TAX-1 and MCF-7_CC cells (data not shown), and IκB levels were also unchanged during selection for paclitaxel resistance (Figure 6). Because only cells exposed to the maximally tolerated dose of paclitaxel were retained during selection of MCF-7_TAX-1 cells, it is likely that cells selected at lower doses could have exhibited elevated production of TNF-α and TNF-α-mediated NF-κB activation. However, survival by circumventing the TNF-α ability to stimulate TNFR1-induced cytotoxicity must lie downstream of the receptor. MCF-7_TAX-1 cells are also high expressors of Abcb1 [24]. Interestingly, another paclitaxel-resistant MCF-7 cell line (MCF-7_TAX-2 cells) [23] retained sensitivity to TNF-α (data not shown), suggesting that defects in the TNF-α pathway are not critical for taxane resistance in vitro. Nevertheless, considering that three of the four taxane-resistant cell lines exhibited alterations in TNF-α signaling and that docetaxel has been shown to increase sTNF-α levels in both breast and ovarian tumor cells, it appears that we have identified a common but unknown mechanism of taxane cytotoxicity and resistance that warrants further study for its potential clinical relevance.

To provide further support for a general involvement of the TNF-α pathway in docetaxel cytotoxicity and in the induction of docetaxel resistance, we also showed in this study that selection of breast and ovarian tumor cells for resistance to docetaxel results in changes in the expression of networks of genes related to TNF-α signaling (Figure 8 and Table 2). Quite strikingly, the vast majority of the upregulated genes depicted in Table 2 code for proteins that are TNF-ligand family members, TNF-receptor family members, TNF receptor-associated proteins, TNF-dependent activators of NF-κB, or proteins that help promote degradation of the inhibitor of NF-κB (IκB). Other upregulated genes are TNF-dependent inhibitors of apoptosis. Downregulated genes code for proteins that inhibit the activation of NF-kB or promote apoptosis. The net effect of the changes in gene expression would thus be to promote the ability of TNF to augment NF-κB-dependent cell survival, while blocking its ability to induce tumor cell death via activation of TNFR1.

The findings of our study may have significant clinical relevance. A presentation at the 26^th annual meeting of the European Association of Urology in 2011 [43] revealed that serum levels of proinflammatory cytokines, including TNF-α, increased 2 days after administration of docetaxel to patients with castration-resistant prostate cancer. Interestingly, these changes in cytokine expression correlated with the induction of apoptosis and with clinical response. In addition, a study presented recently at the American Association for Cancer Research, 101^st Annual Meeting [44], revealed that, in patients with serous epithelial ovarian carcinoma, pretreatment tumor expression of various genes within the TNF-α and NF-κB signaling networks could be used to distinguish between responders and nonresponders to paclitaxel/carboplatin chemotherapy. It also was shown in a small study involving patients with locally advanced breast cancer that tumor levels of nuclear (activated) NF-κB could be used to distinguish between responders and nonresponders to neoadjuvant anthracycline- and/or taxane-based chemotherapy regimens [45]. These and other studies strongly support the likely clinical significance of the findings. For example, because TNF-α has been shown to reduce tumor vascularization in mice through its effects on TNFR1-expressing endothelial cells [46], the reported ability of docetaxel to affect tumor angiogenesis [10] may be through an ability of the drug to promote sTNFα-mediated decreases in tumor vascularization. Moreover, one of the well-established dose-limiting toxicities associated with docetaxel chemotherapy in breast cancer patients is fatigue [47], and high TNF levels have been shown to correlate with fatigue onset in cancer patients [48]. Given our findings of docetaxel-induced TNF-α production, perhaps these two phenomena are linked.

Finally, a previous clinical study used a TNF-decoy receptor (entanercept) to permit patients to tolerate higher doses of docetaxel without significant toxicity [49]; however, given our findings, it is not surprising that these blockers would create a greater tolerance to docetaxel, unfortunately at the likely expense of lesser anti-tumor efficacy. Our findings further question the utility of administering docetaxel to cancer patients on TNF-α blockers for treatment of co-morbid inflammatory diseases.

Our study provides evidence for the first time that taxanes can induce sTNF-α expression in two tumor cell lines of different tissue origin. Although this would promote the cytotoxicity of docetaxel, continued exposure to the drug appears to result in a downregulation of TNF-α-mediated cytotoxicity, while promoting TNF-α-dependent activation of NF-kB-dependent cell-survival pathways and the inhibition of apoptosis. In addition, although drug-resistance studies often involve selection of cells to maximally tolerated drug doses [50, 51], our study illustrates the critical role that the drug-selection dose may play on the mechanisms by which tumor cells acquire chemotherapy resistance. At lower doses of taxanes (3 to 5 nM), TNF-α-mediated activation of NF-κB-dependent cell-survival pathways appears to be an important mechanism of taxane resistance, whereas at selection doses ≥ 15 nM docetaxel, the drug induces overexpression of Abcb1, resulting in reduced accumulation of docetaxel into cells and a consequent reduction in docetaxel-stimulated TNF-α production. Since the concentration of docetaxel within patient tumors is typically lower than that present in the vasculature, perhaps the pathways associated with resistance to lower concentrations of docetaxel are of greater clinical relevance. This may explain why Abcb1 inhibitors have had little ability to reverse resistance to taxanes in cancer patients [52, 53]. In addition, given that some cancer patients with inflammatory diseases may be taking TNF blockers, this newly identified role for TNF in docetaxel cytotoxicity may be of particular importance, suggesting that these blockers may compromise the efficacy of docetaxel chemotherapy.