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. 2013 Aug;12(8):1676-87.
doi: 10.1158/1535-7163.MCT-12-1019. Epub 2013 May 29.

TLR4 is a novel determinant of the response to paclitaxel in breast cancer

Sandeep Rajput  1 , Lisa D Volk-Draper, Sophia Ran
Affiliations

TLR4 is a novel determinant of the response to paclitaxel in breast cancer

Sandeep Rajput et al. Mol Cancer Ther. 2013 Aug.

Abstract

Overexpression of Toll-like receptor-4 (TLR4) in human tumors often correlates with chemoresistance and metastasis. We found that TLR4 is overexpressed in the majority of clinical breast cancer samples and in 68% of the examined breast cancer lines. TLR4 is activated by lipopolysaccharide (LPS) and other ligands including the widely used drug paclitaxel. LPS is frequently used to show a tumor-promoting role of TLR4 although this bacterial component is unlikely to be found in the breast cancer environment. We reasoned that paclitaxel-dependent activation of TLR4 is more relevant to breast cancer chemoresistance that could be mediated by activation of the NF-κB pathway leading to upregulation of prosurvival genes. To test this hypothesis, we correlated TLR4 expression with resistance to paclitaxel in two modified breast cancer lines with either depleted or overexpressed TLR4 protein. Depletion of TLR4 in naturally overexpressing MDA-MB-231 cells downregulated prosurvival genes concomitant with 2- to 3-fold reduced IC(50) to paclitaxel in vitro and a 6-fold decrease in recurrence rate in vivo. Conversely, TLR4 overexpression in a negative cell line HCC1806 significantly increased expression of inflammatory and prosurvival genes along with a 3-fold increase of IC(50) to paclitaxel in vitro and enhanced tumor resistance to paclitaxel therapy in vivo. Importantly, both tumor models showed that many paclitaxel-upregulated inflammatory cytokines were coinduced with their receptors suggesting that this therapy induces autocrine tumor-promoting loops. Collectively, these results show that paclitaxel not only kills tumor cells but also enhances their survival by activating TLR4 pathway. These findings suggest that blocking TLR4 could significantly improve response to paclitaxel therapy.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: Authors declare no conflicting interests.

Figures

Figure 1
Figure 1. Frequency of TLR4 expression in human BC tumors and lines
(A) Analysis of TLR4 mRNA in human normal breast (n=5) and tumors (n=20) by RT-qPCR. (B) RT-qPCR analysis of TLR4 mRNA in BC lines. Insert: Western blot of TLR4 protein expression in MDA-MB-231 and HCC1806 cells. HEK293 and THP1 represent lines with negative and positive TLR4 expression, respectively. (C) RT-PCR analysis of TLR4 mRNA expression in cell lines. Underline and asterisk indicates the cell lines to represent TLR4+ and TLR4− lines. (D, F) Activation of TLR4 pathway by paclitaxel inMDA-MB-231 and HCC1806 cells. RT-qPCR quantification of IL-6, IL-8, TNFα and MCP-1 in cells treated with nab-PXL (0–30nM) for 48hrs. (E, G) MDA-MB-231 and HCC1806 cells were pre-treated with control-IgG or anti-TLR4 antibody (4ug/ml) for 2hrs before nab-PXL treatment (10nM) and analyzed as in 1D. Data are presented as β-actin normalized mRNA expression ±S.D. from two experiments done in duplicate. The P-values represent * <0.05 and ** <0.01 vs. control as determined by Student’s paired t test.
Figure 2
Figure 2. TLR4-positive and negative lines significantly differ in sensitivity to PXL
MDA-MB-231 and HCC1806 cells were treated with PXL (A) or nab-PXL (B) at indicated concentrations (0–100nM) for 48hrs followed by calculating IC50. (C, D) 231Cntrl, 231TLR4− cells, 1806Cntrl and 1806TLR4+ cells were treated and analyzed as described under 2A. (E) MDA-MB-231 cells were pretreated with 4µg/ml of anti-TLR4 or control IgG for 2hrs followed by analysis described under 2A. (F, G) Cells were pretreated with a TLR4 inhibitor TAK-242 (10uM) or with an LPS antagonist LPS-EKUltra for 2hrs followed by analysis described under 2A. Data are presented as percent of viable cells ±S.D. from three experiments done in triplicate. The P-values represent * <0.05 vs. control as determined by Student’s paired t-test.
Figure 3
Figure 3. TLR4 mediates the response of BC cells to nab-PXL
(A, B) Western blot analysis of NF-κB p-p50, NF-κB p50, NF-κB p-p65, NF-κB p65, p-Akt, Akt, Bcl-2, Bcl-xL and β-actin proteins in 231Cntrl, 231TLR4−, 1806Cntrl, and 1806TLR4+ cells treated with nab-PXL (10nM, 48hrs). (C, D) Cell cycle analysis by FACS of PI-stained cells treated with nab-PXL for 48hrs. Data are presented as mean percentage of cells in different stages of cell cycle ±S.D. from three experiments. (E, F) Apoptosis of 231Cntrl, 231TLR4−, 1806Cntrl, and 1806TLR4+ cells treated with nab-PXL for 48hrs determined by FACS measurement of% Annexin-and PI-positive cells. Data are presented as mean percentage of apoptotic cells ±S.D. from three experiments. Asterisks indicate P-values <0.05 vs. control as determined by Student’s unpaired t-test.
Figure 4
Figure 4. PXL upregulates inflammatory cytokines and receptors in TLR4+ lines
(A) Number of inflammatory genes upregulated >2 fold in231Cntr, 231TLR4,1806Cntr and 1806TLR4+ cells in PXL-treated(10nM, 48hrs) vs. untreated cells. Asterisks * and ** indicate P-values<0.05 and <0.01 vs. control determined by Chi-square test. (B) Fold increase of upregulated inflammatory genes in 231Cntrl, 231TLR4−,1806Cntrl, and 1806TLR4+ lines after PXL treatment. Asterisks * indicate P-values <0.05 vs. control as determined by Mann-U-Whitney test comparing number of ligands and receptors upregulated >1.5-fold in 231Cntrl vs. 231TLR4− cells. (C) Main cytokines and corresponding receptors upregulated >2.0 fold in 231Cntrl, 231TLR4, 1806Cntrl and 1806TLR4+ cells.
Figure 5
Figure 5. Inhibition of CXCR2 and CCR4 receptors synergistically increases sensitivity to nab-PXL in MDA-MB-231 cells
Cells were pretreated with 0–1µM of a CXCR2 inhibitor SB225002 (A) or a CCR4 receptor inhibitor C021 dihydrochloride(B) followed by 48hrs treatment with nab-PXL(10nM). IC50 was calculated as described in legend for Fig. 2.(C) Table demonstrating the synergy between nab-PXL and inhibitors of CCR4, CXCR2,or MEK as represented by measured as CI. (D) MDA-MB-231 cells were treated MEK inhibitor (0–100 uM) and nab-PXL (10nM) for 48hrs followed by calculating IC50. Data are presented as percentage of viable cells vs. control ±S.D. from two experiments done in triplicate. (E) MDA-MB-231 cells were pretreated with CXCR2 (1µM) and MEK inhibitors (50µM) alone or in combination for 2hrs followed by nab-PXL (10nM) for 48hrs. Phosphorylation of ERK1/2 and AKT was analyzed by Western blot.
Figure 6
Figure 6. TLR4 determines sensitivity to nab-PXL in breast cancer modelsin vivo
(A) The growth of 231TLR4− and control lines implanted in SCID mice was monitored twice weekly. Each point represents the mean tumor volume ±S.E and the asterisks indicate P-values >0.05. (B) Growth of a231TLR4− clone demonstrating significant delay in establishing tumor mass in all mice per group (n=6) compared with controls. Each line represents tumor growth in individual mouse. (C) Growth of HCC1806TLR4+ and HCC1806Cntrl tumorsin SCID mice. TLR4 significantly increased tumor growth in all clones with * and *** indicating P-values <0.05 and <0.001, respectively. (D) 231TLR4− and control tumors of 150 mm3 were treated with 10mg/kg of nab-PXL i.v. for 8 days. Five out 6 mice (83.3%) bearing 231TLR4− tumors (yellow circles) had complete response (CR) while 0% CRs was achieved in all other groups. (E) Bio-imaging of representative tumors from groups described in D.(F) Tumor growth of control and TLR4+overexpressing HCC1806 lines.
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