Toll-like receptor 3 acts as a suppressor gene in breast cancer initiation and progression: a two-stage association study and functional investigation

Abstract

Toll-like receptor 3 (TLR3) is a receptor recognizing double-stranded RNA (dsRNA) from viruses as well as from lytic mammalian cells. In the present study, we performed a two-stage association study (n = 3,551) and found that the minor alleles of two SNPs (the T-allele of rs5743312 and the T-allele of rs3775296) conferred increased risks of breast cancer incidence. The adjusted odds ratios (ORs) were 2.281 (P = 7.01 × 10^-5) and 2.086 (P = 8.69 × 10^-5), respectively. Specifically, the susceptibility variants within TLR3 were significantly associated with larger tumor size (adjusted P-values: 0.004 for rs5743312 and 0.004 for rs3775296). Furthermore, we investigated the biological function of the TLR3 protein in breast cancer cell lines. Notably, the stable expression of TLR3 directly inhibited cell proliferation both in vitro and in vivo. We also verified that TLR3 conferred less invasive phenotypes on breast cancer cells by regulating the mRNA expression of a panel of genes. TLR3-mediated inhibition of proliferation was caused by downregulation of the EGFR/PI3K/AKT pathway. In summary, our findings strongly suggest that common genetic changes in the TLR3 gene may influence breast cancer susceptibility and development, and TLR3 plays a negative regulatory role in the initiation and progression of human breast cancer cells, at least in part by downregulating the EGFR/PI3K/AKT pathway.

Keywords: Toll-like receptor 3; breast cancer; double-stranded RNA.

Figure 1.

Expression of TLR3 in human breast cancer cell lines and stably transfected cell lines. 2a. Relative expression of TLR3 mRNA was detected by real-time PCR in six human breast cancer cell lines: MCF-7, MDA-MB-468, MDA-MB-231LM, MDA-MB-231HM, T-47D and SUM-149. 2b. Comparison of the relative levels of TLR3 mRNA in the six human breast cancer cell lines by semiquantitative real-time RT-PCR. 2c. Differential expression of the TLR3 protein in the six human breast cancer cell lines. 2d. Relative expression of TLR3 mRNA detected by real-time PCR in TLR3 stable transfectants, mock-transfected and MDA-MB-231 parental cells. 3e. Relative expression of TLR3 protein detected by Western blot is shown in TLR3 stable transfectants, mock-transfected and MDA-MB-231 parental cells. 1–3 represents MDA-MB-231-LM cells, MDA-MB-231-vect cells and MDA-MB-231-TLR3 cells, respectively; N.S. no significance (**, P < 0.01).

Figure 2.

TLR3 overexpression inhibited the growth of breast cancer cells both in vitro and in vivo. 3a. Cellular proliferation assay. The growth of cells was determined by a Cell Counting Kit 8 assay and expressed as an absorbance value (OD) value. Points indicate the mean; bars indicate the standard error; P < 0.05 versus mock-transfected and parental cells. 3b. Tumor growth curves. A total of 1 × 106 viable cells were injected subcutaneously into the right axilla of each mouse. These curves show tumor growth until week 12 (for the BxPC3 group). Points indicate the mean; bars indicate the standard error; P < 0.05 versus mock-transfected.

Figure 3.

TLR3 overexpression resulted in an altered expression profile of genes required for cell cycle arrest, epithelial-mesenchymal transition, invasion and metastasis. Quantitative real-time PCR analyses showed that TLR3 overexpression resulted in the significant downregulation of EGFR (decreased 72.5%), β-catenin (decreased 65.6%), AKT (decreased 61.7%), Cathepsin-D (decreased 83.5%), and MMP9 (decreased 64.2%); significant upregulation of Smad2 (2.35-fold) and TGFβ (2.09-fold); and stabilized expression of ERK, p21, MMP2, MMP7, MMP1, bFGF, c-jun, uPA, Cyclin D, Smad3, E-cadherin, VEGF, vimentin, and fibronectin. GAPDH was used as an internal control. (** represents P＜0.01 for expression in MDA-MB-231-TLR3 versus in MDA-MB-231-vect).

Figure 4.

TLR3 was involved in the EGFR-mediated pathway. EGFR signaling was inhibited by the endogenous expression of TLR3. Western blot analysis of the expression of key factors in the EGFR-mediated pathway in MDA-MB-231 and its TLR3 transfectants. 1–4: MDA-MB-231LM cells transiently transfected with pcDNA3.1(+), MDA-MB-231LM cells transiently transfected with pcDNA3.1(+)-TLR3, MDA-MB-231-Vect cells, and MDA-MB-231-TLR3 cells. GAPDH was used as an internal control.

Figure 5.

Differential TLR3 expression in breast cancer tissues and normal breast tissues. Relative TLR3 mRNA expression was detected in breast cancer tissues (n = 43) and normal breast tissues (n = 35). The horizontal lines represent the mean values. The mean TLR3 mRNA expression level in normal breast tissues was significantly higher than that in breast cancer tissues (P-value of Mann–Whitney test<0.05).

Figure 6.

Candidate SNPs for genotyping in the TLR3 region. Eight candidate SNPs were chosen for genotyping in the 22.3-kb region of TLR3, from 1.5 kb upstream of the 5ʹ-flanking region to 0.5 kb downstream of the 3ʹ-flanking region TLR3. One missense SNP, rs5743316 (row in blue), was excluded because no polymorphism was detected in the Shanghai population. SNPs with red rows entered the second-stage study for replication genotyping.