Low TLR9 expression defines an aggressive subtype of triple-negative breast cancer.

Toll-like receptor-9 (TLR9) is a DNA receptor widely expressed in cancers. Although synthetic TLR9 ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology is unclear. We discovered that low tumor TLR9 expression is associated with significantly shortened disease-specific survival in patients with triple negative but not with ER+ breast cancers. A likely mechanism of this clinical finding involves differential responses to hypoxia. Our pre-clinical studies indicate that while TLR9 expression is hypoxia-regulated, low TLR9 expression has different effects on triple negative and ER+ breast cancer invasion in hypoxia. Hypoxia-induced invasion is augmented by TLR9 siRNA in triple negative, but not in ER+ breast cancer cells. This is possibly due to differential TLR9-regulated TIMP-3 expression, which remains detectable in ER+ cells but disappears from triple-negative TLR9 siRNA cells in hypoxia. Our results demonstrate a novel role for this innate immunity receptor in cancer biology and suggest that TLR9 expression may be a novel marker for triple-negative breast cancer patients who are at a high risk of relapse. Furthermore, these results suggest that interventions or events, which induce hypoxia or down-regulate TLR9 expression in triple-negative breast cancer cells may actually induce their spread.

Expression of toll-like receptor-9 is increased in poorly differentiated prostate tumors.

BACKGROUND: Toll-like receptor-9 (TLR9) is a cellular receptor for bacterial and vertebrate DNA. In addition to cells of the immune system, it is also expressed in various human cancer cell lines, including prostate cancer. We demonstrated previously that synthetic TLR9 ligands induce matrix metalloproteinase-13-mediated invasion in TLR9-expressing prostate cancer cells in vitro. Other studies have suggested possible sex steroid regulation of the function of the various TLRs. The role of TLR9 in the pathophysiology of prostate or any cancer is, however, unknown. METHODS: Expression of TLR9, androgen receptor (AR), or the estrogen receptors alpha (ERalpha) and beta (ERbeta) were studied with immunohistochemistry in prostate cancer (n = 62) and benign prostatic hyperplasia (n = 45) specimens. TLR9 staining scores were compared with tumor stage, Gleason score, prostate-specific antigen (PSA) concentrations before tissue sampling and with the staining scores of AR, ERalpha, and ERbeta. RESULTS: TLR9 expression was statistically significantly increased in prostate cancer epithelium and stroma, as compared with the same cellular compartments in benign hyperplasia. Significantly increased (P = 0.04) TLR9 expression was detected in cancers with high Gleason score (>7, n = 23), as compared with lower Gleason scores (< or =7, n = 39). No statistically significant associations were detected between TLR9 expression scores and PSA concentrations or tumor staging. Prostate adenocarcinoma cells were all positive for TLR9, AR, and ERbeta but negative for ERalpha expression. In cancer stroma cells, increased TLR9 expression was associated with increased ERalpha expression. CONCLUSIONS: Expression of TLR9 is increased in prostate cancer specimens, especially in the most poorly differentiated forms.

Reactive oxygen species-regulating proteins peroxiredoxin 2 and thioredoxin, and glyceraldehyde-3-phosphate dehydrogenase are differentially abundant in induced sputum from smokers with lung cancer or asbestos exposure.

Lung cancer is a deadly disease, typically caused by known risk factors, such as tobacco smoke and asbestos exposure. By triggering cellular oxidative stress and altering the antioxidant pathways eliminating reactive oxygen species (ROS), tobacco smoke and asbestos predispose to cancer. Despite easily recognizable high-risk individuals, lung cancer screening and its early detection are hampered by poor diagnostic tools including the absence of proper biomarkers. This study aimed to recognize potential lung cancer biomarkers using induced sputum noninvasively collected from the lungs of individuals in risk of contracting lung cancer. Study groups composed of current and former smokers, who either were significantly asbestos exposed, had lung cancer, or were unexposed and asymptomatic. Screening of potential biomarkers was performed with 52, and five differentially abundant proteins, peroxiredoxin 2 (PRDX2), thioredoxin (TXN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), extracellular matrix protein 1 (ECM1), and protein S100 A8 (S100A8), were chosen to undergo validation, for their previously known connection with oxidative stress or cancer. Results from the validation in 123 sputa showed that PRDX2, TXN, and GAPDH were differentially abundant in sputa from individuals with lung cancer. TXN had a negative correlation with asbestos exposure, yet a positive correlation with smoking and lung cancer. Thus, tobacco smoking, asbestos exposure, and lung carcinogenesis may disturb the cellular redox state in different ways. A strong correlation was found among PRDX2, TXN, GAPDH, and S100A8, suggesting that these proteins may present a diagnostic biomarker panel to aid recognizing individuals at high risk of contracting lung cancer.

Expression of macrophage inhibitory cytokine-1 in prostate cancer bone metastases induces osteoclast activation and weight loss.

BACKGROUND: Macrophage inhibitory cytokine-1 (MIC-1) belongs to the bone morphogenic protein/transforming growth factor-beta (BMP/TGF-beta) superfamily. Serum MIC-1 concentrations are elevated in patients with advanced prostate cancer. The effects of MIC-1 on prostate cancer bone metastases are unknown. METHODS: In vitro effects of MIC-1 on osteoblast differentiation and activity were analyzed with alkaline phosphatase and mineralization assays; osteoclast numbers were counted microscopically. MIC-1 effects on TLR9 expression were studied with Western blotting. Human Du-145 prostate cancer cells were stably transfected with a cDNA encoding for mature MIC-1 or with an empty vector. The in vivo growth characteristics of the characterized cells were studied with the intra-tibial model of bone metastasis. Tumor associated bone changes were viewed with X-rays, histology, and histomorphometry. Bone formation was assayed by measuring serum PINP. RESULTS: MIC-1 induced osteoblast differentiation and activity and osteoclast formation in vitro. These effects were independent of TLR9 expression, which was promoted by MIC-1. Both MIC-1 and control tumors induced mixed sclerotic/lytic bone lesions, but MIC-1 increased the osteolytic component of tumors. Osteoclast formation at the tumor-bone interface was significantly higher in the MIC-1 tumors, whereas bone formation was significantly higher in the control mice. At sacrifice, the mice bearing MIC-1 tumors were significantly lighter with significantly smaller tumors. CONCLUSIONS: MIC-1 up-regulates TLR9 expression in various cells. MIC-1 stimulates both osteoblast and osteoclast differentiation in vitro, independently of TLR9. MIC-1 over-expressing prostate cancer cells that grow in bone induce osteoclast formation and cachexia.

Toll-like receptor 9 mediates CpG oligonucleotide-induced cellular invasion.

Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13-mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasion-inducing effect in MDA-MB-231 cells. In contrast, methylation of the cytosine residues of the parent CpG oligonucleotide did not affect the TLR9-mediated invasion compared with the unmethylated parent CpG oligonucleotide. Finally, expression of TLR9 was studied in clinical breast cancer samples and normal breast epithelium with immunohistochemistry. TLR9 staining localized in epithelial cells in both cancer and normal samples. The mean TLR9 staining intensity was significantly increased in the breast cancer cells compared with normal breast epithelial cells. In conclusion, our results suggest that TLR9 expression is increased in breast cancer and CpG oligonucleotide-induced cellular invasion is mediated via TLR9 and TRAF6, independent of MyD88. Further, our findings suggest that the structure and/or stability of DNA may influence the induction of TLR9-mediated invasion in breast cancer.

Lower frequency of TLR9 variant associated with protection from breast cancer among African Americans.

INTRODUCTION: Toll-like receptor 9 (TLR9) is an innate immune system DNA-receptor that regulates tumor invasion and immunity in vitro. Low tumor TLR9 expression has been associated with poor survival in Caucasian patients with triple negative breast cancer (TNBC). African American (AA) patients with TNBC have worse prognosis than Caucasians but whether this is due to differences in tumor biology remains controversial. We studied the prognostic significance of tumor Toll like receptor-9 (TLR9) protein expression among African American (AA) triple negative breast cancer (TNBC) patients. Germline TLR9 variants in European Americans (EAs) and AAs were investigated, to determine their contribution to AA breast cancer risk. METHODS: TLR9 expression was studied with immunohistochemistry in archival tumors. Exome Variant Server and The Cancer Genome Atlas were used to determine the genetic variation in the general EA and AA populations, and AA breast cancer cases. Minor allele frequencies (MAFs) were compared between EAs (n = 4300), AAs (n = 2203), and/or AA breast cancer cases (n = 131). RESULTS: Thirty-two TLR9 variants had a statistically significant MAF difference between general EAs and AAs. Twenty-one of them affect a CpG site. Rs352140, a variant previously associated with protection from breast cancer, is more common in EAs than AAs (p = 2.20E-16). EAs had more synonymous alleles, while AAs had more rare coding alleles. Similar analyses comparing AA breast cancer cases with AA controls did not reveal any variant class differences; however, three previously unreported TLR9 variants were associated with late onset breast cancer. Although not statistically significant, rs352140 was observed less frequently in AA cases compared to controls. Tumor TLR9 protein expression was not associated with prognosis. CONCLUSIONS: Tumor TLR9 expression is not associated with prognosis in AA TNBC. Significant differences were detected in TLR9 variant MAFs between EAs and AAs. They may affect TLR9 expression and function. Rs352140, which may protect from breast cancer, is 1.6 X more common among EAs. These findings call for a detailed analysis of the contribution of TLR9 to breast cancer pathophysiology and health disparities.

Tumor infiltrating CD8+ T lymphocyte count is independent of tumor TLR9 status in treatment naïve triple negative breast cancer and renal cell carcinoma.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor of the innate immune system that is widely expressed in cancers. We demonstrated that low tumor TLR9 expression predicts poor disease-specific survival in triple negative breast cancer (TNBC) and renal cell carcinoma (RCC). We hypothesized that this is because TLR9 expression affects tumor immunophenotype. To begin to test this, we compared the number of tumor infiltrating CD8+ T lymphocytes with TLR9 expression in treatment naïve breast cancer (n = 197) and RCC (n = 94) cohorts with known TLR9 expression status. CD8+ T lymphocyte counts were assayed with image analysis after immunohistochemistry (IHC). Tumor TLR9 expression was not correlated with CD8+ T cell counts in breast cancer or RCC. CD8+ T cell counts were significantly associated with tumor proliferation index in TNBC, but not in non-TNBC. CD8+ T cell counts were also significantly associated with tumor grade in non-TNBC, but not in TNBC. In RCC, CD8+ T cell counts were significantly associated with tumor stage. CD8+ T cell counts were significantly associated with prognosis in TNBC and RCC, but the presence of CD8+ T cells in these tumors had opposite effects on disease-specific survival: High CD8+ counts were associated with better prognosis in TNBC and worse prognosis in RCC. Among TNBC patients, those with low tumor TLR9 and low CD8+ T cell counts had the poorest prognosis (log-rank p = 0.0002 vs. high tumor TLR9 and high CD8+ T cell count). In conclusion, pre-treatment tumor TLR9 status is not associated with tumor infiltrating CD8+ T lymphocytes in TNBC or RCC. The combination of TLR9 and CD8+ TIL count might be a novel composite prognostic marker in TNBC.

Expression of Toll-like receptor-9 is associated with poor progression-free survival in prostate cancer.

Toll-like receptor-9 (TLR9) is a member of the innate immune system and recognizes bacterial and vertebrate DNA in cells. In addition to being expressed in cells of the immune system, it is widely expressed in various types of human cancer, including prostate cancer. We have previously demonstrated that synthetic TLR9 ligands induce invasion in TLR9-expressing prostate cancer cells in vitro. However, the role of TLR9 in the pathophysiology of prostate cancer is unclear. The expression of TLR9 in radical prostatectomy samples (n=186) was studied using immunohistochemistry. TLR9 staining scores were compared with tumor stage, Gleason score and prostate-specific antigen (PSA) concentration prior to treatment and progression-free survival. Results revealed that 124 (66.7%) of the tumors were strongly positive, 59 (31.7%) were weakly positive and 3 (1.6%) were negative, for cytoplasmic TLR9 immunostaining in cancer cells. There was no significant association between cytoplasmic TLR9 expression and distributions of pT-class, prostatectomy sample margin status, Gleason score and preoperative PSA value. Prostate cancer-specific progression-free survival was significantly longer for patients whose tumors were graded as negative for cytoplasmic TLR9 expression, as compared with patients whose tumors were strongly immunopositive for cytoplasmic TLR9 (P=0.009). In the Cox regression analysis, high TLR9 expression was an independent marker of poor prognosis in prostate cancer. Expression of TLR9 is associated with poor progression-free survival in prostate cancer patients who were treated by radical prostatectomy with curative intent.

Toll-like receptor 9 is a novel biomarker for esophageal squamous cell dysplasia and squamous cell carcinoma progression.

BACKGROUND: Stimulation of Toll-like receptor 9 (TLR9) has been linked to invasion in various cancer cells in vitro. We investigated TLR9 expression in normal, dysplastic and malignant esophageal squamous epithelium. METHODS: TLR9 expression was analyzed by immunohistochemistry in 46 cases of esophageal squamous cell carcinoma, including 12 cases with adjacent squamous dysplasia and 24 cases with normal esophageal epithelium. TLR9 expression was compared with tumor grade, stage, proliferation, apoptosis and vascular density. RESULTS: In normal esophageal squamous epithelium, TLR9 staining intensity decreased linearly from the basal layers to the superficial layers (p < 0.001). Strong TLR9 expression was detected across full thickness of high-grade dysplasia, the intensity clearly differing from the normal squamous epithelium and squamous cell carcinoma (p < 0.001). All squamous cell carcinomas exhibited TLR9 expression that was positively associated with a high grade (p < 0.05), the presence of lymph node metastases (p < 0.05) and previously undetected distant metastases (p < 0.05). CONCLUSIONS: Expression of TLR9 in the basal parts of normal esophageal epithelium suggests a role related to cell proliferation and differentation. TLR9 upregulation detected in dysplastic epithelium and in disseminated carcinomas indicates that this protein may serve as a novel marker for esophageal squamous dysplasia and carcinoma with metastatic potential.

Absent Toll-like receptor-9 expression predicts poor prognosis in renal cell carcinoma.

BACKGROUND: Toll-like receptor 9 (TLR9) is a cellular DNA-receptor whose activation with cognate ligands triggers an immune reaction, with increased production of inflammatory cytokines. The aim of this study was to examine the expression of TLR9 in renal cell carcinoma (RCC), which is generally renowned of its immunogenic nature. We also evaluated the prognostic value of TLR9 in RCC. METHODS: TLR9 expression in RCC was characterized with immunohistochemistry in a retrospective study population of 152 RCC patients who underwent renal surgery. The TLR9 staining intensity was compared with clinical parameters. RESULTS: Of the studied tumours, 112 (81%) exhibited cytoplasmic TLR9 immunostaining. No association was detected between cytoplasmic TLR9 immunoexpression intensity and stage, nuclear grade, histological subtype or tumour necrosis. Cytoplasmic TLR9 immunoexpression was, however, a marker of favourable RCC specific survival both in univariate analysis and in multivariate regression model. CONCLUSIONS: We conclude that TLR9 expression is an independent prognostic marker of RCC and the absence of TLR9 expression is related to poorer prognosis in RCC.

Toll-like receptor-9 expression is inversely correlated with estrogen receptor status in breast cancer.

Toll-like receptor 9 (TLR9) recognizes microbial and vertebrate DNA. We previously demonstrated TLR9 expression in human breast cancer cell lines and showed that TLR9 ligands stimulate their in vitro invasion. The aim of this study was to characterize TLR9 expression in clinical breast cancer specimens. Immunohistochemical staining intensity was compared with known baseline prognostic factors and distant metastasis-free survival. TLR9 expression was detected in 98% of the tumors studied (n = 141). The mean TLR9 staining intensity was higher in ER- than in the highly ER+ breast cancers (p = 0.039). High-grade tumors had significantly increased TLR9 expression (p = 0.027) compared with lower-grade tumors. The highest TLR9 expression was detected in the mucinous and the lowest in the tubular breast cancers (p = 0.034). Distant metastasis-free survival was higher in the lower TLR9-expressing half of the cohort than in the higher TLR9-expressing group (p = 0.118). TLR9 expression did not correlate with menopausal, PgR or Her2 status, patient age, tumor proliferative or invasive characteristics.