Recruitment of regulatory T cells is correlated with hypoxia-induced CXCR4 expression, and is associated with poor prognosis in basal-like breast cancers.

INTRODUCTION: Basal-like breast cancers behave more aggressively despite the presence of a dense lymphoid infiltrate. We hypothesised that immune suppression in this subtype may be due to T regulatory cells (Treg) recruitment driven by hypoxia-induced up-regulation of CXCR4 in Treg. METHODS: Immunoperoxidase staining for FOXP3 and CXCL12 was performed on tissue microarrays from 491 breast cancers. The hypoxia-associated marker carbonic anhydrase IX (CA9) and double FOXP3/CXCR4 staining were performed on sections from a subset of these cancers including 10 basal-like and 11 luminal cancers matched for tumour grade. RESULTS: High Treg infiltration correlated with tumour CXCL12 positivity (OR 1.89, 95% CI 1.22 to 2.94, P = 0.004) and basal phenotype (OR 3.14, 95% CI 1.08 to 9.17, P = 0.004) in univariate and multivariate analyses. CXCL12 positivity correlated with improved survival (P = 0.005), whereas high Treg correlated with shorter survival for all breast cancers (P = 0.001), luminal cancers (P < 0.001) and basal-like cancers (P = 0.040) that were confirmed in a multivariate analysis (OR 1.61, 95% CI 1.02 to 2.53, P = 0.042). In patients treated with hormone therapy, high Treg were associated with a shorter survival in a multivariate analysis (OR 1.78, 95% CI 1.01 to 3.15, P = 0.040). There was a tendency for luminal cancers to show CXCL12 expression (102/138, 74%) compared to basal-like cancers (16/27, 59%), which verged on statistical significance (P = 0.050). Up-regulation of CXCR4 in Treg correlated with the basal-like phenotype (P = 0.029) and tumour hypoxia, as indicated by CA9 expression (P = 0.049). CONCLUSIONS: Our data show that in the setting of hypoxia and CXCR4 up-regulation in Treg, CXCL12 expression may have the negative consequence of enhancing Treg recruitment and suppressing the anti-tumour immune response.

The presence of tumor-infiltrating FOXP3+ lymphocytes correlates with intratumoral angiogenesis in endometrial cancer.

OBJECTIVES: CD4(+)CD25(+) regulatory T-cells (Tregs), that express the transcription factor FOXP3, suppress effector T-cell populations and can enable tumour cells to evade the host immune response. In this study, we investigated the numbers of FOXP3(+) Tregs in the normal and malignat endometrium and examined potential links with tumor angiogenesis. METHODS: Paraffin-embedded tissues from 79 patients with stage I endometrial adenocarcinoma and 12 samples from normal endometrium were analyzed using immunohistochemistry for the detection of FOXP3(+) lymphocytes. The presence of FOXP3(+) lymphocytic infiltration was correlated with the tumor vascular density, the hypoxia inducible factors HIF-1alpha and HIF-2alpha, VEGF, estrogen and progesterone receptor expression. Survival analysis was also performed. RESULTS: In normal endometrium, FOXP3 was expressed by stroma infiltrating lymphocytes, with a mean number 8 (range 5-11) lymphocytes per x100 optical field. In tumors, 55/79 (69.6%) cases showed little FOXP3(+) lymphocytic infiltration (0-2 per x100 optical field). In the remaining 24/79 (30.4%) cases that were scored as positive the mean score ranged from 3-8 (median 5). Low numbers of FOXP3(+) lymphocytes significantly correlated with tumoral ER negativity and low vascular density. Survival analysis showed no significant impact of FOXP3 lymphocytic infiltration, although there was a trend towards worse prognosis. CONCLUSIONS: The correlation between the presence of FOXP3(+) Tregs and high vessel density in endometrial adenocarcinomas suggests a link between immunity, intratumoral angiogenesis and poor prognosis. However, further studies are required as significantly fewer Tregs were detected in the tumor microenvironment compared to normal endometrium.

Increased frequency of regulatory T cells in peripheral blood and tumour infiltrating lymphocytes in colorectal cancer patients.

Recent results have shown a correlation between survival and frequency of tumour infiltrating T lymphocytes in colorectal cancer patients. However, it remains unclear whether the frequency of regulatory T cells is higher in colorectal cancer as compared to normal colon. To address this question we analysed the frequency and function of regulatory T cells in the peripheral blood and tumour infiltrating lymphocytes of colorectal cancer patients. The proportion of regulatory T cells in the peripheral blood of colorectal cancer patients (mean 8%) was significantly higher than that in normal controls (mean 2.2%). There were significantly more regulatory T cells in tumour infiltrating lymphocytes (mean 19.2%) compared to lymphocytes from an autologous non-malignant portion of the colon (mean 9%). Regulatory T cells from colorectal cancer patients were FOXP3 positive and suppressed the proliferation of autologous CD4+ CD25- cells. A higher density of tumour infiltrating regulatory T cells was found in patients with advanced as compared to early disease. These results support the hypothesis that increased numbers of regulatory T cells in the blood and tumours of colorectal cancer patients may influence the immune response against cancer and suggest that strategies to overcome regulatory T cell activity may be beneficial in the treatment of human colorectal cancer.

Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera.

Estrogen receptor alpha (ERalpha) is a transcription factor that regulates expression of target genes in a ligand-dependent manner. Activation of gene expression is mediated by two transcription activation functions AF-1 and AF-2, which act in a promoter- and cell-specific manner. Whilst AF-2 activity is regulated by estrogen (E2) binding, the activity of AF-1 is additionally modulated by phosphorylation at several sites. One of these phosphorylation sites, serine 118 (S118) is of particular interest as its mutation significantly reduces ERalpha activity. Previous studies have shown that S118 can be phosphorylated by the ERK1/2 mitogen activated protein kinases (MAPK) and by the cyclin-dependent protein kinase Cdk7. In this study we use antisera that specifically recognize ERalpha phosphorylated at S118 to demonstrate that MAPK phosphorylates S118 in a ligand-independent manner, whereas Cdk7 mediates E2-induced phosphorylation of S118. E2 stimulation of S118 phosphorylation was observed within 10 min of its addition and was maximal at 10(-7) M E2. S118 phosphorylation was maximal at 30 min but then declined, such that by 180 min following E2 addition little S118 phosphorylation was evident. S118 phosphorylation was also induced by the partial estrogen antagonist 4-hydroxytamoxifen, but not by the complete antagonist ICI 182, 780. S118 phosphorylation upon addition of the MAPK inducers EGF or PMA followed the expected time courses. Finally, we show that ERalpha is phosphorylated at S118 in vivo using immunoblotting of extracts prepared from a series of ERalpha-positive breast tumours.

The p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner.

BACKGROUND: p68 (Ddx5) and p72 (Ddx17) are highly related members of the DEAD box family and are established RNA helicases. They have been implicated in growth regulation and have been shown to be involved in both pre-mRNA and pre-rRNA processing. More recently, however, these proteins have been reported to act as transcriptional co-activators for estrogen-receptor alpha (ER alpha). Furthermore these proteins were shown to interact with co-activators p300/CBP and the RNA polymerase II holoenzyme. Taken together these reports suggest a role for p68 and p72 in transcriptional activation. RESULTS: In this report we show that p68 and p72 can, in some contexts, act as transcriptional repressors. Targeting of p68 or p72 to constitutive promoters leads to repression of transcription; this repression is promoter-specific. Moreover both p68 and p72 associate with histone deacetylase 1 (HDAC1), a well-established transcriptional repression protein. CONCLUSIONS: It is therefore clear that p68 and p72 are important transcriptional regulators, functioning as co-activators and/or co-repressors depending on the context of the promoter and the transcriptional complex in which they exist.

Expression of the forkhead transcription factor FOXP1 is associated with that of estrogen receptor-beta in primary invasive breast carcinomas.

We previously identified a correlation between estrogen receptor alpha (ERalpha) and the candidate tumour suppressor gene Forkhead Box P1 (FOXP1), whose nuclear protein expression in breast tumours was associated with improved patient survival. However, the expression pattern of FOXP1 in normal breast tissue is more reminiscent of the second receptor, ERbeta, which has an emerging role as a tumour suppressor in breast cancer and critically may underlie the ability of some ERalpha-negative tumours to respond to tamoxifen. In a series of 283 breast cancers, in which ERalpha-positive tumours were treated with tamoxifen, the nuclear expression of ERbeta correlated significantly with ERalpha (p = 0.004), low-tumour grade (p = 0.008) and nuclear FOXP1 (p = 0.01). High-grade tumours exhibited significantly more cytoplasmic ERbeta than the low-grade tumours (p = 0.006). Regression analysis demonstrated that FOXP1 expression was most closely related to nuclear ERbeta (p = 0.021). Neither, nuclear or cytoplasmic ERbeta expression demonstrated prognostic significance. FOXP1 is not estrogen regulated and silencing FOXP1 expression, using siRNA, did not affect ERalpha, ERbeta or progesterone receptor expression, suggesting ER and FOXP1 co-expression may reflect a common regulatory mechanism.

The number of regulatory T cells in prostate cancer is associated with the androgen receptor and hypoxia-inducible factor (HIF)-2alpha but not HIF-1alpha.

BACKGROUND: Regulatory T cells (T(R)) mediate peripheral immunological tolerance and are implicated in tumor progression. Because prostate cancer is being investigated for treatment by immunotherapy, we have assessed tumor T(R) in prostate cancers. METHODS: T(R) cells were identified by FOXP3 in tissue microarrays (TMAs) from 146 radical prostatectomies and correlated with clinicopathological tumor parameters and prostatic specific antigen rise (PSA). RESULTS: Twenty of 146 tumors contained no T(R). The mean of the average for the remaining 146 patients was 7.24. There was a significant correlation between T(R) and androgen receptor (P=0.003) and with hypoxia-inducible factor (HIF)-2alpha (P=0.007) but not HIF-1alpha (P=0.25). There was no significant correlation between T(R) numbers and stage, capsular invasion, urethral margins, vascular invasion, Gleason score, pre-operative PSA, or time to PSA recurrence (all P>0.05). CONCLUSIONS: T(R) in prostate tumors shows significant heterogeneity and may be the result of hormonal and hypoxic signaling. Targeting these may reduce T(R) in tumors allowing more successful immune therapies.

Quantification of regulatory T cells enables the identification of high-risk breast cancer patients and those at risk of late relapse.

PURPOSE: To assess the clinical significance of tumor-infiltrating FOXP3-positive regulatory T cells (TR) in breast cancer patients with long-term follow-up. PATIENTS AND METHODS: FOXP3-positive TR were detected by immunohistochemistry with our new, extensively characterized FOXP3 monoclonal antibody, 236A/E7. Numbers of FOXP3-positive lymphocytes in tissue microarray cores from pure ductal carcinoma in situ (DCIS; n = 62), invasive breast cancer (n = 237) or from comparable areas of normal terminal duct lobular breast tissue (n = 10) were determined. A median cutoff of > or = 15 defined patients with high numbers of TR. RESULTS: TR numbers were significantly higher in in situ and invasive breast carcinomas than in normal breast; invasive tumors have significantly higher numbers than DCIS (P = .001). High numbers of FOXP3-positive TR identified patients with DCIS at increased risk of relapse (P = .04) and patients with invasive tumors with both shorter relapse-free (P = .004) and overall survival (P = .007). High TR numbers were present in high-grade tumors (P < or = .001), in patients with lymph node involvement (P = .01), and in estrogen receptor (ER) -negative tumors (P = .001). Importantly, high numbers of TR within ER-positive tumors identified high-risk patients (P = .005). Unlike conventional clinicopathologic factors, high numbers of FOXP3-positive TR can identify patients at risk of relapse after 5 years. CONCLUSION: These findings indicate that quantification of FOXP3-positive TR in breast tumors is valuable for assessing disease prognosis and progression, and that TR are an important therapeutic target for breast cancer. FOXP3-positive TR represent a novel marker for identifying late-relapse patients who may benefit from aromatase therapy after standard tamoxifen treatment.

The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor.

The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. Here, we show that p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53-dependent promoters and that endogenous p68 and p53 co-immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53-dependent apoptosis, but does not influence p53 stabilisation or expression of non-p53-responsive genes. We also show, by chromatin immunoprecipitation, that p68 is recruited to the p21 promoter in a p53-dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock-down does not significantly affect NF-kappaB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity.