GATA transcription factors in testicular adrenal rest tumours.

Testicular adrenal rest tumours (TARTs) are benign adrenal-like testicular tumours that frequently occur in male patients with congenital adrenal hyperplasia. Recently, GATA transcription factors have been linked to the development of TARTs in mice. The aim of our study was to determine GATA expression in human TARTs and other steroidogenic tissues. We determined GATA expression in TARTs (n = 16), Leydig cell tumours (LCTs; n = 7), adrenal (foetal (n = 6) + adult (n = 10)) and testis (foetal (n = 13) + adult (n = 8)). We found testis-like GATA4, and adrenal-like GATA3 and GATA6 gene expressions by qPCR in human TARTs, indicating mixed testicular and adrenal characteristics of TARTs. Currently, no marker is available to discriminate TARTs from LCTs, leading to misdiagnosis and incorrect treatment. GATA3 and GATA6 mRNAs exhibited excellent discriminative power (area under the curve of 0.908 and 0.816, respectively), while immunohistochemistry did not. GATA genes contain several CREB-binding sites and incubation with 0.1 mM dibutyryl cAMP for 4 h stimulated GATA3, GATA4 and GATA6 expressions in a human foetal testis cell line (hs181.tes). Incubation of adrenocortical cells (H295RA) with ACTH, however, did not induce GATA expression in vitro Although ACTH did not dysregulate GATA expression in the only human ACTH-sensitive in vitro model available, our results do suggest that aberrant expression of GATA transcription factors in human TARTs might be involved in TART formation.

Tribbles homolog 3 denotes a poor prognosis in breast cancer and is involved in hypoxia response.

INTRODUCTION: Hypoxia in solid tumors is associated with treatment resistance, resulting in poor prognosis. Tribbles homolog 3 (TRIB3) is induced during hypoxia and is involved in multiple cellular pathways involved in cell survival. Here, we investigated the role of TRIB3 in breast cancer. METHODS: TRIB3 mRNA expression was measured in breast tumor tissue from 247 patients and correlated with clinicopathological parameters and clinical outcome. Furthermore, we studied TRIB3 expression regulation in cell lines, xenografts tissues and human breast cancer material using Reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Finally, the effect of small interfering RNA (siRNA) mediated TRIB3 knockdown on hypoxia tolerance was assessed. RESULTS: Breast cancer patients with low, intermediate or high TRIB3 expression exhibited a mean disease free survival (DFS) of 80 (95% confidence interval [CI] = 74 to 86), 74 (CI = 67 to 81), and 63 (CI = 55 to 71) months respectively (P = .002, Mantel-Cox log-rank). The prognostic value of TRIB3 was limited to those patients that had received radiotherapy as part of their primary treatment (n = 179, P = .005) and remained statistically significant after correction for other clinicopathological parameters (DFS, Hazard Ratio = 1.90, CI = 1.17 to 3.08, P = .009). In breast cell lines TRIB3 expression was induced by hypoxia, nutrient starvation, and endoplasmic reticulum stress in an hypoxia inducible factor 1 (HIF-1) independent manner. TRIB3 induction after hypoxia did not increase with decreasing oxygen levels. In breast tumor xenografts and human breast cancer tissues TRIB3 co-localized with the hypoxic cell marker pimonidazole. The induction of TRIB3 by hypoxia was shown to be regulated via the PERK/ATF4/CHOP pathway of the unfolded protein response and knockdown of TRIB3 resulted in a dose-dependent increase in hypoxia sensitivity. CONCLUSIONS: TRIB3 is independently associated with poor prognosis of breast cancer patients, possibly through its association with tumor cell hypoxia.

Do the survivin (BIRC5) splice variants modulate or add to the prognostic value of total survivin in breast cancer?

BACKGROUND: A total of 4 additional splice variants (survivin-DeltaEx3, survivin 2alpha, survivin-2B, and survivin-3B) have been described for survivin [baculoviral IAP repeat-containing protein (BIRC-5), approved gene symbol BIRC5], which has been implicated in both inhibition of apoptosis and regulation in mitosis in many tumor types. In this study, we assessed whether the survivin splice variants modulate or add to the prognostic value of total survivin in breast cancer. METHODS: With quantitative reverse transcription-PCR, we measured mRNA concentrations of survivin and all variants in tumor tissue from 275 patients with breast cancer and associated these with clinicopathologic characteristics and relapse-free survival. RESULTS: Total survivin, survivin-DeltaEx3, and survivin 2alpha mRNA levels were associated with young age and ductal histology. Total survivin and survivin-DeltaEx3 were highest in samples with advanced histological grade, whereas patients with 4-9 involved lymph nodes expressed less survivin-2B mRNA than those with 1-3 involved nodes. All variants were higher in tumors negative for steroid hormone receptors. Total survivin, survivin 2alpha, and survivin-3B were associated with poor relapse-free survival in univariate analyses. Survivin 2alpha and survivin-3B added to the prognostic value of total survivin in multivariate analyses. In addition, the prognostic value of total survivin was evident only in the presence of higher expression levels of these 2 variants. CONCLUSIONS: All variants of survivin exhibited particular associations with clinicopathologic characteristics (age, histology, grade, and steroid hormone receptor status) of breast cancer patients. Survival analyses suggest a modulating role of survivin 2alpha and survivin-3B on the biological function of total survivin.

Tissue inhibitors of metalloproteinase expression in human breast cancer: TIMP-3 is associated with adjuvant endocrine therapy success.

Tissue inhibitors of matrix metalloproteinase (TIMPs) may be involved in tumour growth, apoptosis, angiogenesis, invasion, and the development of metastases. This study has evaluated the association of the expression levels of the TIMP forms 1, 2, 3, and 4, measured by quantitative real-time RT-PCR, with classical clinicopathological characteristics, ie age, menopausal status, tumour size, histological grade, number of involved lymph nodes, and steroid hormone receptor status, and with disease progression and treatment sensitivity in 273 breast cancer patients. The mRNA levels of TIMP-1 and TIMP-2 were not associated with any known clinicopathological tumour feature. TIMP-3 and TIMP-4 levels were significantly higher in steroid hormone receptor-positive samples, although the levels of TIMP-4 were much lower than those of the other TIMPs. Only TIMP-3 predicted relapse-free survival (RFS) time differently depending on post-surgical treatment as, in particular, the interaction of TIMP-3 with endocrine therapy (p = 0.008, HR = 0.24, 95% CI = 0.09-0.69) contributed significantly to RFS in multivariate Cox regression analysis. In subgroup analyses, the 107 patients treated with tamoxifen differed greatly in prognosis after dichotomization by the median TIMP-3 level (p = 0.0003). Thus, high tumour levels of the matrix metalloproteinases inhibitor and pro-apoptotic factor TIMP-3 are associated with successful tamoxifen treatment of patients with breast cancer.

Mammaglobin is associated with low-grade, steroid receptor-positive breast tumors from postmenopausal patients, and has independent prognostic value for relapse-free survival time.

PURPOSE: The tumor mRNA expression levels of mammaglobin, a novel breast-specific and breast cancer-associated marker, were correlated with disease outcome in 280 patients with primary breast cancer. PATIENTS AND METHODS: Mammaglobin expression levels were assessed by quantitative reverse transcriptase polymerase chain reaction in frozen tumor tissue from breast cancer patients with a median age of 60 years (range, 30 to 88 years) and a median follow-up of 85 months (range, 2 to 169 months). RESULTS: High expression levels were associated with low-grade tumors (P =.018), with positive estrogen and progesterone receptor status (P <.001), and postmenopausal status (P =.010). In the analysis of all patients, low tumor mammaglobin expression levels predicted an early relapse both in Cox univariate (hazard ratio [HR], 0.52; 95% CI, 0.34 to 0.79; P =.002) and multivariate regression analyses corrected for the traditional prognostic factors (HR, 0.55; 95% CI, 0.35 to 0.88; P =.012). The association of mammaglobin expression with the rate of relapse was particularly favorable in patients who received adjuvant tamoxifen treatment (HR, 0.35; 95% CI, 0.17 to 0.71; P =.004). CONCLUSION: These results demonstrate that the assessment of the tumor mRNA expression level of the breast-specific protein mammaglobin could be useful to stratify patients for individual adjuvant treatment strategies.

Molecular beacon reverse transcription-PCR of human chorionic gonadotropin-beta-3, -5, and -8 mRNAs has prognostic value in breast cancer.

BACKGROUND: The beta-subunit of human chorionic gonadotropin (hCG) is encoded by four genes, of which expression of the hCGbeta-3, -5, and -8 genes could have prognostic value in breast cancer. METHODS: Applying a new, modified Molecular Beacon reverse transcription-PCR assay, we investigated the prognostic value of the hCGbeta-3, -5, and -8 gene transcripts in 129 sporadic unilateral breast cancer samples from patients with a median follow-up of 62.3 months. RESULTS: Expression of hCGbeta-3, -5, -8 was significantly (P = 0.020) associated with relapse-free survival (RFS). In multivariate survival analysis, hCGbeta-3, -5, and -8 maintained prognostic value for RFS, with high expression predicting shorter RFS (P = 0.015; hazard ratio, 2.25; 95% confidence interval, 1.17-4.34). Only 1 of 24 (4%) node-negative patients with low hCGbeta-3, -5, -8 expression relapsed, in contrast to 7 of 26 (27%) patients with high expression (P = 0.046). CONCLUSIONS: Expression of hCGbeta-3, -5, -8, which differ by only one nucleotide from other hCGbeta genes, can be assessed by our modified Molecular Beacon assay in breast cancer tissues. Expression of hCGbeta-3, -5, -8 has independent, prognostic value for RFS in breast cancer and may help identify node-negative patients with poor prognosis.

E1AF expression levels are not associated with prognosis in human breast cancer.

E1AF is a transcription factor involved in regulation of several metastasis-associated genes, and is associated with overexpression of HER2/neu. We were unable to find a clear prognostic value of E1AF expression in human breast cancer. Furthermore, no association of E1AF levels with HER2/neu mRNA levels, hormone receptor status, histological grade, tumor size, or lymph node involvement was found.