Michael P.A. Davies

Estrogen Receptor-Positive Proliferating Cells in the Normal and Precancerous Breast

Recently it has been shown that epithelial cell expression of the estrogen receptor (ER) and that of the proliferation-associated marker Ki-67 are almost mutually exclusive in the normal premenopausal human breast but that coexpression frequently occurs in estrogen receptor-positive (ER+) breast cancers. This coexpression may indicate disordered expression of ER in the cell cycle or failure to suppress division of ER+ cells and could be important in neoplastic transformation. The purpose of this study was to determine whether in situ proliferations known to be associated with different levels of risk for developing breast cancer contain these coexpressing cells and, if so, the stage at which they occur. We found that ER+ proliferating cells were rare in premenopausal lobules but increased with age in the normal breast. There was no difference in nonlesional tissue between cancerous and noncancerous breasts. The percentage of dual-expressing cells was significantly increased, however, in all of the in situ proliferations and correlated positively with the level of risk of developing breast cancer. We suggest that development of at least some human breast cancers is associated with increasing failure to down-regulate ER as cells enter the cycle or to suppress division of ER+ cells. The mechanism may involve the loss of a tumor suppressor gene.

'Declining estrogen receptor-beta expression defines malignant progression of human breast neoplasia'

It has been shown that the risk of breast cancer developing in certain morphologically identifiable benign breast lesions correlates with expression of estrogen receptor alpha (ER-&agr;). Although ER-&agr; and ER-&bgr; genes share a large degree of homology, it is generally thought that their distribution and functions are substantially different in many tissues. Recent development of reliable antibodies to ER-&bgr; has provided this first opportunity to test the hypothesis that the likelihood of malignant transformation in morphologically benign breast lesions can be accurately defined by the distribution and level of ER-&bgr; expression relative to that of ER-&agr;. Using a monoclonal antibody, ER-&bgr; protein expression has been analyzed in 53 normal breasts and compared with a cohort of histologically distinct breast lesions of different prognostic risk (54 hyperplasia of usual type, 35 ductal carcinoma in situ, and 141 invasive cancers). All of these tissues were also assessed for ER-&agr;. Expression of ER-&bgr; protein was also analyzed in an additional spectrum of benign breast lesions with low or negligible risk of progression to malignancy. The median proportion of cells expressing ER-&bgr; was highest in normal breast lobules (median 94.33%, interquartile range 78.25–99.00) but declined significantly through usual ductal hyperplasia (median 76.67, interquartile range 49.17–95.00, P = 0.002) and ductal carcinoma in situ (median 70.00, interquartile range 59.00–85.00, P = 0.009) to invasive cancer (median 60.00, interquartile range 50.00–80.00, P < 0.001). An appreciable proportion (33.81%) of ER-&agr;-negative invasive cancers expressed ER-&bgr;. A high but variable level of ER-&bgr; expression occurred in the benign lesions. The data from the intact histologic tissues were evaluated with respect to the relative expression of ER-&agr; and ER-&bgr; in five mammary cell lines of different behavioral phenotype (MCF7, ZR-75, T47D, MDAMB231, HUMA121). The highly significant differences in expression and distinct tissue distributions of ER-&agr; and ER-&bgr; within the histologic lesions of defined risk, together with the data from the cell lines, support the original hypothesis that the tissue concentration, relative occurrence, and/or interaction of these two types of estrogen receptor may play an important role in modulating mammary tumorigenesis.

SIRT4 Coordinates the Balance between Lipid Synthesis and Catabolism by Repressing Malonyl CoA Decarboxylase

Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.

A Prognostic DNA Methylation Signature for Stage I Non–Small-Cell Lung Cancer

PURPOSE Non-small-cell lung cancer (NSCLC) is a tumor in which only small improvements in clinical outcome have been achieved. The issue is critical for stage I patients for whom there are no available biomarkers that indicate which high-risk patients should receive adjuvant chemotherapy. We aimed to find DNA methylation markers that could be helpful in this regard. PATIENTS AND METHODS A DNA methylation microarray that analyzes 450,000 CpG sites was used to study tumoral DNA obtained from 444 patients with NSCLC that included 237 stage I tumors. The prognostic DNA methylation markers were validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. RESULTS Unsupervised clustering of the 10,000 most variable DNA methylation sites in the discovery cohort identified patients with high-risk stage I NSCLC who had shorter relapse-free survival (RFS; hazard ratio [HR], 2.35; 95% CI, 1.29 to 4.28; P = .004). The study in the validation cohort of the significant methylated sites from the discovery cohort found that hypermethylation of five genes was significantly associated with shorter RFS in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high- and low-risk stage I NSCLC (HR, 3.24; 95% CI, 1.61 to 6.54; P = .001). CONCLUSION The DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.

Frequent mutations in chromatin-remodelling genes in pulmonary carcinoids

Pulmonary carcinoids are rare neuroendocrine tumors of the lung. The molecular alterations underlying the pathogenesis of these tumors have not been systematically studied so far. Here we perform gene copy number analysis (n=54), genome/exome (n=44) and transcriptome (n=69) sequencing of pulmonary carcinoids and observe frequent mutations in chromatin-remodeling genes. Covalent histone modifiers and subunits of the SWI/SNF complex are mutated in 40% and 22.2% of the cases respectively, with MEN1, PSIP1 and ARID1A being recurrently affected. In contrast to small-cell lung cancer and large-cell neuroendocrine tumors, TP53 and RB1 mutations are rare events, suggesting that pulmonary carcinoids are not early progenitor lesions of the highly aggressive lung neuroendocrine tumors but arise through independent cellular mechanisms. These data also suggest that inactivation of chromatin remodeling genes is sufficient to drive transformation in pulmonary carcinoids.

Obesity, metabolic factors and risk of different histological types of lung cancer: A Mendelian randomization study.

Background Assessing the relationship between lung cancer and metabolic conditions is challenging because of the confounding effect of tobacco. Mendelian randomization (MR), or the use of genetic instrumental variables to assess causality, may help to identify the metabolic drivers of lung cancer. Methods and findings We identified genetic instruments for potential metabolic risk factors and evaluated these in relation to risk using 29,266 lung cancer cases (including 11,273 adenocarcinomas, 7,426 squamous cell and 2,664 small cell cases) and 56,450 controls. The MR risk analysis suggested a causal effect of body mass index (BMI) on lung cancer risk for two of the three major histological subtypes, with evidence of a risk increase for squamous cell carcinoma (odds ratio (OR) [95% confidence interval (CI)] = 1.20 [1.01–1.43] and for small cell lung cancer (OR [95%CI] = 1.52 [1.15–2.00]) for each standard deviation (SD) increase in BMI [4.6 kg/m2]), but not for adenocarcinoma (OR [95%CI] = 0.93 [0.79–1.08]) (Pheterogeneity = 4.3x10-3). Additional analysis using a genetic instrument for BMI showed that each SD increase in BMI increased cigarette consumption by 1.27 cigarettes per day (P = 2.1x10-3), providing novel evidence that a genetic susceptibility to obesity influences smoking patterns. There was also evidence that low-density lipoprotein cholesterol was inversely associated with lung cancer overall risk (OR [95%CI] = 0.90 [0.84–0.97] per SD of 38 mg/dl), while fasting insulin was positively associated (OR [95%CI] = 1.63 [1.25–2.13] per SD of 44.4 pmol/l). Sensitivity analyses including a weighted-median approach and MR-Egger test did not detect other pleiotropic effects biasing the main results. Conclusions Our results are consistent with a causal role of fasting insulin and low-density lipoprotein cholesterol in lung cancer etiology, as well as for BMI in squamous cell and small cell carcinoma. The latter relation may be mediated by a previously unrecognized effect of obesity on smoking behavior.

Wild-type oestrogen receptor beta (ERβ1) mRNA and protein expression in Tamoxifen-treated post-menopausal breast cancers

This study has tested the hypothesis that comparison of protein and mRNA expression for ERα and ERβ1 by human breast cancers provides novel information relating to the clinical and pathological characteristics of human breast cancers. Expression of ERα and ERβ1 was identified in 167 invasive cancers from postmenopausal women treated only with endocrine therapy. The cohort included 143 cases receiving only adjuvant Tamoxifen following surgery. ERα and ERβ1 expression was analysed by immunohistochemistry and reverse transcription RT–PCR and compared with clinical progression of individual cancers. ERα protein was closely associated with the corresponding RNA detected by RT–PCR (Chi-square, P<0.001). In contrast, ERβ1 protein and mRNA were inconsistent. Although an association was identified between ERα and ERβ mRNAs (Chi-square, P<0.001) and between ERα protein and ERβ1 mRNA (Chi-square, P<0.027), no association was identified for the ERα and ERβ1 proteins detected by immunohistochemistry. ERβ1 was not associated with outcome. However, in the absence of ERα, ERβ1 protein expression was associated with elevated cell proliferation. There was a trend for the ERβ1 protein-positive cases to have a worse outcome, both within the group as a whole as well as within the ERα-positive Tamoxifen-treated cases. This study has confirmed the hypothesis that expression of ERα is an important determinant of breast cancer progression, and has further demonstrated that ERβ1 may play a role in the response of breast cancers to endocrine therapy.

Epigenetic biomarkers in lung cancer

Lung cancer mortality is strongly associated with the predominant diagnosis of late stage lesions that hampers effective therapy. Molecular biomarkers for early lung cancer detection is an unmet public health need and the lung cancer research community worldwide is putting a lot of effort to utilise major lung cancer population programmes in order to develop such molecular tools. The study of cancer epigenetics in the last decade has radically altered our views in cancer pathogenesis, providing new insights in biomarker development for risk assessment, early detection and therapeutic stratification. DNA methylation and miRNAs have rapidly emerged as potential biomarkers in body fluids showing promise to assist the clinical management of lung cancer. These new developments are exemplified in this review, demonstrating the huge potential of clinical cancer epigenetics, but also critically discussing the necessary validation steps to bring epigenetic biomarkers towards clinical implementation and the weaknesses of current biomarker studies.

Significance of the metastasis-inducing protein AGR2 for outcome in hormonally treated breast cancer patients

The anterior gradient protein-2 (AGR2) is inducible by oestrogen and itself can induce metastasis in a rat model for breast cancer. Here, a rabbit antibody to recombinant human AGR2 was used to assess its prognostic significance in a retrospective cohort of 351 breast cancer patients treated by adjuvant hormonal therapy. The antibody stains 66% of breast carcinomas to varying degrees. The percentage of positive carcinoma cells in tumours directly correlates with the level of AGR2 mRNA (Spearmans rank correlation, P=0.0007) and protein (linear regression analysis r2=0.95, P=0.0002). There is a significant association of staining of carcinomas for AGR2 with oestrogen receptor α (ERα) staining and with low histological grade (both Fishers Exact test P<0.0001). In the ERα-positive cases, but not the ERα-negative cases, when subdivided into the separate staining classes for AGR2, there is a significantly progressive decrease in patient survival with increased staining (log rank test, P=0.006). The significant association of staining for AGR2 with patient death over a 10-year period (log rank test P=0.007, hazard ratio=3) only becomes significant at 6 years of follow-up. This may be due to the cessation of adjuvant hormonal therapy at an earlier time, resulting in adverse re-expression of the metastasis-inducing protein AGR2.

Abnormal regulation of the oestrogen receptor in benign breast lesions

Background—In normal breast tissue the oestrogen receptor (ER) and the proliferation associated antigen Ki67 are negatively associated, indicating that ER+ cells are non-dividing, or that the receptor is downregulated as cells enter cycle. This relation is completely or partially lost in many ER+ breast cancers and in in situ proliferations associated with an increased cancer risk, where coexpression of the two markers is often found. Aims—To determine whether similar changes can be identified in other risk associated breast lesions. Patients/Methods—Paraffin wax blocks from 12 cases of lactational change, 21 apocrine metaplasias, 22 duct ectasias, 20 sclerosing adenosis, 20 fibroadenomas, 19 phyllodes tumours, 20 radial scars, 21 papillomas (15 solitary and six multiple), 15 gynaecomastias, and nine postmortem male breast tissues were retrieved. Immunohistochemistry was used to determine the expression of ER and dual labelling immunofluorescence was used to detect cells expressing both ER and Ki67. Results—Increased numbers of ER+ cells were seen in sclerosing adenosis, radial scars, papillomas, fibroadenomas, and phyllodes tumours but not in apocrine cysts (where no ER+ cells were detected) or duct ectasia (where normal numbers were found). As in the normal breast, the proportion of ER+ cells increased with age in all lesions with the exception of fibroadenomas. Coexpression of ER and Ki67 was found in an increased proportion of cells of all risk associated lesions studied. ER+ cells were less likely to be dividing than ER− cells in all cases, although this was significant only for sclerosing adenosis. The data on sclerosing adenosis, radial scars, papillomas, and fibroadenomas are comparable with those reported previously in hyperplasia of usual type, whereas those in duct ectasia are similar to those of the normal breast. The findings in all lesions, however, differed from those in ductal carcinoma in situ, where proportions of ER+ and ER+/Ki67+ cells are higher and the relation between ER+ cell numbers and age is lost. Thus, the nature and degree of dysregulation of ER in benign breast lesions is broadly in accordance with the degree of risk of developing breast cancer with which they are associated. In gynaecomastia, the proportions of ER+ and ER+/Ki67+ cells were comparable with those seen in benign female breast lesions, but changes with age were not observed. However, the changes in gynaecomastia were similar to those seen in normal male breast. Conclusion—These findings are in keeping with the contention that the dissociation of ER and Ki67 expression is a very early change in the pathway to many breast cancers. However, this change might only have preneoplastic importance in the hormonal milieu of the female breast.