Genome-wide DNA methylation indicates silencing of tumor suppressor genes in uterine leiomyoma

Background

Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown.

Principal Findings

We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels.

Conclusions

These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women.     

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.     

Breast feeding, parity and breast cancer subtypes in a Spanish cohort

Background

Differences in the incidence and outcome of breast cancer among Hispanic women compared with white women are well documented and are likely explained by ethnic differences in genetic composition, lifestyle, or environmental exposures.

Methodolgy/Principal Findings

A population-based study was conducted in Galicia, Spain. A total of 510 women diagnosed with operable invasive breast cancer between 1997 and 2010 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics were collected. The different breast cancer tumor subtypes were compared on their clinico-pathological characteristics and risk factor profiles, particularly reproductive variables and breastfeeding. Among the 501 breast cancer patients (with known ER and PR receptors), 85% were ER+/PR+ and 15% were ER-&PR-. Among the 405 breast cancer with known ER, PR and HER2 status, 71% were ER+/PR+/HER2- (luminal A), 14% were ER+/PR+/HER2+ (luminal B), 10% were ER−/PR−/HER2- (triple negative breast cancer, TNBC), and 5% were ER−/PR−/HER2+ (non-luminal). A lifetime breastfeeding period equal to or longer than 7 months was less frequent in case patients with TNBC (OR = 0.25, 95% CI = 0.08–0.68) compared to luminal A breast cancers. Both a low (2 or fewer pregnancies) and a high (3–4 pregnancies) number of pregnancies combined with a long breastfeeding period were associated with reduced odds of TNBC compared with luminal A breast cancer, although the association seemed to be slightly more pronounced among women with a low number of pregnancies (OR = 0.09, 95% CI = 0.005–0.54).

Conclusions/Significance

In case-case analyses with the luminal A cases as the reference group, we observed a lower proportion of TNBC among women who breastfed 7 or more months. The combination of longer breastfeeding duration and lower parity seemed to further reduce the odds of having a TNBC compared to a luminal A breast cancer.     

Global gene expression analysis of the interaction between cancer cells and osteoblasts to predict bone metastasis in breast cancer

Background

Bone metastasis is a main cause of morbidity in breast cancer. Since breast cancer is a heterogeneous disease, the interactions of cancer cells with the skeletal host cells might also be diverse. We hypothesized that gene expression signatures induced by heterotypic interaction of breast cancer cells and osteoblasts might be of clinical relevance.

Methodology/Principal Findings

We established an ex vivo co-culture model using benign breast epithelial cells or a panel of 5 malignant breast epithelial cells in combination with primary human osteoblasts and determined associated gene expression changes with HEEBO microarrays. Pretreatment gene expression profiles of 295 early stage breast cancers published from the Netherlands Cancer Institute with a median follow up of 12.6 years allowed evaluating in vitro effects in the in vivo situation.The effects of the interaction between osteoblasts and breast cancer cell lines of different origin were very heterogeneous. Hs578T cells started to proliferate in co-culture with osteoblasts, SKBR-3 induced a TGF-β response and MDA-MB231 cells showed two distinct sets of up-regulated genes: A set of interferon response genes associated with an up-regulation of STAT1 was in vivo remarkably coherent providing a basis for segregation of tumors into two groups. In a uni-variate analysis, early stage tumors with high expression levels (n = 136) of this gene set had a significantly lower overall survival rate (p = 0.005) (63% at 10 years) than tumors with low expression levels (n = 159) (overall survival: 77% at 10 years). The second gene set was associated with IL-6 and did not significantly change the overall survival rate (p = 0.165), but was significantly associated with a shorter time to bone metastasis (p = 0.049; 74% vs. 83% at 10 years).

Conclusion/Significance

An IL-6 gene expression pattern induced by heterotypic interaction of breast cancer cells with osteoblasts in vitro is associated with a higher rate of bone metastasis in vivo.     

Functional significance of vitamin D receptor FokI polymorphism in human breast cancer cells

Background

The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)₂D₃, the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)₂D₃ downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined.

Methods/Findings

To elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)₂D₃ treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively.

Conclusions/Significance

These results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.     

High-dose chemotherapy followed by autologous stem cell transplantation as a first-line therapy for high-risk primary breast cancer: a meta-analysis

Background and Objectives

Several trials have generated conflicting results about the results of high-dose chemotherapy followed by autologous stem cell transplantation (HDCT) for primary breast cancer. This meta-analysis summarizes the available evidence from all suitable studies.

Design and Methods

Prospective, randomized trials with HDCT as a first-line therapy for primary breast cancer were included in this meta-analysis. The primary outcome of interest for our analysis was survival (disease-free survival and overall survival); secondary endpoints included treatment-related mortality (TRM) and second (non-breast) cancers. We used a median age of 47, a PR positive rate of 50% and a premenopausal rate of 70% as cutoff values to complete the subgroup analyses, which were pre-planned according to the prepared protocol.

Results

Fourteen trials with 5747 patients were eligible for the meta-analysis. Compared with non-HDCT, non-significant second (non-breast) cancers (RR = 1.28; 95% CI = 0.82–1.98) and higher TRM (RR = 3.42; 95% CI = 1.32–8.86) were associated with HDCT for primary breast cancer. A significant DFS benefit of HDCT was documented (HR = 0.89; 95% CI = 0.79–0.99). No difference in OS (overall survival) was found when the studies were pooled (HR = 0.91; 95% CI = 0.82–1.00, p = 0.062). In subgroup analysis, age and hormone receptor status had a significant interaction with prolonged DFS and OS.

Conclusions

HDCT has a benefit on DFS and OS compared to SDC in some special patients with high-risk primary breast cancer.     

HIV tropism and decreased risk of breast cancer

Background

During the first two decades of the U.S. AIDS epidemic, and unlike some malignancies, breast cancer risk was significantly lower for women with human immunodeficiency virus (HIV) infection compared to the general population. This deficit in HIV-associated breast cancer could not be attributed to differences in survival, immune deficiency, childbearing or other breast cancer risk factors. HIV infects mononuclear immune cells by binding to the CD4 molecule and to CCR5 or CXCR4 chemokine coreceptors. Neoplastic breast cells commonly express CXCR4 but not CCR5. In vitro, binding HIV envelope protein to CXCR4 has been shown to induce apoptosis of neoplastic breast cells. Based on these observations, we hypothesized that breast cancer risk would be lower among women with CXCR4-tropic HIV infection.

Methods and Findings

We conducted a breast cancer nested case-control study among women who participated in the WIHS and HERS HIV cohort studies with longitudinally collected risk factor data and plasma. Cases were HIV-infected women (mean age 46 years) who had stored plasma collected within 24 months of breast cancer diagnosis and an HIV viral load ≥500 copies/mL. Three HIV-infected control women, without breast cancer, were matched to each case based on age and plasma collection date. CXCR4-tropism was determined by a phenotypic tropism assay. Odds ratios (OR) and 95% confidence intervals (CI) for breast cancer were estimated by exact conditional logistic regression. Two (9%) of 23 breast cancer cases had CXCR4-tropic HIV, compared to 19 (28%) of 69 matched controls. Breast cancer risk was significantly and independently reduced with CXCR4 tropism (adjusted odds ratio, 0.10, 95% CI 0.002–0.84) and with menopause (adjusted odds ratio, 0.08, 95% CI 0.001–0.83). Adjustment for CD4⁺ cell count, HIV viral load, and use of antiretroviral therapy did not attenuate the association between infection with CXCR4-tropic HIV and breast cancer.

Conclusions

Low breast cancer risk with HIV is specifically linked to CXCR4-using variants of HIV. These variants are thought to exclusively bind to and signal through a receptor that is commonly expressed on hyperplastic and neoplastic breast duct cells. Additional studies are needed to confirm these observations and to understand how CXCR4 might reduce breast cancer risk.     

Family history and breast cancer hormone receptor status in a Spanish cohort

Background

Breast cancer is a heterogenous disease that impacts racial/ethnic groups differently. Differences in genetic composition, lifestyles, reproductive factors, or environmental exposures may contribute to the differential presentation of breast cancer among Hispanic women.

Materials and Methods

A population-based study was conducted in the city of Santiago de Compostela, Spain. A total of 645 women diagnosed with operable invasive breast cancer between 1992 and 2005 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics of the tumors were collected. Hormone receptor negative tumors were compared with hormone receptor postive tumors on their clinico-pathological characteristics as well as risk factor profiles.

Results

Among the 645 breast cancer patients, 78% were estrogen receptor-positive (ER+) or progesterone receptor-positive (PR+), and 22% were ER−&PR−. Women with a family history of breast cancer were more likely to have ER−&PR− tumors than women without a family history (Odds ratio, 1.43; 95% confidence interval, 0.91–2.26). This association was limited to cancers diagnosed before age 50 (Odds ratio, 2.79; 95% confidence interval, 1.34–5.81).

Conclusions

An increased proportion of ER−&PR− breast cancer was observed among younger Spanish women with a family history of the disease.     

Trends in breast cancer mortality in Sweden before and after implementation of mammography screening

Background

Incidence-based mortality modelling comparing the risk of breast cancer death in screened and unscreened women in nine Swedish counties has suggested a 39% risk reduction in women 40 to 69 years old after introduction of mammography screening in the 1980s and 1990s.

Objective

We evaluated changes in breast cancer mortality in the same nine Swedish counties using a model approach based on official Swedish breast cancer mortality statistics, robust to effects of over-diagnosis and treatment changes. Using mortality data from the NordCan database from 1974 until 2003, we estimated the change in breast cancer mortality before and after introduction of mammography screening in at least the 13 years that followed screening start.

Results

Breast mortality decreased by 16% (95% CI: 9 to 22%) in women 40 to 69, and by 11% (95% CI: 2 to 20%) in women 40 to 79 years of age.

Discussion

Without individual data it is impossible to completely separate the effects of improved treatment and health service organisation from that of screening, which would bias our results in favour of screening. There will also be some contamination of post-screening mortality from breast cancer diagnosed prior to screening, beyond our attempts to adjust for delayed benefit. This would bias against screening. However, our estimates from publicly available data suggest considerably lower benefits than estimates based on comparison of screened versus non-screened women.     

Ethnic differences in survival after breast cancer in South East Asia

Background

The burden of breast cancer in Asia is escalating. We evaluated the impact of ethnicity on survival after breast cancer in the multi-ethnic region of South East Asia.

Methodology/Principal Findings

Using the Singapore-Malaysia hospital-based breast cancer registry, we analyzed the association between ethnicity and mortality following breast cancer in 5,264 patients diagnosed between 1990 and 2007 (Chinese: 71.6%, Malay: 18.4%, Indian: 10.0%). We compared survival rates between ethnic groups and calculated adjusted hazard ratios (HR) to estimate the independent effect of ethnicity on survival. Malays (n = 968) presented at a significantly younger age, with larger tumors, and at later stages than the Chinese and Indians. Malays were also more likely to have axillary lymph node metastasis at similar tumor sizes and to have hormone receptor negative and poorly differentiated tumors. Five year overall survival was highest in the Chinese women (75.8%; 95%CI: 74.4%–77.3%) followed by Indians (68.0%; 95%CI: 63.8%–72.2%), and Malays (58.5%; 95%CI: 55.2%–61.7%). Compared to the Chinese, Malay ethnicity was associated with significantly higher risk of all-cause mortality (HR: 1.34; 95%CI: 1.19–1.51), independent of age, stage, tumor characteristics and treatment. Indian ethnicity was not significantly associated with risk of mortality after breast cancer compared to the Chinese (HR: 1.14; 95%CI: 0.98–1.34).

Conclusion

In South East Asia, Malay ethnicity is independently associated with poorer survival after breast cancer. Research into underlying reasons, potentially including variations in tumor biology, psychosocial factors, treatment responsiveness and lifestyle after diagnosis, is warranted.     

Pretreatment serum concentrations of 25-hydroxyvitamin D and breast cancer prognostic characteristics: a case-control and a case-series study

Background

Results from epidemiologic studies on the relationship between vitamin D and breast cancer risk are inconclusive. It is possible that vitamin D may be effective in reducing risk only of specific subtypes due to disease heterogeneity.

Methods and Findings

In case-control and case-series analyses, we examined serum concentrations of 25-hydroxyvitamin D (25OHD) in relation to breast cancer prognostic characteristics, including histologic grade, estrogen receptor (ER), and molecular subtypes defined by ER, progesterone receptor (PR) and HER2, among 579 women with incident breast cancer and 574 controls matched on age and time of blood draw enrolled in the Roswell Park Cancer Institute from 2003 to 2008. We found that breast cancer cases had significantly lower 25OHD concentrations than controls (adjusted mean, 22.8 versus 26.2 ng/mL, p<0.001). Among premenopausal women, 25OHD concentrations were lower in those with high- versus low-grade tumors, and ER negative versus ER positive tumors (p≤0.03). Levels were lowest among women with triple-negative cancer (17.5 ng/mL), significantly different from those with luminal A cancer (24.5 ng/mL, p = 0.002). In case-control analyses, premenopausal women with 25OHD concentrations above the median had significantly lower odds of having triple-negative cancer (OR = 0.21, 95% CI = 0.08–0.53) than those with levels below the median; and every 10 ng/mL increase in serum 25OHD concentrations was associated with a 64% lower odds of having triple-negative cancer (OR = 0.36, 95% CI = 0.22–0.56). The differential associations by tumor subtypes among premenopausal women were confirmed in case-series analyses.

Conclusion

In our analyses, higher serum levels of 25OHD were associated with reduced risk of breast cancer, with associations strongest for high grade, ER negative or triple negative cancers in premenopausal women. With further confirmation in large prospective studies, these findings could warrant vitamin D supplementation for reducing breast cancer risk, particularly those with poor prognostic characteristics among premenopausal women.     

Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model

Purpose

Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure “transplant conditions” (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01.

Methods

Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts.

Results

Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001).

Conclusion

Pre-confluent cells should be used to maximize graft cell viability.