PGRMC2 is an intracellular haem chaperone critical for adipocyte function.

Haem is an essential prosthetic group of numerous proteins and a central signalling molecule in many physiologic processes1,2. The chemical reactivity of haem means that a network of intracellular chaperone proteins is required to avert the cytotoxic effects of free haem, but the constituents of such trafficking pathways are unknown3,4. Haem synthesis is completed in mitochondria, with ferrochelatase adding iron to protoporphyrin IX. How this vital but highly reactive metabolite is delivered from mitochondria to haemoproteins throughout the cell remains poorly defined3,4. Here we show that progesterone receptor membrane component 2 (PGRMC2) is required for delivery of labile, or signalling haem, to the nucleus. Deletion of PGMRC2 in brown fat, which has a high demand for haem, reduced labile haem in the nucleus and increased stability of the haem-responsive transcriptional repressors Rev-Erbα and BACH1. Ensuing alterations in gene expression caused severe mitochondrial defects that rendered adipose-specific PGRMC2-null mice unable to activate adaptive thermogenesis and prone to greater metabolic deterioration when fed a high-fat diet. By contrast, obese-diabetic mice treated with a small-molecule PGRMC2 activator showed substantial improvement of diabetic features. These studies uncover a role for PGRMC2 in intracellular haem transport, reveal the influence of adipose tissue haem dynamics on physiology and suggest that modulation of PGRMC2 may revert obesity-linked defects in adipocytes.

ARID1A and PGR proteins interact in the endometrium and reveal a positive correlation in endometriosis.

Endometriosis is a disorder in which endometrial cells normally limited to the lining of the uterus proliferate outside the uterine cavity and can cause pelvic pain and infertility. ARID1A levels are significantly reduced in the eutopic endometrium from women with endometriosis. Uterine specific Arid1a knock-out mice were infertile due to loss of epithelial progesterone receptor (PGR) signaling. However, the functional association of ARID1A and PGR in endometriosis has not been studied. We examined the expression patterns and co-localization of ARID1A and PGR in eutopic endometrium from women with and without endometriosis using immunostaining and Western blot analysis. ARID1A and PGR proteins co-localized in the epithelium during the proliferative and the early secretory phases. Our immunoprecipitation analysis and proximity ligation assay (PLA) revealed physical interaction between ARID1A and PGR-A but not PGR-B in the mouse and human endometrium. ARID1A levels positively correlated with PGR levels in the eutopic endometrium of women with endometriosis. Our results bring new perspectives on the molecular mechanisms involved in endometrial receptivity and progesterone resistance in endometriosis. The interrelationship between ARID1A and PGR may contribute to explaining the non-receptive endometrium in endometriosis-related infertility.

Absence of progesterone receptor membrane component 1 reduces migration and metastasis of breast cancer.

BACKGROUND: Progesterone receptor membrane component 1 (Pgrmc1) is a non-classical progesterone receptor associated with the development of the mammary gland and xenograft-induced breast cancer. Importantly, Pgrmc1 is associated with the expression of estrogen receptor alpha and can be used for predicting the prognosis of breast cancer. Whether the genetic deletion of Pgrmc1 affects the progression of breast cancer is still unclear. METHODS: We used MMTV-PyMT transgenic mice that spontaneously develop breast tumors. In backcrossed FVB Pgrmc1 knockout (KO) mice, we monitored the development of the primary tumor and lung metastasis. In MCF-7 and MDA-MB-231 tumor cell lines, the migratory activity was evaluated after Pgrmc1 knockdown. RESULTS: There was no significant difference in the development of breast cancer in terms of tumor size at 13 weeks of age between WT and Pgrmc1 KO mice. However, Pgrmc1 KO mice had a significantly longer survival duration compared with WT mice. Furthermore, Pgrmc1 KO mice exhibited a significantly lower degree of lung metastasis. Compared with those of WT mice, the tumors of Pgrmc1 KO mice had a low expression of focal adhesion kinase and epithelial-mesenchymal transition markers. PGRMC1 knockdown resulted in a significantly reduced migration rate in breast cancer cell lines. CONCLUSIONS: Pgrmc1 KO mice with breast cancer had a prolonged survival, which was accompanied by a low degree of lung metastasis. PGRMC1 showed a significant role in the migration of breast cancer cells, and may serve as a potential therapeutic target in breast cancer. Video Abstract.

FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation.

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, ß-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.

Downregulation of nuclear progestin receptor (Pgr) and subfertility in double knockouts of progestin receptor membrane component 1 (pgrmc1) and pgrmc2 in zebrafish.

The progestin receptor membrane components (Pgrmcs) contain two paralogs, Pgrmc1 and Pgrmc2. Our previous research into single knockout of Pgrmc1 or Pgrmc2 suggests that Pgrmc1 and Pgrmc2 regulate membrane progestin receptor or steroid synthesis and therefore female fertility in zebrafish. Additional roles of Pgrmcs may not be determined in using single Pgrmc knockouts due to compensatory roles between Pgrmc1 and Pgrmc2. To address this question, we crossed single knockout pgrmc1 (pgrmc1-/-) with pgrmc2 (pgrmc2-/-), and generated double knockouts for both pgrmc1 and pgrmc2 (pgrmc1/2-/-) in a vertebrate model, zebrafish. In addition to the delayed oocyte maturation and reduced female fertility, significant reduced ovulation was found in double knockout (pgrmc1/2-/-) in vivo, though not detected in either single knockout of Pgrmc (pgrmc1-/- or pgrmc2-/-). We also found significant down regulation of nuclear progestin receptor (Pgr) protein expression only in pgrmc1/2-/-, which was most likely the cause of reduced ovulation. Lower protein expression of Pgr also resulted in reduced expression of metalloproteinase in pgrmc1/2-/-. With this study, we have provided new evidence for the physiological functions of Pgrmcs in the regulation of female fertility by regulation of ovulation, likely via regulation of Pgr, which affects regulation of metalloproteinase expression and oocyte ovulation.

Paternal developmental toxicant exposure is associated with epigenetic modulation of sperm and placental Pgr and Igf2 in a mouse model.

Preterm birth (PTB), parturition prior to 37 weeks' gestation, is the leading cause of neonatal mortality. The causes of spontaneous PTB are poorly understood; however, recent studies suggest that this condition may arise as a consequence of the parental fetal environment. Specifically, we previously demonstrated that developmental exposure of male mice (F1 animals) to the environmental endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was associated with reduced sperm quantity/quality in adulthood and control female partners frequently delivered preterm. Reproductive defects persisted in the F2 and F3 descendants, and spontaneous PTB was common. Reproductive changes in the F3 males, the first generation without direct TCDD exposure, suggest the occurrence of epigenetic alterations in the sperm, which have the potential to impact placental development. Herein, we conducted an epigenetic microarray analysis of control and F1 male-derived placentae, which identified 2171 differentially methylated regions, including the progesterone receptor (Pgr) and insulin-like growth factor (Igf2). To assess if Pgr and Igf2 DNA methylation changes were present in sperm and persist in future generations, we assessed methylation and expression of these genes in F1/F3 sperm and F3-derived placentae. Although alterations in methylation and gene expression were observed, in most tissues, only Pgr reached statistical significance. Despite the modest gene expression changes in Igf2, offspring of F1 and F3 males consistently exhibited IUGR. Taken together, our data indicate that paternal developmental TCDD exposure is associated with transgenerational placental dysfunction, suggesting epigenetic modifications within the sperm have occurred. An evaluation of additional genes and alternative epigenetic mechanisms is warranted.

The Application of Non-Invasive Apoptosis Detection Sensor (NIADS) on Histone Deacetylation Inhibitor (HDACi)-Induced Breast Cancer Cell Death.

Breast cancer is the most common malignancy in women and the second leading cause of cancer death in women. Triple negative breast cancer (TNBC) subtype is a breast cancer subset without ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2) expression, limiting treatment options and presenting a poorer survival rate. Thus, we investigated whether histone deacetylation inhibitor (HDACi) could be used as potential anti-cancer therapy on breast cancer cells. In this study, we found TNBC and HER2-enriched breast cancers are extremely sensitive to Panobinostat, Belinostat of HDACi via experiments of cell viability assay, apoptotic marker identification and flow cytometry measurement. On the other hand, we developed a bioluminescence-based live cell non-invasive apoptosis detection sensor (NIADS) detection system to evaluate the quantitative and kinetic analyses of apoptotic cell death by HDAC treatment on breast cancer cells. In addition, the use of HDACi may also contribute a synergic anti-cancer effect with co-treatment of chemotherapeutic agent such as doxorubicin on TNBC cells (MDA-MB-231), but not in breast normal epithelia cells (MCF-10A), providing therapeutic benefits against breast tumor in the clinic.

Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization.

STUDY QUESTION: Is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) required for progesterone-driven endometrial stromal cell decidualization? SUMMARY ANSWER: GREB1 is a novel progesterone-responsive gene required for progesterone-driven human endometrial stromal cell (HESC) decidualization. WHAT IS KNOWN ALREADY: Successful establishment of pregnancy requires HESCs to transform from fibroblastic to epithelioid cells in a process called decidualization. This process depends on the hormone progesterone, but the molecular mechanisms by which it occurs have not been determined. STUDY DESIGN, SIZE, DURATION: Primary and transformed HESCs in which GREB1 expression was knocked down were decidualized in culture for up to 6 days. Wild-type and progesterone receptor (PR) knockout mice were treated with progesterone, and their uteri were assessed for levels of GREB1 expression. PARTICIPANTS/MATERIALS, SETTING, METHODS: Analysis of previous data included data mining of expression profile data sets and in silico transcription factor-binding analysis. Endometrial biopsies obtained from healthy women of reproductive age during the proliferative phase (Days 8-12) of their menstrual cycle were used for isolating HESCs. Experiments were carried out with early passage (no more than four passages) HESCs isolated from at least three subjects. Transcript levels of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were detected by quantitative RT-PCR as readouts for HESC decidualization. Cells were also imaged by phase-contrast microscopy. To assess the requirement for GREB1, PR and SRC-2, cells were transfected with specifically targeted small interfering RNAs. Results are shown as mean and SE from three replicates of one representative patient-derived primary endometrial cell line. Experiments were also conducted with transformed HESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Progesterone treatment of mice and transformed HESCs led to an ~5-fold (5.6 ± 0.81, P < 0.05, and 5.2 ± 0.26, P < 0.01, respectively) increase in GREB1 transcript levels. This increase was significantly reduced in the uteri of PR knock-out mice (P < 0.01), in HESCs treated with the PR antagonist RU486 (P < 0.01), or in HESCs in which PR expression was knocked down (P < 0.05). When GREB1 expression was knocked down, progesterone-driven decidualization markers in both immortalized and primary HESCs was significantly reduced (P < 0.05 and P < 0.01). Finally, GREB1 knock down signficantly reduced expression of the PR target genes WNT4 and FOXOA1 (P < 0.05 and P < 0.01, respectively). LARGE SCALE DATA: This study used the Nuclear Receptor Signaling Atlas. LIMITATIONS, REASONS FOR CAUTION: Although in vitro cell culture studies indicate that GREB1 is required for endoemtrial decidualization, the in vivo role of GREB1 in endometrial function and dysfunction should be assessed by using knock-out mouse models. WIDER IMPLICATIONS OF THE FINDINGS: Identification and functional analysis of GREB1 as a key molecular mediator of decidualization may lead to improved diagnosis and clinical management of women with peri-implantation loss due to inadequate endometrial decidualization. STUDY FUNDING AND COMPETING INTEREST(S): This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests.

Prediction of Recurrence and Survival for Triple-Negative Breast Cancer (TNBC) by a Protein Signature in Tissue Samples.

To date, there is no available targeted therapy for patients who are diagnosed with triple-negative breast cancers (TNBC). The aim of this study was to identify a new specific target for specific treatments. Frozen primary tumors were collected from 83 adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling by iTRAQ-OFFGEL-LC-MS/MS approach in two series: a training cohort (n = 42) and a test set (n = 41). Patients who remains free of local or distant metastasis for a minimum of 5 years after surgery were classified in the no-relapse group; the others were in the relapse group. OPLS and Kaplan-Meier analyses were performed to select candidate markers, which were validated by immunohistochemistry. Three proteins were identified in the training set and validated in the test set by Kaplan-Meier method and immunohistochemistry (IHC): TrpRS as a good prognostic markers and DP and TSP1 as bad prognostic markers. We propose the establishment of an IHC test to calculate the score of TrpRS, DP, and TSP1 in TNBC tumors to evaluate the degree of aggressiveness of the tumors. Finally, we propose that DP and TSP1 could provide therapeutic targets for specific treatments.

Infertility associated with the absence of endometrial progesterone receptors in a bitch.

A three-year-old intact female Old English sheepdog was presented for evaluation of infertility. A uterine biopsy was performed during dioestrus, and the microscopic appearance was inconsistent with progesterone stimulation; the glands were sparse, simple and failed to show coiling, while the glandular epithelium was cuboidal instead of columnar. There was very little evidence of glandular activity. Due to the inappropriate appearance of the glands for the stage of the cycle, immunohistochemistry for progesterone receptors was performed. No progesterone receptor-positive immunoreactivity was identified in the endometrial luminal epithelium, glandular epithelium or stroma. Weak intranuclear immunoreactivity was identified within the smooth muscle cells of the myometrium. The absence of progesterone receptors within the endometrial glands is the most likely explanation for the abnormal appearance of the endometrium and for this bitch's infertility. To our knowledge, this is the first report of endometrial progesterone receptor absence in a bitch.

Paracrine-rescued lobulogenesis in chimeric outgrowths comprising progesterone-receptor-null mammary epithelium and redirected wild-type testicular cells.

We have previously shown that non-mammary and tumorigenic cells can respond to the signals of the mammary niche and alter their cell fate to that of mammary epithelial progenitor cells. Here we tested the hypothesis that paracrine signals from mammary epithelial cells expressing progesterone receptor (PR) are dispensable for redirection of testicular cells, and that re-directed wild-type testicular-derived mammary cells can rescue lobulogenesis of PR-null mammary epithelium by paracrine signaling during pregnancy. We injected PR-null epithelial cells mixed with testicular cells from wild-type adult male mice into cleared fat-pads of recipient mice. The testicular cells were redirected in vivo to mammary epithelial cell fate during regeneration of the mammary epithelium, and persisted in second-generation outgrowths. In the process, the redirected testicular cells rescued the developmentally deficient PR-null cells, signaling them through the paracrine factor RANKL to produce alveolar secretory structures during pregnancy. This is the first demonstration that paracrine signaling required for alveolar development is not required for cellular reprogramming in the mammary gland, and that reprogrammed testicular cells can provide paracrine signals to the surrounding mammary epithelium.

Control of mammary stem cell function by steroid hormone signalling.

The ovarian hormones oestrogen and progesterone profoundly influence breast cancer risk, underpinning the benefit of endocrine therapies in the treatment of breast cancer. Modulation of their effects through ovarian ablation or chemoprevention strategies also significantly decreases breast cancer incidence. Conversely, there is an increased risk of breast cancer associated with pregnancy in the short term. The cellular mechanisms underlying these observations, however, are poorly defined. Here we demonstrate that mouse mammary stem cells (MaSCs) are highly responsive to steroid hormone signalling, despite lacking the oestrogen and progesterone receptors. Ovariectomy markedly diminished MaSC number and outgrowth potential in vivo, whereas MaSC activity increased in mice treated with oestrogen plus progesterone. Notably, even three weeks of treatment with the aromatase inhibitor letrozole was sufficient to reduce the MaSC pool. In contrast, pregnancy led to a transient 11-fold increase in MaSC numbers, probably mediated through paracrine signalling from RANK ligand. The augmented MaSC pool indicates a cellular basis for the short-term increase in breast cancer incidence that accompanies pregnancy. These findings further indicate that breast cancer chemoprevention may be achieved, in part, through suppression of MaSC function.

Low ATM protein expression in malignant tumor as well as cancer-associated stroma are independent prognostic factors in a retrospective study of early-stage hormone-negative breast cancer.

INTRODUCTION: The serine/threonine protein kinase ataxia telangiectasia mutated (ATM) is critical in maintaining genomic integrity. Upon DNA double-strand breaks, ATM phosphorylates key downstream proteins including p53 and BRCA1/2, thereby orchestrating complex signaling pathways involved in cell cycle arrest, DNA repair, senescence and apoptosis. Although sporadic mutation of ATM occurs rarely in breast cancer, the status of its protein expression and its clinical significance in breast cancer remain not well established. Our study was designed to investigate the influence of ATM protein in both tumor and cancer-associated stroma on clinical outcome in hormone-positive (HPBC) and hormone-negative (HNBC) early-stage breast cancer (EBC). METHODS: Tissue microarrays (TMAs), containing formalin-fixed, paraffin-embedded resected tumors from two cohorts of patients (HPBC cohort: n=130; HNBC cohort: n=168) diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, were analyzed for ATM protein expression using fluorescence immunohistochemistry (IHC) and automated quantitative analysis (AQUA). ATM expression levels were measured within the tumor as a whole (tATM) as indicated by pan-cytokeratin expression, tumor nuclear compartment (nATM) as indicated by both DAPI and pan-cytokeratin-positive results, and cancer-associated stroma (csATM) as indicated by vimentin-positive and pan-cytokeratin-negative results. ATM expression levels within these compartments were correlated with clinical outcome. RESULTS: While tATM and nATM were significantly lower in tumors compared to normal breast epithelial tissues, csATM was significantly higher than the corresponding normal tissue compartment. In addition, the median expression level of both tATM and nATM were two- to threefold lower (P<0.001) in HNBC than in HPBC. In both HNBC and HPBC cohorts, patients with low tATM, nATM and csATM tumors had significantly poorer survival outcomes than those with a high tATM, nATM and csATM, but this effect was more pronounced in HNBC. A multivariate analysis demonstrates that these biomarkers predict survival independent of tumor size and lymph node status, but only in the HNBC cohort (P<0.001). CONCLUSIONS: Low ATM protein expression in both malignant tumor and stromal compartments likely contributes to the aggressive nature of breast cancer and is an independent prognostic factor associated with worse survival in HNBC patients.

Reduction of global 5-hydroxymethylcytosine is a poor prognostic factor in breast cancer patients, especially for an ER/PR-negative subtype.

DNA methylation at the 5 position of cytosine (5 mC) is an epigenetic hallmark in cancer. The 5 mC can be converted to 5-hydroxymethylcytosine (5 hmC) through a ten-eleven-translocation (TET). We investigated the impact of 5 mC, 5 hmC, TET1, and TET2 on tumorigenesis and prognosis of breast cancer. Immunohistochemistry was used to assess the levels of 5 mC, 5 hmC, TET1, and TET2 in the corresponding tumor adjacent normal (n = 309), ductal carcinoma in situ (DCIS, n = 120), and invasive ductal carcinoma (IDC, n = 309) tissues for 309 breast ductal carcinoma patients. 5 mC, 5 hmC, TET1-n, and TET2-n were significantly decreased during DCIS and IDC progression. In IDC, the decrease of 5 hmC was correlated with the cytoplasmic mislocalization of TET1 (p < 0.001) as well as poor disease-specific survival (DSS) (adjusted hazard ratio [AHR] 1.95, p = 0.003) and disease-free survival (DFS) (AHR 1.91, p = 0.006). The combined decrease of 5 mC and 5 hmC was correlated with worse DSS (AHR 2.19, p = 0.008) and DFS (AHR 1.99, p = 0.036). Stratification analysis revealed that the low level of 5 mC was associated with poor DSS (AHR 1.89, p = 0.044) and DFS (AHR 2.02, p = 0.035) for the ER/PR-positive subtype. Conversely, the low level of 5 hmC was associated with worse DSS (AHR 2.77, p = 0.002) and DFS (AHR 2.69, p = 0.006) for the ER/PR-negative subtype. The decreases of 5 mC, 5 hmC, TET1-n, and TET2-n were biomarkers of tumor development. The global reduction of 5 hmC was a poor prognostic factor for IDC, especially for ER/PR-negative subtype.

Chromatin composition alterations and the critical role of MeCP2 for epigenetic silencing of progesterone receptor-B gene in endometrial cancers.

OBJECTIVE: To understand the epigenetic mechanism underlying the PR-B gene silencing in endometrial cancer (EC) cells, we compared the chromatin composition between transcriptionally active and silenced PR-B genes in EC cell lines and cancer tissues. METHODS: Chromatin Immunoprecipitation (ChIP) assay was performed to measure MBD occupancy and histone acetylation/methylation in transcriptionally active and silenced PR-B genes. PR-B-positive/-negative, as well as epigenetic inhibitor-treated/-untreated EC cells were used as study models. Real-time polymerase chain reaction (PCR) and Western blot analysis were applied to measure the mRNA and protein levels of PR-B, MBD, and histones. RESULTS: A close association among PR-B methylation, MBD binding and PR-B gene silencing was observed. Treatment with epigenetic inhibitors led to dynamic changes in the PR-B chromatin composition and gene expression. Increased H3/H4 acetylation and H3-K4 methylation, and decreased H3-K9 methylation were found to be associated with re-activation of silenced PR-B genes. MeCP2 knockdown resulted in a decreased MeCP2 binding to PR-B genes and an increased PR-B expression. ChIP analysis of MeCP2 binding to PR-B genes in the PR-B-positive/-negative EC samples confirmed the significant role of MeCP2 in PR-B silencing. CONCLUSION: PR-B gene expression is regulated by a concerted action of epigenetic factors including DNA methylation, MBD binding, and histone modifications. MeCP2 occupancy of PR-B genes plays a critical role in PR-B gene silencing. These findings enriched our knowledge of the epigenetic regulation of PR-B expression in EC, and suggested that the epigenetic re-activation of PR-B could be explored as a potential strategy to sensitize the PR-B-negative endometrial cancers to progestational therapy.

Proteomic profiling of triple-negative breast carcinomas in combination with a three-tier orthogonal technology approach identifies Mage-A4 as potential therapeutic target in estrogen receptor negative breast cancer.

Breast cancer is a very heterogeneous disease, encompassing several intrinsic subtypes with various morphological and molecular features, natural history and response to therapy. Currently, molecular targeted therapies are available for estrogen receptor (ER)(-) and human epidermal growth factor receptor 2 (Her2)-positive breast tumors. However, a significant proportion of primary breast cancers are negative for ER, progesterone receptor (PgR), and Her2, comprising the triple negative breast cancer (TNBC) group. Women with TNBC have a poor prognosis because of the aggressive nature of these tumors and current lack of suitable targeted therapies. As a consequence, the identification of novel relevant protein targets for this group of patients is of great importance. Using a systematic two dimensional (2D) gel-based proteomic profiling strategy, applied to the analysis of fresh TNBC tissue biopsies, in combination with a three-tier orthogonal technology (two dimensional PAGE/silver staining coupled with MS, two dimensional Western blotting, and immunohistochemistry) approach, we aimed to identify targetable protein markers that were present in a significant fraction of samples and that could define therapy-amenable sub-groups of TNBCs. We present here our results, including a large cumulative database of proteins based on the analysis of 78 TNBCs, and the identification and validation of one specific protein, Mage-A4, which was expressed in a significant fraction of TNBC and Her2-positive/ER negative lesions. The high level expression of Mage-A4 in the tumors studied allowed the detection of the protein in the tumor interstitial fluids as well as in sera. The existence of immunotherapeutics approaches specifically targeting this protein, or Mage-A protein family members, and the fact that we were able to detect its presence in serum suggest novel management options for TNBC and human epidermal growth factor receptor 2 positive/estrogen receptor negative patients bearing Mage-A4 positive tumors.

Reproductive risk factors and oestrogen/progesterone receptor-negative breast cancer in the Breast Cancer Family Registry.

BACKGROUND: Oestrogen receptor (ER)- and progesterone receptor (PR)-negative (ER-PR-) breast cancer is associated with poorer prognosis compared with other breast cancer subtypes. High parity has been associated with an increased risk of ER-PR- cancer, but emerging evidence suggests that breastfeeding may reduce this risk. Whether this potential breastfeeding benefit extends to women at high risk of breast cancer remains critical to understand for prevention. METHODS: Using population-based ascertained cases (n=4011) and controls (2997) from the Breast Cancer Family Registry, we examined reproductive risk factors in relation to ER and PR status. RESULTS: High parity (≥3 live births) without breastfeeding was positively associated only with ER-PR- tumours (odds ratio (OR)=1.57, 95% confidence interval (CI), 1.10-2.24); there was no association with parity in women who breastfed (OR=0.93, 95% CI 0.71-1.22). Across all race/ethnicities, associations for ER-PR- cancer were higher among women who did not breastfeed than among women who did. Oral contraceptive (OC) use before 1975 was associated with an increased risk of ER-PR- cancer only (OR=1.32, 95% CI 1.04-1.67). For women who began OC use in 1975 or later there was no increased risk. CONCLUSIONS: Our findings support that there are modifiable factors for ER-PR- breast cancer and that breastfeeding in particular may mitigate the increased risk of ER-PR- cancers seen from multiparity.

Progesterone signaling inhibits cervical carcinogenesis in mice.

Human papillomavirus is the main cause of cervical cancer, yet other nonviral cofactors are also required for the disease. The uterine cervix is a hormone-responsive tissue, and female hormones have been implicated in cervical carcinogenesis. A transgenic mouse model expressing human papillomavirus oncogenes E6 and/or E7 has proven useful to study a mechanism of hormone actions in the context of this common malignancy. Estrogen and estrogen receptor α are required for the development of cervical cancer in this mouse model. Estrogen receptor α is known to up-regulate expression of the progesterone receptor, which, on activation by its ligands, either promotes or inhibits carcinogenesis, depending on the tissue context. Here, we report that progesterone receptor inhibits cervical and vaginal epithelial cell proliferation in a ligand-dependent manner. We also report that synthetic progestin medroxyprogesterone acetate promotes regression of cancers and precancerous lesions in the female lower reproductive tracts (ie, cervix and vagina) in the human papillomavirus transgenic mouse model. Our results provide the first experimental evidence that supports the hypothesis that progesterone signaling is inhibitory for cervical carcinogenesis in vivo.

BRACking news on triple-negative/basal-like breast cancers: how BRCA1 deficiency may result in the development of a selective tumor subtype.

Mutations in the BRCA1 tumor suppressor predispose to the development of breast and ovarian cancers. Noticeably, the majority of BRCA1-associated breast cancers are triple-negative (ER-, PR- and HER2-) and display a basal-like phenotype, which are features relatively uncommon among sporadic breast cancers. It is well documented that BRCA1 is involved in a number of cellular functions converging to the maintenance of genomic stability. However, the control over DNA integrity does not seem to account for the peculiar phenotype of BRCA1-associated tumors since mutations in other genes involved in such a function, namely BRCA2, associate to a broader spectrum of breast carcinoma subtypes. Indeed, an increasing body of evidence indicates that BRCA1 is implicated also in the regulation of transcription by impinging upon general components of the transcriptional machinery. Thus, elucidating the complex biochemical network regulated by BRCA1 may allow a better understanding also of the biology of sporadic triple-negative/basal-like tumors and lay down the basis for novel preventive measures and more effective therapeutic strategies. This review summarizes recent findings on the role of BRCA1 in the regulation of transcription and how this might set the ground for the development of cancers with triple-negative/basal-like features.

Increased gene copy number of KIT and VEGFR2 at 4q12 in primary breast cancer is related to an aggressive phenotype and impaired prognosis.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and no targeted treatments are available for TNBC. Drugs inhibiting tyrosine kinases, such as vascular endothelial growth factor receptor 2 (VEGFR2) and KIT, have shown some promising results for patients with TNBC. The aim of the study was to investigate whether gains and/or amplifications of VEGFR2 and KIT, located at 4q12, occur in TNBC. Fluorescence in situ hybridization (FISH) was used to quantify gene copy numbers of VEGFR2 and KIT in 83 primary human breast cancers including 31 TNBCs. Gains were defined as ≥ 4 gene copies in >40% of the cancer cells, whereas amplification was defined as CEP >2 in more than 10% of the cancer cells. A tumor was considered FISH positive for KIT and/or VEGFR2 if it displayed copy number gain and/or amplification. Ten (32%) of the TNBCs were VEGFR2 FISH positive and nine (29%) were KIT FISH positive, whereas non-TNBCs were FISH positive for VEGFR2 and KIT in nine (18%) cases for both genes, but no significant difference between TNBCs and non-TNBCs was found. FISH positivity for VEGFR2 and KIT was significantly correlated (χ(2) test, P < 0.001), and significantly related to ER negativity and high Nottingham histological grade (NHG). A significantly worse 5-year breast cancer specific survival (BCSS) was seen for FISH positive cases. Increased copy number of VEGFR2 and KIT thus has the potential of functioning as a novel predictive biomarker for selected targeted therapy particularly in the difficult-to-treat TNBC patient category.