Estrogenic microenvironment generated by organochlorine residues in adipose mammary tissue modulates biomarker expression in ERα-positive breast carcinomas

Introduction  

Breast cancer is the most frequent malignant disease in women. Exposure to estrogens throughout a woman's life is a risk factor for the development of breast cancer. Organochlorine compounds (OCCs), such as pesticides and polychlorinated biphenyls, are persistent lipophilic chemicals identified as endocrine disruptors, mainly with estrogenic effects. To test the hypothesis that the amount and quality of organochlorine residues in adipose tissue adjacent to breast carcinoma affect the biological behavior of the tumor, we studied biomarker expression in breast carcinoma and the OCC body burden in patients from an urban area adjacent to Paraná fluvial system, Argentina.     

SERMs suppresses the growth of ERα positive cervical cancer xenografts through predominant inhibition of extra-nuclear ERα expression

The role of estrogens and estrogen receptors (ER) in cervical cancer (CC) is not well established. However, epidemiological studies and abundant evidence from genetically engineered mouse models support such hypothesis. In this study, we have addressed estrogen responsiveness in a human CC cell line xenograft mouse model. We assessed the sensitivity of Ethynyl Estradiol (EE), SERMs (fulvestrant, MPP) and a non-SERM (EGCG) to competitively modulate the growth of ERα+^ve MS751 CC xenografts. We also checked the agonistic-antagonistic propensity of the above treatments to alter the histology of ovariectomised mouse uterine cervix. Chronic EE treatment encouraged the growth of ERα+^ve MS751 CC xenografts, while SERMs and EGCG significantly decreased tumor formation. SERMs were found to inhibit ERα expression, localized within cytoplasmic and membrane compartments. Conversely, ERα was not inducible and EE administration suppressed the growth of ERα-^ve HeLa CC xenografts. SERMs competitively induced atrophic features to uterine cervix, with MPP giving rise to mucinous metaplasia in the ectocervix. We have demonstrated that, estrogen sensitivity mediated through ERα has promoted CC tumorigenesis. This in turn was modulated by SERMs, predominantly through inhibition of extra-nuclear ERα expression. Though, induction of hyper-estrogenic status in the ectocervix, might underrate the utility of SERMs in ERα+^ve CC.     

Upregulation of CYP1B1 by hypoxia is mediated by ERα activation in breast cancer cells

Endocrine therapy for breast cancer often leads to drug resistance and tumor recurrence; tumor hypoxia is also associated with mortality and tumor relapse. Cytochrome P450 1B1 (CYP1B1) regulates estrogen metabolism in breast cells and is known to be overexpressed in breast cancer tissue. Although the individual association of hypoxia-induced hypoxia-inducible factor-1-alpha (HIF-1α) and CYP1B1 with tumorigenesis is well known, the association between HIF-1α and CYP1B1 leading to tumorigenesis has not been investigated. Here, we investigated the correlation between hypoxia and CYP1B1 expression in breast cancer cells for tumorigenesis-related mechanisms. Hypoxia was induced in the human breast cancer cell lines MCF-7 (Er-positive) and MDA-MB-231 (triple-negative) and the normal breast epithelial cell line MCF10A; these cell lines were then subjected to immunoblotting, transient transfection, luciferase assays, gene silencing using small interfering RNA, polymerase chain reaction analysis, chromatin immunoprecipitation, co-immunoprecipitation, and mammalian two-hybrid assays. Furthermore, immunofluorescence analysis of the tumor microarrays was performed, and the pub2015 and the Cancer Genome Atlas patient datasets were analyzed. HIF-1α expression in response to hypoxia occurred in both normal and breast cancer cells, whereas CYP1B1 was induced only in estrogen receptor α (ERα)-positive breast cancer cells under hypoxia. HIF-1α activated ERα through direct binding and in a ligand-independent manner to promote CYP1B1 expression. Therefore, we suggested the mechanism by which hypoxia and ER-positivity orchestrate breast cancer relapse.     

Stop! In the name of transforming growth factor-β: keeping estrogen receptor-α-positive mammary epithelial cells from proliferating

Recent genetic and cell biological studies illustrate the importance of active transforming growth factor-β signaling in preventing the proliferation of estrogen receptor-positive cells in the normal mammary gland, and suggest how the loss of this inhibition may be important in early breast cancer progression.     

Targeted inhibition of ERα signaling and PIP5K1α/Akt pathways in castration‐resistant prostate cancer

Selective ERα modulator, tamoxifen, is well tolerated in a heavily pretreated castration‐resistant prostate cancer (PCa) patient cohort. However, its targeted gene network and whether expression of intratumor ERα due to androgen deprivation therapy (ADT) may play a role in PCa progression is unknown. In this study, we examined the inhibitory effect of tamoxifen on castration‐resistant PCa in vitro and in vivo. We found that tamoxifen is a potent compound that induced a high degree of apoptosis and significantly suppressed growth of xenograft tumors in mice, at a degree comparable to ISA‐2011B, an inhibitor of PIP5K1α that acts upstream of PI3K/AKT survival signaling pathway. Moreover, depletion of tumor‐associated macrophages using clodronate in combination with tamoxifen increased inhibitory effect of tamoxifen on aggressive prostate tumors. We showed that both tamoxifen and ISA‐2011B exert their on‐target effects on prostate cancer cells by targeting cyclin D1 and PIP5K1α/AKT network and the interlinked estrogen signaling. Combination treatment using tamoxifen together with ISA‐2011B resulted in tumor regression and had superior inhibitory effect compared with that of tamoxifen or ISA‐2011B alone. We have identified sets of genes that are specifically targeted by tamoxifen, ISA‐2011B or combination of both agents by RNA‐seq. We discovered that alterations in unique gene signatures, in particular estrogen‐related marker genes are associated with poor patient disease‐free survival. We further showed that ERα interacted with PIP5K1α through formation of protein complexes in the nucleus, suggesting a functional link. Our finding is the first to suggest a new therapeutic potential to inhibit or utilize the mechanisms related to ERα, PIP5K1α/AKT network, and MMP9/VEGF signaling axis, providing a strategy to treat castration‐resistant ER‐positive subtype of prostate cancer tumors with metastatic potential.

Abbreviations

CRPC

castration‐resistant prostate cancer

DHT

dihydrotestosterone

E2

estradiol

ERα

estrogen receptor alpha

GO

gene ontology

NG‐CHM

Next‐Generation Clustered Heatmaps

PCa

prostate cancer

TMAs

tissue microarrays

     

Intracellular localization of CK2α as a prognostic factor in invasive breast carcinomas

Overexpression of the ubiquitous protein kinase, CK2α, has been reported in various human cancers. Here, we demonstrate that nuclear and nucleolar CK2α localization in invasive ductal carcinomas of the breast is a reliable predictor of poor prognosis. Cellular localization of CK2α in nuclei and nucleoli was analyzed immunohistochemically using surgical tissue blocks from 112 patients, who had undergone surgery without neoadjuvant chemotherapy. Clinical data collection and median follow‐up period were for more than 5 y. In total, 93.8% of patients demonstrated elevated CK2α expression in nuclei and 36.6% of them displayed elevated expression predominantly in nucleoli. Clinicopathological malignancy was strongly correlated with elevated nuclear and nucleolar CK2α expression. Recurrence‐free survival was significantly worse (P = .0002) in patients with positive nucleolar CK2α staining. The 5‐y survival rate decreased to a roughly 50% in nucleolar CK2α‐positive patients of triple‐negative (P = .0069) and p Stage 3 (P = .0073) groups. In contrast, no patients relapsed or died in the triple‐negative group who exhibited a lack of nucleolar CK2α staining. Evaluation of nucleolar CK2α staining showed a high secondary index with a hazard ratio of 6.629 (P = .001), following lymph node metastasis with a hazard ratio of 14.30 (P = .0008). Multivariate analysis demonstrated that nucleolar CK2α is an independent factor for recurrence‐free survival. Therefore, we propose that histochemical evaluation of nucleolar CK2α‐positive staining may be a new and robust prognostic indicator for patients who need further treatment. Functional consequences of nucleolar CK2 dysfunction may be a starting point to facilitate development of novel treatments for invasive breast carcinoma.     

Involvement of α6β4 integrin in the mechanisms that regulate breast cancer progression

Integrin α₆β₄ is mostly expressed in epithelial tissues and endothelial and Schwann cells. Expression of α₆β₄ is increased in many epithelial tumours, implicating its involvement in tumour malignancy. Moreover, this integrin activates several key signalling molecules in carcinoma cells, but its ability to activate the phosphatidylinositol 3-kinase/Akt pathway is among the mechanisms by which α₆β₄ integrin regulates tumour behaviour. In this review we discuss the biological and clinical features of α₆β₄ integrin that allow it to promote tumour survival and progression of mammary tumours.     

Estrogen receptor-β: why may it influence clinical outcome in estrogen receptor-α positive breast cancer?

In the previous issue of the journal, Lin and coworkers present data demonstrate that increased expression of estrogen receptor (ER)-β in ER-α-positive breast cancer cells antagonizes a defined group of ER-α/estrogen stimulated genes that are involved in cell cycle regulation and DNA replication. Similar expression patterns for these genes were found human ER-α positive breast tumors expressing higher levels or ER-β, and this correlated with better clinical outcome. The implications for these data, which suggest that ER-β is a positive actor and diagnostic marker for therapeutic outcome, are discussed.     

Modulation of BRCA1 mediated DNA damage repair by deregulated ER-α signaling in breast cancers

Abstract: BRCA1 mutation carriers have a greater risk of developing cancers in hormone-responsive tissues like breasts and ovaries. However, this tissue-specific incidence of BRCA1 related cancers remains elusive. The majority of the BRCA1 mutated breast cancers exhibit typical histopathological features of high-grade tumors, with basal epithelial phenotype, classified as triple-negative molecular subtype and have a higher percentage of DNA damage and chromosomal abnormality. Though there are many studies relating BRCA1 with ER-α (Estrogen receptor-α), it has not been reported whether E2 (Estrogen) -ER-α signaling can modulate the DNA repair activities of BRCA1. The present study analyzes whether deregulation of ER-α signaling, arising as a result of E2/ER-α deficiency, could impact the BRCA1 dependent DDR (DNA Damage Response) pathways, predominantly those of DNA-DSB (Double Strand break) repair and oxidative damage response. We demonstrate that E2/E2-stimulated ER-α can augment BRCA1 mediated high fidelity repairs like HRR (Homologous Recombination Repair) and BER (Base Excision Repair) in breast cancer cells. Conversely, a condition of ER-α deficiency itself or any interruption in ligand-dependent ER-α transactivation resulted in delayed DNA damage repair, leading to persistent activation of γH2AX and retention of unrepaired DNA lesions, thereby triggering tumor progression. ER-α deficiency not only limited the HRR in cells but also facilitated the DSB repair through error prone pathways like NHEJ (Non Homologous End Joining). ER-α deficiency associated persistence of DNA lesions and reduced expression of DDR proteins were validated in human mammary tumors.     

Triple therapy with osimertinib,bevacizumab and cetuximab in EGFR-mutant lung cancer with HIF-1α/TGF-α expression

Osimertinib, a third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is the standard treatment for patients with lung cancer harboring EGFR T790M; however, acquired resistance is inevitable due to genetic and epigenetic changes in cancer cells. In addition, a recent randomized clinical trial revealed that the combination of osimertinib and bevacizumab failed to exhibit superior progression-free survival compared with osimertinib alone. The present study aimed to investigate the effect of triple therapy with osimertinib, bevacizumab and cetuximab in xenograft tumors with different initial tumor volumes (conventional model, 200 mm³ and large model, 500 mm³). The results demonstrated that osimertinib significantly inhibited tumor growth in both the conventional and large models; however, maximum tumor regression was attenuated in the large model in which hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor-α (TGF-α) expression levels increased. Although the combination of osimertinib and bevacizumab exerted a greater inhibitory effect on tumor growth compared with osimertinib in the conventional model, the effect of this combination therapy was attenuated in the large model. TGF-α attenuated sensitivity to osimertinib in vitro; however, this negative effect was counteracted by the combination of osimertinib and cetuximab, but not osimertinib and bevacizumab. In the large xenograft tumor model, the triple therapy induced the greatest inhibitory effect on tumor growth compared with osimertinib alone and its combination with bevacizumab. Clinical trials of the triple therapy are required for patients with lung cancer with EGFR mutations and HIF-1α/TGF-α.     

TGF-β1 modulates temozolomide resistance in glioblastoma via altered microRNA processing and elevated MGMT

BackgroundOur previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-β1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-β1 in temozolomide resistance has remained unknown.MethodsThe distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-β1–treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-β1 in temozolomide resistance.ResultsOverexpression of TGF-β1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-β1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-β1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-β1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-β1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-β1 or lncRNA expression benefited from temozolomide therapy.ConclusionsOur results reveal an underlying mechanism by which TGF-β1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-β inhibitor may serve as an effective therapy for glioblastomas.     

Trends in oligomannosylation and α1,2-mannosidase expression in human cancers

Aberrant protein glycosylation is a prominent cancer feature. While many tumour-associated glycoepitopes have been reported, advances in glycoanalytics continue to uncover new associations between glycosylation and cancer. Guided by a comprehensive literature survey suggesting that oligomannosylation (Man_5–9 GlcNAc₂) is a widespread and often regulated glycosignature in human cancers, we here revisit a valuable compilation of nearly 500 porous graphitized carbon LC-MS/MS N-glycomics datasets acquired across 11 human cancer types to systematically test for oligomannose-cancer associations. Firstly, the quantitative glycomics data obtained across 34 cancerous cell lines demonstrated that oligomannosylation is a pan-cancer feature spanning in a wide abundance range. In keeping with literature, our quantitative glycomics data of tumour and matching control tissues and new MALDI-MS imaging data of tissue microarrays showed a strong cancer-associated elevation of oligomannosylation in both basal cell (p = 1.78 × 10^–12) and squamous cell (p = 1.23 × 10^–11) skin cancer and colorectal cancer (p = 8.0 × 10^–4). The glycomics data also indicated that some cancer types including gastric and liver cancer exhibit unchanged or reduced oligomannose levels, observations also supported by literature and MALDI-MS imaging data. Finally, expression data from public cancer repositories indicated that several α1,2-mannosidases are regulated in tumour tissues suggesting that these glycan-processing enzymes may contribute to the cancer-associated modulation of oligomannosylation. This omics-centric study has compiled robust glycomics and enzyme expression data revealing interesting molecular trends that open avenues to better understand the role of oligomannosylation in human cancers.     

Glycoprotein hormone α-subunit promotes cell proliferation and tumorigenesis in breast cancer

The glycoprotein hormone α-subunit (CGA) is implicated in the occurrence and progression of a number of solid tumors. However, its role in breast cancer remains unclear. The present study aimed to investigate the biological functions and mechanisms of action of CGA in breast cancer. CGA protein expression was evaluated in clinical breast cancer specimens using immunohistochemistry. The association between CGA expression and patient prognosis was determined using the Kaplan-Meier method and Mantel-Cox test. At the same time, CGA mRNA and protein expression was explored in a normal mammary epithelial cell line and breast cancer cell lines. Breast cancer cell lines overexpressing or deficient in CGA were established, and the effect of CGA on cell proliferation was evaluated in vitro, and in vivo using a mouse xenograft tumor model. Intracellular signaling pathway activities were evaluated using western blotting in CGA-overexpressing or -depleted cells. Increased CGA protein expression was significantly associated with a poor prognosis in patients with breast cancer. Furthermore, while CGA mRNA and protein expression level was negligible in normal mammary epithelial cells, it was elevated in breast cancer cell lines. In vitro and in vivo experiments showed that CGA overexpression enhanced breast cancer cell proliferation via activation of the epidermal growth factor receptor, extracellular signal-regulated kinase 1/2 and serine/threonine kinase Akt signaling cascades. The present results suggest that CGA is upregulated in breast cancer tissues and that it is associated with a poor prognosis. CGA may serve as a candidate for developing targeted therapies for breast cancer.     

Significance of combined TGF-β1 and survivin expression on the prognosis of patients with triple-negative breast cancer

Compared with other types of breast cancer, triple-negative breast cancer (TNBC) has the characteristics of rapid progression, a lack of specific molecular targets for treatment and a poor prognosis. However, based on previously published studies, TGF-β1 and survivin are potentially meaningful for the prognosis of patients with TNBC. The present study was therefore designed to measure and compare the expression of transforming growth factor-β1 (TGF-β1) and survivin in tissue samples of TNBC and non-TNBC patients in order to evaluate their ability as prognostic indicators. In total, 90 TNBC and 52 non-TNBC tissue specimens were selected, following which immunohistochemistry was used to detect the expression of TGF-β1 and survivin in the cancer tissues. Subsequently, the potential association between the expression levels of these two proteins and the clinicopathological variables was analyzed. The expression levels of TGF-β1 and survivin in TNBC tissues were found to be significantly higher compared with those in the non-TNBC tissues. In addition, the results of the present study demonstrated that TGF-β1 expression was positively associated with survivin expression in the TNBC samples, but no significant correlation was found between TGF-β1 and survivin expression in the non-TNBC samples. Kaplan-Meier (K-M) analysis was performed to assess the levels of TGF-β1 and survivin in regard to patient survival, and univariate and multivariate Cox analyses of TGF-β1 and survivin protein expression were performed to analyze the overall survival (OS) and progression-free survival (PFS) rates of patients with TNBC and non-TNBC. Although multivariate Cox analysis demonstrated that neither TGF-β1 or survivin were independent prognostic predictors of TNBC or non-TNBC, results of the K-M curve revealed that patients with TNBC with TGF-β1- and survivin-positive breast cancer exhibited shorter OS and PFS times. Multivariate Cox analysis demonstrated that in patients with TNBC, the combined expression of TGF-β1 and survivin may yield additional prognostic information, compared with patients with non-TNBC.