Mammary carcinoma in aged gerbil mothers after endocrine disruption in pregnancy and lactation

Compounds that trigger breast cancer onset and establishment are of great interest in biological research. Endocrine disruptors are relevant because they initiate carcinogenesis by changing endocrine pathways. Bisphenol A (BPA), as a ubiquitous xenoestrogen, is largely associated with dysfunctions in the female reproductive system and associated organs. This study proposes an investigation of the mammary gland (MG) in aged Mongolian gerbil (Meriones unguiculatus) mothers after their exposure to BPA in two windows of morphophysiological plasticity: pregnancy and lactation. A low dose (50 μg/kg) and a high dose (5000 μg/kg) of BPA were considered, and results showed few differences between them. As expected, we observed contrasts among control and BPA-exposed MG. The control groups presented a regressive phase with high apoptotic activity and elastic stroma. However, BPA damaged mammary tissue and provoked multifocal carcinoma development supported by an apparent epithelial-mesenchymal transition (EMT) and reactive stroma establishment. BPA remodeled stromal fibers deposition and enhanced the recruitment of tumor-associated cells, contributing to a tumoral microenvironment. Overexpression of TGF-β1 was induced by BPA in the epithelial compartment of exposed MG, and increased expression of metalloproteinases (MMP-2, MMP-3, MMP-9) was present in carcinoma cells. In conclusion, exposure of mothers to BPA during the gestational/lactational window of susceptibility leads to carcinogenic impacts with aging.

P–289 Progesterone receptor is not downregulated in endometrial epithelial compartment during embryo receptivity phase in assisted reproductive cycles

Study question Is progesterone receptor (PGR) downregulation disrupted within endometrial epithelial compartment, during embryo receptivity phase in assisted reproductive technology (ART) cycles? Summary answer PGR is not downregulated in endometrial epithelial cells from ART cycles during embryo receptivity phase. What is known already Progesterone (P4) promotes the downregulation of its own progesterone receptor (PGR). During the mid-luteal phase, PGR is downregulated in endometrial epithelial cells (EEC), a critical process for embryo implantation. Embryos are unable to attach to the maternal surface when PGR expression is sustained in EEC. Non-physiologic ovarian steroid produced or employed in ART cycles may alter endometrial development compromising its receptivity. Scarce information is available whether PGR is downregulated in EEC from ARTs including ovarian stimulation for in vitro fertilization (IVF) cycles or hormonal endometrial preparation for frozen thawed embryo transfer (HEP-FET). Study design, size, duration Cross sectional study including endometrial samples from fertile women during natural cycle (FNC, n = 23), from infertile women submitted to IVF (n = 19) and from infertile women who underwent mock HEP-FET (n = 35). Samples were obtained between 2018–2019. Sample size was calculated considering a power of 90%, alpha error=0.05, an expected PGR expression of 2 and 0.5 in ART and FNC groups, respectively, having a standard deviation=0.9. At least 9 patients would be necessary in each group. Participants/materials, setting, methods Endometrial samples were obtained during mid-luteal phase scheduled 7 days after ovulation in FNC, 5 days after oocyte retrieval in IVF without embryo transfer or 5 days after P4 supplementation in HEP-FET. Immunohistochemistry was employed to quantify PGR using histologic score (Hscore). PGR mRNA levels were determined by qRT-PCR from EEC dissected by laser capture microdissection. Anova test was used for comparing means of Hscore and mRNA among groups. Statistical significance was established as P < 0.05. Main results and the role of chance No statistical differences were found in demographic characteristics including age, body mass index or endometrial thickness. The PGR expression was reduced in FNC compared to IVF and HP-FET endometria (0.6 ± 0.1, 1.9 ± 0.9 and 2.2 ± 0.9 respectively; P < 0.0001). The PGR mRNA levels from ECC dissected by laser capture microdissection were higher in IVF and HP-ET cycles compared to FNC (10.6 ± 3.1, 13.6 ± 2.3 and 0.8 ± 0.1 respectively; P < 0.0001) corroborating the elevated PGR Hscore in EEC from ART cycles. Limitations, reasons for caution This is a descriptive study reporting failure of PGR downregulation in endometria from ART cycles with vaginal P4 supplementation during the luteal-phase. Whether interference or resistance to P4 signal is the mechanism involved in the failure of PGR down regulation in ART cycles needs to be determined Wider implications of the findings: PGR downregulation within EEC was shown in FNC. The retained PGR expression detected in most ART cycles may interfere with embryo implantation and might explain the restricted pregnancy success. Future studies might reveal whether PGR evaluation in EEC can predict embryo implantation. Trial registration number Not Aplicable

Pharyngeal spreading of peri-implant infections under antiresorptive/antiangiogenic therapy

Objectives To assess the influence of antiresorptive/antiangiogenic therapy on the spreading of peri-implant infections in the pharyngeal region. Material and methods This analysis was based on tissue biopsies obtained from a total of twenty-five albino rats having either received (1) amino-bisphosphonate (Zoledronate) (Zo) (n=4), (2) RANKL inhibitor (Denosumab) (De) (n=4), (3) antiangiogenic medication (Bevacizumab) (Be) (n=4), (4) Zo+Be (n=3), (5) De+Be (n=5), or (6) no medication (Co) (n=5). Drug administration was repeated at 12 weeks. Chronic-type peri-implant infections were induced at titanium implants located in the upper jaws. The surface area (%) of infiltrated connective tissue (ICT) and CD68-positive cells was assessed within the lateral pharyngeal/retropharyngeal connective tissue zone. Results Mean (±SD) and median ICT% values and CD68 counts were markedly highest in the De+Be (11.10±6.04; 11.81; 95% CI − 3.89; 26.11) and De (5.70±5.06; 6.19; 95% CI − 2.34; 13.75) groups, reaching statistical significance for De CD68 counts over the Co (0.18±0.25; 0.18; 95% CI −2.14; 2.51) group. In both De+Be and De groups, the ICTs were occasionally associated with an ulceration of the epithelial compartment. Conclusions Induced peri-implant infections were not associated with any inflammatory lesions in pharyngeal tissues. While these findings were similar under Zo and Be medication, De and De+Be had a marked effect on ICT and CD68 values. The clinical relevance of these adverse findings needs further investigation.

Inhibition of stromal biglycan promotes normalization of the tumor microenvironment and enhances chemotherapeutic efficacy

Background Biglycan is a proteoglycan found in the extracellular matrix. We have previously shown that biglycan is secreted from tumor endothelial cells and induces tumor angiogenesis and metastasis. However, the function of stroma biglycan in breast cancer is still unclear. Methods Biglycan gene analysis and its prognostic values in human breast cancers were based on TCGA data. E0771 breast cancer cells were injected into WT and Bgn KO mice, respectively. Results Breast cancer patients with high biglycan expression had worse distant metastasis-free survival. Furthermore, biglycan expression was higher in the tumor stromal compartment compared to the epithelial compartment. Knockout of biglycan in the stroma (Bgn KO) in E0771 tumor-bearing mice inhibited metastasis to the lung. Bgn KO also impaired tumor angiogenesis and normalized tumor vasculature by repressing tumor necrosis factor-ɑ/angiopoietin 2 signaling. Moreover, fibrosis was suppressed and CD8+ T cell infiltration was increased in tumor-bearing Bgn KO mice. Furthermore, chemotherapy drug delivery and efficacy were improved in vivo in Bgn KO mice. Conclusion Our results suggest that targeting stromal biglycan may yield a potent and superior anticancer effect in breast cancer.

The miR-424/503 cluster modulates Wnt/β-catenin signaling in the mammary epithelium by regulating the expression of the LRP6 co-receptor

During the female lifetime, the enlargement of the epithelial compartment dictated by the ovarian cycles is supported by a transient increase in the MaSC population. Notably, activation of Wnt/β-catenin signaling is an important trigger for MaSC expansion. Here, we report that the miR-424/503 cluster is a novel modulator of canonical Wnt-signaling in the mammary epithelium that exerts its function by targeting the LRP6 co-receptor. Additionally, we show that the loss of this microRNA cluster is associated with breast cancers possessing high levels of Wnt/β-catenin signaling.

γδ T cells regulate the intestinal response to nutrient sensing

The intestine is a site of direct encounter with the external environment and must consequently balance barrier defense with nutrient uptake. To investigate how nutrient uptake is regulated in the small intestine, we tested the effect of diets with different macronutrient compositions on epithelial gene expression. We found that enzymes and transporters required for carbohydrate digestion and absorption were regulated by carbohydrate availability. The “on-demand” induction of this machinery required γδ T cells, which regulated this program through the suppression of interleukin-22 production by type 3 innate lymphoid cells. Nutrient availability altered the tissue localization and transcriptome of γδ T cells. Additionally, transcriptional responses to diet involved cellular remodeling of the epithelial compartment. Thus, this work identifies a role for γδ T cells in nutrient sensing.

Full-length native CGRP neuropeptide and its stable analogue SAX, but not CGRP peptide fragments, inhibit mucosal HIV-1 transmission

The vasodilator neuropeptide calcitonin gene-related peptide (CGRP) plays both detrimental and protective roles in different pathologies. CGRP is also an essential component of the neuro-immune dialogue between nociceptors and mucosal immune cells. We previously reported that CGRP strongly inhibits human immunodeficiency virus type 1 (HIV-1) infection, by suprresing Langerhans cells (LCs)-mediated HIV-1 transfer to T-cells during trans-infection. To understand the requirements for CGRP receptor (CGRP-R) activation during inhibition of HIV-1 transmission, we here investigated the anti-HIV-1 activities of full-length native CGRP and its recently developed stable analogue SAX, as well as several CGRP peptide fragments containing its binding C-terminal and activating N-terminal regions. We show that SAX significantly inhibits LCs-mediated HIV-1 trans-infection but with lower potency than CGRP, while all CGRP peptide fragments tested have no effect. In addition, CGRP readily enters the epithelial compartment of mucosal tissues and does not modify the distribution and density of mucosal immune cells. In-vivo, a single CGRP treatment in humanized mice, before vaginal challenge with high-dose HIV-1, restricts the increase in plasma viral load and maintains higher CD4+ T-cell counts. Together, our results call for the optimization and design of CGRP analogues and agonists, which could be harnessed for prevention of mucosal HIV-1 transmission.

Stiffness increases with myofibroblast content and collagen density in mesenchymal high grade serous ovarian cancer

Women diagnosed with high-grade serous ovarian cancers (HGSOC) are still likely to exhibit a bad prognosis, particularly when suffering from HGSOC of the Mesenchymal molecular subtype (50% cases). These tumors show a desmoplastic reaction with accumulation of extracellular matrix proteins and high content of cancer-associated fibroblasts. Using patient-derived xenograft mouse models of Mesenchymal and Non-Mesenchymal HGSOC, we show here that HGSOC exhibit distinct stiffness depending on their molecular subtype. Indeed, tumor stiffness strongly correlates with tumor growth in Mesenchymal HGSOC, while Non-Mesenchymal tumors remain soft. Moreover, we observe that tumor stiffening is associated with high stromal content, collagen network remodeling, and MAPK/MEK pathway activation. Furthermore, tumor stiffness accompanies a glycolytic metabolic switch in the epithelial compartment, as expected based on Warburg’s effect, but also in stromal cells. This effect is restricted to the central part of stiff Mesenchymal tumors. Indeed, stiff Mesenchymal tumors remain softer at the periphery than at the core, with stromal cells secreting high levels of collagens and showing an OXPHOS metabolism. Thus, our study suggests that tumor stiffness could be at the crossroad of three major processes, i.e. matrix remodeling, MEK activation and stromal metabolic switch that might explain at least in part Mesenchymal HGSOC aggressiveness.

AKT3 Expression in Mesenchymal Colorectal Cancer Cells Drives Growth and Is Associated with Epithelial-Mesenchymal Transition

Colorectal cancer (CRC) is a heterogeneous disease that can currently be subdivided into four distinct consensus molecular subtypes (CMS) based on gene expression profiling. The CMS4 subtype is marked by high expression of mesenchymal genes and is associated with a worse overall prognosis compared to other CMSs. Importantly, this subtype responds poorly to the standard therapies currently used to treat CRC. We set out to explore what regulatory signalling networks underlie the CMS4 phenotype of cancer cells, specifically, by analysing which kinases were more highly expressed in this subtype compared to others. We found AKT3 to be expressed in the cancer cell epithelium of CRC specimens, patient derived xenograft (PDX) models and in (primary) cell cultures representing CMS4. Importantly, chemical inhibition or knockout of this gene hampers outgrowth of this subtype, as AKT3 controls expression of the cell cycle regulator p27KIP1. Furthermore, high AKT3 expression was associated with high expression of epithelial-mesenchymal transition (EMT) genes, and this observation could be expanded to cell lines representing other carcinoma types. More importantly, this association allowed for the identification of CRC patients with a high propensity to metastasise and an associated poor prognosis. High AKT3 expression in the tumour epithelial compartment may thus be used as a surrogate marker for EMT and may allow for a selection of CRC patients that could benefit from AKT3-targeted therapy.

Interfollicular epidermal stem-like cells for the recreation of the hair follicle epithelial compartment

Background Hair follicle (HF) development and growth are dependent on epithelial-mesenchymal interactions (EMIs). Dermal papilla (DP) cells are recognized as the key inductive mesenchymal player, but the ideal source of receptive keratinocytes for human HF regeneration is yet to be defined. We herein investigated whether human interfollicular epidermal keratinocytes with stem-like features (EpSlKCs), characterized by a α6bri/CD71dim expression, can replace human hair follicular keratinocytes (HHFKCs) for the recreation of the HF epithelium and respective EMIs. Methods The α6bri/CD71dim cellular fraction was selected from the whole interfollicular keratinocyte population through fluorescence-activated cell sorting and directly compared with follicular keratinocytes in terms of their proliferative capacity and phenotype. The crosstalk with DP cells was studied in an indirect co-culture system, and EpSlKC hair forming capacity tested in a hair reconstitution assay when combined with DP cells. Results EpSlKCs exhibited a phenotypic profile similar to follicular keratinocytes and were capable of increasing DP cell proliferation and, for short co-culture times, the number of alkaline phosphatase-active cells, suggesting an improvement of their inductivity. Moreover, the recreation of immature HFs and sebaceous glands was observed after EpSlKC and DP cell co-grafting in nude mice. Conclusions Our results suggest that EpSlKCs are akin to follicular keratinocytes and can crosstalk with DP cells, contributing to HF morphogenesis in vivo, thus representing an attractive epithelial cell source for hair regeneration strategies.