Oestrogen deprivation induces chemokine production and immune cell recruitment in in vitro and in vivo models of oestrogen receptor-positive breast cancer

Background Oestrogen receptor-positive (ER+) breast cancer is commonly treated using endocrine therapies such as aromatase inhibitors which block synthesis of oestradiol, but the influence of this therapy on the immune composition of breast tumours has not been fully explored. Previous findings suggest that tumour infiltrating lymphocytes and immune-related gene expression may be altered by treatment with aromatase inhibitors. However, whether these changes are a direct result of impacts on the host immune system or mediated through tumour cells is not known. We aimed to investigate the effect of oestrogen deprivation on the expression of chemokines and immune infiltration in vitro and in an ER+ immunocompetent mouse model. Methods RT-qPCR and a bead-based Bioplex system were used to investigate the expression of chemokines in MCF-7 breast cancer cells deprived of oestrogen. A migration assay and flow cytometry were used to measure the migration of human peripheral blood mononuclear cells (PBMCs) to MCF-7 cells grown without the main biologically active oestrogen, oestradiol. Using flow cytometry and immunohistochemistry, we examined the immune cell infiltrate into tumours created by injecting SSM3 ER+ breast cancer cells into wild-type, immunocompetent 129/SvEv mice. Results This study demonstrates that oestrogen deprivation increases breast cancer secretion of TNF, CCL5, IL-6, IL-8, and CCL22 and alters total human peripheral blood mononuclear cell migration in an in vitro assay. Oestrogen deprivation of breast cancer cells increases migration of CD4+ T cells and decreases migration of CD11c+ and CD14+ PBMC towards cancer cells. PBMC migration towards breast cancer cells can be reduced by treatment with the non-steroidal anti-inflammatory drugs, aspirin and celecoxib. Treatment with endocrine therapy using the aromatase inhibitor letrozole increases CD4+ T cell infiltration into ER+ breast cancer tumours in immune competent mice. Conclusions These results suggest that anti-oestrogen treatment of ER+ breast cancer cells can alter cytokine production and immune cells in the area surrounding the cancer cells. These findings may have implications for the combination and timing of anti-oestrogen therapies with other therapies.

SOX2OT Long Noncoding RNA Is Regulated by the UPR in Oestrogen Receptor-Positive Breast Cancer

Endoplasmic reticulum (ENR) stress perturbs cell homeostasis and induces the unfolded protein response (UPR). In breast cancer, this process is activated by oestrogen deprivation and is associated with tamoxifen resistance. We present evidence that the transcription factor SOX2 and the long noncoding RNA SOX2 overlapping transcript (SOX2OT) are upregulated in oestrogen receptor-positive (ER+) breast cancer and in response to oestrogen deprivation. We examined the effect of the UPR on SOX2 and SOX2OT expression and the effect of SOX2OT on UPR pathways in breast cancer cell lines. The induction of the UPR by thapsigargin or glucose deprivation upregulates SOX2OT expression. This upregulation is also shown with the anti-oestrogen 4OH-tamoxifen and mTOR inhibitor everolimus in ER + breast cancer cells that are sensitive to oestrogen deprivation or everolimus treatment. SOX2OT overexpression decreased BiP and PERK expression. This effect of SOX2OT overexpression was confirmed on BiP and PERK pathway by q-PCR. Our results show that a long noncoding RNA regulates the UPR and evince a new function of SOX2OT as a participant of ENR stress reprogramming of breast cancer cells.

Mathematical modelling of breast cancer cells in response to endocrine therapy and Cdk4/6 inhibition

Oestrogen receptor (ER)-positive breast cancer is responsive to a number of targeted therapies used clinically. Unfortunately, the continuous application of any targeted therapy often results in resistance to the therapy. Our ultimate goal is to use mathematical modelling to optimize alternating therapies that not only decrease proliferation but also stave off resistance. Toward this end, we measured levels of key proteins and proliferation over a 7-day time course in ER+ MCF-7 breast cancer cells. Treatments included endocrine therapy, either oestrogen deprivation, which mimics the effects of an aromatase inhibitor, or fulvestrant, an ER degrader. These data were used to calibrate a mathematical model based on key interactions between ER signalling and the cell cycle. We show that the calibrated model is capable of predicting the combination treatment of fulvestrant and oestrogen deprivation. Further, we show that we can add a new drug, palbociclib, to the model by measuring only two key proteins, cMyc and hyperphosphorylated RB1, and adjusting only parameters associated with the drug. The model is then able to predict the combination treatment of oestrogen deprivation and palbociclib. We illustrate the model's potential to explore protocols that limit proliferation and hold off resistance by not depending on any one therapy.

SOX2OT Long Noncoding RNA Is Regulated by the UPR in Oestrogen Receptor-Positive Breast Cancer

Endoplasmic reticulum (ER) stress perturbs cell homeostasis and induces the unfolded protein response (UPR). In breast cancer, this process is activated by oestrogen deprivation and is associated with tamoxifen resistance. We present evidence that the transcription factor SOX2 and the long noncoding RNA SOX2 overlapping transcript (SOX2OT) are up-regulated in oestrogen receptor-positive (ER+) breast cancer and in response to oestrogen deprivation. We examined the effect of the UPR on SOX2 and SOX2OT expression, and the effect of SOX2OT on UPR pathways in breast cancer cell lines. The induction of the UPR by thapsigargin or glucose deprivation up-regulates SOX2OT expression. This up-regulation is also shown with the anti-oestrogen 4OH-tamoxifen and mTOR inhibitor everolimus in ER + breast cancer cells that are sensitive to oestrogen deprivation or everolimus treatment. SOX2OT overexpression decreased BiP and PERK expression. This effect of SOX2OT overexpression was confirmed on BiP and PERK pathway by q-PCR. Our results show that a long noncoding RNA regulates the UPR and evince a new function of SOX2OT as a participant of ER stress reprogramming of breast cancer cells.

Association between subjective olfactory dysfunction and female hormone-related factors in South Korea

An association between olfactory dysfunction and female hormone level has been reported; however, no previous studies have investigated the correlation with life-long female hormone exposure. The aim of this study was to estimate the association between subjective olfactory dysfunction and various endogenous and exogenous female hormone-related factors including age at menarche and menopause, number of pregnancies and deliveries, age at first and last delivery, duration of breastfeeding, use of oral contraceptives, and use of hormone therapy. The study analysed a total of 3863 female participants using data from the Korean National Health and Nutrition Examination Survey V (2010–2012). The prevalence of olfactory dysfunction was 3.5% for premenopausal participants and 6.2% for postmenopausal women. Among premenopausal women (compared to women breastfed less than 12 months), the 12–24-month group (OR = 4.690, 95% CI = 1.431–15.369) and the 25–48-month group (OR = 6.548, 95% CI = 1.758–24.394) had higher rates of olfactory dysfunction. In postmenopausal women, starting menopause at a younger age was positively associated with olfactory dysfunction (OR = 0.939, 95% CI = 0.887–0.993). These data suggest that a longer duration of endogenous oestrogen deprivation is associated with subjective olfactory dysfunction.

Increased long QT and torsade de pointes reporting on tamoxifen compared with aromatase inhibitors

ObjectiveA prolonged QTc (LQT) is a surrogate for the risk of torsade de pointes (TdP). QTc interval duration is influenced by sex hormones: oestradiol prolongs and testosterone shortens QTc. Drugs used in the treatment of breast cancer have divergent effects on hormonal status.MethodsWe performed a disproportionality analysis using the European database of suspected adverse drug reaction (ADR) reports to evaluate the reporting OR (ROR χ2) of LQT, TdP and ventricular arrhythmias associated with selective oestrogen receptor modulators (SERMs: tamoxifen and toremifene) as opposed to aromatase inhibitors (AIs: anastrozole, exemestane and letrozole). When the proportion of an ADR is greater in patients exposed to a drug (SERMs) compared with patients exposed to control drug (AIs), this suggests an association between the specific drug and the reaction and is a potential signal for safety. Clinical and demographic characterisation of patients with SERMs-induced LQT and ventricular arrhythmias was performed.ResultsSERMs were associated with higher proportion of LQT reports versus AIs (26/8318 vs 11/14851, ROR: 4.2 (2.11–8.55), p<0.001). SERMs were also associated with higher proportion of TdP and ventricular arrhythmia reports versus AIs (6/8318 vs 2/14851, ROR: 5.4 (1.29–26.15), p:0.02; 16/8318 vs 12/14851, ROR: 2.38 (1.15–4.94), p:0.02, respectively). Mortality was 38% in patients presenting ventricular arrhythmias associated with SERMs.ConclusionsSERMs are associated with more reports of drug-induced LQT, TdP and ventricular arrhythmias compared with AIs. This finding is consistent with oestradiol-like properties of SERMs on the heart as opposed to effects of oestrogen deprivation and testosterone increase induced by AIs.Trial registration numberNCT03259711.

Dietary whey reduces energy intake and alters hypothalamic gene expression in obese phyto-oestrogen-deprived male rats

Removing dietary phyto-oestrogens in adult male rats causes obesity and diabetes. As whey proteins have been reported to reduce food intake and improve glucose homoeostasis, we investigated whether they could attenuate susceptibility to obesity and diabetes due to phyto-oestrogen deprivation. To this end, thirty male Wistar rats were fed a high-phyto-oestrogen (HP) or a phyto-oestrogen-free (PF) diet for 10 weeks; six rats from each group were killed. The remaining HP animals (six animals) continued receiving the HP diet for 6 weeks. The remaining PF rats (twelve rats) were divided in two groups: one was given the PF diet and the other a variation of the PF diet plus whey protein (PF-W). Body weight, food intake and adipose tissue weights were recorded. Hypothalamic mRNA expressions of orexigenic (neuropeptide Y, agouti-related protein (AgRP)) and anorexigenic (pro-opiomelanocortin (POMC), cocaine-amphetamine-related transcript (CART)) neuropeptides were quantified by real-time PCR. Serum glucose, insulin and total thyroxine (T4), thyroid-stimulating hormone, testosterone and oestradiol were assessed. After 10 weeks of PF diet, increased body weight, adiposity and energy intake, with up-regulation of AgRP and down-regulation of POMC', were observed. Longer treatment exacerbated these results, increased total T4 levels, reduced oestradiol levels and impaired glucose homoeostasis. PF-W reduced energy intake and increased POMC expression; however, body weight and adiposity remained unchanged. PF-W could not prevent the hormonal changes or the high circulating glucose levels induced by phyto-oestrogen deprivation, but reduced fasting insulin. These data demonstrate that, although 6 weeks of whey administration could not prevent obesity in phyto-oestrogen-deprived rats, the reduction in energy intake and circulating insulin could be beneficial with longer treatments.