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Background: Endocrine therapy (ET) has improved the clinical outcomes of Estrogen receptor alpha-positive (ERɑ +) breast cancer (BC) patients, even though resistance to ET remains a clinical issue. Mutations in the hormone-binding domain of ERɑ represent an acquired intrinsic mechanism of ET resistance. However, the latter also depends on the multiple functional interactions between BC cells and the tumor microenvironment (TME). Here, we investigated how the most common Y537S-ERɑ mutation may influence the behavior of fibroblasts, the most prominent component of the TME., Methods: We conducted coculture experiments with normal human foreskin fibroblasts BJ1-hTERT (NFs), cancer-associated fibroblasts (CAFs), isolated from human BC specimens, and Y537S CRISPR-expressing MCF-7 BC cells (MCF-7YS). Mass spectrometry (MS) and Metacore analyses were performed to investigate how the functional interactions between BC cells/fibroblasts may affect their proteomic profile. The impact of fibroblasts on BC tumor growth and metastatic potential was evaluated in nude mice., Results: Mutant BC conditioned medium (CM) affected the morphology/proliferation/migration of both NFs and CAFs. 198 deregulated proteins signed the proteomic similarity profile of NFs exposed to the YS-CM and CAFs. Among the upregulated proteins, Yes-associated protein 1 (YAP1) was the main central hub in the direct interaction network. Increased YAP1 protein expression and activity were confirmed in NFs treated with MCF-7YS-CM. However, YAP1 activation appears to crosstalk with the insulin growth factor-1 receptor (IGF-1R). Higher amount of IGF-1 were noticed in the MCF-7YS-CM cells compared to the MCF-7P, and IGF-1 immunodepletion reversed the enhanced YAP1 expression and activity. Mutant cells upon exposure to the NF- and CAF-CM exhibited an enhanced proliferation/growth/migration/invasion compared to the MCF-7P. MCF-7YS cells when implanted with CAFs showed an early relative increased tumor volume compared to YS alone. No changes were observed when MCF-7P cells were co-implanted with CAFs. Compared with that in MCF-7P cells, the metastatic burden of MCF-7YS cells was intrinsically greater, and this effect was augmented upon treatment with NF-CM and further increased with CAF-CM., Conclusions: YS mutant BC cells induced the conversion of fibroblasts into CAFs, via YAP, which represent a potential therapeutic target which interrupt the functional interactions between mutant cells/TME and to be implemented in the novel therapeutic strategy of a subset of metastatic BC patients carrying the frequent Y537S mutations., Competing Interests: Declarations Ethics approval and consent to participate In the present study we used fibroblast specimens, isolated in accordance with approved guidelines, from patients who had signed informed consent and approved by the Ethic Institutional Committees at Annunziata Hospital, Cosenza, Italy (#149 issued by Comitato Etico Regione Calabria, Sezione Area Nord c/o Azienda Ospedaliera di Cosenza, 28/10/2015). All animals were maintained and handled in accordance with the recommendation of the Guidelines for the Care and Use of Laboratory Animals and experiments were approved by the Animal Care Committee of University of Calabria (OPBA), Italy (454/2023-PR, May 17, 2023). Consent for publication We have obtained consents to publish this paper from all the participants of this study. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Objective: Thioalbamide is a ribosomally synthesized and post-translationally modified peptide (RiPP) belonging to the family of thioamitides, a rare class of microbial specialized metabolites with unusual post-translational modifications and promising biological activities. Recent studies have demonstrated the ability of thioalbamide to exert highly selective cytotoxic effects on tumor cells by affecting their energy metabolism, thus causing abnormal ROS production and triggering apoptosis. This study is aimed to investigate the molecular mechanisms underlying the antitumor activity of thioalbamide in order to identify its exact molecular target., Methods: Wild type MCF-7 and MDA-MB-231 breast cancer cell lines as well as cancer cells deprived of mitochondrial DNA (ρ 0 cells) were employed in order to assess thioalbamide effects on tumor bioenergetics. In this regard, metabolic profile was evaluated by a Seahorse XFe96 analyzer, and the activity of the enzyme complexes involved in oxidative phosphorylation was quantified by spectrophotometric assays. Thioalbamide effects on tumor invasiveness were assessed by gelatin zymography experiments and invasion assays. In vivo experiments were carried out on breast cancer xenograft and "experimental metastasis" mouse models., Results: Experiments carried out on ρ 0 breast cancer cells, together with Seahorse analysis and the application of spectrophotometric enzymatic assays, highlighted the ability of thioalbamide to affect the mitochondrial respiration process, and allowed to propose the F o F 1 -ATPase complex as its main molecular target in breast cancer cells. Additionally, thioalbamide-mediated OXPHOS inhibition was shown, for the first time, to reduce tumor invasiveness by inhibiting metalloproteinase-9 secretion. Furthermore, this study has confirmed the antitumor potential of thioalbamide in two different in vivo models. In particular, experiments on MCF-7 and MDA-MB-231 xenograft mouse models have confirmed in vivo its high anti-proliferative and pro-apoptotic activity, while experiments on MDA-MB-231 ″experimental metastasis" mouse models have highlighted its ability to inhibit breast cancer cell invasiveness., Conclusions: Overall, our results shed more light on the molecular mechanisms underlying the pharmacological potential of thioamidated peptides, thus reducing the gap that separates this rare class of microbial metabolites from clinical studies, which could validate them as effective tools for cancer treatment., Competing Interests: Conflict of Interest The authors declare that they have no conflict of interests., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Adipose tissue is a metabolic and endocrine organ that secretes bioactive molecules called adipocytokines. Among these, adiponectin has a crucial role in obesity-associated breast cancer. The key molecule of adiponectin signaling is AMPK, which is mainly activated by liver kinase B1 (LKB1). Here, we demonstrated that estrogen receptor-α (ERα)/LKB1 interaction may negatively interfere with the LKB1 capability to phosphorylate AMPK and inhibit its downstream signaling TSC2/mTOR/p70S6k. In adiponectin-treated MCF-7 cells, AMPK signaling was not working, resulting in its downstream target acetyl-CoA carboxylase (ACC) being still active. In contrast, in MDA-MB-231 cells, AMPK and ACC phosphorylation was enhanced by adiponectin, inhibiting lipogenesis and cell growth. Upon adiponectin, ERα signaling switched the energy balance of breast cancer cells toward a lipogenic phenotype. Therefore, adiponectin played an inhibitory role on ERα-negative cell growth and progression in vitro and in vivo. In contrast, low adiponectin levels, similar to those circulating in obese patients, acted on ERα-positive cells as a growth factor, stimulating proliferation. The latter effect was blunted in vivo by high adiponectin concentration. All this may have translational relevance, addressing how the handling of adiponectin, as a therapeutic tool in breast cancer treatment, needs to be carefully considered in ERα-positive obese patients, where circulating levels of this adipocytokine are relatively low. In other words, in ERα-positive breast cancer obese patients, higher adiponectin doses should be administered with respect to ERα-negative breast cancer, also opportunely combined with antiestrogen therapy. -Mauro, L., Naimo, G. D., Gelsomino, L., Malivindi, R., Bruno, L., Pellegrino, M., Tarallo, R., Memoli, D., Weisz, A., Panno, M. L., Andò, S. Uncoupling effects of estrogen receptor α on LKB1/AMPK interaction upon adiponectin exposure in breast cancer.

Cancer-associated fibroblasts (CAFs), the principal components of the tumor stroma, play a central role in cancer development and progression. As an important regulator of the crosstalk between breast cancer cells and CAFs, the cytokine leptin has been associated to breast carcinogenesis. The nuclear Farnesoid X Receptor-(FXR) seems to exert an oncosuppressive role in different tumors, including breast cancer. Herein, we demonstrated, for the first time, that the synthetic FXR agonist GW4064, inhibiting leptin signaling, affects the tumor-promoting activities of CAFs in breast malignancy. GW4064 inhibited growth, motility and invasiveness induced by leptin as well as by CAF-conditioned media in different breast cancer cell lines. These effects rely on the ability of activated FXR to increase the expression of the suppressor of the cytokine signaling 3 (SOCS3) leading to inhibition of leptin-activated signaling and downregulation of leptin-target genes. In vivo xenograft studies, using MCF-7 cells alone or co-injected with CAFs, showed that GW4064 administration markedly reduced tumor growth. Interestingly, GW4064-treated tumors exhibited decreased levels of leptin-regulated proteins along with a strong staining intensity for SOCS3. Thus, FXR ligands might represent an emerging potential anti-cancer therapy able to block the tumor supportive role of activated fibroblasts within the breast microenvironment.

The role of the obesity cytokine leptin in breast cancer progression has raised interest in interfering with leptin's actions as a valuable therapeutic strategy. Leptin interacts with its receptor through three different binding sites: I-III. Site I is crucial for the formation of an active leptin-leptin receptor complex and in its subsequent activation. Amino acids 39-42 (Leu-Asp-Phe-Ile- LDFI) were shown to contribute to leptin binding site I and their mutations in alanine resulted in muteins acting as typical antagonists. We synthesized a small peptide based on the wild-type sequence of leptin binding site I (LDFI) and evaluated its efficacy in antagonizing leptin actions in breast cancer using in vitro and in vivo experimental models. The peptide LDFI abolished the leptin-induced anchorage-dependent and -independent growth as well as the migration of ERα-positive (MCF-7) and -negative (SKBR3) breast cancer cells. These results were well correlated with a reduction in the phosphorylation levels of leptin downstream effectors, as JAK2/STAT3/AKT/MAPK. Importantly, the peptide LDFI reversed the leptin-mediated up-regulation of its gene expression, as an additional mechanism able to enhance the peptide antagonistic activity. The described effects were specific for leptin signalling, since the developed peptide was not able to antagonize the other growth factors' actions on signalling activation, proliferation and migration. Finally, we showed that the LDFI pegylated peptide markedly reduced breast tumour growth in xenograft models. The unmodified peptide LDFI acting as a full leptin antagonist could become an attractive option for breast cancer treatment, especially in obese women., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Local estrogen production by aromatase is an important mechanism of autocrine stimulation in hormone-dependent breast cancer. We have previously shown that 17-β estradiol (E(2)) rapidly enhances aromatase enzymatic activity through an increase of tyrosine protein phosphorylation controlled by the activity of the c-Src kinase in breast cancer cells. Here, we investigated the protein tyrosine phosphatase PTP1B (protein tyrosine phosphatase 1B) as a potential regulator of aromatase activity. We demonstrated a specific association between PTP1B and aromatase at protein-protein level and a reduction of aromatase activity in basal and E(2)-treated MCF-7 and ZR75 breast cancer cells when PTP1B was overexpressed. Indeed, a specific tyrosine phosphatase inhibitor increased basal and E(2)-induced enzymatic activity as well as tyrosine phosphorylation status of the purified aromatase protein. Moreover, E(2) through phosphatidylinositol 3 kinase/Akt activation caused a significant decrease of PTP1B catalytic activity along with an increase in its serine phosphorylation. Concomitantly, the phosphatidylinositol 3 kinase inhibitor LY294002 or a dominant negative of Akt was able to reduce the E(2) stimulatory effects on activity and tyrosine phosphorylation levels of aromatase. Taken together, our results suggest that E(2) can impair PTP1B ability to dephosphorylate aromatase, and thus it increases its enzymatic activity, creating a positive feedback mechanism for estradiol signaling in breast cancer.

In situ estrogen production by aromatase conversion from androgens plays an important role in breast tumor promotion. Here, we show that 17beta-estradiol (E2) can rapidly enhance aromatase enzymatic activity through an increase of aromatase protein phosphorylation in breast cancer cell lines. In vivo labeling experiments and site-directed mutagenesis studies demonstrated that phosphorylation of the 361-tyrosine residue is crucial in the up-regulation of aromatase activity under E2 exposure. Our results demonstrated a direct involvement of nonreceptor tyrosine-kinase c-Src in E2-stimulated aromatase activity because inhibition of its signaling abrogated the up-regulatory effects induced by E2 on aromatase activity as well as phosphorylation of aromatase protein. In addition, from our data it emerges that aromatase is a target of cross talk between growth factor receptors and estrogen receptor alpha signaling. These findings show, for the first time, that tyrosine phosphorylation processes play a key role in the rapid changes induced by E2 in aromatase enzymatic activity, revealing the existence of a short nongenomic autocrine loop between E2 and aromatase in breast cancer cells.