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Abstract Background Over the last two decades, tumor-derived RNA expression signatures have been developed for the two most commonly diagnosed tumors worldwide, namely prostate and breast tumors, in order to improve both outcome prediction and treatment decision-making. In this context, molecular signatures gained by main components of the tumor microenvironment, such as cancer-associated fibroblasts (CAFs), have been explored as prognostic and therapeutic tools. Nevertheless, a deeper understanding of the significance of CAFs-related gene signatures in breast and prostate cancers still remains to be disclosed. Methods RNA sequencing technology (RNA-seq) was employed to profile and compare the transcriptome of CAFs isolated from patients affected by breast and prostate tumors. The differentially expressed genes (DEGs) characterizing breast and prostate CAFs were intersected with data from public datasets derived from bulk RNA-seq profiles of breast and prostate tumor patients. Pathway enrichment analyses allowed us to appreciate the biological significance of the DEGs. K-means clustering was applied to construct CAFs-related gene signatures specific for breast and prostate cancer and to stratify independent cohorts of patients into high and low gene expression clusters. Kaplan-Meier survival curves and log-rank tests were employed to predict differences in the outcome parameters of the clusters of patients. Decision-tree analysis was used to validate the clustering results and boosting calculations were then employed to improve the results obtained by the decision-tree algorithm. Results Data obtained in breast CAFs allowed us to assess a signature that includes 8 genes (ITGA11, THBS1, FN1, EMP1, ITGA2, FYN, SPP1, and EMP2) belonging to pro-metastatic signaling routes, such as the focal adhesion pathway. Survival analyses indicated that the cluster of breast cancer patients showing a high expression of the aforementioned genes displays worse clinical outcomes. Next, we identified a prostate CAFs-related signature that includes 11 genes (IL13RA2, GDF7, IL33, CXCL1, TNFRSF19, CXCL6, LIFR, CXCL5, IL7, TSLP, and TNFSF15) associated with immune responses. A low expression of these genes was predictive of poor survival rates in prostate cancer patients. The results obtained were significantly validated through a two-step approach, based on unsupervised (clustering) and supervised (classification) learning techniques, showing a high prediction accuracy (≥ 90%) in independent RNA-seq cohorts. Conclusion We identified a huge heterogeneity in the transcriptional profile of CAFs derived from breast and prostate tumors. Of note, the two novel CAFs-related gene signatures might be considered as reliable prognostic indicators and valuable biomarkers for a better management of breast and prostate cancer patients.

Abstract Background The cyclin D1-cyclin dependent kinases (CDK)4/6 inhibitor palbociclib in combination with endocrine therapy shows remarkable efficacy in the management of estrogen receptor (ER)-positive and HER2-negative advanced breast cancer (BC). Nevertheless, resistance to palbociclib frequently arises, highlighting the need to identify new targets toward more comprehensive therapeutic strategies in BC patients. Methods BC cell lines resistant to palbociclib were generated and used as a model system. Gene silencing techniques and overexpression experiments, real-time PCR, immunoblotting and chromatin immunoprecipitation studies as well as cell viability, colony and 3D spheroid formation assays served to evaluate the involvement of the G protein-coupled estrogen receptor (GPER) in the resistance to palbociclib in BC cells. Molecular docking simulations were also performed to investigate the potential interaction of palbociclib with GPER. Furthermore, BC cells co-cultured with cancer-associated fibroblasts (CAFs) isolated from mammary carcinoma, were used to investigate whether GPER signaling may contribute to functional cell interactions within the tumor microenvironment toward palbociclib resistance. Finally, by bioinformatics analyses and k-means clustering on clinical and expression data of large cohorts of BC patients, the clinical significance of novel mediators of palbociclib resistance was explored. Results Dissecting the molecular events that characterize ER-positive BC cells resistant to palbociclib, the down-regulation of ERα along with the up-regulation of GPER were found. To evaluate the molecular events involved in the up-regulation of GPER, we determined that the epidermal growth factor receptor (EGFR) interacts with the promoter region of GPER and stimulates its expression toward BC cells resistance to palbociclib treatment. Adding further cues to these data, we ascertained that palbociclib does induce pro-inflammatory transcriptional events via GPER signaling in CAFs. Of note, by performing co-culture assays we demonstrated that GPER contributes to the reduced sensitivity to palbociclib also facilitating the functional interaction between BC cells and main components of the tumor microenvironment named CAFs. Conclusions Overall, our results provide novel insights on the molecular events through which GPER may contribute to palbociclib resistance in BC cells. Additional investigations are warranted in order to assess whether targeting the GPER-mediated interactions between BC cells and CAFs may be useful in more comprehensive therapeutic approaches of BC resistant to palbociclib.

Abstract Background Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERβ, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells. Methods The potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients. Results After the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients. Conclusions Overall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.

Abstract The G protein-coupled estrogen receptor (GPER) mediates estrogen action in different pathophysiological conditions, including cancer. GPER expression and signaling have been found to join in the progression of triple-negative breast cancer (TNBC), even though controversial data have been reported. In present study, we aimed at providing new mechanistic and biological discoveries knocking out (KO) GPER expression by CRISPR/Cas9 technology in MDA-MB-231 TNBC cells. GPER KO whole transcriptome respect to wild type (WT) MDA-MB-231 cells was determined through total RNA sequencing (RNA-Seq) and gene ontology (GO) enrichment analysis. We ascertained that anti-proliferative and pro-apoptotic gene signatures characterize GPER KO MDA-MB-231 cells. Thereafter, we determined that these cells exhibit a reduced proliferative, clonogenic and self-renewal potential along with an increased mitochondria-dependent apoptosis phenotype. In addition, we recognized that decreased cAMP levels trigger the JNK/c-Jun/p53/Noxa axis, which in turn orchestrates the pro-apoptotic effects observed in GPER KO cells. In accordance with these data, survival analyses in TNBC patients of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset indicated that high Noxa expression correlates with improved outcomes in TNBC patients. Furthermore, we demonstrated that GPER KO in TNBC cells impairs the expression and secretion of the well-acknowledged GPER target gene named CTGF, thus resulting in the inhibition of migratory effects in cancer-associated fibroblasts (CAFs). Overall, the present study provides novel mechanistic and biological insights on GPER KO in TNBC cells suggesting that GPER may be considered as a valuable target in comprehensive therapeutic approaches halting TNBC progression.

Abstract The receptor for advanced glycation end products (RAGE) is implicated in diabetes and obesity complications, as well as in breast cancer (BC). Herein, we evaluated whether RAGE contributes to the oncogenic actions of Insulin, which plays a key role in BC progression particularly in obese and diabetic patients. Analysis of the publicly available METABRIC study, which collects gene expression and clinical data from a large cohort (n = 1904) of BC patients, revealed that RAGE and the Insulin Receptor (IR) are co-expressed and associated with negative prognostic parameters. In MCF-7, ZR75 and 4T1 BC cells, as well as in patient-derived Cancer-Associated Fibroblasts, the pharmacological inhibition of RAGE as well as its genetic depletion interfered with Insulin-induced activation of the oncogenic pathway IR/IRS1/AKT/CD1. Mechanistically, IR and RAGE directly interacted upon Insulin stimulation, as shown by in situ proximity ligation assays and coimmunoprecipitation studies. Of note, RAGE inhibition halted the activation of both IR and insulin like growth factor 1 receptor (IGF-1R), as demonstrated in MCF-7 cells KO for the IR and the IGF-1R gene via CRISPR-cas9 technology. An unbiased label-free proteomic analysis uncovered proteins and predicted pathways affected by RAGE inhibition in Insulin-stimulated BC cells. Biologically, RAGE inhibition reduced cell proliferation, migration, and patient-derived mammosphere formation triggered by Insulin. In vivo, the pharmacological inhibition of RAGE halted Insulin-induced tumor growth, without affecting blood glucose homeostasis. Together, our findings suggest that targeting RAGE may represent an appealing opportunity to blunt Insulin-induced oncogenic signaling in BC.

Abstract Background The receptor for advanced glycation-end products (RAGE) and its ligands have been implicated in obesity and associated inflammatory processes as well as in metabolic alterations like diabetes. In addition, RAGE-mediated signaling has been reported to contribute to the metastatic progression of breast cancer (BC), although mechanistic insights are still required. Here, we provide novel findings regarding the transcriptomic landscape and the molecular events through which RAGE may prompt aggressive features in estrogen receptor (ER)-positive BC. Methods MCF7 and T47D BC cells stably overexpressing human RAGE were used as a model system to evaluate important changes like cell protrusions, migration, invasion and colony formation both in vitro through scanning electron microscopy, clonogenic, migration and invasion assays and in vivo through zebrafish xenografts experiments. The whole transcriptome of RAGE-overexpressing BC cells was screened by high-throughput RNA sequencing. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses allowed the prediction of potential functions of differentially expressed genes (DEGs). Flow cytometry, real time-PCR, chromatin immunoprecipitation, immunofluorescence and western blot assays were performed to investigate the molecular network involved in the regulation of a novel RAGE target gene namely EphA3. The clinical significance of EphA3 was explored in the TCGA cohort of patients through the survivALL package, whereas the pro-migratory role of EphA3 signaling was ascertained in both BC cells and cancer-associated fibroblasts (CAFs). Statistical analysis was performed by t-tests. Results RNA-seq findings and GSEA analysis revealed that RAGE overexpression leads to a motility-related gene signature in ER-positive BC cells. Accordingly, we found that RAGE-overexpressing BC cells exhibit long filopodia-like membrane protrusions as well as an enhanced dissemination potential, as determined by the diverse experimental assays. Mechanistically, we established for the first time that EphA3 signaling may act as a physical mediator of BC cells and CAFs motility through both homotypic and heterotypic interactions. Conclusions Our data demonstrate that RAGE up-regulation leads to migratory ability in ER-positive BC cells. Noteworthy, our findings suggest that EphA3 may be considered as a novel RAGE target gene facilitating BC invasion and scattering from the primary tumor mass. Overall, the current results may provide useful insights for more comprehensive therapeutic approaches in BC, particularly in obese and diabetic patients that are characterized by high RAGE levels.

“Morzeddhu” in the local dialect of Catanzaro (“Morzello” in Italian) is an official typical dish of the capital of the Calabria region. It is a peasant dish, almost unknown at an international level, that labels, in an extraordinary way, the culinary identity of Catanzaro, a city founded around the X century. After America’s discovery, its preparation was optimized and definitively fixed. Its recipe is strictly based on a cow’s “fifth quarter” combined with spicy and typical Mediterranean vegetables. Remarkably, no pork meat is used, and when all traditional ingredients are included in the complex and quite long preparation of this special dish, it can deserve the title of “Illustrissimo”. This review provides a scientific description of Illustrissimo, emphasizing its unique properties and connection to the circular economy, food security, and the Mediterranean diet. We also highlight its unique quality compared to other alternatives through an analysis of their nutritional facts and bioactive compounds. Nutritionally, offal and fifth quarter components are a rich source of high-quality protein, with lower levels of total fat and saturated fatty acids compared to other meat cuts. In essence, this dish offers a great example of a high-quality yet affordable meal, aligning perfectly with a Mediterranean diet.

Triple-negative breast cancer (TNBC) is an aggressive breast tumor subtype that currently lacks targeted treatment options. The role played by the insulin-like growth factor-1 (IGF-1) and its cognate receptor IGF-1R in TNBC has been reported. Nevertheless, the molecular mechanisms by which the IGF-1/IGF-1R system may contribute to TNBC progression still remains to be fully understood. By computational analysis of the vast cancer genomics information in public databases (TCGA and METABRIC), we obtained evidence that high IGF-1 or IGF-1R levels correlate with a worse clinical outcome in TNBC patients. Further bioinformatics analysis revealed that both the focal adhesion and the Hippo pathways are enriched in TNBC harboring an elevated expression of IGF-1 or IGF-1R. Mechanistically, we found that in TNBC cells, the IGF-1/IGF-1R system promotes the activation of the FAK signal transduction pathway, which in turn regulates the nuclear accumulation of YAP (yes-associated protein/yes-related protein) and the expression of its target genes. At the biological level, we found that the IGF-1/IGF-1R-FAK-YAP network cascade triggers the growth potential of TNBC cells, as evaluated in different experimental systems. Overall, our results suggest that the IGF-1/IGF-1R/FAK/YAP axis may contribute to the progression of the aggressive TNBC subtype.

Arylalkane-derived prodrugs of arylacetic acids are a small group of substances that have long been known for their anti-inflammatory action. Despite their ease of synthesis and good potential for the development of new potent and safe anti-inflammatory agents, this group of substances has not received much attention from researchers so far. Therefore, representative arylalkane derivatives were investigated through molecular docking techniques to verify the possible hepatic activation mode toward active metabolites by CYP1A2. In this regard, arylalkanoic acid prodrugs were docked with a crystallographic structure of human CYP1A2, in which the enzyme is co-crystallized with the selective competitive inhibitor α-naphthoflavone BHF. Of note, all the examined compounds proved capable of interacting with the enzyme active site in a manner similar to Nabumetone, thus confirming that a productive metabolic transformation is feasible. On the basis of these findings, it is possible to argue that subtle differences in the way CYP1A2 accommodates the ligands depend on the fine details of their molecular structures. Overall, these data suggest that compounds simply formed by an aromatic moiety bearing an appropriate alkane-derived chain could lead to innovative anti-inflammatory agents.

Abstract Background Metabolic disorders are associated with increased incidence, aggressive phenotype and poor outcome of breast cancer (BC) patients. For instance, hyperinsulinemia is an independent risk factor for BC and the insulin/insulin receptor (IR) axis is involved in BC growth and metastasis. Of note, the anti-diabetic metformin may be considered in comprehensive therapeutic approaches in BC on the basis of its antiproliferative effects obtained in diverse pre-clinical and clinical studies. Methods Bioinformatics analysis were performed using the information provided by The Invasive Breast Cancer Cohort of The Cancer Genome Atlas (TCGA) project. The naturally immortalized BC cell line, named BCAHC-1, as well as cancer-associated fibroblasts (CAFs) derived from BC patients were used as model systems. In order to identify further mechanisms that characterize the anticancer action of metformin in BC, we performed gene expression and promoter studies as well as western blotting experiments. Moreover, cell cycle analysis, colony and spheroid formation, actin cytoskeleton reorganization, cell migration and matrigel drops evasion assays were carried out to provide novel insights on the anticancer properties of metformin. Results We first assessed that elevated expression and activation of IR correlate with a worse prognostic outcome in estrogen receptor (ER)-positive BC. Thereafter, we established that metformin inhibits the insulin/IR-mediated activation of transduction pathways, gene changes and proliferative responses in BCAHC-1 cells. Then, we found that metformin interferes with the insulin-induced expression of the metastatic gene CXC chemokine receptor 4 (CXCR4), which we found to be associated with poor disease-free survival in BC patients exhibiting high levels of IR. Next, we ascertained that metformin prevents a motile phenotype of BCAHC-1 cells triggered by the paracrine liaison between tumor cells and CAFs upon insulin activated CXCL12/CXCR4 axis. Conclusions Our findings provide novel mechanistic insights regarding the anti-proliferative and anti-migratory effects of metformin in both BC cells and important components of the tumor microenvironment like CAFs. Further investigations are warranted to corroborate the anticancer action of metformin on the tumor mass toward the assessment of more comprehensive strategies halting BC progression, in particular in patients exhibiting metabolic disorders and altered insulin/IR functions.

Background: Few studies have assessed the differences of patterns of Long COVID (L-COVID) with regards to the pathogenetic SARS-CoV-2 strains. Objectives: To investigate the relationship between demographic and clinical characteristics of acute phase of infection and the persistence of L-COVID symptoms and clinical presentation across different SARS-CoV-2 strains. Methods: In this observational-multicenter study we recorded all demographic and clinical characteristics, severity of infection, presence/persistence of symptoms of fatigue, dyspnoea and altered quality of life (QoL) at baseline and after 6 months, in a sample of Italian patients from Liguria between March 2020 and March 2022. Results: 308 patients (mean age 63.2 years; 55.5% men) with previous COVID were enrolled. Obese patients were 21.2% with a significant difference in obesity prevalence across the second and third wave (p = 0.012). Treatment strategies differed between waves (p < 0.001): more patients required invasive mechanical ventilation in the first wave, more patients were treated with high-flow nasal cannula/non-invasive ventilation in the in the second and more patients were treated with oxygen-therapy in the fourth wave. At baseline, a high proportion of patients were symptomatic (dyspnoea and fatigue), with impairment in some QoL indicators. A higher prevalence of patients with pain, were seen in the first wave compared to later infections (p = 0.01). At follow-up, we observed improvement of dyspnoea, fatigue and some dimensions of QoL scale evaluation such as mobility, usual activities, pain evaluations; instead there was no improvement in remaining QoL scale indicators (usual care and anxiety-depression). Conclusions: There were no significant differences in the prevalence of the most frequent L-COVID symptoms, except for QoL pain domain that was especially associated with classical variant. Our results show substantial impact on social and professional life and usual care activities. These findings highlight the importance of multidisciplinary post COVID follow-up care including mental health support and rehabilitation program.

Metabolic disorders, such as obesity, type 2 diabetes (T2D) and metabolic syndrome, have been implicated in breast cancer (BC) progression. In this regard, insulin has been shown to promote mitogenic and metastatic responses in BC through diverse signaling pathways. Moreover, high levels of insulin and elevated expression of its cognate receptor, namely insulin receptor (IR), have been associated with increased BC incidence, resistance to treatments and poor outcomes. Metformin (1,1-dimethylbiguanide hydrochloride) is the most commonly prescribed drug for T2D treatment worldwide. Metformin has been shown to interfere with BC cell growth. In order to provide novel insights through which metformin can elicit anti-cancer responses in BC, we performed bioinformatics analysis as well as TaqMan Gene Expression Assay, flow cytometry, immunofluorescence, immunoblots, 2D and 3D proliferation assays and motility experiments. A naturally immortalized BC cell line (namely BCAHC-1) and important components of the tumor microenvironment, such as cancer-associated fibroblasts (CAFs) derived from BC patients, were used as model systems. We found that metformin inhibits the activation of main transduction pathways, the gene expression changes and the proliferative effects induced by insulin in BCAHC-1 cells. Moreover, metformin prevented the insulin-stimulated induction of CXC chemokine receptor 4 (CXCR4), which has been involved in BC metastatic dissemination. Next, metformin suppressed the invasion of CAFs triggered through CXCR4 via insulin stimulated BCAHC-1 cells. Our findings may suggest novel transduction mechanisms involved in the inhibitory effects elicited by metformin in both BC cells and CAFs.

Current advances in molecular profiling methodologies and the accessibility of multi-omics datasets are paving the way toward a better understanding of heterogeneous diseases, including breast cancer (BC). In this regard, we sought to uncover the transcriptional changes triggered by estrogen and insulin in a primary BC cell line (BCAHC-1), which expresses the 46kDa isoform of the estrogen receptor (ER)α and the insulin receptor, as we have previously ascertained. Raw data from RNA sequencing of BCAHC-1 cells were processed by the Bcl2Fastq 2.20 version of the Illumina pipeline, while in silico analyses were performed in R Studio using the TCGA dataset. Real-time PCR, immunoblotting, ELISA and chromatin immunoprecipitation experiments were used to identify the molecular events triggered by estrogen and insulin in BCAHC-1 cells and cancer-associated fibroblasts (CAFs). Furthermore, migration and invasion assays allowed us to ascertain the mechanisms triggering these biological responses in the presence of the aforementioned hormone treatments. First, we determined that 17β-estradiol (E2) and insulin stimulate a peculiar IL-11 expression and IL-11 secretion in BCAHC-1 cells. Thereafter, bioinformatics analyses confirmed the up-regulation of IL-11 in ER-positive BCs, with respect to adjacent normal tissues, and its association with worse survival. Next, the involvement of IL-11 in pro-metastatic transduction signaling was established via pathway enrichment analyses. Notably, we found that the secretion of IL-11 by BCAHC-1 cells prompts an invasive phenotype of CAFs through the up-regulation of genes belonging to the extracellular matrix organization pathway, namely, the intercellular adhesion molecule 1 and integrin alpha 5. Overall, our findings indicate that IL-11 secretion by BC cells may elicit a paracrine action on the surrounding stroma and introduce invasive properties, suggesting that IL-11 could be considered a valuable target in comprehensive treatments of ER-positive BC patients.

Tumor microenvironment (TME) includes a wide variety of cell types and soluble factors capable of suppressing immune-responses. While the role of NK cells in TME has been analyzed, limited information is available on the presence and the effect of polymorphonuclear (PMN) myeloid-derived suppressor cells, (MDSC). Among the immunomodulatory cells present in TME, MDSC are potentially efficient in counteracting the anti-tumor activity of several effector cells. We show that PMN-MDSC are present in high numbers in the PB of patients with primary or metastatic lung tumor. Their frequency correlated with the overall survival of patients. In addition, it inversely correlated with low frequencies of NK cells both in the PB and in tumor lesions. Moreover, such NK cells displayed an impaired anti-tumor activity, even those isolated from PB. The compromised function of NK cells was consequent to their interaction with PMN-MDSC. Indeed, we show that the expression of major activating NK receptors, the NK cytolytic activity and the cytokine production were inhibited upon co-culture with PMN-MDSC through both cell-to-cell contact and soluble factors. In this context, we show that exosomes derived from PMN-MDSC are responsible of a significant immunosuppressive effect on NK cell-mediated anti-tumor activity. Our data may provide a novel useful tool to implement the tumor immunoscore. Indeed, the detection of PMN-MDSC in the PB may be of prognostic value, providing clues on the presence and extension of both adult and pediatric tumors and information on the efficacy not only of immune response but also of immunotherapy and, possibly, on the clinical outcome.

Abstract Background Hypoxia plays a relevant role in tumor-related inflammation toward the metastatic spread and cancer aggressiveness. The pro-inflammatory cytokine interleukin-1β (IL-β) and its cognate receptor IL1R1 contribute to the initiation and progression of breast cancer determining pro-tumorigenic inflammatory responses. The transcriptional target of the hypoxia inducible factor-1α (HIF-1α) namely the G protein estrogen receptor (GPER) mediates a feedforward loop coupling IL-1β induction by breast cancer-associated fibroblasts (CAFs) to IL1R1 expression by breast cancer cells toward the regulation of target genes and relevant biological responses. Methods In order to ascertain the correlation of IL-β with HIF-1α and further hypoxia-related genes in triple-negative breast cancer (TNBC) patients, a bioinformatics analysis was performed using the information provided by The Invasive Breast Cancer Cohort of The Cancer Genome Atlas (TCGA) project and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) datasets. Gene expression correlation, statistical analysis and gene set enrichment analysis (GSEA) were carried out with R studio packages. Pathway enrichment analysis was evaluated with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. TNBC cells and primary CAFs were used as model system. The molecular mechanisms implicated in the regulation of IL-1β by hypoxia toward a metastatic gene expression profile and invasive properties were assessed performing gene and protein expression studies, PCR arrays, gene silencing and immunofluorescence analysis, co-immunoprecipitation and ChiP assays, ELISA, cell spreading, invasion and spheroid formation. Results We first determined that IL-1β expression correlates with the levels of HIF-1α as well as with a hypoxia-related gene signature in TNBC patients. Next, we demonstrated that hypoxia triggers a functional liaison among HIF-1α, GPER and the IL-1β/IL1R1 signaling toward a metastatic gene signature and a feed-forward loop of IL-1β that leads to proliferative and invasive responses in TNBC cells. Furthermore, we found that the IL-1β released in the conditioned medium of TNBC cells exposed to hypoxic conditions promotes an invasive phenotype of CAFs. Conclusions Our data shed new light on the role of hypoxia in the activation of the IL-1β/IL1R1 signaling, which in turn triggers aggressive features in both TNBC cells and CAFs. Hence, our findings provide novel evidence regarding the mechanisms through which the hypoxic tumor microenvironment may contribute to breast cancer progression and suggest further targets useful in more comprehensive therapeutic strategies.

Abstract Among the prognostic and predictive biomarkers of breast cancer (BC), the role of estrogen receptor (ER)α wild‐type has been acknowledged, although the action of certain ERα splice variants has not been elucidated. Insulin/insulin receptor (IR) axis has also been involved in the progression and metastasis of BC. For instance, hyperinsulinemia, which is often associated with obesity and type 2 diabetes, may be a risk factor for BC. Similarly, an aberrant expression of IR or its hyperactivation may correlate with aggressive BC phenotypes. In the present study, we have shown that a novel naturally immortalized BC cell line (named BCAHC‐1) is characterized by a unique expression of 46 kDa ERα splice variant (ERα46) along with IR. Moreover, we have shown that a multifaceted crosstalk between ERα46 and IR occurs in BCAHC‐1 cells upon estrogen and insulin exposure for growth and pulmonary metastasis. Through high‐throughput RNA sequencing analysis, we have also found that the cytokine interleukin‐11 (IL11) is the main factor linking BCAHC‐1 cells to breast cancer‐associated fibroblasts (CAFs). In particular, we have found that IL11 induced by estrogens and insulin in BCAHC‐1 cells regulates pro‐tumorigenic genes of the “extracellular matrix organization” signaling pathway, such as ICAM‐1 and ITGA5, and promotes both migratory and invasive features in breast CAFs. Overall, our results may open a new scientific avenue to identify additional prognostic and therapeutic targets in BC.

Advanced glycation end products (AGEs) and the cognate receptor, named RAGE, are involved in metabolic disorders characterized by hyperglycemia, type 2 diabetes mellitus (T2DM) and obesity. Moreover, the AGEs/RAGE transduction pathway prompts a dysfunctional interaction between breast cancer cells and tumor stroma toward the acquisition of malignant features. However, the action of the AGEs/RAGE axis in the main players of the tumor microenvironment, named breast cancer-associated fibroblasts (CAFs), remains to be fully explored. In the present study, by chemokine array, we first assessed that interleukin-8 (IL-8) is the most up-regulated pro-inflammatory chemokine upon AGEs/RAGE activation in primary CAFs, obtained from breast tumors. Thereafter, we ascertained that the AGEs/RAGE signaling promotes a network cascade in CAFs, leading to the c-Fos-dependent regulation of IL-8. Next, using a conditioned medium from AGEs-exposed CAFs, we determined that IL-8/CXCR1/2 paracrine activation induces the acquisition of migratory and invasive features in MDA-MB-231 breast cancer cells. Altogether, our data provide new insights on the involvement of IL-8 in the AGEs/RAGE transduction pathway among the intricate connections linking breast cancer cells to the surrounding stroma. Hence, our findings may pave the way for further investigations to define the role of IL-8 as useful target for the better management of breast cancer patients exhibiting metabolic disorders.

Arylalkane-derived prodrugs of arylacetic acids are a small group of substances that have long been known for their anti-inflammatory action. Despite their ease of synthesis and good potential for the development of new potent and safe anti-inflammatory agents, this group of substances has not received much attention from researchers so far. Therefore, representative arylalkane derivatives were investigated through molecular docking techniques to verify the possible hepatic activation mode toward active metabolites by CYP1A2. In this regard, arylalkanoic acid prodrugs were docked with a crystallographic structure of human CYP1A2, in which the enzyme is co-crystallized with the selective competitive inhibitor α-naphthoflavone BHF. Of note, all the examined compounds proved capable of interacting with the enzyme active site in a manner similar to Nabumetone, thus confirming that a productive metabolic transformation is feasible. On the basis of these findings, it is possible to argue that subtle differences in the way CYP1A2 accommodates the ligands depend on the fine details of their molecular structures. Overall, these data suggest that compounds simply formed by an aromatic moiety bearing an appropriate alkane-derived chain could lead to innovative anti-inflammatory agents. [ABSTRACT FROM AUTHOR]