Breast Cancer: From Pathophysiology to Novel Therapeutic Approaches

Hormone-specific anticancer drugs for breast cancer treatment can cause serious side effects. Thus, treatment with natural compounds has been considered a better approach as this minimizes side effects and has multiple targets. 6-Gingerol is an active polyphenol in ginger with various modalities, including anticancer activity, although its mechanism of action remains unknown. Increases in the level of reactive oxygen species (ROS) can lead to DNA damage and the induction of DNA damage response (DDR) mechanism, leading to cell cycle arrest apoptosis and tumorsphere suppression. Epidermal growth factor receptor (EGFR) promotes tumor growth by stimulating signaling of downstream targets that in turn activates tumor protein 53 (p53) to promote apoptosis. Here we assessed the effect of 6-gingerol treatment on MDA-MB-231 and MCF-7 breast cancer cell lines. 6-Gingerol induced cellular and mitochondrial ROS that elevated DDR through ataxia-telangiectasia mutated and p53 activation. 6-Gingerol also induced G0/G1 cell cycle arrest and mitochondrial apoptosis by mediating the BAX/BCL-2 ratio and release of cytochrome c. It also exhibited a suppression ability of tumorsphere formation in breast cancer cells. EGFR/Src/STAT3 signaling was also determined to be responsible for p53 activation and that 6-gingerol induced p53-dependent intrinsic apoptosis in breast cancer cells. Therefore, 6-gingerol may be used as a candidate drug against hormone-dependent breast cancer cells. Full article

Triple-negative breast cancer (TNBC) has a poor prognosis as the therapy has several limitations, most importantly, treatment resistance. In this study we examined the different responses of triple-negative breast cancer line MDA-MB-231 and hormone receptor-positive breast cancer line MCF7 to a combined treatment including olaparib, a poly-(ADP ribose) polymerase (PARP) inhibitor, oxaliplatin, a third-generation platinum compound and LY294002, an Akt pathway inhibitor. We applied the drugs in a single, therapeutically relevant concentration individually and in all possible combinations, and we assessed the viability, type of cell death, reactive oxygen species production, cell-cycle phases, colony formation and invasive growth. In agreement with the literature, the MDA-MB-231 cells were more treatment resistant than the MCF7 cells. However, and in contrast with the findings of others, we detected no synergistic effect between olaparib and oxaliplatin, and we found that the Akt pathway inhibitor augmented the cytostatic properties of the platinum compound and/or prevented the cytoprotective effects of PARP inhibition. Our results suggest that, at therapeutically relevant concentrations, the cytotoxicity of the platinum compound dominated over that of the PARP inhibitor and the PI3K inhibitor, even though a regression-based model could have indicated an overall synergy at lower and/or higher concentrations. Full article

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are involved in various immune cell-mediated diseases. Their role in cancer is poorly investigated, and research focusses on Siglec-expression on immune cells interacting with tumor cells. This study evaluates the role of Siglec-8 in breast cancer (BC). Siglec-8 expression was analyzed immunohistochemically on 235 primary BC cases and was correlated with clinical and pathological parameters and outcome. Cell culture experiments were performed with various BC cell lines. Siglec-8 was expressed in 215 BC cases and expression was lowest in triple-negative BC. It correlated with estrogen receptor-status, grading and the prognostic factors galectin (Gal)-7 and tumor-associated mucin-1 (TA-MUC1). However, Gal-7 and TA-MUC1 were only prognosticators for clinical outcome in the cohort expressing high (Immunoreactivity score IRS > 3) Siglec-8 levels but not in the low-expressing cohort. Siglec-8 knockdown led to a significantly reduced Gal-7 expression in MCF7 cells. All BC cell lines expressed low Siglec-8-levels, that could be elevated in MCF7 by Peroxisome proliferator-activated receptor (PPARγ)-stimulation. This study demonstrates that Siglec-8 is expressed in BC cells and correlates with known clinical and prognostic parameters. It is probably associated with Gal-7 and TA-MUC1 and might be regulated via PPARγ. Further analyses focusing on functional associations will clarify Siglec-8’s eligibility as a possible therapeutic target. Full article

Background: HER2-based retargeted viruses are in advanced phases of preclinical development of breast cancer models. Mesothelin (MSLN) is a cell-surface tumor antigen expressed in different subtypes of breast and non-breast cancer. Its recent identification as a marker of some triple-negative breast tumors renders it an attractive target, presently investigated in clinical trials employing antibody drug conjugates and CAR-T cells. The availability of MSLN-retargeted oncolytic viruses may complement the current immunotherapeutic panel of biological drugs against HER2-negative breast and non-breast tumors. Methods: A fully virulent, tumor-targeted oncolytic Herpes simplex virus-1 (MSLN-THV) with a selectivity for mesothelin-expressing cancer cells was generated. Recombineering technology was used to replace an essential moiety of the viral glycoprotein D with antibody fragments derived from clinically validated MSLN monoclonal antibodies, and to allow IL12 cargo expression in infected cells. Panels of breast and female reproductive system cell lines were used to verify the oncolytic potential of the viral constructs. A platform for production of the retargeted viruses was developed in HEK 293 cells, providing stable expression of a suitable chimeric receptor. Results: We demonstrated the selectivity of viral infection and cytotoxicity by MSLN-retargeted viruses in a panel of mesothelin-positive cancer cells, originating from breast and female reproductive system tumors. We also developed a second-generation oncolytic MSLN-THV, encoding IL12, to enhance the immunotherapeutic potential of the viral backbone. A non-tumor cell line expressing a chimeric MSLN/Nectin-1 receptor, de-sensitized from antiviral responses by genetic inactivation of the Stimulator of Interferon Genes (STING)-dependent pathway was engineered, to optimize viral yields. Conclusions: Our proof-of-concept study proposes MSLN-retargeted herpesviruses as potential cancer immunotherapeutics for assessments in preclinical models of MSLN-positive tumors, complementing the available panel of oncolytic viruses to HER2-negative breast tumors. Full article

A coherence between thyroid dysfunction and breast cancer incidence exists. Thyroid hormone metabolites bind to TAAR1 (trace amine-associated receptor 1) and through that modulate the serotonergic and dopaminergic system. Catecholamines themselves are synthesized by the L-dopa decarboxylase (DDC). The aim of our study was to analyze the influence of catecholamines on the DDC expression in primary breast cancer patients and the role of DDC concerning overall survival (OS). DDC expression was analyzed by immunohistochemistry. The effect of epinephrine on the expression of DDC and the ${\mathrm{G}}_{\mathrm{i}}$- protein was analyzed on the protein level via Western blot. A viability assay was performed to test the metabolic cell viability. The overexpression of DDC in the primary tumor was associated with longer OS (p = 0.03). Stimulation with epinephrine induced the downregulation of DDC (p = 0.038) and significantly increased viability in T47D cells (p = 0.028). In contrast, epinephrine induced an upregulation of DDC and decreased the proliferation of MCF7 cells (p = 0.028). Epinephrine led to an upregulation of $G_i$ protein expression in MCF7 cells (p = 0.008). DDC is a positive prognostic factor for OS in breast cancer patients, and it is regulated through epinephrine differently in MCF7 and T47D. DDC may represent a novel target for the treatment of breast cancer, especially concerning its interaction with epinephrine. Full article

Hyperthermia (HT) treatment is a noninvasive cancer therapy, often used with radiation therapy and chemotherapy. Compared with 37 °C, 42 °C is mild heat stress for cells and produces reactive oxygen species (ROS) from mitochondria. To involve subsequent intracellular accumulation of DOX, we have previously reported that the expression of ATP-binding cassette sub-family G member 2 (ABCG2), an exporter of doxorubicin (DOX), was suppressed by a larger amount of intracellular mitochondrial ROS. We then hypothesized that the additive effect of HT and chemotherapy would be induced by the downregulation of ABCG2 expression via intracellular ROS increase. We used human breast cancer cell lines, MCF-7 and MDA-MB-453, incubated at 37 °C or 42 °C for 1 h to clarify this hypothesis. Intracellular ROS production after HT was detected via electron spin resonance (ESR), and DOX cytotoxicity was calculated. Additionally, ABCG2 expression in whole cells was analyzed using Western blotting. We confirmed that the ESR signal peak with HT became higher than that without HT, indicating that the intracellular ROS level was increased by HT. ABCG2 expression was downregulated by HT, and cells were injured after DOX treatment. DOX cytotoxicity enhancement with HT was considered a result of ABCG2 expression downregulation via the increase of ROS production. HT increased intracellular ROS production and downregulated ABCG2 protein expression, leading to cell damage enhancement via DOX. Full article

Cell plasticity is the ability that cells have to modify their phenotype, adapting to the environment. Cancer progression is under the strict control of the the tumor microenvironment that strongly determines its success by regulating the behavioral changes of tumor cells. The cross-talk between cancer and stromal cells and the interactions with the extracellular matrix, hypoxia and acidosis contribute to trigger a new tumor cell identity and to enhance tumor heterogeneity and metastatic spread. In highly aggressive triple-negative breast cancer, tumor cells show a significant capability to change their phenotype under the pressure of the hypoxic microenvironment. In this study, we investigated whether targeting the hypoxia-induced protein carbonic anhydrase IX (CA IX) could reduce triple-negative breast cancer (TNBC) cell phenotypic switching involved in processes associated with poor prognosis such as vascular mimicry (VM) and cancer stem cells (CSCs). The treatment of two TNBC cell lines (BT-549 and MDA-MB-231) with a specific CA IX siRNA or with a novel inhibitor of carbonic anhydrases (RC44) severely impaired their ability to form a vascular-like network and mammospheres and reduced their metastatic potential. In addition, the RC44 inhibitor was able to hamper the signal pathways involved in triggering VM and CSC formation. These results demonstrate that targeting hypoxia-induced cell plasticity through CA IX inhibition could be a new opportunity to selectively reduce VM and CSCs, thus improving the efficiency of existing therapies in TNBC. Full article

The role of tumour-infiltrating inflammatory cells (TIICs) in the disease progression of hormone-receptor-positive breast cancer (HR+ BC) is largely unclear since it is generally regarded as the least immunogenic BC subtype. This study investigated the prognostic significance of CD1a+ dendritic cells, CD20+ B cells, CD45RO+ memory T cells and CD4+ T-helper cells in HR+ BC. One hundred and forty-six patients were treated for early stage, distant-metastases-free HR+ BC in an accelerated partial breast irradiation (APBI) phase II trial. Immunohistochemistry was used to double-stain two adjoining sets of tissue microarrays from pre-RT (radiotherapy) tumour resection samples for CD1a/CD20 and CD45RO/CD4. Cell densities of CD1a+, CD20+, CD45RO+ and CD4+ TIICs in the stromal and intraepithelial compartment were registered semiautomatically. High densities of CD20+ and CD4+ TIICs were strongly associated with reduced disease-free survival (DFS), while high stromal CD45RO+ TIIC densities were indicators of subsequent successful treatment. An immunoscore based on CD20+ and CD45RO+ TIIC densities identified three different risk groups (p < 0.001). Thus, contrary to current assumptions, intratumoural immune cell composition might be an important prognostic indicator and a possible contributing factor in the outcome of HR+ BC and should be the subject of further research. Specifically, B-cell infiltration entailed an increased relapse rate and could play an important role in disease progression. Full article

Breast cancer (BC) is a heterogeneous and complex disease as witnessed by the existence of different subtypes and clinical characteristics that poses significant challenges in disease management. The complexity of this tumor may rely on the highly interconnected nature of the various biological processes as stated by the new paradigm of Network Medicine. We explored The Cancer Genome Atlas (TCGA)-BRCA data set, by applying the network-based algorithm named SWItch Miner, and mapping the findings on the human interactome to capture the molecular interconnections associated with the disease modules. To characterize BC phenotypes, we constructed protein–protein interaction modules based on “hub genes”, called switch genes, both common and specific to the four tumor subtypes. Transcriptomic profiles of patients were stratified according to both clinical (immunohistochemistry) and genetic (PAM50) classifications. 266 and 372 switch genes were identified from immunohistochemistry and PAM50 classifications, respectively. Moreover, the identified switch genes were functionally characterized to select an interconnected pathway of disease genes. By intersecting the common switch genes of the two classifications, we selected a unique signature of 28 disease genes that were BC subtype-independent and classification subtype-independent. Data were validated both in vitro (10 BC cell lines) and ex vivo (66 BC tissues) experiments. Results showed that four of these hub proteins (AURKA, CDC45, ESPL1, and RAD54L) were over-expressed in all tumor subtypes. Moreover, the inhibition of one of the identified switch genes (AURKA) similarly affected all BC subtypes. In conclusion, using a network-based approach, we identified a common BC disease module which might reflect its pathological signature, suggesting a new vision to face with the disease heterogeneity. Full article

Development of targeted therapies for triple-negative breast cancer (TNBC) is an unmet medical need. Cisplatin has demonstrated its promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with hypoxia that, in turn, promotes cancer stem cell (CSC) enrichment and drug resistance. Therapeutic approaches to attenuate this may lead to increased cisplatin efficacy in the clinic for the treatment of TNBC. In this report we analyzed clinical datasets of TNBC and found that TNBC patients possessed higher levels of EGFR and hypoxia gene expression. A similar expression pattern was also observed in cisplatin-resistant ovarian cancer cells. We, thus, developed a new therapeutic approach to inhibit EGFR and hypoxia by combination treatment with metformin and gefitinib that sensitized TNBC cells to cisplatin and led to the inhibition of both CD44+/CD24− and ALDH+ CSCs. We demonstrated a similar inhibition efficacy on organotypic cultures of TNBC patient samples ex vivo. Since these drugs have already been used frequently in the clinic; this study illustrates a novel, clinically translatable therapeutic approach to treat patients with TNBC. Full article

Collagen type 1 (COL1) is a ubiquitously existing extracellular matrix protein whose high density in breast tissue favors metastasis and chemoresistance. COL1-binding of MDA-MB-231 and MCF-7 breast cancer cells is mainly dependent on β₁-integrins (ITGB1). Here, we elucidate the signaling of chemoresistance in both cell lines and their ITGB1-knockdown mutants and elucidated MAPK pathway to be strongly upregulated upon COL1 binding. Notably, Discoidin Domain Receptor 1 (DDR1) was identified as another important COL1-sensor, which is permanently active but takes over the role of COL1-receptor maintaining MAPK activation in ITGB1-knockdown cells. Consequently, inhibition of DDR1 and ERK1/2 act synergistically, and sensitize the cells for cytostatic treatments using mitoxantrone, or doxorubicin, which was associated with an impaired ABCG2 drug efflux transporter activity. These data favor DDR1 as a promising target for cancer cell sensitization, most likely in combination with MAPK pathway inhibitors to circumvent COL1 induced transporter resistance axis. Since ITGB1-knockdown also induces upregulation of pEGFR in MDA-MB-231 cells, inhibitory approaches including EGFR inhibitors, such as gefitinib appear promising for pharmacological interference. These findings provide evidence for the highly dynamic adaptation of breast cancer cells in maintaining matrix binding to circumvent cytotoxicity and highlight DDR1 signaling as a target for sensitization approaches. Full article

Current methods for the intraoperative determination of breast cancer margins commonly suffer from the insufficient accuracy, specificity and/or low speed of analysis, increasing the time and cost of operation as well the risk of cancer recurrence. The purpose of this study is to develop a method for the rapid and accurate determination of breast cancer margins using direct molecular profiling by mass spectrometry (MS). Direct molecular fingerprinting of tiny pieces of breast tissue (approximately 1 × 1 × 1 mm) is performed using a home-built tissue spray ionization source installed on a Maxis Impact quadrupole time-of-flight mass spectrometer (qTOF MS) (Bruker Daltonics, Hamburg, Germany). Statistical analysis of MS data from 50 samples of both normal and cancer tissue (from 25 patients) was performed using orthogonal projections onto latent structures discriminant analysis (OPLS-DA). Additionally, the results of OPLS classification of new 19 pieces of two tissue samples were compared with the results of histological analysis performed on the same tissues samples. The average time of analysis for one sample was about 5 min. Positive and negative ionization modes are used to provide complementary information and to find out the most informative method for a breast tissue classification. The analysis provides information on 11 lipid classes. OPLS-DA models are created for the classification of normal and cancer tissue based on the various datasets: All mass spectrometric peaks over 300 counts; peaks with a statistically significant difference of intensity determined by the Mann–Whitney U-test (p < 0.05); peaks identified as lipids; both identified and significantly different peaks. The highest values of Q2 have models built on all MS peaks and on significantly different peaks. While such models are useful for classification itself, they are of less value for building explanatory mechanisms of pathophysiology and providing a pathway analysis. Models based on identified peaks are preferable from this point of view. Results obtained by OPLS-DA classification of the tissue spray MS data of a new sample set (n = 19) revealed 100% sensitivity and specificity when compared to histological analysis, the “gold” standard for tissue classification. “All peaks” and “significantly different peaks” datasets in the positive ion mode were ideal for breast cancer tissue classification. Our results indicate the potential of tissue spray mass spectrometry for rapid, accurate and intraoperative diagnostics of breast cancer tissue as a means to reduce surgical intervention. Full article

The aim of this study was to evaluate the prognostic impact of prostaglandin E2 receptor 3 (EP3) receptor expression might have on the two different breast cancer entities: multifocal/multicentric versus unifocal. As the prognosis determining aspects, we investigated the overall- and disease-free survival by uni-and multivariate analysis. To underline the study’s conclusion, we additionally considered the histopathological grading and the tumor node metastasis (TNM) staging system. A retrospective statistical analysis was performed on survival related events in a series of 289 sporadic breast cancer (BC) patients treated at the Department of Obstetrics and Gynecology at the Ludwig–Maximillian’s University in Munich between 2000 and 2002. The EP3 receptor expression was analyzed by immunohistochemistry and showed to have a significantly positive association with breast cancer prognosis for both entities, although with major differences. Patients with unifocal BC with EP3 receptor expression showed a significant improved overall survival, in contrast to the patient cohort with multifocal/multicentric BC. In this group, EP3 expression revealed its positive impact merely five years after initial diagnosis. Underlining the positive influence of EP3 as a positive prognosticator notably for unifocal breast cancer, only this patient cohort showed favorable outcomes in staging and grading. Especially EP3 expression in unifocal breast cancer was identified as an independent prognostic marker for the overall survival, when adjusted for age, grading, and staging. Altogether, our results strengthen the need to further investigate the behavior of EP3 in breast cancer and understand why markers linked to inflammation show different effects on prognosis and clinicopathological parameters on each focality type. Full article

Although medicinal mushroom extracts have been proposed as promising anti-cancer agents, their precise impacts on metastatic breast cancer are still to be clarified. For this purpose, the present study exploited the effect of a novel medicinal mushroom blend, namely Micotherapy U-care, in a 4T1 triple-negative mouse breast cancer model. Mice were orally administered with Micotherapy U-care, consisting of a mixture of Agaricus blazei, Ophiocordyceps sinensis, Ganoderma lucidum, Grifola frondosa, and Lentinula edodes. The syngeneic tumor-bearing mice were generated by injecting 4T1 cells in both supplemented and non-supplemented mice. After sacrifice 35 days later, specific endpoints and pathological outcomes of the murine pulmonary tissue were evaluated. (i) Histopathological and ultrastructural analysis and (ii) immunohistochemical assessment of TGF-ß1, IL-6 and NOS2, COX2, SOD1 as markers of inflammation and oxidative stress were performed. The QoL was comparatively evaluated. Micotherapy U-care supplementation, starting before 4T1 injection and lasting until the end of the experiment, dramatically reduced the pulmonary metastases density, also triggering a decrease of fibrotic response, and reducing IL-6, NOS, and COX2 expression. SOD1 and TGF-ß1 results were also discussed. These findings support the valuable potential of Micotherapy U-care as adjuvant therapy in the critical management of triple-negative breast cancer. Full article

Alterations in the composition and architecture of the extracellular matrix (ECM) can influence cancer growth and dissemination. During epithelial-mesenchymal transition (EMT), epithelial cells assume a mesenchymal cell phenotype, changing their adhesion profiles from cell-cell contacts to cell-matrix interactions, contributing to metastasis. Breast cancer cells present at different stages of differentiation, producing distinct ECMs in the same tumor mass. However, the contribution of ECM derived from metastatic tumor cells to EMT is unclear. Here, we showed the mechanisms involved in the interaction of MCF-7, a low-metastatic, epithelial breast cancer cell line, with the ECM produced by a high metastatic breast tumor cell, MDA-MB-231 (MDA-ECM). MDA-ECM induced morphological changes in MCF-7 cells, decreased the levels of E-cadherin, up-regulated mesenchymal markers, and augmented cell migration. These changes were accompanied by the activation of integrin-associated signaling, with increased phosphorylation of FAK, ERK, and AKT and activation canonical TGF-β receptor signaling, enhancing phosphorylation of SMAD2 and SMAD4 nuclear translocation in MCF-7 cells. Treatment with Kistrin (Kr), a specific ligand of integrin αvβ3 EMT induced by MDA-ECM, inhibited TGF-β receptor signaling in treated MCF-7 cells. Our results revealed that after interaction with the ECM produced by a high metastatic breast cancer cell, MCF-7 cells lost their characteristic epithelial phenotype undergoing EMT, an effect modulated by integrin signaling in crosstalk with TGF-β receptor signaling pathway. The data evidenced novel potential targets for antimetastatic breast cancer therapies. Full article

Crosstalk between neoplastic and immune cells in the tumor microenvironment (TME) influences the progression of disease in human and canine cancer patients. Given that canine mammary tumors are a useful model to study breast cancer biology, we aimed to evaluate the expression of genes associated with T lymphocyte activity in benign, malignant, and metastatic canine mammary tumors. Interestingly, metastatic tumors exhibit increased expression of CXCR3, CCR2, IL-4, IL-12p40, and IL-17. In particular, we focused on IL-17, a key interleukin associated with the Th17 lymphocyte phenotype. Th17 cells have been shown to play a contradictory role in tumor immunity. Although IL-17 showed a high expression in the metastatic tumors, the expression of RORγt, a crucial transcription factor for Th17 differentiation was barely detected. We further investigated IL-17 expression using immunohistochemistry, through which we confirmed the increased expression of this interleukin in malignant and metastatic mammary tumors. Finally, we compared the plasma levels of IL-17 in healthy and malignant mammary tumor-bearing dogs using ELISA but found no differences between the groups. Our data indicate that the IL-17 in metastatic tumors may be produced by other cell types, but not by Th17 lymphocytes. Overall, our results broaden the available knowledge on the interactions in canine mammary tumors and provide insight into the development of new therapeutic strategies, with potential benefits for human immune oncology. Full article

Mismatch repair (MMR) analysis in breast cancer may help to inform immunotherapy decisions but it lacks breast-specific guidelines. Unlike in other neoplasms, MMR protein loss shows intra-tumor heterogeneity and it is not mirrored by microsatellite instability in the breast. Additional biomarkers can improve MMR clinical testing. Phosphatase and tensin homolog (PTEN) inactivation is an early oncogenic event that is associated with MMR deficiency (dMMR) in several tumors. Here, we sought to characterize the diagnostic utility of PTEN expression analysis for MMR status assessment in breast cancer. A total of 608 breast cancers were profiled for their MMR and PTEN status. Proteins expression and distribution were analyzed by immunohistochemistry (IHC) on tissue microarrays and confirmed on full sections; PTEN copy number alterations were detected using a real-time PCR assay. Overall, 78 (12.8%) cases were MMR-heterogeneous (hMMR), while all patterns of PTEN expression showed no intra-tumor heterogeneity. Wild-type PTEN expression was observed in 15 (18.5%) dMMR tumors (p < 0.0001). Survival analyses revealed significant correlations between MMR-proficient (pMMR), PTEN expression, and a better outcome. The positive predictive value of PTEN-retained status for pMMR ranged from 94.6% in estrogen receptor (ER)+/HER2- tumors to 100% in HER2-amplified and ER-/HER2- cases. We propose a novel diagnostic algorithm where PTEN expression analysis can be employed to identify pMMR breast cancers. Full article

Metastasis is considered a major burden in cancer, being responsible for more than 90% of cancer-related deaths. Tumor angiogenesis is one of the main processes that lead to tumor metastasis. Penfluridol is a classic and commonly used antipsychotic drug, which has a great ability to cross the blood–brain barrier. Recent studies have revealed that penfluridol has significant anti-cancer activity in diverse tumors, such as metastatic breast cancer and glioblastoma. Here, we aim to identify the effect of low doses of penfluridol on tumor microenvironment and compare it with its effect on tumor cells. Although low concentration of penfluridol was not toxic for endothelial cells, it blocked angiogenesis in vitro and in vivo. In vitro, penfluridol inhibited VEGF-induced primary endothelial cell migration and tube formation, and in vivo, it blocked VEGF- and FGF-induced angiogenesis in the matrigel plug assay. VEGF-induced VEGFR2 phosphorylation and the downstream p38 and ERK signaling pathways were not affected in endothelial cells, although VEGF-induced Src and Akt activation were abrogated by penfluridol treatment. When cancer cells were treated with the same low concentration of penfluridol, basal Src activation levels were mildly impaired, thus impacting their cell migration and wound healing efficiency. The potential of cancer-induced paracrine effect on endothelial cells was explored, although that did not seem to be a player for angiogenesis. Overall, our data demonstrates that low penfluridol levels, similar to the ones clinically used for anti-psychotic conditions, suppress angiogenic efficiency in the tumor microenvironment. Full article

Breast cancer is the most commonly diagnosed cancer type and the leading cause of cancer-related mortality in women worldwide. Breast cancer is fairly heterogeneous and reveals six molecular subtypes: luminal A, luminal B, HER2+, basal-like subtype (ER−, PR−, and HER2−), normal breast-like, and claudin-low. Breast cancer screening and early diagnosis play critical roles in improving therapeutic outcomes and prognosis. Mammography is currently the main commercially available detection method for breast cancer; however, it has numerous limitations. Therefore, reliable noninvasive diagnostic and prognostic biomarkers are required. Biomarkers used in cancer range from macromolecules, such as DNA, RNA, and proteins, to whole cells. Biomarkers for cancer risk, diagnosis, proliferation, metastasis, drug resistance, and prognosis have been identified in breast cancer. In addition, there is currently a greater demand for personalized or precise treatments; moreover, the identification of novel biomarkers to further the development of new drugs is urgently needed. In this review, we summarize and focus on the recent discoveries of promising macromolecules and cell-based biomarkers for the diagnosis and prognosis of breast cancer and provide implications for therapeutic strategies. Full article

Metabolic reprogramming of cancer is now considered a hallmark of many malignant tumors, including breast cancer, which remains the most commonly diagnosed cancer in women all over the world. One of the main challenges for the effective treatment of breast cancer emanates from the existence of a subpopulation of tumor-initiating cells, known as cancer stem cells (CSCs). Over the years, several pathways involved in the regulation of CSCs have been identified and characterized. Recent research has also shown that CSCs are capable of adopting a metabolic flexibility to survive under various stressors, contributing to chemo-resistance, metastasis, and disease relapse. This review summarizes the links between the metabolic adaptations of breast cancer cells and CSC-associated pathways. Identification of the drivers capable of the metabolic rewiring in breast cancer cells and CSCs and the signaling pathways contributing to metabolic flexibility may lead to the development of effective therapeutic strategies. This review also covers the role of these metabolic adaptation in conferring drug resistance and metastasis in breast CSCs. Full article

The management of breast cancer (BC) has rapidly evolved in the last 20 years. The improvement of systemic therapy allows a remarkable control of extracranial disease. However, brain (BM) and leptomeningeal metastases (LM) are frequent complications of advanced BC and represent a challenging issue for clinicians. Some prognostic scales designed for metastatic BC have been employed to select fit patients for adequate therapy and enrollment in clinical trials. Different systemic drugs, such as targeted therapies with either monoclonal antibodies or small tyrosine kinase molecules, or modified chemotherapeutic agents are under investigation. Major aims are to improve the penetration of active drugs through the blood–brain barrier (BBB) or brain–tumor barrier (BTB), and establish the best sequence and timing of radiotherapy and systemic therapy to avoid neurocognitive impairment. Moreover, pharmacologic prevention is a new concept driven by the efficacy of targeted agents on macrometastases from specific molecular subgroups. This review aims to provide an overview of the clinical and molecular factors involved in the selection of patients for local and/or systemic therapy, as well as the results of clinical trials on advanced BC. Moreover, insight on promising therapeutic options and potential directions of future therapeutic targets against BBB and microenvironment are discussed. Full article

Breast cancer was one of the first malignancies to benefit from targeted therapy, i.e., treatments directed against specific markers. Inhibitors against HER2 are a significant example and they improved the life expectancy of a large cohort of patients. Research on new biomarkers, therefore, is always current and important. AXL, a member of the TYRO-3, AXL and MER (TAM) subfamily, is, today, considered a predictive and prognostic biomarker in many tumor contexts, primarily breast cancer. Its oncogenic implications make it an ideal target for the development of new pharmacological agents; moreover, its recent role as immune-modulator makes AXL particularly attractive to researchers involved in the study of interactions between cancer and the tumor microenvironment (TME). All these peculiarities characterize AXL as compared to other members of the TAM family. In this review, we will illustrate the biological role played by AXL in breast tumor cells, highlighting its molecular and biological features, its involvement in tumor progression and its implication as a target in ongoing clinical trials. Full article

Estrogen nuclear receptors, represented by the canonical forms ERα66 and ERβ1, are the main mediators of the estrogen-dependent pathophysiology in mammals. However, numerous isoforms have been identified, stimulating unconventional estrogen response pathways leading to complex cellular and tissue responses. The estrogen receptor variant, ERα36, was cloned in 2005 and is mainly described in the literature to be involved in the progression of mammary tumors and in the acquired resistance to anti-estrogen drugs, such as tamoxifen. In this review, we will first specify the place that ERα36 currently occupies within the diversity of nuclear and membrane estrogen receptors. We will then report recent data on the impact of ERα36 expression and/or activity in normal breast and testicular cells, but also in different types of tumors including mammary tumors, highlighting why ERα36 can now be considered as a marker of malignancy. Finally, we will explain how studying the regulation of ERα36 expression could provide new clues to counteract resistance to cancer treatments in hormone-sensitive tumors. Full article

Several studies show that triple-negative breast cancer (TNBC) patients have the lowest vitamin D concentration among all breast cancer types, suggesting that this vitamin may induce a protective effect against TNBC. This effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D), can be attributed to its potential to modulate proliferation, differentiation, apoptosis, inflammation, angiogenesis, invasion and metastasis and is supported by many in vitro and animal studies, but its exact mechanism is poorly known. In a fraction of TNBCs that harbor mutations that cause the loss of function of the DNA repair-associated breast cancer type 1 susceptibility (BRCA1) gene, 1,25(OH)2D may induce protective effects by activating its receptor and inactivating cathepsin L-mediated degradation of tumor protein P53 binding protein 1 (TP53BP1), preventing deficiency in DNA double-strand break repair and contributing to genome stability. Similar effects can be induced by the interaction of 1,25(OH)2D with proteins of the growth arrest and DNA damage-inducible 45 (GADD45) family. Further studies on TNBC cell lines with exact molecular characteristics and clinical trials with well-defined cases are needed to determine the mechanism of action of vitamin D in TNBC to assess its preventive and therapeutic potential. Full article

In locally advanced (LA) breast cancer (BC), neoadjuvant treatments have led to major achievements, which hold particular relevance in HER2-positive and triple-negative BC. Conversely, their role in hormone receptor positive (HR+), hormone epidermal growth factor 2 negative (HER2-) BC is still under debate, mainly due to the generally low rates of pathological complete response (pCR) and lower accuracy of pCR as predictors of long-term outcomes in this patient subset. While administration of neoadjuvant chemotherapy (NCT) in LA, HR+, HER2- BC patients is widely used in clinical practice, neoadjuvant endocrine therapy (NET) still retains an unfulfilled potential in the management of these subgroups, particularly in elderly and unfit patients. In addition, NET has gained a central role as a platform to test new drugs and predictive biomarkers in previously untreated patients. We herein present historical data regarding Tamoxifen and/or Aromatase Inhibitors and a debate on recent evidence regarding agents such as CDK4/6 and PI3K/mTOR inhibitors in the neoadjuvant setting. We also discuss key issues concerning the optimal treatment length, appropriate comparisons with NCT efficacy and use of NET in premenopausal patients. Full article

Over the last decades a renewed interest in n−3 very long polyunsaturated fatty acids (PUFAs), derived mainly from fish oils in the human diet, has been observed because of their potential effects against cancer diseases, including breast carcinoma. These n−3 PUFAs mainly consist of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that, alone or in combination with anticancer agents, induce cell cycle arrest, autophagy, apoptosis, and tumor growth inhibition. A large number of molecular targets of n−3 PUFAs have been identified and multiple mechanisms appear to underlie their antineoplastic activities. Evidence exists that EPA and DHA also elicit anticancer effects by the conversion to their corresponding ethanolamide derivatives in cancer cells, by binding and activation of different receptors and distinct signaling pathways. Other conjugates with serotonin or dopamine have been found to exert anti-inflammatory activities in breast tumor microenvironment, indicating the importance of these compounds as modulators of tumor epithelial/stroma interplay. The objective of this review is to provide a general overview and an update of the current n−3 PUFA derivative research and to highlight intriguing aspects of the potential therapeutic benefits of these low-toxicity compounds in breast cancer treatment and care. Full article