Recent Advances in Breast Cancer Research

This Special Issue (SI), titled "Recent Advances in Breast Cancer Research", covers 12 research articles and 1 communication in the field of breast cancer (BC) research [...].

A case series-based exploration of multi-regional expression heterogeneity in triple-negative breast cancer patients • Clinical study: 10 TNBC patients; RNA sequencing analysis of 2-4 regions per tumor; • Gene expression signatures were categorized into three types of variability: between-patient (108 genes); between patient, across region (183 genes); and within-patient, between-region (778 genes) variability; • Extensive intertumoral heterogeneity and regional ITH in gene expression and image-derived features in TNBC were determined.
Prolonged exposure to simulated microgravity changes release of small extracellular vesicle in breast cancer cells • Supernatants of MCF-7 BC cells, which were harvested after 5 or 10 days of simulated microgravity, were created via a random positioning machine (RPM); • Substantial increase in released vesicles following RPM exposure at both time points were recorded; • Altered distribution of subpopulations and their surface protein expression were recorded; • Minimal changes were noted between the time points, thus pointing to early adaption. Research Article [17] Piasna-Słupecka E. et al.
Young shoots of red beet and the root at full maturity inhibit proliferation and induce apoptosis in breast cancer cell lines • Effects of juice from young shoots of beetroot compared to juice from root at full maturity on human BC and normal cells; • Juice from young shoots (native and digested form) was a significantly stronger inhibitor of the proliferation of MCF-7 and MDA-MB-231 BC cells than the native and digested juice from red beetroot; • Regardless of the juice type, a significantly greater antiproliferative effect was measured in estrogen-dependent MCF-7 than in MDA-MB-231 BC cells; • All analyzed types of beetroot juice, as well as juice from young shoots and the root, were subjected to digestion and absorption, exerting antiproliferative and apoptotic effects on both BC cell types. A gene expression-profiling-based approach led to further stratification of FELs.
Communication [12] Fischer et al. [7] investigated 47 patients with metastatic BC and reported that the expression levels of circulating miR-200 family members were significantly increased during disease progression, which was predictive of circulating tumor cell (CTC) status. Both elevated CTCs and increased circulation of miR-200 content in blood plasma were associated with reduced OS and PFS. These factors are promising biomarkers for optimizing the clinical management of metastatic BC [7]. Another study investigated tumor specimens from 10 triple-negative breast cancer (TNBC) patients [8]. This BC type exhibited profound intratumoral heterogeneity. Therefore, single biopsy specimens may show only a portion of genetic aberrations of the entire tumor [8]. Genetic aberrations are involved in various cancer-specific biological processes, like tumorigenicity, induction of cell signaling, senescence, angiogenesis, migration, and response to treatment [8]. The authors concluded that medications used on the basis of the molecular profile of diagnostic biopsies may fail to remove the tumor, thus resulting in tumor recurrence. Knowledge of the molecular mechanisms that drive intratumoral heterogeneity in TNBC supports the development of new therapeutic targets.
A further article reports the results of a retrospective histological analysis of 113 cases of male BC, focusing on sTILs and programmed cell death ligand-1 (PD-L1) expression [9]. The authors demonstrated that a subset of male BC patients harbors an immunological environment characterized by an increase in sTILs with PD-L1 expression. Male BC are not only ER related and endocrine dependent, but also frequently HER2 low. These patients may benefit from immune checkpoint inhibitor therapy. In addition, frequent HER2-low status provides new options for anti-HER2 therapy in male BC patients [9].
Sokolenko et al. [10] investigated 229 BC patients. Among these patients treated via neoadjuvant chemotherapy (NACT) were 25 BRCA1 carriers and 204 women without recurrent BRCA1 alterations. NACT often results in a pathologic complete response (pCR). The authors found a lack of visible tumor cells in the post-NACT tumor bed to be a reliable indicator of the complete elimination of transformed clones [10].
Zaib et al. [11] investigated histological specimens of 97 patients with TNBC, showing that CD22 is highly expressed in this tumor type. The authors suggest that CD22 is a suitable prognostic biomarker in TNBC patients [11].
A further human study published as a communication focused on gene expression profiling of fibro-epithelial lesions (FELs) in the breast [12]. The authors studied the expression of 750 tumor-related genes in 34 FELs (5 fibroadenomas (FAs), 9 cellular FAs, 9 benign phyllodes tumors (PTs), 7 borderline PTs, and 4 malignant PTs). The overall gene expression profiles of benign PTs, cellular FAs, and FAs were similar. Borderline and benign PTs only slightly differed, whereas greater difference was detected between borderline and malignant PTs. This gene-expression-profiling-based approach supports further stratification of FELs, thus improving understanding of pathogenesis and diagnosis of BC [12].
Two studies focused on BC cells exposed to simulated microgravity conditions using an RPM, which is a device designed to create conditions of weightlessness on Earth [13,14]. Known microgravity-induced changes in human cancer cells include alterations in the cytoskeleton and changes in the ECM, adhesion, migration, differentiation, proliferation, survival, and apoptosis [20]. Differential expression of various genes and protein production and secretion were reported in benign and malignant cells [21][22][23].
Wise et al. [13] analyzed the supernatants of MCF-7 BC cells in order to determine extracellular vesicles (EVs). The cells had been exposed to an RPM for 5 or 10 days. A clear rise in released vesicles following RPM exposure was measured at both time points. Moreover, changes in the distribution of subpopulations related to surface protein expression were reported. Studying BC cells under microgravity led to an improved in vitro model that focused on changes in small EVs. Cancer research in space will extend our knowledge of cell communication in the tumor microenvironment and contribute to finding new therapies for BC [13].
Another microgravity experiment studied MCF-7 and MDA-MB-231 BC cells for 14 days using an RPM [14]. Both cell types grew in one of two phenotype forms: (1) adherent two-dimensional monolayers or (2) three-dimensional multicellular spheroids (MCSs). ERK1, AKT1, MAPK14, EGFR, CTNNA1, CTNNB1, ITGB1, COL4A5, ACTB, and TUBB genes of MCSs were differentially regulated in both cell lines. Bioinformatic analyses revealed a positive association between the real metastatic microtumor environment and MCSs regarding EGF/MAP signaling, focal adhesion, cytoskeleton, and the ECM, depending on the BC type. This long-term investigation improved pre-existing knowledge of tumor spheroid formation and growth [14].
The third cell culture study applied different types of BC cells and reported distinct roles for the Grainyhead-like 2 (GRHL2) gene in luminal and basal BC [15]. GRHL2 gene silencing performed via a mouse model revealed a decrease in primary tumor growth and reduced the number and size of lung colonies. Altogether, GRHL2 influences growth and motility. It is negatively associated with patient survival and growth suppression.
Meligova et al. [16] conducted a pharmacological study that investigated MCF-7 BC cells and their responses to antiestrogens and retinoids [16]. The authors showed that ERβ1 is a marker of responsiveness; in contrast, ERβ2 was shown to be an indicator of MCF7 cells' resistance to antiestrogens alone and in combination with all-trans retinoic acid (ATRA) [16]. The authors concluded that the five unique hub genes (PPARG, HIPK2, ZFP36L1, HMGB2, and ALDH1A3) create a gene expression signature that specified the therapeutic response of ERβ1-expressing and ERα-positive BC cells to 4-hydroxytamoxifen and ATRA therapy [16].
Moreover, a BC cell study showed that NUF2 promotes BC development and, thus, serves as a new tumor stem cell indicator [17]. The findings of this study were as follows: the overexpressed NUF2 upregulated the proliferation and tumor stemness ability of BC cell lines MCF-7 and Hs-578T [17].
Piasna-Słupecka et al. [18] found that the young shoots of red beet are a richer source of total polyphenols that have anti-carcinogenic properties and exhibit higher antioxidant activity. The polyphenolic profile of the juice from young shoots of beetroot and the apoptosis mechanisms induced by subjecting the juices to in vitro gastrointestinal digestion and absorption were studied [18]. The authors demonstrated the antiproliferative and apoptotic effects of the evaluated types of beetroot juice, in particular those made of young shoots or roots that were subjected to the process of digestion and absorption in an in vitro gastrointestinal tract model, against BC cells [18].
A combined cell culture and animal study (mice) investigated CCL2's role in mediating stromal interactions [19]. THP-1-derived macrophages and mammary fibroblasts were cocultured for 72 h, which induced an M2 phenotype and a rise in CCL2 gene expression. Mice that overexpressed CCL2 in the mammary glands were analyzed for global gene expression via RNAseq, showing upregulation of cancer-associated gene pathways. The CCL2-overexpressing mice showed enhanced macrophage infiltration and tumorigenesis [19].
Taken together, these 13 publications demonstrate novel findings in the field of BC research. The authors investigated several genes and molecular pathways, increasing our understanding of BC to aid improved diagnosis and the development of novel therapies. Studies that applied modern molecular biological approaches and bioinformatic analyses were published in this SI.
I wish to thank all of the authors who contributed to this SI. I am convinced that application of modern molecular biological technologies, together with a personalized medicine, will enable prevention and diagnosis of and new therapies for BC. Currently, many investigations are applying OMICS technologies and bioinformatics to identify new proteins that may serve as new mortality-decreasing drug targets or detect novel biomarkers that aid improved diagnosis of BC. Funding: D.G. was funded by Deutsches Zentrum für Luft-und Raumfahrt (DLR), BMWK, projects 50WB2219 and 50WK2270G.
Acknowledgments: I would like to thank Herbert Schulz, Otto von Guericke University Magdeburg, Germany, for his help with EndNote and making important suggestions.

Conflicts of Interest:
The author declares no conflict of interest.