Lipid Droplet Biosynthesis Impairment through DGAT2 Inhibition Sensitizes MCF7 Breast Cancer Cells to Radiation

Breast cancer is the most frequent cancer in women worldwide and late diagnosis often adversely affects the prognosis of the disease. Radiotherapy is commonly used to treat breast cancer, reducing the risk of recurrence after surgery. However, the eradication of radioresistant cancer cells, including cancer stem cells, remains the main challenge of radiotherapy. Recently, lipid droplets (LDs) have been proposed as functional markers of cancer stem cells, also being involved in increased cell tumorigenicity. LD biogenesis is a multistep process requiring various enzymes, including Diacylglycerol acyltransferase 2 (DGAT2). In this context, we evaluated the effect of PF-06424439, a selective DGAT2 inhibitor, on MCF7 breast cancer cells exposed to X-rays. Our results demonstrated that 72 h of PF-06424439 treatment reduced LD content and inhibited cell migration, without affecting cell proliferation. Interestingly, PF-06424439 pre-treatment followed by radiation was able to enhance radiosensitivity of MCF7 cells. In addition, the combined treatment negatively interfered with lipid metabolism-related genes, as well as with EMT gene expression, and modulated the expression of typical markers associated with the CSC-like phenotype. These findings suggest that PF-06424439 pre-treatment coupled to X-ray exposure might potentiate breast cancer cell radiosensitivity and potentially improve the radiotherapy effectiveness.     

In Ovarian Cancer Multicellular Spheroids,Platelet Releasate Promotes Growth,Expansion of ALDH+ and CD133+ Cancer Stem Cells,and Protection against the Cytotoxic Effects of Cisplatin,Carboplatin and Paclitaxel

A high platelet count is associated with a poor prognosis in ovarian cancer (OvCa). Despite good clinical responses with platinating agents in combination with taxanes, numerous OvCa patients relapse due to chemotherapy resistance. Here, we report that treatment of OvCa cells A2780, OVCAR5 and MDAH with releasate from activated platelets (PR) promoted multicellular tumor spheroid (MCTS) formation. These OvCa-MCTSs had increased percentages of CD133+ and aldehyde dehydrogenase (ALDH)+ cells, bona fide markers of OvCa cancer stem cells (CSCs). PR increased OVCAR5- and MDAH-MCTS viability and decreased the cytotoxic and pro-apoptotic effects of paclitaxel, cisplatin and carboplatin. PR increased the volume of spontaneously formed OVCAR8-MCTSs and counteracted their size reduction due to cisplatin, carboplatin and paclitaxel treatment. PR promoted the survival of ALDH+ and CD133+ OvCa cells during cisplatin, carboplatin and paclitaxel treatment. In conclusion, molecules and growth factors released by activated platelets (EGF, PDGF, TGF-β, IGF and CCL5) may protect tumor cells from chemotherapy by promoting the expansion of ALDH+ and CD133+ OvCa-CSCs, favoring drug resistance and tumor relapse.     

The Effect of Tau and Taxol on Polymerization of MCF7 Microtubules In Vitro

Overexpression of Tau protein in breast cancer cells is identified as an indicator for potential resistance to taxane-based therapy. As reported findings have been obtained mostly from clinical studies, the undetermined underlying mechanism of such drug resistance needs to be thoroughly explored through comprehensive in vitro evaluations. Tau and Taxol bind to the beta tubulin site in microtubules’ structure. This is of particular interest in breast cancer, as microtubules of these cancer cells are structurally distinct from some other microtubules, such as neuronal microtubules, due to their unique beta tubulin isotype distribution. The observed changes in the in vitro polymerization of breast cancer microtubules, and the different function of some molecular motors along them, leave open the possibility that the drug resistance mechanism can potentially be associated with different responses of these microtubules to Tau and Taxol. We carried out a series of parallel experiments to allow comparison of the in vitro dual effect of Tau and Taxol on the polymerization of MCF7 microtubules. We observed a concentration-dependent demotion-like alteration in the self-polymerization kinetics of Tau-induced MCF7 microtubules. In contrast, microtubules polymerized under the simultaneous effects of Tau and Taxol showed promoted assembly as compared with those observed in Tau-induced microtubules. The analysis of our data obtained from the length of MCF7 microtubules polymerized under the interaction with Tau and Taxol in vitro suggests that the phenomenon known as drug resistance in microtubule-targeted drugs such as Taxol may not be directly linked to the different responses of microtubules to the drug. The effect of the drug may be mitigated due to the simultaneous interactions with other microtubule-associated proteins such as Tau protein. The observed regulatory effect of Tau and Taxol on the polymerization of breast cancer microtubules in vitro points to additional evidence for the possible role of tubulin isotypes in microtubules’ functions.     

A Novel 3D Culture Scaffold to Shorten Development Time for Multicellular Tumor Spheroids

Multicellular tumor spheroids and tumoroids are considered ideal in vitro models that reflect the features of the tumor microenvironment. Biomimetic components resembling the extracellular matrix form scaffolds to provide structure to 3-dimensional (3D) culture systems, supporting the growth of both spheroids and tumoroids. Although Matrigel has long been used to support 3D culture systems, batch variations, component complexity, and the use of components derived from tumors are complicating factors. To address these issues, we developed the ACD 3D culture system to provide better control and consistency. We evaluated spheroid and tumoroid formation using the ACD 3D culture system, including the assessment of cell viability and cancer marker expression. Under ACD 3D culture conditions, spheroids derived from cancer cell lines exhibited cancer stem cell characteristics, including a sphere-forming size and the expression of stem cell marker genes. The ACD 3D culture system was also able to support patient-derived primary cells and organoid cell cultures, displaying adequate cell growth, appropriate morphology, and resistance to oxaliplatin treatment. These spheroids could also be used for drug screening purposes. In conclusion, the ACD 3D culture system represents an efficient tool for basic cancer research and therapeutic development.     

SATB1-Mediated Upregulation of the Oncogenic Receptor Tyrosine Kinase HER3 Antagonizes MET Inhibition in Gastric Cancer Cells

MET-amplified gastric cancer cells are extremely sensitive to MET inhibition in vitro, whereas clinical efficacy of MET inhibitors is disappointing. The compensatory activation of other oncogenic growth factor receptors may serve as an underlying mechanism of resistance. In this study, we analyzed the role of HER receptors, in particular HER3 and its ligand heregulin, in this respect. This also included the chromatin-organizer protein SATB1, as an established regulator of HER expression in other tumor entities. In a panel of MET-amplified gastric carcinoma cell lines, cell growth under anchorage-dependent and independent conditions was studied upon inhibitor treatment or siRNA-mediated knockdown. Expression analyses were performed using RT-qPCR, FACS, and immunoblots. Signal transduction was monitored via antibody arrays and immunoblots. As expected, MET inhibition led to a growth arrest and inhibition of MAPK signaling. Strikingly, however, this was accompanied by a rapid and profound upregulation of the oncogenic receptor HER3. This finding was determined as functionally relevant, since HER3 activation by HRG led to partial MET inhibitor resistance, and MAPK/Akt signaling was even found enhanced upon HRG+MET inhibitor treatment compared to HRG alone. SATB1 was identified as mediator of HER3 upregulation. Concomitantly, SATB1 knockdown prevented upregulation of HER3, thus abrogating the HRG-promoted rescue from MET inhibition. Taken together, our results introduce the combined HER3/MET inhibition as strategy to overcome resistance towards MET inhibitors.     

Divergence of Intracellular Trafficking of Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 3 in MCF-7 Breast Cancer Cells and MCF-7-Derived Stem Cell-Enriched Mammospheres

Breast cancer MCF-7 cell-line-derived mammospheres were shown to be enriched in cells with a CD44+/CD24– surface profile, consistent with breast cancer stem cells (BCSC). These BCSC were previously reported to express key sphingolipid signaling effectors, including pro-oncogenic sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1P3). In this study, we explored intracellular trafficking and localization of SphK1 and S1P3 in parental MCF-7 cells, and MCF-7 derived BCSC-enriched mammospheres treated with growth- or apoptosis-stimulating agents. Intracellular trafficking and localization were assessed using confocal microscopy and cell fractionation, while CD44+/CD24- marker status was confirmed by flow cytometry. Mammospheres expressed significantly higher levels of S1P3 compared to parental MCF-7 cells (p < 0.01). Growth-promoting agents (S1P and estrogen) induced SphK1 and S1P3 translocation from cytoplasm to nuclei, which may facilitate the involvement of SphK1 and S1P3 in gene regulation. In contrast, pro-apoptotic cytokine tumor necrosis factor α (TNFα)-treated MCF-7 cells demonstrated increased apoptosis and no nuclear localization of SphK1 and S1P3, suggesting that TNFα can inhibit nuclear translocation of SphK1 and S1P3. TNFα inhibited mammosphere formation and induced S1P3 internalization and degradation. No nuclear translocation of S1P3 was detected in TNFα-stimulated mammospheres. Notably, SphK1 and S1P3 expression and localization were highly heterogenous in mammospheres, suggesting the potential for a large variety of responses. The findings provide further insights into the understanding of sphingolipid signaling and intracellular trafficking in BCs. Our data indicates that the inhibition of SphK1 and S1P3 nuclear translocation represents a novel method to prevent BCSCs proliferation.     

Oxidative Stress-Mediated Apoptosis Induced by Ethanolic Mango Seed Extract in Cultured Estrogen Receptor Positive Breast Cancer MCF-7 Cells

Breast cancer has become a global health issue requiring huge expenditures for care and treatment of patients. There is a need to discover newer cost-effective alternatives for current therapeutic regimes. Mango kernel is a waste product with potential as a source of anti-cancer phytochemicals, especially since it is non-toxic towards normal breast cell lines at concentrations for which it induces cell death in breast cancer cells. In this study, the anti-cancer effect of mango kernel extract was determined on estrogen receptor-positive human breast carcinoma (MCF-7) cells. The MCF-7 cells were cultured and treated with 5, 10 and 50 μg/mL of mango kernel extract for 12 and 24 h. In response to treatment, there were time- and dose-dependent increases in oxidative stress markers and pro-apoptotic factors; Bcl-2-like protein 4 (BAX), p53, cytochrome c and caspases (7, 8 and 9) in the MCF-7 cells treated with the extract. At the same time, there were decreases in pro-survival markers (Bcl-2 and glutathione) as the result of the treatments. The changes induced in the MCF-7 cells by mango kernel extract treatment suggest that the extract can induce cancer cell apoptosis, likely via the activation of oxidative stress. These findings need to be evaluated further to determine whether mango kernel extract can be developed as an anti-breast cancer agent.     

Cell Fusion of Mesenchymal Stem/Stromal Cells and Breast Cancer Cells Leads to the Formation of Hybrid Cells Exhibiting Diverse and Individual (Stem Cell) Characteristics

Cancer is one of the most common diseases worldwide, and treatment bears many challenges such as drug and radioresistance and formation of metastases. These difficulties are due to tumor heterogeneity, which has many origins. One may be cell fusion, a process that is relevant in both physiological (e.g., wound healing) and pathophysiological (cancer and viral infection) processes. In this study, we examined if cell fusion between mesenchymal stem/stromal cells (MSCs) and breast cancer (BC) cells occurs and if newly generated hybrid cells may exhibit cancer stem/initiating cell (CS/IC) characteristics. Therefore, several methods such as mammosphere assay, AldeRed assay, flow cytometry (CD24, CD44, CD104) and Western blot analysis (of epithelial to mesenchymal transition markers such as SNAIL, SLUG and Twist) were applied. In short, four different hybrid clones, verified by short tandem repeat (STR) analysis, were analyzed; each expressed an individual phenotype that seemed not to be explicitly related to either a more stem cell or cancer cell phenotype. These results show that cancer cells and MSCs are able to fuse spontaneously in vitro, thereby giving rise to hybrid cells with new properties, which likely indicate that cell fusion may be a trigger for tumor heterogeneity.     

The Impact of Human Lipoaspirate and Adipose Tissue-Derived Stem Cells Contact Culture on Breast Cancer Cells: Implications in Breast Reconstruction

Background: Autologous fat transfer in the form of lipoaspirates for the reconstruction of the breast after breast cancer surgery is a commonly used procedure in plastic surgery. However, concerns regarding the oncologic risk of nutrient-rich fat tissue are widely debated. Previous studies have primarily focused on studying the interaction between adipose-derived stem cells (ASCs) and breast cancer cells. Methods: In this study, we performed a comprehensive analysis of the paracrine- and contact-based interactions between lipoaspirates, ASCs and breast cancer cell lines. An inverted flask culture method was used to study the contact-based interaction between lipoaspirates and breast cancer cells, while GFP-expressing breast cancer cell lines were generated to study the cell–cell contact interaction with ASCs. Three different human breast cancer cell lines, MCF-7, MDA-MB-231 and BT-474, were studied. We analyzed the impact of these interactions on the proliferation, cell cycle and epithelial-to-mesenchymal (EMT) transition of the breast cancer cells. Results: Our results revealed that both lipoaspirates and ASCs do not increase the proliferation rate of the breast cancer cells either through paracrine- or contact-dependent interactions. We observed that lipoaspirates selectively inhibit the proliferation of MCF-7 cells in contact co-culture, driven by the retinoblastoma (Rb) protein activity mediating cell cycle arrest. Additionally, ASCs inhibited MDA-MB-231 breast cancer cell proliferation in cell–cell contact-dependent interactions. Quantitative real-time PCR revealed no significant increase in the EMT-related genes in breast cancer cells upon co-culture with ASCs. Conclusion: In conclusion, this study provides evidence of the non-oncogenic character of lipoaspirates and supports the safety of clinical fat grafting in breast reconstruction after oncological surgical procedures. In vivo studies in appropriate animal models and long-term post-operative clinical data from patients are essential to reach the final safety recommendations.     

Dendritic Cells Pulsed with Cytokine-Adjuvanted Tumor Membrane Vesicles Inhibit Tumor Growth in HER2-Positive and Triple Negative Breast Cancer Models

Dendritic cells (DCs) are the most effective antigen presenting cells for the development of T cell responses. The only FDA approved DC-based immunotherapy to date is Sipuleucel-T, which utilizes a fusion protein to stimulate DCs ex vivo with GM-CSF and simultaneously deliver the antigen PAP for prostate cancer. This approach is restricted by the breadth of immunity elicited to a single antigen, and to cancers that have a defined tumor associated antigen. Other multi-antigen approaches have been restricted by poor efficacy of vaccine adjuvants. We have developed a vaccine platform that consists of autologous DCs pulsed with cytokine-adjuvanted tumor membrane vesicles (TMVs) made from tumor tissue, that encapsulate the antigenic landscape of individual tumors. Here we test the efficacy of DCs pulsed with TMVs incorporated with glycolipid-anchored immunostimulatory molecules (GPI-ISMs) in HER2-positive and triple negative breast cancer murine models. Pulsing of DCs with TMVs containing GPI-ISMs results in superior uptake of vesicles, DC activation and cytokine production. Adaptive transfer of TMV-pulsed DCs to tumor bearing mice results in the inhibition of tumor growth, reduction in lung metastasis, and an increase in immune cell infiltration into the tumors. These observations suggest that DCs pulsed with TMVs containing GPI-GM-CSF and GPI-IL-12 can be further developed to be used as a personalized immunotherapy platform for cancer treatment.     

Connexin46 Expression Enhances Cancer Stem Cell and Epithelial-to-Mesenchymal Transition Characteristics of Human Breast Cancer MCF-7 Cells

Connexins (Cxs) are a family of proteins that form two different types of ion channels: hemichannels and gap junction channels. These channels participate in cellular communication, enabling them to share information and act as a synchronized syncytium. This cellular communication has been considered a strong tumor suppressor, but it is now recognized that some type of Cxs can be pro-tumorigenic. For example, Cx46 expression is increased in human breast cancer samples and correlates with cancer stem cell (CSC) characteristics in human glioma. Thus, we explored whether Cx46 and glioma cells, can set up CSC and epithelial-to-mesenchymal transition (EMT) properties in a breast cancer cell line. To this end, we transfected MCF-7 cells with Cx46 attached to a green fluorescent protein (Cx46GFP), and we determined how its expression orchestrates both the gene-expression and functional changes associated with CSC and EMT. We observed that Cx46GFP increased Sox2, Nanog, and OCT4 mRNA levels associated with a high capacity to form monoclonal colonies and tumorspheres. Similarly, Cx46GFP increased the mRNA levels of n-cadherin, Vimentin, Snail and Zeb1 to a higher migratory and invasive capacity. Furthermore, Cx46GFP transfected in MCF-7 cells induced the release of higher amounts of VEGF, which promoted angiogenesis in HUVEC cells. We demonstrated for the first time that Cx46 modulates CSC and EMT properties in breast cancer cells and thus could be relevant in the design of future cancer therapies.     

Anti-Epidermal Growth Factor Receptor (EGFR) Antibodies Overcome Resistance of Ovarian Cancer Cells to Targeted Therapy and Natural Cytotoxicity

The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.     

The Effects of Genistein at Different Concentrations on MCF-7 Breast Cancer Cells and BJ Dermal Fibroblasts

This study aimed to evaluate the safety and potential use of soy isoflavones in the treatment of skin problems, difficult-to-heal wounds and postoperative scars in women after the oncological treatment of breast cancer. The effects of different concentrations of genistein as a representative of soy isoflavonoids on MCF-7 tumor cells and BJ skin fibroblasts cultured in vitro were assessed. Genistein affects both healthy dermal BJ fibroblasts and cancerous MCF-7 cells. The effect of the tested isoflavonoid is closely related to its concentration. High concentrations of genistein destroy MCF-7 cancer cells, regardless of the exposure time, with a much greater effect on reducing cancer cell numbers at longer times (48 h). Lower concentrations of genistein (10 and 20 μM) increase the abundance of dermal fibroblasts. However, higher concentrations of genistein (50 μM and higher) are detrimental to fibroblasts at longer exposure times (48 h). Our studies indicate that although genistein shows high potential for use in the treatment of skin problems, wounds and surgical scars in women during and after breast cancer treatment, it is not completely safe. Introducing isoflavonoids to treatment requires further research into their mechanisms of action at the molecular level, taking into account genetic and immunological aspects. It is also necessary to conduct research in in vivo models, which will allow for eliminating adverse side effects of therapy.