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.     

Pancreatic Cancer and Cellular Senescence: Tumor Microenvironment under the Spotlight

Pancreatic ductal adenocarcinoma (PDAC) has one of the most dismal prognoses of all cancers due to its late manifestation and resistance to current therapies. Accumulating evidence has suggested that the malignant behavior of this cancer is mainly influenced by the associated strongly immunosuppressive, desmoplastic microenvironment and by the relatively low mutational burden. PDAC develops and progresses through a multi-step process. Early in tumorigenesis, cancer cells must evade the effects of cellular senescence, which slows proliferation and promotes the immune-mediated elimination of pre-malignant cells. The role of senescence as a tumor suppressor has been well-established; however, recent evidence has revealed novel pro-tumorigenic paracrine functions of senescent cells towards their microenvironment. Understanding the interactions between tumors and their microenvironment is a growing research field, with evidence having been provided that non-tumoral cells composing the tumor microenvironment (TME) influence tumor proliferation, metabolism, cell death, and therapeutic resistance. Simultaneously, cancer cells shape a tumor-supportive and immunosuppressive environment, influencing both non-tumoral neighboring and distant cells. The overall intention of this review is to provide an overview of the interplay that occurs between senescent and non-senescent cell types and to describe how such interplay may have an impact on PDAC progression. Specifically, the effects and the molecular changes occurring in non-cancerous cells during senescence, and how these may contribute to a tumor-permissive microenvironment, will be discussed. Finally, senescence targeting strategies will be briefly introduced, highlighting their potential in the treatment of PDAC.     

CTI-2 Inhibits Metastasis and Epithelial-Mesenchymal Transition of Breast Cancer Cells by Modulating MAPK Signaling Pathway

Although some breast cancer patients die due to tumor metastasis rather than from the primary tumor, the molecular mechanism of metastasis remains unclear. Therefore, it is necessary to inhibit breast cancer metastasis during cancer treatment. In this case, after designing and synthesizing CTI-2, we found that CTI-2 treatment significantly reduced breast cancer cell metastasis in vivo and in vitro. Notably, with the treatment of CTI-2 in breast cancer cells, the expression level of E-cadherin increased, while the expression level of N-cadherin and vimentin decreased. In addition, after CTI-2 treatment, those outflow levels for p-ERK, p-p38, and p-JNK diminished, while no significant changes in the expression levels of ERK, JNK, or p38 were observed. Our conclusion suggested that CTI-2 inhibits the epithelial-mesenchymal transition (EMT) of breast carcinoma cells by inhibiting the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, thereby inhibiting the metastasis of breast tumor cells. Therefore, we believe that CTI-2 is another candidate for breast tumor medication.     

Role of the NUDT Enzymes in Breast Cancer

Despite global research efforts, breast cancer remains the leading cause of cancer death in women worldwide. The majority of these deaths are due to metastasis occurring years after the initial treatment of the primary tumor and occurs at a higher frequency in hormone receptor-positive (Estrogen and Progesterone; HR+) breast cancers. We have previously described the role of NUDT5 (Nudix-linked to moiety X-5) in HR+ breast cancer progression, specifically with regards to the growth of breast cancer stem cells (BCSCs). BCSCs are known to be the initiators of epithelial-to-mesenchyme transition (EMT), metastatic colonization, and growth. Therefore, a greater understanding of the proteins and signaling pathways involved in the metastatic process may open the door for therapeutic opportunities. In this review, we discuss the role of NUDT5 and other members of the NUDT family of enzymes in breast and other cancer types. We highlight the use of global omics data based on our recent phosphoproteomic analysis of progestin signaling pathways in breast cancer cells and how this experimental approach provides insight into novel crosstalk mechanisms for stratification and drug discovery projects aiming to treat patients with aggressive cancer.     

Resveratrol Sensitizes Tamoxifen in Antiestrogen-Resistant Breast Cancer Cells with Epithelial-Mesenchymal Transition Features

Tamoxifen resistance remains to be a huge obstacle in the treatment of hormone-dependent breast cancer, and this therefore highlights the dire need to explore the underlying mechanisms. The epithelial-mesenchymal transition (EMT) is a molecular process through which an epithelial cell transfers into a mesenchymal phenotype. Roles of EMT in embryo development, cancer invasion and metastasis have been extensively reported. Herein, we established tamoxifen-resistant MCF-7/TR breast cancer cells and showed that MCF-7/TR cells underwent EMT driven by enhanced endogenous TGF-β/Smad signaling. Ectopic supplement of TGF-β promoted in MCF-7 cells a mesenchymal and resistant phenotype. In parallel, we demonstrated that resveratrol was capable of synergizing with tamoxifen and triggering apoptosis in MCF-7/TR cells. Further Western blot analysis indicated that the chemosensitizing effects of resveratrol were conferred with its modulation on endogenous TGF-β production and Smad phosphorylation. In particular, 50 μM resveratrol had minor effects on MCF-7/TR cell proliferation, but could significantly attenuate endogenous TGF-β production and the Smad pathway, ultimately leading to reversion of EMT. Collectively, our study highlighted distinct roles of EMT in tamoxifen resistance and resveratrol as a potential agent to overcome acquired tamoxifen resistance. The molecular mechanism of resveratrol chemosensitizing effects is, at least in part, TGF-β/Smad-dependent.     

Heterogeneous Manifestations of Epithelial–Mesenchymal Plasticity of Circulating Tumor Cells in Breast Cancer Patients

To date, there is indisputable evidence of significant CTC heterogeneity in carcinomas, in particular breast cancer. The heterogeneity of CTCs is manifested in the key characteristics of tumor cells related to metastatic progression – stemness and epithelial–mesenchymal (EMT) plasticity. It is still not clear what markers can characterize the phenomenon of EMT plasticity in the range from epithelial to mesenchymal phenotypes. In this article we examine the manifestations of EMT plasticity in the CTCs in breast cancer. The prospective study included 39 patients with invasive carcinoma of no special type. CTC phenotypes were determined by flow cytometry before any type of treatment. EMT features of CTC were assessed using antibodies against CD45, CD326 (EpCam), CD325 (N-cadherin), CK7, Snail, and Vimentin. Circulating tumor cells in breast cancer are characterized by pronounced heterogeneity of EMT manifestations. The results of the study indicate that the majority of heterogeneous CTC phenotypes (22 out of 24 detectable) exhibit epithelial–mesenchymal plasticity. The variability of EMT manifestations does not prevent intravasation. Co-expression of EpCAM and CK7, regardless of the variant of co-expression of Snail, N-cadherin, and Vimentin, are associated with a low number of CTCs. Intrapersonal heterogeneity is manifested by the detection of several CTC phenotypes in each patient. Interpersonal heterogeneity is manifested by various combinations of CTC phenotypes in patients (from 1 to 17 phenotypes).     

Silver Nanoparticles Modulate the Epithelial-to-Mesenchymal Transition in Estrogen-Dependent Breast Cancer Cells In Vitro

Silver nanoparticles (AgNPs) are frequently detected in many convenience goods, such as cosmetics, that are applied directly to the skin. AgNPs accumulated in cells can modulate a wide range of molecular pathways, causing direct changes in cells. The aim of this study is to assess the capability of AgNPs to modulate the metastasis of breast cancer cells through the induction of epithelial-to-mesenchymal transition (EMT). The effect of the AgNPs on MCF-7 cells was investigated via the sulforhodamine B method, the wound healing test, generation of reactive oxygen species (ROS), the standard cytofluorimetric method of measuring the cell cycle, and the expression of EMT marker proteins and the MTA3 protein via Western blot. To fulfill the results, calcium flux and HDAC activity were measured. Additionally, mitochondrial membrane potential was measured to assess the direct impact of AgNPs on mitochondria. The results indicated that the MCF-7 cells are resistant to the cytotoxic effect of AgNPs and have higher mobility than the control cells. Treatment with AgNPs induced a generation of ROS; however, it did not affect the cell cycle but modulated the expression of EMT marker proteins and the MTA3 protein. Mitochondrial membrane potential and calcium flux were not altered; however, the AgNPs did modulate the total HDAC activity. The presented data support our hypothesis that AgNPs modulate the metastasis of MCF-7 cells through the EMT pathway. These results suggest that AgNPs, by inducing reactive oxygen species generation, alter the metabolism of breast cancer cells and trigger several pathways related to metastasis.     

MMP1 and MMP11 Expression in Peripheral Blood Mononuclear Cells upon Their Interaction with Breast Cancer Cells and Fibroblasts

Tumor-infiltrating immune cells phenotype is associated with tumor progression. However, little is known about the phenotype of the peripheral blood mononuclear cells (PBMC) from breast cancer patients. We investigated MMP1 and MMP11 expression in PBMC from breast cancer patients and we analyzed gene expression changes upon their interaction with cancer cells and cancer-associated fibroblasts (CAF). We measured the impact of PBMC on proinflammatory gene expression in breast cancer cells, normal fibroblast (NF), and CAF and the impact on proliferation and invasiveness capacity of breast cancer cells. Gene expression of MMP1 and MMP11 in PBMC from breast cancer patients (n = 54) and control (n = 28); expression of IL1A, IL6, IL17, IFNβ, and NFĸB in breast cancer cell lines (MCF-7 and MDA-MB-231); and, additionally, IL10 and MMP11 in CAF and NF were analyzed by qRT-PCR before and after co-culture. Our results show the existence of a subpopulation of breast cancer patients (25.9%) with very high levels of MMP11 gene expression in PBMC. Also, gene expression of MMP1 and MMP11 increases in PBMC after co-culture with breast cancer cell lines, NF or CAF. PBMC from healthy or breast cancer patients induce an increased proliferation rate on MCF-7 and an increased invasiveness capacity of MDA-MB-231. Finally, we show a differential expression profile of inflammatory genes in NF and CAF when co-cultured with control or breast cancer PBMC. We have observed that MMPs’ expression in PBMC is regulated by the microenvironment, while the expression of inflammatory genes in NF or CAF is differentially regulated by PBMC. These findings confirm the importance of the crosstalk between stromal cells and suggest that PBMC would play a role in promoting aggressive tumor behavior.     

The Role of MicroRNAs in Breast Cancer Stem Cells

The concept of the existence of a subset of cancer cells with stem cell-like properties, which are thought to play a significant role in tumor formation, metastasis, resistance to anticancer therapies and cancer recurrence, has gained tremendous attraction within the last decade. These cancer stem cells (CSCs) are relatively rare and have been described by different molecular markers and cellular features in different types of cancers. Ten years ago, a novel class of molecules, small non-protein-coding RNAs, was found to be involved in carcinogenesis. These small RNAs, which are called microRNAs (miRNAs), act as endogenous suppressors of gene expression that exert their effect by binding to the 3′-untranslated region (UTR) of large target messenger RNAs (mRNAs). MicroRNAs trigger either translational repression or mRNA cleavage of target mRNAs. Some studies have shown that putative breast cancer stem cells (BCSCs) exhibit a distinct miRNA expression profile compared to non-tumorigenic breast cancer cells. The deregulated miRNAs may contribute to carcinogenesis and self-renewal of BCSCs via several different pathways and can act either as oncomirs or as tumor suppressive miRNAs. It has also been demonstrated that certain miRNAs play an essential role in regulating the stem cell-like phenotype of BCSCs. Some miRNAs control clonal expansion or maintain the self-renewal and anti-apoptotic features of BCSCs. Others are targeting the specific mRNA of their target genes and thereby contribute to the formation and self-renewal process of BCSCs. Several miRNAs are involved in epithelial to mesenchymal transition, which is often implicated in the process of formation of CSCs. Other miRNAs were shown to be involved in the increased chemotherapeutic resistance of BCSCs. This review highlights the recent findings and crucial role of miRNAs in the maintenance, growth and behavior of BCSCs, thus indicating the potential for novel diagnostic, prognostic and therapeutic miRNA-based strategies.     

MicroRNA Regulation of Breast Cancer Stemness

Recent advances in our understanding of breast cancer have demonstrated that cancer stem-like cells (CSCs, also known as tumor-initiating cell (TICs)) are central for progression and recurrence. CSCs are a small subpopulation of cells present in breast tumors that contribute to growth, metastasis, therapy resistance, and recurrence, leading to poor clinical outcome. Data have shown that cancer cells can gain characteristics of CSCs, or stemness, through alterations in key signaling pathways. The dysregulation of miRNA expression and signaling have been well-documented in cancer, and recent studies have shown that miRNAs are associated with breast cancer initiation, progression, and recurrence through regulating CSC characteristics. More specifically, miRNAs directly target central signaling nodes within pathways that can drive the formation, maintenance, and even inhibition of the CSC population. This review aims to summarize these research findings specifically in the context of breast cancer. This review also discusses miRNAs as biomarkers and promising clinical therapeutics, and presents a comprehensive summary of currently validated targets involved in CSC-specific signaling pathways in breast cancer.     

Multifunctional Role of Lipids in Modulating the Tumorigenic Properties of 4T1 Breast Cancer Cells

Tumor growth and progression are linked to an altered lipid metabolism in the tumor microenvironment (TME), including tumor cells and tumor-associated macrophages (TAMs). A growing number of lipid metabolism targeting drugs have shown efficacy in anti-tumor therapy. In addition, exogenously applied lipids and lipid analogues have demonstrated anti-tumor activities in several cancers, including breast cancer. In this study, we investigated the anti-tumor efficacies of the natural lipids palmitic acid (PA), sphingomyelin (SM), ceramide (Cer) and docosahexaenoic acid (DHA) on breast cancer cells. All tested lipids reduced the malignancy of breast cancer cells in vitro by impairing cell proliferation, migration and invasiveness. PA showed superior anti-tumor properties, as it additionally impaired cancer cell viability by inducing apoptosis, without affecting healthy cells. Co-culture experiments further demonstrated that Cer and PA reduced the immunosuppressive phenotype of M2 macrophages and the M2 macrophage-promoted the epithelial–mesenchymal transition (EMT) and migration of breast cancer cells. At the molecular level, this coincided with the up-regulation of E-cadherin. Our results highlight a powerful role for exogenously applied PA and Cer in reducing breast cancer tumorigenicity by simultaneously targeting cancer cells and M2 macrophages. Our findings support the notion that lipids represent alternative biocompatible therapeutic agents for breast cancer.     

Notch Signaling in Breast Tumor Microenvironment as Mediator of Drug Resistance

Notch signaling dysregulation encourages breast cancer progression through different mechanisms such as stem cell maintenance, cell proliferation and migration/invasion. Furthermore, Notch is a crucial driver regulating juxtracrine and paracrine communications between tumor and stroma. The complex interplay between the abnormal Notch pathway orchestrating the activation of other signals and cellular heterogeneity contribute towards remodeling of the tumor microenvironment. These changes, together with tumor evolution and treatment pressure, drive breast cancer drug resistance. Preclinical studies have shown that targeting the Notch pathway can prevent or reverse resistance, reducing or eliminating breast cancer stem cells. In the present review, we will summarize the current scientific evidence that highlights the involvement of Notch activation within the breast tumor microenvironment, angiogenesis, extracellular matrix remodeling, and tumor/stroma/immune system interplay and its involvement in mechanisms of therapy resistance.     

mTOR Activity and Autophagy in Senescent Cells,a Complex Partnership

Cellular senescence is a form of proliferative arrest triggered in response to a wide variety of stimuli and characterized by unique changes in cell morphology and function. Although unable to divide, senescent cells remain metabolically active and acquire the ability to produce and secrete bioactive molecules, some of which have recognized pro-inflammatory and/or pro-tumorigenic actions. As expected, this “senescence-associated secretory phenotype (SASP)” accounts for most of the non-cell-autonomous effects of senescent cells, which can be beneficial or detrimental for tissue homeostasis, depending on the context. It is now evident that many features linked to cellular senescence, including the SASP, reflect complex changes in the activities of mTOR and other metabolic pathways. Indeed, the available evidence indicates that mTOR-dependent signaling is required for the maintenance or implementation of different aspects of cellular senescence. Thus, depending on the cell type and biological context, inhibiting mTOR in cells undergoing senescence can reverse senescence, induce quiescence or cell death, or exacerbate some features of senescent cells while inhibiting others. Interestingly, autophagy—a highly regulated catabolic process—is also commonly upregulated in senescent cells. As mTOR activation leads to repression of autophagy in non-senescent cells (mTOR as an upstream regulator of autophagy), the upregulation of autophagy observed in senescent cells must take place in an mTOR-independent manner. Notably, there is evidence that autophagy provides free amino acids that feed the mTOR complex 1 (mTORC1), which in turn is required to initiate the synthesis of SASP components. Therefore, mTOR activation can follow the induction of autophagy in senescent cells (mTOR as a downstream effector of autophagy). These functional connections suggest the existence of autophagy regulatory pathways in senescent cells that differ from those activated in non-senescence contexts. We envision that untangling these functional connections will be key for the generation of combinatorial anti-cancer therapies involving pro-senescence drugs, mTOR inhibitors, and/or autophagy inhibitors.     

Targeting Breast Cancer Stem Cells Using Naturally Occurring Phytoestrogens

Breast cancer therapies have made significant strides in improving survival for patients over the past decades. However, recurrence and drug resistance continue to challenge long-term recurrence-free and overall survival rates. Mounting evidence supports the cancer stem cell model in which the existence of a small population of breast cancer stem cells (BCSCs) within the tumor enables these cells to evade conventional therapies and repopulate the tumor, giving rise to more aggressive, recurrent tumors. Thus, successful breast cancer therapy would need to target these BCSCs, as well the tumor bulk cells. Since the Women’s Health Initiative study reported an increased risk of breast cancer with the use of conventional hormone replacement therapy in postmenopausal women, many have turned their attention to phytoestrogens as a natural alternative. Phytoestrogens are plant compounds that share structural similarities with human estrogens and can bind to the estrogen receptors to alter the endocrine responses. Recent studies have found that phytoestrogens can also target BCSCs and have the potential to complement conventional therapy eradicating BCSCs. This review summarized the latest findings of different phytoestrogens and their effect on BCSCs, along with their mechanisms of action, including selective estrogen receptor binding and inhibition of molecular pathways used by BCSCs. The latest results of phytoestrogens in clinical trials are also discussed to further evaluate the use of phytoestrogen in the treatment and prevention of breast cancer.     

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.     

Dichloroacetate Radiosensitizes Hypoxic Breast Cancer Cells

Mitochondrial metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways can potentially sensitize tumors with limited treatment options, such as triple-negative breast cancer (TNBC), to chemo- and/or radiotherapy. Dichloroacetate (DCA) is a specific inhibitor of the pyruvate dehydrogenase kinase (PDK), which leads to enhanced reactive oxygen species (ROS) production. ROS are the primary effector molecules of radiation and an increase hereof will enhance the radioresponse. In this study, we evaluated the effects of DCA and radiotherapy on two TNBC cell lines, namely EMT6 and 4T1, under aerobic and hypoxic conditions. As expected, DCA treatment decreased phosphorylated pyruvate dehydrogenase (PDH) and lowered both extracellular acidification rate (ECAR) and lactate production. Remarkably, DCA treatment led to a significant increase in ROS production (up to 15-fold) in hypoxic cancer cells but not in aerobic cells. Consistently, DCA radiosensitized hypoxic tumor cells and 3D spheroids while leaving the intrinsic radiosensitivity of the tumor cells unchanged. Our results suggest that although described as an oxidative phosphorylation (OXPHOS)-promoting drug, DCA can also increase hypoxic radioresponses. This study therefore paves the way for the targeting of mitochondrial metabolism of hypoxic cancer cells, in particular to combat radioresistance.     

Irradiated Triple-Negative Breast Cancer Co-Culture Produces a Less Oncogenic Extracellular Matrix

Triple-negative breast cancer is the most common and most deadly cancer among women. Radiation is a mainstay of treatment, administered after surgery, and used in the hope that any remaining cancer cells will be destroyed. While the cancer cell response is normally the focus of radiation therapy, little is known about the tumor microenvironment response after irradiation. It is widely reported that increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. Aside from the classical fibrotic response, ratios of collagen isoforms have not been studied in a radiated tumor microenvironment. Here, we created one healthy co-culture of stromal fibroblasts and adipose-derived stem cells, and one triple-negative breast cancer co-culture, made of stromal fibroblasts, adipose derived stem cells, and triple-negative breast cancer cells. After irradiation, growth and decellularization of co-cultures, we reseeded the breast cancer cells for 24 h and analyzed the samples using mass spectrometry. Proteomic analysis revealed that collagen VI, a highly oncogenic collagen isoform linked to breast cancer, was decreased in the irradiated cancer co-culture. This indicates that the anti-cancer impact of radiation may be not only cell ablative, but also influential in creating a less oncogenic microenvironment.