Salinomycin disturbs Golgi function and specifically affects cells in epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) gives rise to cells with properties similar to cancer stem cells (CSCs). Targeting the EMT program to selectively eliminate CSCs is a promising way to improve cancer therapy. Salinomycin (Sal), a K+/H+ ionophore, was identified as highly selective towards CSC-like cells, but its mechanism of action and selectivity remains elusive. Here, we show that Sal, similar to monensin and nigericin, disturbs the function of the Golgi. Sal alters the expression of Golgi-related genes and leads to marked changes in Golgi morphology, particularly in cells that have undergone EMT. Moreover, Golgi-disturbing agents severely affect post-translational modifications of proteins, including protein processing, glycosylation and secretion. We discover that the alterations induced by Golgi-disturbing agents specifically affect the viability of EMT cells. Collectively, our work reveals a novel vulnerability related to the EMT, suggesting an important role for the Golgi in the EMT and that targeting the Golgi could represent a novel therapeutic approach against CSCs.

Stroma-derived miR-214 coordinates tumor dissemination.

BACKGROUND: Tumor progression is based on a close interaction between cancer cells and Tumor MicroEnvironment (TME). Here, we focus on the role that Cancer Associated Fibroblasts (CAFs), Mesenchymal Stem Cells (MSCs) and microRNAs (miRs) play in breast cancer and melanoma malignancy. METHODS: We used public databases to investigate miR-214 expression in the stroma compartment of primary human samples and evaluated tumor formation and dissemination following tumor cell injections in miR-214 overexpressing (miR-214over) and knock out (miR-214ko) mice. In addition, we dissected the impact of Conditioned Medium (CM) or Extracellular Vesicles (EVs) derived from miR-214-rich or depleted stroma cells on cell metastatic traits. RESULTS: We evidence that the expression of miR-214 in human cancer or metastasis samples mostly correlates with stroma components and, in particular, with CAFs and MSCs. We present data revealing that the injection of tumor cells in miR-214over mice leads to increased extravasation and metastasis formation. In line, treatment of cancer cells with CM or EVs derived from miR-214-enriched stroma cells potentiate cancer cell migration/invasion in vitro. Conversely, dissemination from tumors grown in miR-214ko mice is impaired and metastatic traits significantly decreased when CM or EVs from miR-214-depleted stroma cells are used to treat cells in culture. Instead, extravasation and metastasis formation are fully re-established when miR-214ko mice are pretreated with miR-214-rich EVs of stroma origin. Mechanistically, we also show that tumor cells are able to induce miR-214 production in stroma cells, following the activation of IL-6/STAT3 signaling, which is then released via EVs subsequently up-taken by cancer cells. Here, a miR-214-dependent pro-metastatic program becomes activated. CONCLUSIONS: Our findings highlight the relevance of stroma-derived miR-214 and its release in EVs for tumor dissemination, which paves the way for miR-214-based therapeutic interventions targeting not only tumor cells but also the TME.

Ancient Origins of Cytoskeletal Crosstalk: Spectraplakin-like Proteins Precede the Emergence of Cortical Microtubule Stabilization Complexes as Crosslinkers.

Adhesion between cells and the extracellular matrix (ECM) is one of the prerequisites for multicellularity, motility, and tissue specialization. Focal adhesions (FAs) are defined as protein complexes that mediate signals from the ECM to major components of the cytoskeleton (microtubules, actin, and intermediate filaments), and their mutual communication determines a variety of cellular processes. In this study, human cytoskeletal crosstalk proteins were identified by comparing datasets with experimentally determined cytoskeletal proteins. The spectraplakin dystonin was the only protein found in all datasets. Other proteins (FAK, RAC1, septin 9, MISP, and ezrin) were detected at the intersections of FAs, microtubules, and actin cytoskeleton. Homology searches for human crosstalk proteins as queries were performed against a predefined dataset of proteomes. This analysis highlighted the importance of FA communication with the actin and microtubule cytoskeleton, as these crosstalk proteins exhibit the highest degree of evolutionary conservation. Finally, phylogenetic analyses elucidated the early evolutionary history of spectraplakins and cortical microtubule stabilization complexes (CMSCs) as model representatives of the human cytoskeletal crosstalk. While spectraplakins probably arose at the onset of opisthokont evolution, the crosstalk between FAs and microtubules is associated with the emergence of metazoans. The multiprotein complexes contributing to cytoskeletal crosstalk in animals gradually gained in complexity from the onset of metazoan evolution.

miRNA-guided reprogramming of glucose and glutamine metabolism and its impact on cell adhesion/migration during solid tumor progression.

MicroRNAs (miRNAs) are small, non-coding RNAs about 22 nucleotides in length that regulate the expression of target genes post-transcriptionally, and are highly involved in cancer progression. They are able to impact a variety of cell processes such as proliferation, apoptosis and differentiation and can consequently control tumor initiation, tumor progression and metastasis formation. miRNAs can regulate, at the same time, metabolic gene expression which, in turn, influences relevant traits of malignancy such as cell adhesion, migration and invasion. Since the interaction between metabolism and adhesion or cell movement has not, to date, been well understood, in this review, we will specifically focus on miRNA alterations that can interfere with some metabolic processes leading to the modulation of cancer cell movement. In addition, we will analyze the signaling pathways connecting metabolism and adhesion/migration, alterations that often affect cancer cell dissemination and metastasis formation.

The Tongue Squamous Carcinoma Cell Line Cal27 Primarily Employs Integrin α6ß4-Containing Type II Hemidesmosomes for Adhesion Which Contribute to Anticancer Drug Sensitivity.

Integrins are heterodimeric cell surface glycoproteins used by cells to bind to the extracellular matrix (ECM) and regulate tumor cell proliferation, migration and survival. A causative relationship between integrin expression and resistance to anticancer drugs has been demonstrated in different tumors, including head and neck squamous cell carcinoma. Using a Cal27 tongue squamous cell carcinoma model, we have previously demonstrated that de novo expression of integrin αVß3 confers resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through a mechanism involving downregulation of active Src, increased cell migration and invasion. In the integrin αVß3 expressing Cal27-derived cell clone 2B1, αVß5 expression was also increased, but unrelated to drug resistance. To identify the integrin adhesion complex (IAC) components that contribute to the changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines. Mass spectrometry (MS)-based proteomics analysis indicated that both cell lines preferentially, but not exclusively, use integrin α6ß4, which is classically found in hemidesmosomes. The anticancer drug resistant cell clone 2B1 demonstrated an increased level of α6ß4 accompanied with increased deposition of a laminin-332-containing ECM. Immunofluorescence and electron microscopy demonstrated the formation of type II hemidesmosomes by both cell types. Furthermore, suppression of α6ß4 expression in both lines conferred resistance to anticancer drugs through a mechanism independent of αVß3, which implies that the cell clone 2B1 would have been even more resistant had the upregulation of α6ß4 not occurred. Taken together, our results identify a key role for α6ß4-containing type II hemidesmosomes in regulating anticancer drug sensitivity.

Integrin Crosstalk Contributes to the Complexity of Signalling and Unpredictable Cancer Cell Fates.

Integrins are heterodimeric cell surface receptors composed of α and ß subunits that control adhesion, proliferation and gene expression. The integrin heterodimer binding to ligand reorganises the cytoskeletal networks and triggers multiple signalling pathways that can cause changes in cell cycle, proliferation, differentiation, survival and motility. In addition, integrins have been identified as targets for many different diseases, including cancer. Integrin crosstalk is a mechanism by which a change in the expression of a certain integrin subunit or the activation of an integrin heterodimer may interfere with the expression and/or activation of other integrin subunit(s) in the very same cell. Here, we review the evidence for integrin crosstalk in a range of cellular systems, with a particular emphasis on cancer. We describe the molecular mechanisms of integrin crosstalk, the effects of cell fate determination, and the contribution of crosstalk to therapeutic outcomes. Our intention is to raise awareness of integrin crosstalk events such that the contribution of the phenomenon can be taken into account when researching the biological or pathophysiological roles of integrins.

Sirt3 Exerts Its Tumor-Suppressive Role by Increasing p53 and Attenuating Response to Estrogen in MCF-7 Cells.

Estrogen (E2) is a major risk factor for the initiation and progression of malignancy in estrogen receptor (ER) positive breast cancers, whereas sirtuin 3 (Sirt3), a major mitochondrial NAD+-dependent deacetylase, has the inhibitory effect on the tumorigenic properties of ER positive MCF-7 breast cancer cells. Since it is unclear if this effect is mediated through the estrogen receptor alpha (ERα) signaling pathway, in this study, we aimed to determine if the tumor-suppressive function of Sirt3 in MCF-7 cells interferes with their response to E2. Although we found that Sirt3 improves the antioxidative response and mitochondrial fitness of the MCF-7 cells, it also increases DNA damage along with p53, AIF, and ERα expression. Moreover, Sirt3 desensitizes cells to the proliferative effect of E2, affects p53 by disruption of the ERα-p53 interaction, and decreases proliferation, colony formation, and migration of the cells. Our observations indicate that these tumor-suppressive effects of Sirt3 could be reversed by E2 treatment only to a limited extent which is not sufficient to recover the tumorigenic properties of the MCF-7 cells. This study provides new and interesting insights with respect to the functional role of Sirt3 in the E2-dependent breast cancers.

KANK2 Links αVß5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration.

Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. We have previously observed decreased cell migration and increased sensitivity to microtubule (MT) poisons, paclitaxel and vincristine, in the melanoma cell line MDA-MB-435S upon transfection with integrin αV-specific siRNA, suggesting a link between adhesion and drug sensitivity. To elucidate the underlying mechanism, we determined αV-dependent changes in IAC composition. Using mass spectrometry (MS)-based proteomics, we analyzed the components of isolated IACs of MDA-MB-435S cells and two MDA-MB-435S-derived integrin αV-specific shRNA-expressing cell clones with decreased expression of integrin αV. MS analysis showed that cells preferentially use integrin αVß5 for the formation of IACs. The differential analysis between MDA-MB-435S cells and clones with decreased expression of integrin αV identified key components of integrin αVß5 adhesion complexes as talins 1 and 2, α-actinins 1 and 4, filamins A and B, plectin and vinculin. The data also revealed decreased levels of several components of the cortical microtubule stabilization complex, which recruits MTs to adhesion sites (notably liprins α and ß, ELKS, LL5ß, MACF1, KANK1, and KANK2), following αV knockdown. KANK2 knockdown in MDA-MB-435S cells mimicked the effect of integrin αV knockdown and resulted in increased sensitivity to MT poisons and decreased migration. Taken together, we conclude that KANK2 is a key molecule linking integrin αVß5 IACs to MTs, and enabling the actin-MT crosstalk that is important for both sensitivity to MT poisons and cell migration.

FADD-deficient mouse embryonic fibroblasts undergo RIPK1-dependent apoptosis and autophagy after NB-UVB irradiation.

Sun or therapy-related ultraviolet B (UVB) irradiation induces different cell death modalities such as apoptosis, necrosis/necroptosis and autophagy. Understanding of mechanisms implicated in regulation and execution of cell death program is imperative for prevention and treatment of skin diseases. An essential component of death-inducing complex is Fas-associated protein with death domain (FADD), involved in conduction of death signals of different death modalities. The purpose of this study was to enlighten the role of FADD in the selection of cell death mode after narrow-band UVB (NB-UVB) irradiation using specific cell death inhibitors (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (zVAD-fmk), Necrostatin-1 and 3-Methyladenine) and FADD-deficient (FADD-/-) mouse embryonic fibroblasts (MEFs) and their wild type (wt) counterparts. The results imply that lack of FADD sensitized MEFs to induction of receptor-interacting protein 1 (RIPK1)-dependent apoptosis by the generation of reactive oxygen species (ROS), but without activation of the proteins p53, Bax and Bcl-2 as well as without the enrolment of calpain-2. Autophagy was established as a contributing factor to NB-UVB-induced death execution. By contrast, wt cells triggered intrinsic apoptotic pathway that was resistant to the inhibition by zVAD-fmk and Necrostatin-1 pointing to the mechanism overcoming the cell survival. These findings support the role of FADD in prevention of autophagy-dependent apoptosis.

Differential Effects of Integrin αv Knockdown and Cilengitide on Sensitization of Triple-Negative Breast Cancer and Melanoma Cells to Microtubule Poisons.

Low survival rates of patients with metastatic triple-negative breast cancer (TNBC) and melanoma, in which current therapies are ineffective, emphasize the need for new therapeutic approaches. Integrin ß1 appears to be a promising target when combined with chemotherapy, but recent data have shown that its inactivation increases metastatic potential owing to the compensatory upregulation of other integrin subunits. Consequently, we analyzed the potential of integrin subunits αv, α3, or α4 as targets for improved therapy in seven TNBC and melanoma cell lines. Experiments performed in an integrin αvß1-negative melanoma cell line, MDA-MB-435S, showed that knockdown of integrin subunit αv increased sensitivity to microtubule poisons vincristine or paclitaxel and decreased migration and invasion. In the MDA-MB-435S cell line, we also identified a phenomenon in which change in the expression of one integrin subunit changes the expression of other integrins, leading to an unpredictable influence on sensitivity to anticancer drugs and cell migration, referred to as the integrin switching effect. In a panel of six TNBCs and melanoma cell lines, the contribution of integrins αv versus integrins αvß3/ß5 was assessed by the combined action of αv-specific small interfering RNA or αvß3/ß5 inhibitor cilengitide with paclitaxel. Our results suggest that, for TNBC, knockdown of integrin αv in combination with paclitaxel presents a better therapeutic option than a combination of cilengitide with paclitaxel; however, in melanoma, neither of these combinations is advisable because a decreased sensitivity to paclitaxel was observed.

De novo expression of transfected sirtuin 3 enhances susceptibility of human MCF-7 breast cancer cells to hyperoxia treatment.

Sirtuin 3 (Sirt3) has a promising role in cancer tumourigenesis and treatment, but there have been controversies about its role as oncogene or tumour suppressor in different types of cancer. Changes in its expression are associated with the excessive production of reactive oxygen species (ROS), thus contributing to mitochondrial dysfunction and age-related pathologies. Hyperoxic treatment (i.e. generator of ROS) was shown to support some tumourigenic properties, but finally suppresses growth of certain mammary carcinoma cells. Due to strikingly reduced Sirt3 level in many breast cancer cell lines, we aimed to clarify the effect of de novo Sirt3 expression upon hyperoxic treatment in the human MCF-7 breast cancer cells. De novo expression of Sirt3 decreased metabolic activity and cellular growth of MCF-7 cells, reduced expression of proangiogenic and epithelial mesenchymal transition genes, induced metabolic switch from glycolysis to oxidative phosphorylation, and decreased abundance of senescent cells. These effects were enhanced upon hyperoxic treatment: induction of DNA damage and upregulation of p53, with an increase of ROS levels followed by mitochondrial and antioxidant dysfunction, resulted in additional reduction of metabolic activity and inhibition of cellular growth and survival. The mitigation of tumorigenic properties and enhancement of the susceptibility of the MCF-7 breast cancer cells to the hyperoxic treatment upon de novo Sirt3 expression indicates that these factors, individually and in combination, should be further explored in vitro and particularly in vivo, as an adjuvant tumour therapy in breast cancer malignancies.

Apigenin, a dietary flavonoid, induces apoptosis, DNA damage, and oxidative stress in human breast cancer MCF-7 and MDA MB-231 cells.

Apigenin is found in several dietary plant foods such as vegetables and fruits. To investigate potential anticancer properties of apigenin on human breast cancer, ER-positive MCF-7 and triple-negative MDA MB-231 cells were used. Moreover, toxicological safety of apigenin towards normal cells was evaluated in human lymphocytes. Cytotoxicity of apigenin towards cancer cells was evaluated by MTT assay whereas further genotoxic and oxidative stress parameters were measured by comet and lipid peroxidation assays, respectively. In order to examine the type of cell death induced by apigenin, several biomarkers were used. Toxicological safety towards normal cells was evaluated by cell viability and comet assays. After the treatment with apigenin, we observed changes in cell morphology in a dose- (10 to 100 µM) and time-dependent manner. Moreover, apigenin caused cell death in both cell lines leading to significant toxicity and dominantly to apoptosis. Furthermore, apigenin proved to be genotoxic towards the selected cancer cells with a potential to induce oxidative damage to lipids. Of great importance is that no significant cytogenotoxic effects were detected in normal cells. The observed cytogenotoxic and pro-cell death activities of apigenin coupled with its low toxicity towards normal cells indicate that this natural product could be used as a future anticancer modality. Therefore, further analysis to determine the exact mechanism of action and in vivo studies on animal models are warranted.

Gene expression in formalin-fixed paraffin-embedded lymph nodes.

Elucidation of molecular pathways involved in development of human lymphoma requires efficient methods for tackling gene expression in lymph nodes. Expression studies of transcription factors in these malignancies facilitate understanding the changes occurring in neoplastic transformation and lymphoma development. Excised lymph nodes are routinely fixed in formalin and embedded in paraffin for diagnosis and stored in many hospitals' pathology archives. These tissues represent a precious resource for research since they allow retrospective studies to cover a broad range of human lymphoma even the less frequent types. Reverse transcription polymerase chain reaction (RT-PCR) is a commonly used method for gene expression analysis and a reproducible protocol for RNA isolation from lymph nodes is an inevitable requirement for these studies. However, formalin fixation and paraffin-embedding interfere with the quality of RNA especially when isolated from lymph nodes being the most fragile lymphatic tissues. We present here a simple and fast method for RNA isolation from formalin-fixed paraffin-embedded lymph nodes that can be successfully applied for RT-PCR as well as for quantitative RT-PCR analysis. We tested diverse isolation reagents and combined a range of factors in order to get a high quality RNA for retrospective studies of gene expression in human lymphoma samples. Our modified method of RNA extraction from FFPE provides superior yields and purity based on qPCR data.

Analysis of Ikaros family splicing variants in human hematopoietic lineages.

Transcription factors from the Ikaros family are involved in lymphocyte differentiation and have a critical role at specific check points of the haemopoietic pathway. However, how developmentally regulated changes are reflected in gene expression programs of lymphocyte differentiation is not well understood. It has been suggested that disregulation of transcription factors from the Ikaros family is associated with the development of different human leukemias. In this work we analyzed the state of Ikaros family members in different leukemic cells with the aim to explore the transcriptional control of human hematopoietic lineages and shed some new light on our understanding of transcription factor significance in human leukemias. By means of RT-PCR and specific primers we investigated the expression of Ikaros, Aiolos and Helios transcription factors and their splicing variants in seven leukemia cell lines derived from different types of leukemia (ALL, CML, AML) and lymphoma (histiocytic lymphoma, Burkitt lymphoma and anaplastic large cell lymphoma). In all of the cell lines examined Ikaros was present in dominant Ik1 to Ik4 isoforms and small Ik6 isoform was absent. Aiolos was expressed in the majority of the cell lines, of both, B and T origin, in the form of the full length Aio1. Helios was also present only in two long isoforms Hel1 and Hel2, and was absent in one third of the lines. Similar distribution of positive and negative expression of Aiolos and Helios found in various types of leukemias could implicate common pathways of their regulation.