Memo1 binds reduced copper ions, interacts with copper chaperone Atox1, and protects against copper-mediated redox activity in vitro.

The protein mediator of ERBB2-driven cell motility 1 (Memo1) is connected to many signaling pathways that play key roles in cancer. Memo1 was recently postulated to bind copper (Cu) ions and thereby promote the generation of reactive oxygen species (ROS) in cancer cells. Since the concentration of Cu as well as ROS are increased in cancer cells, both can be toxic if not well regulated. Here, we investigated the Cu-binding capacity of Memo1 using an array of biophysical methods at reducing as well as oxidizing conditions in vitro. We find that Memo1 coordinates two reduced Cu (Cu(I)) ions per protein, and, by doing so, the metal ions are shielded from ROS generation. In support of biological relevance, we show that the cytoplasmic Cu chaperone Atox1, which delivers Cu(I) in the secretory pathway, can interact with and exchange Cu(I) with Memo1 in vitro and that the two proteins exhibit spatial proximity in breast cancer cells. Thus, Memo1 appears to act as a Cu(I) chelator (perhaps shuttling the metal ion to Atox1 and the secretory path) that protects cells from Cu-mediated toxicity, such as uncontrolled formation of ROS. This Memo1 functionality may be a safety mechanism to cope with the increased demand of Cu ions in cancer cells.

Evaluation of ATOX1 as a Potential Predictive Biomarker for Tetrathiomolybdate Treatment of Breast Cancer Patients with High Risk of Recurrence.

Copper plays a key role in cancer metastasis, which is the most common cause of cancer death. Copper depletion treatment with tetrathiomolybdate (TM) improved disease-free survival in breast cancer patients with high risk of recurrence in a phase II clinical trial. Because the copper metallochaperone ATOX1 was recently reported to drive breast cancer cell migration and breast cancer migration is a critical factor in metastasis, we tested if ATOX1 expression levels in primary tumor tissue could predict the TM treatment outcome of breast cancer patients at high risk of recurrence. We performed ATOX1 immunohistochemical staining of breast tumor material (before TM treatment) of 47 patients enrolled in the phase II TM clinical trial and evaluated ATOX1 expression levels in relation with patient outcome after TM treatment. Our results show that higher ATOX1 levels in the tumor cell cytoplasm correlate with a trend towards better event-free survival after TM treatment for triple-negative breast cancer patients and patients at stage III of disease. In conclusion, ATOX1 may be a potential predictive biomarker for TM treatment of breast cancer patients at high risk of recurrence and should be tested in a larger cohort of patients.

The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration.

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10-15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.

Single-cell tracking demonstrates copper chaperone Atox1 to be required for breast cancer cell migration.

Copper ions are needed for several hallmarks of cancer. However, the involved pathways, mechanisms, and copper-binding proteins are mostly unknown. We recently found that cytoplasmic Antioxidant 1 copper chaperone (Atox1), which is up-regulated in breast cancer, is localized at the lamellipodia edges of aggressive breast cancer cells. To reveal molecular insights into a putative role in cell migration, we here investigated breast cancer cell (MDA-MB-231) migration by video microscopy as a function of Atox1. Tracking of hundreds of individual cells (per condition) over a 9-h time series revealed that cell migration velocity and directionality are significantly reduced upon Atox1 silencing in the cells. Because silencing of the copper transporter ATP7A also reduced cell migration, these proteins appear to be on the same pathway, suggesting that their well-known copper transport activity is involved. In-cell proximity ligation assays demonstrated that Atox1, ATP7A, and the proenzyme of lysyl oxidase (LOX; copper-loaded via ATP7A) are all in close proximity and that LOX activity is reduced upon Atox1 silencing in the cells. Since LOX is an established player in cancer cell migration, our results imply that Atox1 mediates breast cancer cell migration via coordinated copper transport in the ATP7A-LOX axis. Because individual cell migration is an early step in breast cancer metastasis, Atox1 levels in tumor cells may be a predictive measure of metastasis potential and serve as a biomarker for copper depletion therapy.

Evaluation of copper chaperone ATOX1 as prognostic biomarker in breast cancer.

Copper is involved in different hallmarks of cancer, including metastasis, but responsible copper-binding proteins and pathways are not clear. The copper chaperone ATOX1 was recently shown to play a role in breast cancer cell migration, which is a key step in metastasis. Since most cancer-related deaths are due to metastasis, we hypothesized that ATOX1 mRNA expression may be associated with breast cancer disease progression and thus, a prognostic biomarker in breast cancer. We therefore studied the association of ATOX1 expression levels with clinicopathological parameters and survival for 1904 breast cancer patients using the METABRIC data set. Our results indicate ATOX1 expression levels as a potential prognostic biomarker for ER-positive subtypes and early stages of breast cancer. Pre-clinical studies and clinical trials are desired to identify the molecular roles of ATOX1 in these conditions.

Interaction between Copper Chaperone Atox1 and Parkinson's Disease Protein α-Synuclein Includes Metal-Binding Sites and Occurs in Living Cells.

Alterations in copper ion homeostasis appear coupled to neurodegenerative disorders, but mechanisms are unknown. The cytoplasmic copper chaperone Atox1 was recently found to inhibit amyloid formation in vitro of α-synuclein, the amyloidogenic protein in Parkinson's disease. As α-synuclein may have copper-dependent functions, and free copper ions promote α-synuclein amyloid formation, it is important to characterize the Atox1 interaction with α-synuclein on a molecular level. Here we applied solution-state nuclear magnetic resonance spectroscopy, with isotopically labeled α-synuclein and Atox1, to define interaction regions in both proteins. The α-synuclein interaction interface includes the whole N-terminal part up to Gln24; in Atox1, residues around the copper-binding cysteines (positions 11-16) are mostly perturbed, but additional effects are also found for residues elsewhere in both proteins. Because α-synuclein is N-terminally acetylated in vivo, we established that Atox1 also inhibits amyloid formation of this variant in vitro, and proximity ligation in human cell lines demonstrated α-synuclein-Atox1 interactions in situ. Thus, this interaction may provide the direct link between copper homeostasis and amyloid formation in vivo.

The Critical Role of Dysregulated RhoB Signaling Pathway in Radioresistance of Colorectal Cancer.

PURPOSE: To explore whether the Rho protein is involved in the radioresistance of colorectal cancer and investigate the underlying mechanisms. METHODS AND MATERIALS: Rho GTPase expression was measured after radiation treatment in colon cancer cells. RhoB knockout cell lines were established using the CRISPR/Cas9 system. In vitro assays and zebrafish embryos were used for analyzing radiosensitivity and invasive ability. Mass cytometry was used to detect RhoB downstream signaling factors. RhoB and Forkhead box M1 (FOXM1) expression were detected by immunohistochemistry in rectal cancer patients who participated in a radiation therapy trial. RESULTS: RhoB expression was related to radiation resistance. Complete depletion of the RhoB protein increased radiosensitivity and impaired radiation-enhanced metastatic potential in vitro and in zebrafish models. Probing signaling using mass cytometry-based single-cell analysis showed that the Akt phosphorylation level was inhibited by RhoB depletion after radiation. FOXM1 was downregulated in RhoB knockout cells, and the inhibition of FOXM1 led to lower survival rates and attenuated migration and invasion abilities of the cells after radiation. In the patients who underwent radiation therapy, RhoB overexpression was related to high FOXM1, late Tumor, Node, Metastasis stage, high distant recurrence, and poor survival independent of other clinical factors. CONCLUSIONS: RhoB plays a critical role in radioresistance of colorectal cancer through Akt and FOXM1 pathways.

Copper distribution in breast cancer cells detected by time-of-flight secondary ion mass spectrometry with delayed extraction methodology.

Copper (Cu) is an essential transition metal ion that acts as a cofactor in many key enzymes. Cu is also needed for several hallmarks of cancer, and many copper-binding proteins are upregulated in various cancers. However, Cu-dependent cellular mechanisms and molecular pathways involved in cancer progression are not known. Fundamental to a better understanding of such phenomena is the investigation of the Cu subcellular distribution in cancer cells. The authors here show that Time-of-Flight Secondary Ion Mass Spectrometry combined with delayed extraction can be successfully applied to probe Cu localization in fixed MDA-MB-231 breast cancer cells providing subcellular resolution. Interestingly, the authors find Cu to be accumulated at nuclear regions of the cancer cells.

Copper Chaperone Atox1 Interacts with Cell Cycle Proteins.

The anaphase-promoting complex (APC) is involved in several processes in the cell cycle, most prominently it facilitates the separation of the sister chromatids during mitosis, before cell division. Because of the key role in the cell cycle, APC is suggested as a putative target for anticancer agents. We here show that the copper chaperone Atox1, known for shuttling copper in the cytoplasm from Ctr1 to ATP7A/B in the secretory pathway, interacts with several APC subunits. Atox1 interactions with APC subunits were discovered by mass spectrometry of co-immunoprecipitated samples and further confirmed using proximity ligation assays in HEK293T cells. Upon comparing wild-type cells with those in which the Atox1 gene had been knocked out, we found that in the absence of Atox1 protein, cells have prolonged G2/M phases and a slower proliferation rate. Thus, in addition to copper transport for loading of copper-dependent enzymes, Atox1 may modulate the cell cycle by interacting with APC subunits.

Second harmonic generation for collagen I characterization in rectal cancer patients with and without preoperative radiotherapy.

Rectal cancer is treated with preoperative radiotherapy (RT) to downstage the tumor, reduce local recurrence, and improve patient survival. Still, the treatment outcome varies significantly and new biomarkers are desired. Collagen I (Col-I) is a potential biomarker, which can be visualized label-free by second harmonic generation (SHG). Here, we used SHG to identify Col-I changes induced by RT in surgical tissue, with the aim to evaluate the clinical significance of RT-induced Col-I changes. First, we established a procedure for quantitative evaluation of Col-I by SHG in CDX2-stained tissue sections. Next, we evaluated Col-I properties in material from 31 non-RT and 29 RT rectal cancer patients. We discovered that the Col-I intensity and anisotropy were higher in the tumor invasive margin than in the inner tumor and normal mucosa, and RT increased and decreased the intensity in inner tumor and normal mucosa, respectively. Furthermore, higher Col-I intensity in the inner tumor was related to increased distant recurrence in the non-RT group but to longer survival in the RT group. In conclusion, we present a new application of SHG for quantitative analysis of Col-I in surgical material, and the first data suggest Col-I intensity as a putative prognostic biomarker in rectal cancer.

Roles of Copper-Binding Proteins in Breast Cancer.

Copper ions are needed in several steps of cancer progression. However, the underlying mechanisms, and involved copper-binding proteins, are mainly elusive. Since most copper ions in the body (in and outside cells) are protein-bound, it is important to investigate what copper-binding proteins participate and, for these, how they are loaded with copper by copper transport proteins. Mechanistic information for how some copper-binding proteins, such as extracellular lysyl oxidase (LOX), play roles in cancer have been elucidated but there is still much to learn from a biophysical molecular viewpoint. Here we provide a summary of copper-binding proteins and discuss ones reported to have roles in cancer. We specifically focus on how copper-binding proteins such as mediator of cell motility 1 (MEMO1), LOX, LOX-like proteins, and secreted protein acidic and rich in cysteine (SPARC) modulate breast cancer from molecular and clinical aspects. Because of the importance of copper for invasion/migration processes, which are key components of cancer metastasis, further insights into the actions of copper-binding proteins may provide new targets to combat cancer.

Copper chaperone Atox1 plays role in breast cancer cell migration.

Copper (Cu) is an essential transition metal ion required as cofactor in many key enzymes. After cell uptake of Cu, the metal is transported by the cytoplasmic Cu chaperone Atox1 to P1B-type ATPases in the Golgi network for incorporation into Cu-dependent enzymes in the secretory path. Cu is vital for many steps of cancer progression and Atox1 was recently suggested to have additional functionality as a nuclear transcription factor. We here investigated the expression level, cellular localization and role in cell migration of Atox1 in an aggressive breast cancer cell line upon combining immunostaining, microscopy and a wound healing assay. We made the unexpected discovery that Atox1 accumulates at lamellipodia borders of migrating cancer cells and Atox1 silencing resulted in migration defects as evidenced from reduced wound closure. Therefore, we have discovered an unknown role of the Cu chaperone Atox1 in breast cancer cell migration.

X-radiation enhances the collagen type I strap formation and migration potentials of colon cancer cells.

Rectal cancer treatment still fails with local and distant relapses of the disease. It is hypothesized that radiotherapy could stimulate cancer cell dissemination and metastasis. In this study, we evaluated the effect of X-radiation on collagen type I strap formation potential, i.e. matrix remodeling associated with mesenchymal cell migration, and behaviors of SW480, SW620, HCT116 p53+/+ and HCT116 p53-/- colon cancer cells. We determined a radiation-induced increase in collagen type I strap formation and migration potentials of SW480 and HCT116 p53+/+. Further studies with HCT116 p53+/+, indicated that after X-radiation strap forming cells have an increased motility. More, we detected a decrease in adhesion potential and mature integrin ß1 expression, but no change in non-muscle myosin II expression for HCT116 p53+/+ after X-radiation. Integrin ß1 neutralization resulted in a decreased cell adhesion and collagen type I strap formation in both sham and X-radiated conditions. Our study indicates collagen type I strap formation as a potential mechanism of colon cancer cells with increased migration potential after X-radiation, and suggests that other molecules than integrin ß1 and non-muscle myosin II are responsible for the radiation-induced collagen type I strap formation potential of colon cancer cells. This work encourages further molecular investigation of radiation-induced migration to improve rectal cancer treatment outcome.

Radiation-induced myosin IIA expression stimulates collagen type I matrix reorganization.

BACKGROUND AND PURPOSE: Extracellular matrix (ECM) reorganization critically contributes to breast cancer (BC) progression and radiotherapy response. We investigated the molecular background and functional consequences of collagen type I (col-I) reorganization by irradiated breast cancer cells (BCC). MATERIALS AND METHODS: Radiation-induced (RI) col-I reorganization was evaluated for MCF-7/6, MCF-7/AZ, T47D and SK-BR-3 BCC. Phase-contrast microscopy and a stressed matrix contraction assay were used for visualization and quantification of col-I reorganization. Cell-matrix interactions were assessed by the inhibition of ß1 integrin (neutralizing antibody 'P5D2') or focal adhesion kinase (FAK; GSK22560098 small molecule kinase inhibitor). The role of the actomyosin cytoskeleton was explored by western blotting analysis of myosin II expression and activity; and by gene silencing of myosin IIA and pharmacological inhibition of the actomyosin system (blebbistatin, cytochalasin D). BCC death was evaluated by propidium iodide staining. RESULTS: We observed a radiation dose-dependent increase of col-I reorganization by BCC. ß1 Integrin/FAK-mediated cell-matrix interactions are essential for RI col-I reorganization. Irradiated BCC are characterized by increased myosin IIA expression and myosin IIA-dependent col-I reorganization. Moreover, RI col-I reorganization by BCC is associated with decreased BCC death, as suggested by pharmacological targeting of the ß1 integrin/FAK/myosin IIA pathway. CONCLUSIONS: Our data indicate the role of myosin IIA in col-I reorganization by irradiated BCC and reciprocal BCC death.

Unraveling the mechanisms behind the enhanced MTT conversion by irradiated breast cancer cells.

Previously, we described the radiation-induced (RI) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) effect as the increased MTT metabolization at the intermediate dose region after the irradiation of an MCF-7/6 cell monolayer with an X-ray dose gradient. We wondered if the cell monolayer at the intermediate dose region was characterized by an increased metabolic activity. In this study, we unraveled the mechanisms behind the RI MTT effect. Comparison of the MTT, sulforhodamine B (SRB), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium (WST-8), and nitroblue tetrazolium (NBT) assays indicated that the RI MTT effect is not due to an increased cell density, but to an exclusively intracellular MTT conversion. Our results for the MTT and NBT assays after digitonin pretreatment of the irradiated cell monolayer indicated a role of the plasma membrane permeability in the RI MTT effect. Assessment of the radiation impact on the oxidative phosphorylation system by Western blot analysis, spectrophotometric measurement and Blue Native gel electrophoresis showed a dose-dependent downregulation of the oxidative phosphorylation system complexes, whereby the radiosensitivity of each complex was proportional to the number of mitochondrial DNA-encoded subunits. Further, only treatment of the irradiated cell monolayer with a cocktail and not with the individual inhibitors of complexes I, II and IV during the MTT assay prevented the RI MTT effect. In general, our results demonstrate that the RI MTT effect is not due to an increased metabolic activity, but rather to an enhanced cellular MTT entry and mitochondrial MTT conversion.

From clinical observations of intensity-modulated radiotherapy to dedicated in vitro designs.

In this review, an overview of intensity-modulated radiotherapy (IMRT) and related high precision radiation techniques is presented. In addition, the related radiobiological issues are discussed. Hereby, we try to point to the potential differences in radiobiological effect between popular intensity-modulated radiotherapy and related techniques (IMRT+) and conventional or three-dimensional radiotherapy (3D-RT). Further, an overview of the existing in vitro and in vivo radiobiological models to investigate the effect of spatially and/or temporally fractionated dose distributions, as applied in IMRT+, on the biological outcome is given. More in detail, our radiobiological models will be presented. Additionally, we will discuss the (dis)advantages of the presented models, and give some consideration to improve the existing radiobiological models in terms of set-up and clinical relevance.