The Molecular Chaperone CCT Sequesters Gelsolin and Protects it from Cleavage by Caspase-3.

The actin filament severing and capping protein gelsolin plays an important role in modulation of actin filament dynamics by influencing the number of actin filament ends. During apoptosis, gelsolin becomes constitutively active due to cleavage by caspase-3. In non-apoptotic cells gelsolin is activated by the binding of Ca2+. This activated form of gelsolin binds to, but is not a folding substrate of the molecular chaperone CCT/TRiC. Here we demonstrate that in vitro, gelsolin is protected from cleavage by caspase-3 in the presence of CCT. Cryoelectron microscopy and single particle 3D reconstruction of the CCT:gelsolin complex reveals that gelsolin is located in the interior of the chaperonin cavity, with a placement distinct from that of the obligate CCT folding substrates actin and tubulin. In cultured mouse melanoma B16F1 cells, gelsolin co-localises with CCT upon stimulation of actin dynamics at peripheral regions during lamellipodia formation. These data indicate that localised sequestration of gelsolin by CCT may provide spatial control of actin filament dynamics.

The Effect of Hypoxic and Normoxic Culturing Conditions in Different Breast Cancer 3D Model Systems.

The field of 3D cell cultures is currently emerging, and material development is essential in striving toward mimicking the microenvironment of a native tissue. By using the response of reporter cells to a 3D environment, a comparison between materials can be assessed, allowing optimization of material composition and microenvironment. Of particular interest, the response can be different in a normoxic and hypoxic culturing conditions, which in turn may alter the conclusion regarding a successful recreation of the microenvironment. This study aimed at determining the role of such environments to the conclusion of a better resembling cell culture model to native tissue. Here, the breast cancer cell line MCF7 was cultured in normoxic and hypoxic conditions on patient-derived scaffolds and compared at mRNA and protein levels to cells cultured on 3D printed scaffolds, Matrigel, and conventional 2D plastics. Specifically, a wide range of mRNA targets (40), identified as being regulated upon hypoxia and traditional markers for cell traits (cancer stem cells, epithelial-mesenchymal transition, pluripotency, proliferation, and differentiation), were used together with a selection of corresponding protein targets. 3D cultured cells were vastly different to 2D cultured cells in gene expression and protein levels on the majority of the selected targets in both normoxic and hypoxic culturing conditions. By comparing Matrigel and 3DPS-cultured cells to cells cultured on patient-derived scffolds, differences were also noted along all categories of mRNA targets while specifically for the GLUT3 protein. Overall, cells cultured on patient-derived scaffolds closely resembled cells cultured on 3D printed scaffolds, contrasting 2D and Matrigel-cultured cells, regardless of a normoxic or hypoxic culturing condition. Thus, these data support the use of either a normoxic or hypoxic culturing condition in assays using native tissues as a blueprint to optimize material composition.

3D Printed Nanocellulose Scaffolds as a Cancer Cell Culture Model System.

Current conventional cancer drug screening models based on two-dimensional (2D) cell culture have several flaws and there is a large need of more in vivo mimicking preclinical drug screening platforms. The microenvironment is crucial for the cells to adapt relevant in vivo characteristics and here we introduce a new cell culture system based on three-dimensional (3D) printed scaffolds using cellulose nanofibrils (CNF) pre-treated with 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO) as the structural material component. Breast cancer cell lines, MCF7 and MDA-MB-231, were cultured in 3D TEMPO-CNF scaffolds and were shown by scanning electron microscopy (SEM) and histochemistry to grow in multiple layers as a heterogenous cell population with different morphologies, contrasting 2D cultured mono-layered cells with a morphologically homogenous cell population. Gene expression analysis demonstrated that 3D TEMPO-CNF scaffolds induced elevation of the stemness marker CD44 and the migration markers VIM and SNAI1 in MCF7 cells relative to 2D control. T47D cells confirmed the increased level of the stemness marker CD44 and migration marker VIM which was further supported by increased capacity of holoclone formation for 3D cultured cells. Therefore, TEMPO-CNF was shown to represent a promising material for 3D cell culture model systems for cancer cell applications such as drug screening.

Optimized alginate-based 3D printed scaffolds as a model of patient derived breast cancer microenvironments in drug discovery.

The cancer microenvironment influences tumor progression and metastasis and is pivotal to consider when designingin vivo-like cancer models. Current preclinical testing platforms for cancer drug development are mainly limited to 2D cell culture systems that poorly mimic physiological environments and traditional, low throughput animal models. The aim of this work was to produce a tunable testing platform based on 3D printed scaffolds (3DPS) with a simple geometry that, by extracellular components and response of breast cancer reporter cells, mimics patient-derived scaffolds (PDS) of breast cancer. Here, the biocompatible polysaccharide alginate was used as base material to generate scaffolds consisting of a 3D grid containing periostin and hydroxyapatite. Breast cancer cell lines (MCF7 and MDA-MB-231) produced similar phenotypes and gene expression levels of cancer stem cell, epithelial-mesenchymal transition, differentiation and proliferation markers when cultured on 3DPS and PDS, contrasting conventional 2D cultures. Importantly, cells cultured on 3DPS and PDS showed scaffold-specific responses to cytotoxic drugs (doxorubicin and 5-fluorouracil) that were different from 2D cultured cells. In conclusion, the data presented support the use of a tunable alginate-based 3DPS as a tumor model in breast cancer drug discovery.

Breast cancer patient-derived scaffolds as a tool to monitor chemotherapy responses in human tumor microenvironments.

Breast cancer is a heterogeneous disease where the tumor microenvironment, including extracellular components, plays a crucial role in tumor progression, potentially modulating treatment response. Different approaches have been used to develop three-dimensional models able to recapitulate the complexity of the extracellular matrix. Here, we use cell-free patient-derived scaffolds (PDSs) generated from breast cancer samples that were recellularized with cancer cell lines as an in vivo-like culture system for drug testing. We show that PDS cultured MCF7 cancer cells increased their resistance against the front-line chemotherapy drugs 5-fluorouracil, doxorubicin and paclitaxel in comparison to traditional two-dimensional cell cultures. The gene expression of the environmentally adapted cancer cells was modulated in different ways depending on the drug and the concentration used. High doses of doxorubicin reduced cancer stem cell features, whereas 5-fluorouracil increased stemness and decreased the proliferative phenotype. By using PDSs repopulated with other breast cancer cell lines, T-47D and MDA-MB-231, we observed both general and cell line specific drug responses. In summary, PDSs can be used to examine the extracellular matrix influence on cancer drug responses and for testing novel compounds in in vivo-like microenvironments.

Correlated fluorescence microscopy and multi-ion beam secondary ion mass spectrometry imaging reveals phosphatidylethanolamine increases in the membrane of cancer cells over-expressing the molecular chaperone subunit CCTδ.

Changes in the membrane composition of sub-populations of cells can influence different properties with importance to tumour growth, metastasis and treatment efficacy. In this study, we use correlated fluorescence microscopy and ToF-SIMS with C60+ and (CO2)6k+ ion beams to identify and characterise sub-populations of cells based on successful transfection leading to over-expression of CCTδ, a component of the multi-subunit molecular chaperone named chaperonin-containing tailless complex polypeptide 1 (CCT). CCT has been linked to increased cell growth and proliferation and is known to affect cell morphology but corresponding changes in lipid composition of the membrane have not been measured until now. Multivariate analysis of the surface mass spectra from single cells, focused on the intact lipid ions, indicates an enrichment of phosphatidylethanolamine species in the transfected cells. While the lipid changes in this case are driven by the structural changes in the protein cytoskeleton, the consequence of phosphatidylethanolamine enrichment may have additional implications in cancer such as increased membrane fluidity, increased motility and an ability to adapt to a depletion of unsaturated lipids during cancer cell proliferation. This study demonstrates a successful fluorescence microscopy-guided cell by cell membrane lipid analysis with broad application to biological investigation.Graphical abstract.

Characterization of cell-free breast cancer patient-derived scaffolds using liquid chromatography-mass spectrometry/mass spectrometry data and RNA sequencing data.

Patient-derived scaffolds (PDSs) generated from primary breast cancer tumors can be used to model the tumor microenvironment in vitro. Patient-derived scaffolds are generated by repeated detergent washing, removing all cells. Here, we analyzed the protein composition of 15 decellularized PDSs using liquid chromatography-mass spectrometry/mass spectrometry. One hundred forty-three proteins were detected and their relative abundance was calculated using a reference sample generated from all PDSs. We performed heatmap analysis of all the detected proteins to display their expression patterns across different PDSs together with pathway enrichment analysis to reveal which processes that were connected to PDS protein composition. This protein dataset together with clinical information is useful to investigators studying the microenvironment of breast cancers. Further, after repopulating PDSs with either MCF7 or MDA-MB-231 cells, we quantified their gene expression profiles using RNA sequencing. These data were also compared to cells cultured in conventional 2D conditions, as well as to cells cultured as xenografts in immune-deficient mice. We investigated the overlap of genes regulated between these different culture conditions and performed pathway enrichment analysis of genes regulated by both PDS and xenograft cultures compared to 2D in both cell lines to describe common processes associated with both culture conditions. Apart from our described analyses of these systems, these data are useful when comparing different experimental model systems. Downstream data analyses and interpretations can be found in the research article "Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment" [1].

Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment.

Tumor cells interact with the microenvironment that specifically supports and promotes tumor development. Key components in the tumor environment have been linked to various aggressive cancer features and can further influence the presence of subpopulations of cancer cells with specific functions, including cancer stem cells and migratory cells. To model and further understand the influence of specific microenvironments we have developed an experimental platform using cell-free patient-derived scaffolds (PDSs) from primary breast cancers infiltrated with standardized breast cancer cell lines. This PDS culture system induced a series of orchestrated changes in differentiation, epithelial-mesenchymal transition, stemness and proliferation of the cancer cell population, where an increased cancer stem cell pool was confirmed using functional assays. Furthermore, global gene expression profiling showed that PDS cultures were similar to xenograft cultures. Mass spectrometry analyses of cell-free PDSs identified subgroups based on their protein composition that were linked to clinical properties, including tumor grade. Finally, we observed that an induction of epithelial-mesenchymal transition-related genes in cancer cells growing on the PDSs were significantly associated with clinical disease recurrences in breast cancer patients. Patient-derived scaffolds thus mimics in vivo-like growth conditions and uncovers unique information about the malignancy-inducing properties of tumor microenvironment.

The molecular chaperone CCT modulates the activity of the actin filament severing and capping protein gelsolin in vitro.

The oligomeric molecular chaperone CCT is essential for the folding of the highly abundant protein actin, which in its native state forms actin filaments that generate the traction forces required for cell motility. In addition to folding proteins, CCT can provide a platform for protein complex assembly and binds actin filaments assembled in vitro. Some individual subunits of CCT, when monomeric, have been shown to be functionally active, and in particular, the CCTepsilon subunit is involved in the serum response factor pathway that controls actin transcription. Thus, there is a complex interplay between CCT and actin that extends beyond actin folding. CCT has recently been shown to bind gelsolin, an actin filament severing protein that increases actin dynamics by generating filament ends for further actin polymerization. However, the biological significance of the CCT:gelsolin interaction is unknown. Here, using a co-immunoprecipitation assay, we show that CCT binds directly to gelsolin in its calcium-activated, actin-severing conformation. Furthermore, using actin filaments retained from fixed and permeabilized cells, we demonstrate that CCT can inhibit the actin filament severing activity of gelsolin. As our work and that of others shows gelsolin is not folded by CCT, the CCT:gelsolin interaction represents a novel mode of binding where CCT may modulate protein activity. The data presented here reveal an additional level of interplay between CCT and actin mediated via gelsolin, suggesting that CCT may influence processes depending on gelsolin activity, such as cell motility.

Over-Expression Analysis of All Eight Subunits of the Molecular Chaperone CCT in Mammalian Cells Reveals a Novel Function for CCTdelta.

Chaperonin containing tailless complex polypeptide 1 (CCT) forms a classical chaperonin barrel structure where two rings of subunits surround a central cavity. Each ring consists of eight distinct subunits, creating a complex binding interface that makes CCT unique among the chaperonins. In addition to acting as a multimeric chaperonin, there is increasing evidence indicating that the CCT subunits, when monomeric, possess additional functions. Here we assess the role of the CCT subunits individually, using a GFP (green fluorescent protein) tagging approach to express each of the subunits in their monomeric form in cultured mammalian cells. Over-expression of CCTdelta, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface. Two point mutations, one in the apical domain and one in the ATP binding pocket of CCTdelta, that abolish protrusion formation have been identified, consistent with the apical domain containing a novel interaction site that is influenced by the ATPase activity in the equatorial domain. Structured illumination microscopy, together with sub-cellular fractionation, reveals that only the wild-type CCTdelta is associated with the plasma membrane, thus connecting spatial organization with surface protrusion formation. Expression of the equivalent subunit in yeast, GFP-Cct4, rescues growth of the temperature-sensitive strain cct4-1 at the non-permissive temperature, indicative of conserved subunit-specific activities for CCTdelta.

A novel function of the monomeric CCTε subunit connects the serum response factor pathway to chaperone-mediated actin folding.

Correct protein folding is fundamental for maintaining protein homeostasis and avoiding the formation of potentially cytotoxic protein aggregates. Although some proteins appear to fold unaided, actin requires assistance from the oligomeric molecular chaperone CCT. Here we report an additional connection between CCT and actin by identifying one of the CCT subunits, CCTε, as a component of the myocardin-related cotranscription factor-A (MRTF-A)/serum response factor (SRF) pathway. The SRF pathway registers changes in G-actin levels, leading to the transcriptional up-regulation of a large number of genes after actin polymerization. These genes encode numerous actin-binding proteins as well as actin. We show that depletion of the CCTε subunit by siRNA enhances SRF signaling in cultured mammalian cells by an actin assembly-independent mechanism. Overexpression of CCTε in its monomeric form revealed that CCTε binds via its substrate-binding domain to the C-terminal region of MRTF-A and that CCTε is able to alter the nuclear accumulation of MRTF-A after stimulation by serum addition. Given that the levels of monomeric CCTε conversely reflect the levels of CCT oligomer, our results suggest that CCTε provides a connection between the actin-folding capacity of the cell and actin expression.