Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53.

Although the E3 ligase Mdm2 and its homologue and binding partner MdmX are the major regulators of the p53 tumor suppressor protein, it is now evident that Mdm2 and MdmX have multiple functions that do not involve p53. As one example, it is known that Mdm2 can regulate cell migration, although mechanistic insight into this function is still lacking. Here we show in cells lacking p53 expression that knockdown of Mdm2 or MdmX, as well as pharmacological inhibition of the Mdm2/MdmX complex, not only reduces cell migration and invasion, but also impairs cell spreading and focal adhesion formation. In addition, Mdm2 knockdown decreases metastasis in vivo. Interestingly, Mdm2 downregulates the expression of Sprouty4, which is required for the Mdm2 mediated effects on cell migration, focal adhesion formation and metastasis. Further, our findings indicate that Mdm2 dampening of Sprouty4 is a prerequisite for maintaining RhoA levels in the cancer cells that we have studied. Taken together we describe a molecular mechanism whereby the Mdm2/MdmX complex through Sprouty4 regulates cellular processes leading to increase metastatic capability independently of p53.

DISC-3D: dual-hydrogel system enhances optical imaging and enables correlative mass spectrometry imaging of invading multicellular tumor spheroids.

Multicellular tumor spheroids embedded in collagen I matrices are common in vitro systems for the study of solid tumors that reflect the physiological environment and complexities of the in vivo environment. While collagen I environments are physiologically relevant and permissive of cell invasion, studying spheroids in such hydrogels presents challenges to key analytical assays and to a wide array of imaging modalities. While this is largely due to the thickness of the 3D hydrogels that in other samples can typically be overcome by sectioning, because of their highly porous nature, collagen I hydrogels are very challenging to section, especially in a manner that preserves the hydrogel network including cell invasion patterns. Here, we describe a novel method for preparing and cryosectioning invasive spheroids in a two-component (collagen I and gelatin) matrix, a technique we term dual-hydrogel in vitro spheroid cryosectioning of three-dimensional samples (DISC-3D). DISC-3D does not require cell fixation, preserves the architecture of invasive spheroids and their surroundings, eliminates imaging challenges, and allows for use of techniques that have infrequently been applied in three-dimensional spheroid analysis, including super-resolution microscopy and mass spectrometry imaging.

Li-Fraumeni Syndrome-Associated Dimer-Forming Mutant p53 Promotes Transactivation-Independent Mitochondrial Cell Death.

Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li-Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein. SIGNIFICANCE: A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches. See related commentary by Stieg et al., p. 1046. See related article by Gencel-Augusto et al., p. 1230. This article is highlighted in the In This Issue feature, p. 1027.

Delineating the role of membrane blebs in a hybrid mode of cancer cell invasion in three-dimensional environments.

The study of cancer cell invasion in 3D environments in vitro has revealed a variety of invasive modes, including amoeboid migration, characterized by primarily round cells that invade in a protease- and adhesion-independent manner. Here, we delineate a contractility-dependent migratory mode of primarily round breast cancer cells that is associated with extensive integrin-mediated extracellular matrix (ECM) reorganization that occurs at membrane blebs, with bleb necks sites of integrin clustering and integrin-dependent ECM alignment. We show that the spatiotemporal distribution of blebs and their utilization for ECM reorganization is mediated by functional ß1 integrin receptors and other components of focal adhesions. Taken together, the work presented here characterizes a migratory mode of primarily round cancer cells in complex 3D environments and reveals a fundamentally new function for membrane blebs in cancer cell invasion.

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

A novel 3D in vitro metastasis model elucidates differential invasive strategies during and after breaching basement membrane.

Invasive breast cancer and other tumors of epithelial origin must breach a layer of basement membrane (BM) that surrounds the primary tumor before invading into the adjacent extracellular matrix. To analyze invasive strategies of breast cancer cells during BM breaching and subsequent invasion into a collagen I-rich extracellular matrix (ECM), we developed a physiologically relevant 3D in vitro model that recreates the architecture of a solid tumor with an intact, degradable, cell-assembled BM layer embedded in a collagen I environment. Using this model we demonstrate that while the BM layer fully prevents dissemination of non-malignant cells, cancer cells are capable of breaching it and invading into the surrounding collagen, indicating that the developed system recreates a hallmark of invasive disease. We demonstrate that cancer cells exhibiting individual invasion in collagen matrices preferentially adopt a specific mode of collective invasion when transmigrating a cell-assembled BM that is not observed in any other tested fibrillar, non-fibrillar, or composite ECM. Matrix-degrading enzymes are found to be crucial during BM breaching but not during subsequent invasion in the collagen matrix. It is further shown that multicellular transmigration of the BM is less susceptible to pharmacological MMP inhibition than multicellular invasion in composite collagen/basement membrane extract matrices. The newly developed in vitro model of metastasis allows 3D cancer cell invasion to be studied not only as a function of a particular tumor's genetics but also as a function of its heterogeneous environment and the different stages of invasion. As such, this model is a valuable new tool with which to dissect basic mechanisms of invasion and metastasis and develop new therapeutic approaches in a physiologically relevant, yet inexpensive and highly tunable, in vitro setting.

Repression of p63 and induction of EMT by mutant Ras in mammary epithelial cells.

The p53-related transcription factor p63 is required for maintenance of epithelial cell differentiation. We found that activated forms of the Harvey Rat Sarcoma Virus GTPase (H-RAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) oncogenes strongly repress expression of ∆Np63α, the predominant p63 isoform in basal mammary epithelial cells. This regulation occurs at the transcriptional level, and a short region of the ∆Np63 promoter is sufficient for repression induced by H-RasV12. The suppression of ∆Np63α expression by these oncogenes concomitantly leads to an epithelial-to-mesenchymal transition (EMT). In addition, the depletion of ∆Np63α alone is sufficient to induce EMT. Both H-RasV12 expression and ∆Np63α depletion induce individual cell invasion in a 3D collagen gel in vitro system, thereby demonstrating how Ras can drive the mammary epithelial cell state toward greater invasive ability. Together, these results suggest a pathway by which RAS and PIK3CA oncogenes induce EMT through regulation of ∆Np63α.

Breast Cancer Cell Line Aggregate Morphology Does Not Predict Invasive Capacity.

To invade and metastasize to distant loci, breast cancer cells must breach the layer of basement membrane surrounding the tumor and then invade through the dense collagen I-rich extracellular environment of breast tissue. Previous studies have shown that breast cancer cell aggregate morphology in basement membrane extract correlated with cell invasive capacity in some contexts. Moreover, cell lines from the same aggregate morphological class exhibited similarities in gene expression patterns. To further assess the capacity of cell and aggregate morphology to predict invasive capacity in physiologically relevant environments, six cell lines with varied cell aggregate morphologies were assessed in a variety of assays including a 3D multicellular invasion assay that recapitulates cell-cell and cell-environment contacts as they exist in vivo in the context of the primary breast tumor. Migratory and invasive capacities as measured through a 2D gap assay and a 3D spheroid invasion assay reveal that breast cancer cell aggregate morphology alone is insufficient to predict migratory speed in 2D or invasive capacity in 3D. Correlations between the 3D spheroid invasion assay and gene expression profiles suggest this assay as an inexpensive functional method to predict breast cancer invasive capacity.

The effect of fibrillar matrix architecture on tumor cell invasion of physically challenging environments.

Local invasion by and dissemination of cancer cells from a primary tumor are key initial steps of metastasis, the most lethal aspect of cancer. To study these processes in vitro, the invasion of cells from multicellular breast cancer aggregates embedded in three-dimensional (3D) extracellular matrix culture systems was studied. This work showed that in 3D fibrillar environments composed of collagen I, pore size--not the viscoelastic properties of the matrix--was the biophysical characteristic controlling breast cancer cell invasion efficiency. Furthermore, it was shown that fibrillar matrix architecture is a crucial factor that allows for efficient 3D invasion. In a 3D non-fibrillar environment composed of basement membrane extract (BME), invasion efficiency was greatly diminished, the mesenchymal individual mode of 3D invasion was abolished, and establishment of cell polarity and protrusions was compromised. These effects were seen even though the BME matrix has invasion permissive viscoelasticity and suitable adhesion ligands. The altered and limited invasive behavior observed in BME was rescued through introduction of fibrillar collagen into the non-fibrillar matrix. The biophysical cues of fibrillar collagen facilitated efficient invasion of sterically disadvantageous environments through assisting cell polarization and formation of stable cell protrusions. Finally, we suggest the composite matrices employed in this study consisting of fibrillar collagen I and BME in either a liquid-like or gelled state are suitable for a wide range of 3D cell studies, as these matrices combine fibrillar features that require cells to deploy integrin-dependent mechanotransduction machinery and a tunable non-fibrillar component that may require cells to adopt alternative migratory modes.

SMAD versus non-SMAD signaling is determined by lateral mobility of bone morphogenetic protein (BMP) receptors.

Bone (or body) morphogenetic proteins (BMPs) belong to the TGFß superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.

Comprehensive analysis of TGF-ß and BMP receptor interactomes.

An immense number of cellular processes are initiated by cell surface serine/threonine kinase receptors belonging to the TGF-ß/BMP family. Subsequent downstream signalling cascades, as well as their crosstalk results in enormous specificity in terms of phenotypic outcome, e.g. proliferation, differentiation, migration or apoptosis. Such signalling diversity is achieved by the ability of receptors to interact with distinct proteins in a spatio-temporal manner. Following the cloning of the TGF-ß/BMP receptors a variety of different technologies were applied to identify such interacting proteins. Here we present a comprehensive survey of known interactome analyses, including our own data, on these receptors and discuss advantages and disadvantages of the applied technologies.

Noggin.

Metabologens initiate, promote and maintain morphogenesis and adult tissue homeostasis. Bone morphogenetic proteins (BMPs) which belong to the transforming growth factor-ß (TGF-ß) superfamily, represent a major class of metabologens that regulate ectoderm, mesoderm and endoderm derived tissue formation. In order to temporally and spatially control BMP initiated signaling cascades, a tight regulatory network is needed to maintain concinnity. There are a number of ways how BMP signaling can be inhibited or more likely be modified, among which the direct extracellular inhibition through cysteine-knot containing proteins from the DAN-, the twisted gastrulation-, chordin- and noggin-family is a classic. This review focuses on noggin and its impact on the vast array of BMP driven actions and thereby invites the ever-growing BMP research field to (re-) investigate noggin's function in detail.

Glutamate receptor dynamics organizing synapse formation in vivo.

Insight into how glutamatergic synapses form in vivo is important for understanding developmental and experience-triggered changes of excitatory circuits. Here, we imaged postsynaptic densities (PSDs) expressing a functional, GFP-tagged glutamate receptor subunit (GluR-IIA(GFP)) at neuromuscular junctions of Drosophila melanogaster larvae for several days in vivo. New PSDs, associated with functional and structural presynaptic markers, formed independently of existing synapses and grew continuously until reaching a stable size within hours. Both in vivo photoactivation and photobleaching experiments showed that extrasynaptic receptors derived from diffuse, cell-wide pools preferentially entered growing PSDs. After entering PSDs, receptors were largely immobilized. In comparison, other postsynaptic proteins tested (PSD-95, NCAM and PAK homologs) exchanged faster and with no apparent preference for growing synapses. We show here that new glutamatergic synapses form de novo and not by partitioning processes from existing synapses, suggesting that the site-specific entry of particular glutamate receptor complexes directly controls the assembly of individual PSDs.