Tissue fluidification promotes a cGAS-STING cytosolic DNA response in invasive breast cancer.

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.

Is cell migration a selectable trait in the natural evolution of cancer development?

Selective evolutionary pressure shapes the processes and genes that enable cancer survival and expansion in a tumour-suppressive environment. A distinguishing lethal feature of malignant cancer is its dissemination and seeding of metastatic foci. A key requirement for this process is the acquisition of a migratory/invasive ability. However, how the migratory phenotype is selected for during the natural evolution of cancer and what advantage, if any, it might provide to the growing malignant cells remain open issues. In this opinion piece, we discuss three possible answers to these issues. We will examine lines of evidence from mathematical modelling of cancer evolution that indicate that migration is an intrinsic selectable property of malignant cells that directly impacts on growth dynamics and cancer geometry. Second, we will argue that migratory phenotypes can emerge as an adaptive response to unfavourable growth conditions and endow cells not only with the ability to move/invade, but also with specific metastatic traits, including drug resistance, self-renewal and survival. Finally, we will discuss the possibility that migratory phenotypes are coincidental events that emerge by happenstance in the natural evolution of cancer. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma.

During wound repair, branching morphogenesis and carcinoma dissemination, cellular rearrangements are fostered by a solid-to-liquid transition, known as unjamming. The biomolecular machinery behind unjamming and its pathophysiological relevance remain, however, unclear. Here, we study unjamming in a variety of normal and tumorigenic epithelial two-dimensional (2D) and 3D collectives. Biologically, the increased level of the small GTPase RAB5A sparks unjamming by promoting non-clathrin-dependent internalization of epidermal growth factor receptor that leads to hyperactivation of the kinase ERK1/2 and phosphorylation of the actin nucleator WAVE2. This cascade triggers collective motility effects with striking biophysical consequences. Specifically, unjamming in tumour spheroids is accompanied by persistent and coordinated rotations that progressively remodel the extracellular matrix, while simultaneously fluidizing cells at the periphery. This concurrent action results in collective invasion, supporting the concept that the endo-ERK1/2 pathway is a physicochemical switch to initiate collective invasion and dissemination of otherwise jammed carcinoma.

From jamming to collective cell migration through a boundary induced transition.

Cell monolayers provide an interesting example of active matter, exhibiting a phase transition from flowing to jammed states as they age. Here we report experiments and numerical simulations illustrating how a jammed cellular layer rapidly reverts to a flowing state after a wound. Quantitative comparison between experiments and simulations shows that cells change their self-propulsion and alignment strength so that the system crosses a phase transition line, which we characterize by finite-size scaling in an active particle model. This wound-induced unjamming transition is found to occur generically in epithelial, endothelial and cancer cells.

A RAB35-p85/PI3K axis controls oscillatory apical protrusions required for efficient chemotactic migration.

How cells move chemotactically remains a major unmet challenge in cell biology. Emerging evidence indicates that for interpreting noisy, shallow gradients of soluble cues a system must behave as an excitable process. Here, through an RNAi-based, high-content screening approach, we identify RAB35 as necessary for the formation of growth factors (GFs)-induced waves of circular dorsal ruffles (CDRs), apically restricted actin-rich migratory protrusions. RAB35 is sufficient to induce recurrent and polarized CDRs that travel as propagating waves, thus behaving as an excitable system that can be biased to control cell steering. Consistently, RAB35 is essential for promoting directed chemotactic migration and chemoinvasion of various cells in response to gradients of motogenic GFs. Molecularly, RAB35 does so by directly regulating the activity of p85/PI3K polarity axis. We propose that RAB35 is a molecular determinant for the control of an excitable, oscillatory system that acts as a steering wheel for GF-mediated chemotaxis and chemoinvasion.

Endocytic reawakening of motility in jammed epithelia.

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.

Bursts of activity in collective cell migration.

Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media, and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems.

The CDC42-interacting protein 4 controls epithelial cell cohesion and tumor dissemination.

The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-ß1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.

A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination.

The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5-dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and ß3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.

TRAIL-induced apoptosis of FHIT-negative lung cancer cells is inhibited by FHIT re-expression.

Fragile histidine triad (FHIT) is a tumor suppressor gene whose allelic loss is associated to a number of human cancers. FHIT protein acts as a diadenosine oligophosphate hydrolase, but its tumor suppressive activity appears as independent from its enzymatic activity. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) can induce apoptosis in the FHIT-negative non-small lung cancer cell line Calu-1. We generated four FHIT-inducible Calu-1 cell clones and demonstrated that FHIT expression was able to protect cells from TRAIL-induced apoptosis, without affecting TRAIL-receptors surface expression. FHIT-specific small interference RNA transfection of SV40-immortalized normal bronchial BEAS cells that show levels of FHIT protein comparable to those of normal bronchial cells, resulted in a significant increase of TRAIL-induced apoptosis. Of note, suramin-mediated inhibition of FHIT enzymatic activity also enhanced TRAIL-induced apoptosis. We conclude that FHIT expression in lung cancer cells is protective from TRAIL-induced apoptosis. Our data suggest that FHIT exerts this protective effect downstream TRAIL-receptors and likely requires its dinucleoside-triphosphate hydrolase activity. As TRAIL represents in the near future a good candidate for death ligands-based anticancer therapy, its potential therapeutic use should be envisaged as preliminary to molecular genetics interventions or drug-induced epigenetic modulations aimed to restoring FHIT gene expression levels in non-small cells lung tumors.

Phorbol ester-induced PKCepsilon down-modulation sensitizes AML cells to TRAIL-induced apoptosis and cell differentiation.

Despite the relevant therapeutic progresses made in these last 2 decades, the prognosis of acute myeloid leukemia (AML) remains poor. Phorbol esters are used at very low concentrations as differentiating agents in the therapy of myeloid leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in turn, is a death ligand that spares normal cells and is therefore currently under clinical trials for cancer therapy. Emerging evidence, however, suggests that TRAIL is also involved in nonapoptotic functions, like cell differentiation. PKCepsilon is differentially modulated along normal hematopoiesis, and its levels modulate the response of hematopoietic precursors to TRAIL. Here, we investigated the effects of the combination of phorbol esters (phorbol ester 4-beta-phorbol-12,13-dibutyrate [PDBu]) and TRAIL in the survival/differentiation of AML cells. We demonstrate here that PDBu sensitizes primary AML cells to both the apoptogenic and the differentiative effects of TRAIL via PKCepsilon down-modulation, without affecting TRAIL receptor surface expression. We believe that the use of TRAIL in combination with phorbol esters (or possibly more specific PKCepsilon down-modulators) might represent a significative improvement of our therapeutic arsenal against AML.

Aberrant expression of B203.13 antigen in acute lymphoid leukemia of B-cell origin.

The B203.13 monoclonal antibody was developed by immunizing mice with the B/monocyte biphenotypic cell line B1b. During normal hematopoiesis B203.13 is expressed on a fraction of CD34+ cells, while on mature cells it is only present on B-lymphocytes. We tested this antibody as a marker of childhood B-acute lymphoblastic leukemia (B-ALL). Bone marrow aspirates from 139 cases of early B-ALL and 25 controls were studied. About 40% of the B-ALL patients expressed B203.13. In these patients, B203.13 was constantly co-expressed with CD10, but never co-expressed with CD20, contrary to the controls. The CD10(+)/B203.13(+) phenotype was specific to B-ALL, since CD10(+)/CD20(+) cells from common acute lymphoblastic leukemia (c-ALL) did not express B203.13. We concluded that the use of B203.13 in association with CD10 and CD20 provides meaningful information for distinguishing normal residual B-cells from leukemic B-lymphoblasts and that recurrence of a CD10(+)/B203.13(+) phenotype after transplantation may be a very early relapse indicator of early B-acute lymphoblastic leukemia.

Timing and expression level of protein kinase C epsilon regulate the megakaryocytic differentiation of human CD34 cells.

Protein kinase C (PKC)-mediated intracellular signaling participates in several key steps of hematopoietic cell differentiation. The epsilon isoform of PKC has been associated with erythroid differentiation as well as with the early phases of megakaryocytic (MK) lineage commitment. Here, we worked on the hypothesis that PKCepsilon expression levels might be modulated during MK differentiation, with a specific role in the early as well as in the late phases of thrombopoiesis. We demonstrate that--at variance with the erythroid lineage development--PKCepsilon is completely downmodulated in TPO-induced CD34 cells from day 6 onward. The forced expression of PKCepsilon in the late phases of MK differentiation delays the phenotypic differentiation of progenitors likely via Bcl-xL upregulation. Moreover, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), known as a negative regulator of early erythroid expansion, is not apoptogenic for thrombopoietin-induced CD34 cells, but rather accelerates their maturation. However, PKCepsilon levels negatively interfere also with the effects of TRAIL in MK differentiation. PKCepsilon can therefore be considered a signaling intermediate whose expression levels are finely tuned, with a virtually opposite kinetic, in erythroid versus megakaryocytic lineages, to adequately respond to the signaling requirements of the specific hematopoietic lineage.

Exogenous hydrogen sulfide induces functional inhibition and cell death of cytotoxic lymphocytes subsets.

The toxic effects of exogenous hydrogen sulfide on peripheral blood lymphocytes have been investigated in detail. Hydrogen sulfide is now considered as a gasotransmitter with specific functional roles in different cell types, like neurons and vascular smooth muscle. Here we show that exogenous hydrogen sulfide induces a caspase-independent cell death of peripheral blood lymphocytes that depends on their intracellular glutathione levels, with a physiologically relevant subset specificity for CD8+ T cells and NK cells. Although lymphocyte activation does not modify their sensitivity to HS-, after 24 h exposure to hydrogen sulfide surviving lymphocyte subsets show a dramatically decreased proliferation in response to mitogens and a reduced IL-2 production. Overall, our data demonstrate that HS- reduces the cellular cytotoxic response of peripheral blood lymphocytes as well as their production of IL-2, therefore de-activating the major players of local inflammatory responses, adding new basic knowledge to the clinically well known anti-inflammatory effects of sulfur compounds.