Anchorage-independent cell proliferation promoted by fascin's F-actin bundling.

The actin filament (F-actin) bundling protein fascin-1 is highly enriched in many metastatic cancers. Fascin's contribution to metastasis have been ascribed to its enhancement of cell migration and invasion. However, mouse genetic studies clearly point to functions also in tumorigenesis, yet without mechanistic underpinnings. Here, we show that fascin expression promotes the formation of a non-canonical signaling complex that enables anchorage-independent proliferation. This complex shares similarities to focal adhesions and we refer to them as pseudo-adhesion signaling scaffolds (PASS). PASS are enriched with tyrosine phosphorylated proteins and require fascin's F-actin-bundling activity for its assembly. PASS serve as hubs for the Rac1/PAK/JNK proliferation signaling axis, driven by PASS-associated Rac-specific GEFs. Experimental disruption of either fascin or RacGEF function abrogates sustained proliferation of aggressive cancers in vitro and in vivo . These results add a new molecular element to the growing arsenal of metabolic and oncogenic signaling programs regulated by the cytoskeleton architecture.

RhoA activation promotes glucose uptake to elevate proliferation in MAPK inhibitor resistant melanoma cells.

Cutaneous melanomas harboring a B-RafV600E mutation are treated with immune check point inhibitors or kinase inhibitor combination therapies relying on MAPK inhibitors (MAPKi) Dabrafenib and Trametinib (Curti and Faries, 2021). However, cells become resistant to treatments over the timespan of a few months. Resistance to MAPKi has been associated with adoption of an aggressive amoeboid phenotype characterized by elevated RhoA signaling, enhanced contractility and thick cortical filamentous actin (F-actin) structures (Kim et al., 2016; Misek et al., 2020). Targeting active RhoA through Rho-kinase (ROCK) inhibitors, either alone or in combination with immunotherapies, reverts MAPKi-resistance (Misek et al., 2020; Orgaz et al., 2020). Yet, the mechanisms for this behavior remain largely unknown. Given our recent findings of cytoskeleton's role in cancer cell proliferation (Mohan et al., 2019), survival (Weems et al., 2023), and metabolism (Park et al., 2020), we explored possibilities by which RhoA-driven changes in cytoskeleton structure may confer resistance. We confirmed elevated activation of RhoA in a panel of MAPKi-resistant melanoma cell lines, leading to a marked increase in the presence of contractile F-actin bundles. Moreover, these cells had increased glucose uptake and glycolysis, a phenotype disrupted by pharmacological perturbation of ROCK. However, glycolysis was unaffected by disruption of F-actin bundles, indicating that glycolytic stimulation in MAPKi-resistant melanoma is independent of F-actin organization. Instead, our findings highlight a mechanism in which elevated RhoA signaling activates ROCK, leading to the activation of insulin receptor substrate 1 (IRS1) and P85 of the PI3K pathway, which promotes cell surface expression of GLUT1 and elevated glucose uptake. Application of ROCK inhibitor GSK269962A results in reduced glucose uptake and glycolysis, thus impeding cell proliferation. Our study adds a mechanism to the proposed use of ROCK inhibitors for long-term treatments on MAPKi-resistant melanomas.

Actin-Membrane Release Initiates Cell Protrusions.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.

Mechanical regulation of glycolysis via cytoskeleton architecture.

The mechanics of the cellular microenvironment continuously modulates cell functions such as growth, survival, apoptosis, differentiation and morphogenesis via cytoskeletal remodelling and actomyosin contractility1-3. Although all of these processes consume energy4,5, it is unknown whether and how cells adapt their metabolic activity to variable mechanical cues. Here we report that the transfer of human bronchial epithelial cells from stiff to soft substrates causes a downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by the disassembly of stress fibres, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small-cell lung cancer cells, which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress-fibre subset that is insensitive to substrate stiffness. Our data reveal a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, thus coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to tune energy production in variable microenvironments, whereas the resistance of the cytoskeleton in response to mechanical cues enables the persistence of high glycolytic rates in cancer cells despite constant alterations of the tumour tissue.

Robust and automated detection of subcellular morphological motifs in 3D microscopy images.

Rapid developments in live-cell three-dimensional (3D) microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, tools to systematically measure and visualize the intricate relationships between intracellular signaling, cytoskeletal organization and downstream cell morphological outputs do not exist. Here, we introduce u-shape3D, a computer graphics and machine-learning pipeline to probe molecular mechanisms underlying 3D cell morphogenesis and to test the intriguing possibility that morphogenesis itself affects intracellular signaling. We demonstrate a generic morphological motif detector that automatically finds lamellipodia, filopodia, blebs and other motifs. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This indicates that subcellular signaling processes are differentially modulated by local morphological motifs. Overall, our computational workflow enables the objective, 3D analysis of the coupling of cell shape and signaling.

Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1P29S Melanoma Cells.

Actin assembly supplies the structural framework for cell morphology and migration. Beyond structure, this actin framework can also be engaged to drive biochemical signaling programs. Here, we describe how the hyperactivation of Rac1 via the P29S mutation (Rac1P29S) in melanoma hijacks branched actin network assembly to coordinate proliferative cues that facilitate metastasis and drug resistance. Upon growth challenge, Rac1P29S-harboring melanoma cells massively upregulate lamellipodia formation by dendritic actin polymerization. These extended lamellipodia form a signaling microdomain that sequesters and phospho-inactivates the tumor suppressor NF2/Merlin, driving Rac1P29S cell proliferation in growth suppressive conditions. These biochemically active lamellipodia require cell-substrate attachment but not focal adhesion assembly and drive proliferation independently of the ERK/MAPK pathway. These data suggest a critical link between cell morphology and cell signaling and reconcile the dichotomy of Rac1's regulation of both proliferation and actin assembly by revealing a mutual signaling axis wherein actin assembly drives proliferation in melanoma.

SMIFH2 has effects on Formins and p53 that perturb the cell cytoskeleton.

Formin proteins are key regulators of the cytoskeleton involved in developmental and homeostatic programs, and human disease. For these reasons, small molecules interfering with Formins' activity have gained increasing attention. Among them, small molecule inhibitor of Formin Homology 2 domains (SMIFH2) is often used as a pharmacological Formin blocker. Although SMIFH2 inhibits actin polymerization by Formins and affects the actin cytoskeleton, its cellular mechanism of action and target specificity remain unclear. Here we show that SMIFH2 induces remodelling of actin filaments, microtubules and the Golgi complex as a result of its effects on Formins and p53. We found that SMIFH2 triggers alternated depolymerization-repolymerization cycles of actin and tubulin, increases cell migration, causes scattering of the Golgi complex, and also cytotoxicity at high dose. Moreover, SMIFH2 reduces expression and activity of p53 through a post-transcriptional, proteasome-independent mechanism that influences remodelling of the cytoskeleton. As the action of SMIFH2 may go beyond Formin inhibition, only short-term and low-dose SMIFH2 treatments minimize confounding effects induced by loss of p53 and cytotoxicity.

Proteomic analyses uncover a new function and mode of action for mouse homolog of Diaphanous 2 (mDia2).

mDia2 is an auto-inhibited Formin influencing actin dynamics upon conversion to the active conformation. mDia2 regulates actin-based protrusions and cell invasion, cell differentiation, vesicle trafficking, and cytokinesis. However, whether mDia2 has additional functions and how its action is functionally specified remain unknown. Here we draw the interactome of auto-inhibited and constitutively active mDia2 to address these issues. We embed mDia2 in protein networks accounting for its attributed functions and unexpectedly link it to the Ubiquitin Proteasome System. Taking FBXO3 as a test case, we show that mDia2 binds FBXO3 and p53, and regulates p53 transcriptional activity in an actin-nucleation-independent and conformation-insensitive manner. Increased mDia2 and FBXO3 levels elevate p53 activity and expression thereby sensitizing cells to p53-dependent apoptosis, whereas their decrease produces opposite effects. Thus, we discover a new role of mDia2 in p53 regulation suggesting that the closed conformation is biologically active and an FBXO3-based mechanism to functionally specify mDia2's activity.