The role of RAS oncogenes in controlling epithelial mechanics

Activation of RAS oncogenes and the downstream extracellular signal-regulated kinase/mitogen-activated protein kinase pathway alters actomyosin contractility, leading to changes in the mechanical properties of single cells and tissues.Oncogenic RAS alters the ability of cells to sense the stiffness of their environment through changes to cell contractility and substrate adhesion.Oncogenic RAS alters mechanotransduction via the YAP/TAZ signalling pathway.Mechanical changes in RAS-activated cells can drive large-scale deformations in epithelial tissues, including buckling and folding.The balance between elimination and preservation of RAS-transformed cells within a healthy epithelium is influenced by their differential mechanical phenotypes. Mutations in RAS are key oncogenic drivers and therapeutic targets. Oncogenic Ras proteins activate a network of downstream signalling pathways, including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K), promoting cell proliferation and survival. However, there is increasing evidence that RAS oncogenes also alter the mechanical properties of both individual malignant cells and transformed tissues. Here we discuss the role of oncogenic RAS in controlling mechanical cell phenotypes and how these mechanical changes promote oncogenic transformation in single cells and tissues. RAS activation alters actin organisation and actomyosin contractility. These changes alter cell rheology and impact mechanosensing through changes in substrate adhesion and YAP/TAZ-dependent mechanotransduction. We then discuss how these changes play out in cell collectives and epithelial tissues by driving large-scale tissue deformations and the expansion of malignant cells. Uncovering how RAS oncogenes alter cell mechanics will lead to a better understanding of the morphogenetic processes that underlie tumour formation in RAS-mutant cancers. Mutations in RAS are key oncogenic drivers and therapeutic targets. Oncogenic Ras proteins activate a network of downstream signalling pathways, including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K), promoting cell proliferation and survival. However, there is increasing evidence that RAS oncogenes also alter the mechanical properties of both individual malignant cells and transformed tissues. Here we discuss the role of oncogenic RAS in controlling mechanical cell phenotypes and how these mechanical changes promote oncogenic transformation in single cells and tissues. RAS activation alters actin organisation and actomyosin contractility. These changes alter cell rheology and impact mechanosensing through changes in substrate adhesion and YAP/TAZ-dependent mechanotransduction. We then discuss how these changes play out in cell collectives and epithelial tissues by driving large-scale tissue deformations and the expansion of malignant cells. Uncovering how RAS oncogenes alter cell mechanics will lead to a better understanding of the morphogenetic processes that underlie tumour formation in RAS-mutant cancers. RAS was one of the earliest identified human oncogenes [1.Fernández-Medarde A. et al.40 Years of RAS – a historic overview.Genes. 2021; 12: 681Crossref PubMed Scopus (13) Google Scholar] and RAS family genes, KRAS (KRAS4A and KRAS4B), NRAS, or HRAS, the most commonly dysregulated proto-oncogenes in human cancer [2.Sanchez-Vega F. et al.Oncogenic signaling pathways in The Cancer Genome Atlas.Cell. 2018; 173: 321-337.e10Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar]. Ras proteins are small plasma membrane-associated GTPases that, under normal conditions, are activated by extracellular growth factors binding to surface transmembrane receptors [3.Gimple R.C. Wang X. RAS: Striking at the core of the oncogenic circuitry.Front. Oncol. 2019; 9: 965Crossref PubMed Scopus (72) Google Scholar]. Their activation induces multiple downstream signalling pathways, including the extracellular signal-regulated kinase 1/2 (ERK1/2) [4.Lavoie H. et al.ERK signalling: A master regulator of cell behaviour, life and fate.Nat. Rev. Mol. Cell Biol. 2020; 21: 607-632Crossref PubMed Scopus (313) Google Scholar] and phosphatidylinositol 3-kinase (PI3K) cascades [5.Krygowska A.A. Castellano E. PI3K: A crucial piece in the RAS signaling puzzle.Cold Spring Harb. Perspect. Med. 2018; 8a031450Crossref PubMed Scopus (26) Google Scholar], to promote cell growth, cell cycle entry, and cell survival. Oncogenic RAS activation results in the hyperactivation of these pathways in the absence of a ligand or receptor activation to promote signal-independent cell proliferation and ultimately cancer. Thus, components of RAS-activated signalling have long been used as targets of anticancer therapies in the clinic [6.Braicu et al.A comprehensive review on MAPK: A promising therapeutic target in cancer.Cancers. 2019; 11: 1618Crossref PubMed Scopus (402) Google Scholar]. Excitingly, after decades of being considered undruggable, oncogenic KRAS with an oncogenic G12C mutation has recently become a clinically important therapeutic target in its own right following the development of new mutation-specific inhibitors [7.Skoulidis F. et al.Sotorasib for lung cancers with KRAS p.G12C mutation.N. Engl. J. Med. 2021; 384: 2371-2381Crossref PubMed Scopus (526) Google Scholar]. It has long been recognised that, unlike many other oncogenes, RAS signalling also alters the cytoskeleton [8.Soriano O. et al.The crossroads between RAS and RHO signaling pathways in cellular transformation, motility and contraction.Genes. 2021; 12: 819Crossref PubMed Scopus (22) Google Scholar] and cell adhesion [9.Kinbara K. et al.Ras GTPases: Integrins' friends or foes?.Nat. Rev. Mol. Cell Biol. 2003; 4: 767-777Crossref PubMed Google Scholar]. These changes alter both the mechanical properties of cells and their ability to interact with and sense their mechanical environment. While many of these molecular changes have been studied in single cells, the effects on interconnected cells in vivo are profound. Multiple recent studies have demonstrated how RAS-dependent changes in cell mechanics result in large-scale deformations of epithelial tissues, including buckling and folding. These deformations are likely to be crucial in the loss of tissue architecture during tumorigenesis. In this review, we discuss how oncogenic RAS alters cell mechanics and mechanoresponses in single cells and how these changes translate to tissue-level disruption in epithelia and contribute to cancer progression. Cancer cells and tumours have material properties that are very different from those of healthy cells and tissues [10.Guck J. et al.Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence.Biophys. J. 2005; 88: 3689-3698Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar]. It is not clear when during tumorigenesis these internal changes arise and how much they are the product of evolution, because cancer cells adapt through mutation and selection to changes in their environment [11.Vendramin R. et al.Cancer evolution: Darwin and beyond.EMBO J. 2021; 40e108389Crossref PubMed Scopus (61) Google Scholar]. This includes their physical environment because tumours differ mechanically from healthy tissue due to extracellular matrix (ECM) stiffening [12.Kai F. et al.The extracellular matrix modulates the metastatic journey.Dev. Cell. 2019; 49: 332-346Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar], something known to promote invasion and metastasis [13.Acerbi I. et al.Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration.Integr. Biol. 2015; 7: 1120-1134Crossref Google Scholar]. It is also possible that oncogenic signalling itself changes cancer cell mechanics as a prerequisite to cancer cell survival and proliferation within the altered microenvironments they experience during tumour progression [14.Matthews H.K. et al.Oncogenic signaling alters cell shape and mechanics to facilitate cell division under confinement.Dev. Cell. 2020; 52: 563-573.e3Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. Because mutations that alter RAS signalling often occur early in cancer development [15.Saito T. et al.A temporal shift of the evolutionary principle shaping intratumor heterogeneity in colorectal cancer.Nat. Commun. 2018; 9: 2884Crossref PubMed Scopus (71) Google Scholar], early oncogenic RAS mutations could set the stage for future cancer evolution. In support of this idea, oncogenic RAS has been shown to directly alter cell mechanics by altering cytoskeletal organisation and actomyosin contractility [16.Helfman D.M. Pawlak G. Myosin light chain kinase and acto-myosin contractility modulate activation of the ERK cascade downstream of oncogenic Ras.J. Cell. Biochem. 2005; 95: 1069-1080Crossref PubMed Scopus (21) Google Scholar]. Oncogenic KRAS has been shown to increase contractility in mammary epithelial cells and their ability to exert forces on the substrate [17.Panciera T. et al.Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.Nat. Mater. 2020; 19: 797-806Crossref PubMed Scopus (105) Google Scholar]. This was associated with an increase in actomyosin bundles, which are key in the generation and transmission of force. RAS-induced transformation has also been shown to require RhoA [8.Soriano O. et al.The crossroads between RAS and RHO signaling pathways in cellular transformation, motility and contraction.Genes. 2021; 12: 819Crossref PubMed Scopus (22) Google Scholar,18.Sahai E. et al.Cross-talk between Ras and Rho signalling pathways in transformation favours proliferation and increased motility.EMBO J. 2001; 20: 755-766Crossref PubMed Scopus (338) Google Scholar], which increases actomyosin contractility through the downstream effector ROCK and phosphorylation of myosin light chain [19.Amano M. et al.Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1692) Google Scholar]. Consistent with this, the contractile phenotypes seen in studies of RAS-transformed single cells and cell clusters are diminished by ROCK inhibition [17.Panciera T. et al.Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.Nat. Mater. 2020; 19: 797-806Crossref PubMed Scopus (105) Google Scholar,20.Nyga A. et al.Oncogenic RAS instructs morphological transformation of human epithelia via differential tissue mechanics.Sci. Adv. 2021; 7: eabg6467Crossref PubMed Scopus (7) Google Scholar,21.Moruzzi M. et al.Generation of anisotropic strain dysregulates wild-type cell division at the interface between host and oncogenic tissue.Curr. Biol. 2021; 31: 3409-3418.e6Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar]. In addition, signalling pathways downstream of RAS, particularly the ERK/mitogen-activated protein kinase (MAPK) pathway, have been shown to modulate Rho GTPase activity and myosin contractility at multiple levels. Phosphoproteome analyses identified several Rho GTPase–activating proteins and Rho guanine nucleotide exchange factors (Rho GEFs) that are modulated by oncogenic RAS-ERK signalling [22.Kubiniok P. et al.Time-resolved phosphoproteome analysis of paradoxical RAF activation reveals novel targets of ERK.Mol. Cell. Proteomics. 2017; 16: 663-679Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar]. In addition, a downstream substrate of growth factor–induced Ras-ERK signalling, the p90 ribosomal S6 kinase (RSK1 and RSK2), was shown to directly phosphorylate myosin phosphatase-targeting subunit 1 (MYPT1) to regulate cell migration in kidney cell lines [23.Samson S.C. et al.p90 ribosomal S6 kinase (RSK) phosphorylates myosin phosphatase and thereby controls edge dynamics during cell migration.J. Biol. Chem. 2019; 294: 10846-10862Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar]. A similar regulation of cell migration through RSK-MYPT1 and myosin activity was observed in the KRAS-mutant non–small cell lung adenocarcinoma cell line A549 and in the NRAS-mutant fibrosarcoma cell line HT1080 [23.Samson S.C. et al.p90 ribosomal S6 kinase (RSK) phosphorylates myosin phosphatase and thereby controls edge dynamics during cell migration.J. Biol. Chem. 2019; 294: 10846-10862Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar]. In addition, a study of glioblastoma cell lines (RAS wild type) reported a role for serum- and growth factor–induced ERK-RSK2 signalling in changing the cytoskeleton through the activation of RhoA through LARG, a Rho GEF, and actin binding proteins such as filamin A [24.Shi G.-X. et al.RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E190-E199Crossref PubMed Scopus (27) Google Scholar]. RAS-ERK was also shown to promote the nuclear translocation and activity of RSK2, which is required for transformation in this system [25.Cho Y.-Y. et al.Ribosomal S6 kinase 2 is a key regulator in tumor promoter–induced cell transformation.Cancer Res. 2007; 67: 8104-8112Crossref PubMed Scopus (95) Google Scholar]. These data demonstrating an impact of RAS on ROCK and RSK activity provide clear evidence of a direct path from oncogenic RAS-ERK signalling to reorganisation of the actomyosin cytoskeleton and cell contractility. Thus, oncogenic RAS and the deregulation of RAS-ERK signalling alter cell mechanics through multiple different mechanisms (Figure 1), some of which are likely to influence the ability of cells to undergo migration and invasion. Oncogenic RAS has also been shown to alter the material properties of the cytoplasm. Measurements of cytoplasmic viscosity made using particle-tracking microrheology showed a decrease in particle movement within KRAS-transformed MCF10A cells [17.Panciera T. et al.Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.Nat. Mater. 2020; 19: 797-806Crossref PubMed Scopus (105) Google Scholar]. Interestingly, despite this link between oncogenic RAS signalling and increased cytoplasmic viscosity and actomyosin contractility, individual cancer cells have frequently been found to be more compliant than nontransformed cells when mechanically probed [10.Guck J. et al.Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence.Biophys. J. 2005; 88: 3689-3698Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar,26.Xu W. et al.Cell stiffness is a biomarker of the metastatic potential of ovarian cancer cells.PLoS One. 2012; 7e46609Crossref Google Scholar,27.Hayashi K. Iwata M. Stiffness of cancer cells measured with an AFM indentation method.J. Mech. Behav. Biomed. Mater. 2015; 49: 105-111Crossref PubMed Scopus (112) Google Scholar], even in cases where overall tissue stiffness is increased [28.Samuel M.S. et al.Actomyosin-mediated cellular tension drives increased tissue stiffness and β-catenin activation to induce epidermal hyperplasia and tumor growth.Cancer Cell. 2011; 19: 776-791Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar]. Specifically, particle-tracking microrheology of lung adenocarcinoma cells showed that cancer cells within tissues soften, while the surrounding ECM stiffens [29.Panzetta V. et al.Mechanical phenotyping of cells and extracellular matrix as grade and stage markers of lung tumor tissues.Acta Biomater. 2017; 57: 334-341Crossref PubMed Scopus (28) Google Scholar]. In the case of RAS transformation, nontransformed breast epithelial cells (MCF10A) constitutively soften when forced to express oncogenic HRAS [14.Matthews H.K. et al.Oncogenic signaling alters cell shape and mechanics to facilitate cell division under confinement.Dev. Cell. 2020; 52: 563-573.e3Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. Similarly, a 24-h induction of oncogenic HRAS decreased the stiffness of loosely attached or suspended kidney epithelial cells (MDCK) in the absence of cell–cell adhesion [30.Gullekson C. et al.Mechanical mismatch between Ras transformed and untransformed epithelial cells.Soft Matter. 2017; 13: 8483-8491Crossref PubMed Google Scholar]. However, in the same system, this increased the cortical stiffness of the monolayer as a whole. Thus, the effect of RAS on cell stiffness depends on whether RAS-transformed cells are isolated or present within a collective. Because different oncogenic mutations have certain preferences for the downstream effectors, this could also modify the outcome of the RAS signalling network on the regulation of the cell mechanical response [5.Krygowska A.A. Castellano E. PI3K: A crucial piece in the RAS signaling puzzle.Cold Spring Harb. Perspect. Med. 2018; 8a031450Crossref PubMed Scopus (26) Google Scholar]. Cell compliance also depends on the cell adhesion to the substrate and other cells, which is altered by oncogenic mutations in RAS. As an example of this, increasing stiffness of a 3D collagen matrix in which cells are grown resulted in an increase in cytoplasmic elasticity and internal stiffness of single KRAS-mutant metastatic breast cancer cells (MDA-MB-231) measured by monitoring the thermal fluctuations of intracellular tracers with an optical trap [31.Wullkopf L. et al.Cancer cells' ability to mechanically adjust to extracellular matrix stiffness correlates with their invasive potential.Mol. Biol. Cell. 2018; 29: 2378-2385Crossref PubMed Scopus (124) Google Scholar]. Single cells detaching from MDA-MB-231 3D spheroids increased their cytoplasmic viscosity and therefore decreased their internal stiffness to facilitate migration [32.Higgins G. et al.An exploratory study on the role of the stiffness of breast cancer cells in their detachment from spheroids and migration in 3D collagen matrices.bioRxiv. 2022; (Published online April 16, 2022)https://doi.org/10.1101/2021.01.21.427639PubMed Google Scholar]. This suggests a functional role of changes in cell compliance following RAS activation in facilitating processes such as cell migration and metastasis. In addition, specific RAS-dependent changes in cell mechanics have been observed as cells round up and dissociate from the substrate before cell division. Mitotic rounding normally involves an actin-dependent increase in cortical tension [33.Fischer-Friedrich E. et al.Quantification of surface tension and internal pressure generated by single mitotic cells.Sci. Rep. 2015; 4: 6213Crossref Scopus (116) Google Scholar] and cell stiffening [34.Kunda P. et al.Moesin controls cortical rigidity, cell rounding, and spindle morphogenesis during mitosis.Curr. Biol. 2008; 18: 91-101Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar,35.Taubenberger A.V. et al.The mechanics of mitotic cell rounding.Front. Cell Dev. Biol. 2020; 8: 687Crossref PubMed Scopus (50) Google Scholar]. However, the process can be accentuated by RAS transformation [14.Matthews H.K. et al.Oncogenic signaling alters cell shape and mechanics to facilitate cell division under confinement.Dev. Cell. 2020; 52: 563-573.e3Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. As a result, RAS-transformed cells entering mitosis round up better under conditions of physical confinement in a manner that depends on RAS-ERK signalling and the mitogen-activated protein kinase kinase (MEK). Given the important role of the ECM in the regulation of cell mechanics, it is also important to consider the manifold ways in which RAS signalling alters mechanics through its impact on the ECM or cell–ECM attachment. RAS signalling alters the adhesion of cells to the ECM in part by impacting integrin-based substrate attachments at focal adhesion complexes [9.Kinbara K. et al.Ras GTPases: Integrins' friends or foes?.Nat. Rev. Mol. Cell Biol. 2003; 4: 767-777Crossref PubMed Google Scholar,36.Kechagia J.Z. et al.Integrins as biomechanical sensors of the microenvironment.Nat. Rev. Mol. Cell Biol. 2019; 20: 457-473Crossref PubMed Scopus (546) Google Scholar] (Figure 1), altering cell behaviours to aid oncogenesis. As an example of this, in single mammary epithelial cells, oncogenic RAS-generated contractility increases the formation and maturation of focal adhesions and interferes with adhesion-driven mechanosensing through inhibition of focal adhesion kinase (FAK) [17.Panciera T. et al.Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.Nat. Mater. 2020; 19: 797-806Crossref PubMed Scopus (105) Google Scholar]. In fibroblasts, binding of active ERK to focal adhesions by FAK and RACK1 allows their disassembly and facilitates cell migration [37.Vomastek T. et al.RACK1 targets the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility.Mol. Cell. Biol. 2007; 27: 8296-8305Crossref PubMed Scopus (67) Google Scholar]. At the molecular level, ERK associates with paxillin [38.Ishibe S. et al.Paxillin serves as an ERK-regulated scaffold for coordinating FAK and Rac activation in epithelial morphogenesis.Mol. Cell. 2004; 16: 257-267Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar], and transient and sustained RAS-Raf-MEK–mediated ERK activation promotes paxillin phosphorylation [39.Woodrow M. Ras-induced serine phosphorylation of the focal adhesion protein paxillin is mediated by the Raf→MEK→ERK pathway.Exp. Cell Res. 2003; 287: 325-338Crossref PubMed Scopus (25) Google Scholar], which is necessary for its tyrosine phosphorylation and association with FAK at focal adhesions [40.Liu Z.-X. et al.Hepatocyte growth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association.J. Biol. Chem. 2002; 277: 10452-10458Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar]. ERK regulation of the paxillin–FAK complex at focal adhesions also increases the association of FAK with p85, a subunit of PI3K, leading to the activation of downstream kinase Akt. This activation of PI3K results in further activation of Rac GTPases [38.Ishibe S. et al.Paxillin serves as an ERK-regulated scaffold for coordinating FAK and Rac activation in epithelial morphogenesis.Mol. Cell. 2004; 16: 257-267Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar], which play a major role in control of the actin cytoskeleton. Rac activation, together with Cdc42 and myosin II, forms a key response to mechanical stress in KRAS-mutated pancreatic cancer cells, promoting cytoskeletal remodelling, contractility, and migration [41.Kalli M. et al.Mechanical stress signaling in pancreatic cancer cells triggers p38 MAPK- and JNK-dependent cytoskeleton remodeling and promotes cell migration via Rac1/cdc42/myosin II.Mol. Cancer Res. 2022; 20: 485-497Crossref PubMed Scopus (14) Google Scholar]. In the absence of attachment to a substrate anchorage-independent growth of RAS-transformed cells also requires paxillin-regulated FAK phosphorylation [42.Wade R. et al.Paxillin enables attachment-independent tyrosine phosphorylation of focal adhesion kinase and transformation by RAS.J. Biol. Chem. 2011; 286: 37932-37944Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar]. At the same time, active RAS can act as a negative focal adhesion regulator by mediating the dephosphorylation of both paxillin and FAK at the Y397 site, a process that is regulated not by Raf or PI3K but by direct activation of Cdc42 and PAK1 [43.Zheng Y. et al.FAK phosphorylation by ERK primes Ras-induced tyrosine dephosphorylation of FAK mediated by PIN1 and PTP-PEST.Mol. Cell. 2009; 35: 11-25Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar]. Thus, the tightly regulated processes of focal adhesion assembly and disassembly are key to many RAS-induced oncogenic cell behaviours, including cell transformation, migration, and invasion. Because adhesion affects the cytoskeletal remodelling in adherent cells and vice versa, it is hard to dissect the direct cause of RAS-induced changes to the mechanical responses of cells in complex environments. One study looking at the impact of changes in substrate stiffness to the behaviour of KRAS-transformed cells [17.Panciera T. et al.Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.Nat. Mater. 2020; 19: 797-806Crossref PubMed Scopus (105) Google Scholar] showed that cells spread on soft substrates (150 Pa) in an ERK-dependent manner, but not on stiffer substrates (5.7 kPa), closer to those found in fibrotic tumours [44.Plunkett C. et al.H-Ras transformation of mammary epithelial cells induces ERK-mediated spreading on low stiffness matrix.Adv. Healthc. Mater. 2020; 9: 1901366Crossref Scopus (5) Google Scholar]. In this case, the inhibition of myosin by the treatment of cells with blebbistatin did not impact cancer cell spreading, implying a role for myosin-independent regulators of cell spreading, such as cell–substrate adhesion, in this change in cell spreading behaviour. Taken together, these data show that oncogenic RAS has an impact on the material properties of the cytoplasm, the cell cortex, and the extracellular material in a tissue. RAS has also been shown to affect the ability of cells to sense their mechanical environment. Mechanosensation, or mechanosensing, depends in part on integrin-based substrate attachments [36.Kechagia J.Z. et al.Integrins as biomechanical sensors of the microenvironment.Nat. Rev. Mol. Cell Biol. 2019; 20: 457-473Crossref PubMed Scopus (546) Google Scholar] and is important for allowing cells to modify their own stiffness through the reorganisation of the actin cytoskeleton in a manner that is suited to the mechanical environment in which the cells find themselves. It also requires active contractility. As an example of the impact of oncogenic mutations on mechanosensation, one study found that KRAS-transformed cells were more sensitive to changes in substrate stiffness than their nontransformed counterparts [17.Panciera T. et al.Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.Nat. Mater. 2020; 19: 797-806Crossref PubMed Scopus (105) Google Scholar]. This shows that oncogenic signalling does not always disrupt the sensitivity of cancer cells to their environment. RAS-ERK signalling also crosstalks with the YAP/TAZ pathway. YAP/TAZ transcriptional regulators play critical roles in mechanotransduction [45.Dupont S. et al.Role of YAP/TAZ in mechanotransduction.Nature. 2011; 474: 179-183Crossref PubMed Scopus (3479) Google Scholar], and their role in cancer development and progression has been widely studied and previously reviewed [46.Zanconato F. et al.YAP/TAZ at the roots of cancer.Cancer Cell. 2016; 29: 783-803Abstract Full Text Full Text PDF PubMed Scopus (1119) Google Scholar,47.Böttcher R.T. et al.A forceful connection: Mechanoregulation of oncogenic YAP.EMBO J. 2017; 36: 2467-2469Crossref PubMed Scopus (2) Google Scholar]. YAP/TAZ are transcriptional coactivators of the Hippo pathway that shuttle between the cell cytoplasm and nucleus in response to mechanical cues, such as the activation of Rho and cortical tension [45.Dupont S. et al.Role of YAP/TAZ in mechanotransduction.Nature. 2011; 474: 179-183Crossref PubMed Scopus (3479) Google Scholar]. Translocation to the nucleus allows their binding to transcription factors and control of tissue homeostasis through the regulation of cell proliferation, apoptosis, and stem cell renewal. The induction of YAP/TAZ nuclear shuttling by oncogenic RAS suggests a direct link between the RAS and Hippo signalling pathways and a possible synergistic role in oncogenic transformation. This is of particular importance because experiments in mice have shown that relapsed KRAS pancreatic tumours have activated YAP1/TEAD2 transcriptional programs that are required for tumour growth [48.Kapoor A. et al.Yap1 activation enables bypass of oncogenic Kras addiction in pancreatic cancer.Cell. 2014; 158: 185-197Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar]. In addition, the overexpression of YAP1 has been shown to clinically correlate with metastasis and poor prognoses in patients with pancreatic ductal adenocarcinoma (PDAC) [49.Salcedo Allende M.T. et al.Overexpression of Yes associated protein 1, an independent prognostic marker in patients with pancreatic ductal adenocarcinoma, correlated with liver metastasis and poor prognosis.Pancreas. 2017; 46: 913-920Crossref PubMed Scopus (41) Google Scholar]. YAP is also translocated to the nuclei of cells in response to their exposure to a stiffer microenvironment and following a direct application of force on the cell nucleus. In both cases, this results in nuclear flattening, which increased the passive transport through nuclear pores, perhaps as the result of mechanically induced nuclear pore dilation [50.Elosegui-Artola A. et al.Force triggers YAP nuclear entry by regulating transport across nuclear pores.Cell. 2017; 171: 1397-1410.e14Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar]. Whether cell spreading following RAS activation results in the flattening of the nucleus and associated increase in YAP translocation is still not clear. However, in one study, it was shown that once YAP has been activated on substrates stiffer than 1 kPa, ERK inhibition no longer impacts cell spreading. This implies that the two pathways