Ha-RasV12-Induced Multilayer Cellular Aggregates Is Mediated by Rac1 Activation Rather Than YAP Activation

We demonstrate that Ha-RasV12 overexpression induces the nuclear translocation of Hippo effector Yes-associated protein (YAP) in MDCK cells via the hippo-independent pathway at the confluent stage. Ha-RasV12 overexpression leads to the downregulation of Caveolin-1 (Cav1) and the disruption of junction integrity. It has been shown that the disruption of actin belt integrity causes YAP nuclear translocation in epithelial cells at high density. Therefore, we hypothesized that Ha-RasV12-decreased Cav1 leads to the disruption of cell junction integrity, which subsequently facilitates YAP nuclear retention. We revealed that Ha-RasV12 downregulated Cav1 through the ERK pathway. Furthermore, the distribution and expression of Cav1 mediated the cell junction integrity and YAP nuclear localization. This suggests that the downregulation of Cav1 induced by Ha-RasV12 disrupted the cell junction integrity and promoted YAP nuclear translocation. We further indicated the consequence of Ha-RasV12-induced YAP activation. Surprisingly, the activation of YAP is not required for Ha-RasV12-induced multilayer cellular aggregates. Instead, Ha-RasV12 triggered the ERK-Rac pathway to promote cellular aggregate formation. Moreover, the overexpression of constitutively active Rac is sufficient to trigger cellular aggregation in MDCK cells at the confluent stage. This highlights that Rac activity is essential for cellular aggregates.


Introduction
Ras is a small GTPase containing three isoforms, including Harvey-Ras (Ha-Ras), Kirsten-Ras (K-Ras), and N-Ras. More than 30% of human cancers are driven by one or multiple point mutations of the Ras gene [1]. The mutation of Ras at position 12 from glycine to valine results in a GTP-bound state, which is constantly active to trigger the downstream signaling for cell proliferation, cell invasion, cell migration, and cytoskeleton organization, so as to cause cell tumorigenesis [2,3]. The Ha-Ras V12 mutant has been observed in many human cancers, especially in head and neck squamous cell carcinoma and bladder cancer [4].
Around 90% of human cancer is from epithelial tissue. Oncogenes, such as Ras, promote the regulation of the epithelial-mesenchymal transition (EMT) in epithelial cells [5]. To maintain the properties and function of epithelial cells, cell junctions play an important role. In epithelial cells, the intracellular force is provided by actin and actomyosin cytoskeletons, which form stress fibers and adhesion belts on cell-cell contacts and cell-ECM sites [6]. In epithelial cells, there are two types of heavy chains in myosin II, including myosin IIA and myosin IIB. They also have diverse functions in the regulation of cell morphology, cell polarity, and cell orientation [7]. Myosin II has also been reported to contribute to cancer progression. The absence of myosin IIB induces cell proliferation and anti-apoptosis through the upregulation of nuclear YAP in epithelial cells [8].

Plasmids, shRNA, siRNA, and Transfection
The Caveolin-1-Myc-mRFP plasmid was kindly gifted by Dr. Ivan R Nabi [29]. The short hairpin RNA (shRNA) constructs, such as shLacZ, shCav1 #1, and shCav1 #2, were generated as previously described [22]. Constitutively active and dominant-negative Rhofamily GTPase (Rac1) were gifted by Dr. Bing-Cheng Wang [30]. The transfection of the plasmid was performed as previously described [22]. To generate clones expressing stable Cav1, flow cytometry was used to sort the cells to enrich the mRFP-positive cells. Geneticin (G418, Sigma, St. Louis, MO, USA) was also used to select clones expressing stable plasmids. Lentiviral shRNA vectors were used to silence genes as previously described [22]. Cells were selected by using puromycin (Sigma).

RT-PCR
TRIzol reagent (Life Technologies, Carlsbad, CA, USA) was used to extract the total RNA from cells following the manufacturer's instructions. The RNA quality was verified by NanoDrop (Thermo Scientific, Waltham, MA, USA) and reverse-transcribed by using the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer's protocol (Thermo Scientific). cDNA was used as a template for PCR using specific primers for the following genes: dog CTGF (forward: 5 -GAG GAA CTG TGT ACC GGA GC-3 ; reverse: 5 -AAC AGG CAC TCC ACT CTG TG-3 ); dog CYR61 (forward: 5 -CCC AGT TTT TGG ACG GAG C-3 ; reverse: 5 -CAT TTC TTG CCC TTC AGG CT-3 ) and β-actin (forward: 5 -ACT GGG ACG ACA TGG AGA AG-3 ; and reverse: 5 -GGT ACG ACC AGA GGC ATA CAG-3 ). PCR was performed as previously described [22].

Cell Fractionation
To obtain protein from different cell fractions, we used the REAP (rapid, efficient, and practical) method, which is a 2 min cell fractionation method [32]. Cells (2 × 10 6 ) were seeded in a 10 cm dish in an overconfluent experiment. The cells were rinsed with PBS and collected in 1 mL of PBS. After centrifuging at 12,000 rpm for 10 s, the supernatant was removed. Cell pellets were suspended in 0.1% NP40. Lysate (300 µL) was collected as "whole cell lysate", and further lysed using lysis buffer. The remaining lysate (700 µL) was centrifuged at 12,000 rpm for 10 s. Then, the supernatants of the lysate as the "cytosolic fraction" and cell pellet of the lysate as the "nuclear fraction" were treated with lysis buffer in order to quantify the cell concentrations for further Western immunoblot analysis.

Pull-Down Assay
To obtain the Rac1 activity, we used the Rac1 Activation Assay Kit (Merck Millipore, MA, USA). For the overconfluent experiment, 2 × 10 6 cells were plated in a 10 cm dish. After rinsing the cells with PBS, the cells were harvested with 1 mL ice-cold Mg 2+ lysis/wash buffer (MLB) (containing 10% glycerol, 10 µL/mL leupeptin, and 10 µL/mL aprotinin). After quantifying the cell lysate concentration, 10 µL of Rac assay reagent (PAK-1 PBD, agarose) was applied to 2.5 mg of the protein sample. Then, the reaction mixture was gently agitated for 1 h at 4 • C. Next, the agarose beads were collected by centrifuging at 14,000× g Biomedicines 2022, 10, 977 4 of 16 for 10 s. After washing the beads three times with MLB buffer, the sample underwent Western immunoblot analysis.

Crystal Violet Staining
Cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 1 h at room temperature. The paraformaldehyde was washed away with PBS and the cells were stained with 0.025% crystal violet for 10 min. The cells were washed with PBS until they were clear. Phase-contrast images were captured by inverted microscopy (Leica DM IRB) with a 10× lens. The area of multilayer cellular aggregates was analyzed using ImageJ software.

Anchorage-Independent Growth Assay
The ability of anchorage-independent growth was determined by the number of colony formations in the soft agar system. The soft agar system contained 1.5 mL of base agar (0.5% low-melting agarose, 1× high-glucose DMEM, and 5% CCS) suspended with 5 × 10 3 cells in 1.5 mL of top agarose (0.35% low-melting agarose, 1× high-glucose DMEM, and 5% CCS) in a 35 mm dish. After solidification, 2 mL of high-glucose DMEM supplemented with 5% CCS was added to the soft agar, then incubated under a 5% CO 2 atmosphere at 37 • C. The culture medium was replenished every three days. After 14 days, the sample was fixed with 4% paraformaldehyde overnight. Then, the sample was stained with 0.025% crystal violet for a further 1 h. The soft agar was washed with PBS until it became clear. Images were taken using a camera (Alphaimager 2200, Alpha Innotech Co., San Leandro, CA, USA).

Statistical Analysis
Data are expressed as the mean ± S.E.M. and are representative of experiments performed at least three times independently. The quantification results of YAP nuclear localization were defined by the percentage of cells with predominant nuclear YAP localization (N > C, >110%), equal nuclear and cytosol YAP localization (N = C, 90-110%), and predominant cytosol YAP localization (N < C, <90%). The definition of YAP was based on the expression intensity of YAP in the nucleus divided by the expression intensity of YAP in the cytosol. We set a small range (0.9-1.1) as the equal nuclear and cytosol YAP localization. Values outside this range were either classified as predominant nuclear YAP localization or predominant cytosol YAP localization. GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used to calculate the plot mean and standard error of the mean (SEM) of the measured quantities, and significances were assessed by one-way ANOVA. The results were analyzed via ANOVA and t-tests. Statistical significance was determined with p-values < 0.05.

Ha-Ras V12 Facilitates YAP Nuclear Translocation through Downregulated Junctional Myosin IIB
Our previous studies demonstrated that Ha-Ras V12 overexpression results in the cell softening and transformation of MK4 cells (MDCK harboring inducible Ha-Ras V12 gene) [33]. After reaching confluence (>2 days after cell plating), MK4 cells displayed cytoplasmic YAP and well-organized actin belt formation. Upon IPTG induction, the overexpression of Ha-Ras V12 induced YAP nuclear translocation and disrupted the actin belt integrity (Figure 1a-c). Western blot analysis of nuclear/cytoplasmic fractionations confirmed that Ha-Ras V12 overexpression caused the dephosphorylation and nuclear translocation of YAP ( Figure S1a,b). Consequently, the expression of YAP-TEAD downstream genes, such as CTGF, was upregulated in Ha-Ras V12 -overexpressed cells (Figure 1d,e). However, Hippo pathway-associated components were not downregulated after Ha-Ras V12 induction ( Figure S1c,d), suggesting that the Hippo pathway is irrelevant to Ha-Ras V12 -induced YAP activation in MK4 cells at the overconfluent stage. Non-muscle myosin II is a well-known component of the actin belt, which is a dense circumferential belt of actin filaments located on the cytoplasmic face of the adherens junction [7]. Furukawa et al. demonstrated that the global inhibition of myosin with blebbistatin disrupted the actomyosin tension of the actin belt and led to YAP nuclear translocation in high-density cells [8]. To understand whether myosin II is involved in Ha-Ras V12 -induced YAP nuclear translocation in MDCK cells at the confluent stage, we assessed the expression and distribution of myosin IIA and myosin IIB. In normal MK4 cells, myosin IIA was present in the cytosol and myosin IIB was lined with the junctional actin and located at the adherens junction ( Figure 1c and Figure S2). The induction of Ha-Ras V12 did not change the protein levels of myosin IIA and myosin IIB (Figure 1f,g) and the distribution of myosin IIA ( Figure S2a). Meanwhile, the junctional actin belts were disrupted and accompanied by dislocated myosin IIB (Figure 1c). Myosin IIB dislocated from the cell junction to the cytosol after Ha-Ras V12 induction. To further confirm the role of myosin IIB-related actomyosin tension in regulating YAP nuclear translocation, we used myosin II inhibitors, including ML7 (myosin II lightchain kinase inhibitor) and blebbistatin (myosin II heavy-chain inhibitor). As shown in Figure 2a,b, the treatment with blebbistatin did not destroy the junctional actin structure in MK4 cells. Both ML7 and blebbistatin caused the redistribution of myosin IIB from the junction to the cytosol ( Figure 2a) and enhanced nuclear YAP translocation (Figure 2b,c). Taken together, these data revealed that the overexpression of Ha-Ras V12 suppressed junctional myosin IIB, which might be relevant for YAP nuclear translocation.

Cav1 Preserved Junctional Myosin IIB and Restricted Ha-Ras V12 -Induced YAP Nuclear Translocation
Caveolin-1 plays an essential role in maintaining cell junction integrity by recruiting the E-cadherin-catenin complex to caveolae [25]. Our previous study showed that Cav1 was downregulated upon Ha-Ras V12 induction. Moreover, Cav1 abolishes not only Ha-Ras V12 -induced cell scattering, but also the disorganization of junctional protein [22]. In this study, we first knocked down Cav1 with specific shRNA in both MDCK and MK4 cells. The Western blot results showed the knockdown efficiency of Cav1 in MK4 and MDCK cells ( Figure S3a,b). Specifically, the results showed that the knockdown efficiency of Cav1 was higher in MK4 cells compared to that in MDCK cells. The depletion of Cav1 decreased the junctional myosin IIB expression and disturbed the actin belt integrity in MK4 cells (Figure 3a). In contrast, the downregulation of Cav1 in MDCK only resulted in slightly junctional myosin IIB downregulation and actin belt disruption ( Figure S3d). Meanwhile, nuclear YAP retention became prominent at the confluent stage in both

Cav1 Preserved Junctional Myosin IIB and Restricted Ha-Ras V12 -Induced YAP Nuclear Translocation
Caveolin-1 plays an essential role in maintaining cell junction integrity by recruiting the E-cadherin-catenin complex to caveolae [25]. Our previous study showed that Cav1 was downregulated upon Ha-Ras V12 induction. Moreover, Cav1 abolishes not only Ha-Ras V12 -induced cell scattering, but also the disorganization of junctional protein [22]. In this study, we first knocked down Cav1 with specific shRNA in both MDCK and MK4 cells. The Western blot results showed the knockdown efficiency of Cav1 in MK4 and MDCK cells ( Figure S3a,b). Specifically, the results showed that the knockdown efficiency of Cav1 was higher in MK4 cells compared to that in MDCK cells. The depletion of Cav1 decreased the junctional myosin IIB expression and disturbed the actin belt integrity in MK4 cells (Figure 3a). In contrast, the downregulation of Cav1 in MDCK only resulted in slightly junctional myosin IIB downregulation and actin belt disruption ( Figure S3d). Meanwhile, nuclear YAP retention became prominent at the confluent stage in both MDCK and MK4 cells (Figure 3b,c and Figure S3e,f). Collectively, these data suggest that Cav1 is required for stabilizing junctional myosin IIB and actin belt integrity, so as to suppress YAP nuclear translocation.  To confirm the role of Cav1 in modulating the junctional myosin IIB distribution and YAP nuclear translocation, we further overexpressed Cav1-conjugated RFP in MK4 cells. We found that Cav1 overexpression prevented Ha-Ras V12 -induced junctional myosin IIB downregulation. The overexpression of Cav1 in MK4 cells also displayed a well-organized actin belt structure, regardless of Ha-Ras V12 induction (Figure 3d). Furthermore, Cav1 overexpression abolished Ha-Ras V12 -induced YAP nuclear translocation at the overconfluent stage (Figure 3e,f).

Ha-Ras V12 Downregulated Cav1 Expression and YAP Nuclear Retention through MEK Pathway
We already know that Ha-Ras V12 overexpression downregulates the Cav1 expression and changes the Cav1 distribution (22). To investigate the mechanism of Cav1 downregulation triggered by Ha-Ras V12 overexpression, we evaluated the ERK expression, which is a downstream signal of Ras. The results showed that the overexpression of Ha-Ras V12 induced ERK activation (Figure 4b). Moreover, the inhibition of ERK activation using MEK inhibitor U0126 prevented the downregulation of Cav1 and YAP nuclear translocation in Ha-Ras V12 -overexpressed cells (Figure 4). Some studies have shown that the activation of JNK and Rac1-downstream signals of MAPK-is relevant to YAP nuclear retention [34,35]. We next wanted to assess whether the activation of JNK and Rac1 is required for Ha-Ras V12 -induced YAP nuclear retention. Cells were treated with SP600125 (JNK inhibitor) and EHT1864 (Rac1 inhibitor). Both SP600125 and EHT1864 failed to prevent Ha-Ras V12 -induced YAP nuclear translocation (Figure 4d,e). In summary, the results confirmed that Ha-Ras V12 downregulated Cav1 expression through ERK activation and further induced YAP nuclear translocation. To confirm the role of Cav1 in modulating the junctional myosin IIB distribution and YAP nuclear translocation, we further overexpressed Cav1-conjugated RFP in MK4 cells. We found that Cav1 overexpression prevented Ha-Ras V12 -induced junctional myosin IIB downregulation. The overexpression of Cav1 in MK4 cells also displayed a well-organized actin belt structure, regardless of Ha-Ras V12 induction (Figure 3d). Furthermore, Cav1 overexpression abolished Ha-Ras V12 -induced YAP nuclear translocation at the overconfluent stage (Figure 3e,f).

Ha-Ras V12 Downregulated Cav1 Expression and YAP Nuclear Retention through MEK Pathway
We already know that Ha-Ras V12 overexpression downregulates the Cav1 expression and changes the Cav1 distribution (22). To investigate the mechanism of Cav1 downregulation triggered by Ha-Ras V12 overexpression, we evaluated the ERK expression, which is a downstream signal of Ras. The results showed that the overexpression of Ha-Ras V12 induced ERK activation (Figure 4b). Moreover, the inhibition of ERK activation using MEK inhibitor U0126 prevented the downregulation of Cav1 and YAP nuclear translocation in Ha-Ras V12 -overexpressed cells (Figure 4). Some studies have shown that the activation of JNK and Rac1-downstream signals of MAPK-is relevant to YAP nuclear retention [34,35]. We next wanted to assess whether the activation of JNK and Rac1 is required for Ha-Ras V12 -induced YAP nuclear retention. Cells were treated with SP600125 (JNK inhibitor) and EHT1864 (Rac1 inhibitor). Both SP600125 and EHT1864 failed to prevent Ha-Ras V12 -induced YAP nuclear translocation (Figure 4d,e). In summary, the results confirmed that Ha-Ras V12 downregulated Cav1 expression through ERK activation and further induced YAP nuclear translocation.

Nuclear YAP Is Not Involved in Ha-Ras V12 -Induced Cellular Aggregation
After determining the regulation of YAP nuclear translocation in Ha-Ras V12 -overexpressing cells, we sought to investigate the function of Ha-Ras V12 -induced nuclear YAP. To assess the function of nuclear YAP, we employed verteporfin (VP, YAP inhibitor) to suppress YAP nuclear translocation in Ha-Ras V12 -overexpressing cells. VP successfully abolished Ha-Ras V12 -induced YAP activation at the overconfluent stage (Figure 5a,b). VP also downregulated Ha-Ras V12induced YAP-TEAD downstream genes CYR61 mRNA expression (Figure 5c,d). Nuclear YAP has been reported to be involved in cellular aggregation in MDCK cells at the confluent stage [14]. In our study, we also found that the overexpression of Ha-Ras V12 triggered multilayer cellular aggregation at the overconfluent stage (Figure 5e,f). We propose that nuclear YAP induced by Ha-Ras V12 overexpression may require multilayer cellular aggregation. Surprisingly, the inhibition of nuclear YAP did not suppress Ha-Ras V12 -induced multilayer cellular aggregation (Figure 5e,f). Interestingly, one study determined that YAP regulates anchorage-independent growth in prostate cancer cells [36]. We indicated that the overexpression of Ha-Ras V12 induced colony formation, which can be suppressed by VP (Figure 5g,h). This result suggests that nuclear YAP is required for colony formation, instead of multilayer cellular aggregation.

Nuclear YAP Is Not Involved in Ha-Ras V12 -Induced Cellular Aggregation
After determining the regulation of YAP nuclear translocation in Ha-Ras V12 -overexpressing cells, we sought to investigate the function of Ha-Ras V12 -induced nuclear YAP. To assess the function of nuclear YAP, we employed verteporfin (VP, YAP inhibitor) to suppress YAP nuclear translocation in Ha-Ras V12 -overexpressing cells. VP successfully abolished Ha-Ras V12 -induced YAP activation at the overconfluent stage (Figure 5a,b). VP also downregulated Ha-Ras V12 -induced YAP-TEAD downstream genes CYR61 mRNA expression (Figure 5c,d). Nuclear YAP has been reported to be involved in cellular aggregation in MDCK cells at the confluent stage [14]. In our study, we also found that the overexpression of Ha-Ras V12 triggered multilayer cellular aggregation at the overconfluent stage (Figure 5e,f). We propose that nuclear YAP induced by Ha-Ras V12 overexpression may require multilayer cellular aggregation. Surprisingly, the inhibition of nuclear YAP did not suppress Ha-Ras V12 -induced multilayer cellular aggregation (Figure 5e,f). Interestingly, one study determined that YAP regulates anchorage-independent growth in prostate cancer cells [36]. We indicated that the overexpression of Ha-Ras V12 induced colony formation, which can be suppressed by VP (Figure 5g,h). This result suggests that nuclear YAP is required for colony formation, instead of multilayer cellular aggregation.

Rac1 Activity Is Required for Ha-Ras V12 -Induced Cellular Aggregation
In other studies, YAP has been found to be necessary for cellular aggregation in heterogeneous cultures [14]. YAP-activated cells were co-cultured with normal MDCK cells to observe cellular aggregation formation. In our study, we found that the overexpression of Ha-Ras V12 induced multilayer cellular aggregation in both homogeneous and heterogeneous cells (Figure 6a and Figure S4a). Ha-Ras V12 disrupted the cell junction integrity and disturbed actin filament organization. We assumed that cytoskeleton reorganization may be involved in Ha-Ras V12 -induced cellular aggregation. Our previous study revealed that Ha-Ras V12 promotes RhoA activity, which is relevant for cytoskeleton organization [33]. RhoA activates Rho-associated protein kinase (ROCK), which is relevant for myosin lightchain (MLC) activation. The activation of MLC facilitated actin reorganization. Therefore, we applied cells with Y27632 (ROCK inhibitor) to indicate whether the inhibition of ROCK prevented Ha-Ras V12 -induced multilayer cellular aggregate formation. Surprisingly, the inhibition of ROCK triggered multilayer cellular aggregates in MK4 cells without Ha-Ras V12 induction (Figure 6a,b). Some studies have stated that the inhibition of Rho-associated protein kinase (ROCK) facilitated cell extrusion in MDCK cells [18,37]. Moreover, the inhibition of ROCK has also been reported to induce Rac1 activity [38]. We also found that both the inhibition of ROCK and induction of Ha-Ras V12 upregulated Rac activity (Figure 6c,d).
To determine whether Rac activity is required for multilayer cellular aggregate formation, we applied cells with combined treatment, including Y27632 and EHT1864 (Rac inhibitor). It was demonstrated that EHT1864 prevented both Ha-Ras V12 -and Y27632-induced multilayer cellular aggregates. Furthermore, to confirm whether the activation of Rac is sufficient for multilayer cellular aggregation, we transfected different forms of Rac-conjugated GFP in MDCK and MK4 cells, including Rac1-wild type (Rac1-WT), Rac1 constitutive active form (Rac1-L), and dominant-negative form (Rac1-N). The results demonstrated that only cells overexpressing the Rac constitutive active form (Rac1-L) triggered cellular aggregation at the confluent stage (Figure 6e,f and Figure S6b). Collectively, the activation of Rac1 is required and sufficient for cellular aggregation at the confluent stage.

Ha-Ras V12 -Induced Multilayer Cellular Aggregates through ERK-Rac Pathway
We further sought to investigate the mechanism of Ha-Ras V12 -induced Rac activity. The ERK pathway has been reported to activate Rac activity in BRAF-and NRAS-mutant melanoma [39]. To evaluate whether ERK is relevant to Ha-Ras V12 -induced Rac activation and multilayer cellular aggregate formation, the cells were treated with U0126. It was found that Ha-Ras V12 triggered Rac activation and cellular aggregate formation, which was suppressed by U0126 (Figure 7). This suggests that Ha-Ras V12 induced ERK activation and further upregulated Rac activity to trigger multilayer cellular aggregate formation.

Ha-Ras V12 -Induced Multilayer Cellular Aggregates through ERK-Rac Pathway
We further sought to investigate the mechanism of Ha-Ras V12 -induced Rac activity. The ERK pathway has been reported to activate Rac activity in BRAF-and NRAS-mutant melanoma [39]. To evaluate whether ERK is relevant to Ha-Ras V12 -induced Rac activation and multilayer cellular aggregate formation, the cells were treated with U0126. It was found that Ha-Ras V12 triggered Rac activation and cellular aggregate formation, which was suppressed by U0126 (Figure 7). This suggests that Ha-Ras V12 induced ERK activation and further upregulated Rac activity to trigger multilayer cellular aggregate formation.

Discussion
In this study, we demonstrated the mechanism underlying Ha-Ras V12 -induced YAP nuclear translocation and multilayer cellular aggregates. We found that Ha-Ras V12 overexpression induced YAP nuclear translocation through the disturbance of the cell junction and dislocated myosin IIB. Our previous study revealed that Ha-Ras V12 decreased Cav1 expression [22]. Our data showed that the overexpression of Cav1 abolished Ha-Ras V12induced myosin IIB delocalization and YAP nuclear translocation in MK4 cells. Moreover, the knockdown of Cav1 resulted in the downregulation of junctional myosin IIB and YAP nuclear retention in MDCK and MK4 cells. U0126 prevented Ha-Ras V12 -induced Cav1 downregulation. This suggests that Ha-Ras V12 induced Cav1 downregulation and YAP nuclear translocation through the MEK signaling pathway. Nevertheless, the activation of YAP was required for Ha-Ras V12 -induced independent growth, but not multilayer cellular

Discussion
In this study, we demonstrated the mechanism underlying Ha-Ras V12 -induced YAP nuclear translocation and multilayer cellular aggregates. We found that Ha-Ras V12 overexpression induced YAP nuclear translocation through the disturbance of the cell junction and dislocated myosin IIB. Our previous study revealed that Ha-Ras V12 decreased Cav1 expression [22]. Our data showed that the overexpression of Cav1 abolished Ha-Ras V12induced myosin IIB delocalization and YAP nuclear translocation in MK4 cells. Moreover, the knockdown of Cav1 resulted in the downregulation of junctional myosin IIB and YAP nuclear retention in MDCK and MK4 cells. U0126 prevented Ha-Ras V12 -induced Cav1 downregulation. This suggests that Ha-Ras V12 induced Cav1 downregulation and YAP nuclear translocation through the MEK signaling pathway. Nevertheless, the activation of YAP was required for Ha-Ras V12 -induced independent growth, but not multilayer cellular aggregates. Next, we demonstrated Ha-Ras V12 -induced Rac1 activation through the MAPK signaling pathway, resulting in multilayer cellular aggregates in MK4 cells (Figure 8).
aggregates. Next, we demonstrated Ha-Ras V12 -induced Rac1 activation through the MAPK signaling pathway, resulting in multilayer cellular aggregates in MK4 cells ( Figure  8). Figure 8. Mechanism of Ha-Ras V12 -induced YAP nuclear translocation and multilayer cellular aggregates. Ha-Ras V12 downregulated Cav1 through the ERK pathway, and further suppressed junctional myosin IIB so as to promote YAP nuclear retention. Ha-Ras V12 also induced the ERK-Rac pathway to trigger multilayer cellular aggregate formation at the overconfluent stage.↑ indicates upregulation and ↓ indicates downregulation.
The role of YAP in multilayer cellular aggregate formation remains controversial. Bin Zhao et al. demonstrated that the overexpression of YAP-WT constitutively formed (YAP-5SA) in MDCK cells triggered multilayer cellular aggregates when YAP-transformed cells were surrounded by normal cells, whereas the overexpression of YAP-S94A-which eliminated TEAD binding-prevented multilayer cellular aggregate formation [14]. In our study, we observed multilayer cellular aggregate formation in the entire population of Ha-Ras V12 -overexpressed cells. The overexpression of Ha-Ras V12 facilitated cell transformation, which resulted in YAP nuclear translocation in all bottom fractions of cells; however, YAP was located in the cytosol inside the cellular aggregate area ( Figure S5). However, the reason for YAP being located in the cytosol in the cellular aggregate area remains unknown and requires further investigation. Additionally, only a handful of studies support the idea of nuclear YAP not being required for Ha-Ras V12 -induced multilayer cellular aggregate formation. Firstly, VP disturbed YAP/TEAD binding and prevented Ha-Ras V12induced YAP nuclear retention; however, it failed to abolish Ha-Ras V12 -induced multilayer cellular aggregate formation (Figure 5e,f). Secondly, we confirmed that Rac is required for cellular aggregate formation. Lastly, the inhibition of Rac failed to prevent Ha-Ras V12 -induced YAP nuclear translocation.
The activation of YAP can be triggered by diverse mechanotransductions, such as low cell density, stiff ECM, high cell-spreading area, and cell stretching [40]. The underlying mechanism for the regulation of YAP nuclear translocation may differ from various stimulations. Cell junction integrity and actomyosin tension have been shown to regulate YAP nuclear translocation [41,42]. We demonstrated that Cav1 overexpression restored Ha-Ras V12 -induced cell junction disruption and YAP nuclear translocation. Furthermore, Figure 8. Mechanism of Ha-Ras V12 -induced YAP nuclear translocation and multilayer cellular aggregates. Ha-Ras V12 downregulated Cav1 through the ERK pathway, and further suppressed junctional myosin IIB so as to promote YAP nuclear retention. Ha-Ras V12 also induced the ERK-Rac pathway to trigger multilayer cellular aggregate formation at the overconfluent stage. ↑ indicates upregulation and ↓ indicates downregulation.
The role of YAP in multilayer cellular aggregate formation remains controversial. Bin Zhao et al. demonstrated that the overexpression of YAP-WT constitutively formed (YAP-5SA) in MDCK cells triggered multilayer cellular aggregates when YAP-transformed cells were surrounded by normal cells, whereas the overexpression of YAP-S94A-which eliminated TEAD binding-prevented multilayer cellular aggregate formation [14]. In our study, we observed multilayer cellular aggregate formation in the entire population of Ha-Ras V12 -overexpressed cells. The overexpression of Ha-Ras V12 facilitated cell transformation, which resulted in YAP nuclear translocation in all bottom fractions of cells; however, YAP was located in the cytosol inside the cellular aggregate area ( Figure S5). However, the reason for YAP being located in the cytosol in the cellular aggregate area remains unknown and requires further investigation. Additionally, only a handful of studies support the idea of nuclear YAP not being required for Ha-Ras V12 -induced multilayer cellular aggregate formation. Firstly, VP disturbed YAP/TEAD binding and prevented Ha-Ras V12 -induced YAP nuclear retention; however, it failed to abolish Ha-Ras V12 -induced multilayer cellular aggregate formation (Figure 5e,f). Secondly, we confirmed that Rac is required for cellular aggregate formation. Lastly, the inhibition of Rac failed to prevent Ha-Ras V12 -induced YAP nuclear translocation.
The activation of YAP can be triggered by diverse mechanotransductions, such as low cell density, stiff ECM, high cell-spreading area, and cell stretching [40]. The underlying mechanism for the regulation of YAP nuclear translocation may differ from various stimulations. Cell junction integrity and actomyosin tension have been shown to regulate YAP nuclear translocation [41,42]. We demonstrated that Cav1 overexpression restored Ha-Ras V12 -induced cell junction disruption and YAP nuclear translocation. Furthermore, the knockdown of Cav1 and inhibition of myosin II disturbed junctional myosin IIB and subsequently caused YAP nuclear translocation in MK4 cells at the overconfluent stage. Therefore, understanding how Cav1 modulates cell junction integrity and myosin IIB distribution will be an important area for future research. In our study, we found that U0126 prevented Ha-Ras V12 -induced Cav1 downregulation. Cav1 has also been found to play an inhibitory role in the Ras/MAPK cascade [43]. Furthermore, the regulation of the Ras/MAPK cascade is complicated, and the overexpression of Cav1 may prevent the activation of the Ras/MAPK cascade. Considering the lower Cav1 expression in several cancers, these data highlight the role of tumor suppressor Cav1 in the initiation of tumorigenesis.
Actomyosin dynamics are essential for adherens junction formation and functioning during epithelial tissue homeostasis [44,45]. Rho GTPases play an important role in regulating junctional actin and myosin [46]. Rho-associated coiled-coil kinase (ROCK) activates myosin II contractility through the phosphorylated myosin light chain. Rac is a member of the Rho GTPases family. The activation of Rac1 in the initiation of cell-cell contact facilitates cadherin ligation [20]. After cell junction maturation, actin filaments are organized with myosin II into bundles, leading to a functional contractile structure [47]. Moderated Rac1 activity functions to stabilize cadherin-dependent adhesion [21]. However, the overactivation of Rac1 in Ha-Ras V12 -transformed cells results in the disassembly of cadherin contacts. Whether and how Rac1 activity is required for Ha-Ras V12 -induced cellular and mechanical transformation remains to be further elucidated.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10050977/s1, Figure S1: Ha-RasV12-induced YAP nuclear translocation through the Hippo-independent pathway; Figure S2: The distribution of myosin IIA did not change at confluent stage after Ha-RasV12 overexpression; Figure S3: Absence of Cav1 inhibited junctional myosin IIB and induced YAP nuclear translocation in MDCK cells; Figure S4: Activation of Rac is required for multilayer cellular aggregates; Figure S5: YAP translocated to cytosol in the multilayer cellular aggregates area in Ha-RasV12-overexpressing cells.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.