TGF-β1 Facilitates TAp63α Protein Lysosomal Degradation to Promote Pancreatic Cancer Cell Migration

Simple Summary Numerous studies demonstrate that the activation of transforming growth factor-β (TGF-β) signaling is a critical driving force for promoting cancer cell migration and tumor metastasis. Our recent study indicates that TGF-β1 promotes FBXO3-mediated ΔNp63α protein degradation to facilitate cancer metastasis. In this study, we show that TGF-β1 can inhibit TAp63α protein stability in a lysosome-dependent, but canonical Smad pathway-independent manner, which leads to upregulation of p53-R248W expression, and consequently results in increased pancreatic cancer cell migration. Abstract TGF-β signaling plays a pivotal role in promoting tumor cell migration and cancer metastasis. ΔNp63α and TAp63α are two major isoforms of p53-related p63 protein. Our recent study has shown that TGF-β1 promotes ΔNp63α protein degradation to facilitate cancer metastasis. However, whether TAp63α is involved in TGF-β1-induced cancer metastasis remains unclear. In this study, we show that, in human pancreatic cancer MIA PaCa-2 cells harboring p53-R248W allele, TGF-β1 can significantly inhibit TAp63α protein stability in a Smad pathway-independent manner. Lysosome inhibitor, chloroquine, but not proteasome inhibitor MG132, can rescue TGF-β1-induced downregulation of TAp63α protein. In addition, we show that either TGF-β1 treatment or silencing of TAp63α can dramatically increase migration of MIA PaCa-2 cells. Importantly, the restored expression of TAp63α can effectively block TGF-β1-induced migration of MIA PaCa-2 cells. Mechanistically, we show that TGF-β1 promotes TAp63α protein degradation, leading to upregulation of p53-R248W protein expression, and consequently resulting in elevated MIA PaCa-2 cell migration. Together, this study indicates that lysosomal degradation is an important way for regulating TAp63α protein fate and highlights that TGF-β1-TAp63α-mutant p53 axis is critically important in pancreatic cancer metastasis.


Introduction
TGF-β1 is a member of the TGF-β family which plays a pivotal role in a series of biological processes, including cell differentiation, proliferation, epithelial-mesenchymal transition (EMT), cell migration, tumor metastasis and immune escape [1][2][3]. As a ligand for TGF-β receptor, TGF-β1 binds to the TGF-β receptor and transduce signals through canonical Smad pathway or noncanonical Ras-Erk, PI3K/AKT and Rho-like GTPase pathways [4,5]. In the canonical Smad pathway, activated TGF-β receptor phosphorylates Smad2/Smad3, which then bind to Smad4 to form heteromeric Smad complexes translocated into the nucleus to transactivate downstream target genes [4,6,7]. Accumulating evidence indicates that TGF-β1 signaling can also execute its function through the regulation of protein stability. It is reported that TGF-β1 can promote iNOS protein degradation in a ubiquitin-proteasome-dependent manner during inflammatory process [8]. Furthermore, TGF-β1 can promote type I collagen protein lysosomal degradation [9]. Moreover, TGF-β1 can also facilitate MyD88 protein degradation by recruiting Smurf1 and Smurf2 [10].
p63,a p53 family member, which plays an important role in a variety of biological processes, including cell adhesion, proliferation, senescence, survival, tumor metastasis and embryonic development [11]. Derived from the alternative transcriptional start sites at the N termini and alternative splicing sites at the C termini, p63 gene encodes a series of protein isoforms [11]. ∆Np63α and TAp63α are two major isoforms of p63 family proteins. It has been well-documented that ∆Np63α is mainly expressed in epithelial cells and serves as an important tumor metastasis inhibitor [11]. The activation of PI3K/HER2/Ras can inhibit ∆Np63α gene transcription to promote cancer metastasis [12,13]. Oxidative stress suppresses cell motility and tumor metastasis via upregulation of ∆Np63α expression [14]. Our recent study demonstrates that TGF-β1 promotes FBXO3-mediated degradation of ∆Np63α to facilitate tumor metastasis [15]. On the other hand, TAp63α is mainly expressed in testicular tissues, ovarian, spermatocytes, and embryos [16][17][18][19][20][21]. It has been shown that TAp63α also plays a role in tumor metastasis. TAp63α can coordinately regulate Dicer and miR-130b to suppress tumor metastasis [22]. Furthermore, TAp63α can transcriptionally inhibit miR-19 to suppress tobacco smoke-induced lung cancer EMT [23]. Moreover, TAp63α can also transactivate miR-133b to inhibit colon cancer metastasis [24]. However, whether TAp63α is also involved in TGF-β1-induced cancer metastasis remains unclear.
In this paper, we show that TGF-β1 can significantly inhibit TAp63α protein stability in a lysosome-dependent manner, which leads to the upregulation of p53-R248W protein expression, and consequently results in increased MIA PaCa-2 cell migration.

Transwell Assay for Cell Migration
Cell migration was measured as our previous described [26]. In briefly, MIA PaCa-2 cells (3 × 10 5 ) grown in serum-free DMEM medium were seeded into transwell inner chamber. The outer chamber was filled with regular DMEM medium. Cells were incubated for 24 h. Non-migrating cells were carefully removed and migrating cells were stained with 0.1% crystal violet for 10-15 min at room temperature. Cells were photographed using a Nikon light microscope. At least 100 cells from five random fields were counted.

Statistical Analysis
GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) was used to statistical analysis. All experiments were performed at least three times in triplicate. Student's t-test was used to assess significance.

TGF-β1 Promotes TAp63α Protein Degradation in A Lysosome-Dependent Manner
TAp63α and ∆Np63α are two major isoforms of p63 protein. Our recent study demonstrates that TGF-β1 promotes FBXO3-mediated ∆Np63α protein proteasomal degradation to facilitate cancer metastasis [15]. However, whether TAp63α also plays a role in TGF-β1induced tumor metastasis remains unclear. To explore this issue, we explored the effects of TGF-β1 on TAp63α expression. Human pancreatic cancer MIA PaCa-2 cells primarily expressed TAp63α isoform, as evidenced by western blot and RT-PCR (reverse transcriptional PCR) analyses ( Figure 1A,B). As shown in Figure 1C, TGF-β1 treatment significantly upregulated epithelial-mesenchymal transition (EMT) markers Twist1 and Snail1 expression, as expected. Notably, TGF-β1 markedly inhibited TAp63α protein expression. Next, we explored the molecular basis by which TGF-β1 inhibits TAp63α expression. As shown in Figure 1D, TGF-β1 treatment had no effects on TAp63α mRNA levels in MIA PaCa-2 cells, suggesting TGF-β1 can't affect TAp63α transcription. By contrast, TGF-β1 treatment significantly shortened TAp63α protein half-life in MIA PaCa-2 cells ( Figure 1E,F). It is well-known that ubiquitin-proteasome and autophagy/lysosome are two major systems for protein degradation. To explore the role of proteasome and lysosome in TGF-β1-induced TAp63α protein degradation, we used proteasome inhibitor MG132 or lysosome inhibitor chloroquine (CLQ) to treat MIA PaCa-2 cells. As shown in Figure 1G, proteasome inhibitor MG132 treatment had no effect on TGF-β1-induced downregulation of TAp63α protein expression. However, TGF-β1-induced downregulation of TAp63α protein level was completely blocked by lysosome inhibitor chloroquine (CLQ) ( Figure 1H). Next, we investigated whether TGF-β1 promotes TAp63α lysosomal degradation is dependent of canonical Smad pathway. As shown in Figure 1I, treatment with Smad3 inhibitor SIS3 had no effects on TGF-β1-induced downregulation of TAp63α. Moreover, we found that TGF-β1 had little effects on TAp63γ protein expression ( Figure 1J). Together, these results demonstrate that TGF-β1 specifically promotes TAp63α protein lysosomal degradation in a canonical Smad-independent manner in MIA PaCa-2 cells.

TGF-β1 Inhibits TAp63α to Promote Pancreatic Cancer Cell Migration
Our abovementioned data show that TGF-β1 promotes TAp63α protein lysosomal degradation. Since TAp63α also serves as a key tumor metastasis suppressor, we thus, asked whether TGF-β1-induced downregulation of TAp63α contributes to pancreatic cancer cell migration. As shown in Figure 2A,B, TGF-β1 significantly increased MIA PaCa-2 cell migration, as evidenced by transwell assays. In addition, silencing of p63 by short hairpin RNA (shRNA) led to significantly increased MIA PaCa-2 cell migration ( Figure 2C,E). Conversely, ectopic expression of TAp63α suppressed MIA PaCa-2 cell migration ( Figure 2F-H). Importantly, TGF-β1-induced upregulation of MIA PaCa-2 cell migration was totally blocked by ectopic expression of TAp63α ( Figure 2I-K). Together, these results indicate that downregulation of TAp63α plays a causative role in TGF-β1induced MIA PaCa-2 cell migration.

TGF-β1-Induced Downregulation of TAp63α Upregulates Mutant p53 Expression to Promote Pancreatic Cancer Cell Migration
Next, we examined the molecular mechanism by which TAp63α regulates MIA PaCa-2 cell migration. It has been documented that cancer-associated p53 hot-spot mutants, including R175H, R273H and R248W, possesses gain of function in promoting cancer metastasis [26]. Since MIA PaCa-2 cells carry a p53-R248W mutation [27]. We asked whether TGF-β1 affects p53-R248W protein expression in MIA PaCa-2 cells. As shown in Figure 3A, TGF-β1 significantly upregulated expression of EMT markers Twist1 and Snail1, as expected. It markedly elevated p53-R248W protein expression. Furthermore, knockdown of TAp63α by shRNAs dramatically increased p53-R248W protein expression, concomitant with increased expression of Twist1 and Snail1 proteins in MIA PaCa-2 cells ( Figure 3B). qPCR analyses showed that silencing of TAp63α significantly upregulated expression of p53-R248W mRNA ( Figure 3C). Immunofluorescence staining assay showed that knockdown of TAp63α dramatically led to accumulation of p53-R248W in the nucleus ( Figure 3D). On the other hand, ectopic expression of TAp63α significantly inhibited ex-Biology 2021, 10, 597 6 of 10 pression of both p53-R248W mRNA and protein, concomitant with reduced expression of Twist1 and Snail1 proteins in MIA PaCa-2 cells (Figure 3E,F). Importantly, knockdown of TAp63α-induced upregulation of Snail1 and twist1 protein expression and increased MIA PaCa-2 cell migration were totally reversed by simultaneous knockdown of p53-R248W ( Figure 3G,I). Next, we investigated whether TGF-β1 promotes p53-R248W expression via inhibiting TAp63α. As shown in Figure 3J, ectopic expression of TAp63α can totally inhibit TGF-β1-induced upregulation of p53-R248W and Snail1 protein expression. In addition, TGF-β1-induced MIA PaCa-2 cell migration can be completely suppressed by silencing of p53-R248W expression, consistent with ectopic expression of TAp63α ( Figure 2I,K and Figure 3K,M). Together, these results indicate that TGF-β1 up-regulates p53-R248W expression via suppression of TAp63α, thereby facilitating MIA PaCa-2 cell migration.

Discussion
TGF-β signaling plays a pivotal in the regulation of EMT, cell migration and tumor metastasis [1][2][3]. It has been documented that the activation of TGF-β signaling can promote expression of Snail, Twist1, Slug, and ZEB1, which in turn, regulates the expression of N-cadherin, Vimentin and, E-cadherin, proteins critically involved in EMT [28]. Moreover, TGF-β1 can activate PI3K/AKT/mTOR signaling or promote expression of EGFR and FosB to facilitate cancer cell motility, invasion and tumor metastasis [29,30]. Furthermore, TGF-β1 has been shown to promote mutant-p53/Smad complex formation, which antagonizes TAp63 transcriptional activity to facilitate cancer metastasis [31]. Our recent study demonstrates that TGF-β1 promotes FBXO3-mediated ∆Np63α protein degradation to enhance tumor metastasis [15]. In here, we show that TGF-β1 can inhibit TAp63α protein stability in a lysosome-dependent, but canonical Smad pathway-independent manner, leading to upregulation of p53-R248W expression, and consequently resulting in increased MIA PaCa-2 cell migration ( Figure 4). Accumulating evidence indicate that TGF-β signaling regulates a series of genes transcription through canonical Smad pathway or noncanonical Ras-Erk, PI3K/AKT, and Rho-like GTPase pathways [4,5]. Furthermore, it is reported that physiologic levels of TGF-β1 can stimulate paxillin mRNA translation [32]. In recent years, lots of researches demonstrated that TGF-β signaling also can regulate protein degradation. TGF-β1 can promote iNOS and MyD88 protein degradation in a proteasome-dependent manner [8,10]. Furthermore, TGF-β1 can promote type I collagen protein lysosomal degradation [9]. Our recent study demonstrates that TGF-β1 promotes ∆Np63α protein proteasomal degradation [15]. In this study, we show that TGF-β1 can facilitate TAp63α protein degradation in lysosome-dependent manner. Therefore, TGF-β signaling regulates protein expression in multiple levels, including transcription, translation and protein degradation.
Understanding how TGF-β1 facilitates TAp63α protein lysosomal degradation is not yet clear. It has been reported that the chaperone-mediated autophagy (CMA) is one of the intracellular proteolytic machineries for selective protein degradation within lysosome [33]. In the CMA process, CMA substate interacts with heat shock cognate protein 70 (Hsc70), which carries CMA substate to lysosome to degradation via lysosomal membrane protein type 2a (LAMP2A) [33]. Since TGF-β can facilitate Hsc70 and Smad2/3 interaction [34], it is plausible that TGF-β1 may promote TAp63α and Hsc70 interaction, resulting in TAp63α protein degradation in a CMA-dependent manner, a possibility needs to be further investigated.
While ∆Np63α is mainly expressed in epidermis [11], the expression of TAp63α in epidermis is extremely low [35]. Interestingly, it has been shown that the expression of TGF-β1 is high in epidermis [36]. In this study, we show that TGF-β1 inhibit TAp63α expression. Whether high levels of TGF-β1 contribute to low levels of TAp63α expression in epidermis remains to be seen.

Conclusions
Numerous studies demonstrate that TGF-β signaling plays an important role in promoting cancer cell migration and tumor metastasis. In this study, we show that TGF-β1 can specifically inhibit TAp63α, a p53 family member, protein stability in a lysosome-dependent, but canonical Smad pathway-independent, manner, which leads to upregulation of p53-R248W protein expression, and consequently results in pancreatic cancer cell migration. Our study reveals a new molecular mechanism by which TGF-β1 promotes cancer cell migration and demonstrates that lysosomal degradation is a novel way to regulate TAp63α protein fate.