Rasで形質転換されたMDCK細胞の遊走および浸潤におけるPTENの役割

概要

INTRODUCTION
　Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is one of the most crucial tumor suppressor genes involved in the regulation of phosphatidylinositol 3- kinase (PI3K) mediated signal transduction. It plays a vital role in the induction of cell polarity in motile cells, including the single-celled organism Dictyostelium discoideum, and mammalian immune cells such as neutrophils. The segregated localization of PI3K and PTEN which correlate with PI(3,4,5)P3 and PI(4,5)P2 at the plasma membrane has been observed to modulate cell polarity, migration and epithelial-mesenchymal transition. Ras is a small GTPase protein that is frequently mutated in patients with cancer. At the cellular level, it regulates numerous tumor-promoting processes, including enhancement of cell proliferation, suppression of cell death, cell metastasis, and modulation of the tumor microenvironment. One of the most studied pathways downstream of Ras is the PI3K pathway, which regulates actin reorganization to induce membrane ruffling through the Rac family of small GTPases for cell motility.
　In this study, we examined the role of PTEN in the migration and invasion of cancer cells expressing oncogenic Ras (RasV12). Homozygous knockout (KO) of the PTEN gene in RasV 12-transformed MDCK epithelial cells resulted in almost complete suppression of 2D migration, as well as 3D invasion through Matrigel. Here, we propose a migration mechanism for epithelial cancer cells mediated by the balance between Ras and PTEN activities.

RESULTS
1. Synergistic activation of the PI3K pathway in PTEN KO MDCK cell line expressing RasV12
　To investigate the role ofPTEN in epithelial cancer migration and invasion, we used MDCK cells expressing the active form of Ras, RasV12, in a doxycycline (Dox)-inducible manner (RasV12 cells), and established PTEN homozygous KO cells using the CRISPR/Cas9 system. Two independent cell lines, targeted with distinct sgRNAs, were obtained from normal MDCK or RasV12 cells, we analyzed AKT phosphorylation in PTEN KO cell lines to evaluate their PI(3,4,5)P3 levels. AKT was highly phosphorylated in PTEN KO cell lines. Consistent with the role of Ras in the PI3K pathway, RasV12 (+Dox) cells also showed elevated AKT phosphorylation, which was further enhanced by the loss of PTEN. These results indicate that RasV12 and the loss ofPTEN synergistically activate the PI3K signaling pathway.

2. Migratory morphology of RasV 12-transformed MDCK cells depends on PTEN
　The wild-type MDCK cells had a typical epithelial morphology and formed island­ like cell aggregates. Consistent with its roles in cancer migration, the induction of RasV 12 expression transformed MDCK cells to a laterally polarized appearance. Dimensional measurements revealed a higher length/width ratio and a smaller shape factor for RasV 12 (+Dox) cells, reflecting their enhanced ability for directional migration. Smaller cell heights and larger areas also supported their migratory morphology compared to wild-type MDCK cells. Interestingly, further deletion of the PTEN gene in RasV 12 (+Dox) cells resulted in an evenly spread shape. The average length/width ratio and shape factor returned to approximately one, indicating a loss of directionality of migration. These data suggest that PTEN plays an essential role in the directional migration of RasV 12-transformed MDCK cells, thus facilitating cancer migration and invasion.

3. PTEN is required for the 2D migration of RasV 12-transformed MDCK cells
　To examine whether the loss of PTEN had an effect on epithelial cancer migration, we performed a wound-healing assay using RasV 12 (+Dox) or RasV 12 (+Dox) +PTEN KO cells. RasV 12 (+Dox) 4-PTEN KO cells showed reduced migration compared to RasV 12 (+Dox) cells. Quantification of the gap area showed that RasV 12 (+Dox) cells moved faster than RasV 12 (+Dox) +PTEN KO cells. To analyze single-cell speed, we tracked individual cells at the leading edge and found that the loss of PTEN suppressed the speed of movement of RasV 12 (+Dox) cells. The effect of PTEN KO was also investigated in normal (non­ transformed) MDCK cells. We found that PTEN KO cells promoted gap closure as well as single-cell migration. These results demonstrate a unique requirement of the PTEN gene for the migration of RasV 12 (+Dox) cells.

4. PTEN gene is necessary for invasion of RasV 12-transformed MDCK cells through Matrigel
　A series of confocal images and subsequent three-dimensional reconstitution, showed that wild-type MDCK cells formed a relatively globular cluster with a smooth surface. In contrast, RasV 12 (+Dox) cells exhibited extended protrusions, thereby supporting their invasive features. Importantly, RasV 12 (+Dox) +PTEN KO cells showed less aggressive features without forming invasive cell extensions. These data suggest that loss of PTEN also affects the invasiveness of RasV 12-transformed MDCK cells in 3D.
　Next, we performed a Matrigel invasion assay using wild-type, RasV 12 (+Dox), PTEN KO, and RasV12 (+Dox) +PTEN KO MDCK cells. While the wild-type and PTEN KO cells did not migrate through Matrigel, RasV 12 (+Dox) cells showed a significantly higher degree of invasiveness, which was suppressed by KO of PTEN. A 3D migration assay without Matrigel showed similar results.
　RasV 12-promoted invasion through Matrigel was recovered by expressing full- length PTEN. Transfection of PTEN (Cl24A), a catalytically dead mutant, or PTEN (APho), in which the entire phosphatase domain was deleted, failed to rescue the phenotype. A mutant lacking the C2 domain could not recover the invasion ability of RasV12 (+Dox) +PTEN KO cells. Consistently, deletion of the C-terminal tail, which is known to induce an open conformation that exposes the C2 domain for membrane binding, rescued the invasive phenotype. Deletion of PDZ-binding motif, also rescued the invasion of RasV12 cells, indicating that PDZ binding is dispensable. Finally, a constitutive membrane-associated mutant of ΡTΈΝ, PTEN (Myr), efficiently rescued this phenotype. These results collectively demonstrate that membrane recruitment of PTEN is an essential step for its function in epithelial cancer cell invasion driven by RasV12.

5. Localization of PTEN at the cell rear during cancer invasion
　To explore the localization of PTEN in RasV 12 cells, we expressed mCherry-tagged wild-type PTEN in RasV 12 (+Dox) +PTEN KO cells, and performed an invasion assay. Three- dimensional imaging showed that mCherry-PTEN preferentially accumulated at the rear side of cells, penetrating through the membrane pores. We verified our observation by quantifying mCherry-PTEN fluorescence intensity at the cell rear relative to the front. While the fluorescence of GFP-RasV12 was uniformly distributed throughout the plasma membrane, mCherry-PTEN showed a significant preference for the cell rear.

CONCLUSION
　We have shown an unexpected role for PTEN in supporting epithelial cancer cell migration. The mechanism by which PTEN functions to create a balance between PI(4,5)P2 and PI(3,4,5)P3 at the plasma membrane underpins RasV12-driven cancer cell migration and invasion. This study provides important insights into the common mechanisms of cell migration shared between immune cells and metastatic cancer cells.