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. 2019 Oct 28;8(11):63.
doi: 10.1038/s41389-019-0172-9.

PR55α regulatory subunit of PP2A inhibits the MOB1/LATS cascade and activates YAP in pancreatic cancer cells

Ashley L Hein  1 , Nichole D Brandquist  1 , Caroline Y Ouellette  1 , Parthasarathy Seshacharyulu  2 , Charles A Enke  1 , Michel M Ouellette  2   3 , Surinder K Batra  4 , Ying Yan  5   6
Affiliations

PR55α regulatory subunit of PP2A inhibits the MOB1/LATS cascade and activates YAP in pancreatic cancer cells

Ashley L Hein et al. Oncogenesis. .

Abstract

PP2A holoenzyme complexes are responsible for the majority of Ser/Thr phosphatase activities in human cells. Each PP2A consists of a catalytic subunit (C), a scaffold subunit (A), and a regulatory subunit (B). While the A and C subunits each exists only in two highly conserved isoforms, a large number of B subunits share no homology, which determines PP2A substrate specificity and cellular localization. It is anticipated that different PP2A holoenzymes play distinct roles in cellular signaling networks, whereas PP2A has only generally been defined as a putative tumor suppressor, which is mostly based on the loss-of-function studies using pharmacological or biological inhibitors for the highly conserved A or C subunit of PP2A. Recent studies of specific pathways indicate that some PP2A complexes also possess tumor-promoting functions. We have previously reported an essential role of PR55α, a PP2A regulatory subunit, in the support of oncogenic phenotypes, including in vivo tumorigenicity/metastasis of pancreatic cancer cells. In this report, we have elucidated a novel role of PR55α-regulated PP2A in the activation of YAP oncoprotein, whose function is required for anchorage-independent growth during oncogenesis of solid tumors. Our data show two lines of YAP regulation by PR55α: (1) PR55α inhibits the MOB1-triggered autoactivation of LATS1/2 kinases, the core member of the Hippo pathway that inhibits YAP by inducing its proteasomal degradation and cytoplasmic retention and (2) PR55α directly interacts with and regulates YAP itself. Accordingly, PR55α is essential for YAP-promoted gene transcriptions, as well as for anchorage-independent growth, in which YAP plays a key role. In summary, current findings demonstrate a novel YAP activation mechanism based on the PR55α-regulated PP2A phosphatase.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Protein phosphatase 2A (PP2A) heterotrimeric holoenzymes.
One scaffold (PR65 or A) subunit binds to one catalytic (C) subunit to form an A/C heterodimer, which can further complex with one of the regulatory (B) subunits. The A and C subunits each contain two highly conserved isoforms (97% similarity between Cα and Cβ and 87% similarity between Aα and Aβ). A large number of the B subunits are classified into four distinct subfamilies (B, B′, B″, and B‴), which share no homology–
Fig. 2
Fig. 2. Analysis of YAP protein level and YAP-S127 phosphorylation in normal and malignant human pancreatic cells.
YAP protein level is markedly increased in the human pancreatic cancer cells (AsPC-1, Capan-1, CD18/HPAF, and L3.6) compared with human pancreatic ductal cells (HPNE), as determined by immunoblotting. HeLa human cervical cancer cells and SH-SY5Y human neuroblastoma cells serve as a positive and negative control, respectively, for YAP protein expression. GAPDH in the lysates was measured as internal controls. The protein levels of YAP, pYAP-S127, and GAPDH were quantified using ImageJ software. Relative YAP and pYAP-S127 levels in the samples were normalized with GAPDH levels and the ratio of pYAP-S127/YAP determined
Fig. 3
Fig. 3. PR55α-knockdown by shRNA inhibits YAP and activates the MOB1-LATS1/2 cascade in pancreatic cancer cells.
a CD18/HPAF and AsPC-1 cells were stably transduced with Dox-inducible shRNAs targeting various regions of PR55α or, as a control, with nontargeting shRNA. After incubation with Dox (2 µg/ml) for 3 days, the cell lysates (100 µg) were analyzed by immunoblotting for the indicated protein levels and/or phosphorylation. GAPDH served as an internal protein expression control. b Validation of the effects of PR55α on MOB1/LATS/YAP cascade in pancreatic cancer cells. shRNA-transduced CD18/HPAF cells were incubated with Dox (2 µg/ml) for the days indicated and analyzed for PR55α expression and the levels/phosphorylation of MOB1, LATS1/2, and YAP. GAPDH serves as an internal control. c To test the effect of PR55α on LATS2 protein stability, shRNA-transduced CD18/HPAF cells were incubated in medium containing cycloheximide (CHX, 15 μg/ml) to halt protein synthesis for the indicated hours. Whole-cell extracts were analyzed for LATS2 and GAPDH protein levels by immunoblotting. The protein levels of LATS2 and GAPDH were quantified using ImageJ software; relative LATS2 levels were normalized with GAPDH levels in the samples and protein half-life determined using SigmaPlot (version 11.2) analytical program
Fig. 4
Fig. 4. Ectopic PR55α expression in human normal pancreatic ductal cells (HPNE) activates YAP and inhibits the MOB1-LATS1/2 cascade.
a Dox-inducible retroviral vector expressing PR55α (pRevTRE-PR55α) was constructed and retrovirus was produced as described in the “Materials and methods“ section. Subsequently, HPNE cells were transduced with both pRevTet-On (Clontech) that expresses rtTA and pRevTRE-PR55α (or pRevTRE-Control) and selected for resistant cells to both G418 (400 µg/ml) and Hygromycin B (200 µg/ml). Diagram demonstrates the Tet-inducible retroviral vector (pRevTRE-PR55α) expressing PR55α. b The transduced cells were induced for ectopic PR55α expression by incubation with increasing doses of Dox (1 µg/ml) for 3 days and the effect of PR55α on the phosphorylation and level of YAP and LATS1/2 analyzed by immunoblotting. GAPDH served as an internal control. c Control- and PR55α-transduced HPNE cells were incubated with Dox (1 µg/ml) for the indicated days and analyzed for the effect of PR55α on YAP and the Hippo pathway (MST1/2, MOB1, and LATS1/2), by analyzing their phosphorylation and levels by immunoblotting. GAPDH served as an internal control
Fig. 5
Fig. 5. PR55α is essential for maintaining YAP stability.
Cytoplasmic and nuclear extracts were isolated using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Fisher Scientific) and analyzed for YAP protein expression. a α-tubulin and Lamin A/C were used as the cytoplasmic and nuclear markers, respectively. PR55α in cytoplasm and nucleus was analyzed by Western blot analysis. b To inhibit cellular proteasome activity, CD18/HPAF cells transduced with Control-shRNA or PR55α-shRNA were treated with MG132 (25 μM) for the indicated hours. Cytoplasmic and nuclear extracts were isolated from the treated cells and analyzed for YAP expression by immunoblotting. α-tubulin and Lamin A/C served as the internal controls for cytoplasmic and nuclear extract, respectively. c To test the effect of PR55α on YAP protein stability, Control-/PR55α-shRNA-transduced CD18/HPAF cells were treated with CHX (15 μg/ml) to halt protein synthesis for the indicated hours, cytoplasmic and nuclear extracts were isolated from the treated cells, and YAP protein levels analyzed by immunoblotting. α-tubulin and Lamin A/C served as internal controls for cytoplasmic and nuclear extract, respectively
Fig. 6
Fig. 6. Interaction of PR55α and YAP in pancreatic malignant and normal cells.
a PR55α was immunoprecipitated from 1 mg protein lysates of CD18/HPAF cells expressing Control-shRNA or PR55α-shRNA with anti-PR55α (100C1) rabbit IgG and probed by immunoblotting for the presence of PR55α, PP2A-C, and PP2A-A subunits with anti-PR55α (2G9), anti-PP2A-C (ID6), and anti-PP2A-A (H300) antibody, respectively. YAP and GAPDH in the lysate were measured by immunoblotting for YAP protein loading control and internal control for protein quantification, respectively. b PR55α was immunoprecipitated from the indicated protein lysates (1 mg per sample) with anti-PR55 (100C1) antibody. The obtained immunoprecipitates were divided into two halves: one half remained untreated (−) and the other half was treated with Shrimp Alkaline Phosphatase (SAP) at 10 units/ml (+) at 37 °C for 1 h. The resulting precipitates were rinsed once with cell lysis buffer and subjected to immunoblotting analysis for PR55α, YAP, and YAP-S127 with the antibody for PR55α (2G9), YAP (D24E4), and YAP-Ser127 phosphorylation (D9W2I), respectively. c PR55α were immunoprecipitated (IP) with anti-PR55α (2G9) antibody from CD18/HPAF cells stably transduced with empty vector, Flag-YAP, or Flag-YAP (5SA) mutant and immunoblotted (IB) using anti-PR55α (100C1) and anti-Flag (M2) antibodies. Lysates from the indicated cells were probed for Flag-YAP (input) and GAPDH (input) with specific antibodies by Western blotting. Intracellular distribution and co-localization of PR55α and YAP in pancreatic malignant and normal cells. The indicated cells were induced for the expression of PR55α-shRNA (CD18/HPAF) (d) or ectopic PR55α (HPNE) (e) by 2 µg/ml Dox for 3 days, and stained with anti-PR55α (100C1) and anti-YAP (1A12) antibodies, as described in the “Materials and methods” section. Images were analyzed for the cellular distribution of PR55α and YAP using a Zeiss-810 confocal laser-scanning microscope. Co-localization of PR55α and YAP in CD18/HPAF cells with/without PR55α-knockdown and in HPNE cells with/without ectopic PR55α expression were examined and shown as merged images (PR55α/YAP and MERGE). Scale bars, 50 µm
Fig. 7
Fig. 7. Effects of MOB1- and LATS1/2-knockdown by siRNA on the YAP activation promoted by PR55α.
a MOB1 was knocked down by siRNAs in both HPNE and CD18/HPAF cells with/without the expression of ectopic PR55α and PR55α-shRNA, respectively. After 72 h siRNA-transfection, the cells were analyzed for the phosphorylation and/or level of MOB1, LATS1/2, and YAP by immunoblotting. GAPDH served as an internal control. The levels of YAP and GAPDH were quantified using ImageJ software. YAP protein levels were normalized by the corresponding GAPDH levels and relative YAP levels in the samples were analyzed by SigmaPlot software and presented as bar graphs. b LATS1 and/or LATS2 were knocked down by siRNAs in CD18/HPAF and AsPC-1 cells expressing Control-shRNA or PR55α-shRNA. After 72 h incubation in medium containing Dox (2 µg/ml) to maintain shRNA expression, the cells were analyzed for the phosphorylation and/or levels of PR55α, LATS1, LATS2, YAP, and GAPDH. The levels of YAP and GAPDH were quantified using ImageJ software and relative YAP protein levels versus GAPDH levels were indicated under the YAP blots
Fig. 8
Fig. 8. Effects of PR55α on YAP-targeted gene expressions and anchorage-independent growth.
a PR55α promotes gene expressions of YAP targets. Ectopic PR55α and PR55α-shRNA were induced by Dox in HPNE for 3 days and CD18/HPAF cells for 6 days, respectively. The resulting cells were harvested to extract total RNA samples to analyze the mRNA expression of YAP target genes (ANKD1, CTGF, CRY61, and Survivin) by qRT-PCR, as described in the “Materials and methods” section. The study was repeated two times with duplicate samples and the result expressed as mean ± s.d (n = 6), p = 0.02. Statistical analyses were performed using the Student’s t-test. qRT-PCR, quantitative (q) Reverse Transcription (RT) PCR. b PR55α promotes anchorage-independent growth of HPNE cells. Upper panel: HPNE cells (5 × 104) with/without ectopic PR55α expression were incubated with 1 µg/ml Dox for 48 h, plated in soft-agar in six-well plates and incubated for 14 days. Left panels: representative images of the soft-agar assay photographed by phase-contrast optics. Box plot: colonies in soft-agar were counted by ImageJ software and shown as mean ± s.d. of 14 samples. Scale bar represents 100 μm. c PR55α-knockdown by shRNA inhibits anchorage-independent growth of pancreatic cancer cells. shRNA-transduced CD18/HPAF and AsPC-1 cells were incubated with 2 µg/ml Dox for 48 h, plated in soft-agar in six-well plates at 4 × 104 and incubated for 14 days. Left panels: representative images of soft-agar assay photographed by phase-contrast optics. Box plot: colonies in soft-agar were quantified by ImageJ software and shown as mean ± s.d. of two sets of experiments in triplicate samples. Scale bar represents 100 μm
Fig. 9
Fig. 9. A model for the regulation of the Hippo pathway and YAP by PR55α.
Black lines indicate a current understanding of the Hippo signaling cascades that regulate YAP phosphorylation and stability, resulting in YAP cytoplasmic retention by 14-3-3 and proteasomal degradation by SCF(β-TrCP). Red lines indicate the novel findings presented in this report, showing that PR55α inhibits the MOB1-activated LATS1-S909/LATS2-S872 autophosphorylation that prevents YAP activation, while PR55α concomitantly inhibits YAP phosphorylation, both of which lead to YAP activation. Blue dotted lines indicate that PR55α activates MST1/2 levels and phosphorylation through an unknown mechanism
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