Journal: Oncogene

Article Title: PR55α subunit of protein phosphatase 2A supports KRAS G12D -driven tumorigenesis that requires YAP activation

doi: 10.1038/s41388-025-03477-y

Figure Lengend Snippet: A The scheme for generating the HPNE-based in vitro stepwise transformation model system. B Diagram illustrating the retroviral vector that expresses Doxycycline (Dox)-inducible human PR55α. HPNE cells were transduced with Dox-inducible PR55α retroviral vector (pRevTRE-PR55α) or control empty vector and selected with 200 μg/ml Hygromycin for stably transduced cells. The PR55α-transduced cells were induced by 1 μg/ml Dox for 3 days and analyzed for the protein levels of PR55α and GAPDH by Western blotting. C HPNE/Control and HPNE/PR55α cells were transduced with a retroviral vector expressing V5-tagged human p53 R175H mutant or control vector. The stably transduced clones were selected for Blasticidin (4 μg/mL) and verified for the expression of ectopic V5-p53 R175H by Western blot analysis with an anti-V5 antibody. D The indicated HPNE isogenic cell lines were transduced with a retroviral vector expressing the KRAS G12D mutant, the most frequently detected KRAS mutant in human pancreatic cancer (PC), and selected by 800 μg/ml Zeocin for stably transduced cells. The expression of KRAS G12D in the resulting cells was validated by Western blot analysis using a specific antibody.

Article Snippet: The pBABE-zeo/KRAS G12D construct was generated by cloning human KRAS G12D cDNA into the pBABE-zeo retroviral vector (Addgene #1766).

Techniques: Transformation Assay, In Vitro, Retroviral, Plasmid Preparation, Transduction, Control, Stable Transfection, Western Blot, Expressing, Mutagenesis, Clone Assay

Journal: Oncogene

Article Title: PR55α subunit of protein phosphatase 2A supports KRAS G12D -driven tumorigenesis that requires YAP activation

doi: 10.1038/s41388-025-03477-y

Figure Lengend Snippet: A The indicated HPNE isogenic cell lines were subcutaneously implanted into the flanks of athymic mice and monitored for tumor growth over a 10-week period. Tumor size was measured weekly using a digital caliper. For mice implanted with cells expressing Dox-inducible PR55α, Dox (1 mg/ml) was administered in the drinking water and refreshed every 48 h. As a positive control, CD18/HPAF pancreatic cancer cells were implanted into athymic nude mice and monitored for tumor growth in parallel. The table summarizes the tumor formation rates for each cell line. In a separate cohort, CA3 treatment (1 mg/kg) was initiated 10 days after implantation of HPNE/PR55α + KRAS G12D cells and administered via intraperitoneal injection three times per week for three weeks, as described previously [ 41 ]. Following treatment, mice remained in remission for an additional six weeks. B Representative images of tumor-bearing mice and excised tumor samples at the end of the experiment. C Line graph depicts the number of mice with tumors over time. Statistical significance ( p -value) was assessed using Student’s t-test with SigmaPlot software. D Line graph shows average tumor volume measured weekly for 8 weeks following implantation. E At the study endpoint, tumor xenografts from mice implanted with PK and BK cells were excised and weighed. F Tumor xenograft and adjacent normal tissues were analyzed by IHC analysis for expression of Ki67 (a proliferation biomarker), PR55α, and YAP, as described in Materials and Methods . Histological analysis was also performed using H&E staining. Scale bar = 50 μm.

Article Snippet: The pBABE-zeo/KRAS G12D construct was generated by cloning human KRAS G12D cDNA into the pBABE-zeo retroviral vector (Addgene #1766).

Techniques: Expressing, Positive Control, Injection, Software, Biomarker Discovery, Staining

Journal: Oncogene

Article Title: PR55α subunit of protein phosphatase 2A supports KRAS G12D -driven tumorigenesis that requires YAP activation

doi: 10.1038/s41388-025-03477-y

Figure Lengend Snippet: Oncogenic mutations in the KRAS oncogene cause KRAS activation, which is present in 90–95% of PC and serves as the driver of PC. The Raf/MEK/ERK oncogenic pathway is the major downstream effector of KRAS [ 45 ]. PR55α/PP2A was shown to facilitate the activation of the Raf/MEK/ERK signaling pathway by dephosphorylating the inhibitory sites in Raf and KSR1 [ 16 , 17 ]. Raf1 activation can block MST activity in activating LATS1/2. Conversely, the activation of LATS1/2 can suppress Raf1 [ 36 – 38 ]. YAP oncogene activation is required for KRAS-driven PC progression [ 26 – 28 ]. Our studies show that PR55α-associated PP2A promotes YAP activation by inhibiting the LATS/MOB1 auto-activation loop and dephosphorylating YAP, thus stabilizing YAP protein [ 19 ]. Our previous studies also identified a positive feedback regulation of the MST/LATS cascade by PR55α expression [ 19 ]. However, this PR55α-stimulated MST/LATS signaling was abolished by oncogenic KRAS G12D .

Article Snippet: The pBABE-zeo/KRAS G12D construct was generated by cloning human KRAS G12D cDNA into the pBABE-zeo retroviral vector (Addgene #1766).

Techniques: Activation Assay, Blocking Assay, Activity Assay, Expressing