Fig 2: Reactivating PTEN expression and PI3K pathway inhibition through ATXN3 depletion and HDAC inhibition. (a), ATXN3 depletion limits Akt phosphorylation. A549 cells were transfected with siRNAs as indicated (NT1, nontargeting control). After 48 h cells were stimulated with 0.5 ng/ml EGF (Preprotech) for 5 min and lysed in ice-cold NP40 buffer (0.5% NP40, 25 mM Tris pH7.9, 100 mM NaCl, 50 mM NaF, with protease and phosphatase inhibitors (Roche)). Samples were processed for immunoblotting with rabbit anti-phospho-Akt (T308, Cell Signaling, 9275) and other antibodies as before. An asterisk indicates nonspecific bands. (b), PTEN transcription shows a limited biphasic response to HDAC inhibition. A549 cells were treated with increasing concentrations of Vorinostat (Selleck) for 16 h prior to preparation of total mRNA. Transcript levels were determined by qRT-PCR and are shown normalized to the vehicle control (below). SCG3 is included as an example of a transcript that is highly responsive to HDAC inhibition (above). PCR primer pairs are listed in Supplementary Table 1. (c and d), ATXN3 depletion enhances the PTEN response to HDAC inhibition. A549 cells were transfected with the indicated siRNAs for 48 h and treated with vehicle or 2 µM Vorinostat for the final 16 h prior to preparation of total mRNA or protein extracts. (c), Transcripts were quantified by qRT-PCR and are shown normalized to the level in mock-transfected cells. Bars show the mean values obtained from two independent experiments. (d), In a parallel experiment to that shown in panel c, protein was extracted in NP40 buffer and immunoblotted for ATXN3 and PTEN. (e and f), ATXN3 depletion and HDAC inhibition are additive with respect to cell viability. A549 cells were transfected as indicated with siRNA for 72 h and treated with vehicle (0.04% DMSO) or Vorinostat for the final 48 h prior to analysis. Viable cell numbers were determined based on proportionality with the amount of ATP present, measured using the CellTitre-Glo assay (Promega). Mean values are derived from three independent experiments and normalized to vehicle controls; the error bars show standard deviation. (e) In siC-transfected cells the LC50 for Vorinostat is 2.4 µM. (f), ATXN3 depletion reduces viable cell numbers and works additively with HDAC inhibition. In each case, Vorinostat significantly reduced cell viability relative to the vehicle control, and ATXN3 siRNAs significantly reduced cell viability relative to siC (P<0.05).

Fig 3: A siRNA screen identifies Josephin family DUBs as negative regulators of PTEN abundance. (a and b), Identification of ATXN3, ATXN3L and JOSD1 from an unbiased DUB siRNA screen. A549 cells (from ECACC and cultured in DMEM with 10% FBS, 1% NEAA, 100 U/ml penicillin/streptomycin at 37 °C and 5% CO2) were seeded at 9 × 107 cells in 150 mm dishes and transfected with a library of pooled siRNA oligos targeting 92 human DUBs (Qiagen), or AllStars non targeting control siRNA (Qiagen, siC), or no siRNA (Mock), at a final concentration of 40 nM using Oligofectamine (Invitrogen). Proteins were extracted 72 h later and equal amounts of lysates (determined by BCA assay, Thermo Scientific) were arrayed in 96-well plates. Proteins extracted in NP40 buffer (0.5% NP40, 25 mM Tris pH7.9, 150 mM NaCl with protease inhibitors (Roche)) were resolved on four 8% SDS-PAGE gels, transferred to BiotraceNT membrane (VWR) and immunoblotted with mouse anti-PTEN (Santa Cruz, sc-7974) and mouse anti-ß-actin (ab6276, Abcam) antibodies. Proteins were visualized using IRDye-conjugated secondary antibodies and a LI-COR Odyssey 2.1; 16-bit images were quantified using Odyssey analysis software. The PTEN levels were expressed relative to actin and then normalized to the median of each immunoblot, before collation and ranking of the log2 transformed data. (a), The entire data set ranked by PTEN abundance, highlighting DUBs selected for deconvolution (blue), including the Josephins ATXN3, ATXN3L and JOSD1 (shown in maroon); JOSD2 is shown in black. (b), A portion of an immunoblot from the screen, showing PTEN levels after depletion of the Josephin DUBs or the nontargeting control (siC). (c), Josephin DUBs do not coordinately regulate other PI3K pathway components. The entire NP40 lysate library was immunoblotted for p85 (BD Biosciences, 610045), p110a (Cell Signaling, 4249), p110ß (Cell Signaling, 3011), p110? (Cell Signaling, 5405), PDK1 (BD Biosciences, 611071) and total Akt (Cell Signaling, 9272) and expression was quantified as before; log2 transformed data for the Josephin DUBs are represented in a heatmap. (d), Josephin family DUB siRNAs are target specific. A549 cells were seeded in six-well plates at 1 × 105 cells per well and the following day transfected with the indicated siRNA pools (10 nM) using Lipofectamine RNAiMAX (Invitrogen). Total RNA was extracted 48 h later using RNeasy columns with on-column DNase digestion (Qiagen). cDNA was reverse transcribed from 1 µg RNA with RevertAid H-minus M-MuLV reverse transcriptase (Fermentas) using an oligodT primer (Promega). qRT-PCR was performed in triplicate using SYBR Green Supermix and a CFX system (Biorad). PCR primer pairs are listed in Supplementary Table 1. Samples underwent two-step amplification at 94 °C (30 s) and 60 °C (60 s); melt curves were analyzed after 40 cycles. The Ct values for test genes were normalized to actin and relative expression represented as 2-[??Ct]. (e), Deconvolution of siRNA pools supports a role for three Josephin DUBs in the negative regulation of PTEN. A549 cells were seeded in six-well plates at 6 × 104 cells per well and the following day transfected for 72 h with each of the four individual oligos (A-D; 10 nM) that comprised each siRNA pool (P) using Lipofectamine RNAiMAX (Invitrogen). Data are shown for five DUBs whose depletion enhanced PTEN expression in the initial screen. Equal amounts of each lysate were immunoblotted for PTEN and actin. The heatmap summarizes the normalized PTEN expression; scale limits represent 1.5-fold change.

Fig 4: Expression but not stability of PTEN protein is dependent on ATXN3 abundance. (a), Efficient ATXN3 depletion is required to induce PTEN expression. A549 cells in six-well plates were transfected for 72 h with new batches of the indicated ATXN3 siRNAs (10 nM, sequences listed in Supplementary Table 2). After 72 h, cells were lysed in NP40 buffer and equal amounts of lysate immunoblotted using mouse anti-ATXN3 (BioLegend, 650401), PTEN and actin antibodies as before; a representative experiment is shown (top). PTEN and ATXN3 were quantified relative to actin and normalized to the mock control (below); mean data are shown from three (ATXN3) or four (PTEN) independent experiments, and error bars show standard deviation. The Student t-test compared PTEN or ATXN3 abundance in response to each siRNA with that with siC, *P<0.05, **P<0.001. (b), ATXN3 depletion induces PTEN in KRAS wild-type non-small-cell lung cancer. NCI-H322 cells (from CR-UK and cultured in RPMI with 10% FBS) were transfected and analyzed as described above (72 h). (c and d), ATXN3 depletion does not alter the stability of PTEN in the absence of translation. A549 cells were transfected for a total of 72 h with ATXN3, nontargeting (siC), or no (Mock) siRNA for a minimum of 52 h before treatment for the indicated duration with 100 µg/ml cycloheximide (CHX, Sigma). Protein turnover was monitored over an initial 8 h chase up to a maximum of 20 h; cells were lysed in NP40 buffer and processed for immunoblotting. (c), Equal amounts of lysate were immunoblotted for PTEN, ATXN3 or actin. (d), PTEN was quantified relative to actin and normalized to the amount in each sample prior to starting the cycloheximide treatment. The half-life, indicated by a dotted line, is >20 h for PTEN.