Ptenα and ptenβ promote carcinogenesis through wdr5 and h3k4 trimethylation

ABSTRACT PTENα and PTENβ are two longer translational variants of phosphatase and tensin homolog (PTEN) messenger RNA. Their expressional regulations and functions in carcinogenesis remain

largely unknown. Here, we demonstrate that, in contrast with the well-established tumour-suppressive role of canonical PTEN, PTENα and PTENβ promote tumourigenesis by directly interacting

with the histone H3 lysine 4 (H3K4) presenter WDR5 to promote H3K4 trimethylation and maintain a tumour-promoting signature. We also show that USP9X and FBXW11 bind to the amino-terminal

extensions of PTENα/β, and respectively deubiquitinate and ubiquitinate lysines 235 and 239 in PTENα to regulate PTENα/β stability. In accordance, USP9X promotes tumourigenesis and FBXW11

suppresses tumourigenesis through PTENα/β. Taken together, our results indicate that the _Pten_ gene is a double-edged sword for carcinogenesis, and reinterpretation of the importance of the

_Pten_ gene in carcinogenesis is warranted. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

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support SIMILAR CONTENT BEING VIEWED BY OTHERS THE NTE DOMAIN OF PTENΑ/Β PROMOTES CANCER PROGRESSION BY INTERACTING WITH WDR5 VIA ITS SSSRRSS MOTIF Article Open access 14 May 2024 THE

EQUILIBRIUM OF TUMOR SUPPRESSION: DUBS AS ACTIVE REGULATORS OF PTEN Article Open access 16 November 2022 METTL3 PROMOTES TUMOUR DEVELOPMENT BY DECREASING APC EXPRESSION MEDIATED BY _APC_

MRNA _N_6-METHYLADENOSINE-DEPENDENT YTHDF BINDING Article Open access 21 June 2021 DATA AVAILABILITY ChIP-Seq data that support the findings of this study have been deposited in the GEO

under accession codes GSE135478 and GSE126982. RNA-Seq data that support the findings of this study have been deposited in the GEO under accession codes GSE135477 and GSE126983. Previously

published RNA-Seq data that were reanalysed here are available under accession code GSE14520. Mass spectrometry data (Fig. 1e and Supplementary Table 3) have been deposited in

ProteomeXchange with primary accession code PXD013196. The human LIHC, pancreatic cancer and breast cancer data were derived from the TCGA Research Network (http://cancergenome.nih.gov/).

Source data are available online for Figs. 1–8 and Extended Data Figs. 1–8. All other data supporting the findings of this study are available from the corresponding author on reasonable

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ACKNOWLEDGEMENTS We thank Y. Yin at Peking University for providing the materials described in the Methods. This work was supported by the National Key Research Program of China

(NO2015CB910403) and the National Natural Science Foundation (81830091, 91853206, 81430061 and 81972583) and its innovative group support (number 81721004), as well as the Fundamental

Research Funds for the Central Universities. AUTHOR INFORMATION Author notes * These authors contributed equally: Shao-Ming Shen, Cheng Zhang, Meng-Kai Ge, Shuang-Shu Dong. AUTHORS AND

AFFILIATIONS * Department of Pathophysiology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren-Ji Hospital and Key Laboratory of Cell Differentiation and

Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China Shao-Ming Shen, Cheng Zhang, Meng-Kai Ge, Li Xia, Ping He, Na Zhang, Shuo

Yang, Yun Yu, Jun-Ke Zheng & Guo-Qiang Chen * Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of the Chinese Academy of Sciences,

Chinese Academy of Sciences, Shanghai, China Shuang-Shu Dong & Yan Ji * Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and

Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai, China Jian-Xiu Yu * Department of Liver Surgery, Ren-Ji Hospital, Shanghai Jiao Tong University School of Medicine,

Shanghai, China Qiang Xia Authors * Shao-Ming Shen View author publications You can also search for this author inPubMed Google Scholar * Cheng Zhang View author publications You can also

search for this author inPubMed Google Scholar * Meng-Kai Ge View author publications You can also search for this author inPubMed Google Scholar * Shuang-Shu Dong View author publications

You can also search for this author inPubMed Google Scholar * Li Xia View author publications You can also search for this author inPubMed Google Scholar * Ping He View author publications

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publications You can also search for this author inPubMed Google Scholar * Qiang Xia View author publications You can also search for this author inPubMed Google Scholar * Guo-Qiang Chen

View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.-M.S., C.Z., M.-K.G. and S.-S.D. performed most of the experiments. L.X. performed the

LC−MS/MS analyses. P.H., N.Z. and S.Y. conducted partial experiments. Y.J. performed the bioinformatics analysis. Q.X. collected patient samples. Y.Y., J.-K.Z. and J.-X.Y. provided

constructive comments and discussion. G.-Q.C. and S.-M.S. designed and supervised the entire project and prepared the manuscript. CORRESPONDING AUTHORS Correspondence to Shao-Ming Shen or

Guo-Qiang Chen. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 INCONSISTENT EXPRESSIONAL PATTERNS OF PTEN AND PTENΑ/Β IN LIVER CANCER AND

POTENTIAL REGULATORS. (A) Genomic alterations of PTEN gene in TCGA cohorts analyzed by The cBioPortal for Cancer Genomics (http://www.cbioportal.org/). Arrowhead points to the liver cancer

cohort. (B) Representative images of the IHC staining of PTENα/β (top) and PTEN (bottom) in the sections of SMMC-7721xenografts with or without depletion of PTENα/β (PTENα/βKO-1) and PTEN

(PTEN3KO) respectively (n=1 experiment). Scale bar, 100 μm. (C) Numbers of samples in 9 indicated subgroups divided according to PTEN and PTENα/β expressions in tumor tissues compared to

paired adjacent tissues. (D) Representative images of the IHC staining of indicated proteins in the serial sections of tumor tissue (T) and paired adjacent normal tissue (N) isolated from

one liver cancer patient (n=1 experiment). Scale bar, 100 μm. (E) Western blot analysis and quantification of indicated proteins in 293T, Hela and MEF treated with 50 μg/ml CHX for different

time points. (F) Western blot analysis of indicated proteins in 293T cells transduced by shRNAs as indicated. (G) qRT-PCR analysis of PTEN mRNA in 293T cells with gNS or gUSP9X infection.

(H) Analysis of the correlation between USP9X and PTENα/β in human liver cancer samples with decreased PTEN in tumor tissues compared to paired adjacent normal tissues. Samples were divided

into 6 subgroups according to Figure 1J. Data were analyzed by Pearson’s χ2 test and the P value is shown. The experiments in E, F were repeated three times independently with the similar

results, and one representative result was shown. For G, data represent means with bar as S.E.M. of three independent experiments; two-tailed unpaired t-test. NC, negative control. Source

data Source data EXTENDED DATA FIG. 2 USP9X DEUBIQUITINATES AND STABILIZES PTENΑ/Β. (A) Western blot analysis with anti-Flag or anti-HA antibody in the input and immunoprecipitates of 293T

cells co-transfected with HA-tagged PTENβ and Flag-tagged USP9X or its four fragments as shown in the top schematics. (B) Schematics of N-terminal S-tag fused PTEN isoforms and derivatives.

(C, D) 293T and Hela cells with or without USP9X knockdown by shRNA were treated with 50 μg/ml CHX for different time points (C) or 10 μM MG132 for 6 h (D), followed by Western blot analysis

of indicated proteins. (E, F) In vivo ubiquitination assay in 293T cells transfected with the indicated plasmids was followed by Western blot analysis with antibody against total ubiquitin

(E) and antibodies against different types of ubiquitin chain (F). All experiments were repeated at least three times independently with the similar results and one representative result was

shown respectively. NC, negative control; EV, empty vector. Source data EXTENDED DATA FIG. 3 FBXW11 UBIQUITINATES AND DESTABILIZES PTENΑ/Β. (A) 293T cells were transduced by lentiviruses

encoding shRNAs targeting indicated genes, followed by Western blot for PTEN and PTENα/β with β-actin as loading control. Protein bands were quantified and the ratio of PTEN or PTENα/β

versus β-actin was respectively calculated and normalized with shNC cells. The shadowy area presents ratio range of 0.67 to 1.5. (B) The genomic locations of gRNAs targeting FBXW11 (top).

The Sanger sequencing analysis of genomic DNA from 293T cells transduced by lentivirus encoding gRNAs targeting FBXW11, along with gNS (bottom). Purple arrowheads indicate the start sites of

perturbed genomic sequence. (C) qRT-PCR analysis of PTEN mRNA in SMMC-7721 and 293T cells with or without FBXW11 depletion by CRISPR-CAS9. (D) The abundances of PTENα/β and PTEN relative to

loading control according to the experiments in Figure 3D were respectively quantified (n=3 independent biological samples). (E-G) In vivo ubiquitination assay of Flag-tagged PTEN, PTENα

and PTENβ in 293T cells without (E) or with (G) co-transfected of Myc-TR-TUBEs, followed by Western blot analysis with antibody against total ubiquitin (E, G) and antibodies against

different types of ubiquitin chain (F). (H) 293T and SMMC-7721 cells with or without FBXW11 over-expression were treated with 10 μM MG132 for 6 h, followed by Western blot analysis of

indicated proteins. Experiments in E-H were repeated three times independently with similar results and one representative result was shown. For C, D, data represent means with bar as S.E.M.

of three independent experiments; two-tailed unpaired t-test (C) and two-way ANOVA (D). NC, negative control; NS, non-specific control; EV, empty vector. Source data Source data EXTENDED

DATA FIG. 4 CLINICAL SIGNIFICANCE OF PTEN AND PTENΑ/Β, AND THEIR SPECIFIC DEPLETION BY CRISPR-CAS9. (A-F) Patients with liver cancer were divided into two groups respectively with

equal/increased (n=49 samples) and decreased PTEN (n=39 samples) (A-C) or equal/increased (n= 63 samples) and decreased PTENα/β (n=25 samples) (D-F) in tumor tissues compared to paired

adjacent normal tissues. Scatterplot analysis of tumor size (A, D), percentage of tumor stage (B, E), and Kaplan-Meier plot of overall survival (C, F) between the two groups were shown. Data

represent means with bar as S.E.M. (A, D), and were analyzed using Mann–Whitney U test (two-sided) (A, D), Pearson’s χ2 test (B, E), and log-rank test (two-sided) (C, F). (G) Patients with

liver cancer were divided into two groups respectively with equal/increased and decreased PTEN in tumor tissues compared to paired adjacent normal tissues, with the latter being further

divided into two subgroups respectively with equal/increased and decreased PTENα/β. Kaplan-Meier plot of overall survival between the three groups were shown. Data were analyzed using

log-rank test (two-sided), and P value is shown. (H) A schematic illustrating the two gRNAs respectively designed to target all three isoforms of PTEN (gPTEN3) and only the long isoforms of

PTEN (gPTENα/β). (I, J) Western blot analysis of indicated proteins in 293T (I) and MCF-7 (J) cells transduced by lentiviruses encoding CAS9 and gPTEN3 or gPTENα/β. Experiments in I, J were

repeated three times independently with the similar results, and one representative image was shown. NS, non-specific control. Source data Source data EXTENDED DATA FIG. 5 PTENΑ/Β IS CRUCIAL

FOR THE TUMOR-PROMOTING ROLE OF USP9X. (A) Comparison of USP9X expression between tumor tissues and matched non-tumor tissues in LIHC and BRCA cohorts from TCGA. Paired two-tailed Student’s

t-test. (B) Kaplan-Meier plot of overall survival of patients with high or low expression of USP9X in the tumor tissues of BRCA cohort from TCGA. Log-rank test (two-sided). (C-E) A

DOX-inducible expression system encoding wild-type or mutant PTENα/β was introduced into SMMC-7721 USP9XKO cells, followed by Western blot analysis showing induction of indicated proteins by

DOX administration (C). These cells were subcutaneously injected into nude mice (1.5×106 cells per mouse, n=5 mice for each group), followed by administration with or without 0.25 mg/kg DOX

in feed immediately after injection. Tumors were measured at different days (D), and harvested and weighed on day 23 (E). (F-K) Western blot analysis of indicated proteins in two

PTENα/β-knockout SMMC-7721 clones, PTENα/βKO-1 (F-H) and PTENα/βKO-2 (I-K), with gNS or gUSP9X infection (F, I). These cells were subcutaneously injected into nude mice (3×106 cells per

mouse, n=8 or 10 mice). Tumors were measured at different days (G, J), and harvested and weighed on day 17 (H, K). (L, M) Western blot analysis of indicated proteins in 293T (L) and MiaPaCa2

cells (M) with or without USP9X depletion. Experiments in C, F, I, L, M and all animal experiments were respectively repeated three times and at least twice independently, with similar

results, and result from one experiment was shown. For D, E, G, H, J, K data represent means with bar as S.E.M.; two-way ANOVA for D, G, J; two-tailed unpaired t-test for E, H, K. NC,

negative control; NS, non-specific control. Source data Source data EXTENDED DATA FIG. 6 IDENTIFICATION OF PTENΑ/Β-REGULATED GENES AND INVESTIGATION OF THE ROLE OF NOTCH3 IN TUMORIGENESIS.

(A) Schematics of PTENα and PTENβ derivatives with a C-terminal GFP-tag. (B) The workflow for identifying PTENα/β-regulated (activated or inhibited) genes in SMMC-7721 cells. G1-4, group

1-4. (C, D) Cell type-independent PTENα/β-activated genes were identified by cross matching PTENα/β-activated genes identified in (B) with genes down-regulated by PTENα/β depletion in

MiaPaCa2 cells. Workflow (C) and Venn diagram reporting the number of genes (D) are shown. (E-G) Western blot analysis of indicated proteins in SMMC-7721 cells with gNS or gNOTCH3 infection

(E). These cells were subcutaneously injected into nude mice (3×106 cells per mouse, n=8 mice for each group). Tumors were measured at different days (F), and harvested and weighed on day 17

(G). RNA-seq experiment was performed on two independent samples for each cell line in SMMC-7721 and one sample for each cell line in MiaPaCa2. The source data was provided as GSE126983.

E-G were repeated twice with the similar results, and result from one experiment was shown. For F, G, data represent means with bar as S.E.M.; two-way ANOVA for F; two-tailed unpaired t-test

for G. NS, non-specific control. Source data Source data EXTENDED DATA FIG. 7 PTENΑ/Β AND WDR5 INTERACT WITH EACH OTHER AND REGULATE A SIMILAR SUBSET OF GENES. (A, B) Western blot analysis

of indicated proteins (A) and qRT-PCR analysis of NOTCH paralogs (B) in MiaPaCa2 cells with gNS, gWDR5 or gRBBP5 infection. (C) qRT-PCR analysis of indicated genes in MiaPaCa2 cells gNS,

gWDR5 or gRBBP5 infection. (D) Bacterially expressed PTEN and PTENα derivatives with an N-terminal S-tag were incubated with GST-tagged WDR5, followed by S-tag pulldown and Western blot

analysis of indicated proteins. (E) Western blot analysis of indicated proteins in the immunoprecipitates of GFP-tagged proteins transfected in 293T cells. For B, C, data represent means

with bar as S.E.M. of three independent experiments; two-tailed unpaired t-test. All other experiments were repeated three times independently with similar results, and result from one

experiment was shown. NS, non-specific control. Source data Source data EXTENDED DATA FIG. 8 PTENΑ/Β PROMOTES TUMORIGENESIS THROUGH THE WDR5-H3K4ME3 AXIS. (A) Example tracks of WDR5 and

PTENα/β co-occupancy at the NOTCH3 (top) and SLC12A5 (bottom) loci in SMMC-7721 cells. (B) ChIP-qRT-PCR analysis of PTENα/β occupation on the promoters of indicated genes in SMMC-7721 cells

with or without depletion of PTENα/β by CRISPR-CAS9, with IgG used as negative control (n=3 biologically independent experiments). (C) Immunofluorescent staining of H3K4me3 together with

re-staining of DAPI in SMMC-7721 cells with gNS and gPTENα/β infection. Scale bars, 50 μm. (D) Bacterially expressed N-terminal-S-tagged PTEN and PTENα derivatives were incubated with

GST-tagged WDR5, followed by S-tag pulldown and Western blot analysis. (E-G) Western blot analysis of indicated proteins in SMMC-7721 PTEN3KO cells transduced by PTENα or PTENαΔ116-148 (E).

These cells were subcutaneously injected into nude mice (1.5×106 cells per mouse, n=5 mice for each group). Tumors were measured at different days (F), and harvested and weighed on day 21

(G). (H-J) Two WDR5-depleted SMMC-7721 clones (WDR5KO-1 and WDR5KO-2) along with parental cells were transduced by PTENα or EV, followed by Western blot analysis of indicated proteins (H).

These cells were subcutaneously injected into nude mice (1.5×106 cells per mouse, n=7 mice for each group). Tumors were measured at different days (I), and harvested and weighed on day 17

(J). All animal experiments were repeated at least twice, and other experiments except for A were repeated three times independently, with the similar results, and result from one experiment

was shown. For B, F, G, I, J data represent means with bar as S.E.M.; two-tailed unpaired t-test for B, G, J; two-way ANOVA for F, I. NS, non-specific control; EV, empty vector. Source data

Source data SUPPLEMENTARY INFORMATION REPORTING SUMMARY SUPPLEMENTARY TABLE 1 Information on samples from patients with liver cancer (cohort 1). Supplementary Table 2 Information on

microarrays of samples from patients with liver cancer (cohort 2). Supplementary Table 3 N173 and PTENα interacting proteins, including three sections, respectively, with N173, PTENα and

both interacting proteins. Supplementary Table 4 PTENα/β-regulated genes. Supplementary Table 5 Plasmids used in this study. Supplementary Table 6 Target sequences of the shRNAs, gRNAs for

CRISPR–Cas9 and gRNAs for CRISPR–dCas9 used in this study. Supplementary Table 7 Antibodies and reagents used in this study. Supplementary Table 8 Primers used in this study. SOURCE DATA

SOURCE DATA FIG. 1 Statistical Source Data SOURCE DATA FIG. 1 Unprocessed Western Blots and/or gels SOURCE DATA FIG. 2 Unprocessed Western Blots and/or gels SOURCE DATA FIG. 3 Statistical

Source Data SOURCE DATA FIG. 3 Unprocessed Western Blots and/or gels SOURCE DATA FIG. 4 Statistical Source Data SOURCE DATA FIG. 4 Unprocessed Western Blots and/or gels SOURCE DATA FIG. 5

Statistical Source Data SOURCE DATA FIG. 5 Unprocessed Western Blots and/or gels SOURCE DATA FIG. 6 Statistical Source Data SOURCE DATA FIG. 6 Unprocessed Western Blots and/or gels SOURCE

DATA FIG. 7 Statistical Source Data SOURCE DATA FIG. 7 Unprocessed Western Blots and/or gels SOURCE DATA FIG. 8 Statistical Source Data SOURCE DATA FIG. 8 Unprocessed Western Blots and/or

gels SOURCE DATA EXTENDED DATA FIG. 1 Statistical Source Data SOURCE DATA EXTENDED DATA FIG. 1 Unprocessed Western Blots and/or gels SOURCE DATA EXTENDED DATA FIG. 2 Unprocessed Western

Blots and/or gels SOURCE DATA EXTENDED DATA FIG. 3 Statistical Source Data SOURCE DATA EXTENDED DATA FIG. 3 Unprocessed Western Blots and/or gels SOURCE DATA EXTENDED DATA FIG. 4 Statistical

Source Data SOURCE DATA EXTENDED DATA FIG. 4 Unprocessed Western Blots and/or gels SOURCE DATA EXTENDED DATA FIG. 5 Statistical Source Data SOURCE DATA EXTENDED DATA FIG. 5 Unprocessed

Western Blots and/or gels SOURCE DATA EXTENDED DATA FIG. 6 Statistical Source Data SOURCE DATA EXTENDED DATA FIG. 6 Unprocessed Western Blots and/or gels SOURCE DATA EXTENDED DATA FIG. 7

Statistical Source Data SOURCE DATA EXTENDED DATA FIG. 7 Unprocessed Western Blots and/or gels SOURCE DATA EXTENDED DATA FIG. 8 Statistical Source Data SOURCE DATA EXTENDED DATA FIG. 8

Unprocessed Western Blots and/or gels RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Shen, SM., Zhang, C., Ge, MK. _et al._ PTENα and PTENβ promote

carcinogenesis through WDR5 and H3K4 trimethylation. _Nat Cell Biol_ 21, 1436–1448 (2019). https://doi.org/10.1038/s41556-019-0409-z Download citation * Received: 11 March 2019 * Accepted:

20 September 2019 * Published: 04 November 2019 * Issue Date: November 2019 * DOI: https://doi.org/10.1038/s41556-019-0409-z SHARE THIS ARTICLE Anyone you share the following link with will

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