Deubiquitination and stabilization of PTEN by USP13

USP13 regulates PTEN protein level and AKT signaling

Deubiquitinating enzymes (DUBs) are a group of proteases that regulate ubiquitin-dependent pathways by cleaving ubiquitin-protein bonds¹⁹. In order to identify PTEN-interacting deubiquitinases, we screened a panel of DUBs, in which a total of 30 deubiquitinase ORFs were fused with a triple-epitope tag, SFB (S-protein, FLAG tag and streptavidin-binding peptide), and then co-transfected with MYC-tagged PTEN into 293T cells. Immunoblotting assays showed that MYC-PTEN could be detected on S-protein beads conjugated with five DUBs, USP7, USP8, USP10, USP13 or USP39 (Fig. 1a). Moreover, MYC-PTEN transfected into HeLa cells could also be pulled down by each of these five SFB-tagged DUBs (Fig. 1b), further corroborating a physical association.

To examine the effects of these five PTEN-associated DUBs on PTEN expression and the growth of tumor cells, we stably expressed them individually in the MCF7 human breast cancer cell line. Although each of these five DUBs could interact with endogenous PTEN (Supplementary Fig. S1a), only one of them, USP13, significantly increased endogenous PTEN protein expression (Supplementary Fig. S1a). Compared with the control MCF7 cells, cells overexpressing USP7, USP10 or USP13 displayed a pronounced reduction in both proliferation (Supplementary Fig. S1b) and anchorage-independent growth (Supplementary Fig. S1c, d). Therefore, USP13 stood out as the top candidate for a possible PTEN deubiquitinase and a putative tumor suppressor.

As an alternative approach to identify PTEN-associated DUBs, we isolated PTEN-containing protein complexes using SFB-tagged PTEN. Tandem affinity purification using streptavidinsepharose beads and S-protein-agarose beads followed by mass spectrometric analysis identified six DUBs, USP10, USP13, USP7, USP8, USP39 and USP4, as PTEN interactors (Supplementary Table S1). Conversely, purification of SFB-tagged USP13 complexes identified PTEN as a USP13-interacting protein (Supplementary Table S2).

Next, we expressed USP13 in additional human breast cancer cells. This overexpression upregulated PTEN protein and downregulated AKT and FOXO1/3 phosphorylation in the MDAMB-231 cell line (Fig. 1c) which expresses moderate but detectable levels of endogenous PTEN (Supplementary Fig. S2a), while knockdown of PTEN in USP13-overexpressing MDA-MB-231 cells rescued the phosphorylation of both AKT and FOXO1/3 (Supplementary Fig. S2b). In contrast, expression of USP13 did not reduce phospho-AKT and phospho-FOXO levels in BT549 cells (Fig. 1c) which showed no PTEN protein expression (Supplementary Fig. S2a) due to a frameshift mutation^{20, 21}. Overexpression of a catalytically inactive mutant of USP13, C345A²², had no effect on PTEN protein levels and the phosphorylation of AKT and FOXO1/3 in these two cell lines (Fig. 1c).

To further validate regulation of PTEN protein by USP13, we performed loss-of-function analysis in multiple cell lines that express abundant USP13 and PTEN protein levels. Two independent USP13 shRNAs both decreased PTEN protein expression by 80% and increased phospho-AKT and phospho-FOXO1/3 levels by 3- to 5-fold in SUM159 breast cancer cells, while restoration of PTEN or expression of an RNAi-resistant ‘silence mutant’ (i.e., no amino acid change) of USP13 (USP13-RE) in USP13-depleted SUM159 cells completely reversed the effect of USP13 shRNA on upregulating the phosphorylation of AKT and FOXO (Fig. 1d and Supplementary Fig. S2c, d). Similarly, depletion of USP13 downregulated PTEN protein and upregulated AKT and FOXO1/3 phosphorylation in MCF10A (Fig. 1e) and MCF7 (Fig. 1f) mammary epithelial cells and in HCT116 colon cancer cells (Fig. 1g), but not in the isogenic PTEN-null HCT116 cells (Fig. 1g). In addition, USP13 shRNA potentiated insulin-induced AKT phosphorylation in SUM159 cells, which could be reversed by re-expression of PTEN (Supplementary Fig. S2e). We conclude from these data that USP13 inhibits AKT signaling through positive regulation of PTEN protein. It should be noted that neither knockdown nor overexpression of USP13 affected PTEN mRNA levels (Supplementary Fig. S3a, b). Thus, USP13 does not regulate PTEN expression at the transcriptional level.

In contrast to the knockdown effect of USP13, silencing of the other four PTEN-interacting DUBs, USP7, USP8, USP10 or USP39, did not affect PTEN protein levels (Supplementary Fig. S4a-d). USP7 (HAUSP), the only known PTEN deubiquitinase reported to date, regulates PTEN mono-ubiquitination and subcellular localization but not its protein stability¹⁸. In the present study, immunofluorescent staining (Fig. 2a, b) and fractionation assays (Fig. 2c, d) demonstrated that manipulating USP13 levels altered PTEN protein expression but not its localization; moreover, either overexpression or knockdown of USP13 had no effect on the levels of Cyclin A2 and PLK1 (Fig. 1c, d), the key substrates of the APC-CDH1 complex which has been shown to be regulated by nuclear PTEN in a phosphatase-independent manner²³. In contrast, USP7 reduced nuclear localization of PTEN in PC3 cells without affecting total PTEN protein levels (Fig. 2c, d), consistent with previously reported findings¹⁸.

USP13 deubiquitinates and stabilizes PTEN

We sought to determine whether USP13 directly interacts with PTEN and functions as a bona fide PTEN deubiquitinase. Consistent with the interaction observed in the initial screen, coimmunoprecipitation assays confirmed that ectopically expressed MYC-tagged PTEN could be detected in FLAG-tagged wild-type or the C345A mutant of USP13 immunoprecipitates (Fig. 3a), and that endogenous PTEN was present in endogenous USP13 immunoprecipitates (Fig. 3b). Moreover, purified GST-USP13, but not the GST-GFP control, was able to bind to FLAG-tagged PTEN under cell-free conditions (Fig. 3c), which demonstrated a direct interaction between USP13 and PTEN. PTEN consists of an N-terminal phosphatase domain, a C2 domain and a C-terminal PDZ motif²⁴. To map the USP13-binding region on PTEN, we co-expressed MYC-tagged USP13 along with a series of deletion mutants of PTEN¹⁵ (Fig. 3d). Co-immunoprecipitation assays demonstrated that the phosphatase domain of PTEN is essential for its physical interaction with USP13 (Fig. 3e).

We hypothesized that USP13 regulates PTEN through deubiquitination. Indeed, silencing USP13 expression by two independent shRNAs increased PTEN poly-ubiquitination by approximately 3-fold (Fig. 3f). On the other hand, ectopic expression of wild-type USP13, but not the C345A mutant which is still capable of interacting with PTEN (Fig. 3a), reduced the poly-ubiquitination of PTEN by 65% (Fig. 3g), suggesting that the enzymatic activity of USP13 is indispensable for USP13-dependent deubiquitination of PTEN. In order to determine whether PTEN is a direct substrate of USP13, we purified USP13 and ubiquitinated PTEN and then incubated them in a cell-free system. Wild-type USP13 purified from either bacteria or 293T cells, but not its catalytically inactive mutant C345A, decreased PTEN poly-ubiquitination by 64-70% in vitro (Fig. 3h, i). Therefore, USP13 can directly deubiquitinate PTEN.

To determine whether USP13 regulates the stability of PTEN protein, we examined ectopically expressed or endogenous PTEN protein levels in the presence of cycloheximide (CHX), an inhibitor of protein translation. Notably, overexpression of USP13, but not the enzyme-dead mutant, led to a prominent increase in the stability of endogenous or overexpressed PTEN protein, whereas the stability of HSP90 or co-transfected GFP control was not affected (Fig. 4a, b). Conversely, knockdown of USP13 resulted in destabilization of PTEN protein (Fig. 4c, d). Collectively, these results suggest that USP13 is a PTEN deubiquitinase that stabilizes PTEN.

Loss of USP13 promotes tumorigenesis through downregulation of PTEN

We asked whether USP13 functions as a tumor-suppressing protein by regulating PTEN. Two independent USP13 shRNAs (Fig. 1d) both markedly increased the proliferation (Fig. 5a) and anchorage-independent growth (Fig. 5b, c) of SUM159 breast cancer cells, while restoration of PTEN (Fig. 1d) or expression of an RNAi-resistant USP13 mutant (Supplementary Fig. S2c, d) completely reversed the effect of USP13 shRNA (Fig. 5a-d). Moreover, knockdown of USP13 promoted the proliferation of HCT116 colon cancer cells but not the isogenic PTEN-null HCT116 cells (Fig. 5e).

The effect of USP13 shRNA on cell proliferation is AKT-dependent, as treatment with the AKT inhibitor MK-2206 abolished this effect (Fig. 5f). Furthermore, since USP13 regulates AKT phosphorylation through PTEN (Fig. 1c-g), and since AKT plays a critical role in regulating the Warburg effect whereby cancer cells exhibit a high rate of glucose uptake and glycolysis^25-27, we speculated that USP13 might regulate the Warburg effect through PTEN. Indeed, knockdown of USP13 increased glucose uptake and glycolysis, which could be fully reversed by restoration of PTEN, as gauged by lactate production and glucose incorporation assays (Fig. 5g, h).

To investigate the biological function of USP13 in breast cancer cells in vivo, we subcutaneously implanted USP13-depleted SUM159 cells into nude mice and monitored tumor growth for more than nine weeks. Mice bearing USP13 shRNA-expressing SUM159 cells showed increased tumor growth throughout the experiment compared with mice implanted with control shRNA-infected cells (Fig. 5i). At 65 days after tumor cell implantation, we observed a 2.5-fold increase in tumor volume (Fig. 5i) and a 3.5-fold increase in the weight of the tumors formed by USP13-depleted SUM159 cells (Fig. 5j, k). Notably, restoring PTEN expression fully reversed the tumor-promoting effect of USP13 shRNA (Fig. 5i-k). Western blot analysis of tumor lysates confirmed that the effect of USP13 shRNA on PTEN and phospho-AKT was retained in these tumors (Fig. 5l). Therefore, loss of USP13 promotes tumorigenesis through downregulation of PTEN.

The anti-tumor function of USP13 depends on PTEN status

To further determine the dependence of the USP13 function on PTEN status, we compared the PTEN-positive cell line MDA-MB-231 and the PTEN-null cell line BT549 (Fig. 1c). Expression of USP13 (but not the C345A mutant) in MDA-MB-231 cells, which led to upregulation of PTEN and downregulation of phospho-AKT and phospho-FOXO1/3 (Fig. 1c), significantly inhibited cell proliferation (Fig. 6a), colony formation on soft agar (Fig. 6b, c), lactate production (Fig. 6d), glucose uptake (Fig. 6e) and tumor growth (Fig. 6f-h), while knockdown of PTEN (Supplementary Fig. S2b) rescued the proliferation of USP13-overexpressing MDA-MB-231 cells (Fig. 6i). In stark contrast, none of these effects were observed in USP13-overexpressing BT549 cells (Fig. 6a-e and 6j-l), which showed no substantial difference in AKT phosphorylation compared with mock-infected cells (Fig. 1c). Western blot analysis of tumor lysates confirmed that the effect of USP13 on PTEN and phospho-AKT was retained in tumors formed by USP13-overexpressing MDA-MB-231 or BT549 cells (Fig. 6m). Taken together, USP13 has a PTEN-dependent tumor-suppressing function.

USP13 is downregulated in human breast tumors and correlates with PTEN protein levels

PTEN plays a pivotal role in human breast cancer suppression and is dose-dependent. Female patients with Cowden syndrome have partial loss of PTEN due to heterozygous germline PTEN mutations and are estimated to have a 25-50% risk of developing breast cancer²⁸. Genetic analysis of mouse models has revealed Pten haploinsufficiency and dose dependence in breast tumor suppression²⁹. Moreover, while approximately 5% of sporadic breast tumors harbor PTEN mutations⁹, loss of PTEN immunoreactivity is found in nearly 40%³⁰, which indicates that post-transcriptional and post-translational regulation of PTEN may contribute substantially to the development of human breast cancer. To determine the relevance of regulation of PTEN by USP13 in patients, we performed immunohistochemical staining of PTEN and USP13 (Fig. 7a) on the breast cancer progression tissue microarrays (TMAs) from the National Cancer Institute³¹, with antibodies validated for immunohistochemistry (Supplementary Fig. S5). Notably, downregulation of PTEN and USP13 was observed in 73.8% (152 of 206) and 41.3% (83 of 201) of breast tumors, whereas only 31.8% (14 of 44) and 13.2% (5 of 38) of normal mammary tissues exhibited low expression of PTEN and USP13 (Fig. 7b, c), respectively, suggesting that both PTEN and USP13 are downregulated in human breast tumors. Moreover, a significant positive correlation (R = 0.25, P = 4 × 10^-4) between PTEN and USP13 protein levels was observed in these breast carcinomas, in which 88% (73 of 83) of the tumors with low USP13 expression also displayed low PTEN expression (Fig. 7d). However, it should be noted that 38.8% (78 of 201) of total tumor specimens had low PTEN expression but high USP13 expression (Fig. 7d). Collectively, these data suggest that loss of USP13 may contribute to loss of PTEN in a substantial fraction of human tumors, whereas in other tumors PTEN can be inactivated by different mechanisms, including genetic alterations and upregulation of PTEN ubiquitin ligases (such as NEDD4-1¹⁴ and WWP2¹⁵).