The phosphatidylinositol-3-kinase (PI3K) pathway is commonly hyperactivated in cancer. One mechanism by which this occurs is by silencing of the phosphatase and tensin homolog (PTEN), a tumor suppressor and major antagonist of the pathway, through genetic, epigenetic or posttranscriptional mechanisms. Here, we used an unbiased siRNA screen in non-small-cell lung cancer cells to identify deubiquitylases (DUBs) that have an impact on PI3K signaling by regulating the abundance of PTEN. We found that PTEN expression was induced by depleting any of three members of the Josephin family DUBs: ataxin 3 (ATXN3), ataxin 3-like (ATXN3L) and Josephin domain containing 1 (JOSD1). However, this effect is not mediated through altered PTEN protein stability. Instead, depletion of each DUB increases expression of both the PTEN transcript and its competing endogenous RNA, PTENP1. In ATXN3-depleted cells, under conditions of transcriptional inhibition, PTEN and PTENP1 mRNAs rapidly decay, suggesting that ATXN3 acts primarily by repressing their transcription. Importantly, the PTEN induction observed in response to ATXN3 siRNA is sufficient to downregulate Akt phosphorylation and hence PI3K signaling. Histone deacetylase inhibitors (HDACi) have been suggested as potential mediators of PTEN transcriptional reactivation in non-small-cell lung cancer. Although PTEN exhibits a very limited response to the broad-spectrum HDACi Vorinostat (SAHA) in A549 cells, we find that combination with ATXN3 depletion enhances PTEN induction in an additive manner. Similarly, these interventions additively decrease cell viability. Thus, ATXN3 provides an autonomous, complementary therapeutic target in cancers with epigenetic downregulation of PTEN.

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors.

Reversible ubiquitylation of proteins contributes to their integrity, abundance and activity. The RE1-silencing transcription factor (REST) plays key physiological roles and is dysregulated in a spectrum of disease. It is rapidly turned over and is phosphorylated, polyubiquitylated and degraded en masse during neuronal differentiation and cell cycle progression. Through siRNA screening we identified the deubiquitylase USP15 as a key regulator of cellular REST. Both antagonism of REST polyubiquitylation and rescue of endogenous REST levels are dependent on the deubiquitylase activity of USP15. However, USP15 depletion does not destabilize pre-existing REST, but rather specifically impairs de novo REST synthesis. Indeed, we find that a small fraction of endogenous USP15 is associated with polysomes. In accordance with these findings, USP15 does not antagonize the degradation of phosphorylated REST at mitosis. Instead it is required for the rapid accumulation of newly synthesized REST on mitotic exit, thus playing a key role in its cell cycle oscillations. Importantly, this study reveals a novel role for a DUB in specifically promoting new protein synthesis.

Ubiquitin-specific protease 15 (USP15) is a widely expressed deubiquitylase that has been implicated in diverse cellular processes in cancer. Here we identify topoisomerase II (TOP2A) as a novel protein that is regulated by USP15. TOP2A accumulates during G2 and functions to decatenate intertwined sister chromatids at prophase, ensuring the replicated genome can be accurately divided into daughter cells at anaphase. We show that USP15 is required for TOP2A accumulation, and that USP15 depletion leads to the formation of anaphase chromosome bridges. These bridges fail to decatenate, and at mitotic exit form micronuclei that are indicative of genome instability. We also describe the cell cycle-dependent behaviour for two major isoforms of USP15, which differ by a short serine-rich insertion that is retained in isoform-1 but not in isoform-2. Although USP15 is predominantly cytoplasmic in interphase, we show that both isoforms move into the nucleus at prophase, but that isoform-1 is phosphorylated on its unique S229 residue at mitotic entry. The micronuclei phenotype we observe on USP15 depletion can be rescued by either USP15 isoform and requires USP15 catalytic activity. Importantly, however, an S229D phospho-mimetic mutant of USP15 isoform-1 cannot rescue either the micronuclei phenotype, or accumulation of TOP2A. Thus, S229 phosphorylation selectively abrogates this role of USP15 in maintaining genome integrity in an isoform-specific manner. Finally, we show that USP15 isoform-1 is preferentially upregulated in a panel of non-small cell lung cancer cell lines, and propose that isoform imbalance may contribute to genome instability in cancer. Our data provide the first example of isoform-specific deubiquitylase phospho-regulation and reveal a novel role for USP15 in guarding genome integrity.

Post-translational modification of proteins by ubiquitylation is increasingly recognised as a highly complex code that contributes to the regulation of diverse cellular processes. In humans, a family of almost 100 deubiquitylase enzymes (DUBs) are assigned to six subfamilies and many of these DUBs can remove ubiquitin from proteins to reverse signals. Roles for individual DUBs have been delineated within specific cellular processes, including many that are dysregulated in diseases, particularly cancer. As potentially druggable enzymes, disease-associated DUBs are of increasing interest as pharmaceutical targets. The biology, structure and regulation of DUBs have been extensively reviewed elsewhere, so here we focus specifically on roles of DUBs in regulating cell cycle processes in mammalian cells. Over a quarter of all DUBs, representing four different families, have been shown to play roles either in the unidirectional progression of the cell cycle through specific checkpoints, or in the DNA damage response and repair pathways. We catalogue these roles and discuss specific examples. Centrosomes are the major microtubule nucleating centres within a cell and play a key role in forming the bipolar mitotic spindle required to accurately divide genetic material between daughter cells during cell division. To enable this mitotic role, centrosomes undergo a complex replication cycle that is intimately linked to the cell division cycle. Here, we also catalogue and discuss DUBs that have been linked to centrosome replication or function, including centrosome clustering, a mitotic survival strategy unique to cancer cells with supernumerary centrosomes.

Enteric nervous system (ENS) progenitor cells isolated from mouse and human bowel can be cultured in vitro as neurospheres which are aggregates of the proliferating progenitor cells, together with neurons and glial cells derived from them. To investigate the factors regulating progenitor cell proliferation and differentiation, we first characterised cell proliferation in mouse ENS neurospheres by pulse chase experiments using thymidine analogs. We demonstrate rapid and continuous cell proliferation near the neurosphere periphery, after which postmitotic cells move away from the periphery to become distributed throughout the neurosphere. While many proliferating cells expressed glial markers, expression of the neuronal markers β-tubulin III (Tuj1) and nitric oxide synthase was detected in increasing numbers of post-mitotic cells after a delay of several days. Treatment of both mouse and human neurospheres with the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) reduced expression of the transcription factors Hes1 and Hes5, demonstrating inhibition of Notch signaling. DAPT treatment also inhibited progenitor cell proliferation and increased the numbers of differentiating neurons expressing Tuj1 and nitric oxide synthase. To confirm that the cellular effects of DAPT treatment were due to inhibition of Notch signaling, siRNA knockdown of RBPjκ, a key component of the canonical Notch signaling pathway, was demonstrated both to reduce proliferation and to increase neuronal differentiation in neurosphere cells. These observations indicate that Notch signaling promotes progenitor cell proliferation and inhibits neuronal differentiation in ENS neurospheres.

No abstract

USP7 is a nuclear deubiquitylase (DUB) with multiple cancer-associated substrates for which selective inhibitors are available, yet it remains unclear how the pleiotropic effects of USP7 are regulated. We report that S18-phosphorylation does not influence USP7 catalytic activity but instead confers selectivity for protein interactions. In particular, non-S18-phosphorylatable USP7 preferentially interacts with USP11 and TRIM27, together with TCEAL1 and TCEAL4 whose functions are unknown. Intriguingly, USP7 can interact with two cellular forms of TCEAL4, but USP11 only interacts with a lower abundance K142 mono-ubiquitylated form (TCEAL4-Ub), which can scaffold a complex containing both DUBs. Whilst USP11 and TCEAL4 are both USP7 substrates, TCEAL4-Ub levels are specifically maintained by USP11 with their levels positively correlated in cancer cell lines. Together these data illustrate how USP7 phosphorylation and TCEAL4 ubiquitylation combine to define distinct USP7 complexes. As TCEAL4 itself interacts with proteins involved in ubiquitylation and various forms of DNA regulation, these complexes may direct cellular activity of USP7.