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Summary SUPT4H1 is a transcription elongation factor that makes up part of the RNA polymerase II complex. Recent studies propose a selective role for SUPT4H1 in the transcription of repeat-containing DNA, the translated products of which contribute to neurodegenerative disorders such as C9orf72-amyotrophic lateral sclerosis. To investigate the potential of SUPT4H1 as a therapeutic target in repeat-associated neurodegeneration, we depleted SUPT4H1 by RNA interference to inhibit the function of the SUPT4H1/SUPT5H transcription elongation complex. Depletion of SUPT4H1 leads to a global reduction in all cellular RNA, highlighting the significant challenges that are associated with targeting this molecule for the treatment of human disease. Any requirement of SUPT4H1 for transcription of specific transcripts should be interpreted in the context of global modulatory effects on the transcriptome.

SummaryPhosphatidylinositol phosphate (PIP) second messengers relay extracellular growth cues through the phosphorylation status of the inositol sugar, a signal transduction system that is deregulated in cancer. In stark contrast to PIP inositol head-group phosphorylation, changes in phosphatidylinositol (PI) lipid acyl chains in cancer have remained ill-defined. Here, we apply a mass-spectrometry-based method capable of unbiased high-throughput identification and quantification of cellular PI acyl chain composition. Using this approach, we find that PI lipid chains represent a cell-specific fingerprint and are unperturbed by serum-mediated signaling in contrast to the inositol head group. We find that mutation of Trp53 results in PIs containing reduced-length fatty acid moieties. Our results suggest that the anchoring tails of lipid second messengers form an additional layer of PIP signaling in cancer that operates independently of PTEN/PI3-kinase activity but is instead linked to p53.

Gloss Lipids can serve as cellular signaling molecules. Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] is one such lipid that is the key molecular product of the pathway involving phosphoinositide 3-kinase (PI3K), which synthesizes PtdIns(3,4,5)P 3 , and the kinase AKT, which is activated at sites where PtdIns(3,4,5)P 3 is produced. This pathway is referred to as the PI3K/AKT pathway. PtdIns(3,4,5)P 3 triggers cell division, growth, and survival programs, among other cellular processes. The production of PtdIns(3,4,5)P 3 depends on the enzymes responsible for its synthesis, and quenching of PI3K/AKT signaling depends on the enzymes that metabolize PtdIns(3,4,5)P 3 . This Review with 1 figure and 67 references highlights how understanding the cellular locations where PtdIns(3,4,5)P 3 is formed and the kinetics and regulation of the enzymes that produce and metabolize this lipid reveals the spatiotemporal specificity of PI3K/AKT-mediated signaling.

The tumour suppressor APC is the most commonly altered gene in colorectal cancer (CRC). Genetic and epigenetic alterations of APC may therefore be associated with dietary and lifestyle risk factors for CRC. Analysis of APC mutations in the extended mutation cluster region (codons 1276-1556) and APC promoter 1A methylation was performed on 185 archival CRC samples collected from participants of the European Prospective Investigation into Cancer (EPIC)-Norfolk Study, with the aim of relating these to high quality seven-day dietary and lifestyle data collected prospectively. Truncating APC mutations (APC+) and promoter 1A methylation (PM+) were identified in 43% and 23% of CRCs analysed, respectively. Distal CRCs were more likely than proximal CRCs to be APC+ or PM+ (P = 0.04). APC+ CRCs were more likely to be moderately/well differentiated and microsatellite stable than APC- CRCs (P = 0.05 and 0.03). APC+ CRC cases consumed more alcohol than their counterparts (P = 0.01) and PM+ CRC cases consumed lower levels of folate and fibre (P = 0.01 and 0.004). APC+ or PM+ CRC cases consumedhigher levels of processed meat and iron from red meat and red meat products (P=0.007 and 0.006). Specifically, CRC cases harbouring GC to AT transition mutations consumed higher levels of processed meat (35 versus 24 g/day, P = 0.04) and iron from red meat and red meat products (0.8 versus 0.6 mg/day, P = 0.05). In a logistic regression model adjusted for age, sex and cigarette smoking status, each 19g/day (1SD) increment increase in processed meat consumption was associated with cases with GC to AT mutations (OR 1.68, 95% CI 1.03-2.75). In conclusion, APC+ and PM+ CRCs may be influenced by diet and GC to AT mutations in APC are associated with processed meat consumption, suggesting a mechanistic link with dietary alkylating agents, such as N-nitroso compounds.

PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles.

PTEN loss drives many cancers and recent genetic studies reveal that often PTEN is antagonised at the protein level without alteration of DNA or RNA expression. This scenario can already cause malignancy since PTEN is haploinsufficient. We here review normally occurring mechanisms of PTEN protein regulation and discuss three processes where PTEN plasticity is needed: ischaemia, development and wound healing. These situations demand transient PTEN suppression while on the other hand cancer exploits them for continuous proliferation and survival advantages. Therefore increased understanding of PTEN plasticity may help us better interpret tumour development and ultimately lead to drug targets for PTEN supporting cancer therapy.

The tumour suppressor APC is the most commonly altered gene in colorectal cancer (CRC). Genetic and epigenetic alterations of APC may therefore be associated with dietary and lifestyle risk factors for CRC. Analysis of APC mutations in the extended mutation cluster region (codons 1276-1556) and APC promoter 1A methylation was performed on 185 archival CRC samples collected from participants of the European Prospective Investigation into Cancer (EPIC)-Norfolk study, with the aim of relating these to high-quality seven-day dietary and lifestyle data collected prospectively. Truncating APC mutations (APC(+) ) and promoter 1A methylation (PM(+) ) were identified in 43% and 23% of CRCs analysed, respectively. Distal CRCs were more likely than proximal CRCs to be APC(+) or PM(+) (p = 0.04). APC(+) CRCs were more likely to be moderately/well differentiated and microsatellite stable than APC(-) CRCs (p = 0.05 and 0.03). APC(+) CRC cases consumed more alcohol than their counterparts (p = 0.01) and PM(+) CRC cases consumed lower levels of folate and fibre (p = 0.01 and 0.004). APC(+) or PM(+) CRC cases consumed higher levels of processed meat and iron from red meat and red meat products (p = 0.007 and 0.006). Specifically, CRC cases harbouring GC-to-AT transition mutations consumed higher levels of processed meat (35 versus 24 g/day, p = 0.04) and iron from red meat and red meat products (0.8 versus 0.6 mg/day, p = 0.05). In a logistic regression model adjusted for age, sex and cigarette-smoking status, each 19 g/day (1SD) increment increase in processed meat consumption was associated with cases with GC-to-AT mutations (OR 1.68, 95% CI 1.03-2.75). In conclusion, APC(+) and PM(+) CRCs may be influenced by diet and GC-to-AT mutations in APC are associated with processed meat consumption, suggesting a mechanistic link with dietary alkylating agents, such as N-nitroso compounds.

PTEN nuclear entry driven by ubiquitination is mediated by the ligase-interacting protein Ndfip1 and is essential for neuronal survival in mice after cerebral ischemia., PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family–interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1−/− fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.

SummaryHyperactivation of the PI 3-kinase/AKT pathway is a driving force of many cancers. Here we identify the AKT-inactivating phosphatase PHLPP1 as a prostate tumor suppressor. We show that Phlpp1-loss causes neoplasia and, on partial Pten-loss, carcinoma in mouse prostate. This genetic setting initially triggers a growth suppressive response via p53 and the Phlpp2 ortholog, and reveals spontaneous Trp53 inactivation as a condition for full-blown disease. Surprisingly, the codeletion of PTEN and PHLPP1 in patient samples is highly restricted to metastatic disease and tightly correlated to deletion of TP53 and PHLPP2. These data establish a conceptual framework for progression of PTEN mutant prostate cancer to life-threatening disease.

Background The PTEN tumour suppressor gene and PIK3CA proto-oncogene encode proteins which contribute to regulation and propagation of signal transduction through the PI3K/AKT signalling pathway. This study investigates the prevalence of loss of PTEN expression and mutations in both PTEN and PIK3CA in colorectal cancers (CRC) and their associations with tumour clinicopathological features, lifestyle factors and dietary consumptions. Methods 186 adenocarcinomas and 16 adenomas from the EPIC Norfolk study were tested for PTEN and PIK3CA mutations by DNA sequencing and PTEN expression changes by immunohistochemistry. Dietary and lifestyle data were collected prospectively using seven day food diaries and lifestyle questionnaires. Results Mutations in exons 7 and 8 of PTEN were observed in 2.2% of CRC and PTEN loss of expression was identified in 34.9% CRC. Negative PTEN expression was associated with lower blood low-density lipoprotein concentrations (p = 0.05). PIK3CA mutations were observed in 7% of cancers and were more frequent in CRCs in females (p = 0.04). Analysis of dietary intakes demonstrated no link between PTEN expression status and any specific dietary factor. PTEN expression negative, proximal CRC were of more advanced Dukes' stage (p = 0.02) and poor differentiation (p < 0.01). Testing of the prevalence of PIK3CA mutations and loss of PTEN expression demonstrated that these two events were independent (p = 0.55). Conclusion These data demonstrated the frequent occurrence (34.9%) of PTEN loss of expression in colorectal cancers, for which gene mutations do not appear to be the main cause. Furthermore, dietary factors are not associated with loss of PTEN expression. PTEN expression negative CRC were not homogenous, as proximal cancers were associated with a more advanced Dukes' stage and poor differentiation, whereas distal cancers were associated with earlier Dukes' stage.

PTEN is thought to function at the plasma membrane where receptor tyrosine kinases activate PI 3-Kinases. Yet the majority of PTEN is located throughout the cytoplasm so that only a fraction of PTEN could be actively suppressing PI 3-K signaling at any time. Here we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via interaction with phosphatidylinositol 3- phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the C2 domain of PTEN specifically binds PI(3)P via the CBR3-loop. Mutations that render the CBR3-loop incapable of PI(3)P binding abrogate PTEN function in cells but not in vitro. The loss-of-function in cells is rescued by fusion of the canonical PI(3)P vesicle targeting domain, FYVE, to CBR3-loop mutant PTEN, demonstrating the functional relevance of PTEN activity on endosomal membranes. These findings introduce an entirely unexpected site of action of the PTEN tumor suppressor. Furthermore, they introduce the concept of PI 3-K signal activation over the vast surface of the plasma membrane that is contrasted by PTEN-mediated signal termination on the discretely sized and much smaller surfaces of endocytic vesicles. Implications of these results for cancer signaling and growth control will be discussed. Note: This abstract was not presented at the meeting. Citation Format: Adam Naguib, Gylua Bencze, Hyejin Cho, Wu Zheng, Ante Tocilj, Elad Elkayam, Christopher R Faehnle, Nadia Jaber, Christopher Pratt, Muhan Chen, Wei-Xing Zong, Michael S Marks, Leemor Joshua-Tor, Darryl J Pappin, Lloyd C. Trotman. PTEN function is controlled by recruitment to cytoplasmic vesicles. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-063. doi:10.1158/1538-7445.AM2015-LB-063

The PTEN tumor suppressor is among the most frequently altered genes of cancer. PTEN suppression at the protein level is critically associated with disease since PTEN is haploinsufficient in many cancer types, including prostate. We have recently shown strong cooperation between protein phosphatases and PTEN to suppress PI 3-Kinase and AKT signaling in prostate cancer. Intriguingly, our findings showed that PTEN status orchestrates a PHLPP2 response by controlling the protein levels of this phosphatase. Yet, little is known about genes that control the levels and activity of PTEN to a degree that they critically maintain its function in disease. Therefore, it is assumed that PTEN is constitutively active in normal cells. Here we identify the upstream activation mechanism of PTEN and discuss the consequences for cancer diagnosis and therapy with PI 3-Kinase pathway inhibitors. Citation Format: Adam Naguib, Gyula Bencze, Christopher R. Faehnle, Thomas Schalch, Zsolt Lazar, Cristian I. Ruse, Leemor Joshua-Tor, Darryl J. Pappin, Lloyd C. Trotman. Upstream activation of PTEN. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5156. doi:10.1158/1538-7445.AM2013-5156

Hyper-activation of the PI 3-Kinase/ AKT pathway is common in many cancer types. Tumourigenesis through this pathway is prevented by concerted action of multiple tumour suppressor genes. Most notably, PTEN reverts PI 3-Kinase activity whereas excessive pathway activation triggers the p53-mediated senescence arrest. However, it remains ill defined if and at what stage this response acts in human prostate cancer. Here we identify the AKT-inactivating phosphatase PHLPP as a tumour suppressor and demonstrate how the p53-response can antagonise co-deletion of PTEN and PHLPP to form a barrier against prostate cancer progression. We show that Phlpp-loss causes neoplasia and upon partial Pten-loss, carcinoma in mouse prostate. In this setting, Phlpp-deficiency triggers growth arrest via mTorC1-dependent activation of p53 and we find that co-deletion of Pten and Phlpp selects for spontaneous inactivation of p53 in prostate. Validating this conditional gene inactivation scheme in a comprehensive genomic patient data set we find that co-deletion of PTEN and PHLPP is almost exclusively observed in metastatic prostate cancer and tightly correlated to deletion of TP53. Furthermore, PTEN/ PHLPP expression can be used to predict disease outcome in these patients, comparable to the standard histology based method, but adding actionable information on pathway status. Finally, we show that both known PHLPP isoforms compensate for PTEN-suppression in a novel pathway feedback explaining their co-deletion with PTEN in the metastatic samples. Surprisingly, we find that the feedback surge of these genes is sensitive to some pharmacological inhibitors of the PI 3-Kinase pathway. Collectively, our findings emphasise the need for careful evaluation of PI 3-Kinase target therapy effects in prostate cancer and highlight the value of genetically engineered mouse models in this process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2405. doi:10.1158/1538-7445.AM2011-2405

The loss or mutation of tumor suppressor genes is a predominant event in the initiation, progression and metastatic development of cancer. PTEN and p53 are frequently inactivated in lethal metastatic prostate cancer, as they provide a critical growth, proliferation and anti-apoptotic advantage to cells in which their activity is diminished. In mouse prostate cancer models, loss of p53 alone in the prostate does not result in noticeable neoplasia. However, subsequent additional deletion of Pten causes lethal prostatic adenocarcinoma within 6 months, illustrating the need to combat the cooperative power of these two genetic lesions. Although inactivation of these tumor suppressor genes has been widely described at the level of their cancer phenotype, it has recently emerged that one understudied route to combat lesions harboring these alterations is to understand and exploit critical underlying changes in their metabolic makeup. In the present study, we used an array platform to determine the growth phenotype of p53 null Mouse Embryonic Fibroblast (MEFs) under close to 1200 conditions involving different sources of energy, amino acids, hormones, growth factors, chemical ions and responses to chemotherapy agents. with subsequent comparison to the response in p53/Pten double-null MEFs. Our analysis showed that loss of Pten critically altered response, utilization and sensitivity especially to specific hormones, ions and chemotherapy agents. Since this approach revealed Pten-status specific cell sensitivities, we are utilizing this information to establish selective targeting of cells as a precursor to therapeutic intervention in our genetically engineered mouse models of prostate cancer, which harbor the identical tumor suppressor lesions. Collectively, our results establish a rapid screening platform for identification of genotype-specific anti-cancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1269. doi:10.1158/1538-7445.AM2011-1269

The K-RAS oncogene is widely mutated in human cancers. Activating mutations in K-RAS give rise to constitutive signalling through the MAPK/ERK and PI3K/AKT pathways promoting increased cell division, reduced apoptosis and transformation. The majority of activating mutations in K-RAS are located in codons 12 and 13. In a human colorectal cancer we identified a novel K-RAS co-mutation that altered codons 19 and 20 resulting in transitions at both codons (L19F/T20A) in the same allele. Using focus forming transformation assays in vitro , we showed that co-mutation of L19F/T20A in K-RAS demonstrated intermediate transforming ability that was greater than that of individual L19F and T20A mutants, but less than that of G12D and G12V K-RAS mutants. This demonstrated the synergistic effects of co-mutation of codons 19 and 20 and illustrated that co-mutation of these codons is functionally significant.