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Reduced PTEN protein is linked to tumorigenesis. Here, Chen et al. show that the nuclear transport receptor Importin-11 separates PTEN from degradation machinery. IPO11 mutant mice exhibit PTEN degradation, lung adenocarcinoma, and prostate neoplasia. In human prostatectomy patients, IPO11 status predicts disease recurrence and metastasis., Phosphatase and tensin homologue (PTEN) protein levels are critical for tumor suppression. However, the search for a recurrent cancer-associated gene alteration that causes PTEN degradation has remained futile. In this study, we show that Importin-11 (Ipo11) is a transport receptor for PTEN that is required to physically separate PTEN from elements of the PTEN degradation machinery. Mechanistically, we find that the E2 ubiquitin-conjugating enzyme and IPO11 cargo, UBE2E1, is a limiting factor for PTEN degradation. Using in vitro and in vivo gene-targeting methods, we show that Ipo11 loss results in degradation of Pten, lung adenocarcinoma, and neoplasia in mouse prostate with aberrantly high levels of Ube2e1 in the cytoplasm. These findings explain the correlation between loss of IPO11 and PTEN protein in human lung tumors. Furthermore, we find that IPO11 status predicts disease recurrence and progression to metastasis in patients choosing radical prostatectomy. Thus, our data introduce the IPO11 gene as a tumor-suppressor locus, which is of special importance in cancers that still retain at least one intact PTEN allele.

We have recently recapitulated metastasis of human PTEN/TP53–mutant prostate cancer in the mouse using the RapidCaP system. Surprisingly, we found that this metastasis is driven by MYC, and not AKT, activation. Here, we show that cell–cell communication by IL6 drives the AKT–MYC switch through activation of the AKT-suppressing phosphatase PHLPP2, when PTEN and p53 are lost together, but not separately. IL6 then communicates a downstream program of STAT3-mediated MYC activation, which drives cell proliferation. Similarly, in tissues, peak proliferation in Pten/Trp53–mutant primary and metastatic prostate cancer does not correlate with activated AKT, but with STAT3/MYC activation instead. Mechanistically, MYC strongly activates the AKT phosphatase PHLPP2 in primary cells and prostate cancer metastasis. We show genetically that Phlpp2 is essential for dictating the proliferation of MYC-mediated AKT suppression. Collectively, our data reveal competition between two proto-oncogenes, MYC and AKT, which ensnarls the Phlpp2 gene to facilitate MYC-driven prostate cancer metastasis after loss of Pten and Trp53. Significance: Our data identify IL6 detection as a potential causal biomarker for MYC-driven metastasis after loss of PTEN and p53. Second, our finding that MYC then must supersede AKT to drive cell proliferation points to MYC inhibition as a critical part of PI3K pathway therapy in lethal prostate cancer. Cancer Discov; 5(6); 636–51. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 565

Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs.

Background: Amplification of 3q26 is one of the most frequent genetic alterations in many human malignancies. Recently, we isolated a novel oncogene eIF-5A2 within the 3q26 region. Functional study has demonstrated the oncogenic role of eIF-5A2 in the initiation and progression of human cancers. In the present study, we aim to investigate the physiological and pathological effect of eIF-5A2 in an eIF-5A2 transgenic mouse model.Methods: An eIF-5A2 transgenic mouse model was generated using human eIF-5A2 cDNA. The eIF-5A2 transgenic mice were characterized by histological and immunohistochemistry analyses. The aging phenotypes were further characterized by wound healing, bone X-ray imaging and calcification analysis. Mouse embryo fibroblasts (MEF) were isolated to further investigate molecular mechanism of eIF-5A2 in aging.Results: Instead of resulting in spontaneous tumor formation, overexpression of eIF-5A2 accelerated the aging process in adult transgenic mice. This included decreased growth rate and body weight, shortened life span, kyphosis, osteoporosis, delay of wound healing and ossification. Investigation of the correlation between cellular senescence and aging showed that cellular senescence is not required for the aging phenotypes in eIF-5A2 mice. Interestingly, we found that activation of eIF-5A2 repressed p19 level and therefore destabilized p53 in transgenic mouse embryo fibroblast (MEF) cells. This subsequently allowed for the accumulation of chromosomal instability, such as errors in cell dividing during metaphase and anaphase. Additionally, a significantly increase in number of aneuploidy cells (p < 0.05) resulted from an increase in the incidences of misaligned and lagging chromosomal materials, anaphase bridges, and micronuclei in the transgenic mice.Conclusion: These observations suggest that eIF-5A2 mouse models could accelerate organismal aging by increasing chromosome instability. © 2011 Chen et al; licensee BioMed Central Ltd., published_or_final_version

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: Amplification of 1q21 is the most frequent genetic alteration in hepatocellular carcinoma (HCC), which was detected in 58-78% of primary HCC cases by comparative genomic hybridization (CGH). Using chromosome microdissection/ hybrid selection approach we recently isolated a candidate oncogene CHD1L from 1q21 region. Our previous study has demonstrated that CHD1L had strong oncogenic ability, which could be effectively suppressed by siRNA against CHD1L. The molecular mechanism of CHD1L in tumorigenesis has been associated with its role in promoting cell proliferation. Methodology/Principal Findings: To further investigate the in vivo oncogenic role of CHD1L, CHD1L ubiquitous-expression transgenic mouse model was generated. Spontaneous tumor formations were found in 10/41 (24.4%) transgenic mice, including 4 HCCs, but not in their 39 wild-type littermates. In addition, alcohol intoxication was used to induce hepatocyte pathological lesions and results found that overexpression of CHD1L in hepatocytes could promote tumor susceptibility in CHD1L-transgenic mice. To address the mechanism of CHD1L in promoting cell proliferation, DNA content between CHD1Ltransgenic and wildtype mouse embryo fibroblasts (MEFs) was compared by flow cytometry. Flow cytometry results found that CHD1L could facilitate DNA synthesis and G1/ S transition through the up-regulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, and CDK4, and down-regulation of Rb, p27Kip1, and p53. Conclusion/Significance: Taken together, our data strongly support that CHD1L is a novel oncogene and plays an important role in HCC pathogenesis. © 2009 Chen et al., published_or_final_version

Amplification of 1q21 is the most frequent genetic alteration in human hepatocellular carcinoma (HCC), being detected in 58%-78% of primary HCC cases by comparative genomic hybridization. Recently, we isolated a candidate oncogene, Amplified in Liver Cancer 1 (ALC1), from 1q21 by hybrid selection. Here we demonstrate that ALC1 was frequently amplified and overexpressed in HCC. ALC1-transfected cells possessed a strong oncogenic ability, increasing the colony formation in soft agar and increasing the tumorigenicity in nude mice, which could be effectively suppressed by small interfering RNA against ALC1. Functional studies showed that overexpression of ALC1 could promote G1/S phase transition and inhibit apoptosis. Molecular studies revealed that the oncogenic function of ALC1 might be associated with its roles in promoting cell proliferation by down-regulating p53 expression. Conclusion: These results suggest that ALC1 is the target oncogene within the 1q21 amplicon and plays a pivotal role in HCC pathogenesis. (HEPATOLOGY 2007.)

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 sporadic transition from indolent to metastatic disease is a hallmark of prostate cancer (PC) and frequently involves deletion of PTEN and TP53. We recently recapitulated metastasis of Pten/ Trp53-mutant PC in mouse using the RapidCaP system and surprisingly, we found that it is driven by Myc, rather than Akt activation. Here, we show that cell-cell communication by Il6 drives this Akt-Myc switch through activation of the Akt-inactivating phosphatase Phlpp2. Primary cells revealed that loss of Pten/ Trp53 triggers secretion of the Il6 cytokine when these genes are deleted together, but not separately. Il6 then communicates a downstream program of Stat3-mediated Myc activation, which drives cell proliferation. Abrogation of Myc activity by Myc inhibition with the JQ1 bromodomain inhibitor, Myc-RNAi, and Myc-CRISPR/ Cas9 approaches inhibited proliferation. We validated these findings in vivo, where peak proliferation in Pten/ Trp53 mutant primary and metastatic PC did not correlate with activated Akt, but with Stat3/ Myc activation instead. Most notably, we found that Myc strongly activates the Akt phosphatase Phlpp2 in primary cells and RapidCaP metastasis, and showed genetically that Phlpp2 is essential for dictating proliferation and Myc-mediated suppression of Akt. Collectively, our data reveal competition between two proto-oncogenes: Myc and Akt, which ensnarls the Phlpp2 gene to facilitate Myc-driven metastasis. Citation Format: Dawid G. Nowak, Hyejin Cho, Tali Herzka, Victoria M.Y. Wang, Serif Senturk, Daniel V. DeMarco, David Ding, Christof Fellmann, Tumas Beinortas, David Kleinman, Kaitlin Watrud, Muhan Chen, John E. Wilkinson, Mireia Castillo-Martin, Carlos Cordon-Cardo, Brian D. Robinson, Lloyd C. Trotman. Myc drives Pten/ p53-deficient proliferation and metastasis due to Il6-secretion and Akt-suppression via Phlpp2. [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 2258. doi:10.1158/1538-7445.AM2015-2258

Loss of DNA copy number at the short arm of chromosome 3 is one of the most common genetic changes in human lung cancer, suggesting the existence of one or more tumor suppressor genes (TSG) at 3p. To identify most frequently deleted regions and candidate TSGs within these regions, a recently developed single-nucleotide polymorphism (SNP)-mass spectrometry-genotyping (SMSG) technology was applied to investigate the loss of heterozygosity (LOH) in 30 primary non–small-cell lung cancers. A total of 386 SNP markers that spanned a region of 70 Mb at 3p, from 3pter to 3p14.1, were selected for LOH analysis. The average intermarker distance in the present study is ∼180 kb. Several frequently deleted regions, including 3p26.3, 3p25.3, 3p24.1, 3p23, and 3p21.1, were found. Several candidate TSGs within these frequently detected LOH regions have been found, including APG7L at 3p25.3, CLASP2 at 3p23, and CACNA2D3 at 3p21.1. This study also showed that SMSG technology is a very useful approach to rapidly define the minimal deleted region and to identify target TSGs in a given cancer. (Cancer Res 2006; 66(8): 4133-8)

The maintenance of PTEN protein levels is critical for tumor suppression. Yet, the ubiquitination system has been shown to affect PTEN levels both adversely through degradation, as well as positively through nuclear import, and it has remained unclear how these two processes are integrated to prevent cancer. Here we show, that a nuclear import receptor is at the heart of a failsafe system that maintains PTEN levels by mediating its nuclear transport. Loss of import receptor function not only leads to cytoplasmic PTEN accumulation but also prompts PTEN degradation through a novel component of the PTEN ubiquitination system. By testing the consequences of importin loss in vivo, we found that hypomorphic mice developed lung adenocarcinoma, which presented with aberrant cytoplasmic PTEN localization and degradation, as predicted by our in vitro findings. Since the corresponding human locus suffers frequent deletion as well as inactivating mutations in lung cancer, we propose that this import receptor is a novel tumor suppressor that antagonizes PI 3-Kinase signaling in settings with at least one intact PTEN gene. Citation Format: Muhan Chen, Tali M. Herzka, Martha E. Zeeman, Kendra S. Plafker, Matthias Minderer, Mireia Castillo-Martin, Carlos Cordon-Cardo, Scott M. Plafker, Lloyd C. Trotman. PTEN levels are controlled by a nuclear transport receptor in lung cancer. [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 5258. doi:10.1158/1538-7445.AM2013-5258

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