Muhan Chen

Molecular Pathways: PI3K Pathway Phosphatases as Biomarkers for Cancer Prognosis and Therapy

Cancer research has seen tremendous changes over the past decade. Fast progress in sequencing technology has afforded us with landmark genetic alterations, which had immediate impact on clinical science and practice by pointing to new kinase targets, such as phosphoinositide 3-kinase (PI3K), the EGF receptor, or BRAF. The PI3K pathway for growth control has emerged as a prime example for both oncogene activation and tumor suppressor loss in cancer. Here, we discuss how therapy using PI3K pathway inhibitors could benefit from information on specific phosphatases, which naturally antagonize the kinase targets. This PI3K pathway is found mutated in most cancer types, including prostate, breast, colon, and brain tumors. The tumor-suppressing phosphatases operate at two levels. Lipid-level phosphatases, such as PTEN and INPP4B, revert PI3K activity to keep the lipid second messengers inactive. At the protein level, PHLPP1/2 protein phosphatases inactivate AKT kinase, thus antagonizing mTOR complex 2 activity. However, in contrast with their kinase counterparts the phosphatases are unlikely drug targets. They would need to be stimulated by therapy and are commonly deleted and mutated in cancer. Yet, because they occupy critical nodes in preventing cancer initiation and progression, the information on their status has tremendous potential in outcome prediction, and in matching the available kinase inhibitor repertoire with the right patients. Clin Cancer Res; 20(12); 3057–63. ©2014 AACR.

MYC Drives Pten/Trp53-Deficient Proliferation and Metastasis due to IL6 Secretion and AKT Suppression via PHLPP2

UNLABELLED 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.

Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output

Significance This work unveils a previously unidentified function of the tumor suppressor pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) in inhibiting oncogenic signaling by suppressing the steady-state levels of receptor tyrosine kinases such as the EGF receptor. Specifically, PHLPP modifies the histone code to control the transcription of receptor tyrosine kinases. This epigenetic function can account for the upregulation of receptor tyrosine kinases in the multiple cancer types where PHLPP function is compromised. 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.

The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein

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.

Abstract 2405: Identification of PHLPP as a tumour suppressor reveals the role of pathway feedback compensation in PTEN-mutant prostate cancer progression

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

Abstract 2258: Myc drives Pten/ p53-deficient proliferation and metastasis due to Il6-secretion and Akt-suppression via Phlpp2

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

Abstract 5258: PTEN levels are controlled by a nuclear transport receptor in lung cancer.

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.

Abstract 1269: Identifying Pten-sensitive drug therapy through metabolic phenotype-arrays

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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