Supplementary Figure S1 from Activation of Mammalian Target of Rapamycin Signaling Pathway Contributes to Tumor Cell Survival in Anaplastic Lymphoma Kinase–Positive Anaplastic Large Cell Lymphoma

Supplementary Figure 1 from Potential Role of Jun Activation Domain–Binding Protein 1 as a Negative Regulator of p27^kip1 in Pancreatic Adenocarcinoma

Abstract Her2 positive (Her2+) breast cancer (BC) accounts for 20% of all breast cancer subtypes and is associated with high risk of death. Trastuzumab (Ttzm), first FDA-approved targeted therapy for BC, represents a key milestone in the personalized treatment of Her2+ metastatic disease (MBC). However, 75% of Her2+ MBC are or become resistant to Ttzm and experience relapse or disease progression. This suggests that tumors acquire or possess an intrinsic mechanism of resistance that prevents from Her2 inhibition. Therefore, it is important to identify novel pathway to overcome the resistance. Recent studies have proposed various potential mechanisms leading to the resistance, including p27 rapid degradation. Jab1/Csn5 a novel candidate oncogene that we identified contributes to the progression of BC and is correlated with poor prognosis. Our study demonstrated that Jab1/Csn5 degrades p27 in MBC. These findings suggest that Jab1 overexpression contributes to Ttzm-resistance by facilitating p27-degradation. Jab1/Csn5 is overexpressed in 50% of primary and 90% of MBC while its expression is low or absent in normal adult breast tissues. We previously identified that high expression of Jab1 is associated with shorter progression-free survival in MBC patients. In this study, we showed that the knockdown of Jab1 sensitizes to Ttzm-treatment. Mechanistically, we found that Jab1/Csn5 overexpression is significantly correlated with the activation of Akt pathway in Her2+ MBC and xenograft models. Interestingly, activated Akt, due to PTEN-loss or PI3K activating mutation has been widely implicated in the potential mechanism to Ttzm-resistance. Therefore, our results suggest that targeting Jab1/Csn5 overcomes the resistance to Ttzm via interfering with PI3K/Akt pathway. Our study identifies Jab1 as a novel contributor to Ttzm-resistance and elucidates its potential mechanisms of actions. Jab1 could be used as a predictive marker of tumor response to Ttzm, which could assist clinicians in selecting the best treatment modalities for patients with MBC. Note: This abstract was not presented at the meeting. Citation Format: Francois X. Claret, Thuy Vu, Terry J. Shackleford, Jennifer Allensworth, Qingxiu Zhang, Ling Tian, Ronghua Zhang. Jab1/Csn5 a new target in the resistant mechanism to HER2-targeted therapies for breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1825. doi:10.1158/1538-7445.AM2014-1825

Abstract Her2 a key member in the epidermal growth factor receptor (EGFR) family, is one of the most dominant oncogenes in breast cancer. It is overexpressed in approximately 25%-30% of human breast cancers, which confers a more aggressive tumor phenotype and is associated with poor prognosis. The most effective FDA-approved therapy targeting Her2 is the monoclonal antibody trastuzumab (Ttzm; Herceptin). Ttzm inhibits Her2 activation and blocks downregulation of PI3K/Akt activities, which ultimately leads to increased expression of the cyclin-dependent kinase inhibitor p27, cell cycle arrest, and/or apoptosis. However, about 75% of Her2-positive breast tumors are or become resistant to Ttzm and experience relapse or disease progression. This suggests that tumors acquire or possess an intrinsic mechanism of resistance that prevents from Her2 inhibition. Therefore, it is important for the clinic to identify novel pathway to overcome the resistance. Clinicians cannot predict which patients with Her2-positive breast cancer will benefit from this treatment. Despite extensive studies, the mechanisms and genes responsible for resistance to trastuzumab (Ttzm) have not been entirely identified. Thus, novel strategies for treating Her2-positive disease are urgently needed. Recent studies have proposed that resistance to Ttzm is related to IGF-1R overexpression, rapid p27 degradation, PTEN loss, and PI3K mutations, but a clear target for modifying its resistance has yet to be identified. The cytostatic effect of Ttzm correlates with downregulation of Akt activity, which ultimately leads to increased expression of the cyclin-dependent kinase inhibitor p27, cell cycle arrest and/or apoptosis. c-Jun activation domain-binding protein 1 (Jab1/Csn5), a novel candidate oncogene that we identified, contributes to the progression of breast carcinoma and is correlated with poor prognosis. Jab1/Csn5 expression is low or absent in normal adults breast tissues, but over-expressed in 50% of primary breast tumor and 90% of metastatic breast cancer and other tumor types (ovarian cancer, pancreatic, non-small-cell lung carcinoma, and non-Hodgkin's lymphomas). Jab1/Csn5 negatively regulates p27 tumor suppressor gene, mediates p27 nuclear-to-cytoplasmic export and degradation. Jab1 levels are inversely associated with p27 expression in vitro and in vivo, which means that its up-regulation may contribute to trastuzumab (Ttzm) resistance by promoting p27 degradation. This study was conducted to investigate the role of Jab1 in modulating trastuzumab (Ttzm) resistance. We hypothesize that overexpression of Jab1 plays a crucial role in resistance to Ttzm through negative regulation of the cell-cycle inhibitor p27. Using molecular approaches and pre-clinical studies we identified the pathway leading to resistance the Ttzm. In this study, we will test a novel paradigm that inhibition of Jab1 expression renders Ttzm-resistant cells sensitive to Ttzm by stabilizing nuclear p27 expression levels. Finally Jab1 could be used as a predictive marker of tumor response to Ttzm, which could assist clinicians in selecting the best treatment modalities for patients with breast cancer Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B231. Citation Format: Thuy T. Vu, Terry J. Shackleford, Qingxiu Zhang, Francisco J. Esteva, Elias Drakos, Ling Tian, Timothy Kute, Aysegul A. Sahin, George Z. Rassidakis, Francois X. Claret. Jab1/Csn5 a new player driving the resistance to Her2-targeted therapies for breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B231.

Abstract Her2 positive (Her2+) breast cancer (BC) accounts for 18-20% of all breast cancer subtypes and is associated with high risk of death. Trastuzumab, the first Food and Drug Administration-approved targeted therapy for BC, represents a key milestone in the personalized treatment of Her2+ metastatic disease (Her2+ MBC). However, the median duration of response is less than a year, due to either primary or acquired resistance to the therapy. In addition, there is currently no conclusive biomarker for the response of patients to trastuzumab. Therefore, understanding the development of resistance to trastuzumab is of our interest. Recent studies have proposed various potential mechanisms leading to the resistance, including p27 rapid degradation. Previously, our study and other research demonstrated that Jab1 degrades p27 in breast cancer. These findings suggest that Jab1 over-expression contributes to trastuzumab resistance by facilitating p27 degradation. Jab1/CSN5 (c-Jun activation domain-binding protein 1) is over-expressed in 50% of primary and 90% of metastatic breast cancers, while its expression is low or absent in normal adult breast tissues. We previously identified that high expression of Jab1 is associated with shorter progression-free survival in breast cancer patients. In this study, our preliminary data showed that the knockdown of Jab1 sensitizes the breast cancer cells to trastuzumab treatment in a dose-dependent manner. Mechanistically, we found that Jab1 over-expression is significantly correlated with the activation of Akt pathway in Her2+ breast cancer cells and xenograft model. Interestingly, activated Akt, due to PTEN loss or PI3K activating mutation has been widely implicated in the potential mechanism to trastuzumab resistance. Therefore, our results suggest that targeting Jab1 overcomes the resistance to trastuzumab via interfering with PI3K/Akt pathway. In general, our study identifies Jab1 as a novel contributor to trastuzumab resistance and elucidates its potential mechanisms of actions. The successful completion of the study can potentially be translated to the clinic for the benefit of patients refractory to the therapy. Also our study suggests Jab1 expression level can be used as a predictive marker for trastuzumab treatment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-02.

Abstract Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy most common in East Asia and Africa. Previous studies have reported that genetic susceptibility is associated with NPC. Jab1/CSN5 promotes cell growth and proliferation. Recent studies have shown that Jab1/CSN5 is aberrantly expressed, is correlated with low expression of p27, and is associated with advanced tumor stage and poor prognosis in several human cancers. In this study, we examined the functional role and correlation between Jab1/CSN5 and p27 protein expression in NPC tissue samples and cell lines, the association of their expression with clinical outcome and the antitumor effects of cisplatin in NPC cells. Immunohistochemical analysis showed that Jab1/CSN5 aberrant expression was inversely associated with p27 expression in NPC tissue samples, and Jab1/CSN5 overexpression was correlated with poor survival (p<0.01). Further analysis showed that Jab1/CSN5 mediated p27 degradation in a proteasome-dependent manner, and Jab1/CSN5 directly interacted with p27 in NPC cells. In addition, inactivation of Jab1/CSN5 by small interfering RNAs resulted in a remarkable increase in p27 levels and in the inhibition of cell proliferation, thereby indicating that Jab1/CSN5 controls the stability of p27 by targeting it for degradation in NPC. Moreover, suppression of Jab1/CSN5 enhanced the antitumor effects of cisplatin in NPC cells. To our knowledge, these findings are the first to suggest that Jab1/CSN5 overexpression is involved in NPC pathogenesis via Jab1-mediated p27 degradation, thus explaining the low p27 levels seen in NPC. Therefore, Jab1/CSN5 could be a diagnostic marker and a novel therapeutic target in patients with NPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1855. doi:1538-7445.AM2012-1855

Abstract Her2-positive (Her2+) breast cancer has the second worst prognosis of the five subtypes of breast cancer. The most successful targeted therapy for Her2+ breast cancer to date is trastuzumab (Herceptin)-based. However, the median duration of response to trastuzumab is shorter than 1 year, and about 75% of patients who initially responsed, has experienced resistance to the therapy after a year. Thus, better treatment strategies for Her2+ subtype are urgently needed. We and other researchers previously demonstrated that c-Jun activation domain-binding protein 1 (Jab1) negatively regulates p27, mediates p27 nuclear-to-cytoplasmic export and degradation and contributes to the loss of p27 that is seen in >50% of breast tumors and that correlates with poor clinical outcome in breast cancer cells. Thus, implicates the potential role of Jab1 in interfering with trastuzumab. We identified Jab1/Csn5 as a novel oncogene. Jab1 is amplified and overexpressed in elevated in 50% of primary and 90% of metastatic breast tumors, but is low or absent in normal adult breast tissue. In addition, high Jab1 expression is associated with short progression-free survival durations in breast cancer patients. Our preliminary data showed that inactivation of Jab1/Csn5 in trastuzumab-resistant breast cancer cells sensitized the cells to trastuzumab in a dose- and time-dependent manners. However, the ways in which Jab1 expression is up-regulated in Her2+ breast cancer cells and how this up-regulation drives trastuzumab resistance remain largely unclear. In this study, we will seek to analyze the cross-talk between Src/Stat3 or Akt signaling and Jab1 activation in breast carcinoma. We have found that Stat3 is a novel positive regulator of Jab1 expression in breast cancer cells and that Jab1 overexpression driven by the Src/Stat3 pathway compensates for trastuzumab's inhibition of Her2 signaling in Her2+ breast cancer cells. This suggests that activated Stat3, by up-regulating Jab1 expression, inhibits trastuzumab-induced cell-cycle arrest. In general, our proposed study elucidates Jab1 as a novel contributor to trastuzumab resistance and determine its potential as a prognostic and predictive marker as well as an important therapeutic target. The successful completion of our study would benefit breast cancer patients in three ways. 1) Because Jab1 is rarely expressed in mammary epithelial cells, targeting Jab1 would be less toxic to normal epithelial cells, compared to other agents. 2) Combining Jab1 with trastuzumab would interfere with both Src/Stat3 and Akt pathway. This would significantly benefit patients with Her2+ breast cancer refractory to this therapy due to PTEN loss or activating mutant Stat3. 3) Because Jab1 expression is higher in trastuzumab-resistant cells than in trastuzumab-sensitive cells, Jab1 could be used as a marker of tumor response to trastuzumab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1912. doi:1538-7445.AM2012-1912

Abstract Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus–associated malignancy most common in East Asia and Africa. Aberrant expression of Jab1/CSN5, a negative regulator of the cell-cycle inhibitor p27, is correlated with reduced p27 expression and associated with advanced tumor stage and poor prognosis in several human cancers. In this study, we examined the functional relationship between Jab1 and p27 protein expression in NPC. Immunohistochemical analysis showed an inverse association between Jab1 and p27 in NPC tissue samples, and overexpression of Jab1 correlated with poor survival in patients with NPC. Mechanistically, Jab1 and p27 were found to interact directly in NPC cells, with Jab1 mediating p27 degradation in a proteasome-dependent manner. Knockdown of Jab1 resulted in a remarkable increase in p27 levels and inhibition of cell proliferation, indicating that Jab1 targets p27 for degradation, thereby controlling its stability. Jab1 depletion also enhanced the antitumor effects of cisplatin in NPC cells. Together, our findings suggest that Jab1 overexpression plays an important role in the pathogenesis of NPC through Jab1-mediated p27 degradation. Jab1 therefore represents a novel diagnostic marker and therapeutic target in patients with NPC. Cancer Res; 72(7); 1890–900. ©2012 AACR.

Introduction The c-Jun coactivator, Jun activation-domain binding protein 1 (Jab1) also known as the fifth component of the COP9 signalosome complex (CSN5), is a novel candidate oncogene whose aberrant expression contributes to the progression of breast carcinoma and other human cancers. The mechanism of Jab1 gene expression and its deregulation in cancer cells remains to be identified. We therefore investigated the transcriptional regulatory mechanisms of Jab1 expression in human breast carcinoma cells. Methods To identify potential regulators of Jab1 transcription, we cloned the 5' upstream region of the human Jab1 gene and mapped its transcriptional start site. We identified binding sequences for the CCAAT/enhancer binding protein (C/EBP) and GATA, as well as a signal transducer and activator of transcription-3 (Stat3) consensus sequence overlapping the C/EBP site, using 5'- deletion analysis and a gene reporter assay. Mutational analysis of these binding sites was performed to confirm their roles in promoting Jab1 transcription in breast cancer cells. We further confirmed these binding sites using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays. We also analyzed whether the siRNA-mediated inactivation of Stat3 and Src could reduce Jab1 -promoter activity and whether interleukine-6 (IL-6) could mediate increased Jab1 expression through Stat3 signaling. Results We identified binding sequences for C/EBP, GATA, as well as a Stat3 consensus sequence overlapping the C/EBP site in the promoter region of Jab1 . C/EBP-beta2 is a potential transcriptional activator of Jab1 and mutation of the C/EBP/Stat3 binding site significantly reduced Jab1 -promoter activity. In addition, inhibiting Stat3 significantly reduced Jab1 -promoter activation. EMSA and ChIP assays confirmed that C/EBP, GATA1 and Stat3 bind to Jab1 promoter in breast carcinoma cells. We also found that Src, an activator of Stat3, is involved in Jab1 -promoter activation. siRNA knockdown of Src reduced the Jab1 -promoter activity, similar to the results seen when Stat3 was inhibited in breast carcinoma cells. Interestingly, reactivation of Stat3 in normal mammary epithelial cells (MCF-10A, MCF-10F) is sufficient to reactivate Jab1 expression. Treatment with the cytokine IL-6 resulted in increased Jab1 expression that was blocked by inhibition of Stat3. Conclusions These findings reveal a novel mechanism of Jab1 gene regulation and provide functional and mechanistic links between the Src/Stat3 and IL-6/Stat3 signaling axes that are involved in the activation of Jab1 transcription and regulation of this novel oncogenic protein.

Abstract Reduced expression of p27 has been associated with poor prognosis in most human cancers, including pancreatic adenocarcinoma. Jun activation domain–binding protein 1 (JAB1), an activator protein (AP-1) coactivator, previously implicated in p27 degradation, is overexpressed in various tumors and correlates with low p27 expression. We examined JAB1 and p27 in normal and neoplastic pancreatic tissues. Increased JAB1 expression was seen in pancreatic carcinoma samples but not in paired normal pancreatic tissues. Immunohistochemical analysis using tissue microarrays showed that JAB1 was overexpressed in all 32 (100%) pancreatic adenocarcinoma samples tested, predominantly nuclear in 23 (72%) samples and predominantly cytoplasmic in 9 (28%) tumors. When 10% was used as a cutoff for p27 positivity, p27 was expressed in 11 (34%) of tumors; however, p27 expression was localized in the nuclei of tumor cells in only 4 (13%) of the samples. Overexpression of the JAB1 in the pancreatic carcinoma cell lines Panc-1, Mia PaCa-2, and Panc-28 resulted in decreased p27 expression. Conversely, down-regulation of JAB1 by short interfering RNA substantially increased p27 expression and inhibited progression from G1 to S phase of the cell cycle. Interestingly, JAB1-mediated p27 degradation was not impaired when S-phase kinase-interacting protein 2 (Skp2), an F-box protein required for the ubiquitination and consequent degradation of p27, was silenced. Thus, JAB1 may have an Skp2-independent p27 degradation mechanism in pancreatic cancer cells. These findings suggest that JAB1 overexpression is involved in the pathogenesis of pancreatic cancer through JAB1-mediated p27 degradation and that control of JAB1 expression is a novel therapeutic target in patients with pancreatic adenocarcinomas. (Cancer Res 2006; 66(17): 8581-9)

The cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is an important regulator of cell cycle progression controlling the transition from G to S-phase. Low p27 levels or accelerated p27 degradation correlate with excessive cell proliferation and poor prognosis in several forms of cancer. Phosphorylation of p27 at Thr187 by cyclin E-CDK2 is required to initiate the ubiquitination-proteasomal degradation of p27. Protecting p27 from ubiquitin-mediated proteasomal degradation may increase its potential in cancer gene therapy. Here we constructed a non-phosphorylatable, proteolysis-resistant p27 mutant containing a Thr187-to-Ala substitution (T187A) which is not degraded by ubiquitin-mediated proteasome pathway, and compared its effects on cell growth, cell-cycle control, and apoptosis with those of wild-type p27. In muristerone A-inducible cell lines overexpressing wild-type or mutant p27, the p27 mutant was more resistant to proteolysis in vivo and more potent in inducing cell-cycle arrest and other growth-inhibitory effects such as apoptosis. Transduction of p27(T187A) in breast cancer cells with a doxycycline-regulated adenovirus led to greater inhibition of proliferation, more extensive apoptosis, with a markedly reduced protein levels of cyclin E and increased accumulation of cyclin D1, compared with wild-type p27. These findings support the potential effectiveness of a degradation-resistant form of p27 in breast cancer gene therapy.

Abstract Anaplastic lymphoma kinase (ALK)–positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of chimeric nucleophosmin-ALK. Previously, nucleophosmin-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. In this study, we hypothesized that the mammalian target of rapamycin (mTOR) pathway, which functions downstream of AKT, mediates the oncogenic effects of activated PI3K/AKT in ALK+ ALCL. Here, we provide evidence that mTOR signaling phosphoproteins, including mTOR, eukaryotic initiation factor 4E–binding protein-1, p70S6K, and ribosomal protein S6, are highly phosphorylated in ALK+ ALCL cell lines and tumors. We also show that AKT activation contributes to mTOR phosphorylation, at least in part, as forced expression of constitutively active AKT by myristoylated AKT adenovirus results in increased phosphorylation of mTOR and its downstream effectors. Conversely, inhibition of AKT expression or activity results in decreased mTOR phosphorylation. In addition, pharmacologic inhibition of PI3K/AKT down-regulates the activation of the mTOR signaling pathway. We also show that inhibition of mTOR with rapamycin, as well as silencing mTOR gene product expression using mTOR-specific small interfering RNA, decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in ALK+ ALCL cells. Cell cycle arrest was associated with modulation of G1-S-phase regulators, including the cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Apoptosis following inhibition of mTOR expression or function was associated with down-regulation of antiapoptotic proteins, including c-FLIP, MCL-1, and BCL-2. These findings suggest that the mTOR pathway contributes to nucleophosmin-ALK/PI3K/AKT-mediated tumorigenesis and that inhibition of mTOR represents a potential therapeutic strategy in ALK+ ALCL. (Cancer Res 2006; 66(13): 6589-97)