Wild-type PTEN Research Articles

Relationship between PTEN, DNA mismatch repair, and tumor histotype in endometrial carcinoma: retained positive expression of PTEN preferentially identifies sporadic non-endometrioid carcinomas

Loss of PTEN (phosphatase and tensin homolog) expression and microsatellite instability are two of the more common molecular alterations in endometrial carcinoma. From the published literature, it is controversial as to whether there is a relationship between these different molecular mechanisms. Therefore, a cohort of 187 pure endometrioid and non-endometrioid endometrial carcinomas, carefully characterized as to clinical and pathological features, was examined for PTEN sequence abnormalities and the immunohistochemical expression of PTEN and the DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2. MLH1 methylation analysis was performed when tumors had loss of MLH1 protein. Mismatch repair protein loss was more frequent in endometrioid carcinomas compared with non-endometrioid carcinomas, a difference primarily attributable to the presence of MLH1 methylation in a greater proportion of endometrioid tumors. Among the non-endometrioid group, mixed endometrioid/non-endometrioid carcinomas were the histotype that most commonly had loss of a mismatch repair protein. In endometrioid tumors, the frequency of PTEN loss measured by immunohistochemistry and mutation did not differ significantly between the mismatch repair protein intact or mismatch repair protein loss groups, suggesting that PTEN loss is independent of mismatch protein repair status in this group. However, in non-endometrioid carcinomas, both intact positive PTEN immunohistochemical expression and PTEN wild type were highly associated with retained positive expression of mismatch repair proteins in the tumor. Relevant to screening endometrial cancers for Lynch Syndrome, an initial PTEN immunohistochemistry determination may be able to replace the use of four mismatch repair immunohistochemical markers in 63% of patients with non-endometrioid endometrial carcinoma. Therefore, PTEN immunohistochemistry, in combination with tumor histotype, is a useful adjunct in the clinical evaluation of endometrial carcinomas for Lynch Syndrome.

Acid Ceramidase Promotes Nuclear Export of PTEN through Sphingosine 1-Phosphate Mediated Akt Signaling

The tumor suppressor PTEN is now understood to regulate cellular processes at the cytoplasmic membrane, where it classically regulates PI3K signaling, as well as in the nucleus where multiple roles in controlling cell cycle and genome stability have been elucidated. Mechanisms that dictate nuclear import and, less extensively, nuclear export of PTEN have been described, however the relevance of these processes in disease states, particularly cancer, remain largely unknown. We investigated the impact of acid ceramidase on the nuclear-cytoplasmic trafficking of PTEN. Immunohistochemical analysis of a human prostate tissue microarray revealed that nuclear PTEN was lost in patients whose tumors had elevated acid ceramidase. We found that acid ceramidase promotes a reduction in nuclear PTEN that is dependent upon sphingosine 1-phosphate-mediated activation of Akt. We were further able to show that sphingosine 1-phosphate promotes formation of a complex between Crm1 and PTEN, and that leptomycin B prevents acid ceramidase and sphingosine 1-phosphate mediated loss of nuclear PTEN, suggesting an active exportin-mediated event. To investigate whether the tumor promoting aspects of acid ceramidase in prostate cancer depend upon its ability to export PTEN from the nucleus, we used enforced nuclear expression of PTEN to study docetaxel-induced apoptosis and cell killing, proliferation, and xenoengraftment. Interestingly, while acid ceramidase was able to protect cells expressing wild type PTEN from docetaxel, promote proliferation and xenoengraftment, acid ceramidase had no impact in cells expressing PTEN-NLS. These findings suggest that acid ceramidase, through sphingosine 1-phosphate, promotes nuclear export of PTEN as a means of promoting tumor formation, cell proliferation, and resistance to therapy.

PTEN deficiency is associated with reduced sensitivity to mTOR inhibitor in human bladder cancer through the unhampered feedback loop driving PI3K/Akt activation

Background:Preclinical studies have shown that PTEN loss enhances sensitivity to mammalian target of Rapamycin (mTOR) inhibitors because of facilitated PI3K (phosphatidylinositol-3 kinase)/Akt activation and consecutive stimulation of the mTOR pathway. In patients with advanced transitional cell carcinoma (TCC) treated with the mTOR inhibitor everolimus, PTEN loss was, however, associated with resistance to treatment.Methods:Transitional cell carcinoma specimens, human bladder cancer cells and derived mouse xenografts were used to evaluate how the PTEN status influences the activity of mTOR inhibitors.Results:Transitional cell carcinoma patients with a shorter progression-free survival under everolimus exhibited PTEN deficiency and increased Akt activation. Moreover, PTEN-deficient bladder cancer cells were less sensitive to rapamycin than cells expressing wild-type PTEN, and rapamycin strikingly induced Akt activation in the absence of functional PTEN. Inhibition of Akt activation by the PI3K inhibitor wortmannin interrupted this rapamycin-induced feedback loop, thereby enhancing the antiproliferative effects of the mTOR inhibitor both in vitro and in vivo.Conclusion:Facilitation of Akt activation upon PTEN loss can have a more prominent role in driving the feedback loop in response to mTOR inhibition than in promoting the mTOR pathway. These data support the use of both PI3K and mTOR inhibitors to treat urothelial carcinoma, in particular in the absence of functional PTEN.

Multinucleation regulated by the Akt/PTEN signaling pathway is a survival strategy for HepG2 cells

Hepatocellular carcinoma (HCC) is non-responsive to many chemotherapeutic agents including etoposide. The aim of this study was to examine the survival strategy of the HCC cell line HepG2 after etoposide treatment. Here we analyzed and compared spontaneous and etoposide-induced DNA damage in HepG2 (α-fetoprotein (AFP)-positive) and Chang Liver (AFP-negative) cell lines. Compared to Chang Liver cells, HepG2 cells exhibited a significantly higher degree of micronucleation and a higher nuclear division index, as determined by the cytokinesis-block micronucleus assay, following exposure to etoposide. HepG2 cells were also more resistant to etoposide-induced cytotoxicity compared to Chang Liver cells. We also establish that increased etoposide-induced multinucleation in HepG2 cells is dependent on the catalytic activity of Akt, as phosphatidylinositol-3-kinase inhibitors as well as the overexpression of kinase-defective Akt reversed this phenotype. Moreover, ectopic expression of wild type PTEN reduced the frequency of etoposide-induced multinucleated HepG2 cells, and restored HepG2 etoposide sensitivity. Taken together, these results implicate the Akt/PTEN cellular axis as a major determinant of the etoposide resistance of HCC cells.

The positive correlation between DJ‐1 and β‐catenin expression shows prognostic value for patients with glioma

The relationship between DJ-1 and β-catenin, and its impact on the prognosis for glioma patients has not been fully understood. This study determined the effect of DJ-1 on β-catenin and the prognostic significance of this interaction in glioma patients. We collected tumor specimens from 88 glioma patients and determined the expression of DJ-1, β-catenin and PTEN by using immunohistochemical staining. The involvement of DJ-1 and β-catenin in glioma cell lines was evaluated by immunohistochemistry and Western blotting. High DJ-1 expression (37.5%) and high β-catenin expression (34.1%) in glioma specimens were significantly associated with high grade and poor prognosis in glioma patients. However, only high levels of DJ-1 (P = 0.014) was a strong independent prognostic factor, correlated with a reduced overall survival time. In vitro DJ-1 expression was positively correlated with the expression levels of β-catenin and p-Akt, and negatively correlated with PTEN expression in U87, U251 MG, SWO-38 and SHG44 human glioma cell lines. After the knockdown of DJ-1, Akt, p-Akt or β-catenin expression levels were not affected in the PTEN-null cell lines (U87 and U251 MG). However, in the SWO-38 cell line, which has wild-type PTEN protein, the level of PTEN increased while Akt/p-Akt and β-catenin levels were reduced. Furthermore, β-catenin staining weakened in SWO-38 cells after DJ-1 levels decreased according to immunocytochemical analysis. In conclusion, DJ-1 and β-catenin may contribute to the development and recurrence of glioma and are valuable prognostic factors for glioma patients. DJ-1 may regulate β-catenin expression via PTEN and p-Akt.

Second malignant neoplasms (SMN) in Cowden syndrome patients with underlying germline PTEN mutations.

1527 Background: Cowden syndrome (CS) patients with underlying germline PTEN mutations are at significantly increased risks of breast, thyroid, endometrial and renal cancers. Current estimates of lifetime cancer risks do not provide the majority of patients who present after their first cancer a meaningful gauge of their risk of a SMN. Quantification of the risk of a SMN will allow patients to be better informed and provide clinicians a much needed guide to finally address patients’ question of “how likely am I to have a second cancer and will it likely be the same primary?” Methods: We conducted a 5-year, multi-center prospective study of CS and CS-like patients, all of whom had comprehensive PTEN analysis done. Patients’ records were reviewed for prior history of invasive cancers. Invasive SMNs were confirmed by pathology reports or physician reports where possible. All metastatic disease, synchronous primary cancer at the same site and non-melanoma skin cancers were excluded. Incidences of SMNs were compared between patients with pathogenic PTEN mutations (PTENmut+) and wildtype PTEN (PTENwt). Standardized incidence ratios (SIR), cumulative incidences and excess absolute risks for SMNs were calculated comparing against SEER-derived data. Results: Of 2,792 CS/CS-like patients who present with 1 or more cancers, there were 100 (4%) PTENmut+patients. Compared with PTENwt CS/CS-like patients with SMNs (223/2039; 11%), PTENmut+cases (42/100; 42%) were more likely to develop SMNs (p<0.0001). PTENmut+ cases were also younger at median age of SMN diagnosis (48yrs vs 54 yrs; p=0.30). PTENmut+ patients were 56-fold more likely to develop any SMN compared with the general population (95%CI 41-55); corresponding to an excess absolute risk of 731/10000 person years. Breast cancer was the most common SMN encountered in PTENmut+CS/CS-like patients (SIR 73; 95%CI 45-122) followed by endometrial (SIR 44; 95% CI 18-92), renal (SIR 47; 95% CI 15-113) and thyroid cancers (SIR 222; 95% CI 37-734). Conclusions: Our study shows that PTENmut+CS/CS-like patients are significantly more likely to present with a SMN compared to PTENwt CS/CS-like patients and the general population, and should be advised and surveillanced accordingly.

Abstract 4312: Grb10, a negative regulator of insulin signaling, is a tumor suppressor in prostate cancer.

Abstract Prostate cancer (PCa) is manageable in its initial stages and when localized, but as it progresses it metastasizes and also becomes resistant to conventional chemotherapies. Identifying mechanisms that are related to the rapid progression of the disease could result in development of appropriate intervention strategies. We analyzed protein and mRNA expression of Grb10 (also known as insulin receptor-binding protein) in various PCa cell lines and found a significant decrease when compared to normal prostate epithelial cells. We also analyzed the expression pattern of Grb10 in a human PCa tissue array and found significant loss of Grb10 mRNA in more than 90% of the samples. Based on Gleason Score, the loss of Grb10 mRNA correlated with the stage and grade of the disease. These initial observations led us to hypothesize that Grb10 may act as a tumor suppressor whose expression is lost during the progression of human PCa. Using immuno-histochemical analysis of human PCa tissue samples we found loss of Grb10 protein with increasing tumor grade. We next analyzed various components of insulin signaling, based on the evidence that Grb10 expression is negatively correlated with insulin signaling. We observed significant increase at both mRNA and protein levels of IR, IGF-1R, IRS-1 and IRS-2 in PCa cells and tissues that directly correlated with loss of Grb10. Recently, Grb10 expression was shown to be negatively correlated with PTEN levels in different cancer types including PCa. We determined the expression pattern of Grb10 mRNA in prostate tissue samples from 15-weeks old PTEN wild type (WT) and PTEN knockout (KO) mice and observed that Grb10 levels were significantly down-regulated in PTEN-KO mice suggesting that loss of PTEN results in loss of Grb10. Further analysis suggested increased insulin signaling in PTEN-KO mice compared to PTEN-WT mice confirming negative regulation of Grb10 with insulin signaling. To further understand the role of Grb10 in PCa, we overexpressed Grb10 in human PCa DU145 (PTEN-mutated) cells. Overexpression of Grb10 resulted in decrease in colony formation, migration and invasion that was associated with down-regulation of insulin signaling. In summary, our findings suggest that Grb10 is a negative regulator of insulin signaling whose expression is lost in PCa in humans and correlates well with disease progression and aggressiveness. We conclude that Grb10 is a tumor suppressor in human PCa whose loss promotes rapid progression of the disease. Identification of this novel role of Grb10 could open up new strategies for the management of human PCa. Citation Format: Mohammad Imran Khan, Vaqar M. Adhami, Rahul Kumar Lall, Deeba Nadeem Syed, Imtiaz Ahmad Siddiqui, Bilal Bin Hafeez, Ajit Kumar Verma, Hasan Mukhtar. Grb10, a negative regulator of insulin signaling, is a tumor suppressor in prostate 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 4312. doi:10.1158/1538-7445.AM2013-4312

Abstract 3135: In vitro and in vivo synthetic lethal screens to identify novel targets in the context of PTEN deficiency.

Abstract Functional genomic screens have been employed by many groups to identify novel targets for cancer therapeutics. However, translation of these data sets into new drug discovery programs has proved challenging, in part due to difficulties in obtaining strong on-target knockdown, coupled with misleading off-target effects. Most commercially available pooled shRNA screening libraries provide 5-6 shRNAs per gene, with little knowledge of knock-down efficiency. In an effort to overcome these issues, we designed a custom shRNA library targeting 580 human genes, including key nodes in signal transduction pathways and genes of interest for oncology drug development. This pathway-centric design allows us to determine critical pathways for cancer cell growth in addition to individual gene phenotypes. For each gene, we have total of 11 shRNAs, a subset of which have been previously shown high efficiency knock-down. In particular, we emphasized genes where tool or lead compounds were available for rapid follow-up. Both in size and scope, this library was designed to quickly identify important signaling pathways that are essential on their own or when perturbed in the presence of small molecule drugs in our pipeline. With this focused library, we performed functional genomic shRNA screens in PTEN mutant or null lines across cancer types in vitro, including four cell lines that are PTEN deficient (UACC62, MDA-MB-468, U87-MG and PC3), and one cell line, (HCT-116) that is PTEN wild type. Meanwhile, we performed in vivo screens with PC3 cells. To identify hits from our screens, we used an internally developed algorithm, c-FOLD, to calculate p-value and fold changes for each shRNA. As confirmation that our library and methods can identify essential genes in a context specific manner, we observe BRAF as the top essential gene hit in the cell line which contains an activated BRAF allele, UACC62, but not in other lines tested. We then examined the concordance of hits in our in vitro and in vivo experiments with PC3 cells. In general, individual shRNAs were highly correlated between in vitro and in vivo assays, but a subset is specific to one setting, and may present particularly interesting targets. To discover synthetic lethal partners to PTEN deficiency, we required that a gene is essential in all four PTEN deficient lines in vitro, as well as in PC3 in vivo, but not essential in HCT-116. Among the top hits are multiple shRNAs against ENTPD5, an ER enzyme hydrolyzing UDP to UMP. In addition, using our pathway approach, we identified components in JNK pathways as synthetic lethal partners to PTEN deficiency. The results obtained from these studies established a strong platform for in vivo and in vitro identification of novel targets and novel combination partners. These approaches can also be integrated with other Omics data, such as mutation and expression, to ultimately find promising targets to develop novel cancer therapeutics. Citation Format: Jing-xin Zhang, Joshua Murtie, Oleg Iartchouk, Hui Cao, Gary Shapiro, Zhifang Li, Hongyun Wang, Zhihu Ding, Yu-an Zhang, Madelyn Light, Dietmar Hoffman, May Cindhuchao, Saurin Jani, Richard Newcombe, Eva Bric-Furlong, Bin Wu, Angela Virone-Oddos, Stephan Reiling, Joachim Theihaber, Christoph Lengauer, Jack Pollard, James Watters, Serena Silver, Venkat Reddy. In vitro and in vivo synthetic lethal screens to identify novel targets in the context of PTEN deficiency. [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 3135. doi:10.1158/1538-7445.AM2013-3135

Abstract 3151: High-throughput RNAi screening identifies a novel metastatic melanoma drug target in the context of PTEN mutation.

Abstract It has become increasingly important to identify targeted therapy in a defined molecular context. Here we will present our data which suggests TERT, a telomerase reverse transcriptase, serves as a novel drug target in melanoma in the context of PTEN mutation. PTEN is mutated in ∼15% of melanoma patients. Genomic RNA-interference (RNAi) technology was applied for synthetic lethality screens in an effort to identify the targets which upon knock-down significantly decrease cell viability specifically in the context of somatic mutations of the PTEN gene. We carried out such screen in a panel of 15 melanoma cell lines, including four PTEN mutated cell lines, against an siRNA library covering the kinome (∼700 genes with 4 siRNA per target) using CellTiter Glo as the endpoint to measure cell viability. Lethal genes, which decreased cell viability significantly upon knockdown, were initially identified for each cell line. Then the hit lists were compared between wild-type PTEN and mutant PTEN using a chi-square test, a statistical analysis. 29 gene candidates were selected for hit confirmation using the same set of siRNA, among which 24 genes were confirmed as potential PTEN synthetic lethal candidates, this results in a high confirmation rate of 83%. Based upon its association with melanoma risk (Law et al, J Invest Dermatol, 2012), We further validated TERT using a commercially available small molecule inhibitor. This inhibitor has significantly higherpotency in the PTEN mutant context (average IC50 of 0.34uM) compared to PTEN wild-type background (average IC50 of 0.75uM). This result both validates our RNAi screening results, but more importantly, this compound can serve as an initial structure scaffold for further optimization towards development as a novel drug for metastatic melanoma patients who carry PTEN mutations. Evaluation of more TERT inhibitors in a diverse panel of melanoma cell lines is presently underway. Citation Format: Holly Yin, Donald Chow, Chris Sereduk, Meraj Aziz, Seungchan Kim, Kevin M. Brown, Jeffrey M. Trent. High-throughput RNAi screening identifies a novel metastatic melanoma drug target in the context of PTEN mutation. [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 3151. doi:10.1158/1538-7445.AM2013-3151

Abstract 2048: PTEN deficient lung cancer cells are sensitive to the combination of olaparib with cisplatin through suppressing DNA damage signaling.

Abstract PARP inhibition in combination with DNA-damaging agents is one of the most promising new therapeutic approaches to cancer. PTEN is a tumor-suppressor gene deactivating PI3K downstream of EGFR signaling. In H1650 cells harboring EGFR deletion mutation in exon 19 with PTEN loss, we reported that synergistic effects of a PARP inhibitor (olaparib) and a DNA-damaging agent (cisplatin) were observed in vitro and in vivo (#4686, AACR 2012). We further clarified how PTEN loss affected DNA damage signaling. H1299 and A549 are lung cancer cells harboring wild type PTEN. Antagonistic effects of olaparib with cisplatin were shown in H1299 and A549 cells according to combination index (CI). Sensitivity to a PARP inhibitor was reported to be associated with a defect in Mre11 expression. H1650 cells exhibited lower levels of Mre11 compared with PTEN-transfected H1650 cells (designated H1650PTEN+ cells). To address how lower expression levels of Mre11 affects the synergism between cisplatin and olaparib, Mre11 expression vector was transfected into H1650 cells (designated H1650Mre11+ cells). CIs were 0.23, 0.20, 0.57 and 0.29 when concentration ratios of cisplatin and olaparib were designed to be molar ratios of 1:1, 1:2, 1:3 and 1:5, respectively, in H1650 cells, while CIs were 0.76, 0.91, 0.97 and 0.77 in H1650Mre11+ cells. Although these CIs in H1650Mre11+ were somewhat elevated compared with those observed in original H1650 cells, these results indicated that lower levels of Mre11 alone could not be the sole reason for the synergism. On the other hand, PTEN has other nuclear functions, including transcriptional regulation of the RAD51 gene, whose product is essential for homologous recombination (HR) repair of DNA breaks. RPA is displaced from single stranded DNA by RAD51 to initiate HR. There were no significant differences of RAD51 and RPA levels by Western blotting. Meanwhile, PTEN deficiency resulted in significant reduction in RAD51 and RPA focus formation after drug-exposure or irradiation compared to H1650PTEN+ cells although γH2AX was similarly increased in both cells. As RPA binds to single stranded DNA, the RPA focus could be a marker for end resection at the double stranded DNA ends. Thus, inactivation of PTEN might lead to suppression of DNA damage signaling, leading to the lower levels of end resection and, hence, less RPA focus formation. As shown above, reduced levels of Mre11 alone could not provide a sufficient explanation for this, though Mre11 is involved in the molecular mechanisms of the end resection. The combination of cisplatin with olaparib in PTEN deficient lung tumors might be pursued in clinical trials although further investigations should be required to clarify whether and to what extent the molecular events including Mre11, RAD51 and RPA are responsible for the synergic effect. Citation Format: Daisuke Minami, Nagio Takigawa, Hiromasa Takeda, Minoru Takata, Nobuaki Ochi, Eiki Ichihara, Akiko Hisamoto, Katsuyuki Hotta, Mitsune Tanimoto, Katsuyuki Kiura. PTEN deficient lung cancer cells are sensitive to the combination of olaparib with cisplatin through suppressing DNA damage signaling. [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 2048. doi:10.1158/1538-7445.AM2013-2048

Abstract 235: Oncogene addiction switch from NOTCH to PI3K/AKT requires simultaneous targeting of NOTCH and PI3K pathway inhibition in glioblastoma.

Abstract Notch signaling pathway regulates normal stem cells in the brain and glioma stem cells (GSCs) with high Notch activity. However, blocking the proteolytic activation of NOTCH with γ-secretase inhibitors (GSIs) fails to alter the growth of some GSCs as GSIs seem to be active in only a fraction of GSCs lines with constitutive NOTCH activity and limiting the efficacy of this strategy. Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identification of new therapeutic applications. Here we report loss of PTEN as a critical event leading to resistance to NOTCH inhibition, which causes the transfer of “oncogene addiction” from the NOTCH to the phosphoinositol-3 kinase (PI3K)/AKT pathway. This novel observation suggests the need to simultaneous inhibition of both pathways as a means to improve therapeutic efficacy in human GBMs. We investigated the effects of Notch pathway inhibition in GSC using GSI. Drug cytotoxicity test on 16 GSCs show differential growth response to GSI stratifying GSCs into two groups: responders (6 cell lines) vs non-responders (10 cell lines). Active Notch signaling seems to be important features for the GSC as Notch inhibition only affected GSCs defined as having increased endogenous Notch activity. However in the responder group GSCs with the PTEN mutation seems to be less sensitive to GSI treatment. Here we show that NOTCH regulates the expression of PTEN and the activity of the PI3K/AKT signaling pathway in GSCs since treatment with GSI attenuated Notch intracellular domain (NICD), Hes-1, Hes-3, and Hes-5 and increases PTEN expression. NOTCH regulates PTEN expression via Hes-1 as knockdown of either Notch or Hes1 led to increase expression of PTEN. In contrast, PTEN knockdown decreases the response of GSCs to GSI in PTEN wild type GSCs. Our data shows that PTEN is an important mediator of GSI induced attenuation of cell growth suggesting that there might be a regulatory circuit-linking Notch signaling with PTEN/PI3K/Akt pathway, providing the basis for the design of new therapeutic strategies and suggests the need to simultaneously inhibit both pathways as a means to improve therapeutic efficacy in human GBMs. Our preliminary data reveal the synergistic attenuation of cell growth using combination of GSI and PI3K inhibitors in PTEN mutant GSCs. Our results warrant further studies to test the efficacy of combined PI3K/AKT- and NOTCH-directed therapies in human cancer. Citation Format: Norihiko Saito, Jun Fu, Shuzhen Wang, Erik P. Sulman, Frederick F. Lang, Wk Alfred Yung, Dimpy Koul. Oncogene addiction switch from NOTCH to PI3K/AKT requires simultaneous targeting of NOTCH and PI3K pathway inhibition in glioblastoma. [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 235. doi:10.1158/1538-7445.AM2013-235

Abstract 3736: IL6 feedback loop regulates breast cancer stem cells which mediate trastuzumab resistance.

Abstract The development of HER2 targeting agents such as trastuzumab and lapatinib represents one of the greatest achievements in clinical oncology demonstrating the effectiveness of molecularly targeted therapeutics (2). Despite the clinical benefits of trastuzumab in improving progression free and overall survival, majority of patients demonstrate “intrinsic” resistance or develop “acquired” resistance within one-to-two years. Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain to be investigated. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrated that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of breast cancer stem cell (CSC) population. We and others previously demonstrated that cytokines including IL6 and IL8 regulates breast cancer stem cell self-renewal. Furthermore, elevated serum levels of these cytokines in breast cancer patients strongly correlated with metastatic disease and therapeutic resistance. We therefore hypothesized that activation of IL6 feedback loop may mediate trastuzumab resistance. Utilizing these models, we demonstrated that long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. Recombinant IL6 or conditioned medium from high-IL6 secreting cells resulted in trastuzumab resistance in parental PTEN wild type cells expanding CSCs with EMT phenotype characterized by the CD44+/CD24- expression and EMT gene signature. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies have important clinical implications demonstrating that trastuzumab resistance may be mediated by an IL-6 inflammatory loop and suggest that blocking this loop represents a novel strategy to overcome trastuzumab resistance. . Citation Format: April L. Davis, Hasan Korkaya, Maria Ouzounova, Fayaz Malik, Gwangil Kim, Ahmed A. Quraishi, Suling Liu, Shawn G. Clouthier, Max S. Wicha. IL6 feedback loop regulates breast cancer stem cells which mediate trastuzumab resistance. [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 3736. doi:10.1158/1538-7445.AM2013-3736

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.

Alterations in Wnt–β‐catenin and Pten signalling play distinct roles in endometrial cancer initiation and progression

Endometrioid endometrial cancer arises through a gradual series of histological changes, each accompanied by specific alterations in gene expression and activity. Activation of the Wnt-β-catenin pathway and loss of PTEN activity are frequently observed in endometrial cancers. However, the specific roles played by alterations in these pathways in the initiation and progression of endometrial cancer are currently unclear. Here, we investigated the effects of loss of Pten and Apc gene function in the mouse endometrium by employing tissue-specific and inducible mutant alleles, followed by immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of their corresponding cancerous lesions. Loss of the Apc function in the endometrium leads to cytoplasmic and nuclear β-catenin accumulation in association with uterine hyperplasia and squamous cell metaplasia, but without malignant transformation. Loss of Pten function also resulted in squamous metaplasia but, in contrast to loss of Apc function, it initiates endometrial cancer. On the other hand, loss of Apc function in the endometrium accelerates Pten-driven endometrial tumourigenesis. Analysis of compound heterozygous mice confirmed that somatic loss of the wild-type Pten allele represents the rate-limiting initiation step in endometrial cancer. Simultaneous loss of Pten and Apc resulted in endometrial cancer characterized by earlier onset and a more aggressive malignant behaviour. These observations are indicative of the synergistic action between the Wnt-β-catenin and Pten signalling pathways in endometrial cancer onset and progression.

Recommendations from the EGAPP Working Group: can testing of tumor tissue for mutations in EGFR pathway downstream effector genes in patients with metastatic colorectal cancer improve health outcomes by guiding decisions regarding anti-EGFR therapy?

The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group (EWG) found that, for patients with metastatic colorectal cancer (mCRC) who are being considered for treatment with cetuximab or panitumumab, there is convincing evidence to recommend clinical use of KRAS mutation analysis to determine which patients are KRAS mutation positive and therefore unlikely to benefit from these agents before initiation of therapy. The level of certainty of the evidence was deemed high, and the magnitude of net health benefit from avoiding potentially ineffective and harmful treatment, along with promoting more immediate access to what could be the next most effective treatment, is at least moderate.The EWG found insufficient evidence to recommend for or against BRAF V600E testing for the same clinical scenario. The level of certainty for BRAF V600E testing to guide antiepidermal growth factor receptor (EGFR) therapy was deemed low. The EWG encourages further studies of the potential value of testing in patients with mCRC who were found to have tumors that are wild type (mutation negative) for KRAS to predict responsiveness to therapy.The EWG found insufficient evidence to recommend for or against testing for mutations in NRAS, or PIK3CA, and/or loss of expression of PTEN or AKT proteins. The level of certainty for this evidence was low. In the absence of supporting evidence, and with consideration of other contextual issues, the EWG discourages the use of these tests in guiding decisions on initiating anti-EGFR therapy with cetuximab or panitumumab unless further evidence supports improved clinical outcomes. It has been suggested that patients with mCRC whose tumors harbor certain mutations affecting EGFR pathway signaling are typically unresponsive to therapy with anti-EGFR antibodies (cetuximab and panitumumab). The EWG identified recent evidence reviews that have addressed this topic, and this recommendation statement is based on results of these reviews. In developing these recommendations the EWG considered evidence in the areas described below. Although no research syntheses that have formally evaluated analytic validity of these tests were found, the EWG was able to draw the following conclusions from assessments included in the evidence reviews under consideration. There is adequate evidence that KRAS mutation analysis reliably and accurately detects common mutations (codons 12 and 13), whereas evidence was inadequate for less frequent KRAS mutations (e.g., codon 61). There is also adequate evidence that testing for BRAF V600E accurately and reliably detects the mutation. For common mutations in NRAS, PIK3CA, and expression of PTEN AKT, there is adequate evidence of accurate and reliable detection. However, much less data exist in support. Furthermore, in the specific context of mCRC, no evidence was found on the analytic validity of immunohistochemistry (IHC) assays for PTEN or AKT expression. For KRAS mutation analysis, the EWG found convincing evidence for association with treatment response to anti-EGFR therapy, independent of prognostic association. For BRAF V600E mutation testing, the EWG found insufficient evidence for association with treatment response to anti-EGFR therapy independent of prognostic association. The EWG found insufficient evidence for association of results of testing for mutations in NRAS or PIK3CA, and loss of expression of PTEN or ATK proteins, with treatment response to anti-EGFR therapy. For KRAS mutation analysis, the EWG found adequate evidence that improved health outcomes are achieved by avoiding ineffective chemotherapy and potential side effects and expediting access to the next most effective treatment. Inadequate evidence was found regarding association of BRAF V600E mutation testing or loss of PTEN expression with improved health outcomes among patients with mCRC undergoing anti-EGFR therapy as compared with patients with tumors bearing wild-type BRAF sequence and PTEN expression levels, respectively. No evidence was found to support improved health outcomes associated with testing results for NRAS or PIK3CA variants, or AKT protein expression levels in this clinical scenario. CRC is an important and highly prevalent health problem. Improvements in mCRC outcomes associated with pharmacogenetic testing could have important clinical, and potentially public health, impacts. Adverse events related to cancer chemotherapy can be common and severe. Therefore, successfully optimizing treatment to maximize efficacy and minimize side effects is important for reducing mCRC-related morbidity and mortality.

PTEN regulation by the Akt/GSK-3β axis during RANKL signaling

Phosphatase and tensin homolog (PTEN) negatively regulates phosphoinositide 3-kinase (PI3K)/Akt signaling as a lipid phosphatase for the second messenger phosphatidylinositol 3,4,5-triphosphate. We discovered recently that inactivating glycogen synthase kinase-3β (GSK-3β) via Akt plays an important role in receptor activator of nuclear factor κb ligand (RANKL)-induced osteoclastogenesis. However, the signaling link between GSK-3β and PTEN in RANKL signaling has not been revealed. Downregulating PTEN by RNA interference increases Akt and GSK-3β phosphorylation levels by RANKL, thereby promoting the formation of osteoclasts. PTEN phosphorylation at threonine 366 (T366) decreased gradually during RANKL-induced osteoclastogenesis, whereas PTEN protein levels were unaffected. Interestingly, the PTEN phosphorylation defective mutant (T366A) showed increased osteoclastogenesis, which is consistent with its lower phosphatase activity, compared to that of wild-type PTEN. Moreover, treatment with the GSK-3 inhibitor SB216763 suppressed PTEN phosphorylation levels and phosphatase activity and enhanced Akt phosphorylation. These data suggest that inhibiting GSK-3β during RANKL-induced osteoclastogenesis decreases PTEN phosphorylation, leading to enhanced osteoclast differentiation through Akt activation.

Effects of indomethacin on expression of PTEN tumour suppressor in human cancers

Background:Previous studies reported that Non-steroidal Anti-inflammatory Drugs (NSAIDs), chemicals, and food supplements can be used to up-regulate the PTEN mRNA and protein expression, suggesting that these substances may be used in prevention and/or treatment of various human cancers like spinal, brain, colon, breast, prostate, bladder and endometrial cancers.Aim:This was to study expression and sub-cellular localisation of PTEN protein, and review the effect(s) of indomethacin on PTEN's expression in cultured Human Endometrial Cancer (HEC 1B) cell line, which is known to express significant amounts of the wild-type PTEN.Materials and Methods:This involves culture and incubation of artificial HEC 1B cells. All procedures were undertaken in the cell culture hood under the recommended sterile conditions. The cells were then incubated with different concentrations of indomethacin solution, for variable durations and finally fixed (with paraformaldehyde) and stained with fluorescein-labelled diluted secondary antibody (FITC). Immunocytochemistry (IHC) and fluorescent microscopy were then employed for the detection and localisation of the specific antigen (PTEN), using antibodies.Results:The HEC 1B cells, which were cultured and incubated with different concentrations of indomethacin solution, expressed the PTEN protein, most of which was localised to the nucleus with minimal cytoplasmic expression. Increased PTEN expression was observed following treatment of the cells with various concentrations of the solution for variable durations, although there was cell death at higher concentrations and longer duration. This procedure was repeated several times, in order to have consistency and to validate the results.Conclusion:This study agrees with previous studies in similar human cell lines and supports the idea that NSAIDs and other drugs may be used in the future for prevention of human cancers. However, more studies need to be carried out to substantiate these observations.

Vandetanib is effective in EGFR-mutant lung cancer cells with PTEN deficiency

The effectiveness of vandetanib, an agent that targets RET, VEGFR and EGFR signaling, against EGFR-mutant lung cancer cells with PTEN loss was investigated. Two EGFR mutant non-small cell lung cancer (NSCLC) cell lines, PC-9 (PTEN wild type) and NCI-H1650 (PTEN null), were used. We transfected an intact PTEN gene into H1650 cells and knocked down PTEN expression in PC-9 cells using shRNA. The effectiveness of gefitinib and vandetanib was assessed using a xenograft model. While PC-9 cells were more resistant to vandetanib than gefitinib, H1650 cells were more sensitive to vandetanib than gefitinib. Both gefitinib and vandetanib suppressed the activation of EGFR and MAPK in H1650 cells, although phosphorylated AKT levels were not affected. In an H1650 cell xenograft model, vandetanib was also more effective than gefitinib. Although PTEN-transfected H1650 cells did not show restoration of sensitivity to gefitinib in vitro, the xenograft tumors responded to gefitinib and vandetanib. Knockdown of PTEN in PC-9 cells caused resistance to gefitinib. In conclusion, vandetanib might be effective in NSCLC with EGFR mutations that lack PTEN expression. The contribution of PTEN absence to vandetanib activity in NSCLC cells harboring EGFR mutations should be further examined.

Effects of epidermal growth factor receptor and phosphatase and tensin homologue gene expression on the inhibition of U87MG glioblastoma cell proliferation induced by protein kinase inhibitors

The aim of the present study was to analyse the antiproliferative effects and mechanisms of action of protein kinase inhibitors (PKIs) in human glioblastoma multiforme (GBM) cells with different epidermal growth factor receptor (EGFR) and phosphatase and tensin homologue (PTEN) status. The GBM cell models were established by transfection of plasmids carrying wild-type EGFR, mutated EGFRvIII or PTEN and clonal selection in U87MG cells. Phosphatidylinositol 3-kinase (PI3-K)/AKT pathway-focused gene profiles were examined by real-time polymerase chain reaction-based assays, protein expression was evaluated by western blotting and the antiproliferative effects of PKI treatment were determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in GBM cells. The cell model with intact PTEN and low EGFR levels was the most sensitive to treatment with the EGFR inhibitor erlotinib, whereas the model with EGFRvIII was the most resistant to treatment with the mitogen-activated protein kinase kinase inhibitor U0126. The dual PI3-K and mammalian target of rapamycin (mTOR) inhibitor PI103 had the most potent antiproliferative effects against all GBM cells tested. Following simultaneous stimulation of AKT and extracellular signal-regulated kinase, rapamycin concentrations > 0.5 nmol/L failed to exhibit a further growth inhibitory effect. Concurrent inhibition of mTOR and ribosomal protein s6 activity may underlie the inhibition of GBM proliferation by PKI. In conclusion, overexpression of EGFR or EGFRvIII, accompanied by a loss of PTEN, contributed to the activation of multiple intracellular signalling pathways in GBM cells. Rigorous examination of biomarkers in tumour tissues before and after treatment may be necessary to determine the efficacy of PKI therapy in patients with GBM.

Abstract PD01-01: Overcoming endocrine therapy resistance related to PTEN loss by strategic combinations with mTOR, AKT, or MEK inhibitors

Abstract Background: Hyperactive PI3K signaling is associated with a more aggressive subtype of estrogen receptor (ER) positive breast cancer (BC) and with endocrine resistance. Loss or downregulation of PI3K's inhibitor PTEN is more common in basal and luminal B vs. luminal A BC. However, the role of PTEN in modulating response to various endocrine therapies is unclear. Here we investigated the effects of PTEN knockdown (KD) on endocrine sensitivity and the potential of multiple kinase inhibitors to restore and improve responses. Methods: Nude mice bearing ER+ BC xenograft tumors of MCF7 cells stably expressing a doxycycline (Dox)-inducible PTEN-shRNA were randomized to four endocrine treatment groups [continued estrogen (E2) supplementation, or E2-deprivation (ED) alone or in combination with tamoxifen (Tam) or fulvestrant (Ful)]; all -/+ Dox. The effects of single or combined kinase inhibitors on these endocrine treatments -/+ Dox were studied in vitro using inhibitors (i) to mTOR (AZD2014, 0.2 μM), AKT (AZD5363, 1 μM), or MEK (Selumetinib/ARRY-142886, 1 μM). Cell growth, apoptosis, and ER and progesterone receptor (PR) signaling were analyzed using cell cytometry, qRT/PCR, and Western blotting. Synergism tests were used to examine the growth effects of the most promising combinatorial therapy with multiple kinase inhibitors in different endocrine settings. Results: In wild-type (WT) PTEN xenograft tumors, endocrine therapies were very effective, inducing frequent tumor regression. In PTEN KD tumors endocrine therapies were less effective — PTEN KD delayed tumor regression in all endocrine regimens and accelerated tumor progression in the Tam treated group. Furthermore, at day 250, only 1/8 and 0/7 tumors had developed resistance in the ED and the Ful (−Dox) groups, respectively, while with PTEN KD (+Dox), 7/15 and 5/15 tumors developed resistance to ED and to Ful. In vitro PTEN KD also induced resistance to all endocrine therapies. mRNA and/or protein levels of ER and PR were suppressed by PTEN KD and restored by mTORi and AKTi. In cells with WT PTEN, mTORi was highly effective with or without endocrine therapy. However, AKTi and MEKi were more effective in combination with endocrine therapy. All three inhibitors were less effective upon PTEN KD. The mTORi plus AKTi combination resulted in a potent synergistic inhibition in PTEN KD cells in the presence of E2 or with ED. In contrast, in the presence of Tam, AKTi plus MEKi, independent of PTEN status, was the most effective combination at the doses chosen. Finally, these inhibitors and combinations were more effective in the presence of Ful than ED or Tam in WT PTEN cells. AKTi combined with Ful was still highly effective even in PTEN KD cells, but mTORi and MEKi were less effective. Conclusions: Our results suggest that PTEN loss renders endocrine therapy less effective in in vitro and in vivo experimental models. Single AKT/MEK kinase inhibitors are more potent in the presence of endocrine therapy. In PTEN KD cells, the activity of all three kinase inhibitors is largely diminished, except for AKTi in the presence of fulvestrant. Kinase inhibitor combinations are generally more effective, but the optimal combinations vary by PTEN status and type of endocrine therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-01.