Re-expression Of PTEN Research Articles

PTEN-restoration abrogates brain colonisation and perivascular niche invasion by melanoma cells.

Melanoma brain metastases (MBM) continue to be a significant clinical problem with limited treatment options. Highly invasive melanoma cells migrate along the vasculature and perivascular cells may contribute to residual disease and recurrence. PTEN loss and hyperactivation of AKT occur in MBM; however, a role for PTEN/AKT in perivascular invasion has not been described. We used in vivo intracranial injections of murine melanoma and bulk RNA sequencing of melanoma cells co-cultured with brain endothelial cells (brECs) to investigate brain colonisation and perivascular invasion. We found that PTEN-null melanoma cells were highly efficient at colonising the perivascular niche relative to PTEN-expressing counterparts. PTEN re-expression (PTEN-RE) in melanomacells significantly reduced brain colonisation and migration along the vasculature. We hypothesised this phenotype was mediated through vascular-induced TGFβ secretion, which drives AKT phosphorylation. Disabling TGFβ signalling in melanoma cells reduced colonisation and perivascular invasion; however, the introduction of constitutively active myristolated-AKT (myrAKT) restored overall tumour size but not perivascular invasion. PTEN loss facilitates perivascular brain colonisation and invasion of melanoma. TGFβ-AKT signalling partially contributes to this phenotype, but further studies are needed to determine the complementary mechanisms that enable melanoma cells to both survive and spread along the brain vasculature.

Cholesterol biogenesis is a PTEN-dependent actionable node for the treatment of endocrine therapy-refractory cancers.

PTEN and PIK3CA mutations are the most prevalent PI3K pathway alterations in prostate, breast, colorectal, and endometrial cancers. p110β becomes the prominent PI3K isoform upon PTEN loss. In this study, we aimed to understand the molecular mechanisms of PI3K dependence in the absence of PTEN. Using online bioinformatical tools, we examined two publicly available microarray datasets with aberrant PI3K activation. We found that the rate-limiting enzyme of cholesterol biogenesis, SQLE, was significantly upregulated in p110β-hyperactivated or PTEN-deficient mouse prostate tumors. Concomitantly, the expression of cholesterol biosynthesis pathway enzymes was directly correlated with PI3K activation status in microarray datasets and diminished upon PTEN re-expression in PTEN-null prostate cancer cells. Particularly, PTEN re-expression decreased SQLE protein levels in PTEN-deficient prostate cancer cells. We performed targeted metabolomics and detected reduced levels of cholesteryl esters as well as free cholesterol upon PTEN re-expression. Notably, PTEN-null prostate and breast cancer cell lines were more sensitive to pharmacological intervention with the cholesterol pathway than PTEN-replete cancer cells. Since steroid hormones use sterols as structural precursors, we studied whether cholesterol biosynthesis may be a metabolic vulnerability that enhances antihormone therapy in PTEN-null castration-resistant prostate cancer cells. Coinhibition of cholesterol biosynthesis and the androgen receptor enhanced their sensitivity. Moreover, PTEN suppression in endocrine therapy-resistant luminal-A breast cancer cells leads to an increase in SQLE expression and a corresponding sensitization to the inhibition of cholesterol synthesis. According to our data, targeting cholesterol biosynthesis in combination with the hormone receptor signaling axis can potentially treat hormone-resistant prostate and breast cancers.

Abstract 3643: PTEN/STAT3 pathway in cancer-associated fibroblasts in pancreatic cancer

Abstract This study aims to test the hypothesis that direct interactions and auto-regulatory loop between PTEN and STAT3 in cancer-associated fibroblasts (CAFs) contribute to an immunosuppressive tumor microenvironment in pancreatic ductal carcinoma (PDAC). Pancreatic cancer accounts for almost as many deaths as cases because of its poor prognosis. Many studies have led to the paradigm that stromal elements contribute to pancreatic tumor growth. Cancer-associated fibroblasts (CAFs) are a primary cell type in the desmoplastic stroma in PDAC, and targeting them to improve PDAC treatment is thought to be a promising strategy, however; studies have shown both tumor-promoting and tumor-limiting activities associated with PDAC CAFs. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most well-described tumor suppressor genes and can regulate essential transcription factors in stromal fibroblasts. On the other hand, STAT3 signaling in pancreatic CAFs contributes to the immunosuppressive and fibrotic stroma observed with disease progression. In this study, we used novel, dual recombinase mouse models of PDAC, with Kras and p53 mutations engineered in the pancreatic epithelium (Flp-based) and Stat3 deleted in a subpopulation of CAFS (Cre). CAFs isolated from these mice were used to probe PTEN-STAT3 interaction in vitro using the Proximity Ligation Assay (PLA) and by ChIP-seq using an RNA seq. Mouse and Human pancreatic tumor samples were analyzed and quantified using multiplex-immunofluorescence and multispectral imaging. In the mouse genetic model, we found PTEN downregulated in ~80% of CAFs, consistent with previous work from our group that demonstrated PTEN loss in 25% of PDAC patient samples. At the same time, pY705-STAT3 was activated in CAFs, and deletion of STAT3 in CAFs correlated with increased survival, decreased M2-like macrophages, and increased T-cell infiltration. STAT3 ablation in CAFS resulted in the re-expression of PTEN in CAFS, implying an autoregulatory loop. PLA on CAFs isolated revealed that IL6 treatment resulted in the transfer of a STAT3-PTEN protein complex into the nucleus ChIP-seq on CAFs with PTEN STAT3 antibodies revealed a significant overlap in PTEN and STAT3 occupation of gene promoters. This result is consistent with in silico analysis on the PTEN and STAT3 ChIP-Seq data in human cell lines. ChIP-Seq data is being interacted with RNA data to identify essential target genes. Human PDAC tissue microarrays are stained with pSTAT3, PTEN, CD3, and CD8.Our study suggests an autoregulatory look between PTEN and STAT3 in PDAC CAFs. PTEN may suppress specific STAT3 target genes through direct interactions with chromatin. Alternatively, PTEN/STAT3 complexes may target CAF genes that suppress tumor growth or activate anti-tumor immune responses. These investigations could identify potential targets in CAFs for restoring the anti-tumor immune responses in PDAC. Citation Format: Samaneh Saberi, Sudarshana Sharma, Cameron Bumbleburg, Julia E. Lefler, Michael C. Ostrowski. PTEN/STAT3 pathway in cancer-associated fibroblasts in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3643.

BAP1 suppresses prostate cancer progression by deubiquitinating and stabilizing PTEN.

Deubiquitinase BAP1 is an important tumor suppressor in several malignancies, but its functions and critical substrates in prostate cancer (PCa) remain unclear. Here, we report that the mRNA and protein expression levels of BAP1 are downregulated in clinical PCa specimens. BAP1 can physically bind to and deubiquitinate PTEN, which inhibits the ubiquitination-mediated degradation of PTEN and thus stabilizes PTEN protein. Ectopically expressed BAP1 in PCa cells increases PTEN protein level and subsequently inhibits the AKT signaling pathway, thus suppressing PCa progression. Conversely, knockdown of BAP1 in PCa cells leads to the decrease in PTEN protein level and the activation of the Akt signaling pathway, therefore promoting malignant transformation and cancer metastasis. However, these can be reversed by the re-expression of PTEN. More importantly, we found that BAP1 protein level positively correlates with PTEN in a substantial fraction of human cancers. These findings demonstrate that BAP1 is an important deubiquitinase of PTEN for its stability and the BAP1-PTEN signaling axis plays a crucial role in tumor suppression.

MiR-217 Promotes Cardiac Hypertrophy and Dysfunction by Targeting PTEN

Previously, we found that the miR-217 expression level was increased in hearts from chronic heart failure (CHF) patients by using miRNA profile analysis. This study aimed to explore the role of miR-217 in cardiac dysfunction. Heart tissue samples from CHF patients were used to detect miR-217 expression levels. A type 9 recombinant adeno-associated virus (rAAV9) was employed to manipulate miR-217 expression in mice with thoracic aortic constriction (TAC)-induced cardiac dysfunction. Cardiac structure and function were measured by echocardiography and invasive pressure-volume analysis. The expression levels of miR-217 were increased in hearts from both CHF patients and TAC mice. Overexpression of miR-217 in vivo aggravated pressure overload-induced cardiac hypertrophy, fibrosis, and cardiac dysfunction, whereas miR-217-TUD-mediated downregulation of miR-217 reversed these effects. PTEN was predicted and validated as a direct target of miR-217, and re-expression of PTEN attenuated miR-217-mediated cardiac hypertrophy and cardiac dysfunction. Importantly, cardiomyocyte-derived miR-217-containing exosomes enhanced proliferation of fibroblasts in vitro. All of these findings show that miR-217 participates in cardiac hypertrophy and cardiac fibrosis processes through regulating PTEN, which suggests a promising therapeutic target for CHF.

Impaired caudal fin-fold regeneration in zebrafish deficient for the tumor suppressor Pten.

Zebrafish are able to completely regrow their caudal fin‐folds after amputation. Following injury, wound healing occurs, followed by the formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. Here we show that, surprisingly, the phosphatase and tumor suppressor Pten, an antagonist of phosphoinositide‐3‐kinase (PI3K) signaling, is required for zebrafish caudal fin‐fold regeneration. We found that homozygous knock‐out mutant (ptena−/−ptenb−/−) zebrafish embryos, lacking functional Pten, did not regenerate their caudal fin‐folds. AKT phosphorylation was enhanced, which is consistent with the function of Pten. Reexpression of Pten, but not catalytically inactive mutant Pten‐C124S, rescued regeneration, as did pharmacological inhibition of PI3K. Blastema formation, determined by in situ hybridization for the blastema marker junbb, appeared normal upon caudal fin‐fold amputation of ptena−/−ptenb−/− zebrafish embryos. Whole‐mount immunohistochemistry using specific markers indicated that proliferation was arrested in embryos lacking functional Pten, and that apoptosis was enhanced. Together, these results suggest a critical role for Pten by limiting PI3K signaling during the regenerative outgrowth phase of zebrafish caudal fin‐fold regeneration.

Abstract 1339: Role of PTEN loss in basal-like 2 triple negative breast cancer

Abstract Basal-like 2 (BL2) triple negative breast cancer (TNBC) is an aggressive molecular subtype of breast cancer with a poor clinical outcome. These cancers are characterized by overexpression of epidermal growth factor receptor (EGFR), loss of PTEN protein expression, and presence of mutated TP53. Despite overexpression of EGFR, EGFR-targeted therapies have performed poorly in clinical settings. Therefore, understanding the mechanisms of resistance in these cells is critical for improved patient care. To understand the oncogenic signaling landscape in BL2 TNBC, our lab utilizes the BL2 cell lines SUM-149 and SUM-229, which overexpress EGFR activated by an amphiregulin-mediated autocrine loop, and are PTEN null. In addition, we utilize the BL1 subtype cell line MDA-MB-468, which also exhibits overexpression of EGFR and loss of PTEN. Both the BL1 and BL2 cell lines exhibit elevated levels of phosphorylated AKT (p-AKT) compared to control MCF-10A cells. However, while treatment of SUM-149 and SUM-229 cells with inhibitors targeting EGFR (Gefitinib), type-1 PI3K (BKM120, BKM), mTOR (KU-0063794, KU) and pan-AKT (MK2206, MK) did not reduce p-AKT levels, MCF-10A and MDA-MB-468 cell lines treated with the same panel of targeted inhibitors showed marked inhibition of p-AKT. In addition, re-expression of PTEN in SUM-149 and SUM-229 cells had little effect on p-AKT but dramatically reduced p-AKT in MDA-MB-468 cells. Although PTEN re-expression alone had little effect on p-AKT in SUM-149 and SUM-229 cells, these cells did display decreased AKT phosphorylation following treatment with Gefitinib, BKM, KU, and MK. There are three isoforms of AKT (AKT1, AKT2 and AKT3) of which, AKT3 has been implicated in the aggressive nature of some cancers. Previous studies in our lab showed that SUM-149 and SUM-229 cell lines have higher levels of AKT3 message and protein compared to the other AKT isoforms. AKT3 expression was also higher in the BL2 cell lines compared to BL1 and control cells. This differential expression of AKT isoforms led us to examine the impact of AKT isoform-specific signaling. Using PTEN re-expressing SUM-149 cells, we showed that PTEN re-expression resulted in decreased phosphorylation of AKT1 but not of AKT3. However, PTEN re-expression combined with Gefitinib treatment dramatically reduced phosphorylation of both AKT1 and AKT3. Interestingly, while neither re-expression of PTEN nor Gefitinib expression alone had an effect on colony-forming efficiency, PTEN re-expression coupled with Gefitinib treatment was able to reduce colony formation efficiency in both SUM-149 and SUM-229 cell lines. Taken together, these data indicate that BL2 breast cancers, with overexpression of EGFR and loss of PTEN expression, have an oncogenic signaling network involving AKT3 that contributes to the aggressive and drug-resistant phenotype observed in this subset of TNBC. Citation Format: Ericka L. Smith, Christiana S. Kappler, Stephen P. Ethier. Role of PTEN loss in basal-like 2 triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1339. doi:10.1158/1538-7445.AM2017-1339

Abstract 4449: TET1 inhibits gastric cancer metastasis by demethylation and re-expression of PTEN

Abstract TET1 is a member of ten eleven translocation enzymes, which can convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), eventually to promote CpG islands demethylation of specific gene.. In this study, we found that TET1 expression and 5-hmC content were low in tumor tissues compared to its adjacent non-tumor tissues of gastric cancer. Cell proliferation, migration and invasion were enhanced when TET1 knocked down in gastric cancer cell in vitro and in vivo, while it was suppressed when TET1 over-expressed. We also found that PTENis one of the target genes of TET1. TET1 directly binds to the promoter region of PTEN, activates its transcription through demethylating its CpG islands. Knockdown of TET1 results in hypermethylation of PTEN promoter and low expression of PTEN. PTEN knockdown activates AKT and FAK pathway, which are suppressed by PTEN. The activation of PTEN downstream AKT and FAK facilitates tumor migration, invasion and accelerates cell growth. In conclution, we found a novel mechanism that TET1 suppresses tumor cell growth, migration and invasion through conversion PTEN promoter CpG island methylation to demehylation and then re-expression PTEN. Citation Format: Bingya Liu, Yao-fei Pei, Jian-fang Li, Liping Su, Ran Tao, Min Yan, Zhang-gang Zhu. TET1 inhibits gastric cancer metastasis by demethylation and re-expression of PTEN. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4449.

Abstract 4596: Regulation of epidermal growth factor receptor signaling in triple-negative breast cancer

Abstract Although the basal-like 1 (BL1) and BL2 subtypes of triple negative breast cancer (TNBC) share similar genetic and biological characteristics, patients with BL2 type tumors have markedly poorer prognosis. In addition, despite the overexpression of epidermal growth factor receptor (EGFR) characteristically observed in basal-like TNBC, EGFR inhibitors have performed poorly in clinical trials. In order to understand the mechanism of resistance to EGFR inhibition in these tumors, our laboratory utilized cell line models of BL2 (SUM-149 and SUM-229) and BL1 (MDA-MB-468; MM468) TNBC, all of which overexpress EGFR, are PTEN null and exhibit elevated AKT phosphorylation (p-AKT). Compared to MCF10A and MM-468 cells, AKT phosphorylation in the SUM-149 and SUM-229 cell lines was resistant to treatment with the EGFR inhibitor gefitinib, as well as to the PI3K inhibitor BKM120 (BKM) and the mTOR inhibitor KU-0063794 (KU). Even treatment with the allosteric AKT inhibitor MK2206 (MK) only partially reduced p-AKT. RT-PCR and immunoblot analysis of AKT isoform expression showed that in SUM-149 and SUM-229 cells, AKT3 is expressed at higher levels than AKT1 or AKT2 at both the protein and message level. Because AKT3 has a higher IC50 for MK than AKT1 or AKT2, increasing doses of MK were used in an effort to abrogate AKT phosphorylation. The results showed that MK treatment at higher concentrations (5 μM) decreased p-AKT. In addition, shRNA-mediated knock-down of AKT3 sensitized the cells to inhibition of AKT by BKM, KU and MK. Interestingly, MK treatment alone had little effect on colony forming efficiency, even at higher doses, but when added in combination with gefitinib, MK dramatically reduced colony formation. We then investigated the effect of PTEN re-expression on signaling in these cell lines. PTEN re-expression alone had little effect on p-AKT in SUM-149 and SUM-229 cells, but resulted in a dramatic reduction in p-AKT in MM-468 cells. Although PTEN expression alone had little effect on p-AKT in SUM-149 and 229 cells, these cells exhibited decreased p-AKT following treatment with gefitinib and were sensitive to inhibition of AKT by MK. In addition, treatment of SUM-149/PTEN cells with gefitinib markedly reduced colony-forming efficiency. Our laboratory has previously demonstrated that in SUM-149 cells, a set of genes that are essential for survival, including PLK1, are uncoupled from regulation by EGFR. Thus, we examined the effect of AKT inhibition on expression of PLK1 and found that inhibition of AKT or re-expression of PTEN was able to recouple EGFR-mediated regulation of PLK1 expression. Our results demonstrate that PI-3 kinase and mTORC2 drive phosphorylation of AKT1 but not AKT3 in SUM-149 cells, which explains their relative resistance to these targeted drugs. Citation Format: Christiana S. Kappler, Ericka L. Smith, Stephen Ethier. Regulation of epidermal growth factor receptor signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4596.

EBV-miR-BART7-3p promotes the EMT and metastasis of nasopharyngeal carcinoma cells by suppressing the tumor suppressor PTEN.

The epithelial-mesenchymal transition (EMT) is crucial to cancer progression and metastasis. Although multiple cellular miRNAs have been identified to regulate the EMT and metastasis in cancers, the role of viral miRNAs in cancer progression remains largely unknown. Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy typically characterized by its early metastasis. In the present study, we have discovered the involvement of a viral miRNA, EBV-miR-BART7-3p, in the EMT and metastasis of NPC cells. Initially, we observed that EBV-miR-BART7-3p was highly expressed in NPC and positively correlated with lymph node metastasis and clinical stage of NPC. Subsequently, we demonstrated that EBV-miR-BART7-3p enhanced cell migration/invasion in vitro, cancer metastasis in vivo, and particularly the EMT characterized by loss of epithelial markers and gain of mesenchymal features in NPC cells. Furthermore, mechanistic studies disclosed that EBV-miR-BART7-3p targeted a major human tumor suppressor PTEN, modulating PI3K/Akt/GSK-3β signaling and eventually leading to the high expression and nuclear accumulation of Snail and β-catenin, which favor EMT. Knockdown of PTEN could phenocopy the effect of EBV-miR-BART7-3p, whereas re-expression of PTEN resulted in a phenotypic reversion. Moreover, these findings were supported by an observation of an EBV-positive cell model in which silencing of endogenous EBV-miR-BART7-3p partially attenuated cell migration/invasion and altered EMT protein expression pattern via reverting PI3K/Akt, Snail and β-catenin expression. Thus, this study suggests a novel mechanism by which EBV-miR-BART7-3p modulates the EMT and metastasis of NPC cells, and a clinical implication of EBV-miR-BART7-3p as a potential biomarker or therapeutic target.

PTEN loss represses glioblastoma tumor initiating cell differentiation via inactivation of Lgl1

Glioblastoma multiforme is an aggressive and incurable type of brain tumor. A subset of undifferentiated glioblastoma cells, known as glioblastoma tumor initiating cells (GTICs), has an essential role in the malignancy of this disease and also appears to mediate resistance to radiation therapy and chemotherapy. GTICs retain the ability to differentiate into cells with reduced malignant potential, but the signaling pathways controlling differentiation are not fully understood at this time. PTEN loss is a very common in glioblastoma multiforme and leads to aberrant activation of the phosphoinositide 3-kinase pathway. Increased signalling through this pathway leads to activation of multiple protein kinases, including atypical protein kinase C. In Drosophila, active atypical protein kinase C has been shown to promote the self-renewal of neuroblasts, inhibiting their differentiation along a neuronal lineage. This effect is mediated by atypical protein kinase c-mediated phosphorylation and inactivation of Lgl, a protein that was first characterized as a tumour suppressor in Drosophila. The effects of the atypical protein kinase C/Lgl pathway on the differentiation status of GTICs, and its potential link to PTEN loss, have not been assessed previously. Here we show that PTEN loss leads to the phosphorylation and inactivation of Lgl by atypical protein kinase C in glioblastoma cells. Re-expression of PTEN in GTICs promoted their differentiation along a neuronal lineage. This effect was also seen when atypical protein kinase C was knocked down using RNA interference, and when a non-phosphorylatable, constitutively active form of Lgl was expressed in GTICs. Thus PTEN loss, acting via atypical protein kinase C activation and Lgl inactivation, helps to maintain GTICs in an undifferentiated state.

PTEN loss defines a PI3K/AKT pathway-dependent germinal center subtype of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category with distinct molecular subtypes that can be defined by gene expression profiling. However, even within these defined subtypes, heterogeneity prevails. To further elucidate the pathogenesis of these entities, we determined the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in 248 primary DLBCL patient samples. These analyses revealed that loss of PTEN was detectable in 55% of germinal center B-cell-like (GCB) DLBCLs, whereas this abnormality was found in only 14% of non-GCB DLBCL patient samples. In GCB DLBCL, the PTEN status was inversely correlated with activation of the oncogenic PI3K/protein kinase B (AKT) pathway in both DLBCL cell lines and primary patient samples. Reexpression of PTEN induced cytotoxicity in PTEN-deficient GCB DLBCL cell line models by inhibiting PI3K/AKT signaling, indicating an addiction to this pathway in this subset of GCB DLBCLs. PI3K/AKT inhibition induced down-regulation of the transcription factor MYC. Reexpression of MYC rescued GCB DLBCL cells from PTEN-induced toxicity, identifying a regulatory mechanism of MYC expression in DLBCL. Finally, pharmacologic PI3K inhibition resulted in toxicity selectively in PTEN-deficient GCB DLBCL lines. Collectively, our results indicate that PTEN loss defines a PI3K/AKT-dependent GCB DLBCL subtype that is addicted to PI3K and MYC signaling and suggest that pharmacologic inhibition of PI3K might represent a promising therapeutic approach in these lymphomas.

Abstract 2269: Lapatinib and obatoclax kill cns tumor cells through blockade of erbb1 / 3 / 4 and through inhibition of bcl-xl and mcl-1

Abstract Previous studies in breast cancer cells have shown that Lapatinib (Lap) and Obatoclax (GX) interact in a greater than additive manner to cause cell death. The present studies sought to expand our analyses to CNS (Glioblastoma & Medulloblastoma) tumor cells and to further advance our understanding of the mechanisms of drug action. Combinational treatment with Lap / GX resulted in enhanced tumor cell death in multiple CNS tumor types. Moreover, it was discovered that cells lacking PTEN function were resistant to drug combination lethality and re-expression of PTEN in PTEN null cells restored drug sensitivity. Furthermore, PTEN expressing cancer cells, sensitive to Lap / GX treatment, demonstrated drug resistance upon knockdown of PTEN. Additionally, we established that inhibition of ERBB1 / 3 / 4 receptors was most significant for enhancing GX lethality, and that knockdown of BCL-XL and particularly MCL-1, interacted to facilitate Lap lethality. Pre-treatment of tumor cells in vitro and in vivo with GX enhanced the lethality of Lap to a greater extent than concomitant treatment and suppressed tumor growth. Treatment of animals carrying orthotopic CNS tumor isolates and those implanted with anoikis resistant breast tumors in the brain with Lap / GX prolonged survival. Collectively our data argues that combinational Lap / GX therapy could be of use in the treatment of tumors located in the CNS. Further studies must be carried out in order to elucidate whether autophagy or apoptosis plays the most predominant role in cell death in response to Lap / GX treatment. 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 2269. doi:1538-7445.AM2012-2269

OSU-03012 suppresses GRP78/BiP expression that causes PERK-dependent increases in tumor cell killing

We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills tumor cells. OSU lethality was suppressed by knock down of PERK and enhanced by knock down of ATF6 and IRE1α. OSU treatment suppressed expression of the chaperone, BiP/GRP78, and did so through reduced stability of the protein. Knock down of BiP/GRP78 further enhanced OSU lethality. Overexpression of BiP/GRP78 abolished OSU toxicity. Pre-treatment of cells with OSU enhanced radiosensitivity to a greater extent than concomitant or sequential drug treatment with radiation exposure. Expression of a mutant active p110 PI3K, or mutant active forms of the EGFR in GBM cells did not differentially suppress OSU killing. In contrast loss of PTEN function reduced OSU lethality, without altering AKT, p70 S6K or mTOR activity, or the drug’s ability to radiosensitize GBM cells. Knock down of PTEN protected cells from OSU and radiation treatment whereas re-expression of PTEN facilitated drug lethality and radiosensitization. In a dose-dependent fashion OSU prolonged the survival of mice carrying GBM tumors and interacted with radiotherapy to further prolong survival. Collectively, our data show that reduced BiP/GRP78 levels play a key role in OSU-3012 toxicity in GBM cells, and that this drug has in vivo activity against an invasive primary human GBM isolate.

Determining PTEN Functional Status by Network Component Deduced Transcription Factor Activities

PTEN-controlled PI3K-AKT-mTOR pathway represents one of the most deregulated signaling pathways in human cancers. With many small molecule inhibitors that target PI3K-AKT-mTOR pathway being exploited clinically, sensitive and reliable ways of stratifying patients according to their PTEN functional status and determining treatment outcomes are urgently needed. Heterogeneous loss of PTEN is commonly associated with human cancers and yet PTEN can also be regulated on epigenetic, transcriptional or post-translational levels, which makes the use of simple protein or gene expression-based analyses in determining PTEN status less accurate. In this study, we used network component analysis to identify 20 transcription factors (TFs) whose activities deduced from their target gene expressions were immediately altered upon the re-expression of PTEN in a PTEN-inducible system. Interestingly, PTEN controls the activities (TFA) rather than the expression levels of majority of these TFs and these PTEN-controlled TFAs are substantially altered in prostate cancer mouse models. Importantly, the activities of these TFs can be used to predict PTEN status in human prostate, breast and brain tumor samples with enhanced reliability when compared to straightforward IHC-based or expression-based analysis. Furthermore, our analysis indicates that unique sets of PTEN-controlled TFAs significantly contribute to specific tumor types. Together, our findings reveal that TFAs may be used as “signatures” for predicting PTEN functional status and elucidate the transcriptional architectures underlying human cancers caused by PTEN loss.

The mechanism involved in the loss of PTEN expression in NSCLC tumor cells

Loss of PTEN expression is observed in most non-small cell lung cancers (NSCLC). However, the mechanism by which PTEN expression is regulated in NSCLC has not been fully elucidated. In this study, we investigated the role of DNA methyltransferases (Dnmts), microRNA-29b (miR-29b), and anti-miR-29b inhibitor in PTEN promoter methylation and PTEN gene expression in H358 NSCLC cells in vitro and in vivo. PTEN mRNA was measured by RT-PCR. PTEN and Dnmts protein levels were measured by Western blot. miR-29b expression was detected by Northern blot. A xenograft H358 tumor mouse model was established by subcutaneously inoculating H358 cells into the right hind limbs of nude mice. We found that radiation induced cell apoptosis and hypomethylation in PTEN promoter, PTEN and miR-29b expression, and downregulation of Dnmt1, 3a and 3b expression in H358 tumor cells. The effect of radiation on gene expression and apoptosis was blocked by anti-miR-29b inhibitor. In the xenograft H358 tumor model, anti-miR-29b inhibitor reversed radiation-induced tumor growth delay, PTEN reexpression and downregulation of Dnmts expression. Our study suggested that miR-29b is an upstream molecule of PTEN. miR-29b regulates PTEN gene expression through downregulating Dnmts expression and subsequently induces hypomethylation in PTEN promoter. Targeting therapy could be established in NSCLC by upregulating miR-29b expression.

Abstract LB-228: Resistance to BRAF inhibition in BRAF V600E colorectal cancer is associated with PI3K/AKT activation and hypermethylation

Abstract BRAF V600E mutation has been detected in approximately 8% of metastatic colorectal cancers (CRC) and results in constitutive activation of BRAF and the downstream MEK-ERK1/2 pathway. PLX4032, a selective inhibitor of BRAF V600E, has shown meaningful activity in melanoma (greater than 70% response) but only modest activity in BRAF V600E CRC (5%). The resistance of BRAF V600E CRC to BRAF inhibitors has been poorly understood. To explore these differences, BRAF V600E CRC cell lines were compared to BRAF V600E melanoma cell lines by reverse-phase proteomic array, which demonstrated higher levels of phospho-AKT and associated downstream pathway activation in CRC cell lines. Both colorectal cancer cells inherently resistant to PLX4720, an analog of PLX4032, and resistant subclones that were derived from BRAF V600E positive cells, showed increased expression of phospho-AKT and phospho-GSK3β, despite the sustained ability of PLX4720 to effectively inhibit MEK signaling. Silencing of PTEN, which in turn induced AKT activation, desensitized cells to the BRAF inhibitor. Furthermore, combined treatment of PLX4720 with two different PI3K inhibitors, LY294002 and GDC-0941, synergistically inhibited growth in a panel of BRAF V600E cells through both apoptosis and G1 phase arrest. It has been previously shown that BRAF V600E colorectal cancers are commonly associated with the CpG island methylation phenotype (CIMP), which is associated with epigenetically diminished PTEN expression. Combination treatment with the demethylating agent azacitadine and PLX4032 was also synergistic in cell lines with intact but suppressed PTEN, and was associated with PTEN re-expression. This was not seen in non-CIMP or PTEN BRAF V600E cell lines. Thus, colorectal cancer cells appear to escape BRAF targeting through a mechanism of increased PI3K/AKT activity, providing evidence to support investigation of new combination therapies with PI3K/AKT pathway inhibitors or demethylating 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 LB-228. doi:10.1158/1538-7445.AM2011-LB-228

Preclinical evaluation of PARP inhibition as a treatment for endometrioid endometrial carcinomas.

5065 Background: Loss of function of PTEN is the most common genetic defect in endometroid endometrial carcinomas (EEC). PTEN plays a pivotal role in the regulation of the AKT and mTOR signalling pathways and is reported to be inactivated in up to 80% of EECs. PTEN loss of function has recently been shown to cause genetic instability through defects in homologous recombination (HR) DNA repair of double strand breaks. Cancer cells with defective HR have an exquisite sensitivity to inhibitors of the poly(ADP) ribose polymerase (PARP) inhibitors. Recent data suggest PTEN deficiency may also lead to sensitivity to PARP inhibition. Methods: PTEN expression was tested in a panel of eight EEC cell lines by western blotting. Cells were tested for their ability to elicit phospho-γ-H2AX foci (a marker of induction of DNA damage) and RAD51 foci (a marker of competent HR DNA repair) when challenged with ionising radiation. The sensitivity of these cell lines to the PARP inhibitor KU0058948 was tested in vitro. PTEN was silenced and re-expressed in cell lines by generating stably transfected cell lines with either two short hairpin-RNAs against PTEN or with a pBABE retroviral PTEN expression vector, respectively. Results: Out of the 8 EEC cell lines, two cell lines expressed PTEN (Hec-1b, EFE-184). ECC cells lacking PTEN expression (Nou-1, Ishikawa, RL95-2, EN, AN3-CA, Hec-59) were unable to elicit HR DNA repair. PTEN deficient EEC cells showed a significantly greater sensitivity to KU0058948 than PTEN wild-type EEC cells (IC50 10−8M vs 10−4M, respectively). PTEN silencing in Hec-1b cells resulted in a reduced RAD51 foci formation and in increased sensitivity to PARP inhibition. PTEN re-expression in Ishikawa and Nou-1 cells resulted in enhanced RAD51 foci formation and in a lower sensitivity to KU0058948. Conclusions: Given that most EECs lack PTEN expression and that EEC cell lines lacking PTEN expression show an exquisite sensitivity to KU0058948, our results suggest that PARP inhibitors may constitute a novel therapeutic approach for the majority of EECs. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merck Merck AstraZeneca, Pfizer AstraZeneca

Activation of the PI3K-AKT pathway in human malignant mesothelioma cells

Malignant mesothelioma (MM) is a highly aggressive neoplasm, which is associated with asbestos exposure. The dysregulated phosphatidylinositol 3-kinase (PI3K)-AKT pathway plays an important role in cell proliferation, survival and motility in various cancers. In this study, we analyzed the activation status and underlying mechanisms of this pathway in MM cells using 21 cell lines. AKT activation was observed in 13 (62%) of the 21 MM cell lines under serum-starved conditions. Two cell lines, ACC-MESO-1 and Y-MESO-25, showed no expression of PTEN protein, while 7 other cell lines showed low expression of PTEN mRNA and protein compared to expression levels in an immortalized normal mesothelial cell line, MeT-5A. We found that PTEN inactivation in the ACC-MESO-1 and Y-MESO-25 lines was due to a 39.4-kb deletion including PTEN exon 2, and to a 7.7-kb deletion including exon 1, respectively. Re-expression of PTEN in these cells reduced the activity of colony formation in vitro. In contrast, no mutation of PIK3CA or LKB1 was found in any of the MM cell lines. These findings suggest that AKT is frequently activated in MM cells, in part due to the downregulation of PTEN. Thus, the PI3K-AKT signaling pathway is a potential therapeutic target for MM.

Cdc6 and Cyclin E2 Are PTEN-Regulated Genes Associated with Human Prostate Cancer Metastasis

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is frequently inactivated in metastatic prostate cancer, yet the molecular consequences of this and their association with the metastatic phenotype are incompletely understood. We performed transcriptomic analysis and identified genes altered by conditional PTEN reexpression in C4-2, a human metastatic prostate cancer cell line with inactive PTEN. PTEN-regulated genes were disproportionately represented among genes altered in human prostate cancer progression and metastasis but not among those associated with tumorigenesis. From the former set, we identified two novel putative PTEN targets, cdc6 and cyclin E2, which were overexpressed in metastatic human prostate cancer and up-regulated as a function of PTEN depletion in poorly metastatic DU145 human prostate cancer cells harboring a wild type PTEN. Inhibition of cdc6 and cyclin E2 levels as a consequence of PTEN expression was associated with cell cycle G(1) arrest, whereas use of PTEN activity mutants revealed that regulation of these genes was dependent on PTEN lipid phosphatase activity. Computational and promoter-reporter evaluations implicated the E2F transcription factor in PTEN regulation of cdc6 and cyclin E2 expression. Our results suggest a hypothetical model whereby PTEN loss upregulates cell cycle genes such as cdc6 and cyclin E2 that in turn promote metastatic colonization at distant sites.