O6‐methylguanine DNA methyltransferase immunoexpression in nonfunctioning pituitary adenomas

BACKGROUND:

Currently, no effective alternative treatment exists for progressive, regrowing, nonfunctioning pituitary adenomas (NFPA) that are resistant to conventional multimodality therapy. Temozolomide (TMZ) was proposed as a treatment option for pituitary carcinomas and aggressive pituitary adenomas. Recently, it was suggested that the responsiveness of pituitary tumors to TMZ depends on the immunoexpression of O⁶‐methylguanine DNA methyltransferase (MGMT). Therefore, the authors of this report assessed MGMT expression in a series of patients with progressive, regrowing NFPAs to evaluate whether TMZ may serve as alternative treatment option.

METHODS:

On the basis of postoperative magnetic resonance imaging, 45 patients with NFPAs were allocated to either a group with progressive, regrowing tumors (n = 24) or a tumor‐free group (n = 21), which served as a control. MGMT expression was assessed semiquantitatively by immunohistochemistry (low expression was defined as ≤50% immunostained adenoma cells, and high expression was defined as >50% immunostained adenoma cells) and was compared between the 2 groups.

RESULTS:

At the time of initial surgery, low MGMT expression was observed in 12 of 24 patients (50%) in the study group with progressive, regrowing NFPAs. In the control group of tumor‐free patients, only 5 of 21 patients (24%) exhibited low MGMT expression. A comparable distribution of MGMT expression was observed in the specimens from repeat surgeries. A shorter interval to second surgery was observed in patients who had low MGMT expression.

CONCLUSIONS:

The current data has suggested that half of the patients with progressive, regrowing NFPAs exhibit low MGMT expression and are potential candidates for treatment with TMZ. These findings provide a rationale for the use of TMZ as an alternative treatment approach in this subgroup if conventional therapy, including reoperation, radiosurgery, and radiotherapy, fails. Cancer 2009. © 2009 American Cancer Society.     

侵袭性垂体瘤的MRI诊断与影像学特征

目的:探讨MRI对侵袭性垂体瘤的诊断价值与影像特征.方法:回顾性分析经手术病理证实的36例侵袭性垂体瘤的临床资料及其MRI表现.结果:36例侵袭性垂体瘤均向鞍区多个方向生长致周围组织结构受侵,其中以海绵窦和颅底骨质侵犯为主要特征,表现为包绕颈动脉,鞍底受侵下陷,部分肿瘤突破鞍隔,突入蝶窦等,且常伴有坏死或囊变、出血.在T1WI上肿瘤呈低、等信号及混杂信号,T2WI肿瘤呈等信号或不均匀高信号.T1WI增强扫描后肿瘤多呈不均匀强化.结论:MRI检查可清楚显示侵袭性垂体瘤形态、大小、生长方式,以及肿瘤与周围组织的关系,对临床治疗方案的制定具有重要价值.     

垂体腺瘤的血管生成、c-myc表达与侵袭性的相关意义

目的　探讨微血管密度 (microvesseldensity,MVD)、c -myc表达与肿瘤侵袭性的关系。方法　应用EnVision二步法免疫组化技术检测 5 8例垂体腺瘤的MVD、c-myc表达情况 ;分为侵袭组和非侵袭组进行统计学分析 ,并对MVD与c-myc的表达强度进行相关性分析。结果侵袭性垂体腺瘤MVD为 6 .78± 1.6 7;非侵袭性垂体腺瘤MVD为 3.72± 1.0 2 ;差异有显著性 (P <0 .0 0 1)。各型垂体腺瘤中c -myc均有表达 ,总阳性率为 37.9% ;其中侵袭性垂体腺瘤的阳性率为 5 5 .5 6 % ,非侵袭性垂体腺瘤的阳性率为 2 2 .5 8%。差异有显著性 (P <0 .0 5 ) ,侵袭性垂体腺瘤c -myc高表达。直线相关分析显示 ,c -myc表达强度与MVD呈显著正相关 (r=0 .5 5 4 ,P <0 .0 1)。结论　侵袭性垂体腺瘤MVD增高、c -myc高表达 ,是反映垂体瘤侵袭能力及预后的有用指标。侵袭性垂体腺瘤MVD与c -myc表达强度密切相关 ,提示c -myc表达强度与垂体腺瘤MVD在判定垂体腺瘤侵袭性方面有意义     

Thioridazine inhibits autophagy and sensitizes glioblastoma cells to temozolomide

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14–15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome-wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late-stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy-lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.     

Metalloproteinases ADAM12 and MMP‐14 are associated with cavernous sinus invasion in pituitary adenomas

Invasion of tumor cells critically depends on cell‐cell or cell‐extracellular matrix interactions. Enzymes capable of modulating these interactions belong to the proteinase families of ADAM (a disintegrin and metalloprotease) and MMP (matrix metalloprotease) proteins. Our objective is to examine their expression levels and evaluate the relationship between expression levels and cavernous sinus invasion in pituitary adenomas. Tissue samples from 35 patients with pituitary adenomas were analyzed. Quantitative real‐time polymerase chain reaction (qPCR) was employed to assess mRNA expression levels for ADAM and MMP genes. Protein levels were examined using immunohistochemistry and Western Blot. Correlation analyses between expression levels and clinical parameters were performed. By silencing ADAM12 and MMP‐14 with siRNA in a mouse pituitary adenoma cell line (TtT/GF), their cellular effects were investigated. In our study, nine women and 26 men were included, with a mean age of 53.1 years (range 15–84 years) at the time of surgery. There were 19 cases with cavernous sinus invasion. The proteins ADAM12 and MMP‐14 were significantly up‐regulated in invasive adenomas compared to noninvasive adenomas. Both human isoforms of ADAM12 (ADAM12L and ADAM12s) were involved in tumor invasion; moreover, ADAM12L was found to correlate positively with Ki‐67 proliferation index in pituitary adenomas. In TtT/GF pituitary adenoma cells, silencing of ADAM12 and MMP‐14 significantly inhibited cell invasion and migration, respectively, whereas only silencing of ADAM12 suppressed cell proliferation. We conclude that ADAM12 and MMP‐14 are associated with cavernous sinus invasion in pituitary adenomas, which qualifies these proteins in diagnosis and therapy.     

μ‐Calpain regulates caspase‐dependent and apoptosis inducing factor‐mediated caspase‐independent apoptotic pathways in cisplatin‐induced apoptosis

Cisplatin, an effective anticancer agent, can induce tumor cell apoptosis via caspase‐dependent and‐independent pathways. However, the precise mechanism that regulates the pathways remains unclear. In this study, we showed that μ‐calpain mediated both caspase‐dependent and‐independent pathways during cisplatin‐induced apoptosis in human lung adenocarcinoma cells. After cisplatin treatment, calpain activation, as measured by a fluorescent substrate, was an early event, taking place well before apoptosis inducing factor (AIF) release and caspase‐9/‐3 activation. Confocal imaging of cells transfected with AIF‐GFP demonstrated that AIF release occurred about 9 hr after cisplatin treatment. The increase of μ‐calpain activity proved to be a crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples suppressed the endogenous μ‐calpain expression level, as well as cotreated with the calpain inhibitors, calpeptin and PD150606. Inhibition of μ‐calpain not only significantly reduced caspase‐9/‐3 activities but also completely blocked AIF redistribution. Our study also showed that endogenous mitochondrial μ‐calpain could directly induce the truncation and release of AIF, while caspases and cathepsins were not necessary for this process. In conclusion, the study demonstrated that activation of μ‐calpain played an essential role in regulating both caspase‐dependent and AIF‐mediated caspase‐independent apoptotic pathways in cisplatin‐induced apoptosis. © 2009 UICC     

Levetiracetam enhances p53-mediated MGMT inhibition and sensitizes glioblastoma cells to temozolomide

Antiepileptic drugs (AEDs) are frequently used to treat seizures in glioma patients. AEDs may have an unrecognized impact in modulating O⁶-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that has an important role in tumor cell resistance to alkylating agents. We report that levetiracetam (LEV) is the most potent MGMT inhibitor among several AEDs with diverse MGMT regulatory actions. In vitro, when used at concentrations within the human therapeutic range for seizure prophylaxis, LEV decreases MGMT protein and mRNA expression levels. Chromatin immunoprecipitation analysis reveals that LEV enhances p53 binding on the MGMT promoter by recruiting the mSin3A/histone deacetylase 1 (HDAC1) corepressor complex. However, LEV does not exert any MGMT inhibitory activity when the expression of either p53, mSin3A, or HDAC1 is abrogated. LEV inhibits malignant glioma cell proliferation and increases glioma cell sensitivity to the monofunctional alkylating agent temozolomide. In 4 newly diagnosed patients who had 2 craniotomies 7–14 days apart, prior to the initiation of any tumor-specific treatment, samples obtained before and after LEV treatment showed the inhibition of MGMT expression. Our results suggest that the choice of AED in patients with malignant gliomas may have an unrecognized impact in clinical practice and research trial design.     

Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway

Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Here we show for the first time that both enzalutamide and abiraterone render prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT.     

Adenosine 5'‐monophosphate‐activated protein kinase‐dependent mTOR pathway is involved in flavokawain B‐induced autophagy in thyroid cancer cells

Flavokawain B (FKB), a natural kava chalcone, shows potent antitumor activity in various types of cancer, although the mechanism of action remains unclear. In this study, we report that FKB has profound effects on the metabolic state of human thyroid cancer (TCa) cells, leading to high autophagy flux through upregulation of AMP‐activated protein kinase, which in turn inhibits mTOR and activates Beclin‐1 in TCa cells. We further report that the autophagy induced by FKB plays a prosurvival role in TCa cells both in vitro and in vivo. In conclusion, our findings provide evidence that combination treatment with FKB and pharmacological autophagy inhibitors will be a potential therapeutic strategy for the treatment of TCa.     

Targeting cancer stem‐like cells in glioblastoma and colorectal cancer through metabolic pathways

Cancer stem‐like cells (CSCs) are thought to be the main cause of tumor occurrence, progression and therapeutic resistance. Strong research efforts in the last decade have led to the development of several tailored approaches to target CSCs with some very promising clinical trials underway; however, until now no anti‐CSC therapy has been approved for clinical use. Given the recent improvement in our understanding of how onco‐proteins can manipulate cellular metabolic networks to promote tumorigenesis, cancer metabolism research may well lead to innovative strategies to identify novel regulators and downstream mediators of CSC maintenance. Interfering with distinct stages of CSC‐associated metabolics may elucidate novel, more efficient strategies to target this highly malignant cell population. Here recent discoveries regarding the metabolic properties attributed to CSCs in glioblastoma (GBM) and malignant colorectal cancer (CRC) were summarized. The association between stem cell markers, the response to hypoxia and other environmental stresses including therapeutic insults as well as developmentally conserved signaling pathways with alterations in cellular bioenergetic networks were also discussed. The recent developments in metabolic imaging to identify CSCs were also summarized. This summary should comprehensively update basic and clinical scientists on the metabolic traits of CSCs in GBM and malignant CRC.     

DNMT1 and DNMT3b silencing sensitizes human hepatoma cells to TRAIL‐mediated apoptosis via up‐regulation of TRAIL‐R2/DR5 and caspase‐8

DNA methylation plays a critical role in chromatin remodeling and gene expression. DNA methyltransferases (DNMTs) are hypothesized to mediate cellular DNA methylation status and gene expression during mammalian development and in malignant diseases. In this study, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b in cell proliferation and survival of hepatocellular carcinoma (HCC) cells. Gene silencing of both DNMT1 and DNMT3b by targeted siRNA knockdown reduces cell proliferation and sensitizes the cells to tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐mediated cell death. The proapoptotic protein caspase‐8 demonstrated promoter hypermethylation in HCC cells and was up‐regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. In addition, death receptor TRAIL‐R2/DR5 (TRAIL receptor 2/death receptor 5) did not exhibit promoter hypermethylation in HCC cells but was also up‐regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. Consistent with this observation, the combined transfection of DNMT1‐siRNA plus DNMT3b‐siRNA enhanced formation of the TRAIL‐death‐inducing signaling complex formation in HCC cells. In conclusion, our data suggest that DNA methylation of specific genomic regions maintained by DNMT1 and DNMT3b plays a critical role in survival of HCC cells, and a simultaneous knockdown of both DNMT1 and DNMT3b may be a novel anticancer strategy for the treatment of HCC. (Cancer Sci 2010)     

Bortezomib sensitizes human osteosarcoma cells to adriamycin‐induced apoptosis through ROS‐dependent activation of p‐eIF2α/ATF4/CHOP axis

Osteosarcoma is the most common bone cancer, and chemotherapy is currently indispensable for its treatment. Adriamycin has been claimed to be the most effective agent for osteosarcoma, however, the outcome of adriamycin chemotherapy remains unsatisfactory. Here, we reported a potent combination therapy that bortezomib, a proteasome inhibitor, enhances adriamycin‐induced apoptosis to eliminate osteosarcoma cells and we revealed that the activation of p‐eIF2α/ATF4/CHOP axis is the underlying associated mechanisms. First, we observed that bortezomib enhances adriamycin‐mediated inhibition of cell proliferation and enhances the apoptosis in osteosarcoma cell lines. Moreover, this drug combination produced more potent tumor‐growth inhibitory effects in human osteosarcoma cell line KHOS/NP xenografts. Our study showed that reactive oxygen species (ROS) plays an important role in apoptosis induced by adriamycin plus bortezomib, whereas ROS scavenger NAC could almost completely block the apoptosis induced by the combination treatment. Meanwhile, p‐eIF2α is remarkably elevated in the combination group. As a result, ATF4 exhibits strong activation which consequently induces the activation of CHOP and leads to the cell death. Finally, 13 primary osteosarcoma cells demonstrated potent response to the combination treatment. In a human osteosarcoma patient‐derived xenograft (PDX) model, our finding suggests that when combined with bortezomib, a relatively low dose of adriamycin produced more potent tumor‐growth inhibitory effects without increased toxicity. Thus, our findings not only provide a promising combination strategy to overcome osteosarcoma but also shed new light on the strategy of combining increased ROS and inhibited proteasome to open up new opportunities for the clinical development of chemotherapy regimens.     

Alpha fetoprotein is a novel protein‐binding partner for caspase‐3 and blocks the apoptotic signaling pathway in human hepatoma cells

Although there is increasing evidence that alpha fetoprotein (AFP) may function as regulatory factor in the growth of tumor cells, the precise mechanism is still unclear. In the current study, we investigated the role of the cytoplasmic AFP in caspase‐3‐mediated signaling of apoptosis. Our results showed that low doses of TNF‐related apoptosis‐inducing ligand (TRAIL) elevated the activity of caspase‐8, but not caspase‐3. Caspase‐3 colocalized and interacted with AFP in the cytoplasm of Bel 7402 cells, and translocated into nuclei in association with the occurrence of apoptosis while cells were under cotreatment with all‐trans retinoic acid (ATRA) or TRAIL. AFP was able to form complexes with caspase‐3 and block onward transmission of signaling from caspase‐8. Knockdown of AFP increased the sensitivity of Bel 7402 cells to TRAIL, and thereby, triggered caspase‐3 signaling. No intermolecule interaction occurred between AFP and caspase‐8, nor was caspase‐8 activity altered after AFP knockdown, demonstrating the selectivity of AFP in interfering with the apoptotic signaling pathway. The effect of AFP on caspase‐3 was further confirmed by transfection of the AFP gene into HLE cells (AFP negative). We conclude that ATRA or TRAIL resistance in AFP producing hepatoma is at least, in part, attributable to the high level of the cytoplasmic AFP. Therefore, it is possible that the combination of AFP gene silencing together with ATRA/TRAIL cotreatment will benefit the enhancement of the chemotherapeutic efficiency of these agents on tumors. © 2009 UICC     

Effect of AKT3 expression on MYC‐ and caspase‐8‐dependent apoptosis caused by polo‐like kinase inhibitors in HCT 116 cells

Polo‐like kinase (PLK) is a cell‐cycle regulator that is overexpressed in several cancer cell types. Polo‐like kinase is considered a novel target for cancer therapies, and several PLK inhibitors (PLKis), including BI 2536, BI 6727, and GSK461364, have been developed. In this study, we established five BI 2536‐resistant cell lines from human colorectal cancer HCT 116 cells, to explore the resistance mechanism and identify predictable biomarkers of PLKis. We showed that PLKi‐induced caspase‐8 activation was attenuated in the BI 2536‐resistant cell lines. We also showed that the expression of P‐glycoprotein (P‐GP) and AKT3 was upregulated, whereas that of MYC was downregulated in some BI 2536‐resistant cell lines. Expression of P‐GP conferred resistance to PLKis, and PLKi‐induced apoptosis was dependent on MYC and caspase‐8 in HCT 116 cells. We also showed for the first time that AKT3 suppressed BI 6727‐induced caspase‐8 activation and conferred resistance to PLKis. Collectively, these results indicate that MYC, caspase‐8, P‐GP, and AKT3 play critical roles in PLKi‐induced apoptosis. Therefore, they are candidate biomarkers of the pharmacological efficacy of PLKis.     

Inositol hexaphosphate downregulates both constitutive and ligand‐induced mitogenic and cell survival signaling,and causes caspase‐mediated apoptotic death of human prostate carcinoma PC‐3 cells

Constitutively active mitogenic and prosurvival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). Epidermal growth factor (EGF) and insulin‐like growth factor‐1 (IGF‐1) are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both mitogen‐activated protein kinase (MAPK)‐ and Akt‐mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF‐1)‐induced biological responses and associated signaling cascades in advanced and androgen‐independent human PCa PC‐3 cells. Treatment of PC‐3 cells with 2 mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC‐3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand‐induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs extra cellular signal‐regulated kinase 1/2 (ERK1/2), c‐Jun N‐terminal protein kinase (JNK1/2), and p38 under constitutive and ligand‐activated conditions. Under similar condition, IP6 also inhibited AP‐1 DNA‐binding activity and decreased nuclear levels of both phospho and total c‐Fos and c‐Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGF receptor (EGFR) or IGF‐1 receptor (IGF‐1R) pathway‐mediated sustained growth promoting and survival signaling cascades in advanced and androgen‐independent human PCa PC‐3 cells, which might have translational implications in advanced human PCa control and management. © 2009 Wiley‐Liss, Inc.     

Angiopoietin‐like protein 2 renders colorectal cancer cells resistant to chemotherapy by activating spleen tyrosine kinase–phosphoinositide 3‐kinase‐dependent anti‐apoptotic signaling

Angiopoietin‐like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis by activating tumor angiogenesis and tumor cell chemotaxis and invasiveness. However, it is unclear whether ANGPTL2 expression has an effect on tumor cell survival. Here, we explored that possibility by determining whether ANGPTL2 expression altered survival of human colorectal cancer cell lines treated with antineoplastic drugs. To do so, we generated SW480 cells expressing ANGPTL2 (SW480/ANGPTL2) and control (SW480/Ctrl) cells. Apoptosis induced by antineoplastic drug treatment was significantly decreased in SW480/ANGPTL2 compared to control cells. Expression of anti‐apoptotic BCL‐2 family genes was upregulated in SW480/ANGPTL2 compared to SW480/Ctrl cells. To assess signaling downstream of ANGPTL2 underlying this effect, we carried out RNA sequencing analysis of SW480/ANGPTL2 and SW480/Ctrl cells. That analysis, combined with in vitro experiments, indicated that Syk‐PI3K signaling induced expression of BCL‐2 family genes in SW480/ANGPTL2 cells. Furthermore, ANGPTL2 increased its own expression in a feedback loop by activating the spleen tyrosine kinase–nuclear factor of activated T cells (Syk–NFAT) pathway. Finally, we observed a correlation between higher ANGPTL2 expression in primary unresectable tumors from colorectal cancer patients who underwent chemotherapy with a lower objective response rate. These findings suggest that attenuating ANGPTL2 signaling in tumor cells may block tumor cell resistance to antineoplastic therapies.