Evaluation of AKT phosphorylation and PTEN loss and their correlation with the resistance of rituximab in DLBCL

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma with quite high mortality. PTEN/PI3K/AKT signal pathway is constitutively activated and plays an oncogenic role in most tumors including non-Hodgkin’s lymphoma (NHL). Since rituximab used in chemotherapy has been proved to improve the survival of DLBCL patients, rituximab resistance is a common clinical challenge in the treatment of DLBCL. The aims of the present study are to determine the different levels of several important biomarkers of PTEN/PI3K/AKT pathway in DLBCL patients who are resistant or sensitive to rituximab treatment, and investigate the potential clinical application of these biomarkers. Methods: 48 patients with DLBCL who were treated by rituximab-based chemotherapy were divided into 2 groups according to their reactions to rituximab. The expression of p-AKT, PTEN, and Ki-67 protein in 48 DLBCL tissues were detected using immunohistochemistry and analyzed for the clinical pathological significance and the resistance to rituximab. Meanwhile, PTEN gene deletion was detected also by FISH, and mutation of PIK3CA was performed by sequencing analysis. Results: Activation of p-AKT in 12 of 48 (25.0%) and loss expression of PTEN in 15 of 48 (31.3%) DLBCL species were observed. P-AKT activation (P<0.05) and loss of PTEN expression (P<0.05) were significantly associative with high Ki-67 index. P-AKT and PTEN expression showed a significant negative correlation in all 48 DLBCL patients (r=-0.450, P<0.05), and the Spearman correlation coefficient in the resistant group (r=-0.769, P<0.05) was greater than in the sensitive group (r=-0.691, P<0.05). Conclusion: Regulation of PTEN/PI3K/AKT signal pathway participates in the progression of DLBCL, and may be involved in the development of the resistance to rituximab for some DLBCL patients.     

Expression and significance of leptin receptor,p-STAT3 and p-AKT in diffuse large B-cell lymphoma

We investigated the expression and clinical significance of leptin receptor (OBR), p-STAT3 and p-AKT in patients with diffuse large B-cell lymphoma (DLBCL) by immunohistochemical analysis. Immunohistochemistry revealed high expression of OBR, p-STAT3 and p-AKT in 45.0% (36/80), 28.8% (23/80) and 18.8% (15/80) cases of DLBCL, respectively, and minimal staining in 100% (20/20) cases of RLH (P < 0.05). Compared with GCB group, the non-GCB group had higher p-STAT3 expression rate (21/57 vs. 2/23, P < 0.01). The expression of OBR was positively related with that of p-STAT3 and p-AKT in DLBCL patients (P < 0.05). Our data suggest that OBR stimulates the JAK/STAT and PI3K/AKT signaling pathway and induces the phosphorylation of STAT3 and AKT. This may be involved in carcinogenesis and prognosis of DLBCL. The specific inhibitions could interfere in the combination of leptin with OBR and obstruct the JAK/STAT and PI3K/AKT signaling pathways, which could lead to new research and treatment strategies for DLBCL treatment.     

The B‐cell‐activating factor signalling pathway is associated with Helicobacter pylori independence in gastric mucosa‐associated lymphoid tissue lymphoma without t(11;18)(q21;q21)

We previously reported that activation of the B‐cell‐activating factor (BAFF) pathway upregulates nuclear factor‐κB (NF‐κB) and induces BCL3 and BCL10 nuclear translocation in Helicobacter pylori (HP)‐independent gastric diffuse large B‐cell lymphoma (DLBCL) tumours with evidence of mucosa‐associated lymphoid tissue (MALT). However, the significance of BAFF expression in HP independence of gastric low‐grade MALT lymphomas without t(11;18)(q21;q21) remains unexplored. Sixty‐four patients who underwent successful HP eradication for localized HP‐positive gastric MALT lymphomas without t(11;18)(q21;q21) were studied. BAFF expression was significantly higher in the HP‐independent group than in the HP‐dependent group [22/26 (84.6%) versus 8/38 (21.1%); p < 0.001]. Similarly, BAFF receptor (BAFF‐R) expression (p = 0.004) and nuclear BCL3 (p = 0.004), BCL10 (p < 0.001), NF‐κB (p65) (p = 0.001) and NF‐κB (p52) (p = 0.005) expression were closely correlated with the HP independence of these tumours. Moreover, BAFF overexpression was significantly associated with BAFF‐R expression and nuclear BCL3, BCL10, NF‐κB (p65) and NF‐κB (p52) expression. These findings were further validated in an independent cohort, including 40 HP‐dependent cases and 18 HP‐independent cases of gastric MALT lymphoma without t(11;18)(q21;q21). The biological significance of BAFF signalling in t(11;18)(q21;q21)‐negative lymphoma cells was further studied in two types of lymphoma B cell: OCI‐Ly3 [non‐germinal centre B‐cell origin DLBCL without t(11;18)(q21;q21) cell line] and MA‐1 [t(14;18)(q32;q21)/IGH‐MALT1‐positive DLBCL cell line]. In both cell lines, we found that BAFF activated the canonical NF‐κB and AKT pathways, and induced the formation of BCL10–BCL3 complexes, which translocated to the nucleus. BCL10 and BCL3 nuclear translocation and NF‐κB (p65) transactivation were inhibited by either LY294002 or by silencing BCL3 or BCL10 with small interfering RNA. BAFF also activated non‐canonical NF‐κB pathways (p52) through tumour necrosis factor receptor‐associated factor 3 degradation, NF‐κB‐inducing kinase accumulation, inhibitor of κB kinase (IKK) α/β phosphorylation and NF‐κB p100 processing in both cell lines. Our data indicate that the autocrine BAFF signal transduction pathway contributes to HP independence in gastric MALT lymphomas without the t(11;18)(q21;q21) translocation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

AKT activation predicts outcome in breast cancer patients treated with tamoxifen

Oestrogen receptor (ERalpha) expression is a strong predictor of response to endocrine therapy. The PI3K/AKT/mTOR signal transduction pathway has been implicated in endocrine resistance in vitro. The present study was carried out to test the hypothesis that AKT activation mediates tamoxifen resistance in clinical breast cancer. Immunohistochemistry (IHC) using AKT1-3, pan-AKT, pAKT (Thr-308), pAKT (Ser-473), pER (Ser-167), and pHER2 antibodies was performed on 402 ERalpha-positive breast carcinomas from patients treated with tamoxifen. High pAKT (Ser-473) activity (p = 0.0406) and low AKT2 expression (p = 0.0115) alone, or in combination [high pAKT (Ser-473)/low AKT2; 'high-risk' patient group] (p = 0.0014), predicted decreased overall survival in tamoxifen-treated patients with ERalpha-positive breast cancers. There was no significant association between tumour levels of AKT expression or activity and disease-free survival (DFS); however, the 'high-risk' patient group was significantly more likely to relapse (p = 0.0491). During tamoxifen treatment, neither AKT2 nor pAKT predicted DFS. Finally, activation of AKT, via phosphorylation, was linked to activation of both HER2 and ERalpha in this patient cohort. The data presented here show that the PI3K/AKT/mTOR pathway is associated with relapse and death in ERalpha-positive breast cancer patients treated with tamoxifen, supporting in vitro evidence that AKT mediates tamoxifen resistance. Patients with a 'high-risk' expression profile were at increased risk of death (hazard ratio 3.22, p = 0.002) relative to 'low-risk' patients, highlighting the potential that tumour profiling, with multiple IHC markers predictive of therapeutic response, may improve patient selection for endocrine therapies, eg tamoxifen or aromatase inhibitor-based treatments.     

The density of CD8+ T-cell infiltration and expression of BCL2 predicts outcome of primary diffuse large B-cell lymphoma of bone

Primary bone lymphoma (PBL) comprises 5 % of all extranodal non-Hodgkin's lymphomas (NHLs). Diffuse large B-cell lymphoma (DLBCL) accounts for the majority of cases, which is the most heterogeneous group of lymphomas. Previous studies suggested that besides the tumor cell phenotype, phosphatidylinositol 3-kinase/acutely transforming retrovirus/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway activity and the composition of the immune-microenvironment of DLBCL influence the clinical behavior of the disease. The aim of our study was to determine the relationship between clinical factors, tumor cell phenotype, microenvironment, PI3K/AKT/mTOR pathway activity, and disease outcome in primary bone diffuse large B-cell lymphoma (PB-DLBCL). We constructed tissue-microarrays from 41 cases of PB-DLBCL. To characterize tumor cell phenotype, T-cell subsets, macrophages, and PI3K/AKT/mTOR pathway activity immunohistochemical stainings were evaluated. Kaplan–Meier survival analysis provided evidence that age (≤65), CD3 and CD8+ T cell infiltrations >5 %, low BCL2 expression of the tumor cells (≤30 %), and low proliferation index (Ki67?≤?57 %) were associated with favorable outcome of PB-DLBCL patients. Multivariate analysis revealed that CD8+ T cell infiltration >5 % and low BCL2 expression (≤30 %) were independent predictors of survival. Increased macrophage infiltration (>10 %) showed tendency toward an adverse prognostic effect. International prognostic index, tumor cell phenotype (GCB or ABC), MYC protein expression, and activation of PI3K/AKT/mTOR pathway had no significant impact on survival. However, mTOR activity showed a significant correlation with activated B-cell phenotype. We conclude that CD8 and BCL2 expressions are potential prognostic markers for PB-DLBCL patients and the PI3K/AKT/mTOR pathway appears to be an additional therapeutic target in PB-DLBCL with activated-B-cell phenotype.     

Glycogen synthase kinase-3β inactivation is an intracellular marker and regulator for endotoxemic neutrophilia

Neutrophilia, defined as a large number of neutrophils in the circulating blood, is caused by increased differentiation and survival from activation-induced apoptosis. Regulation of apoptosis is essential for neutrophil homeostasis; however, the molecular signaling that regulates this process needs further investigation. Unlike TLR4 wild-type C3H/HeN mice, TLR4 mutated C3H/HeJ mice were insusceptible to LPS-induced blood neutrophilia. LPS prevented constitutive apoptosis in neutrophils and partly involved a blockade of the mitochondrial pathway including mitochondria transmembrane potential loss, myeloid cell leukemia sequence (Mcl) 1 degradation, and caspase-3 activation. In apoptotic neutrophils, glycogen synthase kinase (GSK)-3β was activated, and inhibiting GSK-3β decreased Mcl-1 degradation and apoptosis. LPS caused p38 MAPK-, JNK-, and PI3K/AKT-mediated Mcl-1 stabilization and prevented apoptosis, and LPS induced GSK-3β inactivation mainly through p38 MAPK and PI3K/AKT. Neutrophils in the neutrophilia showed increased GSK-3β inactivation and Mcl-1 stabilization accompanied by activation of p38 MAPK, JNK, and AKT. Notably, LPS-induced ROS generation can partly facilitate p38 MAPK/JNK/AKT activation to regulate GSK-3β-mediated Mcl-1 stability, apoptosis, and neutrophilia. These results demonstrate that the molecular basis of endotoxemic neutrophilia is through a direct action on neutrophils involving GSK-3β inactivation to prevent constitutive apoptosis.     

低强度脉冲超声通过PI3K/AKT/mTOR信号通路抑制骨肉瘤细胞的增殖

目的:阐述低强度脉冲超声（LIPUS）对骨肉瘤细胞增殖的调控作用和机制,探索低强度脉冲超声治疗骨肉瘤的潜在价值。方法:在时间梯度实验中,将MG-63细胞分为LIPUS组和Control组,对LIPUS组加载1、6、12、18和24 h的低强度脉冲超声,CCK-8检测各组细胞增殖活力,Western Blots检测24 h低强度脉冲超声对PI3K/AKT/mTOR信号通路的影响。在机制实验中,将MG-63细胞分为Control组、LY294002组、LIPUS组和LIPUS+740Y-P组,对各组施加对应的干预措施后使用CCK-8检测各组细胞增殖能力,Western Blots检测各组增殖相关蛋白PCNA和Cyclin D1的表达情况。所有数据重复3次后使用单因素方差分析统计。结果:加载低强度脉冲超声18和24 h后,LIPUS组的细胞增殖活力较对照组显著降低（P<0.010, P<0.001）,这些结果表明LIPUS可抑制MG-63细胞的增殖。LIPUS可抑制p-PI3K、p-AKT和p-mTOR的表达（P<0.001）,即抑制PI3K/AKT/mTOR信号通路的激活,而不影响PI3K、AKT和mTOR的表达。使用LIPUS就如同使用PI3K的抑制剂（LY294002）一样,可以抑制MG-63细胞的增殖（P<0.001）,在使用PI3K的激动剂740Y-P后,可逆转LIPUS对MG-63细胞增殖的抑制（P<0.001）。这些结果表明LIPUS可以通过抑制PI3K/AKT/mTOR信号通路的激活来抑制骨肉瘤细胞的增殖。结论:低强度脉冲超声通过PI3K/AKT/mTOR信号通路抑制骨肉瘤细胞的增殖,它可能是单独或联合放化疗治疗骨肉瘤的有效方法之一。     

Prognostic clinicopathologic factors, including immunologic expression in diffuse large B-cell lymphomas

The aim of this study was to assess the clinical significance and potential prognostic value of the expression of a panel of surface markers, proliferating, suppressor and oncogenic proteins in diffuse large B-cell lymphomas (DLBCL). Biopsies were collected from 158 patients with DLBCL and analyzed immunohistochemically for p53, p21/WAF1, bcl-2, cyclin-D1, bcl-6, mdr, CD5, CD30, epithelial membrane antigen (EMA), Ki-67 and c-myc positive tumor cells. Among these, 76 young and middle-aged patients (20-65 years) were selected to investigate the relationship between protein expression, clinical features, and survival. Survival analysis showed that advanced stage, high lactic dehydrogenase level, and high International Prognostic Index (IPI) were poor prognostic factors associated with a shorter overall survival (OS) and disease-free survival (DFS) times. A high p53 expression and low bcl-6 expression were associated with a shorter DFS time. The histological variant type, cyclin-D1+ CD5+ DLBCL, positive epithelial membrane antigen (EMA+) CD30- DLBCL, high bcl-2 expression, and low Ki-67 proliferation activity tended to be associated with worse survival, but the correlations were not statistically significant. In the multivariate analysis, the most significant factors were age, followed by IPI and last p53. The expression of p21/WAF1, mdr, and c-myc proteins did not influence OS and DFS. The expression of p53 and bcl-6 proteins may be useful prognostic indicators in DLBCL. Cyclin-D1+ CD5+ or EMA+ CD30- DLBCL tended to predict a worse survival and may probably bear a significant prognostic value worthy of consideration. Overall, clinical factors appeared to be more important than biologic parameters in determining the prognosis of diffuse large B-cell lymphomas.     

Prognostic significance of XIAP expression in DLBCL and effect of its inhibition on AKT signalling

The inhibitor of apoptosis protein (IAP) family member X‐linked inhibitor of apoptosis protein (XIAP) is essential for cell survival in lymphoma. However, the role of XIAP overexpression in diffuse large B‐cell lymphoma (DLBCL) is not fully elucidated. Therefore, we analysed the expression of XIAP protein and its clinicopathological correlation in a large cohort of DLBCLs by immunohistochemistry in a tissue micro‐array format. XIAP was found to be overexpressed in 55% of DLBCLs and significantly associated with poor clinical outcome (p = 0.0421). To further elucidate the role of XIAP in DLBCL and the inter‐relationship with PI3‐kinase/AKT signalling, we conducted several in vitro studies using a panel of DLBCL cell lines. We found that pharmacological inhibition of XIAP led to caspase‐dependent apoptosis in DLBCL cells. We also detected an inter‐relationship between XIAP expression and activated AKT in DLBCL cells that may explain cellular resistance to PI3‐kinase/AKT inhibition‐mediated apoptosis. Finally, this anti‐apoptotic effect was overcome by simultaneous pharmacological inhibition of XIAP and PI3‐kinase/AKT signalling leading to a more potent synergistically induced apoptosis. In summary, our data suggest that XIAP expression is a poor prognostic factor in DLBCL and the XIAP‐AKT relationship should be explored further as a potential therapeutic target in DLBCL. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Activation of the NF-kappaB signalling pathway in diffuse large B-cell lymphoma: clinical implications

Aim: To characterize the activation of the nuclear factor (NF)‐κB pathway in diffuse large B‐cell lymphoma (DLBCL) by immunohistochemistry. Methods and results: Sixty‐six DLBCLs treated with anthracycline‐containing chemotherapy were evaluated with antibodies against phosphorylated p65 (P‐p65), p65, p50, p52, IKKα, and phosphorylated IκB (P‐IκB). NF‐κB activation was based on the expression of P‐p65, P‐IκB, and nuclear expression of p65 or p52 in the tumour cells. P‐p65 and P‐IκB were expressed in 13 (20%) and 17 cases (26%), respectively. p65, p52 and IKKα were found in the cytoplasm. A correlation was found between expression of P‐p65 and P‐IκB (P < 0.0001), but not between the two subtypes of DLBCL [germinal centre B cell and non‐germinal centre (GC)]. P‐p65+ tumours showed a better response to chemotherapy (P = 0.025) and a trend to increased event‐free survival (P = 0.08). However, P‐IκB expression was not associated with either clinical response or survival. Bcl‐2 was not preferentially expressed on DLBCL tumours with NF‐κB activation, as determined by expression of P‐p65 and P‐IκB proteins. Conclusions: NF‐κB activation in DLBCL is preferentially mediated through the classical pathway and a novel mechanism involving phosphorylation of p65. Activation of NF‐κB by P‐p65 is associated with good prognosis. NF‐κB activation is not confined to non‐GC DLBCL exclusively.     

CAPG facilitates diffuse large B-cell lymphoma cell progression through PI3K/AKT signaling pathway

ObjectiveDiffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous tumor. Currently, macrophage-capping protein (CAPG) has been discovered to play a crucial part as a regulator in various cancers. However, it still remains unclear regarding its regulatory mechanism in DLBCL. Therefore, this study focused on revealing the mechanism underlying CAPG in DLBCL.MethodsThe bioinformatics analysis was conducted to predict the expression of CAPG in DLBCL and its downstream target genes. qRT-PCR was utilized to detect mRNA expression levels of CAPG and CEBPA. Western blot was performed to examine the expression levels of related proteins. Then we employed flow cytometry for the analysis of cell cycle and apoptosis. We also used MTT assay to measure cell survival and IHC assay to detect CAPG expression in DLBLC tissues. Then, ChIP was used to determine the binding of CEBPA and CAPG. For in vivo experiments, xenograft models were employed to investigate the effect of CAPG on DLBCL.ResultsHigh-level of CAPG expression was found in DLBCL cells and tissues. CAPG could strengthen the proliferative and invasive abilities of DLBCL cells, inhibit cell apoptosis, and activate PI3K/AKT signaling pathway. CAPG overexpression accelerated malignant progression of DLBCL cells. In addition, CAPG expression was regulated by CEBPA.ConclusionCAPG enhances the proliferation and invasion of DLBCL cells by promoting PI3K/AKT signaling pathway, which is expected to be a promising target for DLBCL.     

S-phase kinase protein 2 is an attractive therapeutic target in a subset of diffuse large B-cell lymphoma

S-phase kinase protein 2 (SKP2), an F-box protein, targets cell-cycle regulators including cycle-dependent kinase inhibitor p27KiP1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in a variety of cancer cells and has been implicated in oncogenesis; however, its role in diffuse large B-cell lymphoma (DLBCL) has not been elucidated. Therefore, we investigated the role of SKP2 and its ubiquitin-proteasome pathway in a large series (301) of DLBCL patient samples and a panel of DLBCL cell lines. Using immunohistochemistry, SKP2 was detected in 41.6% of DLBCL tumours and was inversely associated with p27Kip1 protein level. The DLBCL subset with high SKP2 and low p27Kip1 showed a strong correlation with the proliferating index marker Ki-67 (p < 0.0001) and also with the germinal centre phenotype (p = 0.0147). Treatment of DLBCL cell lines with bortezomib or expression of SKP2-specific siRNA causes down-regulation of SKP2 and accumulation of p27Kip1, leading to suppression of growth by inducing apoptosis. Furthermore, treatment of DLBCL cells with bortezomib causes apoptosis via involving the mitochondrial pathway and activation of caspases. Finally, treatment of DLBCL cells with bortezomib down-regulated the expression of XIAP, cIAP1, and survivin. Altogether, these results suggest that SKP2 and the ubiquitin-proteasome pathway may be a potential target for therapeutic intervention in DLBCL.     

Aberrant IGF1–PI3K/AKT/MTOR signaling pathway regulates the local immunity of oral lichen planus

ObjectivesOral lichen planus (OLP) is a T cell-mediated immune-related chronic disease, featured by accumulation of T cells and apoptosis of keratinocytes. Insulin-like growth factors 1 (IGF1) signaling, in combination with its downstream PI3K/AKT/MTOR cascade, plays pivotal roles in the regulation of inflammation and immune response. Meanwhile, TRB3 acts as a connective protein in the pathway. This study investigated the possible function of IGF1–PI3K/AKT/MTOR pathway in the local immunity of OLP.MethodsThe expression of phosphorylated IGF1R (p-IGF1R) and TRB3 in lesional tissues of OLP was measured. The effects of T cells pretreated with PI3K inhibitor LY294002, MTOR antagonist rapamycin and exogenous IGF1 on the cell proliferation and apoptosis, as well as supernatant inflammatory cytokine levels were detected in co-culture system of activated T cells and oral keratinocytes, respectively.ResultsThe expression of p-IGF1R and TRB3 in OLP lesions was significantly increased when compared with controls (P < 0.001). Rapamycin-treated T cells displayed enhanced apoptosis rate and promoted proliferation of their keratinocytes in the co-culture system. Notably, abnormal expression of IFN-γ and IL-4 were detected in supernatant of T cell alone and co-culture system in response to pharmacological modulators of IGF1–PI3K/MTOR pathway.ConclusionsThe aberrant IGF1–PI3K/AKT/MTOR signaling may participate in the immunoregulatory mechanism of OLP, via regulation on the crosstalk between T cells and keratinocytes, as well as imbalanced cytokine networks.     

Resistance exercise, but not endurance exercise, induces IKKβ phosphorylation in human skeletal muscle of training-accustomed individuals

The mammalian target of rapamycin complex 1 (mTORC1) is considered an important role in the muscular adaptations to exercise. It has been proposed that exercise-induced signaling to mTORC1 do not require classic growth factor PI3K/Akt signaling. Activation of IKKβ and the mitogen-activated protein kinases (MAPKs) Erk1/2 and p38 has been suggested to link inflammation and cellular stress to activation of mTORC1 through the tuberous sclerosis 1 (TSC1)/tuberous sclerosis 2 (TSC2) complex. Consequently, activation of these proteins constitutes potential alternative mechanisms of mTORC1 activation following exercise. Previously, we demonstrated that mTOR is preferentially activated in response to resistance exercise compared to endurance exercise in trained individuals without concomitant activation of Akt. In the present study, we extended this investigation by examining IκB kinase complex (IKK), TSC1, MAPK, and upstream Akt activators, along with gene expression of selected cytokines, in skeletal muscles from these subjects. Biopsies were sampled prior to, immediately after, and in the recovery period following resistance exercise, endurance exercise, and control interventions. The major finding was that IKKβ phosphorylation increased exclusively after resistance exercise. No changes in TSC1, Erk1/2, insulin receptor, or insulin receptor substrate 1 phosphorylation were observed in any of the groups, while p38 phosphorylation was higher in the resistance exercise group compared to both other groups immediately after the intervention. Resistance and endurance exercise increased IL6, IL8, and TNFα gene expression immediately after exercise. The non-exercise control group demonstrated that cytokine gene expression is also sensitive to repeated biopsy sampling, whereas no effect of repeated biopsy sampling on protein expression and phosphorylation was observed. In conclusion, resistance exercise, but not endurance exercise, increases IKKβ phosphorylation in trained human subjects, which support the idea that IKKβ can influence the activation of mTORC1 in human skeletal muscle.     

PI3KCA mutation status is of limited prognostic relevance in ER-positive breast cancer patients treated with hormone therapy

PI3K/AKT/mTOR pathway alterations are frequent in patients with infiltrating breast cancer (IBC). Their clinical and pathological relevance has been insufficiently documented. We evaluated PI3KCA for mutations and the expression of PTEN, AKT, mTOR and p70S6K by immunohistochemistry in 246 IBC patients treated with hormone therapy (median follow-up, 97 months). A PI3KCA mutation was observed in 50 out of 229 informative cases (21.8 %), PTEN loss in 107 out of 210 (51 %), moderate/high level of expression of AKT in 133 out of 188 (71 %), moderate/high level of expression of mTOR in 173 out of 218 (79 %) and moderate/high level of expression of p70S6K in 111 out of 192 cases (58 %). PI3KCA mutation was associated with the absence of Her2/neu amplification/overexpression and a low level of MIB1/Ki-67 labelling. The expression of p70S6K was associated with a high level of mTOR immunoreactivity, and high PTEN expression was associated with high AKT expression level. Univariate analysis showed that PI3KCA mutation status was not associated with clinical outcome in the series as a whole or in the node-negative subgroup. However, in the node-positive subgroup, exon 9 PI3KCA mutation was associated with unfavourable overall survival (OS), although its impact on the final model in multivariate analysis seemed to be limited. Of the other markers, only high p70S6K expression was associated with a significantly prolonged OS. PI3KCA mutation status is of limited prognostic relevance in oestrogen receptor-positive breast cancer patients treated with hormone therapy.     

p52 Activation in monomorphic B-cell posttransplant lymphoproliferative disorder/diffuse large B-cell lymphoma without BAFF-R expression

Posttransplantation lymphoproliferative disorders (PTLD) are associated with Epstein-Barr virus (EBV) and activate the NF-κB pathway. B-cell activating factor (BAFF) modulates cell growth and survival in non-Hodgkin's lymphomas. However, there are few studies of EBV, BAFF/BAFF-R signaling, and NF-κB1 and NF-κB2 pathway activation in PTLD. Diffuse large B-cell lymphomas (DLBCL) in two different clinical contexts, immunocompetent patients (DLBCL/IC; n = 30) or posttransplantation solid-organ recipients (DLBCL/PTLD; n = 21), were characterized histogenically as germinal center (GC) or non-germinal center (NGC). Expression of BAFF, BAFF-R, and NF-κB proteins p50 and p52 and the presence or absence of EBV were compared in these clinical contexts. Regardless of the GC or NGC pattern of DLBCL, BAFF-R was expressed in 37% of DLBCL/IC but in only 4.8% of DLBCL/PTLD. p52 was expressed in DLBCL/PTLD/NGC (12 of 19 cases) as compared with DLBCL/IC/NGC (0 of 18 cases). This pattern might be related to the presence of EBV and latent membrane protein 1 because p52 expression was observed primarily in EBV-positive DLBCL/PTLD cases expressing latent membrane protein 1. Thus, the activation profile or NGC pattern of DLBCL/PTLD was not associated with BAFF/BAFF-R expression, whereas nuclear p52 related to NF-κB2 pathway activation might be linked to EBV.     

Prognostic value of PIK3CA and phosphorylated AKT expression in ovarian cancer

Disrupted phosphatidylinositol 3-kinase (PI3K) activity and its effect on the downstream target AKT plays an important role in malignant diseases. Gain and/or amplification of PIK3CA gene, encoding the catalytic subunit of phosphatidylinositol 3-kinase (p110α) and its increased expression are associated with enhanced PI3K activity in ovarian cancer cell lines. In this study, ovarian carcinomas with documented clinical outcome were assessed for genetic aberrations at the 3q26.3 locus, including PIK3CA, by fluorescence in situ hybridization. PIK3CA amplification was evaluated by quantitative real-time PCR with respect to a control gene situated at 3q13. The expression of p110α, phosphorylated AKT (pAKT) and the proliferation marker Ki-67 were immunohistochemically investigated. PIK3CA amplification and Ki-67 index were strong predictors for an early tumour-associated death. p110α expression correlated with 3q26.3 gain and Ki-67 index but not with the patient outcome. No relationship could be observed between p110α and pAKT or between pAKT and disease outcome. It is interesting to note that cases with a nuclear pAKT immunoreactivity showed a trend of improved overall survival. Our results underline the prognostic significance of PIK3CA in ovarian carcinoma and argue against a simple linear model of PIK3CA gain/amplification followed by PI3K activation and consecutive AKT phosphorylation in ovarian carcinoma.     

Regulation of SHP2 by PTEN/AKT/GSK-3β signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer

Oncogenic activation accompanied by escape from immune surveillance, such as IFN-γ resistance, is critical for cancer cell growth and survival. In this study, we investigated the crosstalk signaling between IFN-γ resistance and signaling of hyperproliferation in gastric cancer cells. IFN-γ inhibited the cell growth of MKN45 cells but not hyperproliferating AGS cells. AGS cells did not respond to IFN-γ because of a decrease in STAT1 but not due to dysfunctional IFN-γ receptors. Signaling of PI3K/AKT, as well as MEK/ERK, was required for the hyperproliferation; notably, PI3K/AKT alone mediated the IFN-γ resistance. Aberrant Src homology-2 domain-containing phosphatase (SHP) 2 determined IFN-γ resistance but unexpectedly had no effects on hyperproliferation or ERK activation. In the IFN-γ resistant cells, inactivation of glycogen synthase kinase (GSK)-3β by PI3K/AKT was important for SHP2 activation but not for hyperproliferation. An imbalance of AKT/GSK-3β/SHP2 caused by a reduction of PTEN was important for the crosstalk between IFN-γ resistance and hyperproliferation. PI3K is constitutively expressed in AGS cells and immunohistochemical staining showed a correlation between hyperproliferation and expression of SHP2 and STAT1 in gastric tumors. These results demonstrate the effects of PTEN/AKT/GSK-3β/SHP2 signaling on IFN-γ resistance in hyperproliferating gastric cancer cells.     

Immunohistochemical analysis of cleaved caspase-3 detects high level of apoptosis frequently in diffuse large B-cell lymphomas of the central nervous system

The purpose of the present paper was to examine the level of apoptosis and the relationships among apoptosis, apoptosis-associated proteins, and proliferating potential in lymphoma tissues to clarify the characteristics of apoptosis in diffuse large B-cell lymphomas (DLBCL) of the central nervous system (CNS). The formalin-fixed, paraffin-embedded tissues of CNS and non-CNS DLBCL (20 cases each) were studied by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) and immunohistochemistry, using antibodies against single-stranded DNA (ssDNA), cleaved caspase-3, bcl-2, bax, p53, Fas and Ki-67. The cleaved caspase-3 immunohistochemistry detected apoptosis of the lymphoma cells most sensitively compared to TUNEL and ssDNA immunohistochemistry. High expression (grade + + or + + +) of cleaved caspase-3 was found more frequently in CNS DLBCL (11 cases, 55%) than non-CNS DLBCL (three cases, 15%; P = 0.009). Bax-positivity of lymphoma cells was increased in six cases of CNS DLBCL, which also showed high positivity of cleaved caspase-3. There was no significant correlation between the cleaved caspase-3-positivity and the Ki-67 positivity. The present study indicates that the number of apoptotic cells and expression level of cleaved caspase-3 were significantly higher in CNS DLBCL than non-CNS DLBCL, and that the correlation of bax and cleaved caspase-3 expression was often present in CNS DLBCL.