HLA-class-I expression loss, tumor microenvironment and breast cancer prognosis.

Loss of HLA-class-I molecule expression by cancer cells is a frequent event in human tumors that may lead to immune evasion from cytotoxic T-cells. We examined the expression patterns of HLA-class-I molecules in a series of 175 patients with operable breast cancer (BCa). Extensive loss of BCa cell HLA-class-I expression was noted 76.6 % of patients (27.5 % complete loss). A significant association of HLA-preservation with high TIL-density (p = 0.001) was documented. Preservation of HLA was evident only in BCa carcinomas with low HIF1α expression and high TIL-density. Cell line experiments (MCF7 and T47D) showed that induction of HLAs in cancer cells following incubation with lymphocytes or IFNγ, was abrogated under hypoxic conditions. HLA-preservation was linked with better distant metastasis-free survival (p = 0.01), which was confirmed also in multivariate analysis (p = 0.02, HR 3.17). Studying the expression of HLA-class-I molecules in parallel with TIL-density and HIF1α expression may identify subgroups of BCa patients who would benefit from immunotherapy.

Irradiation-induced IFN-type-I pathway activation in prostate cancer cell lines.

The Interferon (ΙFN) Type-I pathway has an important role in the activation of an anti-tumor immune response. We investigated the effects of two different dose fractionations of radiation (3 daily 8 Gy fractions vs. one fraction of 20 Gy) on the activation of the Type-I IFN-pathway in three hormone-dependent (22Rv1) and independent (DU145, PC3), prostate cancer (PC) cell lines. Regardless of the dose schedules, radiation-induced the expression of IFN-stimulated genes in all PC cell lines, with a strong up-regulation of the IFI6v2 and IFI44 genes. In addition, strong up-regulation of the MX1 and MX2 genes was noted in the PC3 cell line. This effect was independent of the expression of IFNß, cGAS, or TREX1 levels. It is suggested that the RT-induced IFN type-I response could be exploited for the development of immuno-RT policies for localized and metastatic PC.

Hypoxia and acidity regulate immune checkpoint molecule and IFN-ß expression in non-small cell lung cancer cell lines.

PURPOSE: Non-small cell lung cancer (NSCLC) is one of the most lethal tumors in humans. Immunotherapy with immune checkpoint inhibitors (ICIs) has revolutionized the treatment of patients with advanced diseases. Tumor microenvironment conditions like hypoxia and low pH may compromise the efficacy of ICIs. MATERIALS AND METHODS: We report the effect of hypoxia and acidity on the expression levels of the major checkpoint molecules, namely PD-L1, CD80, and CD47, in the A549 and H1299 NSCLC cell lines. RESULTS: Hypoxia induces PD-L1 protein and mRNA expression, represses CD80 mRNA levels, and enhances IFNß protein expression. An opposite effect was noticed when cells were exposed to acidic conditions. Hypoxia-induced the CD47 molecule at protein and mRNA levels. It is concluded that hypoxia and acidity are important regulators of the expression of PD-L1 and CD80 immune checkpoint molecules. Acidity contributes to the suppression of the interferon type I pathway. CONCLUSIONS: These findings suggest that hypoxia and acidity assist cancer cells in the escape from immune surveillance through direct effects on cancer cells' ability to present immune checkpoint molecules and release type I interferons. Targeting hypoxia and acidity may enhance the activity of ICIs in NSCLC.

Tertiary Lymphoid Structures, Immune Response, and Prognostic Relevance in Non-Small Cell Lung Cancer.

We assessed the presence of 'tertiary lymphoid structures' (TLS) in a series of surgically treated non-small cell lung carcinomas (NSCLC). The TLS-density in the tumor periphery (pTLS) ranged from 0 to 1.8 (median 0.45), while in inner tumor areas (iTLS) ranged from 0 to 1.0 (median 0); (p < 0.0001). High pTLS-density was linked with early stage of the disease. Glycolysis-related enzyme expression (MCT1, Hexokinase 2) was linked with high pTLS-density (p < 0.05). High pTLS and iTLS densities were linked with better postoperative prognosis (p = 0.02 and p = 0.01, respectively). Assessment of TLS is a useful prognostic marker in NSCLC.

Effect of Arylazo Sulfones on DNA: Binding, Cleavage, Photocleavage, Molecular Docking Studies and Interaction with A375 Melanoma and Non-Cancer Cells.

A set of arylazo sulfones, known to undergo N-S bond cleavage upon light exposure, has been synthesized, and their activity in the dark and upon irradiation towards DNA has been investigated. Their interaction with calf-thymus DNA has been examined, and the significant affinity observed (most probably due to DNA intercalation) was analyzed by means of molecular docking "in silico" calculations that pointed out polar contacts, mainly via the sulfonyl moiety. Incubation with plasmid pBluescript KS II revealed DNA cleavage that has been studied over time and concentration. UV-A irradiation considerably improved DNA damage for most of the compounds, whereas under visible light the effect was slightly lower. Moving to in vitro experiments, irradiation was found to slightly enhance the death of the cells in the majority of the compounds. Naphthylazosulfone 1 showed photo-disruptive effect under UV-A irradiation (IC50 ~13 µΜ) followed by derivatives 14 and 17 (IC50 ~100 µΜ). Those compounds were irradiated in the presence of two non-cancer cell lines and were found equally toxic only upon irradiation and not in the dark. The temporal and spatial control of light, therefore, might provide a chance for these novel scaffolds to be useful for the development of phototoxic pharmaceuticals.

Hypoxia and anaerobic metabolism relate with immunologically cold breast cancer and poor prognosis.

PURPOSE: Hypoxia-Inducible Factor HIF1α and lactate dehydrogenase LDHA drive anaerobic tumor metabolism and define clinical aggressiveness. We investigated their expression in breast cancer and their role in immune response and prognosis of breast cancer. METHODS: Tissue material from 175 breast cancer patients treated in a prospective study were analyzed with immunohistochemistry for HIF1α and LDH5 expression, in parallel with the tumor-infiltrating lymphocyte TIL-density and tertiary lymphoid structure TLS-density. RESULTS: High LDH5 expression was noted in 48/175 tumors, and this was related to HIF1α overexpression (p < 0.0001), triple-negative TNBC histology (p = 0.01), poor disease-specific survival (p < 0.007), metastasis (p < 0.01), and locoregional recurrence (p = 0.03). High HIF1α expression, noted in 39/175 cases, was linked with low steroid receptor expression (p < 0.05), her2 overexpression (p = 0.01), poor survival (p < 0.04), and high metastasis rates (p < 0.004). High TIL-density in the invading tumor front (TILinv) was linked with low LDH5 and HIF expression (p < 0.0001) and better prognosis (p < 0.02). High TIL-density in inner tumor areas (TILinn) was significantly linked with TNBC. Multivariate analysis showed that PgR-status (p = 0.003, HR 2.99, 95% CI 1.4-6.0), TILinv (p = 0.02, HR 2.31, 95% CI 1.1-4.8), LDH5 (p = 0.01, HR 2.43, 95% CI 1.2-5.0), N-stage (p = 0.04, HR 2.42, 95% CI 1.0-5.8), T-stage (p = 0.04, HR 2.31, 95% CI 1.0-5.1), and her2 status (p = 0.05, HR 2.01, 95% CI 1.0-4.2) were independent variables defining death events. CONCLUSION: Overexpression of LDH5, an event directly related to HIF1α overexpression, characterizes a third of breast tumors, which is more frequent in TNBC. Both HIF1α and LDH5 define cold breast cancer microenvironment and poor prognosis. A rational is provided to study further whether metabolic manipulations targeting HIF and LDH5 may enhance the antitumor immune response in breast cancer.

Loss of HLA-class-I expression in non-small-cell lung cancer: Association with prognosis and anaerobic metabolism.

Cancer immuno-editing frequently leads to loss of HLA-class-I molecule (HLA) expression and impaired immune surveillance. We investigated the expression of HLAs in a series of operable non-small-cell lung carcinomas (NSCLCs). Complete loss and extensive loss of expression was noted in 41.5% and 23.4% of cases, respectively. Low CD8 + and FOXP3 + TIL-density was significantly associated with loss of HLAs (p < 0.05 and p = 0.003, respectively). High PD-L1 expression was linked with sustained expression of HLAs. A significant association of loss of HLA-expression with overexpression of LDH5 (p = 0.01) and marginally with HIF1α was recorded. Cell line experiments confirmed that hypoxia and acidity down-regulate the expression of HLAs. A direct association between HLAs and Beclin-1 expression was also noted (p = 0.01). Loss of HLA-expression was linked with poorer survival (p < 0.01), independent of stage. It is concluded that loss of HLA-class-I molecules is frequent in NSCLC and directly linked to micro-environmental hypoxia and acidity.

Postoperative hypofractionated-accelerated radiotherapy (HypoAR) for locally advanced rectal cancer.

BACKGROUND: despite the advances in preoperative hypofractionated-accelerated radiotherapy for patients with locally advanced rectal cancer, postoperative radiotherapy delivered with standard fractionation (46-50 Gy in 5 weeks) remains a standard adjuvant schedule. The role of hypofractionated-accelerated radiotherapy in a postoperative setting remains largely unexplored. METHODS: eighty-eight patients with rectal cancer infiltrating the rectal wall and/or having metastasis to the perirectal lymph nodes were treated with surgery followed by adjuvant chemotherapy and, subsequently, with hypofractionated-accelerated radiotherapy. Ten fractions of 3.4 Gy were delivered to the pelvis for 10 consecutive fractions, within 12 days. The follow-up of patients alive at the time of analysis ranges from 12-120 months (median 48). RESULTS: mild abdominal discomfort and diarrhoea were frequent, but medical medication was demanded in 14/88 (15.9%) of patients. The incidence of late toxicities was low; 4/88 (3.5%) patients complained for intermittent intestinal urgency. Locoregional recurrence occurred in 8/88 patients (9%). The 5-year locoregional relapse-free survival was achieved in 89.7% of patients, and this dropped to 84% in node-positive patients (P = 0.45). The 5-year disease-specific overall survival was 72.4%. Nodal involvement showed a trend to negatively affect prognosis (5-year overall survival 68.2 vs. 79.6%; P = 0.23). CONCLUSION: postoperative hypofractionated-accelerated radiotherapy has minimal early and late toxicity. The locoregional control and disease-specific survival rates are similar to the expected from conventional postoperative chemoradiotherapy. The 2.5-fold decrease of radiotherapy treatment time, reduction of waiting lists and the lower overall cost of radiotherapy are additional benefits associated with hypofractionated-accelerated radiotherapy.

Apalutamide radio-sensitisation of prostate cancer.

INTRODUCTION: The combination of radiotherapy with bicalutamide is the standard treatment of prostate cancer patients with high-risk or locally advanced disease. Whether new-generation anti-androgens, like apalutamide, can improve the radio-curability of these patients is an emerging challenge. MATERIALS AND METHODS: We comparatively examined the radio-sensitising activity of apalutamide and bicalutamide in hormone-sensitive (22Rv1) and hormone-resistant (PC3, DU145) prostate cancer cell lines. Experiments with xenografts were performed for the 22Rv1 cell line. RESULTS: Radiation dose-response viability and clonogenic assays showed that apalutamide had a stronger radio-sensitising activity for all three cell lines. Confocal imaging for γΗ2Αx showed similar DNA double-strand break repair kinetics for apalutamide and bicalutamide. No difference was noted in the apoptotic pathway. A striking cell death pattern involving nuclear karyorrhexis and cell pyknosis in the G1/S phase was exclusively noted when radiation was combined with apalutamide. In vivo experiments in SCID and R2G2 mice showed significantly higher efficacy of radiotherapy (2 and 4 Gy) when combined with apalutamide, resulting in extensive xenograft necrosis. CONCLUSIONS: In vitro and in vivo experiments support the superiority of apalutamide over bicalutamide in combination with radiotherapy in prostate cancer. Clinical studies are encouraged to show whether replacement of bicalutamide with apalutamide may improve the curability rates.

Successful Treatment of a Locally Recurrent and Metastatic Malignant Phyllodes Tumor with Accelerated Radiotherapy and Nab-Paclitaxel, Cisplatin, and Liposomal Doxorubicin Chemotherapy.

Phyllodes tumors are rare breast lesions of fibroepithelial origin. Malignant transformation with metastases is linked with poor prognosis. We present a case of a 62-year-old woman with a recurrent malignant phyllodes tumor of the breast and lung metastases. The patient was originally presented with a borderline phyllodes tumor (7.4 cm) of the left breast, treated with wide local excision. A year later, the patient returned with palpable left breast masses. On PET-CT, increased uptake of 18F-FDG by large breast tumors was evident. A right lung lesion of metastatic origin was also present. A simple left breast mastectomy was performed. Histopathological report described 2 malignant phyllodes tumors (7 cm and 6.5 cm). One month later, during the CT simulation for radiotherapy planning, encysted fluid in the chest wall and 2 additional pulmonary lesions of the right lung were identified, confirming progressive lung metastatic disease. Both the chest wall and the regional lymph node area were irradiated with hypofractionated and accelerated radiotherapy. Biweekly chemotherapy with albumin-bound paclitaxel, cisplatin, and liposomal doxorubicin was also prescribed at the start of radiotherapy for 12 cycles. At the end of chemotherapy, complete regression of lung metastases was achieved, and there was no evidence of local recurrence. Within 2 years of follow-up, the patient is free of disease and treatment-related toxicities. Accelerated hypofractionated radiotherapy is effective in the locoregional control of malignant phyllodes tumors. The combination of cisplatin with nab-paclitaxel and liposomal doxorubicin chemotherapy has acceptable toxicity and is highly effective in eradicating metastatic lesions.

Carbonic anhydrase 9 (CA9) expression in non-small-cell lung cancer: correlation with regulatory FOXP3+T-cell tumour stroma infiltration.

BACKGROUND: Low pH suppresses the proliferation and cytotoxic activity of CD8+ cytotoxic and natural killer lymphocytes. The hypoxia-regulated transmembrane protein, carbonic anhydrase CA9, converts carbon dioxide produced by the Krebs cycle to bicarbonate and protons that acidify the extracellular milieu. We examined whether CA9 is also involved in intratumoural immunosuppression pathways. METHODS: A series of 98 tissue samples of primary non-small-cell lung carcinomas (NSCLC) from patients treated with surgery were analysed for the expression of CA9 and programmed-death ligand PD-L1 by cancer cells, and of FOXP3 by tumour-infiltrating lymphocytes (TILs). RESULTS: There was no direct association of CA9 with PD-L1 expression or the density of TILs in the tumour stroma, but CA9 was directly related to the extent of FOXP3+ TIL density (p = 0.008). Double-stratification survival analysis showed that patients with high CA9 expression and low TIL score had significantly poorer survival compared with all other groups (p < 0.04). In a multivariate analysis stage (p < 0.0001, HR 1.95, 95% CI: 1.3-2.7), TIL score (p = 0.05, HR 0.55, 95% CI: 0.2-1.0) was an independent prognostic variable of death events. CA9 expression by cancer cells is associated significantly with FOXP3+ regulatory T-cell abundance in the tumour stroma of NSCLC. CONCLUSION: The study provides a basis for testing CA9 as a marker of resistance to immune-checkpoint inhibitors and as a therapeutic target to enhance the efficacy of immunotherapy.

FOXP3 infiltrating lymphocyte density and PD-L1 expression in operable non-small cell lung carcinoma.

Purpose/Aim: Regulatory FOXP3+ T-cells control the cytotoxic activity of effector cells and may have an essential role in the development of immune tolerance in cancer patients. Programed death ligand 1 PD-L1, expressed on cancer cell membranes also blocks the cytotoxic activity of PD1+ cytotoxic lymphocytes. Materials and Methods: We assessed the immunohistochemical detection of these immune-tolerance related markers in a series of 98 non-small cell lung carcinomas (NSCLC) treated with surgery. The Tumor Infiltration Lymphocyte TIL density (mean number per x400 optical field) and the percentage of FOXP3+ TILs were assessed. Results: PD-L1 expression was directly linked with the TIL density (p = 0.01) and with the extent of infiltration with FOXP3+ TILs, named as the FIL-score (p = 0.01). FIL-score was significantly higher in stage I disease (p = 0.04). IL6 expression was linked with high TIL-score. A low TIL-score, characterizing immune deficient tumors defined a significantly poorer prognosis subgroup of patients (p = 0.03). Stratification of these tumors according to the FIL-score showed that FOXP3 expression by TILs correlated with an even a poorer prognosis in univariate (p = 0.007; median survival 14 vs. 44 months, respectively) and in multivariate analysis (p = 0.01, hazard ratio 4.3). Conclusion: Tumor stroma infiltration by FOXP3+ Tregs is an early event in the progression of NSCLC. Low lymphocytic infiltration defines poor prognosis, which becomes worse when the small numbers of infiltrating lymphocytes characterizing these tumors contain FOXP3 + Tregs. Furthermore, the direct association of FOXP3+ Treg infiltration density with PD-L1 expression by cancer cells implies a co-ordinated immune-suppressive activity in NSCLC.

Angiogenic regeneration defines loco-regional recurrence following pre-operative radio-chemotherapy for rectal cancer: a pilot study.

Previous studies from our group have brought forward the concept of angiogenic regeneration during radiotherapy (RT), as a major cause of RT failure. This process was examined herein in rectal cancer patients undergoing preoperative chemo-radiotherapy. Out of 25 patients with stage II/III rectal adenocarcinoma, 15 had incomplete response (pIR) after preoperative chemo-radiotherapy. The MIB1 proliferation index, the vascular density (VD) assessed with the anti-CD31 antibody and the Hypoxia Inducible Factor HIF1α was assessed. High VD before RT was related with poor local relapse free survival LRFS (p = 0.04), in cases with pIR. Pre-RT values of MIB1 and of HIF1α were not related with LRFS. High MIB1 index and intensification of VD beyond pre-treatment levels in post-RT samples, features indicative of angiogenic regeneration, defined poor LRFS (p = 0.04 and p = 0.0008, respectively). Angiogenic regeneration is strongly related to failure of RT and surgery to control loco-regional disease in rectal cancer patients. Addition of anti-angiogenic agents in the preoperative chemo-radiotherapy regimens may prove beneficial in subgroups of patients.

SMER28 is a mTOR-independent small molecule enhancer of autophagy that protects mouse bone marrow and liver against radiotherapy.

Effective cytoprotectors that are selective for normal tissues could decrease radiotherapy and chemotherapy sequelae and facilitate the safe administration of higher radiation doses. This could improve the cure rates of radiotherapy for cancer patients. Autophagy is a cytoplasmic cellular process that is necessary for the clearance of damaged or aged proteins and organelles. It is a strong determinant of post-irradiation cell fate. In this study, we investigated the effect of the mTOR-independent small molecule enhancer of autophagy (SMER28) on mouse liver autophagy and post-irradiation recovery of mouse bone marrow and liver. SMER28 enhanced the autophagy flux and improved the survival of normal hepatocytes. This effect was specific for normal cells because SMER28 had no protective effect on hepatoma or other cancer cell line survival in vitro. In vivo subcutaneous administration of SMER28 protected mouse liver and bone marrow against radiation damage and facilitated survival of mice after lethal whole body or abdominal irradiation. These findings open a new field of research on autophagy-targeting radioprotectors with clinical applications in oncology, occupational, and space medicine.

Comparison of the effect of the antiandrogen apalutamide (ARN-509) versus bicalutamide on the androgen receptor pathway in prostate cancer cell lines.

Apalutamide (ARN-509) is an antiandrogen that binds selectively to androgen receptors (AR) and does not show antagonist-to-agonist switch like bicalutamide. We compared the activity of ARN versus bicalutamide on prostate cancer cell lines. The 22Rv1, PC3, and DU145 cell lines were used to study the effect of ARN and bicalutamide on the expression cytoplasmic/nuclear kinetics of AR, AR-V7 variant, phosphorylated AR, as well as the levels of the AR downstream proteins prostate-specific antigen and TMPRSS2, under exposure to testosterone and/or hypoxia. The effects on autophagic flux (LC3A, p62, TFEB, LAMP2a, cathepsin D) and cell metabolism-related enzymes (hypoxia-inducible factor 1α/2α, BNIP3, carbonic anhydrase 9, LDHA, PDH, PDH-kinase) were also studied. The 22Rv1 cell line responded to testosterone by increasing the nuclear entry of AR, AR-V7, and phosphorylated AR and by increasing the levels of prostate-specific antigen and TMPRSS2. This effect was strongly abrogated by ARN and to a clearly lower extent by bicalutamide at 10 µmol/l, both in normoxia and in hypoxia. ARN had a stronger antiproliferative effect than bicalutamide, which was prominent in the 22Rv1 hormone-responsive cell line, and completely repressed cell proliferation at a concentration of 100 µmol/l. No effect of testosterone or of antiandrogens on autophagy flux, hypoxia-related proteins, or metabolism enzyme levels was noted. The PC3 and DU145 cell lines showed poor expression of the proteins and were not responsive to testosterone. On the basis of in-vitro studies, evidence has been reported that ARN is more potent than bicalutamide in blocking the AR pathway in normoxia and in hypoxia. This reflects a more robust, dose-dependent, repressive effect on cell proliferation.

Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.

Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

Differential effect of hypoxia and acidity on lung cancer cell and fibroblast metabolism.

This study examined the metabolic response of lung cancer cells and normal lung fibroblasts to hypoxia and acidity. GLUT1 and HXKII mRNA/protein expression was up-regulated under hypoxia in the MRC5 fibroblasts and in the A549 and H1299 lung cancer cell lines, indicating intensified glucose absorption and glycolysis. Under hypoxia, the LDHA mRNA and LDH5 protein levels increased in the cancer cells but not in the fibroblasts. Acidity suppressed the above-mentioned hypoxia effect. PDH-kinase-1 (PDK1 mRNA and protein) and inactive phosphorylated-PDH protein levels were induced under hypoxia in the cancer cells, whereas these were reduced in the MRC5 lung fibroblasts. In human tissue sections, the prevalent expression patterns supported the contrasting metabolic behavior of cancer cells vs. tumor fibroblasts. The monocarboxylate/lactate transporter 1 (MCT1) was up-regulated in all the cell lines under hypoxic conditions, but it was suppressed under acidic conditions. The mitochondrial DNA (mtDNA) content per cell decreased significantly in the A549 cancer cell line under hypoxia, but it increased in the MRC5 fibroblasts. Taking into account these findings, we suggest that, under hypoxia, cancer cells intensify the anaerobic direction in glycolysis, while normal fibroblasts prefer to seek energy by intensifying the aerobic use of the available oxygen.

Blocking LDHA glycolytic pathway sensitizes glioblastoma cells to radiation and temozolomide.

PURPOSE: Up-regulation of lactate dehydrogenase LDHA, is a frequent event in human malignancies and relate to poor postoperative outcome. In the current study we examined the hypothesis that LDHA and anaerobic glycolysis, may contribute to the resistance of glioblastoma to radiotherapy and to temozolomide. METHODS AND MATERIALS: The expression of LDH5 isoenzyme (fully encoded by the LDHA gene) was assessed in human glioblastoma tissues. Experimental in vitro studies involved the T98 and U87 glioblastoma cell lines. Their sensitivity to radiotherapy and to temozolomide, following silencing of LDHA gene or following exposure to the LDHA chemical inhibitor 'oxamate' and to the glycolysis inhibitor '2-deoxy-d-glucose' (2DG), was studied. RESULTS: Glioblastoma tissues showed strong cytoplasmic and nuclear LDH5 expression in 0-90% (median 20%) of the neoplastic cells. T98 and U87 cell lines showed that blocking glycolysis, either with LDHA gene silencing or exposure to oxamate (30 mM) and blockage of glycolysis with 2DG (500 µM), results in enhanced radiation sensitivity, an effect that was more robust in the T98 radioresistant cell line. Furthermore, all three glycolysis targeting methods, significantly sensitized both cell lines to Temozolomide. CONCLUSIONS: The current study provides evidence that a large subgroup of human glioblastomas are highly glycolytic, and that inhibitors of glycolysis, like LDHA targeting agents, may prove of therapeutic importance by enhancing the efficacy of radiotherapy and temozolomide against this lethal disease.

Expression of enzymes related to glucose metabolism in non-small cell lung cancer and prognosis.

Purpose/Aim: Cancer cells are addicted to glycolytic anaerobic pathways, in presence or in absence of a functional Krebs' cycle (phenomenon Warburg). This metabolic predilection relies on both extracellular (impaired vascularization and oxygenation) and intracellular (oncogenic activation of genes) causes. MATERIALS AND METHODS: We investigated the expression and prognostic relevance of enzymes involved in the glucose absorption and metabolism, monocarboxylate transporter (MCT) expression, MCT1 and MCT2, pentose pathway (Glucose-6-phospahte dehydrogenase G6PD), glycogene synthesis (glycogene synthase GYS1), glycolysis (Hexokinase HXKII, phosphofructokinase PFK1, fructose biphosphate aldolase), fate of pyruvate (pyruvate dehydrogenase PDH, phosphorylated pPDH, PDH kinase PDK1, lactate dehydrogenase LDH5 and LDH1) and key Kreb's cycle enzymes (citrate synthase CSynth and isocitrate dehydrogenase IDH). RESULTS: A strong overexpression of the above enzymes/proteins was noted in a varying percentage of cases examined. An interesting significant correlation between the enzymes involved in glycolysis and with the LDH5 was noted. Adenocarcinomas expressed higher levels of GLUT1 and MCT2 compared to other subtypes. Stage (p = 0.0001), aldolase (p = 0.004), LDH5 (p = 0.008), GLUT2 (p = 0.008), MCT2 (p = 0.009), GSYS1 (p = 0.04), and GLUT1 (p = 0.05) were significantly related with poor disease specific overall survival. In multivariate analysis stage (p = 0.001), LDH5 (p = 0.04), pPDH (p = 0.04), and aldolase (p = 0.04) were independent prognostic variables. CONCLUSION: It is concluded that an orchestrated activation of glucose absorption and metabolism towards anaerobic pathways characterize the majority of NSCLC, and this phenotype is strongly linked with an aggressive clinical behavior. This glycolytic addiction of lung cancer cell is revealed as a key therapeutic target.

Therapeutic interactions of autophagy with radiation and temozolomide in glioblastoma: evidence and issues to resolve.

Glioblastoma is a unique model of non-metastasising disease that kills the vast majority of patients through local growth, despite surgery and local irradiation. Glioblastoma cells are resistant to apoptotic stimuli, and their death occurs through autophagy. This review aims to critically present our knowledge regarding the autophagic response of glioblastoma cells to radiation and temozolomide (TMZ) and to delineate eventual research directions to follow, in the quest of improving the curability of this incurable, as yet, disease. Radiation and TMZ interfere with the autophagic machinery, but whether cell response is driven to autophagy flux acceleration or blockage is disputable and may depend on both cell individuality and radiotherapy fractionation or TMZ schedules. Potent agents that block autophagy at an early phase of initiation or at a late phase of autolysosomal fusion are available aside to agents that induce functional autophagy, or even demethylating agents that may unblock the function of autophagy-initiating genes in a subset of tumours. All these create a maze, which if properly investigated can open new insights for the application of novel radio- and chemosensitising policies, exploiting the autophagic pathways that glioblastomas use to escape death.