Hypoxia inducible factor (HIF-1a and HIF-2a) expression in early esophageal cancer and response to photodynamic therapy and radiotherapy

Hypoxia inducible factor 1a and 2a (HIF-1a and HIF-2a) are key proteins regulating cellular response to hypoxia. Because the efficacy of photodynamic therapy (PDT) is dependent on the presence of oxygen, the assessment of HIF-1a and HIF-2a expression may be of value in predicting clinical response to PDT. Using recently produced MoAbs, we examined the expression of HIF1a and HIF2a in a series of 37 early-stage esophageal cancers treated with PDT and with additional radiotherapy in case of incomplete response after PDT. Strong expression of the HIF1a and of HIF2a proteins in all optical fields examined was noted in 51% and in 13% of cases, respectively. High expression was associated with a low complete response (CR) rate and with the absence of bcl-2 protein expression. On the contrary, bcl-2 expression was associated with a high CR rate. Combined analysis of HIF1a and bcl-2 protein expression revealed that of 16 cases with high HIF1a expression and the absence of bcl-2 reactivity, only 1 (7%) responded completely to PDT (P = 0.007). Bivariate analysis showed that HIF1a expression was independently related to response to PDT (P = 0.04; t ratio = 2.8), whereas bcl-2 approached significance (P = 0.07; t-ratio = 1.8). The final response to radiotherapy was high (70%) and independent of the HIF and bcl-2 status, which may be a result of reoxygenation after cellular depletion mediated by PDT. The present study suggests that assessment of HIF and of bcl-2 expression are important predictors of in vivo sensitivity to PDT. Modulation of PDT response with bioreductive drugs and/or drugs targeting bcl-2 (i.e., taxanes) may prove of significant therapeutic importance in a subgroup of patients with high HIF expression.     

The angiogenic "vascular endothelial growth factor/flk-1(KDR) receptor" pathway in patients with endometrial carcinoma: prognostic and therapeutic implications.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an important endothelial cell mitogen associated with increased angiogenesis and aggressive tumor behavior. Its stimulating effect on endothelial cells basically is dependent on the presence of specific VEGF receptors, such as the flk-1(KDR) receptor. This study investigates the roles of VEGF and of a functionally intact angiogenic pathway, "VEGF/flk-1(KDR)," in patients with endometrial carcinoma and their significance in prognosis and therapy. METHODS: A series of 121 endometrial carcinomas were studied. The expression of VEGF by endometrial tumor cells was assessed using the monoclonal antibody (MoAb) VG1. VEGF/KDR complexes on tumor endothelium or activated microvessel density (aMVD) were identified using the MoAb 11B5. In addition, the standard microvessel density (sMVD) was assessed with anti-CD31. In all tumors, the alkaline phosphatase/antialkaline phosphatase technique was employed. A Fisher exact test or an unpaired, two-tailed t test was used for testing correlations between categoric tumor variables, whereas a log-rank test was used to determine statistical differences between life tables. A Cox proportional hazards model was used to assess the effect of tumor variables on overall survival. RESULTS: Cytoplasmic VEGF expression in > 50% of tumor cells was associated significantly with aMVD (P < 0.0001) and with sMVD (P < 0.003). In univariate survival analysis, VEGF (P = 0.0002), aMVD (P = 0.001), and sMVD (P = 0.0009) were significant prognostic variables. Equally important were the histologic parameters tumor type (P = 0.03), tumor grade (P = 0.003), and disease stage (P < 0.0001). In multivariate analysis, disease stage was the most important independent prognostic factor (P < 0.0001), followed by VEGF/KDR (P < 0.01), and VEGF (P < 0.04). Furthermore, VEGF and VEGF/KDR were the only independent prognostic variables for patients with Stage I endometrioid adenocarcinoma. CONCLUSIONS: sMVD and the angiogenic factor VEGF are important indicators of a poor prognosis in patients with endometrial carcinoma. VEGF/KDR complexes define a subgroup of patients with endometrial carcinoma with an even worse prognosis.     

Thymidine phosphorylase expression in normal, hyperplastic and neoplastic prostates: correlation with tumour associated macrophages, infiltrating lymphocytes, and angiogenesis

Thymidine phosphorylase is an angiogenic factor primarily expressed by cancer cells, stromal cells and tumour-associated macrophages in many human malignancies. These different types of thymidine phosphorylase-expressing cells, however, may have a distinct place in the angiogenic process, and this question was addressed in the present study. A series of 20 normal/hyperplastic prostate glands and 60 prostate carcinomas was investigated by immunohistochemistry, using specific antibodies for thymidine phosphorylase (P-GF.44C), tumour-associated macrophages (CD68), endothelium (CD31) and prostate specific antigen (ER-PR8). Thymidine phosphorylase expression by normal and hyperplastic epithelial or stromal cells occurred almost exclusively in the context of an intense lymphocytic infiltrate. High thymidine phosphorylase cancer cells and thymidine phosphorylase stromal cells expression was associated with high angiogenesis in prostate carcinomas, and this significant association was extended to include both tumour-associated macrophages and tumour-infiltrating lymphocytes. Thymidine phosphorylase expression and tumour-infiltrating lymphocytes were related inversely with prostate specific antigen reactivity. In conclusion, thymidine phosphorylase is a major angiogenic factor in prostate carcinomas and its up-regulation is likely to occur in the context of a host immune response.     

Neutrophil extracellular traps promote differentiation and function of fibroblasts

Neutrophil activation by inflammatory stimuli and the release of extracellular chromatin structures (neutrophil extracellular traps – NETs) have been implicated in inflammatory disorders. Herein, we demonstrate that NETs released by neutrophils treated either with fibrosis‐related agents, such as cigarette smoke, magnesium silicate, bleomycin, or with generic NET inducers, such as phorbol 12‐myristate 13‐acetate, induced activation of lung fibroblasts (LFs) and differentiation into myofibroblast (MF) phenotype. Interestingly, the aforementioned agents or IL‐17 (a primary initiator of inflammation/fibrosis) had no direct effect on LF activation and differentiation. MFs treated with NETs demonstrated increased connective tissue growth factor expression, collagen production, and proliferation/migration. These fibrotic effects were significantly decreased after degradation of NETs with DNase1, heparin or myeloperoxidase inhibitor, indicating the key role of NET‐derived components in LF differentiation and function. Furthermore, IL‐17 was expressed in NETs and promoted the fibrotic activity of differentiated LFs but not their differentiation, suggesting that priming by DNA and histones is essential for IL‐17‐driven fibrosis. Additionally, autophagy was identified as the orchestrator of NET formation, as shown by inhibition studies using bafilomycin A1 or wortmannin. The above findings were further supported by the detection of NETs in close proximity to alpha‐smooth muscle actin (α‐SMA)‐expressing fibroblasts in biopsies from patients with fibrotic interstitial lung disease or from skin scar tissue. Together, these data suggest that both autophagy and NETs are involved not only in inflammation but also in the ensuing fibrosis and thus may represent potential therapeutic targets in human fibrotic diseases. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

LC3A-Positive Light Microscopy Detected Patterns of Autophagy and Prognosis in Operable Breast Carcinomas

Autophagy is a self-degradation mechanism by which cells recycle their own cytoplasmic constituents and dispose of excess or defective organelles after starvation and oxygen deprivation. An antibody to the microtubule-associated protein 1 light chain 3 (LC3A), recognizing both the soluble (LC3A-I) and the membrane-bound form (LC3A-II) of the protein, was used to detect autophagic activity in 102 breast carcinomas. Three distinct patterns were recognized: (1) diffuse cytoplasmic, (2) cytoplasmic/juxta-nuclear, and (3) “stone-like” pattern – dense, rounded, amorphous structures, 5 μm on average, typically enclosed within cytoplasmic vacuoles. The diffuse cytoplasmic pattern showed a direct association with estrogen and progesterone receptor expression. The juxta-nuclear pattern indicated a similar association with hormone receptors, an inverse association with tumor size, and a favorable prognosis. By contrast, an increased number of stone-like structures, probably representing an excessive autophagic response, was related to high-grade tumors and a less favorable outcome. Interestingly, 60 additional epithelial tumors of nonbreast origin disclosed identical autophagic patterns, and so did MDA231 breast cancer xenografts and HCT116 colon tumor spheroids (also analyzed by electron microscopy). Moreover, MCF-7 human breast cancer cell lines confirmed induction of LC3A by anoxia and Thapsigargin. It is concluded that autophagy can be readily recognized in breast carcinomas by light microscopy, after immunohistochemical staining with LC3A, but the significance of the various patterns expressed would need further evaluation.Cancer cells survive the adverse conditions of the extracellular milieu (i.e., hypoxia, nutrient deprivation, and reduced growth factors) through angiogenesis and anaerobic glycolysis.¹ Yet rapidly proliferating malignant neoplasms, having high metabolic demands, are not sufficiently supplied by these processes.^2,3,4,5,6 An alternative metabolic pathway for providing energy, when both oxygen and glucose are depleted, is autophagy—a self-degradation mechanism by which cells recycle their own cytoplasmic constituents and dispose of excess or defective organelles resulting in protein and ATP synthesis.^7,8,9 However, autophagy when prolonged can also cause cell death, but how the outcome is determined and the relationship of autophagy to clinical outcome is, at present, poorly understood.Angiogenesis and anaerobic glycolysis (i.e., the shifting from oxidative phosphorylation to anaerobic metabolism) have both been studied extensively in relation to malignant disease, including breast cancer.^{10,11,12,13,14,15} The activation of antiapoptotic pathways, as a complementary cell surviving mechanism, has also been considered in a number of studies.^16,17 By contrast, the phenomenon of autophagy only recently received attention for its biological significance in human malignancies. It is now known, for example, that under suboptimal microenvironmental conditions, cytoplasmic constituents are first entrapped by a membrane sac, called the isolation membrane, which subsequently closes to form double membrane structures, called autophagosomes or autophagic vacuoles. These, and specifically their outer membrane, are fused with lysosomes producing autolysosomes.^8,18 Lysosomal hydrolases degrade the cytoplasmic contents of the autophagosome, together with its inner membrane,^8,18 and the resulting macromolecules are recycled. Failure to accomplish this self-degradation process, probably because of overload of cytoplasmic components, enzyme exhaustion, or lysosomal enzyme defects, leads to accumulation of debris and massive destruction of tumor cells.^18,19,20,21Increased new blood vessel formation is associated with rapidly advancing tumors.^10,12,13 Equally, high levels of hypoxia are indicative of tumor aggressiveness.^11,22,23,24 Besides, an activated antiapoptotic pathway contributes to tumor growth and progression.²⁵ Tumor hypoxia and accelerated angiogenesis have both been used as markers of poor prognosis for a variety of tumors, including breast cancer.^{10,11,12,13,22,26} Nonetheless, the role of autophagy remains, by and large, obscure in this respect. Is it a favorable or an unfavorable prognostic indicator in human breast malignancies?This study was designed to investigate this question in a series of 102 operable breast carcinomas. However, rather than using electron or fluorescence microscopy, this study uses light microscopy after immunohistochemical staining with an antibody recognizing the autophagy-related protein Atg8 (microtubule associated protein 1 light chain 3 MAP1LC3A or LC3A), an essential component of the autophagic machinery.^27,28 LC3A-II is derived from a proLC3 30-KDa protein after cleavage by autophagin Atg4 to produce the active cytosolic form LC3A-I (18 kDa). After activation by Atg7, LC3A-I is transferred to Atg3 and subsequently converted into the membrane-bound form LC3A-II.^8,28 The latter localizes on the isolation membranes and the complete spherical autophagosomal and autolysosomal membranes, forming a suitable marker of autophagic activity.     

Lung cancer: a comparative study of metabolism related protein expression in cancer cells and tumor associated stroma

Immunohistochemical evaluation of lung carcinomas for key enzymes involved in cellular metabolism (lactate dehydrogenase LDH 1 and 5, pyruvate dehydrogenase PDH, pyruvate dehydrogenase kinase PDHK-1, monocarboxylate transporters MCT 1, 2 and 4, glucose transporter GLUT1, hypoxia inducible factors HIF1alpha and 2alpha) show a complementary metabolic profile between cancer cells and tumor-associated stroma. Cancer cells share enzyme/transporter activities suggestive of an anaerobic metabolism with high affinity for glucose absorption, anaerobic glycolysis and lactate extrusion. On the other hand, the tumour-associated fibroblasts express patterns involved in aerobic pathways and lactate oxidation. These findings bring forward the hypothesis that tumor associated stroma is an accomplice in tumor growth and survival sustaining an independent cellular and metabolic tumor domain. The development of agents exploiting such cancer specific metabolic pathways may prove of importance in the treatment of lung cancer.