Why some tumours trigger neovascularisation and others don't: the story thus far.

BACKGROUND: Angiogenesis is not essential for tumours to develop and expand, as cancer can also grow in a non-angiogenic fashion, but why this type of growth occurs is unknown. Surprisingly, our data from mRNA transcription profiling did not show any differences in the classical angiogenic pathways, but differences were observed in mitochondrial metabolic pathways, suggesting a key role for metabolic reprogramming. We then validated these results with mRNA profiling by investigating differential protein expression via immunohistochemistry in angiogenic and non-angiogenic non-small cell lung cancers (NSCLCs). METHODS: Immunohistochemical staining for 35 angiogenesis- and hypoxia-related biomarkers were performed on a collection of 194 angiogenic and 73 non-angiogenic NSCLCs arranged on tissue microarrays. Sequencing of P53 was performed with frozen tissue samples of NSCLC. RESULTS: The non-angiogenic tumours were distinguished from the angiogenic ones by having higher levels of proteins associated with ephrin pathways, mitochondria, cell biogenesis, and hypoxia-inducible factor 1 (HIF1) regulation by oxygen and transcription of HIF-controlled genes but lower levels of proteins involved in the stroma, cell-cell signaling and adhesion, integrins, and Delta-Notch and epidermal growth factor (EGF)-related signaling. However, proteins classically associated with angiogenesis were present in both types of tumours at very comparable levels. Cytoplasmic expression of P53 was strongly associated with non-angiogenic tumours. A pilot investigation showed that P53 mutations were observed in 32.0% of angiogenic cases but in 71.4% of non-angiogenic tumours. CONCLUSIONS: Our observations thus far indicate that both angiogenic and non-angiogenic tumours experience hypoxia/HIF and vascular endothelial growth factor (VEGF) pathway protein expression in a comparable fashion. However, angiogenesis does not ensue in the non-angiogenic tumours. Surprisingly, metabolic reprogramming seems to distinguish these two types of neoplastic growth. On the basis of these results, we raise the hypothesis that in some, but not in all cases, initial tissue remodeling and/or inflammation could be one of the secondary steps necessary to trigger angiogenesis. In the non-angiogenic tumours, in which neovascularisation fails to occur, HIF pathway activation could be the driving force toward metabolic reprogramming.

Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines.

LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

Increased expression of transcription factor EB (TFEB) is associated with autophagy, migratory phenotype and poor prognosis in non-small cell lung cancer.

OBJECTIVES: We investigated the role of lysosomal biogenesis and hydrolase activity in the clinical behavior and postoperative outcome of lung cancer. MATERIALS AND METHODS: Using immunohistochemistry we investigated the expression of the transcription factor EB (TFEB) which orchestrates lysosomal biogenesis, the lysosome membrane protein LAMP2a and of the lysosomal hydrolase cathepsin D in a series of 98 non-small cell lung carcinomas (NSCLC) treated with surgery alone. In vitro experiments with the A549 and H1299 lung cancer cell lines were also performed. RESULTS: Overexpression of TFEB, LAMP2a and Cathepsin D was noted in 47/98 (47.9%), 43/98 (43.9%) and 39/98 (39.8%) cases, respectively, and were significantly correlated with each other and with adenocarcinomas. High LAMP2a was related to high histology grade. Linear regression analysis confirmed significant association of TFEB with BNIP3 (p=0.0003, r=0.35) and LC3A with LAMP2a expression (p=0.0002, r=0.37). An inverse association of Cathepsin D expression with stone-like structures (SLS) was recorded (p=0.02, r=0.22). On univariate analysis all three lyososomal variables were associated with poor prognosis (p=0.05, 0.04 and 0.01, for TFEB, Cathepsin D and LAMP2a, respectively). Multivariate analysis showed that the SLS number (p=0.0001, HR5.37), Cathepsin D expression (p=0.01, HR=2.2) and stage (p=0.01, HR=1.5) were independent prognostic variables. Silencing of TFEB with siRNAs in the A549 and H1299 lung cancer cell lines did not affect proliferation but resulted in reduced migration ability. CONCLUSION: Lysosomal biogenesis is linked to autophagosomal protein expression in NSCLC and characterizes subgroups of high risk patients after complete surgical lung tumor resection.

Autophagy and lysosomal related protein expression patterns in human glioblastoma.

Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.

Proline-hydroxylated hypoxia-inducible factor 1α (HIF-1α) upregulation in human tumours.

The stabilisation of HIF-α is central to the transcriptional response of animals to hypoxia, regulating the expression of hundreds of genes including those involved in angiogenesis, metabolism and metastasis. HIF-α is degraded under normoxic conditions by proline hydroxylation, which allows for recognition and ubiquitination by the von-Hippel-Lindau (VHL) E3 ligase complex. The aim of our study was to investigate the posttranslational modification of HIF-1α in tumours, to assess whether there are additional mechanisms besides reduced hydroxylation leading to stability. To this end we optimised antibodies against the proline-hydroxylated forms of HIF-1α for use in formalin fixed paraffin embedded (FFPE) immunohistochemistry to assess effects in tumour cells in vivo. We found that HIF-1α proline-hydroxylated at both VHL binding sites (Pro402 and Pro564), was present in hypoxic regions of a wide range of tumours, tumour xenografts and in moderately hypoxic cells in vitro. Staining for hydroxylated HIF-1α can identify a subset of breast cancer patients with poorer prognosis and may be a better marker than total HIF-1α levels. The expression of unhydroxylated HIF-1α positively correlates with VHL in breast cancer suggesting that VHL may be rate-limiting for HIF degradation. Our conclusions are that the degradation of proline-hydroxylated HIF-1α may be rate-limited in tumours and therefore provides new insights into mechanisms of HIF upregulation. Persistence of proline-hydroxylated HIF-1α in perinecrotic areas suggests there is adequate oxygen to support prolyl hydroxylase domain (PHD) activity and proline-hydroxylated HIF-1α may be the predominant form associated with the poorer prognosis that higher levels of HIF-1α confer.

Blood vessel invasion and other variables as predictors of long-term survival in Japanese and British patients with primary invasive breast cancer.

This study was undertaken to investigate the associations of blood vessel invasion (BVI), lymphatic vessel invasion (LVI) or other variables and long-term survival in 173 Japanese and 184 British patients with primary invasive breast cancer, and whether they are associated with survival differences between Japanese and British patients. BVI was detected by objective methods, using both factor VIII-related antigen (F-VIII) staining and elastica van Gieson (E v G) staining. BVI was classified into three subtypes. 1) BVI e, BVI detected by E v G staining alone, 2) BVI f, BVI detected by F-VIII staining alone, 3) BVIef, BVI evaluated by combining BVIf and BVIe. LVI was also detected by objective methods, using lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) staining alone. There was a borderline significance between the frequencies for BVIef of British patients and those of Japanese patients (8.2% vs 3.5%; P = 0.06) but not for LVI (P = 0.36). British patients had a significantly worse relapse-free survival (RFS) and overall survival (OS) than Japanese patients (P < 0.01, P < 0.01, respectively) even though their tumors were smaller and more ER-positive with a similar prevalence of lymph-node involvement. LVI was not significantly associated with RFS and OS, however, BVIef positive tumors had a significantly worse RFS and OS compared with BVIef negative patients, after statistical adjustment for the other variables (P = 0.02, P = 0.01, respectively). The present study shows that BVIef variability might contribute to the Japanese and British disparities in breast cancer outcomes.

Vessel co-option in primary human tumors and metastases: an obstacle to effective anti-angiogenic treatment?

Angiogenesis has been regarded as essential for tumor growth and progression. Studies of many human tumors, however, suggest that their microcirculation may be provided by nonsprouting vessels and that a variety of tumors can grow and metastasize without angiogenesis. Vessel co-option, where tumor cells migrate along the preexisting vessels of the host organ, is regarded as an alternative tumor blood supply. Vessel co-option may occur in many malignancies, but so far mostly reported in highly vascularized tissues such as brain, lung, and liver. In primary and metastatic lung cancer and liver metastasis from different primary origins, as much as 10-30% of the tumors are reported to use this alternative blood supply. In addition, vessel co-option is introduced as a potential explanation of antiangiogenic drug resistance, although the impact of vessel co-option in this clinical setting is still to be further explored. In this review we discuss tumor vessel co-option with specific examples of vessel co-option in primary and secondary tumors and a consideration of the clinical implications of this alternative tumor blood supply.

Overexpression of LC3A autophagy protein in follicular and diffuse large B-cell lymphomas.

BACKGROUND AND OBJECTIVES: Autophagy is a self-degradation mechanism induced under stress conditions in all eukaryotic cells. Its activity in human lymphomas has not been studied as yet. METHODS: In this study, the autophagic activity of lymphoid cells was investigated in follicular lymphomas (FL; 48 cases), diffuse large B-cell lymphomas (DLBCL; 78 cases), and in reactive follicular hyperplasias (41 cases), using the light chain 3A (LC3A) antibody and a standard immunohistochemical technique. RESULTS: In all cases, the pattern of LC3A reactivity was uniformly diffuse cytoplasmic, but expressed more frequently in FLs (68.8%) than in DLBCLs (41%) (p=0.02), and much more commonly in DLBCLs than in reactive lymph nodes (24.3%) (p<0.006). Interestingly, FLs expressing LC3A in >10% of lymphoid cells (high reactivity) were associated with the hypoxia-related protein HIF1α and the enzyme of anaerobic metabolism lactate dehydrogenase LDH5 (p=0.004 and p=0.003, respectively). Such associations, however, were not a feature in DLBCLs of increased LC3A activity. CONCLUSIONS: LC3A expression in FLs is hypoxia-induced, whereas its expression in DLBCLs may be regulated by other molecular mechanisms. The current study provides a tool for further assessment of autophagic activity in translational and autophagy targeting therapy studies.

High DLL4 expression in tumour-associated vessels predicts for favorable radiotherapy outcome in locally advanced squamous cell head-neck cancer (HNSCC).

INTRODUCTION: Expression of the DLL4 (a notch pathway ligand) by tumor-associated endothelium is a postulated marker of vascular maturity and functionality. As vascular functionality is an important parameter defining chemotherapy and oxygen intra-tumoral distribution, we investigated the role of DLL4 expression in tumour vasculature in the efficacy of radio-chemotherapy for HNSCC patients. MATERIALS AND METHODS: Sixty-five biopsy specimens from HNSCC patients with inoperable disease were immunohistochemically examined using anti-CD31 (pan-endothelial cell marker) and anti-DLL4 antibodies and the vascular density (VD) was recorded. Patients were treated with platinum based hypofractionated accelerated conformal radiotherapy. The median follow-up period was 24 months (4-80 months). RESULTS: Using the 33rd and 66th percentiles cases were grouped in three categories of low, medium and high CD31+ or DLL4+ VD. The percentage of vessels expressing DLL4 (DLL4-ratio) ranged from 17 to 100 % (mean 71 %), showing substantial variation among cases. In accordance with previous published studies, a biphasic pattern of association of CD31+ VD with poor outcome was noted. Cases with a medium VD had a significantly better local relapse free survival (LRFS) compared to cases of high VD (p = 0.0005, HR 0.15) and of low VD (p = 0.02, HR 0.28). High DLL4/CD31 ratio defined improved LRFS in both these subgroups of poor prognosis. CONCLUSIONS: The expression of DLL4 is associated with reduced radio-resistance, presumably by reducing hypoxia and improving chemotherapy accessibility. Using the combination of CD31 and DLL4 staining, a classification is suggested so that HNSCCs are categorized in sub-groups to be targeted by different anti-angiogenic and hypoxia targeting agents.

CD31 angiogenesis and combined expression of HIF-1α and HIF-2α are prognostic in primary clear-cell renal cell carcinoma (CC-RCC), but HIFα transcriptional products are not: implications for antiangiogenic trials and HIFα biomarker studies in primary CC-RCC.

Hypoxia-inducible factors, HIF-1α and HIF-2α, are expressed in the majority of clear-cell renal cell carcinoma (CC-RCC). In vitro, HIFα isoforms regulate a differential set of genes, and their effects in vivo within CC-RCC tumours may affect outcome. The role of angiogenesis and HIFα transcriptional products, including those involved in cell metabolism and morphological dedifferentiation have not been extensively investigated and might have relevance to the development of antiangiogenic or anti-HIFα trials in primary CC-RCC, either before or after radical nephrectomy. We analysed 168 consecutive clear-cell renal tumours from 1983 to 1999 within tissue microarrays and assessed expression of HIF-1α and HIF-2α together with the protein expression of seven of their target genes (BNIP3, CA9, Cyclin D1, GLUT-1, LDH5, Oct-4 and VEGF). The expression of these factors was compared with patient overall survival and CD31 angiogenesis. We found that HIFα antigenicity deteriorated with the age of the paraffin block (P < 0.0001) and in tumours from 1983 to 1992 was deemed not to be reliable. Similar findings were found in aged archival osteosarcoma samples. This might have important implications for retrospective biomarker studies that rely on archival tissue material. HIF-1α(HIGH)/HIF-2α(LOW) tumours had a worse overall survival compared with HIF-1α(LOW)/HIF-2α(LOW) tumours (P = 0.04). Surprisingly, on multivariate analysis, high levels of CD31(+) angiogenesis was shown to be an independent prognostic marker of increased overall survival (P = 0.003). We propose that better differentiation of vascular endothelium may be a reflection of a greater production of vessel stabilization factors versus pro-angiogenic factors, and therefore a less aggressive phenotype.

Phosphorylated pVEGFR2/KDR receptor expression in uveal melanomas: relation with HIF2α and survival.

Hypoxia and its down-stream activated pathways are commonly involved in tumor progression. Genes involved in angiogenesis and glycolysis, i.e. vascular endothelial growth factor (VEGF) and lactase dehydrogenase A (LDHA), respectively, are transcriptionally controlled by the hypoxia inducible factors 1α and 2α (HIF1α and HIF2α). A series of 60 uveal melanomas were immunohistochemically assessed for the expression of VEGF and the phosphorylated/activated form of VEGF receptor 2 (pVEGFR2/KDR), after binding to VEGF. The expression of HIF1α, HIF2α and LDH5 was also investigated. Uveal melanomas overexpressing HIF2α (but not that of HIF1α) were significantly associated with high VEGF (P = 0.005), pVEGFR2/KDR (P < 0.0001) and LDH5 (P ≤ 0.0001). High LDH5 was linked with tumor necrosis (P = 0.01) and increased tumor size (P = 0.03). High VEGF was linked with phosphorylated pVEGFR2/KDR receptors. In univariate analysis high pVEGFR2/KDR receptor expression was significantly related with poor prognosis (P = 0.02). It is concluded that HIF2α plays an important role in the progression of uveal melanomas possibly by promoting the autocrine loop VEGF-pVEGFR2/KDR, and by enhancing the expression of LDHA gene, conferring thus a growth advantage. As pVEGFR2/KDR expression was significantly related with poor prognosis, inhibitors of this receptor may improve the clinical outcome of patients with pVEGFR2/KDR overexpressing uveal melanomas.

Prognostic and predictive role of lactate dehydrogenase 5 expression in colorectal cancer patients treated with PTK787/ZK 222584 (vatalanib) antiangiogenic therapy.

PURPOSE: The Colorectal Oral Novel therapy For the Inhibition of angiogenesis and Retarding of Metastases (CONFIRM)-randomized trials, investigating the role of the VEGF-receptor inhibitor PTK787/ZK 222584 (vatalanib) in colorectal cancer (FOLFOX 4 ± vatalanib), showed some benefit in patients with high serum lactate dehydrogenase (LDH) levels. Here, we investigated the expression of LDH5 (encoded entirely by the LDHA gene, regulated by the hypoxia inducible factors) in cancer tissues from patients recruited in the CONFIRM trials and relationship to response. EXPERIMENTAL DESIGN: Paraffin-embedded materials from 179 patients recruited in the CONFIRM trials were analyzed by immunohistochemistry for the expression of the LDH5 protein. Correlations with serum LDH, response, and survival were assessed. RESULTS: A significant association of tumor burden and of poor performance status (PS) with serum LDH was noted. Poor PS and high tumor LDH5 expression predicted for poor response rates. High tissue LDH5 was related to poor progression-free survival (PFS) only in the placebo group of patients, whereas the addition of vatalanib seemed to improved response and PFS in this subgroup. High serum LDH levels were linked with significantly poorer overall survival, which however was not sustained in multivariate analysis. CONCLUSIONS: Serum LDH and tissue LDH5 levels are complementary features that help to characterize the activity of LDH in colorectal cancer and have a potent value in predicting response to chemotherapy. The addition of vatalanib diminished the impact of LDH expression on the prognosis of patients.

Prognostic relevance of light chain 3 (LC3A) autophagy patterns in colorectal adenocarcinomas.

AIMS: The microtubule-associated protein 1 light chain 3 (LC3A) is an essential component of the autophagic vacuoles, forming a reliable marker of autophagic activity. In a previous study, the authors showed that LC3A immunohistochemistry renders three patterns of autophagic expression in breast carcinomas: diffuse cytoplasmic, perinuclear and 'stone-like' intracellular structures (SLS), each with a distinct prognostic relevance. METHODS: Tumour tissues from 155 patients with stage IIA-III colorectal adenocarcinomas, treated with surgery alone, were assessed immunohistochemically for LC3A. Median values were used as cut-off points to separate groups into low and high autophagic activity. Associations with prognosis and with lactate dehydrogenase-5 (LDH5) were sought. RESULTS: High SLS counts were associated with metastases and poor prognosis, while the prominence of the perinuclear pattern was linked to localised disease and good prognosis. The cytoplasmic pattern was irrelevant. Furthermore, patients with increased SLS numbers, but suppressed perinuclear expression, were associated with LDH5 overexpression and had an extremely poor prognosis (3-year survival 16.5%). The prognosis improved considerably when high SLS counts were accompanied by intense perinuclear expression (3-year survival 67%) and were optimal when SLS numbers dropped below median values, irrespective of perinuclear status (3-year survival 94-100%). Multivariate analysis showed that SLS and perinuclear patterns were independent predictors of death events. CONCLUSIONS: Perinuclear LC3A accumulation in colorectal tumour cells is a marker of good prognosis, presumably reflecting a basal autophagic activity. An abnormal or excessive autophagic response, as indicated by increased numbers of SLS, is linked to metastasis and poor prognosis.

Hypoxia and activated VEGF/receptor pathway in multiple myeloma.

BACKGROUND/AIM: Intensified angiogenic pathways are associated with poor prognosis and resistance of multiple myeloma (MM) cells to therapy. The links of the VEGF pathway with the hypoxia inducible factor (HIF) expression in MM are herein investigated. MATERIALS AND METHODS: The vascular density (VD) and the HIF/VEGF/VEGF-receptor expression in the bone marrows of 106 MM cases were studied using immunohistochemistry. RESULTS: HIF1alpha and HIF2alpha were expressed strongly in 33% and 13.2% of the cases, respectively. VEGFR and the phosphorylated (active) form of VEGFR2/KDR receptors were up-regulated in 42.5% and 36.8% of cases, respectively. Both HIF1alpha and HIF2alpha were significantly linked with high VD and VEGF expression. Moreover, the expression of the phosphorylated (active) form of VEGFR2/KDR was significantly linked with VEGF and HIF1alpha expression. The HIF/VEGF/VEGF-receptor pathway is up-regulated in approximately 40% of MM cases and linked with increased angiogenesis. Survival analysis in 37 evaluable patients showed a significantly worse prognosis in cases with high VD. CONCLUSION: HIFs and VEGF are up-regulated in a significant percentage of MM and are strongly related to each other. Targeting HIFs and the VEGF/receptor autocrine loop may prove of importance in the treatment of the disease.

The histone demethylase JMJD2B is regulated by estrogen receptor alpha and hypoxia, and is a key mediator of estrogen induced growth.

Estrogen receptor alpha (ERalpha) plays an important role in breast cancer. Upregulation of HIF-1alpha in ER(alpha)-positive cancers suggests that HIF-1alpha may cooperate with ERalpha to promote breast cancer progression and consequently affect breast cancer treatment. Here, we show the histone demethylase JMJD2B is regulated by both ERalpha and HIF-1alpha, drives breast cancer cell proliferation in normoxia and hypoxia, and epigenetically regulates the expression of cell cycle genes such as CCND1, CCNA1, and WEE1. We also show that JMJD2B and the hypoxia marker CA9 together stratify a subclass of breast cancer patients and predict a worse outcome of these breast cancers. Our findings provide a biological rationale to support the therapeutic targeting of histone demethylases in breast cancer patients.

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 microm 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.

Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer.

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.

Aberrant expression of the neuronal transcription factor FOXP2 in neoplastic plasma cells.

FOXP2 mutation causes a severe inherited speech and language defect, while the related transcription factors FOXP1, FOXP3 and FOXP4 are implicated in cancer. FOXP2 mRNA and protein expression were characterised in normal human tissues, haematological cell lines and multiple myeloma (MM) patients' samples. FOXP2 mRNA and protein were absent in mononuclear cells from different anatomical sites, lineages and stages of differentiation. However, FOXP2 mRNA and protein was detected in several lymphoma (8/20) and all MM-derived cell lines (n = 4). FOXP2 mRNA was expressed in bone marrow samples from 96% of MM patients (24/25), 66.7% of patients with the pre-neoplastic plasma cell proliferation monoclonal gammopathy of undetermined significance (MGUS) (6/9), but not in reactive plasma cells. The frequency of FOXP2 protein expression in CD138(+) plasma cells was significantly higher in MGUS (P = 0.0005; mean 46.4%) and MM patients (P < or = 0.0001; mean 57.3%) than in reactive marrows (mean 2.5%). FOXP2 (>10% nuclear positivity) was detectable in 90.2% of MM (55/61) and 90.9% of MGUS (10/11) patients, showing more frequent expression than CD56 and labelling 75% of CD56-negative MM (9/12). FOXP2 represents the first transcription factor whose expression consistently differentiates normal and abnormal plasma cells and FOXP2 target genes are implicated in MM pathogenesis.

Role of hypoxia-inducible factors in epigenetic regulation via histone demethylases.

Eukaryotic chromatin is subject to multiple posttranslational histone modifications such as acetylation, methylation, phosphorylation, and ubiquitination. These various covalent modifications have been proposed to constitute a "histone code," playing important roles in the establishment of global chromatin environments, transcription, DNA repair, and DNA replication. Among these modifications, histone methylation specifies regulatory marks that delineate transcriptionally active and inactive chromatin. These histone methyl marks were considered irreversible; however, recent identification of site-specific histone demethylases demonstrates that histone methylation is dynamically regulated, which may allow cells to rapidly change chromatin conformation to adapt to environmental stresses or intrinsic stimuli. Of major interest is the observation that these histone demethylase enzymes, which are in the Jumonji gene family, require oxygen to function and, in some cases, are induced by hypoxia in an HIFalpha-dependent manner. This provides a new mechanism for regulation of the response to hypoxia.