Comparative protein profiling identifies elongation factor-1beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis.

Parasites of the Leishmania Viannia subgenus are major causative agents of mucocutaneous leishmaniasis (MCL), a disease characterised by parasite dissemination (metastasis) from the original cutaneous lesion to form debilitating secondary lesions in the nasopharyngeal mucosa. We employed a protein profiling approach to identify potential metastasis factors in laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) through infrequently metastatic (M+/M-) to non-metastatic (M-). Comparison of the soluble proteomes of promastigotes by two-dimensional electrophoresis revealed two abundant protein spots specifically associated with M+ and M+/M- clones (Met2 and Met3) and two others exclusively expressed in M- parasites (Met1 and Met4). The association between clinical disease phenotype and differential expression of Met1-Met4 was less clear in L. Viannia strains from mucosal (M+) or cutaneous (M-) lesions of patients. Identification of Met1-Met4 by biological mass spectrometry (LC-ES-MS/MS) and bioinformatics revealed that M+ and M- clones express distinct acidic and neutral isoforms of both elongation factor-1 subunit beta (EF-1beta) and cytosolic tryparedoxin peroxidase (TXNPx). This interchange of isoforms may relate to the mechanisms by which the activities of EF-1beta and TXNPx are modulated, and/or differential post-translational modification of the gene product(s). The multiple metabolic functions of EF-1 and TXNPx support the plausibility of their participation in parasite survival and persistence and thereby, metastatic disease. Both polypeptides are active in resistance to chemical and oxidant stress, providing a basis for further elucidation of the importance of antioxidant defence in the pathogenesis underlying MCL.

Integrated Analysis of High-Resolution Gene Expression and Copy Number Profiling Identified Biallelic Deletion of CDKN2A/2B Tumor Suppressor Locus As the Most Frequent and Unique Genomic Abnormality in Diffuse Large B-Cell Lymphoma (DLBCL) with Strong Prognostic Value in Both GCB and ABC Subtypes and Not Overcome by a Dose-Intensive Immunochemotherapy Regimen Plus Rituximab. Results of a Prospective GELA Clinical Trial Program

Background and aim of the study: Genomic gains and losses play a crucial role in the development and progression of DLBCL and are closely related to gene expression profiles (GEP), including the germinal center B-cell like (GCB) and activated B-cell like (ABC) cell of origin (COO) molecular signatures. To identify new oncogenes or tumor suppressor genes (TSG) involved in DLBCL pathogenesis and to determine their prognostic values, an integrated analysis of high-resolution gene expression and copy number profiling was performed. Patients and methods: Two hundred and eight adult patients with de novo CD20+ DLBCL enrolled in the prospective multicentric randomized LNH-03 GELA trials (LNH03-1B, -2B, -3B, 39B, -5B, -6B, -7B) with available frozen tumour samples, centralized reviewing and adequate DNA/RNA quality were selected. 116 patients were treated by Rituximab(R)-CHOP/R-miniCHOP and 92 patients were treated by the high dose (R)-ACVBP regimen dedicated to patients younger than 60 years (y) in frontline. Tumour samples were simultaneously analysed by high resolution comparative genomic hybridization (CGH, Agilent, 144K) and gene expression arrays (Affymetrix, U133+2). Minimal common regions (MCR), as defined by segments that affect the same chromosomal region in different cases, were delineated. Gene expression and MCR data sets were merged using Gene expression and dosage integrator algorithm (GEDI, Lenz et al. PNAS 2008) to identify new potential driver genes. Results: A total of 1363 recurrent (defined by a penetrance > 5%) MCRs within the DLBCL data set, ranging in size from 386 bp, affecting a single gene, to more than 24 Mb were identified by CGH. Of these MCRs, 756 (55%) showed a significant association with gene expression: 396 (59%) gains, 354 (52%) single-copy deletions, and 6 (67%) homozygous deletions. By this integrated approach, in addition to previously reported genes (CDKN2A/2B, PTEN, DLEU2, TNFAIP3, B2M, CD58, TNFRSF14, FOXP1, REL...), several genes targeted by gene copy abnormalities with a dosage effect and potential physiopathological impact were identified, including genes with TSG activity involved in cell cycle (HACE1, CDKN2C) immune response (CD68, CD177, CD70, TNFSF9, IRAK2), DNA integrity (XRCC2, BRCA1, NCOR1, NF1, FHIT) or oncogenic functions (CD79b, PTPRT, MALT1, AUTS2, MCL1, PTTG1...) with distinct distribution according to COO signature. The CDKN2A/2B tumor suppressor locus (9p21) was deleted homozygously in 27% of cases and hemizygously in 9% of cases. Biallelic loss was observed in 49% of ABC DLBCL and in 10% of GCB DLBCL. This deletion was strongly correlated to age and associated to a limited number of additional genetic abnormalities including trisomy 3, 18 and short gains/losses of Chr. 1, 2, 19 regions (FDR < 0.01), allowing to identify genes that may have synergistic effects with CDKN2A/2B inactivation. With a median follow-up of 42.9 months, only CDKN2A/2B biallelic deletion strongly correlates (FDR p.value < 0.01) to a poor outcome in the entire cohort (4y PFS = 44% [32-61] respectively vs. 74% [66-82] for patients in germline configuration; 4y OS = 53% [39-72] vs 83% [76-90]). In a Cox proportional hazard prediction of the PFS, CDKN2A/2B deletion remains predictive (HR = 1.9 [1.1-3.2], p = 0.02) when combined with IPI (HR = 2.4 [1.4-4.1], p = 0.001) and GCB status (HR = 1.3 [0.8-2.3], p = 0.31). This difference remains predictive in the subgroup of patients treated by R-CHOP (4y PFS = 43% [29-63] vs. 66% [55-78], p=0.02), in patients treated by R-ACVBP (4y PFS = 49% [28-84] vs. 83% [74-92], p=0.003), and in GCB (4y PFS = 50% [27-93] vs. 81% [73-90], p=0.02), or ABC/unclassified (5y PFS = 42% [28-61] vs. 67% [55-82] p = 0.009) molecular subtypes (Figure 1). Conclusion: We report for the first time an integrated genetic analysis of a large cohort of DLBCL patients included in a prospective multicentric clinical trial program allowing identifying new potential driver genes with pathogenic impact. However CDKN2A/2B deletion constitutes the strongest and unique prognostic factor of chemoresistance to R-CHOP, regardless the COO signature, which is not overcome by a more intensified immunochemotherapy. Patients displaying this frequent genomic abnormality warrant new and dedicated therapeutic approaches.

Frequencies of circulating MDSC correlate with clinical outcome of melanoma patients treated with ipilimumab.

Metastatic melanoma has a poor prognosis with high resistance to chemotherapy and radiation. Recently, the anti-CTLA-4 antibody ipilimumab has demonstrated clinical efficacy, being the first agent to significantly prolong the overall survival of inoperable stage III/IV melanoma patients. A major aim of patient immune monitoring is the identification of biomarkers that predict clinical outcome. We studied circulating myeloid-derived suppressor cells (MDSC) in ipilimumab-treated patients to detect alterations in the myeloid cell compartment and possible correlations with clinical outcome. Lin(-) CD14(+) HLA-DR(-) monocytic MDSC were enriched in peripheral blood of melanoma patients compared to healthy donors (HD). Tumor resection did not significantly alter MDSC frequencies. During ipilimumab treatment, MDSC frequencies did not change significantly compared to baseline levels. We observed high inter-patient differences. MDSC frequencies in ipilimumab-treated patients were independent of baseline serum lactate dehydrogenase levels but tended to increase in patients with severe metastatic disease (M1c) compared to patients with metastases in skin or lymph nodes only (M1a), who had frequencies comparable to HD. Interestingly, clinical responders to ipilimumab therapy showed significantly less lin(-) CD14(+) HLA-DR(-) cells as compared to non-responders. The data suggest that the frequency of monocytic MDSC may be used as predictive marker of response, as low frequencies identify patients more likely benefitting from ipilimumab treatment. Prospective clinical trials assessing MDSC frequencies as potential biomarkers are warranted to validate these observations.

The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1.

The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis.

Brain metastasis: opportunity for drug development?

PURPOSE OF REVIEW: Brain metastases are a common clinical problem, and only limited treatment options exist. We review recent advances in medical brain metastasis research with a focus on the most common tumor types associated with secondary brain colonization: melanoma, breast cancer and lung cancer. We speculate on opportunities for drug development in patients with brain metastases, both as a treatment of established disease and as an adjuvant and prophylactic strategy. RECENT FINDINGS: BRAF inhibitors and the immunomodulatory anticytotoxic T-lymphocyte-associated antigen 4 antibody ipilimumab have shown clinically meaningful activity in melanoma patients with brain metastases. In breast cancer, current studies on drug treatment of brain metastases are mainly focusing on human epidermal growth factor receptor 2 targeting agents such as lapatinib. Emerging data seem to implicate a potential role of targeted agents including antiangiogenic compounds, pazopanib, and epithelial growth factor receptor inhibitors for prevention of brain metastasis formation in breast cancer or nonsmall cell lung cancer. SUMMARY: Novel drugs are beginning to enter clinical practice for selected patients with brain metastases. The promising findings from recent studies may fuel more research on brain metastases and their optimal drug treatment.

Inhibition of cell migration and invasion mediated by the TAT-RasGAP317-326 peptide requires the DLC1 tumor suppressor.

TAT-RasGAP317-326, a peptide corresponding to the 317-326 sequence of p120 RasGAP coupled with a cell-permeable TAT-derived peptide, sensitizes the death response of various tumor cells to several anticancer treatments. We now report that this peptide is also able to increase cell adherence, prevent cell migration and inhibit matrix invasion. This is accompanied by a marked modification of the actin cytoskeleton and focal adhesion redistribution. Interestingly, integrins and the small Rho GTP-binding protein, which are well-characterized proteins modulating actin fibers, adhesion and migration, do not appear to be required for the pro-adhesive properties of TAT-RasGAP317-326. In contrast, deleted in liver cancer-1, a tumor suppressor protein, the expression of which is often deregulated in cancer cells, was found to be required for TAT-RasGAP317-326 to promote cell adherence and inhibit migration. These results show that TAT-RasGAP317-326, besides its ability to favor tumor cell death, hampers cell migration and invasion.

Radiation-induced modifications of the tumor microenvironment promote metastasis.

Radiotherapy is successfully used to treat cancer. Emerging evidence, however, indicates that recurrences after radiotherapy are associated with increased local invasion, metastatic spreading and poor prognosis. Radiation-induced modifications of the tumor microenvironment have been proposed to contribute to increased aggressive tumor behavior, an effect also referred to as tumor bed effect, but the putative mechanisms involved have remained largely elusive. We have recently demonstrated that irradiation of the prospective tumor stroma impairs de novo angiogenesis through sustained inhibition of proliferation, migration and sprouting of endothelial cells. Experimental tumors growing within a pre-irradiated field have reduced tumor angiogenesis and tumor growth, increased hypoxia, necrosis, local invasion and distant metastasis. Mechanisms of progression involve adaptation of tumor cells to local hypoxic conditions as well as selection of cells with invasive and metastatic capacities. The matricellular protein CYR61 and integrin αVβ5 emerged as molecules that cooperate to mediate lung metastasis. Cilengitide, a small molecular inhibitor of αV integrins prevented lung metastasis formation. These results represent a conceptual advance to the understanding of the tumor bed effect and indicate that αV integrin inhibition might be a potential therapeutic approach for preventing metastasis in patients at risk for post-radiation recurrences.

MAP/ERK kinase kinase 1 (MEKK1) mediates transcriptional repression by interacting with polycystic kidney disease-1 (PKD1) promoter-bound p53 tumor suppressor protein.

Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.

Molecular pathways: emerging pathways mediating growth, invasion, and metastasis of tumors progressing in an irradiated microenvironment.

Radiotherapy is a well-established therapeutic modality in oncology. It provides survival benefits in several different cancer types. However, cancers relapsing after radiotherapy often develop into more aggressive conditions that are difficult to treat and are associated with poor prognosis. Cumulative experimental evidence indicates that the irradiated tumor bed contributes to such aggressive behavior. The involved mechanisms have for long remained elusive. Recent progress in the field revealed previously unrecognized cellular and molecular events promoting growth, invasion, and metastasis of tumors progressing in an irradiated microenvironment. Cellular mechanisms include inhibition of sprouting angiogenesis, formation of hypoxia, activation and differentiation of stromal cells, and recruitment of bone marrow-derived cells with vasculogenic and prometastatic activities. Identified pathways include TGF-β/ALK5, CXCL12/CXCR4, KITL/KIT, and CYR61/αVβ5 integrin. The availability of pharmacologic inhibitors impinging on these pathways opens novel opportunities for translational and clinical studies. These experimental results and ongoing work highlight the importance of the irradiated microenvironment in modulating the tumor response to radiotherapy and open new opportunities for the development of novel therapeutic strategies for patients with cancer who relapse after radiotherapy. Here, we review and discuss recent advances in the field and their translational and therapeutic implications to human cancer treatment.

Evaluating bone marrow metastasis of neuroblastoma with iodine-123-MIBG scintigraphy and MRI.

Of 10 patients with neuroblastoma who had both 123I-MIBG scintigraphy and MRI at diagnosis, four presented with bone marrow metastasis that was diagnosed by both imaging modalities and confirmed by bone marrow biopsy and smears. This report focuses on the follow up of the four patients with bone marrow metastasis. MIBG scintigraphy and MRI were concordant in two patients, a case of normalization and a case of relapse in the seventh dorsal vertebra confirmed by surgical biopsy. The last two patients presented a normalized MIBG scan for marrow infiltration after chemotherapy but persistent abnormal MRI signal of several vertebrae, suggesting marrow infiltration, up to 27 mo after the end of chemotherapy in one case. In the second patient, MRI bone marrow aspect returned to normal 4 mo after the end of chemotherapy. Bone marrow biopsy remained negative in these two MIBG-negative patients. These cases suggest that in presence of complete normalization of the MIBG scan after chemotherapy, the persistence of a hypointense signal on bone marrow on T1WI does not necessarily indicate persistence of disease but may be due to delayed normalization. Therefore, attention must be paid to the delay of signal normalization on MRI (which can be as long as more than 2 yr after the end of chemotherapy) in order to avoid false-positive interpretation.

Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice.

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.

Transcriptional control of the Notch1 tumour suppressor gene

Abstract : The Notch pathway is an important regulator of differentiation and carcinogenesis. In keratinocytes and possibly other specific epithelial cell types, it acts as tumour suppressor. Expression of endogenous Notch1 gene is markedly reduced in keratinocyte-derived squamous cell carcinoma (SCC) and cervical cancer cells, as well as in prostate cancer cell lines, and this difference is, at least in part, at the transcriptional level. Little is known on transcriptional control of the Notch1 gene with the exception that it is a p53-target. Our work focused on the mechanisms involved in the different transcription level of the Notch1 gene in normal versus cancer cells. We show that the fully active minimal Notch1 promoter is differentially controlled in normal versus cancer cells. It consists of two distinct regions, one downstream of the transcription start site, which is likely to bind the basic transcription apparatus, and one upstream region characterized by highly GC-rich sequence. This latter region binds Sp/KLF family members, specifically Spa and KLF4, which is upregulated in cancer cells. This is functionally significant as KLF4 overexpression is sufficient to downmodulate Notchl gene transcription, while KLF4 knockdown, in combination with Spa, results in Notch1 upregulation. Control of Notch1 by KLF4/Sp3 is independent of p53. Biochemically, KLF4/Sp3 seem to affect preferentially the initiation step of Notch1 gene transcription, while p53 controls both initiation and elongation steps. Thus, the Notch1 gene is a negative Sp3/KLF4-target and this mechanism contributes, in parallel with p53, to Notch1 downregulation in cancer. Résumé : La voie de signalisation induite par Notch est considérablement impliquée dans la différenciation des cellules et dans la carcinogénèse. Dans les kératinocytes ainsi que dans d'autres types cellulaires de l'épithelium, il agit comme suppresseur de tumeur. L'expression endogène de Notch1 est remarquablement réduite dans les cellules du carcinome spino-cellulaire et du cancer du col de l'utérus ou dans les lignées cellulaires du cancer de la prostate. Cette différence s'explique, du moins en partie, par le niveau de transcription. Peu de choses sont connues sur le contrôle transcriptionnel de Notch1 à l'exception du fait qu'il soit une cible de p53. Notre travail s'est concentré sur les mécanismes impliqués dans la transcription de Notch1, mécanismes qui diffèrent entre les cellules normales et les cellules cancéreuses. Nous avons trouvé la plus petite région du promoteur de Notch1 qui est suffisante pour induire un haut niveau transcriptionnel et qui est contrôlée différemment dans les cellules normales et les cellules cancéreuses. Elle est constituée de deux régions distinctes: une en aval du site de départ de la transcription, qui lie probablement le complexe de base pour la transcription, et une en amont caractérisée par une séquence riche en GC. Cette région lie les membres de la famille Sp/KLF, spécifiquement Sp3 et KLF4, qui sont surexprimés dans les cellules cancéreuses. Ceci est fonctionnellement significatif car la surexpression de KLF4 dans les kératinocytes est suffisante pour diminuer la transcription de Notch1, alors que l'inhibition de KLF4 et de Spa, résulte en une augmentation de Notch1. En outre, le contrôle de Notch1 par KLF4 et Spa est indépendant de p53. Biochimiquement, KLF4 et Spa semblent plutôt affecter l'initiation de la transcription de Notch1 alors que p53 contrôle aussi bien l'initiation que l'élongation. En conclusion, le gène Notch1 est inhibé par Spa et KLF4: ce mécanisme contribue, en parallèle à p53, à diminuer l'expression de Notch1 dans les cellules cancéreuses.

MYC and metastasis.

Aggressive primary tumors express transcriptional signatures that correlate with their metastatic propensity. A number of these signatures have been deployed in the clinic as risk stratification tools. However, the molecular basis of these clinically useful prognostic signatures has remained a largely unresolved area of controversy. We recently found that many prognostic signatures reflect the activity of the MYC oncogene, which in turn regulates tumor metastasis through specific effects on cancer cell invasion and migration. These findings offer a general framework for understanding the molecular basis of clinically prognostic transcriptional signatures and suggest potentially new avenues for studying metastasis.