Mechanistic Study on the Antitumor Potential of the Endocannabinoid Reuptake Inhibitor OMDM-2

Introduction: Endocannabinoid signalling has been shown to be enhanced in several cancer tissues. Previous studies propose that this up-regulation contributes to the inhibition of the cancer cell proliferation by affecting different hallmarks of cancer. However, this anti-proliferative activity is suppressed by the rapid uptake and metabolism of endocannabinoids. An inhibition of endocannabinoid metabolism at the site of tumor growth might prolong their selective antitumor activity and avoid the side effects of direct cannabinoid agonists. The present study was undertaken to investigate the anti-tumor activity of OMDM-2, an endocannabinoid reuptake inhibitor, in-vivo and in-vitro. The molecular mechanisms involved in this activity were investigated. In addition we investigated the possible optimization of the treatment by a combination of OMDM-2 with natural or benchmark chemotherapeutics. Methods: Ehrlich ascites carcinoma bearing mice were used for the in-vivo experiments (n= 287). In these experiments we evaluated the effect of a systemic administration of OMDM-2 on tumor volume, mean survival time and increase in the life span. Time course effects of OMDM-2 on tumor weights, serum transforming growth factor beta-1, and the intratumoral expression of the endoglin receptor were observed. Also the effect of OMDM-2 on angiogenesis and hematological parameters were investigated. The in-vitro anti-proliferative activity of OMDM-2 was analyzed against human breast cancer (MCF-7) and glioma cells (U-87). The involvement of the CB-1 and vanilloid (TPRV1) receptors were evaluated by applying adequate pharmacological inhibitors. The type of cell death was investigated by ELISA, DNA electrophoresis, and morphological observations. Signaling pathways were analyzed by western blots for the MAP-kinases ERK 1 and 2 as well as for AKT. The combination of OMDM-2 with curcumin or temozolomide (TMZ) or paclitaxel was evaluated by isobole, combination index, sensitization factor, and dose reduction index methods. Results: OMDM-2 showed in our in-vivo study promising anticancer and antiangiogenic activities. Both activities were not mediated by the CB-1 receptor. The TGF-B1/endoglin system appears to be involved in the mediation of the anti-tumor activity of OMDM-2 at early stages of tumor development. OMDM-2 showed significant anti-proliferative activity against both - MCF-7 and U-87 in-vitro. The receptors, the mechanisms of action, and the signaling pathways involved in this activity differ according to the type of cancer. Co-incubation of curcumin with OMDM-2 showed potent synergistic activity against MCF-7 while this drug combination may be synergistic or antagonistic against U-87 according to the ratio. Combinations of OMDM-2 with TMZ or paclitaxel were able to decrease their individual doses and to increase the sensitivity of cancer cells. Conclusions: We provided for the first time data on the possible use of OMDM-2 as an anti-cancer drug to be an alternative to direct cannabinoid receptor agonists to avoid their psychotropic and immunosuppressive side effects. In particular, combination therapies utilizing both - molecules targeting the endocannabinoid system and natural anticancer agents or benchmark chemotherapeutic agents- are a worthwhile option to be further systematically explored in the treatment of cancer

Genetic, Cellular and Molecular Defects in Mouse Mutants with Severe Neural Tube Defects

Neural tube defects are one of the most common birth defects. This thesis includes genetic, molecular and cellular analysis of three mouse mutants with neural tube defects. chuzhoi was identified from an ENU G3 screen and exhibits craniorachischisis through failure to initiate neural tube closure. In this thesis, I show that chuzhoi carries a point mutation affecting a splice-site in the Ptk7 gene, leading to addition of three extra amino acids in the protein. Through phenotypic analysis I show that chuzhoi has a wider midline and a smaller length to width ratio, suggesting a defect in convergent extension. Previous work has shown that the Planar Cell Polarity (PCP) signalling pathway is required for the initiation of neural tube closure. Through genetic crosses between chuzhoi and mutants of PCP signalling, I show that Ptk7 can influence the PCP pathway without being a direct component of the pathway. Carrying a mutation in Scribble, Circletail is another mutant displaying craniorachischisis. Scribble in Drosophila is required for the establishment of apical-basal polarity and to control the rate of cell proliferation. I show that mouse Scribble is not required for the establishment of apical-basal polarity nor to control proliferation, during neural tube closure. Previous work in zebrafish has shown that the axis elongation of an embryo requires PCP-dependent orientation of cell division. Here, I show that the orientation of cell division is random in mice during the shaping of the neural plate prior to the initiation of the neural tube closure. Mouse mutants of Tulp3 exhibit spina bifida and exencephaly. The molecular role of Tulp3 is largely unknown; however, domains present in the Tulp3 protein suggest that it may act as a transcription factor and/or be involved in protein-protein interactions. I provide evidence to suggest that Tulp3 is not likely to function as a transcription factor but may participate in many protein-protein interactions to deliver its role during mouse embryogenesis

The role of GLI2 in human basal cell carcinoma tumourigenesis

PhDAbnormal Sonic Hedgehog (SHH) signalling leads to increased transcriptional activation of its downstream effector, GLI2, which is implicated in the pathogenesis of a variety of human tumours, including human basal cell carcinoma (BCC). However, little is known about the molecular mechanisms underlying the tumourigenic role of GLI2 in human skin keratinocytes. This study examines the effects of inducible and stable expression of constitutively active GLI2 (GLI2:N) oncogenic transcription factor, on immortalised human epidermal keratinocytes. It is shown here that GLI2:N overexpressing N/TERT keratinocytes display gene expression patterns and phenotypic characteristics reminiscent of those observed in human BCC in vivo. It is also shown for first time, that expression of GLI2:N in N/TERT keratinocytes is sufficient to induce accumulation of binucleated/tetraploid cells as evidenced by an increase in G2/M phase of the cell cycle and binucleate cell counting, and to promote polyploidy and aneuploidy, in the absence of increased cell death or apoptosis. This cell cycle deregulation is accompanied by strong activation of anti-apoptotic protein BCL-2 and simultaneous suppression of important cell-cycle regulators such as 14-3-3σ and CDK inhibitor p21WAF1/CIP1, with no change in p53 protein levels, indicating uncontrolled proliferation of cells with ploidy abnormalities and/or DNA damage. Consistently, it is shown that p21WAF1/CIP1 protein is also absent in human BCC tumours and that forced overexpression of GLI2:N renders human keratinocytes resistant to apoptosis mediated by ultraviolet B (UVB, 290-320 nm), one of the most important etiological factors in BCC formation. Karyotype analysis of GLI2:N N/TERT keratinocytes further demonstrates that overexpression of GLI2:N induces numerical (tetraploidy, polyploidy, aneuploidy) and structural instability in N/TERT keratinocytes including chromosomal translocations and double minute chromosomes. Furthermore, β-catenin activation is the most common alteration observed during aberrant WNT signalling, and is often implicated in the development of human carcinogenesis and metastasis. In this study it is shown that GLI2:N induction induces nuclear accumulation of β-catenin in keratinocyte cell culture and in the basal layer of organotypic skin rafts, similar to human BCCs. In addition, several WNT genes were found to be upregulated upon GLI2:N induction, while β-catenin transcriptional activity is increased upon stable and conditional expression of GLI2:N. Overall these data give new insights for the possible mechanisms that mediate the tumourigenic potential of GLI2

Deciphering the interplay of molecular alterations underpinning renal cell carcinoma by label-free mass spectrometry and clinical proteomics: A systems medicine approach for precision diagnosis

Renal neoplasia is the 14th most common tumor type diagnosed worldwide. With a vast heterogeneity, renal neoplasia encompasses different subtypes. 90% of the neoplasms arise from the epithelial layer of the nephron and vary from benign renal masses (renal oncocytoma, RO) to more indolent or aggressive cancers (renal cell carcinomas, RCC). As RCC subtypes, clear cell (ccRCC) subtype is the most predominant subtype, followed by papillary (pRCC) and chromophobe (chRCC). Despite the different outcomes, some overlapped histological and morphological features difficult their differentiation and diagnosis. Therefore, new approaches for a clear and accurate diagnosis are still needed. To achieve this goal, renal tissue biopsies diagnosed with ccRCC (n = 7), pRCC (n = 5), chRCC (n = 5), RO (n = 5) and normal adjacent tissue (NAT, n= 5) were enrolled in this study. As a very resourceful tool for proteome analysis and biomarker discovery, mass spectrometry (MS)-based methods were used to interrogate the proteome of each tumor in order to undisclosed differences trough which to develop faster and accurate diagnostics. The results achieved with this doctoral thesis include i) the accomplishment of an effective ultrasonic workflow to recover the proteome of optimal cutting temperature (OCT)-embedded tissues, ii) a novel analytical approach based on MALDI-MS profiling to distinguish chRCC from RO, iii) a 109-protein panel to discriminate between chRCC and RO and NAT, iv) a top 24-protein panel to diagnose ccRCC, pRCC, chRCC and RO based on absolute concentration values, v) the translation and validation of three promising biomarkers by immunohistochemical analysis, and vi) an approach for phosphopeptide enrichment. This work brings new insights into the different mechanisms underlying formation of these tumors as well as it provides valuable information to improve clinical diagnosis by opening new avenues for immunohistochemistry and mass spectrometry-based approaches