Olav Engebraaten

Expression of extracellular matrix components in a highly infiltrative in vivo glioma model

Abstract. This work demonstrates the expression of extracellular matrix (ECM) components in a highly infiltrative brain tumor model developed by simple inoculation of spheroids from five human glioma biopsy tissues directly into the brains of immunodeficient rats. Non-invasive tumors derived from one glioblastoma biopsy specimen and two glioma cell lines (D-54MG and U-251MG) were also included in this study. The extent of tumor cell infiltration was studied using a pan-human monoclonal anti-vimentin antibody. The cellular origin for several of these ECM components was identified using human-specific monoclonal antibodies and polyclonal antibodies detecting epitopes from both species. Immunostaining revealed a diffuse parenchymal staining of glioma-produced tenascin, whereas vitronectin was produced mainly by the invading glioma cells. ECM components such as laminin, fibronectin and collagen type IV were most probably produced by the host and were mainly associated with the blood vessels in the tumors. However, some parenchymal staining with regional variations was observed. The expression pattern of these components was different in cell lines tumors as compared to the biopsy specimen tumors. The α3 and β1 integrin subunits were mainly observed in areas of tumor cell invasion in the invasive tumors. In conclusion, the observed staining patterns clarify the cellular origin and indicate the possible biological function of tenascin, vitronectin, laminin, fibronectin and collagen type IV in these highly invasive malignant tumors of glial origin.

Involvement of the pRb/p16/cdk4/cyclin D1 pathway in the tumorigenesis of sporadic malignant melanomas.

Biopsies from 61 sporadic metastatic malignant melanomas and five melanoma cell lines were examined for homozygous deletions and mutations in the CDKN2 gene (p16). As the p16 protein is involved in a cell cycle regulatory pathway consisting of at least pRb, cdk4 and cyclin D1, the tumours were also screened for amplifications of the last two genes. Moreover, the transcript levels of the genes were determined and the results compared with the immunohistochemically assessed expression of pRb. Altogether, homozygous deletions of CDKN2 were found in seven tumours (11%) and two of five cell lines, whereas a mutation was detected in only one biopsy, indicating that in sporadic melanomas the former mechanism is predominant for inactivating this gene. Notably, in total 59% of the metastatic lesions lacked detectable expression of p16 mRNA, whereas all the biopsies were found to express pRb. In accordance with the postulated negative feedback loop between p16 and pRb, one melanoma cell line showed overexpression of CDKN2 mRNA together with very low levels of the Rb protein. Amplification of the other two genes may not be important in the tumorigenesis of melanomas, as only one CDK4 and no CCND1 amplification was observed. However, highly elevated CDK4 mRNA levels, compared with that seen in a panel of normal tissues, were observed in 76% of the tumours, accompanied in 71% of the cases by high expression of the CCND1 cyclin activator. Although a low frequency of CDKN2 DNA aberrations was observed, the high number of tumours that lacked CDKN2 expression but showed overexpression of CDK4 and/or CCND1, suggest that functional inactivation of pRb through this pathway may be involved in the development or progression of sporadic human melanomas.

Distinct choline metabolic profiles are associated with differences in gene expression for basal-like and luminal-like breast cancer xenograft models

BackgroundIncreased concentrations of choline-containing compounds are frequently observed in breast carcinomas, and may serve as biomarkers for both diagnostic and treatment monitoring purposes. However, underlying mechanisms for the abnormal choline metabolism are poorly understood.MethodsThe concentrations of choline-derived metabolites were determined in xenografted primary human breast carcinomas, representing basal-like and luminal-like subtypes. Quantification of metabolites in fresh frozen tissue was performed using high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS).The expression of genes involved in phosphatidylcholine (PtdCho) metabolism was retrieved from whole genome expression microarray analyses.The metabolite profiles from xenografts were compared with profiles from human breast cancer, sampled from patients with estrogen/progesterone receptor positive (ER+/PgR+) or triple negative (ER-/PgR-/HER2-) breast cancer.ResultsIn basal-like xenografts, glycerophosphocholine (GPC) concentrations were higher than phosphocholine (PCho) concentrations, whereas this pattern was reversed in luminal-like xenografts. These differences may be explained by lower choline kinase (CHKA, CHKB) expression as well as higher PtdCho degradation mediated by higher expression of phospholipase A2 group 4A (PLA2G4A) and phospholipase B1 (PLB1) in the basal-like model. The glycine concentration was higher in the basal-like model. Although glycine could be derived from energy metabolism pathways, the gene expression data suggested a metabolic shift from PtdCho synthesis to glycine formation in basal-like xenografts. In agreement with results from the xenograft models, tissue samples from triple negative breast carcinomas had higher GPC/PCho ratio than samples from ER+/PgR+ carcinomas, suggesting that the choline metabolism in the experimental models is representative for luminal-like and basal-like human breast cancer.ConclusionsThe differences in choline metabolite concentrations corresponded well with differences in gene expression, demonstrating distinct metabolic profiles in the xenograft models representing basal-like and luminal-like breast cancer. The same characteristics of choline metabolite profiles were also observed in patient material from ER+/PgR+ and triple-negative breast cancer, suggesting that the xenografts are relevant model systems for studies of choline metabolism in luminal-like and basal-like breast cancer.

Molecular profiling and characterization of luminal-like and basal-like in vivo breast cancer xenograft models

The number of relevant and well‐characterized cell lines and xenograft models for studying human breast cancer are few, and may represent a limitation for this field of research. With the aim of developing new breast cancer model systems for in vivo studies of hormone dependent and independent tumor growth, progression and invasion, and for in vivo experimental therapy studies, we collected primary mammary tumor specimens from patients, and implanted them in immunodeficient mice. Primary tumor tissue from 29 patients with breast cancer was implanted subcutaneously with matrigel in SCID mice, in the presence of continuous release of estradiol. The tumors were transferred into new animals when reaching a diameter of 15mm and engrafted tumors were harvested for morphological and molecular characterization from passage six. Further, gene expression profiling was performed using Agilent Human Whole Genome Oligo Microarrays, as well as DNA copy number analysis using Agilent Human Genome CGH 244K Microarrays. Of the 30 primary tumors implanted into mice (including two implants from the same patient), two gave rise to viable tumors beyond passage ten. One showed high expression levels of estrogen receptor‐α protein (ER) while the other was negative. Histopathological evaluation of xenograft tumors was carried out at passage 10–12; both xenografts maintained the morphological characteristics of the original tumors (classified as invasive grade III ductal carcinomas). The genomic profile of the ER‐positive xenograft tumor resembled the profile of the primary tumor, while the profile obtained from the ER‐negative parental tumor was different from the xenograft. However, the ER‐negative parental tumor and xenograft clustered on the same branch using unsupervised hierarchical clustering analysis on RNA microarray expression data of “intrinsic genes”. A significant variation was observed in the expression of extracellular matrix (ECM)‐related genes, which were found downregulated in the engrafted tumors compared to the primary tumor. By IHC and qRT‐PCR we found that the downregulation of stroma‐related genes was compensated by the overexpression of such molecules by the mouse host tissue. The two established breast cancer xenograft models showed different histopathological characteristics and profound diversity in gene expression patterns that in part can be associated to their ER status and here described as basal‐like and luminal‐like phenotype, respectively. These two new breast cancer xenografts represent useful preclinical tools for developing and testing of new therapies and improving our knowledge on breast cancer biology.

Triple-Negative Breast Cancer and the Need for New Therapeutic Targets

Triple-negative breast cancers (TNBCs) are a diverse and heterogeneous group of tumors that by definition lack estrogen and progesterone receptors and amplification of the HER2 gene. The majority of the tumors classified as TNBCs are highly malignant, and only a subgroup responds to conventional chemotherapy with a favorable prognosis. Results from decades of research have identified important molecular characteristics that can subdivide this group of breast cancers further. High-throughput molecular analyses including sequencing, pathway analyses, and integrated analyses of alterations at the genomic and transcriptomic levels have improved our understanding of the molecular alterations involved in tumor development and progression. How this knowledge should be used for rational selection of therapy is a challenging task and the subject of numerous ongoing research programs. This review summarizes the current knowledge on the clinical characteristics and molecular alterations of TNBCs. Currently used conventional therapeutic strategies and targeted therapy studies are discussed, with references to recently published results on the molecular characterization of TNBCs.

Interaction between human brain tumour biopsies and fetal rat brain tissue in vitro

SummaryThe invasiveness of human intracranial tumours was studied in an organ culture system. Biopsies from six glioblastomas, four astrocytomas, two mixed gliomas, one ependymoma, four meningiomas and two carcinoma metastases were cut into fragments of 0.5 mm diameter, and placed in agar overlay tissue culture. The tumour specimens formed spheroids which were co-cultured with cell aggregates or fragments from fetal rat brain for up to 10 days in vitro. The invasiveness of the glioblastoma spheroids was characterised by a gradual destruction of normal brain tissue by tumour cells, followed by replacement of normal tissue by these cells. Cocultures from two glioblastomas showed lesions in the normal brain tissue in areas removed from the tumour cells. Tumour spheroids from four glioblastomas totally destroyed the normal brain tissue without any change in the original tumour spheroid configuration. The lowgrade gliomas were less invasive than the glioblastomas. The meningiomas and the metastases were non-invasive. This organ culture assay appeared to reflect the in situ invasive behaviour of the brain tumours examined. It is suggested that it may be used for evaluating the aggressiveness of individual brain tumours with the specific aim of correlating clinical data with the biological character of the tumour.

Combined treatment with Ad-hTRAIL and DTIC or SAHA is associated with increased mitochondrial-mediated apoptosis in human melanoma cell lines

Currently, dacarbazine (DTIC) is the only approved systemic treatment for metastatic malignant melanoma. However, the modest treatment effect encourages studies on novel therapeutic molecules, delivery systems and combination therapies. Full‐length TRAIL, delivered from an adenoviral vector (Ad‐hTRAIL), was studied in combination with DTIC or the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in human melanoma cell lines.

Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

Intratumoral immunotoxin treatment of human malignant brain tumors in immunodeficient animals

Treatment of malignant brain tumors remains a clinical challenge. New treatment modalities are under investigation and among these are intratumoral infusion of immunotoxins that bind to specific cell surface molecules on the malignant cells. We have compared the efficacy of the 425.3‐PE immunotoxin (which targets the epidermal growth factor [EGF] receptor) with the well‐known immunotoxin Tfn‐CRM107 (which targets the transferrin receptor), for the treatment of subcutaneous and intracranial human gliomas in nude animals. Bolus intratumoral administration of 1 μg Tfn‐CRM107 or 425.3‐PE into sc U87Mg tumors in nude mice reduced the tumor volume to 29 and 79%, respectively, of that in the control group 18 days after start of treatment. Higher doses of Tfn‐CRM107 were toxic to the animals, whereas 425.3‐PE was tolerated, with a dose‐response relationship of up to 8 μg, a dose that reduced the tumor volume to 2% of control. In nude rats, treatment of intracerebral U87Mg tumors with Tfn‐CRM107 proved ineffective and doses above 10 ng/animal were toxic to tumor‐bearing rats. In contrast, intratumoral administration of 4 μg 425.3‐PE increased symptom‐free survival from 23 days to 40 days, with 2/9 surviving more than 90 days. We have recently shown that immunodeficient rats inoculated intracerebrally with precultured glioblastoma biopsy specimens develop highly infiltrative brain tumors. Direct interstitial infusion of immunotoxins into such tumors reduced the number of animals with detectable tumors at autopsy after 3 months, from 8/9 in the control animals to 4/6 and 2/6 in animals treated with Tfn‐CRM107 and 425.3‐PE, respectively. In conclusion, the anti‐EGF receptor immunotoxin 425.3‐PE exhibited promising efficacy, comparable to or better than that of Tfn‐CRM107, an immunotoxin that in early clinical trials has been found to give responses in patients with brain tumors.

Low‐molecular contrast agent dynamic contrast‐enhanced (DCE)‐MRI and diffusion‐weighted (DW)‐MRI in early assessment of bevacizumab treatment in breast cancer xenografts

To investigate the effect of bevacizumab treatment on vascular architecture and function in two xenograft models with different angiogenic properties using diffusion‐weighted magnetic resonance imaging (DW‐MRI) and dynamic contrast‐enhanced MRI (DCE‐MRI).