The molecular basis of maternal control in seed development : genetic and molecular analysis of maternal effects in seed development: molecular mapping of cap2 and expression and functional analyses of AtLDC and AtHD2C

The female gametophyte of higher plants gives rise to the diploid embryo and the triploid endosperm which develop to produce the mature seed. Seed development is a concerted interplay of the embryo, endosperm and the surrounding diploid maternal tissue. In addition, it is highly dependent on the contribution from genetic programs executed in the gametophytic generations. What role the gametophytic maternal factors play in this process is still largely unknown. This thesis describes two approaches to identify novel genes involved in seed development. A forward genetic approach addresses the molecular nature of the maternal effect mutant capulet2 (cap2) by molecular mapping and a reverse genetics approach analyze the role in seed development of candidate genes from a promoter trap screen. The capulet2 gametophytic maternal-effect mutant was found in a linkage based screen preformed to identify gametophytic mutants in Arabidopsis (Grini et al., 1999). cap2 embryo and endosperm development is blocked at a very early stages, and heterozygous plants display a 50% reduced seed set. To investigate the molecular nature of the CAP2 gene, a map-based cloning approach was performed. Using PCR-based molecular markers the cap2 mutation was mapped to a genetic interval of 4238 basepairs, on the tip of the right arm of chromosome 1. This interval spanned parts of two genes, one involved in monoterpenoid biosynthesis and the other putatively involved in triterpenoid biosynthesis. Neither of these two genes could be verified to be responsible for the cap2 phenotype by complementation analysis. However the mapping interval of cap2 was reduced from more than 1 Mb to less than 100 kb. In a reverse genetic approach two candidate genes (AtHD2C and AtLDC) selected from a collection of promoter trap lines were analyzed to elucidate their role in seed development. Reporter gene expression studies, expression analysis, and the analysis of T-DNA insertion lines revealed that the candidate genes were expressed in the seed, but also in other organs. The promoter reporter line of AtLDC was found to have a similar but in some respects also different expression patterns in the seed than the original promoter trap line. The AtHD2C gene was found to be redundant as no phenotype could be observed in knock out alleles of the gene

Differential in vivo tumorigenicity of distinct subpopulations from a luminal-like breast cancer xenograft

Intratumor heterogeneity caused by genetic, phenotypic or functional differences between cancer cell subpopulations is a considerable clinical challenge. Understanding subpopulation dynamics is therefore central for both optimization of existing therapy and for development of new treatment. The aim of this study was to isolate subpopulations from a primary tumor and by comparing molecular characteristics of these subpopulations, find explanations to their differing tumorigenicity. Cell subpopulations from two patient derived in vivo models of primary breast cancer, ER+ and ER-, were identified. EpCAM+ cells from the ER+ model gave rise to tumors independently of stroma cell support. The tumorigenic fraction was further divided based on SSEA-4 and CD24 expression. Both markers were expressed in ER+ breast cancer biopsies. FAC-sorted cells based on EpCAM, SSEA-4 and CD24 expression were subsequently tested for differences in functionality by in vivo tumorigenicity assay. Three out of four subpopulations of cells were tumorigenic and showed variable ability to recapitulate the marker expression of the original tumor. Whole genome expression analysis of the sorted populations disclosed high similarity in the transcriptional profiles between the tumorigenic populations. Comparing the non-tumorigenic vs the tumorigenic populations, 44 transcripts were, however, significantly differentially expressed. A subset of these, 26 identified and named genes, highly expressed in the non-tumorigenic population, predicted longer overall survival (N = 737, p<0.0001) and distant metastasis free survival (DMFS) (N = 1379, p<0.0001) when performing Kaplan-Meier survival analysis using the GOBO online database. The 26 gene set correlated with longer DMFS in multiple breast cancer subgroups. Copy number profiling revealed no aberrations that could explain the observed differences in tumorigenicity. This study emphasizes the functional variability among cell populations that are otherwise genomically similar, and that the risk of breast cancer recurrence can only be eliminated if the tumorigenic abilities in multiple cancer cell subpopulations are inhibited

Aldehyde Dehydrogenase (ALDH) Activity Does Not Select for Cells with Enhanced Aggressive Properties in Malignant Melanoma

Malignant melanoma is an exceptionally aggressive, drug-resistant and heterogeneous cancer. Recently it has been shown that melanoma cells with high clonogenic and tumourigenic abilities are common, but markers distinguishing such cells from cells lacking these abilities have not been identified. There is therefore no definite evidence that an exclusive cell subpopulation, i.e. cancer stem cells (CSC), exists in malignant melanoma. Rather, it is suggested that multiple cell populations are implicated in initiation and progression of the disease, making it of importance to identify subpopulations with elevated aggressive properties.. Furthermore, both subpopulations showed similar sensitivity to the anti-melanoma drugs, dacarbazine and lexatumumab.These findings suggest that ALDH does not distinguish tumour-initiating and/or therapy-resistant cells, implying that the ALDH phenotype is not associated with more-aggressive subpopulations in malignant melanoma, and arguing against ALDH as a “universal” marker. Besides, it was shown that the ability to reestablish tumour heterogeneity is not necessarily linked to the more aggressive phenotype

Expression of Cell Surface Markers and Aldefluoractivity in EpCAM Positive Cells from Two Breast Cancer Xenografts Models, measured by flow cytometry.

<p>++++  =  all EpCAM positive cells positive, +++ =  40-90% were positive, ++ =  5–39% were positive, +  =  less than 5% positive cells, 0  =  no cells expressed marker. *  =  was highly positive when xenografts tumor was digested with trypsin.</p><p>Expression of Cell Surface Markers and Aldefluoractivity in EpCAM Positive Cells from Two Breast Cancer Xenografts Models, measured by flow cytometry.</p

Interplay of choline metabolites and genes in patient-derived breast cancer xenografts

Introduction Dysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism. Methods Tumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analyzed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analyzed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson’s correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups. Results Most of the xenograft models were classified as basal-like (N = 19) or luminal B (N = 7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). These characteristics were similar to those found in human tumor samples. Conclusion The higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathway-encoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo

Whole genome expression analyses of sorted tumor cell subpopulations. EpCAM positive cells from the luminal xenografts were separated based on expression of SSEA-4 and CD24 using FACS.

<p>A) Normalized gene expression data from all 15 samples were subjected to t-test comparison of two groups (dbl.high subopoulations vs. the tumorigenic subpopulations) with p≤0.004 and FDR = 0.2. The figure shows a cluster heatmap of the 44 significantly differentially expressed genes. Probes in yellow frames are not included in B, either because they are not annotated, the genes could not be found in GOBO, or they showed lower expression in the dbl.high population. The A_32_P188263 probe maps to the C1QB gene, which is already represented in the 26 gene list. B) Kaplan-Meier analysis using overall survival (OS) and distant metastasis free survival (DMFS) as endpoint and 10-year censoring as displayed in GOBO. C) Total RNA was isolated from FAC-sorted subpopulations and RT-qPCR was performed using primers against CD24. The bars illustrate the fold difference.</p