Understanding the mechanisms and nature of tumour heterogeneity is a key focus of current cancer research. Tumour heterogeneity can arise from clonal evolution and/or differentiation. This thesis investigated the role of methylation in dynamic regulation of CD24 based heterogeneity in colorectal cancer cell lines. First, E-Cadherin variation between the cell lines LS174T and LS180 was investigated to find out whether E-Cadherin had any causal role in the difference in lumen formation between these two cell lines derived from the same tumour. These studies found no evidence of a causal role of E-Cadherin. However, morphological heterogeneity in LS174T was observed during the E-Cadherin study, suggesting that this cell line might be a mixture of two different clones. Single cell sorting by FACS allowed to isolate and establish these clones which were stable in the culture for long enough (until passage ~30) to allow their characterisations. Between the two clones, the CD24⁺ clone (named the LS174T_Clone 1^CD24+) was found to have shorter doubling time (23 hours) than the CD24- clone (named the LS174T_Clone 2CD24-; 30 hours). When cultured in matrigel, the LS174T_Clone 1^CD24+ showed higher clonogenicity (Chapter 4). Microarray analysis further revealed differences in gene expression including LAPTM4B, CXCR4, TGFIB and IL8 between these clones. Interestingly, when maintained for a long time in culture (around passage 50, which is equivalent to ~7 months), CD24 expression went through a gradual change in these clones, which became more evident from subclones of LS174T_Clone 2^CD24- (Chapter 6) and clones from another CRC cell line SW480 (chapter 5 and 6). To understand the mechanism of this dynamic change in the expression of CD24, it was first shown that no mutation could be responsible for this phenomenon. This suggested that promoter methylation of CD24 might be the mechanism of the observed dynamic changes in CD24 expression. Bisulfite (BS) modification of DNA from the LS174T clones and CD24 differentially expressing CRC cell lines (such as CD24- cell lines: CC20, RKO vs CD24+ cell lines: DLD1, NCIH716) followed by Sanger sequencing showed that direct methylation of seven CpG positions in the CD24 promoter region Chr6:106975560-106975834 is strongly correlated with the expression of CD24. Further subcloning and sequencing revealed that changes in the methylation of only two out of the seven CpG positions might be the main contributor to the CD24 expression differences. This is the first evidence of direct methylation-mediated regulation of CD24, showing, more broadly, how methylation can contribute to and maintain dynamic heterogeneity in cancer cells. Finally, a mixed culture experiment with the CD24+ and CD24- clones was conducted to test a simple mathematical model, which aimed to explain the interaction between the clones that are stably present in the LS174T cell line (Chapter 7). Altogether, these experiments suggest that genes such as REG1A (an inducer of angiogenesis) might be expressed because of synergistic interactions between the clones, whereas CXCR4 and TFF2 might be involved in a receptor-ligand complementary relationship. These findings have set a ground for future studies to confirm such interactions between co-existing subpopulations within a heterogeneous milieu of cancer cells.

Squamous cell carcinoma of the head and neck region (HNSCC) is an aggressive malignancy with generally poor prognosis and high mortality. The Special AT-rich binding protein 1 (SATB1) is a genome organizer protein that participates in regulating gene expression by acting as a trans-acting element as well as by recruiting chromatin remodeling complexes and enzymes. SATB1 is often overexpressed in cancer, and its possible role in tumour progression has been explored in several types of cancers and also suggested in HNSCC. However, its influence on molecular and cellular processes in HNSCC has not been examined, and, using primary cell lines, provided the basis of this thesis. This is a comprehensive study of molecular and cellular processes being affected upon siRNA-mediated SATB1 knockdown in vitro and in vivo. 15 HNSCC primary cell lines were obtained from the University of Turku and screened for SATB1 mRNA levels. The comparison of SATB1 mRNA levels with location, lymph node metastasis, disease staging (TNM) or SATB2 mRNA levels revealed no association. Hence, for deeper analysis 7 primary cell lines were selected based on growth inhibitory effects upon transient SATB1 knockdown, rather than their initial SATB1 mRNA levels. Growth inhibition upon SATB1 depletion was shown in monolayer (viable cell quantitation and colony forming ability) as well as non-adherent (spheroid assay) culture conditions. In some cell lines, cell death induced by apoptosis or retardation of cell cycle progression was observed as well. Parallel to this, using the FLAVINO assay, colony forming abilities of tumour cells from patient biopsies obtained from the University Hospital of Leipzig (Department of Otorhinolaryngology, Head and Neck Surgery) were tested post SATB1 knockdown. For molecular analysis, effects of SATB1 knockdown on transcription rates of selected oncogenes were analyzed. Among EMT markers, N-cadherin and beta catenin levels were found reduced upon SATB1 knockdown. The transcription of HER3 and its ligands Heregulin α & β was attenuated in all the seven primary cell lines, irrespectively of the growth inhibitory effects of SATB1 knockdown. These results demonstrated the role of SATB1 in the process of EMT and in autocrine signalling. Effects of HER3 inhibition on transcription rates of SATB1 were tested as well. HER3 inhibition was achieved by Patritumab, a novel monoclonal antibody against HER3. While SATB1 transcription rates remained unchanged upon HER3 inhibition, growth inhibition assays (2D and 3D) revealed that the combined use of HER1 and HER3 inhibitory antibodies provides better tumour cell inhibition over the single treatment. Finally, antitumor effects of SATB1 knockdown were monitored in vivo in two xenograft models (UT-SCC-14 and UT-SCC-42B). Treatment of tumor xenograft-bearing mice with siRNAs formulated in polymeric nanoparticles revealed reduced tumour growth, based on the knockdown of SATB1 as demonstrated on the protein level. Taken together, in this work SATB1 knockdown is demonstrated to mediate growth inhibition, induction of apoptosis, cell cycle retardation, negative impact on EMT and autocrine signaling and in vivo anti-tumour effects, thus highlighting the relevance of SATB1 in HNSCC.:156 pages

No description available.