Browsing by Subject "Mitotic progression"

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Open Access
Elucidating the mechanisms of T-DM1 resistance in in vitro models of HER2 overexpressing breast cancer
(2016-09) Saatci, Özge
Despite the presence of plethora of anti-cancer therapeutics with a variety of different mechanisms of action, it is still not possible to completely eradicate cancer due largely to the occurrence of refractory tumors even years after completion of the treatment. Such “resistant” tumors are formed over time as few cells, which have gained some advantageous genomic alterations eventually populate the entire organ. A lot of in vitro and in vivo studies are currently being done in order to identify the ways by which cancer cells become resistant to given therapy. This would decipher the weaknesses of the resistant tumors and would provide a means to combat drug resistance. T-DM1 is an anti-HER2 therapeutics, being used in refractory HER2-positive breast cancer patients since 2013. It initially generated a huge excitement owing to the highly favorable clinical findings; however, resistance was developed rapidly after 5-6 months following the initial treatment. Currently; very little is known about the mechanisms of acquired resistance against T-DM1, and therefore, identification of novel targets for the treatment of T-DM1 refractory patients would be highly beneficial. In this thesis, I have developed and characterized the acquired T-DM1 resistance phenotypically, and demonstrated abrogation of drug induced mitotic arrest and apoptosis as two novel mechanisms of resistance. I have further analyzed the genomic landscape of resistance in terms of the enrichment of cancer related processes. Cell cycle was found to be the most significantly enriched process among genes deregulated in T-DM1 resistance as identified by next-generation RNA sequencing. Cell cycle was also shown to be activated in TCGA patients expressing high levels of the TDM1 resistance signature and further supported the importance of rewiring cell cycle for the acquisition of T-DM1 resistance in patients as well. I have further identified two important mitotic genes; PLK1 and TACC3 as the common mediators of resistance in different HER2-overexpressing models by a targeted siRNA screen. I have showed that their genomic or pharmacological inhibition confers sensitization to T-DM1 induced growth inhibition, partially through re-induction of apoptotic cell death. I further uncovered a BCL2 dependency in T-DM1 resistant models which was also found to be associated with T-DM1 resistance as inhibition of Bcl2 enhanced T-DM1 induced growth inhibition. Since T-DM1 refractory HER2-positive breast cancer patients are currently not curable, these pre-clinical findings might guide the future clinical test to improve the survival of this patient subgroup via the usage of PLK1 or BCL2 inhibitors in combination with T-DM1.
Open Access
Role of histone variant H3.3 in transcription and mitotic progression
(2017-04) Örs, Ayşegul
Chromatin structure needs to be dynamic and flexible in order for the eukaryotic cellular processes to function correctly. Incorporation of histone variants into chromatin serves to increase epigenetic plasticity by conferring new structural and functional properties to chromatin. Histone variants are implicated in many cellular processes such as transcription or cell division and their deregulation is involved in tumorigenesis. H3.3 is an evolutionarily well conserved histone variant that differs by only a few amino-acids from its replicationdependent counterparts. With the aim of determining H3.3 function, novel knockin/ conditional knock-out mouse models were established and characterized. In these models, one of the two genes coding for H3.3, H3f3a or H3f3b has been modified to code for an N-terminal FLAG-FLAG-HA tagged H3.3A or H3.3B which can be depleted upon Cre expression. Nucleosome resolution genome-wide mapping FH-H3.3A and FH-H3.3B determined that H3.3A and H3.3B were similarly enriched at promoter regions and their enrichment levels positively correlated with high expression and gene body enrichment. They were also found enriched in telomeres and some repetitive DNA sequences. In a subset of these repetitive regions H3.3A and H3.3B showed differential enrichment properties. As double H3.3-KO mouse generation resulted lethal, mouse embryonic fibroblasts (MEFs) were isolated from FH-H3.3 mice and transformed. Using a combination of Cre recombinase mediated knock-out and RNA interference technology, a new cellular model was established where H3.3 expression was essentially depleted. Although H3.3 enrichment profiles were indicative of a role in active transcription, whole transcriptome analysis upon single H3.3 depletion in livers and an almost complete H3.3 depletion in MEFs yielded very few differentially regulated genes. Interestingly, H3.3 depleted MEFs showed a high increase in mitotic defects and abnormal nuclear structures. Thus, an important yet often understudied role for H3.3 in genomic maintenance during mitotic progression was highlighted.