Browsing by Author "Bal, Hilal"
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Item Open Access Identification of long non-coding RNAs overcoming tamoxifen resistance in estrogen receptor alpha positive breast cancer(Bilkent University, 2017-09) Bal, HilalMost of the breast cancer incidences all over the world fall into Estrogen Receptor alpha (ERα)-positive breast cancer subtype, which are treated with endocrine therapy. Tamoxifen, a selective ER modulator drug, is the most prescribed endocrine therapy option for the patients, providing a decreased mortality rate. Although patients respond to tamoxifen well initially they may lose their sensitivity to tamoxifen and develop resistance which is a major obstacle when tackling ERα-positive breast cancer. Global transcriptome analyses performed in recent years demonstrated that most parts of the genomic DNA that are transcribed into RNA are not further translated into proteins. RNA molecules that are not converted into proteins and are therefore called non-coding RNAs (ncRNA) were found to be involved in cellular processes like sequence-specific chromosome modifications, gene silencing and regulation of protein signaling pathways. While the roles of protein and microRNA (miRNA) regulators in the tamoxifen resistance have been identified, the roles of long non-coding RNAs in tamoxifen resistance are still elusive. To elucidate the impact of the long non-coding transcripts in tamoxifen resistance, I have developed acquired tamoxifen resistant ERα-positive cell line models and examined alterations in their transcriptome with respect to long non-coding RNA expression. The results of whole genome RNA-Seq analysis showed that 330 long non-coding transcripts were differentially expressed in the tamoxifen resistant cell line compared to its parental counterpart. I filtered-out ncRNAs according to criteria based on fold change, cancer association, and being a validated lncRNA, and I ended up with two candidate lncRNAs. Here, I continued with the upregulated candidate lncRNA and confirmed its elevated expression by qRT-PCR in both of the in vitro acquired tamoxifen resistant cell line models I used. Moreover, I showed that knockdown of the candidate lncRNA using antisense oligonucleotide (ASO) re-sensitizes resistant cells to tamoxifen. This sensitization effect of candidate lncRNA was achieved via induction of autophagy shown by increased LC3 II/LC3 I ratio followed by apoptosis evidenced by cleaved Caspase 7 when the lncRNA was targeted. Finally, analysis of tamoxifen-treated, ERα-positive breast cancer patient data sets suggested that higher expression of the candidate lncRNA was associated with poor overall, relapse-free and disease-free survival of the patients. Overall, in this thesis, I identified a novel lncRNA regulator of tamoxifen resistance and a potential biomarker of therapy response.Item Open Access Reactivation of cAMP pathway by PDE4D inhibition represents a novel druggable axis for overcoming tamoxifen resistance in ER-positive breast cancer(American Association for Cancer Research, 2018) Mishra, Rasmi R.; Belder, Nevin; Ansari, Suhail A.; Kayhan, Merve; Bal, Hilal; Raza, Umar; Ersan, Pelin G.; Tokat, Ünal M.; Eyüpoğlu, Erol; Saatçi, Özge; Jandaghi, P.; Wiemann, S.; Üner, A.; Çekiç, Çağlar; Riazalhosseini, Y.; Şahin, ÖzgürPurpose: Tamoxifen remains an important hormonal therapy for ER-positive breast cancer; however, development of resistance is a major obstacle in clinics. Here, we aimed to identify novel mechanisms of tamoxifen resistance and provide actionable drug targets overcoming resistance. Experimental Design: Whole-transcriptome sequencing, downstream pathway analysis, and drug repositioning approaches were used to identify novel modulators [here: phosphodiesterase 4D (PDE4D)] of tamoxifen resistance. Clinical data involving tamoxifen-treated patients with ER-positive breast cancer were used to assess the impact of PDE4D in tamoxifen resistance. Tamoxifen sensitization role of PDE4D was tested in vitro and in vivo. Cytobiology, biochemistry, and functional genomics tools were used to elucidate the mechanisms of PDE4D-mediated tamoxifen resistance. Results: PDE4D, which hydrolyzes cyclic AMP (cAMP), was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. Higher PDE4D expression predicted worse survival in tamoxifen-treated patients with breast cancer (n ¼ 469, P ¼ 0.0036 for DMFS; n ¼ 561, P ¼ 0.0229 for RFS) and remained an independent prognostic factor for RFS in multivariate analysis (n ¼ 132, P ¼ 0.049). Inhibition of PDE4D by either siRNAs or pharmacologic inhibitors (dipyridamole and Gebr-7b) restored tamoxifen sensitivity. Sensitization to tamoxifen is achieved via cAMP-mediated induction of unfolded protein response/ER stress pathway leading to activation of p38/JNK signaling and apoptosis. Remarkably, acetylsalicylic acid (aspirin) was predicted to be a tamoxifen sensitizer using a drug repositioning approach and was shown to reverse resistance by targeting PDE4D/ cAMP/ER stress axis. Finally, combining PDE4D inhibitors and tamoxifen suppressed tumor growth better than individual groups in vivo. Conclusions: PDE4D plays a pivotal role in acquired tamoxifen resistance via blocking cAMP/ER stress/p38-JNK signaling and apoptosis.