Role of UPR-regulated long non-coding RNAs in breast cancer progression and response to therapy

Breast cancer accounted for about 23.8% of all new cancer cases in women and 11.6% of all new cancers globally, making it the world’s most prevalent cancer among women. Approximately 70% to 80% of breast cancers are classified as estrogen receptor -positive (ER+) breast cancer that relies on ER signalling. Endocrine therapies that aim to block or reduce the action of estrogen, which is the fire line for crucial for ER+ breast cancer patients, while up to 40% of patients develop resistance over time, leading to a big clinical challenge. This can occur via gain-of-function mutations in estrogen receptor α (ER), altered interactions of ER with coactivators/corepressors, or engagement of compensatory crosstalk between ER and growth factor receptors and unfolded protein response (UPR). UPR is a cellular stress response initiated at the endoplasmic reticulum (EnR) with three sensors: inositol requiring enzyme1α (IRE1α), activating transcription factor-6 (ATF6), and protein kinase RNA-like endoplasmic reticulum kinase (PERK). The UPR aims to restore normal EnR function by halting protein translation, degrading misfolded proteins, and activating signalling pathways that lead to increased production of EnR-resident molecular chaperones. Estrogen signalling and UPR are two crucial pathways that interact in various cellular processes, particularly in the context of ER+ breast cancer. Additionally, in breast cancer, the crosstalk between ER and UPR components can contribute to resistance against anti-estrogen therapies and chemotherapeutics. long non-coding RNAs (lncRNAs) are important regulators of cellular processes. lncRNAs can coordinate UPR by modulating other cellular pathways, such as autophagy, apoptosis, and cell cycle regulation. Recent studies have highlighted the role of interaction between the UPR and the estrogen signalling pathway in contributing to endocrine resistance. I hypothesize that lncRNAs that modulate both the UPR and ER signalling may contribute to the development of endocrine resistance. However, not much is known about UPR-regulated lncRNAs and their effect on EnR stress responses and cancer development. Here, I have identified H19 and Linc01588 as two UPR-regulated lncRNAs and characterized their effect on ER signalling and breast cancer. In the first chapter of results, a systematic bioinformatics approach to identify differential expression of non-coding RNAs (ncRNAs) regulated by the XBP1 was used. From this analysis, three candidate UPR-regulated ncRNAs—H19, MINCR, and LINC01588 were selected for further validation. Among them, H19 and LINC01588 were significantly downregulated upon EnR stress induction, supporting their potential role in UPR regulation. Then the expression profiles of LINC01588 and H19 across all cancer types and breast cancer subtypes showed a significantly elevated expression of LINC01588, while H19 showed a decreased expression in multiple human cancers. Additionally, the prognosis value of LINC01588 and H19 across different breast cancer subtypes showed that increased LINC01588 expression was associated with poor overall survival in ER+ and HER2+ breast cancer patients but not in triple-negative breast cancer (TNBC). High expression of H19 was associated with a higher hazard ratio in overall survival in TNBC patients. Further analysis revealed a strong link between LINC01588 and estrogen signalling pathways, suggesting its possible role in hormone-responsive tumour biology. In the second chapter of results, we show that the expression of the lncRNA H19 is downregulated under conditions of EnR stress. In addition, we selected spiperone, which was the only compound found to downregulate H19 expression, from a group of UPR-inducing compounds including the FDA-approved drug bortezomib and the clinical trial drug fenretinide. H19 expression potentiated the activation of ATF6 and PERK pathways while attenuating the IRE1-XBP1 signalling during conditions of UPR. Furthermore, H19 overexpression was found to enhance cell viability and inhibit EnR stress-induced cell death. Our results suggest that lncRNA H19 constitutes a pivotal regulatory node in the UPR, where PERK-mediated repression of H19 governs cell fate decisions under EnR stress, underscoring its potential as a promising therapeutic target in breast cancer. In the third and fourth chapters of results, we report increased Linc01588 expression in primary and metastatic breast cancer as compared with normal breast tissue. There are 5 isoforms of LINC01588 variant 4 (Linc01588 V4) and variant 5 (Linc01588 V5) were two predominantly expressed variants in breast cancer cell lines. The expression of Linc01588 V4 was markedly elevated in ER+ breast cancer cell lines upon E2-stimulation, while Linc01588 V5 was not. Tamoxifen treatment increased Linc01588 V4 expression in MCF7 cells but not in T47D cells. Knockdown of Linc01588 V4 in MCF7 and T47D cells reduced cell proliferation and migration but had no effect on the expression of ERα protein and induction of E2-responsive genes. Furthermore, Knockdown of Linc01588 V4 sensitized the cells to DNA-damage-induced apoptosis. Moreover, Linc01588 V4 expression was downregulated upon treatment with UPR inducers in ER+ breast cancer cell lines in a PERK-dependent manner. Linc01588 V4 expression enhanced the activation of ATF6 pathway while inhibiting the signalling via IRE1-XBP1 and PERK-ATF4 arm during conditions of UPR. Knockdown of Linc01588 V4 sensitized the cells to UPR-induced apoptosis. Our results provide new insights into potential treatment strategies to overcome endocrine resistance in ER+ breast cancer patients by targeting the lncRNAs that play key roles in endocrine resistance and UPR signalling.

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University of Galway

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CC BY-NC-ND

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University of Galway Theses (PhD Theses)