One of the most abundant constituents of the extracellular matrix is hyaluronan (HA). Being distributed almost in all vertebrates’ tissues, it plays a crucial role in the regulation of cell behaviours in both physiological and pathological conditions, including cancer. Tumour progression occurs through the crosstalk between cancer cells signalling networks and host stroma factors such as TGFβ, PDGF-BB and bFGF, all stimulating HA synthesis and creating an HA-rich stroma. Carcinoma cell-associated microenvironment is characterized by aberrant HA levels of a polydisperse size altering the composition and organization of the ECM and playing a key role in creating an environment in which enhanced inflammation and increased angiogenesis sustain cell growth, survival, and metastasis. HA influences cell behaviours through the interaction with CD44 and RHAMM, eventually triggering protein tyrosine kinases, Src, focal adhesion kinase and Erk kinases. The most aggressive breast cancers are called triple-negative (TNBC), as they lack the expression of oestrogen, progesterone and HER2 receptors. Currently, no efficient targeted therapy is available for TNBCs. Thus, the identification of novel therapeutic targets is of critical importance. HA and hyaluronan synthase 2 (HAS2) play a crucial role in regulating ER-negative breast cancer cells, as HAS2 silencing or HA synthesis inhibition both reduce tumour aggressiveness. The natural antisense transcript HAS2-AS1, a long non-coding RNA, is critical to epigenetically control HAS2 transcription and HA synthesis. Since HAS2-AS1 favours cell growth, invasion, and chemotherapy resistance in several cancers, including among others brain, ovary, and lung tumours, its high expression levels generally correlate with a poor tumour prognosis. However, the role of HAS2-AS1 in breast cancers has not yet been thoroughly investigated and the results of this thesis seem to tell a different story. First, we observed that ER-positive breast cancers had lower HAS2-AS1 expression compared to ER-negative tumours. Moreover, the survival of patients with ER-negative tumours was higher when the expression of HAS2-AS1 was elevated. Experiments with ER-negative cell lines as MDA-MB-231 and Hs 578T revealed that the overexpression of either the full-length HAS2-AS1 or its exon 2 long or short isoforms alone, strongly reduced cell viability, migration, and invasion, whereas HAS2-AS1 silencing increased cell aggressiveness. Unexpectedly, in these ER-negative cell lines, HAS2-AS1 is involved neither in the regulation of HAS2 nor in HA deposition. Transcriptome analysis revealed that HAS2-AS1 modulation affected several pathways, including apoptosis, proliferation, motility, adhesion and signalling, describing this long non-coding RNA as an important regulator of breast cancer cells aggressiveness. In deep investigation of the molecular mechanisms driving HAS2-AS1 modulation of diverse cancer-related pathways, revealed that HAS2-AS1 can bind and sequestrate miR-186-3p, working as a competing endogenous RNA (ceRNA; sponge effect), thus altering the expression of thousand genes. Finally, we found a strict involvement of HAS2-AS1 in modulating apoptosis in MDA-MB-231 cells, probably via miR-186-3p sponging, as its target mRNA of the P2RX7 gene was found profoundly induced upon HAS2-AS1 overexpression. Interestingly, also involvement in mesenchymal-to-epithelial transition and cell cycle regulation was suggested by some preliminary results we obtained. These new scenarios suggest that HAS2-AS1 can be involved in a more complex network of interactions that are critical for several cellular pathways. Nowadays lncRNAs are considered pivotal factors able to alter the landscape of gene expression in many tissues and several of them have been described to inhibit breast cancer tumorigenesis. In this light, HAS2-AS1 could be considered a new tumour suppressor specific for ER-negative breast cancer.

A new role for HAS2-AS1 as competing endogenous RNA for miR-186-3p in a cellular model of triple-negative breast cancer / Arianna Parnigoni , 2022 Mar 17. 34. ciclo, Anno Accademico 2020/2021.

A new role for HAS2-AS1 as competing endogenous RNA for miR-186-3p in a cellular model of triple-negative breast cancer.

PARNIGONI, ARIANNA
2022-03-17

Abstract

One of the most abundant constituents of the extracellular matrix is hyaluronan (HA). Being distributed almost in all vertebrates’ tissues, it plays a crucial role in the regulation of cell behaviours in both physiological and pathological conditions, including cancer. Tumour progression occurs through the crosstalk between cancer cells signalling networks and host stroma factors such as TGFβ, PDGF-BB and bFGF, all stimulating HA synthesis and creating an HA-rich stroma. Carcinoma cell-associated microenvironment is characterized by aberrant HA levels of a polydisperse size altering the composition and organization of the ECM and playing a key role in creating an environment in which enhanced inflammation and increased angiogenesis sustain cell growth, survival, and metastasis. HA influences cell behaviours through the interaction with CD44 and RHAMM, eventually triggering protein tyrosine kinases, Src, focal adhesion kinase and Erk kinases. The most aggressive breast cancers are called triple-negative (TNBC), as they lack the expression of oestrogen, progesterone and HER2 receptors. Currently, no efficient targeted therapy is available for TNBCs. Thus, the identification of novel therapeutic targets is of critical importance. HA and hyaluronan synthase 2 (HAS2) play a crucial role in regulating ER-negative breast cancer cells, as HAS2 silencing or HA synthesis inhibition both reduce tumour aggressiveness. The natural antisense transcript HAS2-AS1, a long non-coding RNA, is critical to epigenetically control HAS2 transcription and HA synthesis. Since HAS2-AS1 favours cell growth, invasion, and chemotherapy resistance in several cancers, including among others brain, ovary, and lung tumours, its high expression levels generally correlate with a poor tumour prognosis. However, the role of HAS2-AS1 in breast cancers has not yet been thoroughly investigated and the results of this thesis seem to tell a different story. First, we observed that ER-positive breast cancers had lower HAS2-AS1 expression compared to ER-negative tumours. Moreover, the survival of patients with ER-negative tumours was higher when the expression of HAS2-AS1 was elevated. Experiments with ER-negative cell lines as MDA-MB-231 and Hs 578T revealed that the overexpression of either the full-length HAS2-AS1 or its exon 2 long or short isoforms alone, strongly reduced cell viability, migration, and invasion, whereas HAS2-AS1 silencing increased cell aggressiveness. Unexpectedly, in these ER-negative cell lines, HAS2-AS1 is involved neither in the regulation of HAS2 nor in HA deposition. Transcriptome analysis revealed that HAS2-AS1 modulation affected several pathways, including apoptosis, proliferation, motility, adhesion and signalling, describing this long non-coding RNA as an important regulator of breast cancer cells aggressiveness. In deep investigation of the molecular mechanisms driving HAS2-AS1 modulation of diverse cancer-related pathways, revealed that HAS2-AS1 can bind and sequestrate miR-186-3p, working as a competing endogenous RNA (ceRNA; sponge effect), thus altering the expression of thousand genes. Finally, we found a strict involvement of HAS2-AS1 in modulating apoptosis in MDA-MB-231 cells, probably via miR-186-3p sponging, as its target mRNA of the P2RX7 gene was found profoundly induced upon HAS2-AS1 overexpression. Interestingly, also involvement in mesenchymal-to-epithelial transition and cell cycle regulation was suggested by some preliminary results we obtained. These new scenarios suggest that HAS2-AS1 can be involved in a more complex network of interactions that are critical for several cellular pathways. Nowadays lncRNAs are considered pivotal factors able to alter the landscape of gene expression in many tissues and several of them have been described to inhibit breast cancer tumorigenesis. In this light, HAS2-AS1 could be considered a new tumour suppressor specific for ER-negative breast cancer.
17-mar-2022
HAS2-AS1; Breast cancer
A new role for HAS2-AS1 as competing endogenous RNA for miR-186-3p in a cellular model of triple-negative breast cancer / Arianna Parnigoni , 2022 Mar 17. 34. ciclo, Anno Accademico 2020/2021.
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Descrizione: A new role for HAS2-AS1 as competing endogenous RNA for miR-186-3p in a cellular model of triple-negative breast cancer.
Tipologia: Tesi di dottorato
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11383/2133584
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