Triple-negative breast cancer (TNBC) represents one of the most aggressive breast tumour subtypes but the molecular mechanisms promoting tumour aggressiveness is still undefined. Several findings indicate that cancer cells undergo a complex metabolic reprogramming to satisfy the increased requirement of macromolecules and energy necessary for proliferation, and mitochondria have a fundamental role in this mechanism. Indeed, mitochondria are producers of intermediates for lipid, nucleic acid, and protein synthesis but also players of control of the cell fate. In these processes, mitochondrial calcium plays a pivotal role: in physiological conditions, it directly regulates three enzymes of the TCA cycle, i.e. pyruvate-, α-ketoglutarate- and isocitrate-dehydrogenases (Berridge et al., 2000); while in pathological conditions, mitochondrial Ca2+ overload sensitizes cells to apoptotic challenges by triggering the opening of the mitochondrial permeability transition pore (mPTP) (Basso et al., 2005). The channel responsible for entry into the mitochondria is the mitochondrial calcium uniporter (MCU) (De Stefani et al., 2011) (Baughman et al., 2011) that is composed of both channel-forming subunits and regulatory proteins. In addition to MCU, MCUb, the dominant-negative isoform of MCU (Raffaello et al., 2013), and the essential MCU regulator EMRE (Sancak et al., 2013) contribute to channel formation. The MICU protein family, comprising MICU1, MICU2 and MICU3, regulates the sophisticated mechanism of mitochondrial calcium uptake by modulating MCU function (De Stefani et al., 2016). While MICU3 expression is mainly confined to the nervous system, MICU1 and MICU2 are ubiquitous. MICU1 and MICU2 form a regulatory heterodimer that finely tunes MCU activity. Within the heterodimer, MICU1 increases and MICU2 reduces MCU activity. Both MICU1 and MICU2 possess EF-hand domains that bind Ca2+ ions. At low extra-mitochondrial [Ca2+], MICU2 plays a dominant effect thus inhibiting MCU activity. At higher extra-mitochondrial [Ca2+], MICU1 exerts a stimulatory effect allowing the prompt response of mitochondria to cytosolic [Ca2+] rises. It was already demonstrated that genetic inhibition of MCU expression causes a significant decline in TNBC metastatic cell motility and invasiveness (Tosatto et al., 2016) and hampers metastasis formation in vivo. To further understand the role of the mitochondrial Ca2+ uptake in TNBC, and to explore possible therapeutic targets, we aimed to study the role of the MICU1 and MICU2 in TNBC progression, both in vitro and in vivo. Firstly, we detected a decrease in MICU1 expression levels in tumour samples compared to normal breast samples underlining the importance of mitochondrial Ca2+ uptake in breast tumour development. Then, we demonstrated that the mitochondrial Ca2+ uptake inhibition, by overexpression ofMICU1 and MICU2 isoforms in which the EF-hands are mutated in order to hamper Ca2+ binding, causes a significant decline in TNBC cell motility in vitro and in tumour growth and metastasis formation in vivo. In these conditions, mROS production was significantly reduced, suggesting that mROS might play a crucial role in MICU1/2 dependent control of malignancy. In the second part of the thesis, we studied the role of MCUb, the dominant-negative isoform of MCU. While MCU expression increases with tumour progression, the expression of MCUb decreases (Tosatto et al., 2016). Thus, we decided to overexpress MCUb in a TNBC cell line. Overexpression of MCUb partially inhibits tumor cell growth. Moreover, MCUb overexpression causes a significant decline in TNBC cell motility. Our results highlight a crucial role of the mitochondrial Ca2+ uptake in the control of TNBC metastatic potential and indicate that the MCU regulatory subunits and MCUb could represent novel therapeutic target for clinical intervention

The mitochondrial Ca2+ homeostasis in breast cancer / Monticelli, Halenya. - (2019 Dec 02).

The mitochondrial Ca2+ homeostasis in breast cancer

Monticelli, Halenya
2019

Abstract

Triple-negative breast cancer (TNBC) represents one of the most aggressive breast tumour subtypes but the molecular mechanisms promoting tumour aggressiveness is still undefined. Several findings indicate that cancer cells undergo a complex metabolic reprogramming to satisfy the increased requirement of macromolecules and energy necessary for proliferation, and mitochondria have a fundamental role in this mechanism. Indeed, mitochondria are producers of intermediates for lipid, nucleic acid, and protein synthesis but also players of control of the cell fate. In these processes, mitochondrial calcium plays a pivotal role: in physiological conditions, it directly regulates three enzymes of the TCA cycle, i.e. pyruvate-, α-ketoglutarate- and isocitrate-dehydrogenases (Berridge et al., 2000); while in pathological conditions, mitochondrial Ca2+ overload sensitizes cells to apoptotic challenges by triggering the opening of the mitochondrial permeability transition pore (mPTP) (Basso et al., 2005). The channel responsible for entry into the mitochondria is the mitochondrial calcium uniporter (MCU) (De Stefani et al., 2011) (Baughman et al., 2011) that is composed of both channel-forming subunits and regulatory proteins. In addition to MCU, MCUb, the dominant-negative isoform of MCU (Raffaello et al., 2013), and the essential MCU regulator EMRE (Sancak et al., 2013) contribute to channel formation. The MICU protein family, comprising MICU1, MICU2 and MICU3, regulates the sophisticated mechanism of mitochondrial calcium uptake by modulating MCU function (De Stefani et al., 2016). While MICU3 expression is mainly confined to the nervous system, MICU1 and MICU2 are ubiquitous. MICU1 and MICU2 form a regulatory heterodimer that finely tunes MCU activity. Within the heterodimer, MICU1 increases and MICU2 reduces MCU activity. Both MICU1 and MICU2 possess EF-hand domains that bind Ca2+ ions. At low extra-mitochondrial [Ca2+], MICU2 plays a dominant effect thus inhibiting MCU activity. At higher extra-mitochondrial [Ca2+], MICU1 exerts a stimulatory effect allowing the prompt response of mitochondria to cytosolic [Ca2+] rises. It was already demonstrated that genetic inhibition of MCU expression causes a significant decline in TNBC metastatic cell motility and invasiveness (Tosatto et al., 2016) and hampers metastasis formation in vivo. To further understand the role of the mitochondrial Ca2+ uptake in TNBC, and to explore possible therapeutic targets, we aimed to study the role of the MICU1 and MICU2 in TNBC progression, both in vitro and in vivo. Firstly, we detected a decrease in MICU1 expression levels in tumour samples compared to normal breast samples underlining the importance of mitochondrial Ca2+ uptake in breast tumour development. Then, we demonstrated that the mitochondrial Ca2+ uptake inhibition, by overexpression ofMICU1 and MICU2 isoforms in which the EF-hands are mutated in order to hamper Ca2+ binding, causes a significant decline in TNBC cell motility in vitro and in tumour growth and metastasis formation in vivo. In these conditions, mROS production was significantly reduced, suggesting that mROS might play a crucial role in MICU1/2 dependent control of malignancy. In the second part of the thesis, we studied the role of MCUb, the dominant-negative isoform of MCU. While MCU expression increases with tumour progression, the expression of MCUb decreases (Tosatto et al., 2016). Thus, we decided to overexpress MCUb in a TNBC cell line. Overexpression of MCUb partially inhibits tumor cell growth. Moreover, MCUb overexpression causes a significant decline in TNBC cell motility. Our results highlight a crucial role of the mitochondrial Ca2+ uptake in the control of TNBC metastatic potential and indicate that the MCU regulatory subunits and MCUb could represent novel therapeutic target for clinical intervention
Breast Cancer, Metastasis, Mitochondrial Calcium uptake, MCU complex, MCU regolatory subunits
The mitochondrial Ca2+ homeostasis in breast cancer / Monticelli, Halenya. - (2019 Dec 02).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3424834
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