Paola Zacchi

A local disruption of iron homeostasis leading to oxidative stress is considered one of the main mechanisms of asbestos-related genotoxicity. Another aspect contributing to the risk of developing pathological consequences upon asbestos exposure is individual genetic factors. In a previous study, we identified a coding SNP in the hephaestin gene (HEPH) that protects against developing asbestos-related thoracic cancer. Heph is a ferroxidase that promotes iron export in concert with the permease ferroportin (Fpn1). Here, we performed an in-depth functional characterization of the HephD568H variant to gain insights into the molecular basis of its protective activity. We showed that HephD568H forms a complex with Fpn1 and possesses full ferroxidase activity. Although HephD568H is more efficiently recruited to the plasma membrane, it is impaired in binding iron-deficient Tfn, whose interaction with wild-type (WT) ferroxidase emerged as a novel mechanism to perceive brain iron needs. Heph is expressed in the human lung by pericytes and fibroblasts, and lung pericytes were shown to respond to iron demand by upregulating the iron exporter pair. These results extend the paradigm of local iron regulation discovered at the blood–brain barrier to the pulmonary vasculature. Furthermore, they establish a mechanistic link between changes in iron sensing and the risk of developing asbestos-related malignancies.

Local disruption of iron homeostasis leading to oxidative stress is considered one of the main mechanisms of asbestos-dependent cyto-/genotoxicity. Another aspect contributing to the risk of developing pathological consequences upon asbestos exposure is due to individual genetic fac-tors. In a previous study we identified a coding SNP in the hephaestin gene (HEPH) exerting a protective activity against the development of asbestos related thoracic cancers. Heph is a fer-roxidase which promotes iron export in concert with the permease ferroportin (Fpn1). Here, we performed an in-depth functional characterization of the HephD568H variant to gain insights on the molecular basis of its protective activity in asbestos-related carcinogenesis. We demonstrat-ed that HephD568H complexes with Fpn1 and possesses a full ferroxidase activity. Despite being more efficiently recruited at the plasma membrane, HephD568H is severely hampered in (iron-depleted) apo-Tfn binding, whose interaction with the WT ferroxidase emerged as a novel mechanism to perceive brain iron needs. Heph in human lung is expressed by pericytes and fi-broblasts and lung pericytes were shown to respond to iron request by up-regulating the iron exporter pair. These findings extend to the pulmonary vascular unit the paradigm of local iron regulation uncovered at the blood-brain barrier. Moreover, they mechanistically correlate alter-ations in iron sensing with the risk of developing asbestos-dependent malignancies.

TMEM16A is a Ca²⁺-activated Cl⁻ channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.

TMEM16A is a Ca2+-activated Cl- channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 antagonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.

Background: The interaction of asbestos fibers with target cell membranes is still poorly investigated. Here, we detected and characterized an enhancement of chloride conductance in Xenopus oocyte cell membranes induced by exposure to crocidolite (Croc) asbestos fibers. Methods: A two-microelectrode voltage clamp technique was used to test the effect of Croc fiber suspensions on outward chloride currents evoked by step membrane depolarization. Calcium imaging experiments were also performed to investigate the variation of ‘resting’ oocyte [Ca²⁺]_i following asbestos exposure. Results: The increase in chloride current after asbestos treatment, was sensitive to [Ca²⁺]_e, and to specific blockers of TMEM16A Ca²⁺-activated chloride channels, MONNA and Ani9. Furthermore, asbestos treatment elevated the ‘resting’ [Ca²⁺]_i likelihood by increasing the cell membrane permeability to Ca² in favor of a tonic activation of TMEME16A channels_. Western blot analysis confirmed that TMEME16A protein was endogenously present in the oocyte cell membrane and absorbed by Croc. Conclusion: the TMEM16A channels endogenously expressed by Xenopus oocytes are targets for asbestos fibers and represent a powerful tool for asbestos–membrane interaction studies. Interestingly, TMEM16A channels are highly expressed in many types of tumors, including some asbestos-related cancers, suggesting them, for the first time, as a possible early target of crocidolite-mediated tumorigenic effects on target cell membranes.

The variant enrichment analysis (VEA), a recently developed bioinformatic workflow, has been shown to be a valuable tool for whole-exome sequencing data analysis, allowing finding differences between the number of genetic variants in a given pathway compared to a reference dataset. In a previous study, using VEA, we identified different pathway signatures associated with the development of pulmonary toxicities in mesothelioma patients treated with radical hemithoracic radiation therapy. Here, we used VEA to discover novel pathways altered in individuals exposed to asbestos who developed or not asbestos-related diseases (lung cancer or mesothelioma). A population-based autopsy study was designed in which asbestos exposure was evaluated and quantitated by investigating objective signs of exposure. We selected patients with similar exposure to asbestos. Formalin-fixed paraffin-embedded (FFPE) tissues were used as a source of DNA and whole-exome sequencing analysis was performed, running VEA to identify potentially disrupted pathways in individuals who developed thoracic cancers induced by asbestos exposure. By using VEA analysis, we confirmed the involvement of pathways considered as the main culprits for asbestos-induced carcinogenesis: oxidative stress and chromosome instability. Furthermore, we identified protective genetic assets preserving genome stability and susceptibility assets predisposing to a worst outcome.

Editorial: Iron Metabolism at the Crossroad of Innate Immune Response and Cancer Progression

Increased hyaluronic acid (HA) production is often associated with cancer progression. In malignant pleural mesothelioma (MPM), HA is found at elevated levels in pleural effusions and sera of patients, and it has been widely debated whether MPM cells are able to produce HA by themselves or through the release of growth factors stimulating other cells. Another key component of the MPM microenvironment is C1q, which can act as a pro-tumorigenic factor favoring cell adhesion, migration and proliferation. The aim of the current study was to prove that MPM primary cells are able to synthesize HA and to inquire the stimulus given by C1q–HA matrix to HA synthesis. We confirmed the presence of a HA coat and cable-like structures around MPM primary cells, as well as an intracellular pool, mainly localized in the cytoplasmic and perinuclear region. After evaluating HA synthase (HAS) enzymes’ basal expression in MPM primary cells, we found that C1q bound to HA was able to impinge upon HA homeostasis by upregulating HAS3 both at the mRNA and the protein levels. High expression of HAS3 has been correlated with a shorter life expectancy in MPM by bioinformatical analysis. These data confirmed that C1q bound to HA may exert pro-tumorigenic activity and identified HAS3 as a potential target in MPM.