The incidence of melanoma is increasing over the years with a still poor prognosis and the lack of a cure able to guarantee an adequate survival of patients. Although the new immuno-based coupled to target therapeutic strategy is encouraging, the appearance of targeted/cross-resistance and/or side effects such as autoimmune disorders could limit its clinical use. Alternative therapeutic strategies are therefore urgently needed to efficiently kill melanoma cells. Ferroptosis induction and execution were evaluated in metastasis-derived wild-type and oncogenic BRAF melanoma cells, and the process responsible for the resistance has been dissected at molecular level. Although efficiently induced in all cells, in an oncogenic BRAF- and ER stress-independent way, most cells were resistant to ferroptosis execution. At molecular level we found that: resistant cells efficiently activate NRF2 which in turn upregulates the early ferroptotic marker CHAC1, in an ER stress-independent manner, and the aldo-keto reductases AKR1C1 ÷ 3 which degrades the 12/15-LOX-generated lipid peroxides thus resulting in ferroptotic cell death resistance. However, inhibiting AKRs activity/expression completely resensitizes resistant melanoma cells to ferroptosis execution. Finally, we found that the ferroptotic susceptibility associated with the differentiation of melanoma cells cannot be applied to metastatic-derived cells, due to the EMT-associated gene expression reprogramming process. However, we identified SCL7A11 as a valuable marker to predict the susceptibility of metastatic melanoma cells to ferroptosis. Our results identify the use of pro-ferroptotic drugs coupled to AKRs inhibitors as a new valuable strategy to efficiently kill human skin melanoma cells.

The function of Kv4.3 (KCND3) channels, which underlie the transient outward current I(to) in human heart, can be modulated by several accessory subunits such as KChIP2 and KCNE1-KCNE5. Here we aimed to determine the regional expression of Kv4.3, KChIP2, and KCNE mRNAs in non-failing and failing human hearts and to investigate the functional consequences of subunit coexpression in heterologous expression systems. We quantified mRNA levels for two Kv4.3 isoforms, Kv4.3-S and Kv4.3-L, and for KChIP2 as well as KCNE1-KCNE5 with real-time RT-PCR. We also studied the effects of KCNEs on Kv4.3+KChIP2 current characteristics in CHO cells with the whole-cell voltage-clamp method. In non-failing hearts, low expression was found for KCNE1, KCNE3, and KCNE5, three times higher expression for KCNE2, and 60 times higher for KCNE4. Transmural gradients were detected only for KChIP2 in left and right ventricles. Compared to non-failing tissue, failing hearts showed higher expression of Kv4.3-L and KCNE1 and lower of Kv4.3-S, KChIP2, KCNE4, and KCNE5. In CHO cells, Kv4.3+KChIP2 currents were differentially modified by co-expressed KCNEs: time constants of inactivation were shorter with KCNE1 and KCNE3-5 while time-to-peak was decreased, and V(0.5) of steady-state inactivation was shifted to more negative potentials by all KCNE subunits. Importantly, KCNE2 induced a unique and prominent 'overshoot' of peak current during recovery from inactivation similar to that described for human I(to) while other KCNE subunits induced little (KCNE4,5) or no overshoot. All KCNEs are expressed in the human heart at the transcript level. Compared to I(to) in native human myocytes, none of the combination of KChIP2 and KCNE produced an ideal congruency in current characteristics, suggesting that additional factors contribute to the regulation of the native I(to) channel.

Dipeptidyl‐aminopeptidase‐like protein 6 (DPPX) was recently shown in the brain to modulate the kinetics of transient A‐type currents by accelerating inactivation and recovery from inactivation. Since the kinetics of human cardiac transient outward current (I_to) are not mimicked by coexpression of the α‐subunit Kv4.3 with its known β‐subunit KChIP2, we have tested the hypothesis that DPPX may serve as an additional β‐subunit in the human heart. With quantitative real‐time RT‐PCR strong mRNA expression of DPPX was detected in human ventricles and was verified at the protein level in human but not in rat heart by a DPPX‐specific antibody. Co‐expression of DPPX with Kv4.3 in Chinese hamster ovary cells produced I_to‐like currents, but compared with expression of KChIP2a and Kv4.3, the time constant of inactivation was faster, the potential of half‐maximum steady‐state inactivation was more negative and recovery from inactivation was delayed. Co‐expression of DPPX in addition to Kv4.3 and KChIP2a produced similar current kinetics as in human ventricular myocytes. We therefore propose that DPPX is an essential component of the native cardiac I_to channel complex in human heart.

Over the past 10 years, it has emerged that pervasive transcription in mammalian genomes has a tremendous impact on several biological functions. Most of transcribed RNAs are lncRNAs and repetitive elements. In this review, we will detail the discovery of a new functional class of natural and synthetic antisense lncRNAs that stimulate translation of sense mRNAs. These molecules have been named SINEUPs since their function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation. Natural SINEUPs suggest that embedded Transposable Elements may represent functional domains in long non-coding RNAs. Synthetic SINEUPs may be designed by targeting the antisense sequence to the mRNA of choice representing the first scalable tool to increase protein synthesis of potentially any gene of interest. We will discuss potential applications of SINEUP technology in the field of molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson’s disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed towards N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson’s disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Potassium (K⁺) channels are essential to neuronal signaling and survival. Here we show that these proteins are targets of reactive oxygen species in mammalian brain and that their oxidation contributes to neuropathy. Thus, the KCNB1 (Kv2.1) channel, which is abundantly expressed in cortex and hippocampus, formed oligomers upon exposure to oxidizing agents. These oligomers were ∼10-fold more abundant in the brain of old than young mice. Oxidant-induced oligomerization of wild-type KCNB1 enhanced apoptosis in neuronal cells subject to oxidative insults. Consequently, a KCNB1 variant resistant to oxidation, obtained by mutating a conserved cysteine to alanine, (C73A), was neuroprotective. The fact that oxidation of KCNB1 is toxic, argues that this mechanism may contribute to neuropathy in conditions characterized by high levels of oxidative stress, such as Alzheimer's disease (AD). Accordingly, oxidation of KCNB1 channels was exacerbated in the brain of a triple transgenic mouse model of AD (3xTg-AD). The C73A variant protected neuronal cells from apoptosis induced by incubation with β-amyloid peptide (Aβ_1–42). In an invertebrate model (Caenorhabditis elegans) that mimics aspects of AD, a C73A-KCNB1 homolog (C113S-KVS-1) protected specific neurons from apoptotic death induced by ectopic expression of human Aβ_1–42. Together, these data underscore a novel mechanism of toxicity in neurodegenerative disease.