Quantitative MRI Evaluation of Ferritin Overexpression in Non-Small-Cell Lung Cancer.

Cancer cells frequently present elevated intracellular iron levels, which are thought to facilitate an enhanced proliferative capacity. Targeting iron metabolism within cancer cells presents an avenue to enhance therapeutic responses, necessitating the use of non-invasive models to modulate iron manipulation to predict responses. Moreover, the ubiquitous nature of iron necessitates the development of unique, non-invasive markers of metabolic disruptions to develop more personalized approaches and enhance the clinical utility of these approaches. Ferritin, an iron storage enzyme that is often upregulated as a response to iron accumulation, plays a central role in iron metabolism and has been frequently associated with unfavorable clinical outcomes in cancer. Herein, we demonstrate the successful utility, validation, and functionality of a doxycycline-inducible ferritin heavy chain (FtH) overexpression model in H1299T non-small-cell lung cancer (NSCLC) cells. Treatment with doxycycline increased the protein expression of FtH with a corresponding decrease in labile iron in vitro and in vivo, as determined by calcein-AM staining and EPR, respectively. Moreover, a subsequent increase in TfR expression was observed. Furthermore, T2* MR mapping effectively detected FtH expression in our in vivo model. These results demonstrate that T2* relaxation times can be used to monitor changes in FtH expression in tumors with bidirectional correlations depending on the model system. Overall, this study describes the development of an FtH overexpression NSCLC model and its correlation with T2* mapping for potential use in patients to interrogate iron metabolic alterations and predict clinical outcomes.

Auranofin Inhibition of Thioredoxin Reductase Sensitizes Lung Neuroendocrine Tumor Cells (NETs) and Small Cell Lung Cancer (SCLC) Cells to Sorafenib as well as Inhibiting SCLC Xenograft Growth.

Thioredoxin Reductase (TrxR) is a key enzyme in hydroperoxide detoxification through peroxiredoxin enzymes and in thiol-mediated redox regulation of cell signaling. Because cancer cells produce increased steady-state levels of reactive oxygen species (ROS; i.e., superoxide and hydrogen peroxide), TrxR is currently being targeted in clinical trials using the anti-rheumatic drug, auranofin (AF). AF treatment decreased TrxR activity and clonogenic survival in small cell lung cancer (SCLC) cell lines (DMS273 and DMS53) as well as the lung atypical (neuroendocrine tumor) NET cell line H727. AF treatment also significantly sensitized DMS273 and H727 cell lines in vitro to sorafenib, a multi-kinase inhibitor that was shown to decrease intracellular glutathione. The pharmacokinetic and pharmacodynamic properties of AF treatment in a mouse SCLC xenograft model was examined to maximize inhibition of TrxR activity without causing toxicity. AF was administered intraperitoneally at 2 mg/kg or 4 mg/kg (IP) once (QD) or twice daily (BID) for 1 to 5 days in mice with DMS273 xenografts. Plasma levels of AF were 10-20 µM (determined by mass spectrometry of gold) and the optimal inhibition of TrxR (50 %) was obtained at 4 mg/kg once daily, with no effect on glutathione peroxidase 1 activity. When this daily AF treatment was extended for 14 days a significant prolongation of median survival from 19 to 23 days (p=0.04, N=30 controls, 28 AF) was observed without causing changes in animal bodyweight, CBCs, bone marrow toxicity, blood urea nitrogen, or creatinine. These results show that AF is an effective inhibitor of TrxR both in vitro and in vivo in SCLC, capable of sensitizing NETs and SCLC to sorafenib, and supports the hypothesis that AF could be used as an adjuvant therapy with agents known to induce disruptions in thiol metabolism to enhance therapeutic efficacy.

Mosaicism in BRPF1-Related Neurodevelopmental Disorder: Report of Two Sisters and Literature Review.

Bromodomain and PHD finger containing 1 (BRPF1)-related neurodevelopmental disorder is characterized by intellectual disability, developmental delay, hypotonia, dysmorphic facial features, ptosis, and blepharophimosis. Both de novo and inherited pathogenic variants have been previously reported in association with this disorder. We report two affected female siblings with a novel variant in BRPF1 c.2420_2433del (p.Q807Lfs∗27) identified through whole-exome sequencing. Their history of mild intellectual disability, speech delay, attention deficient hyperactivity disorder (ADHD), and ptosis align with the features previously reported in the literature. The absence of the BRPF1 variant in parental buccal samples provides evidence of a de novo frameshift pathogenic variant, most likely as a result of parental gonadal mosaicism, which has not been previously reported. The frameshift pathogenic variant reported here lends further support to haploinsufficiency as the underlying mechanism of disease. We review the literature, compare the clinical features seen in our patients with others reported, and explore the possibility of genotype-phenotype correlation based on the location of pathogenic variants in BRPF1. Our study helps to summarize available knowledge and report the first case of a de novo frameshift pathogenic variant in BRPF1 in two siblings with this neurodevelopmental disorder.

Depletion of Labile Iron Induces Replication Stress and Enhances Responses to Chemoradiation in Non-Small-Cell Lung Cancer.

The intracellular redox-active labile iron pool (LIP) is weakly chelated and available for integration into the iron metalloproteins that are involved in diverse cellular processes, including cancer cell-specific metabolic oxidative stress. Abnormal iron metabolism and elevated LIP levels are linked to the poor survival of lung cancer patients, yet the underlying mechanisms remain unclear. Depletion of the LIP in non-small-cell lung cancer cell lines using the doxycycline-inducible overexpression of the ferritin heavy chain (Ft-H) (H1299 and H292), or treatment with deferoxamine (DFO) (H1299 and A549), inhibited cell growth and decreased clonogenic survival. The Ft-H overexpression-induced inhibition of H1299 and H292 cell growth was also accompanied by a significant delay in transit through the S-phase. In addition, both Ft-H overexpression and DFO in H1299 resulted in increased single- and double-strand DNA breaks, supporting the involvement of replication stress in the response to LIP depletion. The Ft-H and DFO treatment also sensitized H1299 to VE-821, an inhibitor of ataxia telangiectasis and Rad2-related (ATR) kinase, highlighting the potential of LIP depletion, combined with DNA damage response modifiers, to alter lung cancer cell responses. In contrast, only DFO treatment effectively reduced the LIP, clonogenic survival, cell growth, and sensitivity to VE-821 in A549 non-small-cell lung cancer cells. Importantly, the Ft-H and DFO sensitized both H1299 and A549 to chemoradiation in vitro, and Ft-H overexpression increased the efficacy of chemoradiation in vivo in H1299. These results support the hypothesis that the depletion of the LIP can induce genomic instability, cell death, and potentiate therapeutic responses to chemoradiation in NSCLC.

Evaluating the iron chelator function of sirtinol in non-small cell lung cancer.

A distinctive feature of cancer is the upregulation of sirtuin proteins. Sirtuins are class III NAD+-dependent deacetylases involved in cellular processes such as proliferation and protection against oxidative stress. SIRTs 1 and 2 are also overexpressed in several types of cancers including non-small cell lung cancer (NSCLC). Sirtinol, a sirtuin (SIRT) 1 and 2 specific inhibitor, is a recent anti-cancer agent that is cytotoxic against several types of cancers including NSCLC. Thus, sirtuins 1 and 2 represent valuable targets for cancer therapy. Recent studies show that sirtinol functions as a tridentate iron chelator by binding Fe3+ with 3:1 stoichiometry. However, the biological consequences of this function remain unexplored. Consistent with preliminary literature, we show that sirtinol can deplete intracellular labile iron pools in both A549 and H1299 non-small cell lung cancer cells acutely. Interestingly, a temporal adaptive response occurs in A549 cells as sirtinol enhances transferrin receptor stability and represses ferritin heavy chain translation through impaired aconitase activity and apparent IRP1 activation. This effect was not observed in H1299 cells. Holo-transferrin supplementation significantly enhanced colony formation in A549 cells while increasing sirtinol toxicity. This effect was not observed in H1299 cells. The results highlight the fundamental genetic differences that may exist between H1299 and A549 cells and offer a novel mechanism of how sirtinol kills NSCLC cells.

Redox sensitive miR-27a/b/Nrf2 signaling in Cr(VI)-induced carcinogenesis.

Hexavalent chromium [Cr(VI)] is a well-known carcinogen that can cause several types of cancer including lung cancer. NF-E2-related factor 2 (Nrf2), the redox sensitive transcription factor, can protect normal cells from a variety of toxicants and carcinogens by inducing the expression of cellular protective genes and maintaining redox balance. However, Nrf2 also protects cancer cells from radio- and chemo-therapies and facilitates cancer progression. Although Cr(VI) treatment has been demonstrated to upregulate Nrf2 expression, the mechanisms for Nrf2 regulation upon chronic Cr(VI) exposure remain to be elucidated. We found that Nrf2 was upregulated in BEAS-2B cells exposed to Cr(VI) from 1 to 5 months, and also in Cr(VI)-induced transformed (Cr-T) cells with Cr(VI) treatment for 6 months. We showed that KEAP1, the classic negative regulator of Nrf2, was downregulated after Cr(VI) exposure for 4 months, suggesting that Nrf2 induction by Cr(VI) treatment is through KEAP1 decrease at late stage. To further decipher the mechanisms of Nrf2 upregulation at early stage of Cr(VI) exposure, we demonstrated that miR-27a and miR-27b were redox sensitive miRNAs, since reactive oxygen species (ROS) scavengers induced miR-27a/b expression. After Cr(VI) exposure for 1 month, the expression levels of miR-27a/b was dramatically decreased. The changes of miR-27a/b and their target Nrf2 were confirmed in vivo by mouse model intranasally exposed to Cr(VI) for 12 weeks. Nrf2 was a direct target of miR-27a/b, which acted as tumor suppressors in vitro and in vivo to inhibit tumorigenesis and cancer development of Cr-T cells. The results suggested that the inhibition of miR-27a/b was responsible for Nrf2 upregulation at both early stage and late stage of Cr(VI) exposure. This novel regulation of Nrf2 upon chronic Cr(VI) exposure through redox-regulated miR-27a/b will provide potential targets for preventing and treating Cr(VI)-induced carcinogenesis in the future.