Complementary anti-cancer pathways triggered by inhibition of sideroflexin 4 in ovarian cancer.

DNA damaging agents are a mainstay of standard chemotherapy for ovarian cancer. Unfortunately, resistance to such DNA damaging agents frequently develops, often due to increased activity of DNA repair pathways. Sideroflexin 4 (SFXN4) is a little-studied inner mitochondrial membrane protein. Here we demonstrate that SFXN4 plays a role in synthesis of iron sulfur clusters (Fe-S) in ovarian cancer cells and ovarian cancer tumor-initiating cells, and that knockdown of SFXN4 inhibits Fe-S biogenesis in ovarian cancer cells. We demonstrate that this has two important consequences that may be useful in anti-cancer therapy. First, inhibition of Fe-S biogenesis triggers the accumulation of excess iron, leading to oxidative stress. Second, because enzymes critical to multiple DNA repair pathways require Fe-S clusters for their function, DNA repair enzymes and DNA repair itself are inhibited by reduction of SFXN4. Through this dual mechanism, SFXN4 inhibition heightens ovarian cancer cell sensitivity to DNA-damaging drugs and DNA repair inhibitors used in ovarian cancer therapy, such as cisplatin and PARP inhibitors. Sensitization is achieved even in drug resistant ovarian cancer cells. Further, knockout of SFXN4 decreases DNA repair and profoundly inhibits tumor growth in a mouse model of ovarian cancer metastasis. Collectively, these results suggest that SFXN4 may represent a new target in ovarian cancer therapy.

Systems biology of ferroptosis: A modeling approach.

Ferroptosis is a recently discovered form of iron-dependent regulated cell death (RCD) that occurs via peroxidation of phospholipids containing polyunsaturated fatty acid (PUFA) moieties. Activating this form of cell death is an emerging strategy in cancer treatment. Because multiple pathways and molecular species contribute to the ferroptotic process, predicting which tumors will be sensitive to ferroptosis is a challenge. We thus develop a mathematical model of several critical pathways to ferroptosis in order to perform a systems-level analysis of the process. We show that sensitivity to ferroptosis depends on the activity of multiple upstream cascades, including PUFA incorporation into the phospholipid membrane, and the balance between levels of pro-oxidant factors (reactive oxygen species, lipoxogynases) and antioxidant factors (GPX4). We perform a systems-level analysis of ferroptosis sensitivity as an outcome of five input variables (ACSL4, SCD1, ferroportin, transferrin receptor, and p53) and organize the resulting simulations into 'high' and 'low' ferroptosis sensitivity groups. We make a novel prediction corresponding to the combinatorial requirements of ferroptosis sensitivity to SCD1 and ACSL4 activity. To validate our prediction, we model the ferroptotic response of an ovarian cancer stem cell line following single- and double-knockdown of SCD1 and ACSL4. We find that the experimental outcomes are consistent with our simulated predictions. This work suggests that a systems-level approach is beneficial for understanding the complex combined effects of ferroptotic input, and in predicting cancer susceptibility to ferroptosis.

The Immunosuppressive Microenvironment in BRCA1-IRIS-Overexpressing TNBC Tumors Is Induced by Bidirectional Interaction with Tumor-Associated Macrophages.

Tumor-associated macrophages (TAM) promote triple-negative breast cancer (TNBC) progression. Here, we report BRCA1-IRIS-overexpressing (IRISOE) TNBC cells secrete high levels of GM-CSF in a hypoxia-inducible factor-1α (HIF1α)- and a NF-κB-dependent manner to recruit macrophages to IRISOE cells and polarize them to protumor M2 TAMs. GM-CSF triggered TGFß1 expression by M2 TAMs by activating STAT5, NF-κB, and/or ERK signaling. Despite expressing high levels of TGFß1 receptors on their surface, IRISOE TNBC cells channeled TGFß1/TßRI/II signaling toward AKT, not SMAD, which activated stemness/EMT phenotypes. In orthotopic and syngeneic mouse models, silencing or inactivating IRIS in TNBC cells lowered the levels of circulating GM-CSF, suppressed TAM recruitment, and decreased the levels of circulating TGFß1. Coinjecting macrophages with IRISOE TNBC cells induced earlier metastasis in athymic mice accompanied by high levels of circulating GM-CSF and TGFß1. IRISOE TNBC cells expressed low levels of calreticulin (the "eat me" signal for macrophages) and high levels of CD47 (the "do not eat me" signal for macrophages) and PD-L1 (a T-cell inactivator) on their surface. Accordingly, IRISOE TNBC tumors had significantly few CD8+/PD-1+ cytotoxic T cells and more CD25+/FOXP3+ regulatory T cells. These data show that the bidirectional interaction between IRISOE cells and macrophages triggers an immunosuppressive microenvironment within TNBC tumors that is favorable for the generation of immune-evading/stem-like/IRISOE TNBC metastatic precursors. Inhibiting this interaction may inhibit disease progression and enhance patients' overall survival. SIGNIFICANCE: The BRCA1-IRIS oncogene promotes breast cancer aggressiveness by recruiting macrophages and promoting their M2 polarization.

Sideroflexin 4 affects Fe-S cluster biogenesis, iron metabolism, mitochondrial respiration and heme biosynthetic enzymes.

Sideroflexin4 (SFXN4) is a member of a family of nuclear-encoded mitochondrial proteins. Rare germline mutations in SFXN4 lead to phenotypic characteristics of mitochondrial disease including impaired mitochondrial respiration and hematopoetic abnormalities. We sought to explore the function of this protein. We show that knockout of SFXN4 has profound effects on Fe-S cluster formation. This in turn diminishes mitochondrial respiratory chain complexes and mitochondrial respiration and causes a shift to glycolytic metabolism. SFXN4 knockdown reduces the stability and activity of cellular Fe-S proteins, affects iron metabolism by influencing the cytosolic aconitase-IRP1 switch, redistributes iron from the cytosol to mitochondria, and impacts heme synthesis by reducing levels of ferrochelatase and inhibiting translation of ALAS2. We conclude that SFXN4 is essential for normal functioning of mitochondria, is necessary for Fe-S cluster biogenesis and iron homeostasis, and plays a critical role in mitochondrial respiration and synthesis of heme.

Stearoyl-CoA Desaturase 1 Protects Ovarian Cancer Cells from Ferroptotic Cell Death.

Activation of ferroptosis, a recently described mechanism of regulated cell death, dramatically inhibits growth of ovarian cancer cells. Given the importance of lipid metabolism in ferroptosis and the key role of lipids in ovarian cancer, we examined the contribution to ferroptosis of stearoyl-CoA desaturase (SCD1, SCD), an enzyme that catalyzes the rate-limiting step in monounsaturated fatty acid synthesis in ovarian cancer cells. SCD1 was highly expressed in ovarian cancer tissue, cell lines, and a genetic model of ovarian cancer stem cells. Inhibition of SCD1 induced lipid oxidation and cell death. Conversely, overexpression of SCD or exogenous administration of its C16:1 and C18:1 products, palmitoleic acid or oleate, protected cells from death. Inhibition of SCD1 induced both ferroptosis and apoptosis. Inhibition of SCD1 decreased CoQ10, an endogenous membrane antioxidant whose depletion has been linked to ferroptosis, while concomitantly decreasing unsaturated fatty acyl chains in membrane phospholipids and increasing long-chain saturated ceramides, changes previously linked to apoptosis. Simultaneous triggering of two death pathways suggests SCD1 inhibition may be an effective component of antitumor therapy, because overcoming this dual mechanism of cell death may present a significant barrier to the emergence of drug resistance. Supporting this concept, we observed that inhibition of SCD1 significantly potentiated the antitumor effect of ferroptosis inducers in both ovarian cancer cell lines and a mouse orthotopic xenograft model. Our results suggest that the use of combined treatment with SCD1 inhibitors and ferroptosis inducers may provide a new therapeutic strategy for patients with ovarian cancer. SIGNIFICANCE: The combination of SCD1 inhibitors and ferroptosis inducers may provide a new therapeutic strategy for the treatment of ovarian cancer patients.See related commentary by Carbone and Melino, p. 5149.

The pro- and anti-tumor roles of mesenchymal stem cells toward BRCA1-IRIS-overexpressing TNBC cells.

BACKGROUND: To evaluate the cross-talk between BRCA1-IRIS (IRIS)-overexpressing (IRISOE) TNBC cells and tumor-resident mesenchymal stem cells (MSCs) that triggers the aggressiveness or elimination of IRISOE TNBC tumors. METHODS: We analyzed the effect of silencing or inactivating IRIS on the bi-directional interaction between IRISOE TNBC cells and MSCs on tumor formation and progression. We analyzed the downstream signaling in MSCs induced by IL-6 secreted from IRISOE TNBC cells. We compared the effect of MSCs on the formation and progression of IRIS-proficient and deficient-TNBC cells/tumors using in vitro and in vivo models. Finally, we analyzed the association between IL-6, PTGER2, and PTGER4 overexpression and breast cancer subtype; hormone receptor status; and distant metastasis-free or overall survival. RESULTS: We show high-level IL-6 secreted from IRISOE TNBC cells that enhances expression of its receptor (IL-6R) in MSCs, their proliferation, and migration toward IRISOE, in vitro, and recruitment into IRISOE TNBC tumors, in vivo. In serum-free medium, recombinant IL-6 and the IL-6-rich IRISOE TNBC cell condition media (CM) decreased STAT3Y705 phosphorylation (p-STAT3Y705) in MSCs. Inhibiting IRIS expression or activity prolonged STAT3Y705 phosphorylation in MSCs. The interaction with IRISOE TNBC cells skewed MSC differentiation toward prostaglandin E2 (PGE2)-secreting pro-aggressiveness cancer-associated fibroblasts (CAFs). Accordingly, co-injecting human or mouse MSCs with IRISOE TNBC tumor cells promoted the formation of aggressive mammary tumors, high circulating IL-6 and PGE2 levels, and reduced overall survival. In contrast, IRIS-silenced or inactivated cells showed reduced tumor formation ability, limited MSC recruitment into tumors, reduced circulating IL-6 and PGE2 levels, and prolonged overall survival. A positive correlation between IL-6, PTGER2, and PTGER4 expression and basal phenotype; ER-negativity; distant metastasis-free and overall survival in basal; or BRCAmutant carriers was observed. Finally, the bi-directional interaction with MSCs triggered death rather than growth of IRIS-silenced TNBC cells, in vitro and in vivo. CONCLUSIONS: The IL-6/PGE2-positive feedback loop between IRISOE TNBC tumor cells and MSCs enhances tumor aggressiveness. Inhibiting IRIS expression limits TNBC tumor growth and progression through an MSC-induced death of IRIS-silenced/inactivated TNBC cells.

Iron and Cancer.

This review explores the multifaceted role that iron has in cancer biology. Epidemiological studies have demonstrated an association between excess iron and increased cancer incidence and risk, while experimental studies have implicated iron in cancer initiation, tumor growth, and metastasis. The roles of iron in proliferation, metabolism, and metastasis underpin the association of iron with tumor growth and progression. Cancer cells exhibit an iron-seeking phenotype achieved through dysregulation of iron metabolic proteins. These changes are mediated, at least in part, by oncogenes and tumor suppressors. The dependence of cancer cells on iron has implications in a number of cell death pathways, including ferroptosis, an iron-dependent form of cell death. Uniquely, both iron excess and iron depletion can be utilized in anticancer therapies. Investigating the efficacy of these therapeutic approaches is an area of active research that promises substantial clinical impact.

DCYTB is a predictor of outcome in breast cancer that functions via iron-independent mechanisms.

BACKGROUND: Duodenal cytochrome b (DCYTB) is a ferrireductase that functions together with divalent metal transporter 1 (DMT1) to mediate dietary iron reduction and uptake in the duodenum. DCYTB is also a member of a 16-gene iron regulatory gene signature (IRGS) that predicts metastasis-free survival in breast cancer patients. To better understand the relationship between DCYTB and breast cancer, we explored in detail the prognostic significance and molecular function of DCYTB in breast cancer. METHODS: The prognostic significance of DCYTB expression was evaluated using publicly available microarray data. Signaling Pathway Impact Analysis (SPIA) of microarray data was used to identify potential novel functions of DCYTB. The role of DCYTB was assessed using immunohistochemistry and measurements of iron uptake, iron metabolism, and FAK signaling. RESULTS: High DCYTB expression was associated with prolonged survival in two large independent cohorts, together totaling 1610 patients (cohort #1, p = 1.6e-11, n = 741; cohort #2, p = 1.2e-05, n = 869; log-rank test) as well as in the Gene expression-based Outcome for Breast cancer Online (GOBO) cohort (p < 1.0e-05, n = 1379). High DCYTB expression was also associated with increased survival in homogeneously treated groups of patients who received either tamoxifen or chemotherapy. Immunohistochemistry revealed that DCYTB is localized on the plasma membrane of breast epithelial cells, and that expression is dramatically reduced in high-grade tumors. Surprisingly, neither overexpression nor knockdown of DCYTB affected levels of ferritin H, transferrin receptor, labile iron or total cellular iron in breast cancer cells. Because SPIA pathway analysis of patient microarray data revealed an association between DCYTB and the focal adhesion pathway, we examined the influence of DCYTB on FAK activation in breast cancer cells. These experiments reveal that DCYTB reduces adhesion and activation of focal adhesion kinase (FAK) and its adapter protein paxillin. CONCLUSIONS: DCYTB is an important predictor of outcome and is associated with response to therapy in breast cancer patients. DCYTB does not affect intracellular iron in breast cancer cells. Instead, DCYTB may retard cancer progression by reducing activation of FAK, a kinase that plays a central role in tumor cell adhesion and metastasis.

BRCA1-IRIS overexpression promotes and maintains the tumor initiating phenotype: implications for triple negative breast cancer early lesions.

Tumor-initiating cells (TICs) are cancer cells endowed with self-renewal, multi-lineage differentiation, increased chemo-resistance, and in breast cancers the CD44+/CD24-/ALDH1+ phenotype. Triple negative breast cancers show lack of BRCA1 expression in addition to enhanced basal, epithelial-to-mesenchymal transition (EMT), and TIC phenotypes. BRCA1-IRIS (hereafter IRIS) is an oncogene produced by the alternative usage of the BRCA1 locus. IRIS is involved in induction of replication, transcription of selected oncogenes, and promoting breast cancer cells aggressiveness. Here, we demonstrate that IRIS overexpression (IRISOE) promotes TNBCs through suppressing BRCA1 expression, enhancing basal-biomarkers, EMT-inducers, and stemness-enforcers expression. IRISOE also activates the TIC phenotype in TNBC cells through elevating CD44 and ALDH1 expression/activity and preventing CD24 surface presentation by activating the internalization pathway EGFR→c-Src→cortactin. We show that the intrinsic sensitivity to an anti-CD24 cross-linking antibody-induced cell death in membranous CD24 expressing/luminal A cells could be acquired in cytoplasmic CD24 expressing IRISOE TNBC/TIC cells through IRIS silencing or inactivation. We show that fewer IRISOE TNBC/TICs cells form large tumors composed of TICs, resembling TNBCs early lesions in patients that contain metastatic precursors capable of disseminating and metastasizing at an early stage of the disease. IRIS-inhibitory peptide killed these IRISOE TNBC/TICs, in vivo and prevented their dissemination and metastasis. We propose IRIS inactivation could be pursued to prevent dissemination and metastasis from early TNBC tumor lesions in patients.

Mitochondria and Iron: current questions.

INTRODUCTION: Mitochondria are cellular organelles that perform numerous bioenergetic, biosynthetic, and regulatory functions and play a central role in iron metabolism. Extracellular iron is taken up by cells and transported to the mitochondria, where it is utilized for synthesis of cofactors essential to the function of enzymes involved in oxidation-reduction reactions, DNA synthesis and repair, and a variety of other cellular processes. Areas covered: This article reviews the trafficking of iron to the mitochondria and normal mitochondrial iron metabolism, including heme synthesis and iron-sulfur cluster biogenesis. Much of our understanding of mitochondrial iron metabolism has been revealed by pathologies that disrupt normal iron metabolism. These conditions affect not only iron metabolism but mitochondrial function and systemic health. Therefore, this article also discusses these pathologies, including conditions of systemic and mitochondrial iron dysregulation as well as cancer. Literature covering these areas was identified via PubMed searches using keywords: Iron, mitochondria, Heme Synthesis, Iron-sulfur Cluster, and Cancer. References cited by publications retrieved using this search strategy were also consulted. Expert commentary: While much has been learned about mitochondrial and its iron, key questions remain. Developing a better understanding of mitochondrial iron and its regulation will be paramount in developing therapies for syndromes that affect mitochondrial iron.

Iron and cancer: recent insights.

Iron is an essential dietary element. However, the ability of iron to cycle between oxidized and reduced forms also renders it capable of contributing to free radical formation, which can have deleterious effects, including promutagenic effects that can potentiate tumor formation. Dysregulation of iron metabolism can increase cancer risk and promote tumor growth. Cancer cells exhibit an enhanced dependence on iron relative to their normal counterparts, a phenomenon we have termed iron addiction. Work conducted in the past few years has revealed new cellular processes and mechanisms that deepen our understanding of the link between iron and cancer. Control of iron efflux through the combined action of ferroportin, an iron efflux pump, and its regulator hepcidin appears to play an important role in tumorigenesis. Ferroptosis is a form of iron-dependent cell death involving the production of reactive oxygen species. Specific mechanisms involved in ferroptosis, including depletion of glutathione and inhibition of glutathione peroxidase 4, have been uncovered. Ferritinophagy is a newly identified mechanism for degradation of the iron storage protein ferritin. Perturbations of mechanisms that control transcripts encoding proteins that regulate iron have been observed in cancer cells, including differences in miRNA, methylation, and acetylation. These new insights may ultimately provide new therapeutic opportunities for treating cancer.

BRCA1-IRIS inactivation overcomes paclitaxel resistance in triple negative breast cancers.

INTRODUCTION: Intrinsic or acquired chemoresistance is a major problem in oncology. Although highly responsive to chemotherapies such as paclitaxel, most triple negative breast cancer (TNBC) patients develop chemoresistance. Here we investigate the role of BRCA1-IRIS as a novel treatment target for TNBCs and their paclitaxel-resistant recurrences. METHODS: We analyzed the response of BRCA1-IRIS overexpressing normal mammary cells or established TNBC cells silenced from BRCA1-IRIS to paclitaxel in vitro and in vivo. We analyzed BRCA1-IRIS downstream signaling pathways in relation to paclitaxel treatment. We also analyzed a large cohort of breast tumor samples for BRCA1-IRIS, Forkhead box class O3a (FOXO3a) and survivin expression. Finally, we analyzed the effect of BRCA1-IRIS silencing or inactivation on TNBCs formation, maintenance and response to paclitaxel in an orthotopic model. RESULTS: We show that low concentrations of paclitaxel triggers BRCA1-IRIS expression in vitro and in vivo, and that BRCA1-IRIS activates two autocrine signaling loops (epidermal growth factor (EGF)/EGF receptor 1 (EGFR)-EGF receptor 2 (ErbB2) and neurogulin 1 (NRG1)/ErbB2-EGF receptor 3 (ErbB3), which enhances protein kinase B (AKT) and thus survivin expression/activation through promoting FOXO3a degradation. This signaling pathway is intact in TNBCs endogenously overexpressing BRCA1-IRIS. These events trigger the intrinsic and acquired paclitaxel resistance phenotype known for BRCA1-IRIS-overexpressing TNBCs. Inactivating BRCA1-IRIS signaling using a novel inhibitory mimetic peptide inactivates these autocrine loops, AKT and survivin activity/expression, in part by restoring FOXO3a expression, and sensitizes TNBC cells to low paclitaxel concentrations in vitro and in vivo. Finally, we show BRCA1-IRIS and survivin overexpression is correlated with lack of FOXO3a expression in a large cohort of primary tumor samples, and that BRCA1-IRIS overexpression-induced signature is associated with decreased disease free survival in heavily treated estrogen receptor alpha-negative patients. CONCLUSIONS: In addition to driving TNBC tumor formation, BRCA1-IRIS overexpression drives their intrinsic and acquired paclitaxel resistance, partly by activating autocrine signaling loops EGF/EGFR-ErbB2 and NRG1/ErbB2-ErbB3. These loops activate AKT, causing FOXO3a degradation and survivin overexpression. Taken together, this underscores the need for BRCA1-IRIS-specific therapy and strongly suggests that BRCA1-IRIS and/or signaling loops activated by it could be rational therapeutic targets for advanced TNBCs.

Biophysical evaluation of two red-shifted hypocrellin B derivatives as novel PDT agents.

Two novel cyclohexane-1,2-diamino and N,N dimethyl amino-propyl substituted hypocrellin B derivatives, abbreviated as CHA2HB and DMAHB, respectively were synthesized. These derivatives exhibited enhanced absorption in phototherapeutic window. Photodynamic action of these derivatives, investigated using optical and electron spin resonance methods, depended on both Type I and Type II mechanisms. Gel electrophoresis indicated 1O2/O2(.-) mediated DNA damage. CHA2HB displayed 20 fold increase in light dependent cytotoxicity on colon cancer cell line (HCT 116) than the well-known hypocrellin B (HB). The light induced, LD(50) values for CHA2HB and DMAHB were found to be 0.1 microM and 1.5 microM, respectively. The singlet oxygen generating efficiency followed the order HB>CHA(2)HB>DMAHB. But, the enhanced red absorption as well as the hydrophilicity renders the CHA2HB a better photodynamic therapeutic agent.