Applying Multimodal Mass Spectrometry to Image Tumors Undergoing Ferroptosis Following In Vivo Treatment with a Ferroptosis Inducer.

Epithelial ovarian cancer (EOC) is the most common form of ovarian cancer. The poor prognosis generally associated with this disease has led to the search for improved therapies such as ferroptosis-inducing agents. Ferroptosis is a form of regulated cell death that is dependent on iron and is characterized by lipid peroxidation. Precise mapping of lipids and iron within tumors exposed to ferroptosis-inducing agents may provide insight into processes of ferroptosis in vivo and ultimately assist in the optimal deployment of ferroptosis inducers in cancer therapy. In this work, we present a method for combining matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) with secondary ion mass spectrometry (SIMS) to analyze changes in spatial lipidomics and metal composition, respectively, in ovarian tumors following exposure to a ferroptosis inducer. Tumors were obtained by injecting human ovarian cancer tumor-initiating cells into mice, followed by treatment with the ferroptosis inducer erastin. SIMS imaging detected iron accumulation in the tumor tissue, and sequential MALDI-MS imaging of the same tissue section displayed two chemically distinct regions of lipids. One region was associated with the iron-rich area detected with SIMS, and the other region encompassed the remainder of the tissue section. Bulk lipidomics confirmed the lipid assignments putatively assigned from the MALDI-MS data. Overall, we demonstrate the ability of multimodal MSI to identify the spatial locations of iron and lipids in the same tissue section and associate these regions with clinical pathology.

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.

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.

A Targeted Mass Spectrometric Assay for Reliable Sensitive Hepcidin Quantification.

Hepcidin, a cysteine-rich peptide hormone, secreted mainly by the liver, plays a central role in iron metabolism regulation. Emerging evidence suggests that disordered iron metabolism is a risk factor for various types of diseases including cancers. However, it remains challenging to apply current mass spectrometry (MS)-based hepcidin assays for precise quantification due to the low fragmentation efficiency of intact hepcidin as well as synthesis difficulties for the intact hepcidin standard. To address these issues we recently developed a reliable sensitive targeted MS assay for hepcidin quantification from clinical samples that uses fully alkylated rather than intact hepcidin as the internal standard. Limits of detection and quantification were determined to be <0.5 ng/mL and 1 ng/mL, respectively. Application of the alkylated hepcidin assay to 70 clinical plasma samples (42 non-cancerous and 28 ovarian cancer patient samples) enabled reliable detection of endogenous hepcidin from the plasma samples, as well as conditioned culture media. The hepcidin concentrations ranged from 0.0 to 95.6 ng/mL across non-cancerous and cancer plasma specimens. Interestingly, cancer patients were found to have significantly higher hepcidin concentrations compared to non-cancerous patients (mean: 20.6 ng/ml for cancer; 5.94 ng/ml for non-cancerous) (p value < 0.001). Our results represent the first application of the alkylated hepcidin assay to clinical samples and demonstrate that the developed assay has better sensitivity and quantification accuracy than current MS-based hepcidin assays without the challenges in synthesis of intact hepcidin standard and accurately determining its absolute amount.

Contribution of three-dimensional architecture and tumor-associated fibroblasts to hepcidin regulation in breast cancer.

Hepcidin is a peptide hormone that negatively regulates iron efflux and plays an important role in controlling the growth of breast tumors. In patients with breast cancer, the combined expression of hepcidin and its membrane target, ferroportin, predict disease outcome. However, mechanisms that control hepcidin expression in breast cancer cells remain largely unknown. Here, we use three-dimensional breast cancer spheroids derived from cell lines and breast cancer patients to probe mechanisms of hepcidin regulation in breast cancer. We observe that the extent of hepcidin induction and pathways of its regulation are markedly changed in breast cancer cells grown in three dimensions. In monolayer culture, BMPs, particularly BMP6, regulate hepcidin transcription. When breast cancer cells are grown as spheroids, there is a >10-fold induction in hepcidin transcripts. Microarray analysis combined with knockdown experiments reveal that GDF-15 is the primary mediator of this change. The increase in hepcidin as breast cells develop a three-dimensional architecture increases intracellular iron, as indicated by an increase in the iron storage protein ferritin. Immunohistochemical staining of human breast tumors confirms that both GDF-15 and hepcidin are expressed in breast cancer specimens. Further, levels of GDF-15 are significantly correlated with levels of hepcidin at both the mRNA and protein level in patient samples, consistent with a role for GDF-15 in control of hepcidin in human breast tumors. Inclusion of tumor-associated fibroblasts in breast cancer spheroids further induces hepcidin. This induction is mediated by fibroblast-dependent secretion of IL-6. Breast cancer cells grown as spheroids are uniquely receptive to IL-6-dependent induction of hepcidin by tumor-associated fibroblasts, since IL-6 does not induce hepcidin in cells grown as monolayers. Collectively, our results suggest a new paradigm for tumor-mediated control of iron through the control of hepcidin by tumor architecture and the breast tumor microenvironment.

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.

Receptor tyrosine kinase Met promotes cell survival via kinase-independent maintenance of integrin α3ß1.

Matrix adhesion via integrins is required for cell survival. Adhesion of epithelial cells to laminin via integrin α3ß1 was previously shown to activate at least two independent survival pathways. First, integrin α3ß1 is required for autophagy-induced cell survival after growth factor deprivation. Second, integrin α3ß1 independently activates two receptor tyrosine kinases, EGFR and Met, in the absence of ligands. EGFR signaling to Erk promotes survival independently of autophagy. To determine how Met promotes cell survival, we inhibited Met kinase activity or blocked its expression with RNA interference. Loss of Met expression, but not inhibition of Met kinase activity, induced apoptosis by reducing integrin α3ß1 levels, activating anoikis, and blocking autophagy. Met was specifically required for the assembly of autophagosomes downstream of LC3II processing. Reexpression of wild-type Met, kinase-dead Met, or integrin α3 was sufficient to rescue death upon removal of endogenous Met. Integrin α3ß1 coprecipitated and colocalized with Met in cells. The extracellular and transmembrane domain of Met was required to fully rescue cell death and restore integrin α3 expression. Thus Met promotes survival of laminin-adherent cells by maintaining integrin α3ß1 via a kinase-independent mechanism.

Hepcidin regulation in prostate and its disruption in prostate cancer.

Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression.

IRP2 regulates breast tumor growth.

Experimental and epidemiologic evidence suggests that dysregulation of proteins involved in iron metabolism plays a critical role in cancer. The mechanisms by which cancer cells alter homeostatic iron regulation are just beginning to be understood. Here, we demonstrate that iron regulatory protein 2 (IRP2) plays a key role in iron accumulation in breast cancer. Although both IRP1 and IRP2 are overexpressed in breast cancer, the overexpression of IRP2, but not IRP1, is associated with decreased ferritin H and increased transferrin receptor 1 (TfR1). Knockdown of IRP2 in triple-negative MDA-MB-231 human breast cancer cells increases ferritin H expression and decreases TfR1 expression, resulting in a decrease in the labile iron pool. Further, IRP2 knockdown reduces growth of MDA-MB-231 cells in the mouse mammary fat pad. Gene expression microarray profiles of patients with breast cancer demonstrate that increased IRP2 expression is associated with high-grade cancer. Increased IRP2 expression is observed in luminal A, luminal B, and basal breast cancer subtypes, but not in breast tumors of the ERBB2 molecular subtype. These results suggest that dysregulation of IRP2 is an early nodal point underlying altered iron metabolism in breast cancer and may contribute to poor outcome of some patients with breast cancer.

Ferritin blocks inhibitory effects of two-chain high molecular weight kininogen (HKa) on adhesion and survival signaling in endothelial cells.

Angiogenesis is tightly regulated through complex crosstalk between pro- and anti-angiogenic signals. High molecular weight kininogen (HK) is an endogenous protein that is proteolytically cleaved in plasma and on endothelial cell surfaces to HKa, an anti-angiogenic protein. Ferritin binds to HKa and blocks its anti-angiogenic activity. Here, we explore mechanisms underlying the cytoprotective effect of ferritin in endothelial cells exposed to HKa. We observe that ferritin promotes adhesion and survival of HKa-treated cells and restores key survival and adhesion signaling pathways mediated by Erk, Akt, FAK and paxillin. We further elucidate structural motifs of both HKa and ferritin that are required for effects on endothelial cells. We identify an histidine-glycine-lysine (HGK) -rich antiproliferative region within domain 5 of HK as the target of ferritin, and demonstrate that both ferritin subunits of the H and L type regulate HKa activity. We further demonstrate that ferritin reduces binding of HKa to endothelial cells and restores the association of uPAR with α5ß1 integrin. We propose that ferritin blocks the anti-angiogenic activity of HKa by reducing binding of HKa to UPAR and interfering with anti-adhesive and anti-proliferative signaling of HKa.

Ferroportin and iron regulation in breast cancer progression and prognosis.

Ferroportin and hepcidin are critical proteins for the regulation of systemic iron homeostasis. Ferroportin is the only known mechanism for export of intracellular non-heme-associated iron; its stability is regulated by the hormone hepcidin. Although ferroportin profoundly affects concentrations of intracellular iron in tissues important for systemic iron absorption and trafficking, ferroportin concentrations in breast cancer and their influence on growth and prognosis have not been examined. We demonstrate here that both ferroportin and hepcidin are expressed in cultured human breast epithelial cells and that hepcidin regulates ferroportin in these cells. Further, ferroportin protein is substantially reduced in breast cancer cells compared to nonmalignant breast epithelial cells; ferroportin protein abundance correlates with metabolically available iron. Ferroportin protein is also present in normal human mammary tissue and markedly decreased in breast cancer tissue, with the highest degree of anaplasia associated with lowest ferroportin expression. Transfection of breast cancer cells with ferroportin significantly reduces their growth after orthotopic implantation in the mouse mammary fat pad. Gene expression profiles in breast cancers from >800 women reveal that decreased ferroportin gene expression is associated with a significant reduction in metastasis-free and disease-specific survival that is independent of other breast cancer risk factors. High ferroportin and low hepcidin gene expression identifies an extremely favorable cohort of breast cancer patients who have a 10-year survival of >90%. Ferroportin is a pivotal protein in breast biology and a strong and independent predictor of prognosis in breast cancer.

Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death.

In vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via alpha3beta1 and alpha6beta4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via alpha3beta1, but not alpha6beta4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through alpha3beta1 and alpha6beta4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.

The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells.

Tks5/Fish is a scaffolding protein with five SH3 domains and one PX domain. In Src-transformed cells, Tks5/Fish localizes to podosomes, discrete protrusions of the ventral membrane. We generated Src-transformed cells with reduced Tks5/Fish levels. They no longer formed podosomes, did not degrade gelatin, and were poorly invasive. We detected Tks5/Fish expression in podosomes in invasive cancer cells, as well as in human breast cancer and melanoma samples. Tks5/Fish expression was also required for protease-driven matrigel invasion in human cancer cells. Finally, coexpression of Tks5/Fish and Src in epithelial cells resulted in the appearance of podosomes. Thus, Tks5/Fish appears to be required for podosome formation, for degradation of the extracellular matrix, and for invasion of some cancer cells.

Chimeric and pseudotyped parvoviruses minimize the contamination of recombinant stocks with replication-competent viruses and identify a DNA sequence that restricts parvovirus H-1 in mouse cells.

Recent studies demonstrated the ability of the recombinant autonomous parvoviruses MVMp (fibrotropic variant of the minute virus of mice) and H-1 to transduce therapeutic genes in tumor cells. However, recombinant vector stocks are contaminated by replication-competent viruses (RCVs) generated during the production procedure. To reduce the levels of RCVs, chimeric recombinant vector genomes were designed by replacing the right-hand region of H-1 virus DNA with that of the closely related MVMp virus DNA and conversely. Recombinant H-1 and MVMp virus pseudotypes were also produced with this aim. In both cases, the levels of RCVs contaminating the virus stocks were considerably reduced (virus was not detected in pseudotyped virus stocks, even after two amplification steps), while the yields of vector viruses produced were not affected. H-1 virus could be distinguished from MVMp virus by its restriction in mouse cells at an early stage of infection prior to detectable viral DNA replication and gene expression. The analysis of the composite viruses showed that this restriction could be assigned to a specific genomic determinant(s). Unlike MVMp virus, H-1 virus capsids were found to be a major determinant of the greater permissiveness of various human cell lines for this virus.