INTRODUCTION
Cancer continues to be the major public health concern with 20 million new cases diagnosed and ten million deaths reported in 2022 worldwide[1]. Despite the monumental strides in understanding its biology and development of sophisticated technologies in diagnostics and therapeutics, its burden continues to grow. Tumor heterogeneity, toxicity and acquired resistance limit the efficacy of widely used cancer therapies such as radiotherapy, chemotherapy, gene therapy, and immunotherapy.
Regulated cell death pathways including anoikis, apoptosis, entosis, necroptosis, pyroptosis, ferroptosis, and autophagy critically maintain the cellular homeostasis under different dysregulated conditions in intracellular and extracellular microenvironment[2]. Among various death modes of tumor cells, ferroptosis, an iron-driven non-apoptotic programmed cell death characterized by depletion of antioxidants [glutathione (GSH) and GSH peroxidase 4 (GPX4)], glutamate overload, iron accumulation and excessive lipid peroxidation has been extensively studied since its discovery in 2012 by Dixon et al[3].
Exogenous pathways induce ferroptosis by inhibiting cystine-glutamate antiporter (solute carrier family 7, member 11, system Xc-), affecting GSH synthesis and its activity, accumulating lipid reactive oxygen species (ROS) and increasing oxidative damage. The endogenous pathway network comprises of lipid metabolism, iron metabolism and mitochondria-related pathways. Many important metabolic pathways such as the production of adenosine triphosphate through oxidative phosphorylation, fatty acid beta oxidation, tricarboxylic acid cycling, iron and calcium homeostasis, ROS production, cell signaling and apoptosis take place in mitochondria. This makes mitochondrial dynamics to play crucial role in ferroptosis[4]. Increased oxidative stress induces irreversible damage to mitochondria, diminishes organelle integrity, and elimination of dysfunctional mitochondria through specific form of autophagy known as mitophagy. Mitochondria undergo fusion and fission to maintain their integrity and homeostasis. Mitochondrial fusion is regulated by mitofusin (MFN) 1, MFN2 and optic atrophy protein 1 whereas dynamin-related protein 1 regulates the fission process. The balance between mitochondrial fission and fusion and hence the mitochondrial dynamics is disrupted by iron overload, thereby regulates mitophagy. It is mainly mediated by phosphatase and TENsin homolog deleted on chromosome 10 induced putative kinase 1/ parkin and Bcl2 interacting protein 3 (BNIP3) pathways[5,6]. Accumulation of putative kinase 1 on the outer mitochondrial membrane recruits and activates parkin which ubiquitinates voltage-dependent anion channel 1 and MFN1/2 proteins to induce mitophagy. BNIP3 receptor, an external mitochondria-associated atypical BH3 member of the Bcl-2 family interacts with LC3B interaction region motif and targets damaged mitochondria for lysosomal degradation[6]. This ultimately leads to energy depletion and cell death. Morphological features during ferroptosis include membrane shrinkage with increased mitochondrial membrane density, decrease in mitochondrial volume and a reduced number or loss of mitochondrial cristae[7].
Complex role of autophagy in promoting or suppressing ferroptosis depending on the specific cellular context is well investigated. Ferritinophagy, special form of autophagy selectively degrades ferritin, an iron storage protein by allowing its binding to the cargo receptor, nuclear receptor coactivator 4, and delivering it to the lysosome for breakdown. As a result, stored iron is released into the cytosol, increasing the labile iron pool which leads to ferroptosis[8]. Autophagic/chaperone mediated autophagic degradation of anti-ferroptotic proteins, GPX4, makes cells vulnerable to ferroptosis. Mitophagy induces ferroptosis by affecting mitochondrial function, including ROS production and iron homeostasis. Non-selective autophagy inhibits ferroptosis by removing damaged organelles and misfolded proteins and reducing oxidative stress. Lipophagy-mediated removal of lipid droplets results in inhibition of ferroptosis.
Deeper understanding of the complex interplay of ferroptosis with mitochondrial dynamics and autophagy is important for developing targeted therapies that can modulate ferroptosis for therapeutic benefit.
This article comments on the article by Rana and Prajapati[9] published in the recent issue, focusing on detailed mechanism of an interplay among ferroptosis, mitochondrial dynamics and autophagy, with an emphasis on its application in treating tumor cells, significant challenges and future perspectives exploring new effective treatment strategies with improved safety profiles.
TARGETING FERROPTOSIS, MITOCHONDRIAL DYNAMICS AND AUTOPHAGY CROSSTALK: PRECLINICAL STUDIES TO EVALUATE ANTICANCER POTENTIAL
Owing to the important role of ferroptosis in the pathological mechanism of tumor development, treatment with ferroptosis targeting inducers effectively eliminates tumor cells through its tumor-suppressor function and thereby holds great therapeutic potential. Among class I ferroptosis inducers (FINs), erastin directly inhibited system Xc, reduced GSH level and inhibited the growth of cervical cancer and ovarian cancer cells[10]. It enhanced the chemotherapeutic potential of anti-cancer drugs such as doxorubicin, cisplatin, temozolomide, cytarabine, etc. in cancer cell lines by targeting voltage-dependent anion channel and RAS genes[11]. However, its poor solubility and unstable metabolism limit its application. Erastin derivatives piperazine erastin and imidazolone erastin exhibit better solubility and stability and are studied to effectively suppress tumor cell growth in experimental models of fibrosarcoma and diffuse large B cell lymphoma[12,13]. Poor pharmacokinetics, lower potency and metabolic stability of Food and Drug Administration-approved immunosuppressant sulfasalazine (inhibits system Xc-) limit its clinical application. However, it is shown to increase the therapeutic potential of standard chemotherapeutic and autophagy-inducing agent temozolomide in the treatment of glioma-induced brain edema[14]. Sorafenib, a well-known inhibitor of receptor tyrosine kinases and system Xc-, is an Food and Drug Administration approved anti-cancer drug to treat thyroid cancer, hepatocellular carcinoma and renal cellular carcinoma[15]. Buthionine sulfoximine is examined to block the synthesis of GSH and suppress the mouse breast cancer growth and escalate the melphalan chemosensitivity of melanoma and neuroblastoma cells[16-18]. Another study reported the ability and efficacy of engineered human cystathionine gamma lyase in impeding the prostate and breast cancer xenografts’ growth and improving the mouse survival in a chronic lymphocytic leukemia by degrading cysteine and cystine with a higher kinetic rate[15,18]. Up-regulation of the heat shock protein is shown to enhance the resistance of cancer cells to the treatment with class I FINs. Targeted inactivation of the activity of GPX4 by class II FINs, including RAS-selective lethal 3 (RSL3), withaferin A and altretamine inhibited the growth of fibrosarcoma, neuroblastoma xenografts and ovarian cancer cells respectively by inducing ferroptosis through GPX4 inhibition[11]. Class III FINs such as ferroptosis suppressor protein 1 inhibitor blocked ferroptosis suppressor protein 1 and reduced the content of ubiquinone to increase lipid peroxidation; whereas another FIN, FIN56 resulted in depletion of GPX4 and coenzyme Q10 through the squalene synthase-mevalonate pathway[11]. Class IV FINs such as BAY87-2243, FINO2 induced ferroptosis by promoting the synthesis of lipid peroxides by expanding the intracellular labile iron pool or the amounts of iron oxide and may provide new opportunities for cancer treatment. High heterogeneity of the sensitivity of tumors to ferroptosis limits its curative potential. This emphasizes the importance of screening ferroptosis-sensitive tumors with anticancer drugs.
Context dependent dual role of mitophagy in enhancing or suppressing ferroptosis is well examined. Increased mitophagy-dependent ROS production and ferroptosis following the inhibition of mitochondrial complex I is observed in melanoma cells[19]. Sequestration of iron in mitophagosomes and thus reduction in the amounts of source materials for ROS in ferroptosis is noted under mild stress or in the early stages of iron overload[20]. Moderate induction of mitophagy could preserve mitochondrial number and function and thus suppresses ferroptosis. On the other hand, extensive mitophagy results in accumulation of additional iron, which amplifies lipid peroxidation and ferroptosis.
Targeting the synergistic and antagonistic relationship between autophagy and ferroptosis in tumor type and stage-dependent manner opens up noble opportunities in cancer treatment. Study by Sandoval-Acuña et al[21] investigated the anti-tumor therapeutic efficacy of deferoxamine via suppressing growth of tumor cells and metastasis by targeting mitochondrial iron metabolism and inducing mitochondrial dysfunction and mitophagy. Interplay between mechanistic target of rapamycin signaling and GPX4 has been shown to modulate autophagy-dependent ferroptosis and increase the sensitivity of radiation therapy in xenograft models of pancreatic cancer cells, suggesting novel therapeutic avenues[22]. Pharmacological inhibition of myoferlin, an oncoprotein (highly expressed in pancreatic ductal adenocarcinoma and regulates the cell membrane biology), via WJ460 could induce mitophagy, release of ROS and its accumulation, lipid peroxidation and apoptosis independent cell death. A synergic effect between FINs, erastin/RSL3, and WJ460 make this combinational regimen a promising strategy for anti-tumor intervention in pancreatic cancer cells[23]. On the contrary, mitochondrial inhibitor, mitochondrial division inhibitor 1 and an iron chelator inhibits ROS production and restores cell proliferation[23]. Induction of autophagy-mediated ferroptosis by polyphyllin VII (a pennogenin isolated from the rhizomes of Paris polyphylla) suppressed growth of gastric cancer[24]. Combinational approach based on use of tamoxifen as an autophagy inducer and sulfasalazine as FINs could effectively treat triple negative breast cancer cells[25]. Recent studies decipher the selective inhibition of the growth and distant metastasis of mitophagy-deficient tumors in vivo upon treatment with ferroptosis-inducers. Defects in mitochondrial degradation or mitophagy mediated by reduced expression or deletion of Unc-51-like kinase 1 promoted nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome activation and interleukin-1 secretion and malignant progression of breast cancer. Another study by Chourasia et al[26] reported that defective mitophagy due to BNIP3 deficiency enhanced hypoxia inducible factor-1 stabilization by increasing mitochondrial ROS, and promoted lung metastasis of breast cancer cells. Study by Liu et al[5] screened and established the patient-derived organoid models of colorectal cancer for ferroptosis-sensitive mitophagy-deficient tumors. Lipid peroxidation-activating transcription factor 4-parkin pathway mediated mitophagy resulted in lipid peroxidation and suppressed ferroptosis in cancer. Combinational approach based on induced ferroptosis in autophagy/mitophagy deficient tumor cells by erastin, RSL3, cysteine deprivation, radiotherapy or immunotherapy provides new therapeutic strategies for malignantly progressing tumors.
Regulation of ferroptosis through autophagy is examined as a promising anti-cancer strategy in overcoming drug resistance. Treatment with lysosomal inhibitors plays an instrumental role in reducing the autophagic degradation of ferritin, partially blocks intracellular iron transport and the burst of ROS associated with ferroptosis. Study by Liu et al[27] reported the effect of 4-octyl itaconate on ferroptosis induction by targeting nuclear receptor coactivator 4-mediated ferritin autophagy and thereby on the death of multi-drug resistant human retinoblastoma cells. Combinational approach based on the application of autophagy inhibitor hydroxychloroquine and the mitogen-activated protein kinase kinase inhibitor trametinib resulted in enhanced antiproliferative activity against KrasG12D/+; Lkb1-/- (KL) lung cancer cells by impairing glucose metabolism. This cotreatment resulted in mitochondrial dysfunction, destructive oxidative stress and triggered ferroptosis[28]. Ultrasmall iron oxide nanoparticles mediated upregulation of autophagy via Beclin1/autophagy related 5-related pathways and ferroptosis induction could treat glioblastoma cells[29].
