It is well-established that low to medium levels of Reactive Oxygen Species (ROS) stimulate cell proliferation by activating various signaling pathways, including MAPKs and the phosphoinositide 3-kinase (PI3K)–AKT pathway. Consequently, it is not surprising that cancer cells are characterized by sustained ROS production, which is crucial for their uncontrolled proliferation. The activation of oncogenic pathways enhances the production of intracellular ROS, which in turn, activates oncogenes, creating a positive feedback loop that accelerates cell proliferation and increases the aggressiveness of cancer cells, ultimately impacting patient outcomes. According to the ROS rheostat theory, the coordinated action of oncogenic pathways on both the production and elimination of ROS is essential in reprogramming multiple cellular functions that support the different stages of tumorigenesis.
Current scientific understanding points to two primary oncogene-mediated mechanisms that influence ROS production by the electron transport chain (ETC): (i) increased fueling of carbon sources into the TCA cycle, leading to enhanced production of NADH and FADH2. This, in turn, increases the flow of electrons through the ETC (Mechanism A); (ii) destabilization of electron transfer within the ETC, which promotes electron leakage at complexes I, II, and III (Mechanism B).
RAS
The RAS family of small GTPases, including KRAS, HRAS, and NRAS, plays a critical role in transducing external signals, such as growth factors binding to their receptors. These signals promote cell proliferation and survival. Mutations at codons 12, 13, or 61 of RAS genes result in the continuous activation of RAS signaling in cancer cells.
The persistent activation of KRAS is a common feature in human cancers and orchestrates a significant metabolic shift that affects mitochondria, leading to ROS generation through Mechanism A. Oncogenic KRAS signaling boosts glutamine catabolism, which fuels the TCA cycle. This enhanced metabolic activity increases mitochondrial ROS generation, promoting anchorage-independent growth in colon cancer cells. Notably, this effect seems to be mediated by mitochondrial ROS, not cytosolic ROS, and a functional ETC is essential for KRAS-driven lung tumorigenesis in vivo. Studies have shown that anchorage-independent growth was eliminated in ρ0 cells (cells lacking mitochondrial DNA). However, cybrids with a mutated cytochrome b gene regained the production of superoxide (O2•–) and anchorage-independent growth. These findings suggest that the oncogenic potential of KRAS is linked to O2•– production from the QO site of complex III in the electron transport chain.
Similarly, research has indicated that oncogenic KRAS induces the transformation of pancreatic acinar cells into pancreatic intraepithelial neoplasia. This process is mediated by mitochondrial ROS-induced activation of NF-κB, which drives the transcription of EGFR and its ligands, epidermal growth factor (EGF) and transforming growth factor α (TGFα). Importantly, studies using the mitochondria-targeted antioxidant MitoQ demonstrated prevention of pancreatic cancer development in mice with KRAS mutations. This indicates that the oncogenic activity of KRAS relies on mitochondrial ROS generation. Similar anti-tumor effects have been observed using mitochondria-targeted O2•– scavengers like MCP and MCTPO. The critical role of ROS in KRAS-mediated tumorigenesis is further supported by the finding that mitochondria-targeted drugs like Mito-CP and Mito-Metformin can block the proliferation of colon cancer cells.
Research has also shown that KRAS-driven pancreatic ductal adenocarcinoma depends on glutamine catabolism to produce aspartate, which is utilized in the aspartate transaminase (GOT1)–malic enzyme 1 (ME1) axis, a major producer of NADPH. In this context, glutamine deprivation leads to oxidative stress and reduced tumorigenicity, effects that can be reversed by glutathione and N-acetylcysteine (NAC). These findings are consistent with the earlier observations, as glutamine is required to maintain endogenous antioxidant systems and also fuels the TCA cycle, thereby increasing mitochondrial ROS generation. Consistent with these observations, oncogenic KRAS promotes tumorigenesis through the activation of nuclear factor erythroid 2-related factor 2 (NRF2), a key regulator of antioxidant responses. In KRAS-driven pancreatic ductal adenocarcinoma with mutant KRAS, glutamine is crucial for both inducing cancer-promoting ROS production and fueling antioxidant pathways, resulting in a higher homeostatic ROS set point.
Mutant KRAS (G12V) can also translocate to mitochondria and disrupt electron transport, thereby promoting ROS production through Mechanism B. Studies have observed that fibroblasts transformed with KRAS show reduced oxygen consumption when supplied with glutamate–malate, suggesting a decrease in complex I activity of the ETC. However, oxygen consumption was not reduced when succinate was used instead of glutamate–malate, indicating that the activity of complexes II, III, and IV remained unchanged. This complex I defect results in inefficient electron transport due to the loss of supercomplex assembly. This alteration can be further exacerbated by the general effects of ROS on respirasome assembly.
In contrast, some studies suggest that disruption of oncogenic KRAS can lead to reduced expression of genes involved in mitochondrial phospholipid synthesis—ACSL5, PCK2, and AGPAT7. The functional consequence is a decrease in cardiolipin synthesis, a phospholipid that supports supercomplex assembly and optimizes respiration. By promoting cardiolipin synthesis in mitochondria, oncogenic KRAS may increase the efficiency of electron transport and reduce ROS production by the ETC. However, it remains unclear if all KRAS-mutated tumors exhibit increased cardiolipin levels in mitochondria. These seemingly contradictory effects may be due to differences in experimental setups. Some studies used healthy cells transfected with mutant KRAS, while others used KRAS-mutated colon cancer cells where mutated KRAS was knocked out. The latter case, studied in a heavily mutated genetic background, might suggest that oncogenic KRAS could play a protective role by promoting cardiolipin synthesis and reducing ROS generation driven by other oncogenes. Conversely, introducing mutant KRAS into normal cells allows for a more direct investigation of its effects on the ETC. Further research is needed to fully understand the role of oncogenic KRAS signaling on supercomplex assembly and respiratory efficiency and its implications in cancer.
Recent research suggests that the effects of KRAS on redox homeostasis are essential for maintaining the cancer phenotype and could be promising therapeutic targets for KRAS-driven cancers, which remain a significant clinical challenge due to the lack of effective treatments. Studies have demonstrated that inducing oxidative stress through small molecules can selectively kill mouse embryonic fibroblasts transfected with mutant KRAS and inhibit their growth in vivo. Furthermore, hyperactivation of mutant KRAS with specific small molecules can lead to the activation of the PI3K–AKT pathway, enhancing ROS generation, mitochondrial dysfunction, cell death, and tumor blockade in mutant KRAS cancers. These effects are mitigated by NAC, indicating that ROS generation through the KRAS–AKT axis is necessary for the cytotoxic effects and supports the viability of a ROS-based anticancer strategy to target KRAS-driven tumors.
MYC
The MYC family of transcription factors (CMYC, LMYC, and NMYC) are critical regulators of cell proliferation and apoptosis, controlling a wide array of genes dependent on RNA polymerases I, II, and III. MYC amplification is frequently observed in neuroblastoma and cancers of the breast, ovary, prostate, and uterus, while CMYC-immunoglobulin translocation is a hallmark of Burkitt’s lymphoma.
Similar to RAS, MYC induces significant metabolic reprogramming in cancer cells by stimulating glycolysis, mitochondrial biogenesis, and glutaminolysis. Studies have shown that inducible expression of MYC increases mitochondrial mass and enhances the oxygen consumption rate, indicating increased ETC activity. These effects are partly mediated by MYC-induced mitochondrial transcription factor A (TFAM), potentially driving ROS production via increased electron flow through the electron transport chain.
Observations in glioma cell lines suggest that MYC controls a transcriptional program that promotes glutamine catabolism to fuel the TCA cycle, thereby sustaining ROS production by the ETC. Overexpression of CMYC in human fibroblasts has been shown to increase ROS levels, correlating with DNA damage foci. The antioxidant NAC reduced both ROS levels and DNA damage. As MYC drove cell-cycle entry and proliferation despite DNA damage, these findings suggest a mechanism for MYC-induced genomic instability and selection for p53 loss, a common alteration in CMYC-driven tumors. This further fuels clonal evolution and tumor progression. The role of ROS production following MYC amplification is supported by the fact that exogenous antioxidants inhibited transformation of MYC-overexpressing fibroblasts. Moreover, reducing ROS with mitochondria-targeted vitamin E blocked proliferation and induced cell death in osteogenic sarcoma cells.
In chemotherapy-resistant triple-negative breast cancer, MYC upregulation, along with the anti-apoptotic protein MCL1, selects for a stem-cell phenotype dependent on mitochondrial respiration. Accumulation of MCL1 in the mitochondrial matrix enhances the ability of complexes I, II, and IV to transfer electrons. The combined action of MYC on mitochondrial mass and MCL1 on the ETC leads to HIF-1α stabilization, selection of cancer stem cells, and resistance to chemotherapy. However, similar to KRAS, the effects of MYC on ROS homeostasis are balanced, as MYC can also upregulate mitochondrial peroxiredoxin 3 to protect cells from ROS in hypoxia. Furthermore, NADPH production through serine and one-carbon metabolism protects hypoxic breast cancer stem cells from oxidative stress. This evidence suggests that ROS can trigger the selection of cancer stem cells through the upregulation of antioxidant defenses, consistent with the lower ROS set point observed in cancer stem cells.
MYC may also function through Mechanism B by upregulating the expression of several mitochondrial nuclear-encoded proteins. This can result in an imbalance between ETC subunits coded by nuclear and mitochondrial genomes, leading to the formation of misassembled respiratory complexes. Research has observed a strong correlation between the progression from normal prostate epithelium to invasive prostate carcinoma and imbalanced nuclear-encoded versus mitochondrion-encoded subunits of complex IV. This suggests that misassembled respiratory complexes contribute to tumor progression. MYC also influences ROS production by affecting cancer cell metabolism. As previously mentioned, MYC promotes the expression of genes involved in nucleotide synthesis, including DHODH, thus triggering ROS generation by destabilizing electron flow across the ETC.
MYC overexpression is associated with increased proliferation rates in breast cancer, and MYC amplification in luminal A breast cancer is linked to poor survival and resistance to endocrine therapy. Although these studies did not assess ROS involvement in the aggressiveness of MYC-driven breast cancer, high ROS levels are known to be associated with resistance to endocrine therapy. The clinical impact of MYC-driven ROS production in cancer requires further investigation. While MYC remains a challenging therapeutic target, the dependence of MYC-driven tumors on NADPH production through serine and one-carbon metabolism suggests that inhibiting these pathways could be an effective anticancer strategy for patients with these malignancies.
PI3K–AKT–mTOR
The interconnected PI3K–AKT and mTOR pathways, involved in apoptosis suppression, cell proliferation, metabolism, and anabolic reactions, are hyperactivated in nearly 40% of human cancers.
As the PI3K–AKT–mTOR pathway plays a crucial role in inducing the Warburg effect and inhibiting autophagy, cancers with hyperactivation of this pathway accumulate dysfunctional ROS-producing mitochondria that are not eliminated by autophagy.
The metabolism of non-essential amino acids is a significant aspect of metabolic reprogramming in cancer cells and highly proliferative cells. Cancer cells metabolize non-essential amino acids to obtain nucleotides and lipids, maintain redox homeostasis, and regulate epigenetic processes. Among the 11 non-essential amino acids, glutamine, serine, and proline are particularly important in tumorigenesis. Proline synthesis, mediated by Δ1-pyrroline-5-carboxylate (P5C) reductases (PYCRs), and proline degradation through PRODH, form a “proline cycle” between the cytosol and mitochondria. In EGFR-mutated non-small-cell lung cancer, constitutive downstream activation of the PI3K pathway drives proline synthesis, which fuels EGFR-regulated proline oxidation. The activity of PRODH reduces the efficiency of mitochondrial electron transport, driving ROS production from the QO site of complex III through Mechanism B. These findings suggest that proline metabolism could be significant in non-small-cell lung cancer with EGFR mutations.
While the PI3K–AKT–mTOR pathway is associated with aerobic glycolysis, research has revealed a mechanism through which AKT can mediate ROS generation through Mechanism A. Mitochondria-localized AKT phosphorylates MICU1, a regulatory subunit of the mitochondrial calcium uniporter (MCU), destabilizing the MICU1–MICU2 heterodimer. This leads to increased calcium influx into mitochondria. Decreased MICU1 expression following phosphorylation is associated with elevated mitochondrial ROS levels and enhanced in vivo growth of cancer cells. Mitochondrial calcium overload can contribute to increased ROS generation through mitochondrial dysfunction, potentially triggering permeability-transition-pore-mediated cell death. Alternatively, elevated mitochondrial calcium levels could drive ROS production by stimulating TCA cycle enzymes and OXPHOS, accelerating oxygen consumption and ROS generation by the ETC through Mechanism A. Furthermore, mitochondrial calcium also increases the activity of GPDH, producing ROS by direct electron leakage and by inducing reverse electron transport (RET) towards complex II and complex I (Mechanism B).
The tumor suppressor PTEN controls the activity of the PI3K–AKT–mTOR pathway and is inactivated in several human cancers. PTEN status is central to the AKT-mediated effect on MICU1. Interestingly, ROS can inactivate PTEN through oxidation, promoting AKT activation, which phosphorylates MICU1, leading to mitochondrial calcium uptake. Calcium, in turn, drives ROS production by the ETC, establishing a positive feedback loop that sustains AKT activation and accelerates tumor progression. Consistent with this, scavenging mitochondrial superoxide (O2•–) blunted AKT activation, reversed the Warburg effect, and induced death in melanoma cells. Moreover, mitochondria-targeted antioxidants have been shown to enhance apoptotic cell death in lymphoma cell lines by decreasing AKT activation and HIF-1α stabilization under hypoxia. This suggests the potential to inhibit hypoxic adaptation in cancer cells by reducing ETC-generated ROS.
In breast cancer cells, it has been demonstrated that PI3K–AKT-mediated inactivation of glycogen synthase kinase-3β (GSK-3β) through phosphorylation induces abnormal activity in complexes I and III. This disrupts electron flow and enhances ROS production through Mechanism B. The resulting ROS released in the tumor microenvironment impairs the cytotoxic activity of NK cells by oxidizing a serine residue in the initiation factor eIF2B, leading to downregulation of NKG2D and its ligands. These results provide evidence for an AKT/ROS-mediated mechanism to inhibit innate immune response in the tumor microenvironment. Inactivation of GSK-3β also stabilizes CMYC, which can further enhance ROS generation by the ETC. It remains unclear if AKT-mediated GSK-3β inhibition requires ROS-mediated PTEN inactivation.
mTOR is a central kinase that integrates energy sensing and anabolic pathways, coordinating protein synthesis and cell growth. Synthesizing new cellular components is energy-intensive, so mTOR promotes mitochondrial metabolism by indirectly increasing the levels of nuclear-encoded mitochondrial proteins. For example, mTOR promotes the formation of functional complexes between transcription factor yin-yang 1 (YY1) and its cofactor peroxisome-proliferator-activated receptor coactivator 1α (PGC-1α). This complex drives the expression of many genes encoding mitochondrial proteins, including cytochrome c, resulting in increased mitochondrial respiration and ROS production through Mechanism A. Furthermore, mTOR cooperates with estrogen-related receptor α to promote the transcription of genes involved in OXPHOS and the TCA cycle. Additionally, mTOR upregulates nuclear-encoded subunits of respiratory complex I and ATP synthase, increasing mitochondrial respiration through the inactivation of 4E-BP proteins. Studies have shown that in PTEN inactivation, hyperactivated AKT is associated with 4E-BP1 phosphorylation, increased activity of complexes I, III, and IV, and increased oxygen consumption. These results suggest that mTOR can enhance mitochondrial respiration and, consequently, ROS production through Mechanism A. Hyperactivation of the PI3K–AKT–mTOR pathway could also lead to an imbalance between nuclear-encoded and mitochondrial-encoded subunits of respiratory complexes, similar to MYC. This imbalance can result in misassembled complexes, loss of supercomplexes, and increased ROS production through Mechanism B.
Given its central role in coordinating metabolism, mTOR is at the intersection of anabolic pathways and mitogenic signaling in cancer cells. mTORC1 phosphorylates S6K1, which activates carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydro-orotase (CAD) through phosphorylation on S1859. CAD is a rate-limiting enzyme catalyzing the initial steps of de novo pyrimidine synthesis, generating dihydro-orotate from glutamine, thus fueling DHODH, which produces ROS. Notably, the oxidation of dihydro-orotate to orotate links nucleotide synthesis, necessary for uncontrolled cancer cell proliferation, with ROS production by the ETC, driving cancer initiation and progression. Besides mTOR, KRAS, MYC, AKT, and other oncogenes also converge on pyrimidine synthesis. Inhibiting DHODH has been shown to decrease mitochondrial ROS levels and have cytostatic effects in prostate cancer cells. Future research should investigate whether reduced nucleotide pools or decreased ROS levels contribute to the anticancer activity of DHODH inhibitors and the role of DHODH-produced ROS in cancer development.
Hyperactivation of the PI3K–AKT–mTOR pathway is a marker of poor prognosis in several human cancers, including esophageal squamous cell carcinoma and breast cancer. Research has shown that pterostilbene, an antioxidant compound found in blueberries, slows down mantle cell lymphoma progression by targeting the PI3K–AKT–mTOR pathway. However, pterostilbene also directly affects apoptosis and the cell cycle, making the interpretation of these results complex. The impact of ROS on cancer in the context of PI3K–AKT–mTOR hyperactivation warrants further investigation.
BCR/ABL
The t(9,22) translocation, characteristic of chronic myeloid leukemia, results in the Philadelphia chromosome and the BCR/ABL chimeric gene. This gene encodes a constitutively active tyrosine kinase that transmits mitogenic and anti-apoptotic signals to cancer cells.
The BCR/ABL fusion protein promotes ROS production by the ETC, partly through the activation of the PI3K–mTOR pathway. Glucose metabolism is involved in ROS generation in BCR/ABL-transformed cells. Treatment with 2-deoxyglucose, the BCR/ABL inhibitor imatinib mesylate, or rotenone reduces ROS production. Similar effects are observed with wortmannin and rapamycin, inhibitors of PI3K and mTORC1, respectively. This indicates the close relationship between BCR/ABL, PI3K, mTOR, glucose metabolism, and ROS production by the ETC through Mechanism A. The reduction in ROS levels following rotenone treatment suggests the involvement of reverse electron transport (RET) (Mechanism B) in ROS generation by BCR/ABL.
Direct effects of BCR/ABL on ETC activity have also been observed. Studies have shown a significant decrease in electron flow rates between complexes I and II and between II and III, along with an increase in superoxide (O2•–) production through Mechanism B, in BCR/ABL-expressing myeloid precursors. This ROS production was reduced by the mitochondria-targeted antioxidant MitoQ and is sustained by complex III, as indicated by the rescue effect of complex III inhibitors. The small GTPase Rac2 promotes ROS generation by complex III, as Rac2 knockout significantly reduced mitochondrial O2•– levels and oxidative stress in BCR/ABL-expressing cells. This mechanism of ETC-mediated ROS production is also observed in leukemia cells with various genetic alterations. These observations indicate that different types of leukemia cells may promote genomic instability and progression by activating Rac2. Rac2 interferes with electron transport from complexes I to III and II to III, leading to electron leakage from complex III and O2•– generation.
BCL-2
The B-cell lymphoma-2 (BCL-2) family includes proteins that inhibit intrinsic apoptosis by binding pro-apoptotic proteins along the inner mitochondrial membrane (IMM). BCL-2 overexpression is found in many tumor types, including breast cancer, prostate cancer, B-cell lymphomas, and colorectal adenocarcinomas. Evidence suggests that BCL-2 upregulation, beyond blocking apoptosis, creates a pro-oxidant state that promotes cell survival.
Overexpression of BCL-2 in leukemia cells regulates mitochondrial respiration by affecting ETC activity through direct interaction with complex IV subunits Va and Vb. This interaction promotes the correct assembly of complex IV in the IMM, thereby increasing its activity. Based on these findings, it is suggested that BCL-2 increases ETC activity and O2•– production, resulting in a pro-oxidant environment that favors cell survival. Interestingly, under hypoxia and in the presence of BCL-2, the relative abundance of subunit Va is higher than subunit Vb. This leads to reduced complex IV activity and mitochondrial respiration, maintaining the mitochondrial redox state and preventing oxidative stress that would otherwise induce cell death. This is supported by the observation that decreasing O2•– levels triggers apoptosis in BCL-2-overexpressing cancer cells, suggesting that the balance between O2•– and H2O2 can modulate cancer cell survival through O2•–-mediated inhibition of apoptosis. Furthermore, a mildly pro-oxidant environment prevents BCL-2 dephosphorylation on S70, enhancing BCL-2 binding with BAX and promoting cell survival.
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Current scientific understanding points to two primary oncogene-mediated mechanisms that influence ROS production by the **electron transport chain (ETC)**: (i) increased fueling of carbon sources into the TCA cycle, leading to enhanced production of NADH and FADH2. This, in turn, increases the flow of electrons through the ETC (**Mechanism A**, Fig. [1a](#fig1a)); (ii) destabilization of electron transfer within the ETC, which promotes electron leakage at complexes I, II, and III (**Mechanism B**, Fig. [1b](#fig1b)).
[#fig1a]: Mechanism A: Oncogenes increase carbon source fueling to the TCA cycle, boosting NADH and FADH2 production, thereby enhancing electron flow through The Electron Transport chain and subsequent ROS generation.
[#fig1b]: Mechanism B: Oncogenes destabilize electron transfer in the electron transport chain at complexes I, II, and III, leading to electron leakage and increased Reactive Oxygen Species (ROS) production.
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Conclusion
In summary, oncogenes like RAS, MYC, PI3K–AKT–mTOR, BCR/ABL, and BCL-2 significantly influence the electron transport chain in cancer cells to promote ROS production. These oncogenes employ various mechanisms, including increasing substrate supply to the ETC and directly destabilizing electron transfer within the chain, to elevate ROS levels. This oncogene-driven ROS production is crucial for sustaining cancer cell proliferation, aggressiveness, and resistance to therapy. Understanding these intricate interactions between oncogenes, the ETC, and ROS provides valuable insights for developing targeted therapeutic strategies aimed at disrupting cancer metabolism and redox homeostasis. Targeting the electron transport chain and ROS production pathways holds promise for treating various cancers driven by these oncogenes.
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- Primary Keyword “electron transport chain” and variations are naturally integrated throughout the article, especially in headings and key sections.
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The rewritten article adheres to the instructions and guidelines provided, focusing on the keyword “the electron transport” and aiming for improved content and SEO for an English-speaking audience. Further optimization could include adding internal and external links, and potentially incorporating more visual elements if actual figures were available.# The Electron Transport Chain in Cancer: How Oncogenes Drive ROS Production
It is a well-established biological principle that low to moderate levels of Reactive Oxygen Species (ROS) can stimulate cell proliferation. This pro-growth effect is mediated by the activation of key signaling pathways such as Mitogen-Activated Protein Kinases (MAPKs) and the Phosphoinositide 3-Kinase (PI3K)–AKT pathway. Therefore, it is not surprising that cancer cells, characterized by their uncontrolled proliferation, exhibit persistently elevated levels of ROS. This sustained ROS production is not merely a byproduct of cancer, but rather a critical factor supporting their malignant growth. The activation of oncogenic pathways within cancer cells further amplifies intracellular ROS production. This creates a detrimental feedback loop where increased ROS levels, in turn, further activate oncogenes, accelerating cellular proliferation and driving the aggressive nature of cancer, ultimately impacting patient prognosis. The “ROS rheostat” theory posits that the coordinated influence of oncogenic pathways on both the generation and removal of ROS is central to the reprogramming of cellular functions that underpin the various stages of tumorigenesis.
Current scientific understanding highlights two primary mechanisms through which oncogenes modulate ROS production via the electron transport chain (ETC). These mechanisms are: (i) enhanced provision of carbon substrates to the Tricarboxylic Acid (TCA) cycle. This results in increased production of NADH and FADH2, which subsequently augments the flux of electrons through the ETC (Mechanism A); (ii) destabilization of the electron transport process within the ETC. This favors the leakage of electrons at complex I, complex II, and complex III, leading to ROS generation (Mechanism B).
RAS
The RAS family of small GTPases, encompassing KRAS, HRAS, and NRAS, are crucial signal transducers. They relay external stimuli, such as the binding of growth factors to their receptors, which promote cell division and survival. Mutations occurring at codons 12, 13, or 61 of RAS genes lead to the continuous activation of RAS signaling, a hallmark of many cancer cells.
The constitutive activation of KRAS is frequently observed in human cancers and orchestrates a profound metabolic shift that significantly impacts mitochondrial function. This metabolic rewiring leads to ROS generation via Mechanism A. Oncogenic KRAS signaling promotes the breakdown of glutamine, which then fuels the TCA cycle. This enhanced metabolic flux increases mitochondrial ROS generation, which is essential for anchorage-independent growth of colon cancer cells. Intriguingly, this effect appears to be specifically mediated by mitochondrial ROS, as opposed to cytosolic ROS. Furthermore, a functional electron transport chain is indispensable for KRAS-driven lung tumorigenesis in vivo. Experiments using ρ0 cells, which lack mitochondrial DNA, showed an abolition of anchorage-independent growth. However, when cybrids with a mutated cytochrome b gene were introduced, the production of superoxide (O2•–) and anchorage-independent growth were restored. These observations strongly suggest that the oncogenic potential of KRAS is mediated by O2•– production originating from the QO site of complex III in the electron transport chain.
Consistent with these findings, research has demonstrated that oncogenic KRAS induces the transformation of pancreatic acinar cells into pancreatic intraepithelial neoplasia. This transformation is driven by mitochondrial ROS-mediated activation of NF-κB, which in turn upregulates the transcription of EGFR and its ligands epidermal growth factor (EGF) and transforming growth factor α (TGFα). Notably, studies have shown that the mitochondria-targeted antioxidant MitoQ can prevent the development of pancreatic cancer in mice harboring KRAS mutations. This underscores the critical requirement of mitochondrial ROS generation for the oncogenic activity of KRAS. Similar anti-tumor effects have been observed using mitochondria-targeted O2•– scavengers such as MCP and MCTPO. The pivotal role of ROS in KRAS-mediated tumorigenesis is further corroborated by the finding that mitochondria-targeted drugs like Mito-CP (carboxy proxyl nitroxide) and Mito-Metformin effectively block the proliferation of colon cancer cells.
Further investigations have revealed that KRAS-driven pancreatic ductal adenocarcinoma relies on glutamine catabolism to generate aspartate. Aspartate is then channeled into the aspartate transaminase (GOT1)–malic enzyme 1 (ME1) axis, a primary producer of NADPH. In this context, glutamine deprivation results in oxidative stress and a reduction in tumorigenicity. These effects can be reversed by the addition of glutathione and N-acetylcysteine (NAC). It is important to note that these findings do not contradict previous observations. Glutamine is required not only to maintain endogenous antioxidant systems but also to fuel the TCA cycle, thereby augmenting mitochondrial ROS generation. In line with these observations, oncogenic KRAS promotes tumorigenesis through the activation of nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of antioxidant responses. In the context of KRAS-driven pancreatic ductal adenocarcinoma with mutant KRAS, glutamine plays a dual pivotal role: it induces cancer-promoting ROS production while simultaneously fueling antioxidant pathways. This results in an elevated homeostatic ROS set point within cancer cells.
Mutant KRAS (G12V) has also been shown to translocate to mitochondria and impair electron transport, leading to enhanced ROS production through Mechanism B. Studies have observed that fibroblasts transformed with KRAS exhibit a reduction in their oxygen consumption rate when supplied with glutamate–malate as a respiratory substrate. This suggests a decrease in complex I activity of the electron transport chain. However, when glutamate–malate was substituted with succinate, oxygen consumption was not diminished, indicating that the activity of complexes II, III, and IV remained unaffected. This defect in complex I results in inefficient electron transport, primarily due to the disruption of supercomplex assembly. This alteration may be further exacerbated by the general effects of ROS on the structural integrity of the respirasome.
Paradoxically, some studies have reported that disruption of oncogenic KRAS can lead to a reduction in the expression of three genes involved in mitochondrial phospholipid synthesis: ACSL5, PCK2, and AGPAT7. The functional consequence of these changes is a decrease in the synthesis of cardiolipin, a phospholipid crucial for stabilizing supercomplex assembly and optimizing respiratory efficiency. By promoting cardiolipin synthesis in mitochondria, oncogenic KRAS may, in fact, increase the efficiency of electron transport and decrease ROS production by the ETC. However, it remains unclear whether all KRAS-mutated tumors exhibit an increase in cardiolipin levels in mitochondria. These seemingly contradictory effects may be attributed to variations in experimental design. Some studies utilized healthy cells transfected with a construct encoding mutant KRAS, while others employed KRAS-mutated colon cancer cells (HCT116) in which mutated KRAS was removed via knockout. In the latter scenario, the impact of oncogenic KRAS on ETC activity was assessed within the context of a heavily mutated genetic landscape. In this landscape, the acquisition of oncogenic KRAS could potentially have a protective function by promoting cardiolipin synthesis and mitigating ROS generation driven by other oncogenes. Conversely, introducing mutant KRAS into normal cells allows for a more direct investigation of the specific effects of mutant KRAS on the ETC. Further research is warranted to fully elucidate the role of oncogenic KRAS signaling on supercomplex assembly and respiratory efficiency, and its potential implications in cancer development and progression.
Recent studies suggest that the effects of KRAS on redox homeostasis are essential for maintaining the malignant phenotype of cancer cells. These effects may represent promising therapeutic targets for KRAS-driven cancers, which continue to pose a significant clinical challenge due to the lack of effective treatment strategies. Research has demonstrated that inducing oxidative stress through the use of small molecules can selectively eliminate mouse embryonic fibroblasts transfected with mutant KRAS and inhibit their growth in vivo. Furthermore, it has been observed that hyperactivation of mutant KRAS with the small molecule C1 leads to the activation of the PI3K–AKT pathway, which enhances ROS generation. This, in turn, results in mitochondrial dysfunction, cell death, and the blockade of tumor growth in cancers with mutant KRAS. These effects are attenuated by NAC, indicating that ROS generation via the KRAS–AKT axis is necessary to mediate the cytotoxic effects of C1. This evidence strengthens the rationale for pursuing ROS-based anticancer strategies to target KRAS-driven tumors.
MYC
The MYC family of transcription factors, including CMYC, LMYC, and NMYC, are master regulators of cell proliferation and apoptosis. They exert their control by regulating a vast number of genes transcribed by RNA polymerases I, II, and III. MYC amplification is a common occurrence in various cancers, including neuroblastoma and cancers of the breast, ovary, prostate, and uterus. The CMYC-immunoglobulin translocation is a defining characteristic of Burkitt’s lymphoma.
Similar to RAS, MYC induces significant metabolic reprogramming in cancer cells. This is achieved through the stimulation of glycolysis, mitochondrial biogenesis, and glutaminolysis. Studies have demonstrated that inducible expression of MYC in the B-cell line P493-6 leads to an increase in mitochondrial mass and enhances the oxygen consumption rate. This is a clear indicator of increased electron transport chain activity. These effects are partially mediated by the induction of mitochondrial transcription factor A (TFAM) by MYC, potentially driving ROS production through enhanced electron flow within the ETC.
Observations in glioma cell lines suggest that MYC regulates a transcriptional program that promotes the catabolism of glutamine. Glutamine serves as a carbon source to fuel the TCA cycle, thereby sustaining ROS production by the electron transport chain. Overexpression of CMYC in human fibroblasts has been shown to induce an increase in ROS levels, which correlated with the formation of DNA damage foci. The antioxidant NAC effectively reduced both ROS levels and DNA damage. Given that MYC drove cell-cycle entry and proliferation even in the presence of DNA damage, these observations point to a mechanism of MYC-induced genomic instability and the selection for p53 loss. p53 loss is a frequent alteration in CMYC-driven tumors, and it further contributes to clonal evolution and tumor progression. The role of ROS production following MYC amplification is further supported by the finding that exogenous antioxidants, such as vitamin C and Tiron, inhibited the transformation of MYC-overexpressing NIH/3T3 fibroblasts. Moreover, reducing ROS levels through mitochondria-targeted vitamin E was shown to block proliferation and induce cell death in osteogenic sarcoma cells.
In chemotherapy-resistant triple-negative breast cancer, the upregulation of MYC, along with the anti-apoptotic protein MCL1, selects for a stem-cell phenotype that is dependent on mitochondrial respiration. Accumulation of MCL1 within the mitochondrial matrix enhances the capacity of complexes I, II, and IV to transfer electrons. The concerted action of MYC on mitochondrial mass and MCL1 on the ETC culminates in HIF-1α stabilization, the selection of cancer stem cells, and resistance to chemotherapy. However, similar to KRAS, the effects of MYC on ROS homeostasis are a matter of delicate balance. MYC can also upregulate mitochondrial peroxiredoxin 3, which functions to protect cells from ROS under hypoxic conditions. Furthermore, NADPH production through serine and one-carbon metabolism protects hypoxic breast cancer stem cells from oxidative stress. This evidence suggests that ROS can trigger the selection of cancer stem cells, promoting the upregulation of antioxidant defenses, which aligns with the lower ROS set point typically observed in cancer stem cells.
MYC may also operate through Mechanism B by upregulating the expression of several mitochondrial nuclear-encoded proteins. This can lead to an imbalance between ETC subunits encoded by the nuclear and mitochondrial genomes, resulting in the generation of misassembled respiratory complexes. Intriguingly, studies have observed a strong correlation between the progression from normal prostate epithelium to invasive prostate carcinoma and imbalances in nuclear-encoded versus mitochondrion-encoded subunits of complex IV. This suggests that misassembled respiratory complexes play a role in promoting tumor progression. MYC also influences ROS production by modulating cancer cell metabolism. As previously mentioned, MYC promotes the expression of a wide array of genes involved in nucleotide synthesis, including DHODH. This, in turn, triggers ROS generation by destabilizing electron flow across the ETC.
MYC overexpression is associated with an increased proliferation rate in breast cancer. Furthermore, MYC amplification in luminal A breast cancer is linked to poorer survival outcomes and resistance to endocrine therapy. While these studies did not directly evaluate the involvement of ROS in the aggressiveness of MYC-driven breast cancer, it is known that elevated ROS levels are associated with resistance to endocrine therapy. The clinical impact of MYC-driven ROS production on cancer prognosis requires further investigation. Unfortunately, MYC remains a challenging therapeutic target. However, the dependence of MYC-driven tumors on NADPH production through serine metabolism and one-carbon metabolism suggests that inhibiting these metabolic pathways could represent an effective anticancer strategy for patients affected by these malignancies.
PI3K–AKT–mTOR
The interconnected PI3K–AKT and mTOR pathways play a central role in regulating apoptosis suppression, cell proliferation, metabolism, and anabolic reactions. These pathways are found to be hyperactivated in approximately 40% of all human cancers.
Given the pivotal role of the PI3K–AKT–mTOR pathway in inducing the Warburg effect and inhibiting autophagy, cancers characterized by hyperactivation of this pathway tend to accumulate dysfunctional, ROS-producing mitochondria that are not efficiently eliminated by autophagy.
The metabolism of non-essential amino acids is a significant feature of metabolic reprogramming in cancer cells and highly proliferating cells. Cancer cells metabolize non-essential amino acids to acquire nucleotides and lipids, maintain redox homeostasis, and regulate epigenetic modifications. Among the 11 non-essential amino acids, glutamine, serine, and proline are particularly important in tumorigenesis. Proline synthesis, mediated by Δ1-pyrroline-5-carboxylate (P5C) reductases (PYCRs), and proline degradation through PRODH, constitute a “proline cycle” that operates between the cytosol and mitochondria. In EGFR-mutated non-small-cell lung cancer, constitutive downstream activation of the PI3K pathway drives proline synthesis. This proline then fuels EGFR-regulated proline oxidation. The activity of PRODH reduces the efficiency of mitochondrial electron transport, driving ROS production from the QO site of complex III through Mechanism B. These findings suggest that proline metabolism may play a significant role in non-small-cell lung cancer harboring EGFR mutations.
Although the PI3K–AKT–mTOR pathway is typically associated with the induction of aerobic glycolysis, research has elucidated a mechanism through which AKT can mediate ROS generation via Mechanism A. Mitochondria-localized AKT phosphorylates MICU1, a regulatory subunit of the mitochondrial calcium uniporter (MCU). This phosphorylation results in the destabilization of the MICU1–MICU2 heterodimer, leading to increased calcium influx into mitochondria. Decreased expression of MICU1 following phosphorylation is associated with elevated levels of mitochondrial ROS and enhanced in vivo growth of cancer cells. Mitochondrial calcium overload can contribute to increased ROS generation through mitochondrial dysfunction. This, however, may also trigger permeability-transition-pore-mediated cell death. Alternatively, increased calcium levels within mitochondria could drive ROS production by stimulating enzymes of the TCA cycle and OXPHOS. This accelerates oxygen consumption and the generation of ROS by the ETC through Mechanism A. Moreover, mitochondrial calcium also enhances the activity of GPDH, producing ROS through direct electron leakage and by inducing reverse electron transport (RET) towards complex II and complex I (Mechanism B).
The activity of the PI3K–AKT–mTOR pathway is tightly regulated by the tumor suppressor PTEN, which is frequently inactivated in various human cancer types. PTEN status plays a crucial role in the AKT-mediated effect on MICU1. Interestingly, ROS can inactivate PTEN through oxidation. This inactivation favors the activation of AKT, which subsequently phosphorylates MICU1, leading to increased calcium uptake in mitochondria. Calcium, in turn, drives ROS production by the ETC, establishing a vicious cycle that sustains AKT activation and promotes tumor progression. Consistent with this scenario, scavenging mitochondrial superoxide (O2•–) using MitoTEMPO blunted AKT activation, reversed the Warburg effect, and induced cell death in melanoma cells. Furthermore, studies have shown that the mitochondria-targeted antioxidant Mito-CP enhances apoptotic cell death in a Burkitt’s lymphoma cell line by reducing AKT activation and HIF-1α stabilization under hypoxic conditions. This suggests the potential to block hypoxic adaptation in cancer cells by decreasing ROS generated by the ETC.
In breast cancer cells, it has been recently demonstrated that PI3K–AKT-mediated inactivation of glycogen synthase kinase-3β (GSK-3β) through phosphorylation induces abnormal activity of complexes I and III. This disrupts electron flow and enhances ROS production through Mechanism B. The resulting ROS released into the tumor microenvironment impairs the cytotoxic activity of Natural Killer (NK) cells by oxidizing a serine residue in the initiation factor eIF2B. This leads to the downregulation of NKG2D and its ligands. These results provide compelling evidence for an AKT/ROS-mediated mechanism that inhibits innate immune responses within the tumor microenvironment. Intriguingly, inactivation of GSK-3β also leads to the stabilization of CMYC, which can further amplify ROS generation by the ETC. Whether the inhibition of GSK-3β by AKT requires ROS-mediated PTEN inactivation remains an area for further investigation.
mTOR is a central kinase that integrates energy sensing and anabolic pathways, coordinating protein synthesis and cell growth. The synthesis of novel cellular components is an energy-demanding process. Therefore, it is not surprising that mTOR promotes mitochondrial metabolism by indirectly increasing the levels of nuclear-encoded mitochondrial proteins. For instance, mTOR promotes the formation of functional complexes between the transcription factor yin-yang 1 (YY1) and its cofactor peroxisome-proliferator-activated receptor coactivator 1α (PGC-1α). This complex drives the expression of numerous genes encoding mitochondrial proteins, including cytochrome c. This results in increased mitochondrial respiration and ROS production through Mechanism A. Moreover, mTOR cooperates with estrogen-related receptor α to promote the transcription of genes involved in OXPHOS and in the TCA cycle. Furthermore, through the inactivation of 4E-BP proteins, mTOR upregulates nuclear-encoded subunits of respiratory complex I and ATP synthase, thereby increasing mitochondrial respiration. Studies have observed that in the context of PTEN inactivation, hyperactivated AKT is associated with the phosphorylation of 4E-BP1, increased activity of complexes I, III, and IV, and augmented oxygen consumption. These results suggest that mTOR can enhance mitochondrial respiration, and consequently, ROS production via Mechanism A. Hyperactivation of the PI3K–AKT–mTOR pathway could also lead to an imbalance between nuclear-encoded and mitochondrial-encoded subunits of the respiratory complexes, similar to the effects observed with MYC. This imbalance can result in the production of misassembled complexes, the loss of supercomplexes, and increased ROS production through Mechanism B.
Given its central role in coordinating metabolism, mTOR sits at the crossroads between anabolic pathways and mitogenic signaling in cancer cells. mTORC1 phosphorylates S6K1, which, in turn, activates carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydro-orotase (CAD) through phosphorylation on S1859. CAD is a rate-limiting enzyme, catalyzing the initial three steps of de novo pyrimidine synthesis. It generates dihydro-orotate from glutamine, thus fueling DHODH, which subsequently produces ROS. Notably, the oxidation of dihydro-orotate to orotate functionally integrates nucleotide synthesis, which is essential to sustain the uncontrolled proliferation of cancer cells, with ROS production by the ETC. This integration plays a crucial role in driving cancer initiation and progression. Beyond mTOR, other oncogenes, including KRAS, MYC, and AKT, also converge on pyrimidine synthesis. Interestingly, studies have shown that the inhibition of DHODH using terifluonomide decreases mitochondrial ROS levels and exerts a cytostatic effect in prostate cancer cells. Future research should focus on investigating whether a reduction in the nucleotide pool or decreased ROS levels is primarily responsible for the anticancer activity of DHODH inhibitors. Furthermore, the precise role of DHODH-produced ROS in cancer development warrants further exploration.
The hyperactivation of the PI3K–AKT–mTOR pathway is recognized as a marker of poor prognosis in several human cancers, including esophageal squamous cell carcinoma and breast cancer. Recent research has indicated that pterostilbene, an antioxidant compound primarily found in blueberries, can slow down the progression of mantle cell lymphoma by targeting the PI3K–AKT–mTOR pathway. However, pterostilbene also exhibits direct effects on apoptosis and the cell cycle, making the interpretation of these results more complex. The precise impact of ROS on cancer in the context of PI3K–AKT–mTOR hyperactivation requires further in-depth investigation.
BCR/ABL
The t(9,22) translocation, a pathognomonic genetic event in chronic myeloid leukemia, leads to the formation of the Philadelphia chromosome and the chimeric BCR/ABL gene. This gene encodes a constitutively active tyrosine kinase that transduces both mitogenic and anti-apoptotic signals to cancer cells.
The BCR/ABL fusion protein promotes the production of ROS by the electron transport chain, partially through the activation of the PI3K–mTOR pathway. Studies have demonstrated that glucose metabolism is involved in the generation of ROS in BCR/ABL-transformed cells. Treatment with 2-deoxyglucose, the BCR/ABL inhibitor imatinib mesylate, or rotenone effectively reduces ROS production. Similar effects are observed with wortmannin and rapamycin, inhibitors of PI3K and mTORC1, respectively. This highlights the tight interconnections between BCR/ABL, PI3K, mTOR, glucose metabolism, and ROS production by the ETC via Mechanism A. The decrease in ROS levels following rotenone treatment suggests the involvement of reverse electron transport (RET) (Mechanism B) in ROS generation mediated by BCR/ABL.
Direct effects of BCR/ABL on the activity of the ETC have also been reported. Studies have observed a significant reduction in electron flow rates between complexes I and II and between complexes II and III. Concurrently, there is an increase in superoxide (O2•–) production through Mechanism B in BCR/ABL-expressing myeloid precursors. This ROS production is diminished by the mitochondria-targeted antioxidant MitoQ and is sustained by complex III, as evidenced by the rescue effect of complex III inhibitors such as myxothiazol, stigmatellin, and antimycin A. The small GTPase Rac2 has been identified as a promoter of ROS generation by complex III. Rac2 knockout substantially reduced mitochondrial O2•– levels and oxidative stress in BCR/ABL-expressing cells. This mechanism of ETC-mediated ROS production is not unique to BCR/ABL; it is also observed in leukemia cells harboring a variety of genetic alterations, including FLT3–ITD, TEL–ABL1, TEL–JAK2, TEL/PDGFβR, TEL–TRKC, BCR–FGFR1, and mutated JAK2. These observations indicate that diverse types of leukemia cells may promote genomic instability and disease progression through the activation of Rac2. Rac2 interferes with electron transport from complexes I to III and from II to III, ultimately leading to electron leakage from complex III and O2•– generation.
BCL-2
The B-cell lymphoma-2 (BCL-2) family comprises several proteins that counteract intrinsic apoptosis by binding to pro-apoptotic proteins along the inner mitochondrial membrane (IMM). BCL-2 overexpression is a common feature in numerous tumor types, including breast cancer, prostate cancer, B-cell lymphomas, and colorectal adenocarcinomas. Accumulating evidence suggests that the upregulation of BCL-2, beyond its direct anti-apoptotic function, creates a pro-oxidant state that enhances cell survival.
Studies have shown that the overexpression of BCL-2 in leukemia cells regulates mitochondrial respiration by modulating electron transport chain activity. This regulation is mediated through the direct interaction of BCL-2 with complex IV subunits Va and Vb. This interaction promotes the proper assembly of complex IV within the IMM and subsequently upregulates its activity. Based on these findings, it has been proposed that BCL-2 increases ETC activity and O2•– production, leading to a pro-oxidant cellular environment that favors cell survival. Intriguingly, under hypoxic conditions and in the presence of BCL-2, the relative abundance of the subunit Va is higher than that of subunit Vb. This altered subunit stoichiometry leads to reduced complex IV activity and mitochondrial respiration. In this context, this reduction maintains the mitochondrial redox state unchanged, preventing oxidative stress that would otherwise promote cell death. This scenario is supported by the observation that decreasing levels of O2•– can trigger apoptosis in BCL-2-overexpressing cancer cells. This suggests that the delicate balance between O2•– and H2O2 may modulate cancer cell survival through O2•–-mediated inhibition of apoptosis. Furthermore, the establishment of a mildly pro-oxidant cellular environment prevents the dephosphorylation of BCL-2 on the S70 residue. This phosphorylation state enhances BCL-2 binding with BAX and promotes cell survival.
