Defective mitochondrial unfolded protein response in cancer acts as a lifeline for tumor growth and survival.

Introduction
Under physiological conditions, the mitochondrial unfolded protein response (UPRmt) serves as an important mechanism for mitochondrial quality control and preserves cellular fitness.1, 2 When cells are exposed to proteotoxic or metabolic stress, UPRmt activates the transcription of molecular chaperones, including heat shock proteins (HSP60, HSP70, HSP90, and HSP10) and mitochondrial proteolytic enzymes such as caseinolytic peptidase ATP-dependent, proteolytic subunit (ClpP), and Lon peptidase 1 (LONP1).3, 4 However, in cancer cells, the UPRmt is reported to be frequently defective. Consequently, instead of restoring protein homeostasis, this altered stress response enables tumor cells to tolerate mitochondrial damage and metabolic dysregulation and allows these cells to evade cell death and become resistant to therapy.5, 6
Recent studies have shown that chaperones, such as HSP60, HSP70, and HSP10, are overexpressed in cancer cells. These chaperones facilitate folding of aberrant proteins, thus conferring resistance to proteotoxic stress.7, 8, 9 Cellular proteases such as ClpP and LONP1 participate in the clearance of damaged proteins. Cancer cells make use of these proteases to preserve mitochondrial structure under adverse circumstances.10 When these regulatory mechanisms are upregulated, cancer cells are able to efficiently clear damaged mitochondrial proteins that accumulate due to proteotoxic and metabolic stress, without triggering cell death. Munch et al. suggested that pharmacologic inhibition of the molecular chaperone, mtHSP90, or the protease, LONP1, could induce acute protein-folding stress in human cells, thus generating an effective UPRmt. During this process, mitochondrial translation is also temporarily inhibited as a result of abnormalities in pre-RNA processing and degradation of the mitochondrial RNase P protein 3. These findings indicate that mitochondrial proteotoxic stress triggers both increased chaperone synthesis and translational attenuation as coordinated methods for restoring proteostasis.11
The transcriptional regulators of UPRmt include activating transcription factor 5 (ATF5), C/EBP homologous protein (CHOP), and forkhead box protein O3a (FOXO3a), which are components of signaling axes involved in UPRmt, such as the SIRT3–FOXO3a axis, the protein kinase B (AKT)–estrogen receptor alpha (ERα) pathway, and the ATF5–ATF4–CHOP axis. These axes are involved in modulating stress responses and in adapting to stress.12, 13, 14, 15, 16 The dysregulation of UPRmt networks has been associated with many pathological conditions ranging from neurodegenerative diseases to cancer.17, 18
This review describes how defective UPRmt can be utilized by tumors to facilitate treatment resistance. We discuss major molecular mediators, including chaperones, proteases, and transcription factors, and the signaling axes that sustain aberrant UPRmt activation in cancer. Furthermore, we present the possibility of therapeutically targeting defective UPRmt in different cancers.

Molecular components and regulators of UPRmt in various cancers

Molecular chaperones
Molecular chaperones are vital parts of the mitochondrial proteostasis network. They help newly imported mitochondrial proteins to fold correctly and assist in refolding stress-denatured proteins within the organelle.19 The basic molecular chaperones involved in UPRmt-HSP60, HSP10, and mtHSP70 are described in the section below.

Heat shock protein 60
The HSPD1 gene encodes heat shock protein 60 (HSP60), a mitochondrial chaperonin that enables cells to adapt to mitochondrial stress, with significant implications for tumor survival and response to treatment.20, 21 It has been noted that breast cancer cell lines treated with cisplatin (MCF7 and MDA-MB-231) show upregulation of HSP60, leading to therapeutic resistance. Nicotinamide ribose (NR), an inducer of UPRmt can further elevate HSP60 levels via sirtuin 3 (SIRT3), a mitochondrial deacetylase. SIRT3 inhibition reduces HSP60 expression and eliminates the protective effects of NR, thus increasing susceptibility to cisplatin (Table 1) (Fig. 1).22 Sonodynamic therapy (SDT) with chlorin e6 and ultrasound reduces HSP60 expression in prostate (PC-3) and breast (MDA-MB-231) cancer cell lines, especially at high ultrasound intensities. This disrupts HSP60-mediated proteostasis and promotes apoptosis, suggesting that sonodynamic therapy may have therapeutic value in certain cancers (Table 1).23, 24 HSP60 is overexpressed in hepatocellular carcinoma compared to normal tissues. The multi-tyrosine kinase inhibitor sorafenib reduces HSPD1 expression and exhibits cytotoxicity, indicating that its anticancer effects may be atleast partially mediated through the regulation of HSP60 expression and UPRmt signaling (Fig. 1).25 It has been observed that HSP60 levels increase rapidly following the induction of the oncogene RAS in non-tumorigenic MCF10A breast epithelial cell lines. This suggests that HSP60 may be activated in the early stages of oncogenic transformation.13 The elevated expression of HSP60 and other UPRmt markers has been seen to be a poor prognostic factor in patients with lung adenocarcinoma (Table 1).26

Heat shock protein 10
Heat shock protein 10 (HSP10) is encoded by the HSPE1 gene (3.3 kb transcript), located on chromosome 2q33.1, and functions in tandem with HSP60. Under normal physiological situations, HSP10 and HSP60 create an efficient machinery called the folding cage for proper protein folding.27 HSP10 is seen to be significantly overexpressed in breast cancer tissues as compared to their adjacent normal counterparts (Table 1).28

Heat shock protein 70
Another chaperone that maintains mitochondrial proteostasis is mtHSP70, encoded by the HSPA9 gene (∼2.8 kb transcript) located on chromosome 5q31.1. mtHsp70 also known as mortalin, PBP74 or Grp75, is a 74 kDa mitochondrial protein belonging to the heat shock protein 70 (HSP70) family.29, 30 Similar to HSP60, elevated tissue expression of mtHSP70 is seen in glioblastomas, and in advanced colorectal cancers, increased plasma levels of this protein are seen in patients with localized prostate cancer (Table 1, Fig. 1). Furthermore, elevated tissue expression of mtHSP70 has been associated with adverse clinical outcomes in lung adenocarcinomas and glioblastomas (Table 1).26, 31, 32, 33 On the other hand, low HSP70 expression was noted to be associated with poorer overall survival in a The Cancer Genome Atlas analysis of laryngeal squamous cell carcinomas.34

Proteases
Proteases break down damaged, misfolded, or poorly organized proteins to maintain mitochondrial integrity.35 Forty-five proteases are found in the matrix, inner membrane, outer membrane, and intermembrane gap of the mitochondrial proteolytic system.36 ATP-depedent proteases are particularly important for protein degradation. The LONP1 protease is an ATP-dependent found in the mitochondrial matrix. On the other hand, the i-AAA and m-AAA proteases are found in the inner membrane with their catalytic domains pointed toward the matrix or the intermembrane space (IMS). The ClpXP complex is made up of the serine protease ClpP and its matrix-attached AAA+ ATPase ClpX. These enzymes work together to break down structurally abnormal polypeptides, respiratory chain components, and protein translocases throughout the many sub-compartments of the mitochondria. The discussion below will focus on ClpP and LONP1, two proteases involved in UPRmt that share critical metabolic targets.37

Caseinolytic peptidase ATP-dependent, proteolytic subunit
ClpP is a tetradecameric serine protease that is localized within the mitochondrial matrix.10, 38 It has a cylindrical structure and functions in conjunction with ClpX, a hexameric ATPase chaperone, to form the ClpXP complex. ClpX identifies, unfolds, and translocates substrate proteins to the proteolytic core ClpP, which then degrades them.38 ClpXP exhibits substrate specificity for enzymes associated with the TCA cycle, elements of the respiratory chain, proteins involved in amino acid degradation, and factors that aid in mitochondrial DNA (mtDNA) transcription and translation.38 Thus, ClpP plays a major role in maintaining stable OXPHOS. Consequently, overexpression of ClpP leads to increased OXPHOS, linking its activity to proliferation and therapy resistance in OXPHOS-dependent cancers.10
ClpXP modulates mitochondrial RNA stability by decreasing ERA-like 12S mitochondrial rRNA chaperone 1 levels, thereby influencing mitochondrial protein synthesis. ClpP protein overexpression has been noted in multiple solid tumors, including those of the lung, breast, prostate, as well as in hematological malignancies such as AML (Table 1).38, 39, 40, 41 Elevated levels of ClpP reduce cellular sensitivity to cisplatin, indicating that targeting ClpP may enhance cisplatin efficacy (Table 1).42 ClpP depletion leads to the accumulation of misfolded succinate dehydrogenase B, disruption of the respiratory complex II, impaired OXPHOS, decreased ATP production, and increased cellular oxidative stress.40 The knockdown of ClpP expression inhibits proliferation in prostate cancer cell lines (PC-3). On the other hand, excessive ClpP activation can lead to cell death by causing proteolysis of enzymes and proteins involved in essential metabolic pathways, including OXPHOS and the respiratory transport chain (Table 1).40 ClpP overexpression has been noted to lead to reduced proliferation in BxPC-3 pancreatic cancer cell lines.43 Thus, the manner in which cancer cell lines respond to ClpP activation is context-dependent and both ClpP activators and inhibitors may potentially have anti-tumor activity in different tumors.44, 45 Nonetheless, the comprehensive consequences of ClpP hyperactivation, including effects extending beyond mitochondrial proteostasis, remain incompletely understood and require further investigation.

Lon peptidase 1
LONP1 is a mitochondrial ATP-dependent protease. It helps in the assembly of the mitochondrial respiratory OXPHOS system.46 LONP1 has an ATP-dependent chaperone function, wherein it interacts with mtHSP70 to fold mitochondrial proteins. The nature of their interaction is substrate-dependent, so that the two act synergistically when their substrate is oxidase assembly 1-like protein and independently when their substrate is ClpX. In tumor cells that are exposed to conditions of mitochondrial stress, LONP1 is phosphorylated by AKT kinase on the Ser173 and Ser181 residues, thus promoting its protease activity. If the function of LONP1 is disrupted, OXPHOS is inhibited due to aggregation of proteins and enzymes involved in the pathway, leading to excessive ROS generation and mitochondrial dysfunction (Fig. 1).46, 47 LONP1 is overexpressed in lymphoma cell lines and protein samples, and inhibiting this protease has been seen to cause lymphoma cell death in-vitro, indicating therapeutic potential.48 LONP1 is commonly upregulated across malignancies and has been associated with elevated metastatic potential in in-vitro studies.49

Transcription factors
ATF5 and CHOP are the key transcription factors involved in UPRmt, and these in turn associate with C/EBPβ and FOXO3a to regulate the expression of genes involved in UPRmt (Table 1). Under conditions of oxidative or proteotoxic stress, these factors serve as molecular communicators between the nucleus and mitochondria. The outcome of regulator functioning depends on the intensity and duration of mitochondrial stress. Thus, these mediators can either facilitate cell survival or trigger apoptotic pathways. This section will focus on the regulatory roles of ATF5, CHOP, and FOXO3a in the activation and modulation of UPRmt.

Activating transcription factor 5
In the model organism Caenorhabditis elegans, the UPRmt mechanism is regulated by the basic leucine zipper transcription factor, ATFS-1. In the presence of mitochondrial oxidative stress, ATFS-1 does not get imported into the mitochondria and is instead translocated into the nucleus, where it activates the transcription of genes involved in UPRmt. ATF5 is the mammalian analogue of ATFS-1. It operates through a similar mechanism and contains both mitochondrial and nuclear targeting sequences. The functional conservation of ATF5 is demonstrated by its ability to rescue UPRmt signaling in ATFS-1-deficient worms when using the same promoter elements.3 ATF5 translation is increased in response to mitochondrial stress through a process mediated by eukaryotic translation initiation factor 2 subunit alpha (eIF2α) phosphorylation (Table 1).50
ATF5-driven UPRmt promotes treatment resistance by enhancing mitochondrial stress resilience. For instance, in the A549 lung carcinoma cell line, Maf1 inhibition following ionizing radiation leads to ATF5 upregulation and UPRmt activation, thus potentially facilitating radio resistance (Table 1, Fig. 1).26 ATF5 plays a key role in regulating the expression of genes involved in mitochondrial dynamics and metabolism, such as dynamin-related protein 1c and glycolysis-related glycerol 3-phosphate dehydrogenase 2.51 Aside from UPRmt regulation, the anti-apoptotic role of ATF5 also encompasses an increase in BCL2 transcription. The anti-apoptotic action of ATF5 is exemplified by its protective effect on glioblastoma cells, so that loss of ATF5 function in these cells leads to cell death in mice models (Table 1).52 These findings suggest that ATF5 could potentially serve as an important therapeutic target.

C/EBP homologous protein and CCAAT/enhancer-binding proteins
CCAAT/enhancer-proteins-CHOP and CCAAT/enhancer-binding proteins (C/EBP) are also basic leucine zipper transcription factors involved in UPRmt. UPRmt is activated by the increased transcription of CHOP and C/EBP, which is mediated by the AP-1 elements located in their respective promoter regions. The two transcription factors then form heterodimers with each other, facilitating DNA-binding and activation of UPRmt gene transcription (Fig. 1).53, 54, 55 Conversely, mutations in the regulatory regions of these transcription factors lead to depletion of the UPRmt response in cell lines (Fig. 1). The role of CHOP and C/EBP with respect to UPRmt is complex and context-dependent in different cancers. These transcription factors behave differently in response to stress generated by the endoplasmic reticulum and mitochondria. During endoplasmic reticulum stress, CHOP exhibits a pro-apoptotic effect, whereas during mitochondrial stress, CHOP promotes anti-apoptotic effects, leading to the upregulation of mitochondrial protective genes. In conclusion, CHOP and C/EBP serve as key regulators of UPRmt in cancer and may serve a dual role depending on the nature and severity of the stress.4, 55, 56

Forkhead box protein O3a
FOXO3a belongs to the forkhead box O (FOXO) family of transcription factors. Under metabolic stress conditions, FOXO3a is phosphorylated via mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) and AMP-activated protein kinase pathways and is translocated to mitochondria. Within the mitochondria, SIRT3, which is a key mitochondrial deacetylase, interacts with phosphorylated FOXO3a and binds to mtDNA alongside with mitochondrial transcrption factor A (TFAM) and mitochondrial RNA polymerase. These interactions promote the expression of mitochondrial genes that encode components of the respiratory chain complex and the OXPHOS system and reestablish the normal energetic state in conditions of metabolic stress (Fig. 1). The nuclear form of FOXO3a upregulates antioxidant genes such as superoxide dismutase (SOD) and catalase, thus protecting cells from oxidative damage. The inhibition of the cellular myelocytomatosis oncogene (c-Myc) by FOXO3a represents an SOD2-independent mechanism through which FOXO3a may reduce mitochondrial ROS levels.57 In gastric cancer cell lines, FOXO3a has been seen to increase cathepsin L-mediated EMT, thus potentially promoting tumor invasion and metastasis. On the other hand, FOXO3a activation is thought to be a mediator in the anti-proliferative effect of metformin cancer cells.58, 59 Thus, FOXO3a has a dual role in cancer growth and may serve as a tumor promoter or suppressor in different contexts.

UPRmt activation axes
The signaling axes that coordinate UPRmt are influenced by the nature of the stress and the cellular environment. There are three principal axes involved in this process-the ATF4-CHOP-ATF5 axis, SIRT3-FOXO3a axis, and AKT-ERα axis (Fig. 2). In response to stress, the ATF4-CHOP-ATF5 axis promotes the expression of mitochondrial chaperones and proteases through the phosphorylation of eIF2α. The AKT-ERα axis connects ROS to estrogen signaling, and the functioning of this axis is particularly evident in breast cancer. The SIRT3-FOXO3a axis protects cells from oxidative stress by controlling ROS levels, triggering mitophagy, and maintaining mitochondrial integrity. These axes show how the mitochondrial stress response network can adapt to allow cancer cells to adjust to harsh conditions. In the following sub-sections, we will learn more about these axes.

ATF5-ATF4-CHOP axis
The ATF5-ATF4-CHOP axis is a key transcriptional regulator of the UPRmt across different cancers (Table 1, Fig. 2).11, 12, 13 This axis connects nuclear gene expression, global translational regulation, and mitochondrial function. The phosphorylation of eIF2α activates stress responses, including UPRmt, and is a crucial upstream event in this pathway (Table 1).60 eIF2α phosphorylation suppresses global cap-dependent mRNA translation and leads to the accumulation of untranslated mRNAs, RNA-binding proteins, and messenger ribonucleoprotein complexes in SGs. These SGs rapidly shift translation priorities and help buffer cells against acute stress.12 Although global translation is restricted, certain mRNAs, such as those for the transcription factors ATF4, CHOP, and ATF5, are translated more under these conditions (Table 1).12, 61 These transcription factors are central components of the integrated stress response, which links cytoplasmic translation control and nuclear gene regulation (Table 1, Fig. 2).3, 12, 62 Since the integrated stress respons and UPRmt are closely interconnected, mitochondrial dysfunction can lead to persistent eIF2α phosphorylation, which can further reinforce this adaptive transcriptional response.3
MMP11 overexpression in breast cancer mouse models has been linked to UPRmt activation through modulation of the CHOP/HSP/ClpP pathway (Table 1).63 This indirect activation of UPRmt by MMP11 may form a part of a broader adaptive resistance mechanism in tumors. In conclusion, the ATF4-CHOP-ATF5 axis is an important part of the mammalian UPRmt and can potentially promote therapeutic resistance in cancer cells.

SIRT3-FOXO3a axis
The SIRT3-FOXO3a axis represents a distinct UPRmt regulatory. This pathway is particularly active in ERα-negative breast cancer cells when exposed to oxidative and mitochondrial stress. This pathway integrates redox control, mitochondrial protein quality regulation, and metabolic adaptation to promote the survival of tumor cells, unlike the CHOP and ERα. The SIRT3 axis comprises three core components: SIRT3, FOXO3a, and SOD2. This axis may be activated by mitochondrial stressors such as proteotoxic or oxidative stress or mitochondrial depolarization.13, 14
SIRT3 levels increase upon exposure to mitochondrial stress at both the mRNA and protein levels (Table 1). This increase may be dependent upon ROS, since coadministration with the antioxidant N-acetylcysteine may eliminate the rise in SIRT3 levels.14 The enzymatic function of SIRT3 depends upon the presence of NAD+. SIRT3 is essential for the activation of FOXO3a via deacetylation. This deacetylation is crucial for the appropriate nuclear localization of FOXO3a, which then binds to DNA and initiates transcription (Fig. 2).13, 14 Upon entering the nucleus, FOXO3a transcribes essential antioxidant genes (Fig. 2).13 Aside from the transcriptional regulation mediated by FOXO3a, SIRT3 also directly activates the SOD2 protein by deacetylation.13, 14 Additionally, under partial control by LC3IIB, the SIRT3 axis regulates mitophagy. It also influences mitochondrial biogenesis through the transcription factor nuclear respiratory factor 1 (NRF1).14 This coordinated response promotes general mitochondrial function and helps lower ROS levels below a threshold suitable for mitochondrial function and cell survival (Fig. 2). SOD2 expression is a good surrogate marker of the functioning of this axis.
In the early stages of tumor growth, lowering the levels of SIRT3 and SOD2 may be required to enable an increase in ROS and thus support the shift to glycolysis, known as the Warburg effect. On the other hand, SOD2 expression has been noted to be higher in metastatic lesions than in primary tumors.13, 14 Genomic alterations in the mtDNA that promote ROS generation have been shown to activate the UPRmt by stimulating the SIRT3- FOXO3a pathway, and such activation is more evident in more invasive breast cancer cell lines rather than in their non-invasive counterparts.13 This suggests that this axis of the UPRmt may be more active in cancer cells with higher metastatic potential, and the axis may have different roles at different stages of tumor growth. As discussed earlier in Section “Heat shock protein 60,” SIRT3 silencing makes breast cancer cell lines more sensitive to cisplatin through downregulation of UPRmt.22 Thus, the SIRT3-FOXO3a axis may play a role in tumor invasiveness, and its targeted inhibition may hold therapeutic potential.

AKT-ERα axis
The UPRmt AKT-ERα axis is activated by the ROS-induced accumulation of misfolded proteins in the mitochondrial IMS in ERα-positive breast cancer cells and is distinct from the UPRmt observed following matrix stress. ROS-induced AKT activation and phosphorylation promotes ERα activity (Fig. 2). The transcriptional program regulated by the ERα axis extends beyond typical cell cycle genes to incorporate genes involved in mitochondrial biogenesis and cytosolic proteostasis.16 ERα activation upregulates NRF1 which is a key transcription factor involved in mitochondrial biogenesis and metabolism, as depicted in Figure 2. This axis also upregulates the activity of the proteasome and the protease, OMI, to limit the accumulation of misfolded proteins.15, 16 The activation of this axis is unique to IMS stress and does not occur with matrix stress. However, in cells that lack ERα, IMS stress also activates alternative UPRmt axes and triggers CHOP and HSP60 overexpression, similar to that observed in matrix stress (Table 1).16 The AKT-ERα axis is not well explored in cancers other than breast cancer, and further research is needed to elucidate its role in cancer pathogenesis.

Current emerging therapeutics for modulation of UPRmt pathways

Inhibitors of UPRmt
Due to the critical function of UPRmt in cancer cell survival and stress adaptation, targeting UPRmt may serve as a potential therapeutic vulnerability for cancer cells.39 Inhibiting UPRmt components or the mechanisms that regulate them are evolving strategies for cancer treatment.

Inhibition of chaperones and proteases
The mitochondrial chaperone, tumor necrosis factor receptor-associated protein 1 (TRAP1) promotes metabolic reprogramming and augments glutamine consumption to produce ATP. Study showed that the TRAP1 inhibitor gamitrinib-triphenylphosphonium may serve as a potential therapeutic agent either alone or in combination with chemotherapy and dysregulate tumor metabolism.64 Silencing of the mitochondrial proteases HClpP and HClpX enhances the sensitivity of human cervical cancer cells to cisplatin as a result of elevated production of cisplatin-mtDNA adducts (Table 1).42
The interaction between HSP60 and ClpP has been seen to be important in carcinogenesis in prostate cancer (Table 1). A recent study identified that inhibition of the physical interaction between these two UPRmt mediators using the compound DCEM1 may serve as a potential treatment approach. Functionally, DCEM1 disrupts mitochondrial proteostasis, resulting in mitochondrial malfunction and metabolic stress, and obstructs signaling axes that are active in prostate cancer growth. Mechanistically, DCEM1 binds to the apical domain of HSP60; as a result, ClpP becomes inaccessible, thus preventing the refolding of damaged proteins. Administration of DCEM1 induces a dose-dependent elevation of ROS and an augmented accumulation of poly-ubiquitinated proteins, leading to apoptotic cell death. In a preclinical study in prostate cancer xenograft models, DCEM1 was noted to suppress oncogenic signaling pathways, including c-Myc and enhancer of zeste homolog 2, therefore inhibiting tumor development. Taken together, disrupting the HSP60/ClpP axis may be a viable treatment strategy worth exploring in prostate cancer.39

Inhibiting TRPV1 in multiple myeloma
The TRPV1 calcium channel is present in both multiple myeloma cell lines and primary multiple myeloma cells (Table 1). AMG9810 (an antagonist of TRPV1) and the anti-multiple myeloma drug bortezomib act synergistically to induce the death of multiple myeloma cells. Bortezomib promotes the formation of misfolded or unfolded proteins, thereby activating the UPRmt. Thus, bortezomib-induced mtHSP70 upregulation mitigates ROS-induced mitochondrial damage and enables cancer cells to evade apoptosis. Notably, AMG9810 depletes mtHSP70 in bortezomib-treated cells, thus sensitizing myeloma cells to the latter drug (Fig. 1). AMG9810 therapy enhances mitophagy, as indicated by elevated PINK1 and reduced VDAC levels, leading to significant mitochondrial impairment and decreased mitochondrial mass. Consequently, AMG9810 exacerbates mitochondrial damage and improves the efficacy of bortezomib.61

Obstructing mitochondrial protein translation in hematopoietic cancers
Human mitochondrial peptide deformylase (HsPDF) is an enzyme responsible for proper protein maturation in the mitochondria through removing the N-terminal formyl group from the newly synthesized protein.65 HsPDF levels have been noted to be elevated in several cancer cell lines and primary myeloid leukemias, with its expression regulated by c-Myc (Table 1). All mtDNA proteins are processed by HsPDF. HsPDF inhibition leads to translation impairment and triggers metabolic stress, thus initiating a UPRmt that leads to cell death. The inhibition of HsPDF by actinonin has been shown to lead to apoptotic cell death in Burkitt lymphoma cell lines. Notably, actinonin activates UPRmt through the upregulation of CHOP, CEBP/B, and LONP1. Mechanistically, actinonin exhibits high selectivity and specificity for c-Myc-positive cells and triggers an imbalance in the mitochondrial membrane potential, which ultimately leads to caspase activation. Thus, targeting HsPDF may have therapeutic potential in certain hematologic malignancies.66

Agonists of UPRmt
As described in the text previously, UPRmt has context-dependent roles, and in certain situations, stimulating this pathway may also have therapeutic utility. ClpP activators have shown great promise as therapeutic agents for addressing mitochondrial dysfunction, particularly in cancer cells that heavily rely on OXPHOS for survival and energy. There are three broad categories of ClpP agonists: acyldepsipeptide antibiotics (ADEPs), ONC201 and its analogs (imipridones), and TR compounds.

Acyldepsipeptide antibiotics
Acyldepsipeptide (ADEPs) were first isolated from the fermentation broth of Streptomyces hawaiiensis during antibacterial screening efforts. Although they were initially classified as antibiotics, subsequent genetic analyses revealed bacterial ClpP to be their primary targets. Functionally, ADEPs cause hyperactivation upon interaction with ClpP and lead to uncontrolled ClpP-mediated proteolysis and, eventually, bacterial cell death.67
Further studies by Wong et al found that ADEPs and their synthetic derivatives bind to hydrophobic pockets of ClpP and dissociate ClpX, the regulatory partner of ClpP, from the ClpXP complex. This interaction interferes with the association between ClpP and ClpX, thereby inducing constitutive protease activation (Fig. 1).68
Remarkably, the synthetic analog ADEP-41 was found to activate human ClpP as well, initiating intrinsic apoptotic signaling pathways and disrupting mitochondrial morphology and function in HeLa (cervical cancer), SH-SY5Y (neuroblastoma), and U2OS (osteosarcoma) cell lines.38 In line with this, another analog, ADEP-28, drives ClpP to transition from a heptameric to a tetradecameric assembly. As a result, the resulting ClpP–ADEP28 complex exhibits an expanded axial pore and a more accessible proteolytic chamber compared with the inactive apo form, thereby enhancing substrate entry and degradation, ultimately leading to the triggering of apoptotic signaling pathways.38, 69

ONC201 and its analogs (imipridones)
ONC201 is a small molecule from the imipridone family that was discovered by scientists while screening for compounds that could activate tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).70 The activation of TRAIL helps trigger autocrine-mediated apoptosis in cancer cells. Initially, ONC201 was thought to work by blocking the ERK and AKT pathways, triggering the nuclear translocation of FOXO3a and subsequent upregulation of TRAIL gene expression (Fig. 1).70, 71 However, recent studies have shown that ONC201 is a strong activator of the mitochondrial protease ClpP. Following treatment with ONC201, triple-negative breast cancer cell lines such as MDA-MB-231 and SUM159 have been noted to show significant increase in the expression of CHOP and ATF4.72 The activation of ClpP by ONC201 leads to the degradation of mitochondrial proteins critical for respiration and translation, resulting in the suppression of mitochondrial metabolism and lipid biosynthesis.38 ClpP activation by ONC201 also lowers c-Myc levels, blocks mTORC1 signaling, increases the expression of death receptor 5, and triggers the activation of caspase-8. These effects work together to cause cell death and slow down tumor growth (Fig. 1).73 Clinically, ONC201 has shown antitumor efficacy across multiple solid and hematologic tumors, and clinical trials of this agent are currently ongoing in the refractory setting in different cancers. It can cross the blood-brain barrier effectively and is now FDA-approved for the treatment of H3K27M-mutant diffuse gliomas. Following the success of ONC201, ONC206 and ONC212 were developed as next-generation imipridones with enhanced potency.73, 74, 75

TR compounds
TR compounds are highly effective and selective small molecules that function by dissociating ClpX from the ClpXP complex; as a result, ClpX is dysregulated, leading to uncontrolled, non-specific proteolytic activity of ClpP within the mitochondria. The uncontrolled ClpP proteolysis destroys crucial mitochondrial proteins and decreases the rate of mtDNA transcription and translation inside the cell. Following TR-107 administration, cellular stress responses are generated that activate mitophagy and ferroptosis pathways. Preclinical studies revealed that TR-107 reduces cell viability and proliferation in human colorectal cancer cell lines (Table 1).10 TR-57 is another small molecule ClpP activator that has been shown to have in-vitro efficacy as a single agent and in combination with venetoclax in chronic lymphocytic leukemia (CLL). Mechanistically, this is thought to be due to activation of the UPRmt, decrease in anti-apoptotic proteins, and activation of the AKT and ERK1/2 pathways.20 Thus, this drug combination has the potential to counter the proliferative and migratory capabilities of CLL cells and may have potential therapeutic value in resistant disease.

Conclusion and future direction of the study
Unlike normal cells, cancer cells can sustain metabolic stress and avoid apoptosis by altering mitochondrial activity, thus facilitating tumor growth and therapy resistance. The UPRmt restores mitochondrial proteostasis by activating nuclear genes that encode mitochondrial chaperones, proteases, antioxidant machinery components, and regulators of mtDNA biogenesis. Central molecular constituents of UPRmt include molecular chaperones (HSP60, HSP10, and mtHSP70), proteases (ClpP, and LONP1), and transcription factors (ATF5, CHOP, C/EBP, and FOXO3a) (Fig. 1). These components carry out signaling through the ATF5-ATF4-CHOP axis, the AKT-ERα, and the SIRT3-FOXO3a axes (Fig. 2). Targeting this complex network, either by inhibiting protective mechanisms or by hyperactivating essential components, opens up avenues for new and potent strategies for cancer treatment. Further preclinical and clinical investigations are warranted to understand the underpinnings of UPRmt to reveal novel approaches for targeting cancer.

Ethics statement
Since it's a review article, this article does not contain any studies with human participants or animals performed by any of the authors.

Funding and support
The authors would like to acknowledge the financial support from the Department of Biotechnology for providing the DBT-JRF fellowship (DBT/2024–25/AIIMS/2566) to N.A. U.S. acknowledge the DBT-RA fellowship (DBT-RA/2024–25/Call-I/RA/41) and the ANRF N-PDF (PDF/2025/000648). Also, the authors would like to acknowledge the financial support from the UGC-SRF grant (211610188930) to R.R.K. and the DST-SERB grant (CRG/2021/001887) and the ICMR grant (ICMR-2023–7664; ICMR-2021–9712-F1) to S.B.

CRediT authorship contribution statement
Uttam Sharma: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization. Vaishnavi Vishwas: Writing – original draft, Data curation. Rajiv Ranjan Kumar: Writing – review & editing, Writing – original draft, Funding acquisition, Data curation. Nikita Agarwal: Writing – review & editing, Writing – original draft. Akshi Shree: Writing – review & editing, Writing – original draft. Jaya Kanta Gorain: Writing – review & editing, Writing – original draft. Archana Sasi: Writing – review & editing. Surender K. Sharawat: Conceptualization, Writing – review & editing. Archna Singh: Writing – review & editing, Resources. Jayanth Kumar Palanichamy: Writing – review & editing, Resources. Sameer Bakhshi: Supervision, Resources, Project administration, Funding acquisition.

Declaration of interest
The authors declared no conflict of interest.