ER-mitochondria tethering and its signaling: A novel therapeutic target in breast cancer.

Introduction

Breast cancer (BC) remains a primary cause of cancer-related mortality among women. Mortality is reduced in high-income countries and higher in low- to middle-income countries. BC incidence from 2000 to 2020 is higher in Asian and African countries, is rapidly increasing in China and South Korea, but is declining in the USA. 1 Mortality rates vary by BC subtype and socio-economic conditions. Patients with the triple-negative subtype (triple-negative BC [TNBC]) are higher compared to other subtypes due to higher metastasis rates in visceral organs and the brain. 2 Metastasis is the major challenge in the treatment of highly fatal subtypes of BC, particularly in TNBC, due to a lack of early symptoms, late diagnosis, and limited treatment options. Targeted therapies have shown promise in treating hormone receptor (HR)-positive BC with hormone receptor inhibitors and HER2-positive BC with HER2 antibodies; however, targeted therapies are limited for TNBC. 3 Therapeutic resistance is also a key contributor to mortality.

Each BC tumor microenvironment (TME) is unique and includes multiple components: BC cells including BC stem cells (BCSCs), stromal cells (e.g., fibroblasts, immune cells, and endothelial cells), and soluble components (e.g., chemokines, cytokines, and oxygen). The TME phenotype abnormally contributes to aggressive proliferation, expanded angiogenesis, immune escape, resistance to apoptosis, drug resistance, and metastasis, 4 some of which are mediated through organelle dynamics. BCSCs are thought to be drug resistant as well as responsible for metastasis development. In the TME, cancer-associated fibroblasts (CAFs) form tunneling nanotubes by contact with BCs and transfer mitochondria and mitochondrial genes. 5 , 6 As a result, BCSCs are recognized as favorable targets.

Mitochondria are key organelles, contributing to energy production, calcium homeostasis, generation of ROS, and induction of apoptosis. 7 They are dynamic organelles, constantly undergoing fusion and fission to modulate energy and macromolecule dynamics in cells. 8 , 9 Those mitochondrial changes also result in differential ROS generation, altered intracellular signaling, and induction of epithelial-to-mesenchymal transition (EMT), 10 , 11 which is frequently implicated in BC cell acquisition of invasive and/or metastatic potential. 12 , 13 , 14 In general, abnormal mitochondrial fission increases proliferation, invasiveness, self-renewal, and drug resistance in BCSCs 15 by adjusting Warburg and reverse Warburg effects based on the energy requirement of cancer cells in the TME. 16 Pioneering work by Danny and colleagues has illuminated the multifaceted role of mitochondria in cancer metastasis, including contributions relating to mitochondrial genetics and polymorphisms, as well as the interplay between mitochondrial and nuclear DNA. 17 , 18 , 19 , 20 , 21 , 22 Their studies have specifically highlighted the importance of these mechanisms in BC progression.

The endoplasmic reticulum (ER), another dynamic organelle, mediates communication between organelles, including mitochondria, 23 and controls EMT and self-renewal of BCSCs by regulating stress mediators. 24 ER-mitochondrial contact sites control expression of genes that regulate the energy production, calcium homeostasis, and the fate of BCSCs. 25 ER also engages mitochondria to regulate cell functions through specialized domains and associated signaling pathways in tumorigenesis. The mitochondrial protein mitoguardin (Miga) facilitates lipid transfer through mitochondria-ER contacts at mitochondria-associated ER membranes (MAMs), 26 supports mitochondrial fusion, and regulates the localization as well as phosphorylation of Yes-associated protein 1 (YAP1), a downstream mediator in the Hippo pathway. 27 These data provide evidence for the critical role of ER stress and mitochondrial dynamics in BC.

Emerging evidence highlights the importance of ER-mitochondria communications in drug resistance and metastasis of BC. 28 , 29 Here, we review connections between mitochondrial dynamics, fusion, fission, mitophagy, caveolin-1 (Cav-1), and YAP pathways. Our focus will be on ER-mitochondrial communications, including ER stress responses in pathophysiological conditions connecting mitochondria, cell survival, or cell death. We also evaluate existing anticancer drugs, clinical inhibitors, and natural compounds that target mitochondrial dynamics in BC. Data are presented demonstrating targeting dynamin-related protein 1 (Drp1) by natural compounds, their mechanisms of action in mitochondrial fission, fusion, and mitophagy, and how this crosstalk contributes to metastatic dissemination and resistance to therapies.

Mitochondrial dynamic mechanisms in BC

The mitochondrial quality is maintained by fission, fusion, and mitophagy. 30 They are the vital mechanisms, highly balanced under normal physiological conditions, and influence the shape and activity of mitochondria. In general, the elongation of mitochondrial scaffolds generated by fusion increases ATP generation and transfers different metabolites. Fission, conversely, creates tiny mitochondria, which are important for oxidative maintenance. 31 Nonetheless, fission and fusion mechanisms depend on the degree of framework interaction. Aberrant changes in the processes of mitochondrial dynamics promote survival, proliferation, and metastasis. 32 Mitochondrial dynamics integrate into the survival of patients with cancer by controlling the cell cycle and cellular metabolism. 33 Moreover, the molecules derived from mitochondria, such as oncometabolites, also integrate stress response pathway proteins with cancer progression. 34 Mitophagy is a mechanism that selectively eliminates damaged mitochondria, lowers the production of mitochondria-derived ROS and inflammation, and can increase cell survival. 35

Mitochondrial fission machinery

Fission is controlled by two categories of fission proteins ( Figure 1 ). They include cytosolic soluble proteins, Drp1, mitochondrial-bound fission 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics protein of 49 kDa (MiD49), as well as mitochondrial elongation factor 1 (MIEF1). 36 , 37 Drp-1 is a cytosolic protein that controls mitochondrial fission. Drp1 is activated by phosphorylation at S616 in a context-dependent manner by cyclin-dependent kinase 1 (CDK1) during cell division, extracellular signal-regulated kinase (ERK) in response to growth factor stimuli, calcium/calmodulin-dependent protein kinase II (CAMKII) in response to calcium stimuli, and Rho-associated kinase during the reorganization of the cytoskeleton. 38 This phosphorylation causes the translocation of Drp-1 to the outer membrane of mitochondria (OMM), where it associates with the adaptor proteins, Fis1 and Mff, and establishes a spiral ring around mitochondria, progressing to constriction and consequent abscission of mitochondria. In addition, it also promotes ER and peroxisome fission. 39 In contrast, the phosphorylation of Drp1 at S637 by protein kinase A (PKA) 40 and the mammalian target of rapamycin (mTOR) 41 inhibits fission. S637 phosphorylation inhibits the shifting of Drp1 from the cytoplasm to the OMM, preventing Drp1 from associating with mitochondria. 42 This phosphorylation also reduces the GTPase activity of Drp1.

In cancer cells, elevated mitochondrial fission supports tumor growth and metastasis. BC cells with high metastatic ability exhibit elongated mitochondria. Functional studies using small interfering RNA (siRNA) to silence Drp-1 suppress mitochondrial recruitment at the migratory front of cancer cells by reducing the formation of lamellipodia. 43 Likewise, silencing of Drp-1 diminished the metastasis of BC cells to the brain by reprogramming lipid metabolism and enhancing the accumulation of lipid droplets by impairing fatty acid oxidation. 44

Mitochondrial periphery fission proteins are also implicated in cancer cell migration and metastasis. Fis1 is an anchored protein on OMM and controls the recruitment of Drp-1 to mitochondria. 45 The phosphorylated form of Fis1 (Y38) stimulates mitochondrial fragmentation. Fis1 facilitates translocation of mitochondria to the migratory leading edge to promote the F-actin-mediated formation of lamellipodia, either by interacting with the Drp-1 or inhibiting mitofusin-2 (Mfn2). 46 Mff, an integral membrane protein localized in OMM, controls the fission and facilitates the formation of inter-mitochondrial networks by physically interacting with the receptors of Drp-1. Mff also reduces the self-renewal of BCSCs by inhibiting glycolysis via interfering with glycolytic pathway genes. 47 MID49 and MIEF1 are adaptor proteins of Drp-1. Like Mff, both are found on the OMM and support mitochondrial fission. They are upregulated in invasive BC types and are positively correlated with tumor grade and size. Silencing them using siRNA induces cell-cycle arrest by augmenting the expression of p27 Kip1 and p21 Waf1 . MID49 and MIEF1 inhibit Drp-1 and the Akt-mTOR-p70S6K pathway to stimulate apoptosis in MCF-7 cells. 48

The phosphorylation site of Drp-1 determines its coupling with pro-fission adaptor proteins. Phosphorylation of Drp-1 at Ser-616 favors mitochondrial fission by coupling with Mff/MiD51, while phosphorylation at Ser-367 favors mitophagy by coupling with MiD49/Fis1 in mitosis. 49 Fission and fusion of mitochondria are also controlled by cardiolipin and phosphatidic acid, respectively. Collectively, mitochondrial fission machinery via Drp1 and its binding partners emerges as a therapeutic target due to their upregulation and overactivation.

Mitochondrial fusion machinery

Mitochondrial fusion is involved in the exchange of metabolites, DNA, lipids, and proteins between mitochondria. Fusion also supports cellular respiration as well as homeostasis of intracellular calcium. Fusion machinery comprises large GTPase fusogens such as mitofusin-1 (Mfn1) and Mfn2 as well as optic atrophy 1 (OPA1). 50

The process of mitochondrial fusion begins with the fusion of OMM and subsequent fusion of IMM ( Figure 2 A). OMM fusion is largely driven by Mfn1 and Mfn2, which are located on the OMM. 51 These proteins interact in trans between two adjacent mitochondria, forming homo- (Mfn1–Mfn1 or Mfn2–Mfn2) as well as heteromeric (Mfn1–Mfn2) complexes that span the OMM twice with the amino and carboxy ends toward the cytoplasmic side. Recent structural studies suggest that Mfns can form dimeric complexes with their HR2 domains interacting and their GTPase domain driving membrane fusion. This tethering brings the OMM into proximity and facilitates docking. 52 Further, tethering of Mfns enables the tethered hydrolysis of GTP, which promotes mitochondrial fusion. 53

Once OMMs are fused, IMM fusion is mainly mediated by OPA1, another large GTPase residing in the IMM. OPA1 employs a specific lipid-binding module, the “paddle domain,” to engage with the membranes enriched in cardiolipin. 54 The paddle domain facilitates the initial association of OPA1 with the lipid bilayer. A highly conserved, intramembrane loop within the paddle domain further anchors OPA1 by deeply penetrating the hydrophobic core of the lipid bilayer, specifically within cardiolipin-rich regions. This insertion significantly enhances the stability of the protein-membrane interaction. Through interactions involving their paddle domains, OPA1 monomers dimerize and promote formation of higher-order helical oligomeric lattices on the membrane surface. This assembly, characterized by its dynamic and flexible nature, imparts the mechanical force required for membrane fusion.

Furthermore, this membrane-associated OPA1 oligomer exhibits structural plasticity, undergoing conformational rearrangements that induce movement of the membrane-inserting loop from the outer to inner membrane leaflets. These dynamic rearrangements directly contribute to lipid bilayer physical remodeling, which is essential for successful fusion. 55 The coordinated effects of these molecular changes of OPA1 ultimately propel the membrane fusion of mitochondria during cellular activity and homeostasis. Mfn2 is also associated with tethering sites of ER and mitochondria. 56

Metabolic stress negatively regulates mitochondrial fusion by activating the transcriptional regulator TRIM28 ( Figure 2 B). Metabolic stress limits oxidative phosphorylation (OXPHOS) and generates ROS, which, in turn, triggers phosphorylation of TRIM28 at S473. Phosphorylated TRIM28 inhibits mitochondrial fusion by promoting the degradation of Mfn2. 57

Both Mfn1 and Mfn2 are underexpressed in many tumors. Mfn2 inhibits angiogenesis and tumor growth by regulating the expression of VEGFA 58 ( Figure 2 C). As a result, both molecules are considered tumor suppressors. Overexpression of Mfn2 also sensitizes MCF-7 cells to camptothecin. 59 Purohit et al. reported that miR-195 negatively modulates mitochondrial fusion in BC cells by binding to the UTR region of Mfn2 ( Figure 2 D). 60 The fusion mechanism of mitochondria is negatively regulated by mitochondrial fission process 1 (MTFP1), which is enriched in the subdomain of IMM. 61 Mechanistically, MTFP1 prevents the fusion of IMM subdomains by reducing the oligomerization of OPA1 via downregulating its expression.

Mitophagy machinery

Mitophagy maintains healthy mitochondria through selectively eliminating impaired, damaged, or malfunctioning mitochondria ( Figure 3 ). It also plays an important role in apoptosis. It is receiving research attention due to its role in age-related diseases including cancer. 62 The mechanism of mitophagy begins with priming mitochondria, formation of autophagosomes via specific OMM receptors or surface proteins conjugated with ubiquitin on mitochondria, and subsequent degradation in lysosomes. 63 This mechanism is highly complex and involves a network of proteins. It is tightly regulated by a PINK1-PRKN-dependent mechanism in response to iron and calcium levels and the dynamics of metabolites from carbohydrates, amino acids, and fatty acid metabolism. 64 In the PINK1-PRKN-dependent mechanism, mitochondria are ubiquitinated, which is mediated by the sensor of mitochondrial damage (PINK1), signal amplifier (PRKN), and signal effector (ubiquitin). Additionally, Mfn2 promotes the recruitment of Parkin onto the OMM of damaged mitochondria, where Mfn2 serves as a receptor for tethering Parkin to PINK1. 65

The PINK1-PRKN-independent mechanism is mediated by different proteins and lipid receptors. 66 In one of the protein-dependent mechanisms, PINK1 initiates the mitophagy by activating Ariadne RBR E3 ubiquitin protein ligase 1 (ARIH1), an E3 ligase, which in turn promotes the ubiquitination of proteins on OMM of damaged mitochondria. Another study reported that mitophagy is induced by an activating molecule in Beclin 1-regulated autophagy protein 1 (AMBRA1) that binds to the adapter LC3 on autophagosomes. 67 The lipid-mediated induction of mitophagy is initiated with the reorganization of cardiolipin in the OMM. It is then recognized as an “eat me” signal and facilitates the binding of the LC3 receptor on autophagosomes, leading to the lysosomal-mediated degradation of defective mitochondria. 68

Genes involved in the biogenesis of lysosomes, a key component of mitophagy, are upregulated by Parkin via promoting translocation of transcription factor EB (TEEB). 69 However, mitophagy is negatively regulated by deubiquitinating enzymes. These studies emphasize the interdependent relationship between mitophagy and mitochondrial dynamics, mechanisms necessary for preserving fully functional mitochondria through the selective degradation of defective and damaged organelles.

Regulation of mitochondrial dynamics

Plasma membrane-localized Cav-1, a key protein of caveolae, modulates mitochondrial fusion and fission, in part, by interacting with Mfn2 and Drp1 through various mechanisms ( Figure 4 A). Phosphorylation of Cav-1 at tyrosine 14 (Y14) reduces Mfn2 translocation to mitochondria, thus reducing the PINK1:Parkin complex. This leads to the accumulation of damaged mitochondria and augments the levels of mitochondrial ROS, which impedes mitophagy. 70 Conversely, under cellular stress, Cav-1 promotes mitochondrial fission by activating PKA. Its activation promotes the activity of a protein phosphatase, which leads to dephosphorylation of Drp1 (Ser637). This dephosphorylation enhances Drp1 translocation to mitochondria.

Cav-1 also functions independently of caveolae within the MAM compartment to regulate mitochondria-ER contacts and, consequently, mitochondrial dynamics in MDA-MB 231 TNBC cells, which relatively express a high amount of Cav-1. 71 Bravo-Sagua et al. used MDA-MB 231 cells as a model and demonstrated that Cav-1 translocates to the ER-mitochondria interface, where it weakens ER-mitochondria tethering, reduces calcium transfer, and makes mitochondrial bioenergetics less sensitive to ER stress by reducing PKA activity. 72 It has been reported that Cav-1 activity enhances mitochondrial membrane potential and reduces ROS generation by activating Rho-associated kinases 1 and 2 (ROCK1/2) signaling in MDA-MB-231 TNBC cells. The ROCK1/2 pathway inhibits AMPK, thus decreasing basal mitophagy and regulating TNBC cell metabolism. 73 Additionally, Cav-1 promotes TNBC cell migration as well as invasion by activating Rac1 in an ROS-dependent manner, thereby promoting F-actin assembly at lamellipodia. 74 Together, these studies highlight the multifaceted role of Cav-1 in regulating mitochondrial dynamics in BC.

The mitochondrial dynamics are also controlled by the Hippo pathway ( Figure 4 B). The transcriptional coactivator YAP1 plays a significant role in BC. 75 Blocking the YAP pathway promotes mitochondrial fission through increased Drp1 activity and reduced Mfn1/2 levels in specific cell types. 76 Conversely, YAP overexpression reduces Drp1-dependent mitochondrial fission through activation of ERK in HER2-positive BC. 77 Furthermore, YAP inhibits mitochondrial fission by regulating Drp1 recruitment to mitochondria. Consequently, high-temperature requirement protein A2 and Omi stress-regulated endoprotease (HtrA2/Omi), a serine protease, is released into the cytosol from the mitochondrial intermembrane space (MIS). In the cytosol, HtrA2/Omi prevents phosphorylation of cofilin, allows F-actin polymerization and lamellipodia formation, and induces cell migration and invasion. 78 Drp1 promotes chemotherapy resistance by regulating nuclear respiratory factor 2 (NRF2), 79 TAZ, 80 and SREBP1/2. 81 Moreover, the YAP-TEAD1 axis promotes focal adhesion (FA) gene transcription in TNBC cells, 82 thereby stabilizing the FA-cytoskeleton, which promotes drug resistance. 83 YAP also transcriptionally regulates mitophagy proteins PINK1 and PARK2 83 and inhibits ER stress-induced apoptosis by suppressing caspase activity. 84 These findings suggest the importance of YAP1 in mitochondrial dynamics, specifically within the context of TNBC and its response to therapy.

ER-mitochondria communication in BC

ER-mitochondrial communication is essential for maintaining cellular fitness. Bidirectional interactions regulate proteostasis as well as the trafficking of metabolites, lipids, and proteins across organelle membranes. This dynamic communication loop ensures proper organelle functionality and overall cellular survival. Disrupted mitochondrial-ER communication, commonly observed during aging, is implicated in age-related diseases, including cancer. 85

ER stress

The ER is mainly associated with protein synthesis, folding and quality control, calcium homeostasis, and lipid biosynthesis ( Figure 5 A). However, this physiological state of proteostasis is disturbed by overloading of unfolded or misfolded proteins and calcium, nutrient deprivation, oxidative stress (OS), and drugs, leading to ER stress. 86 Conversely, mitochondria signal to the ER when experiencing protein or metabolite overload.

The unfolded protein response (UPR) pathway handles the ER stress. 87 Mechanistically, three key ER stress-mediating sensors facilitate the UPR, including protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6). ER stress sensing begins with X-box-binding protein 1 (XBP1) mRNA spliced by IRE1. 88 Further, XBP1 transcriptionally controls protein expression associated with maintaining protein quality and folding, like HSPA5, which assists protein folding and ER-associated degradation (ERAD) component, which facilitates clearance of misfolded proteins. 89

ER stress can be triggered by various pathophysiological conditions, including carcinogenic, transcriptional, and biochemical aberrations. 90 In the TME, protein folding and ER stress are altered in cancer cells and immune cells beyond some tolerable threshold. This leads to the elevation of unfolded and misfolded proteins in the ER, which stimulates ER stress sensors. Mild ER stress can control immunologic and other tumor-associated cells which can promote neoplastic development and progression. 91 In early stages, ER stress communicates to other organelles via MAMs and triggers the adaptive mechanisms to promote cell survival.

Chronic ER stress interferes with functionality in epithelial cells and stroma, including cancer-promoting immune cells. Consequently, activated ER stress receptors and their regulatory cascades trigger abnormal expression of genes mediating metastasis and drug resistance. 91 On the other hand, severe and fatal ER stress triggers immunogenic cell death (ICD) and therapeutic immune responses against tumors. 92 These studies highlight the context-dependent role of ER stress in TME cells.

MAMs and tethering

ER stress also impacts mitochondrial function through MAMs. 93 MAMs are contact regions that primarily tether ER to mitochondria. 94 In higher eukaryotes, Mega2 forms contact sites in MAMs ( Figure 5 B). It forms a light complex with VAPB and increases lipid transport, with a strong preference for trafficking phosphatidyl serine. 95 Cancer cells have irregular MAMs and aberrantly expressed MAM-constitutive proteins. 96 Additionally, MAMs contain both cancer-causing and suppressor proteins, suggesting that complex MAM regulation is linked to tumorigenesis and tumor growth. 97

ER-mitochondria crosstalk can thus facilitate transferring calcium to mitochondria in order to activate pro-survival mechanisms or induce apoptosis. 98 For example, pro-survival proteins or peptides such as Bax inhibitor-1 (BI-1), Bcl-2 proteins, and Ras use the reduction of calcium stores in the ER as a survival strategy. 99 Calcium signaling also governs BC growth and drug resistance. 100 , 101 In addition, the localization of calcium flux also correlates with carcinogenesis. 102 In contrast, many chemotherapeutic agents trigger apoptosis by disrupting ER-mitochondria interactions and inducing calcium dysregulation. 103 Bax and Bid, normally found in the cytoplasm, translocate to both the mitochondria and the ER depending on the stimuli where they trigger cytochrome c release and mitochondrial-mediated apoptosis.

ER-mitochondria crosstalk regulates ROS generation and antioxidant defense. Elevated ROS levels, 104 along with poor antioxidant activity, 105 have been noted in patients with cancer. ROS represents a signaling process controlling mitogen-activated protein kinase (MAPK) stimulation, 106 while antioxidants inhibit MAPK stimulation. 107 This leads to the induction of cell death in cancer cells. Similarly, ROS scavenging has been shown to reduce metastasis. 108

PDZD8 is localized in the ER, primarily at MAMs, and facilitates tethering of the ER to the mitochondria. It mainly regulates the trafficking of lipids from the ER to mitochondria and the levels of cytoplasmic calcium by facilitating the uptake of calcium by mitochondria. A recent study demonstrated that Cdk5 found in MAMs promotes BC by regulating calcium homeostasis in mitochondria via negatively controlling the opening of mitochondrial permeability transition pore and facilitating the uptake of calcium by mitochondria. 109 Abnormal ER stress, ER-mitochondrial tethering, and calcium signaling remodel BC cells and upregulate genes mediating drug resistance and metastasis. Signals like cytochrome c and calcium transfer to the ER by undergoing prolonged opening of IPR6 or recruiting F1SI, which forms the caspase activation complex, acrosome. 110

Strategies for targeting mitochondrial-ER dynamics

The imbalance between mitochondrial fission and fusion causes altered mitochondrial dynamics that support cell survival, progression, and adaptation. Key strategies include the inhibition of mitochondrial fission and the induction of mitochondrial fusion, mitophagy, and ROS generation by exploring chemotherapeutic drugs, clinical metabolites, and natural compounds to efficiently target drug resistance and metastasis in BC.

Targeting mitochondrial dynamics in BC

Recently, several anticancer agents have been explored to target mitochondrial and ER dynamics in BC. Capivasertib (AZD5363), a competitive blocker of the ATP-binding site of AKT, is approved for treating HER2 type BC. It has also been reported to sensitize TNBC stem cells to doxorubicin (DOX) by targeting mitofusion and DOX-induced expression of CD133 and CD44. 111

Recently approved bromodomain inhibitors, birabresib (OTX-015) and JQ1 (thienotriazolodiazepine), reduce mitochondrial fission, mitochondrial mass, and mitochondrial DNA. Treatment with OTX-015 for 48 h reduced the phosphorylated Drp1 and MFF in TNBC cells. The combination treatment also induced mitochondrial elongation by promoting fusion. 112

Flubendazole (FBZ), an anthelminthic drug, has been reported to induce apoptosis and mitophagy in TNBC cells by enhancing the expression of eva-1 homolog A (EVA1A). It impairs the mitochondrial functions by affecting the OMM permeability. Mechanistically, FBZ induces mitophagy by enhancing the expression of Drp1, which in turn promotes the accumulation of PINK1. The accumulated PINK1 causes mitochondrial dysfunction by promoting mitophagy via increasing the expression of EVA1A and inhibiting the progression of BC growth. 113 A recent study demonstrated that sulfhydryl group donor, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), induces mitochondrial fusion by reducing Drp1 expression and enhancing Mfn2 expression. Simultaneously, it reduces the mitophagy flux and inhibits mitochondrial function, ultimately reducing metastasis of TNBC cells. 114

Isotoosendanin (ITSN), a plant-derived triterpenoid, reportedly inhibits TNBC metastasis by blocking TGFβR1. Mechanistically, ITSN reduces the expression of GOT2 by abolishing transforming growth factor β (TGF-β)-Smad2/3 signaling via interacting with TGFβR1. GOT2 promotes translocation of MYH9 to mitochondria, where it triggers phosphorylation of Drp1 and promotes mitochondrial fission, subsequently increasing metastasis lamellipodia formation. 115 Micheliolide (MCL) is a sesquiterpene lactone that selectively abrogates the proliferation of MCF-7 and MDA-MB-231 cells and induces apoptosis by increasing mitochondrial fission via upregulating Drp1. This upregulation of Drp1 induces apoptosis by promoting the loss of mitochondrial membrane potential, generation of ROS, release of cytochrome c , and cleavage of PARP. However, N-acetyl-L-cysteine reversed the MCL-induced apoptosis and downregulated Drp1, indicating the role of Drp1 in MCL-induced apoptosis via the generation of mitochondrial ROS. 116

Hu et al. demonstrated that exposure of TNBC cells to chloroquine (CQ), a quinoline derivative, and isorhamnetin (IH), an O-methylated flavonol, induces apoptosis by enhancing mitochondrial fission. 117 Mechanistically, the combination causes the ROS generation and subsequent activation of CaMKII by phosphorylation at Thr286. The activated CaMKII, in turn, phosphorylates Drp1 at S616 and promotes its transfer to mitochondria, facilitating fission.

Yakobov et al. showed that ellagic acid, a polyphenol, alone or in combination with DOX, induces cell-cycle arrest of MCF-7 cells. 118 This study illustrated that the combination impaired mitochondrial metabolism by impacting mitochondrial fission and inducing mitochondrial fusion and loss of mitochondrial membrane potential by reducing the expression of Drp1.

Erucin (ERN) is an isothiocyanate derivative and structural analog of the anticancer drug sulforaphane. It induces apoptosis, loss of mitochondrial membrane potential, and mitochondrial fission in MCF-7 and MDA-MB-231 cells. Mechanistically, ERN causes mitochondrial fission by promoting transfer of Drp1 and cofilin from the cytosol to mitochondria. Additionally, it stimulates the colocalization and subsequent interaction of Drp1 with cofilin at the OMM by inducing dephosphorylation. This study showed that the dephosphorylation of Drp1 and cofilin is mediated by specific phosphatases via activating the ROCK1 signaling pathway. 119 Algebaly et al. demonstrated that an aqueous extract of Origanum majorana (AEOM) enriched with polyphenols inhibited proliferation and induced apoptosis in MCF-7 cells 120 Mechanistically, it hinders the uptake of glucose and mitochondrial fusion via inducing the expression of genes associated with OS and ROS generation.

Inhibitors of mitochondrial fission

Mitochondrial division inhibitor 1 (Mdivi-1) inhibits mitochondrial fission and induces mitochondrial elongation by targeting the GTPase activity of Drp1. Mechanistically, it enhances the levels of ROS, which causes OS that is fatal to cancer cells. 121 Another high-affinity inhibitor of Drp1 is Driptor1a, which prevents mitochondrial fission. 122 It targets the GTPase domain of Drp1 and blocks ROS generation.

Salt-inducible kinase 2 (SIK2), a serine/threonine kinase tumor suppressor, phosphorylates Drp1 at Ser616 and prevents OXPHOS in ovarian cancer. 123 It also acts by blocking the Ras/ERK pathway as well as phosphatidylinositol 3-kinase/AKT pathway, thus reducing the proliferation and survival of BC cells. 124 Additionally, it regulates EMT, thus preventing metastasis.

Luteolin (LUT) is a natural flavonoid that inhibits the activity of serum/glucocorticoid-regulated kinase (SGK1) and Akt pathways. It targets the SGK1-FOXO3a-BNIP3 signaling pathway, where inhibiting SGK1 activates FOXO3a, thereby upregulating BNIP3. It also induces apoptosis by plummeting the levels of BECN-1 and LC3-II, thereby reducing p62. 125 It also affects the YAP pathway.

Cepharanthine, a bis-benzyl-iso-quinoline alkaloid, inhibits the nuclear factor κB (NF-κB) pathway and AKT/mTOR signaling pathway, inducing apoptosis and the STAT3 pathway, thus suppressing tumor growth. 126 It prevents the fusion of autophagosomes with lysosomes by downregulating MY01C.

Malla et al. demonstrated two novel thieno[2,3-d] pyrimidine derivatives that induced cell death independent of apoptosis in TNBC cells without DNA damage, cellular shrinkage, and activation of caspases. This study found that both compounds inhibited mitochondrial fission by downregulating Drp1 expression. Further, the downregulation of Drp1 expression was confirmed by partial as well as complete DRP1 knockout. 127 These studies imply that targeting mitochondrial fission is a promising strategy for treating BC.

Targeting mitochondrial fusion

Another promising approach is targeting mitochondrial fusion. Braganza et al. demonstrated that myoglobin, an oxygen storage protein, positively regulates mitochondrial fusion and inhibits BC growth under hypoxic stress by modulating PARKN. 128 This study, using cell lines and xenograft murine models, observed that myoglobin protects against BC by attenuating growth via upregulating expression of fusion proteins Mfn1 and Mfn2. Mfn1 and Mfn2 caused cell-cycle arrest and inhibition of growth by inducing ROS generation, which resulted in oxidative degradation of PARKN.

Silibinin (silybin, SIL) is a natural flavonoid that inhibited BC growth in preclinical models. SIL enhanced mitochondrial fusion by reducing Drp1 expression and augmenting OPA1, Mfn1, and Mfn2 expression. It also reduced ROS generation in MDA-MB 231 cells. Furthermore, SIL inhibits the biogenesis of mitochondria by reducing expression of NRF2, mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor gamma coactivator-α (PGC1-α). 129 Together, these results suggest that SIL inhibits the growth of TNBC by reducing ROS, enhancing mitochondrial fusion, and inhibiting mitochondrial biogenesis.

Chang et al. demonstrated that adjuvant treatment with Pep-1-conjugated mitochondria (P-Mito) sensitized TNBC cells to DOX by inducing mitochondrial fusion as well as mitophagy by enhancing Mfn2 and OPA1 expression and restoring the biogenesis of mitochondria by reducing the expression of PARKN. 130 Similar results were observed by the same group in TNBC cells implanted in a mouse model with P-Mito treatment. 131 Additionally, P-Mito treatment caused DNA fragmentation and necrosis in TNBC cells and remodeled the TME by reducing the OS and size of CAFs and increasing the infiltration of immune cells.

Targeting mitophagy

Targeting PINK1/PARKN is the most studied approach to understand mitochondrial dynamics by targeting mitophagy. Compounds like betulinic acid, a pentacyclic triterpenoid, target PINK1 on the OMM and recruit PARKN to ubiquitinate various mitochondrial proteins. Thus, it promotes the clearance of dysfunctional mitochondria, reduces OS, and inhibits drug-resistant cancer cell proliferation. 132 Polyphyllin-I (PP-I), a natural saponin, induces mitophagy by enhancing the stability of PINK1 at OMM and facilitating the recruitment of PARKN and ubiquitin, assisting LC3-dependent tethering of damaged mitochondria to autophagosomes. 133 A non-steroidal isoflavone, formononetin, induces mitophagy. 134 It triggers the mitochondrial damage and stimulates p53-dependent PINK1/PARKN axis via reducing the expression of BACH1 in TNBC cells.

A natural alkaloid, cyclovirobuxine D (CVB-D), induces mitophagy in TNBC cells by targeting the YAP-dependent Drp1 pathway and triggering activation of both PINK1-PARKN-dependent and independent mechanisms. 135 A synthetic analog of coenzyme Q10, idebenone, induces mitophagy and disruption of mitochondrial membrane potential and inhibits the growth of TNBC cells via inhibiting the GADD45/CyclinB/CDK1 axis. 83 Another study demonstrated that a quercetin derivative (TL-2-8) induces mitophagy in MDA-MB 231 cells. 136 Mechanistically, it induces mitophagy by diminishing lysosomal-associated membrane protein 2 (LAMP2) expression and inhibiting the formation of the Hsp90-AHA1 complex in the ER-mitochondria interface via reducing the expression of AHA1.

Therapeutics targeting ER stress

ER stress inducers and UPR modulators reportedly have anticancer activity against BC. Thapsigargin targets the Sarco/ER Ca2+-ATPase (SERCA) pump and depletes calcium stores, resulting in the elevation of the misfolded proteins and activating UPR, which, in turn, causes ER stress. This leads to the activation of PERK, IRE1, ATF4, and CHOP, which activate caspases-3 and 8 and lead to apoptosis. 137 Tunicamycin inhibits N-glycosylation by blocking the enzyme N-acetylglucosamine-1-phosphate transferase (GlcNAc1PT). 138 This blocking results in the elevation of improperly folded glycoproteins in the ER, provoking ER stress and the UPR. It induces ER stress and triggers apoptosis by activating the Wnt/β-catenin signaling pathway. 139 Bortezomib (BTZ), a protease inhibitor, disrupts protein degradation and elevates misfolded proteins in the ER, thus triggering ER stress and stimulating the UPR, which can result in apoptosis in cancer cells ( Table 1 ). 140

Prospectives of targeting mitochondria-ER interface in BC

The ER-mitochondria interface has been extensively studied in various cancers, including BC, highlighting its prospects as a therapeutic target. However, its role in targeting cancer growth, drug resistance, and metastasis is relatively underexplored. Ciscato et al. demonstrated that a selective peptide induced apoptosis and inhibited BC cell growth by inducing mitochondrial calcium overload via displacing hexokinase-2 from MAMs. While this study revealed a promising approach, the detailed mechanisms involved remain to be fully elucidated. 141 Another study explored PS89, an inhibitor of protein disulfide isomerase, to target the ER-mitochondrial interface in leukemia cells, in combination with chemotherapeutic anticancer drugs. This research showed that PS89 sensitizes leukemia cells by reversing death signals from the mitochondria to the ER, inducing degradation of B cell receptor-associated protein 31 at the ER-mitochondrial interface. This degradation leads to ER-mediated apoptosis through increased ROS generation and activation of caspase-8. 142 Miga 2, a mitochondrial lipid transporter protein, regulates cancer progression via the YAP pathway by facilitating the exchange of lipids between the ER and mitochondria and plays an important role in maintaining mitochondrial morphology by controlling mitochondrial fusion and fission through Toll-like receptors. 143 Interestingly, pharmacological targeting of Miga 2 triggers interleukin-12 production and the activation of T cells and antitumor immune cells.

Translating fundamental discoveries in mitochondrial biology into viable clinical strategies is a key goal in oncology, and targeting mitochondrial fusion represents a particularly compelling avenue for clinical translation in cancer research. Targeting mitochondrial dynamics represents an emerging frontier in cancer therapy, with the modulation of mitochondrial fusion emerging as a particularly promising clinical approach. The intricate balance between mitochondrial fusion, mediated primarily by MFN1, MFN2, and OPA1, and fission is crucial for maintaining mitochondrial health, metabolic function, and cellular homeostasis—processes frequently dysregulated in cancer cells to support their growth, survival, and resistance to therapy. 144 , 145 Compelling evidence suggests that enhancing mitochondrial fusion can exert anticancer effects. For instance, reduced expression of fusion proteins like MFN2 has been linked to increased metastatic potential in certain cancers, implying that restoring fusion could suppress tumor progression. 146 Furthermore, impairments in mitochondrial fusion, often driven by OPA1 downregulation or dysfunction, have been associated with increased chemoresistance, suggesting that pharmacologically promoting OPA1 activity or fusion processes might re-sensitize cancer cells to existing treatments. 147 Therefore, the development of pharmacological agents or genetic approaches specifically designed to promote mitochondrial fusion in cancer cells is an active area of research poised for clinical translation, offering a novel therapeutic axis to disrupt cancer cell metabolism, limit progression, and overcome treatment failure.

Conclusion and future directions

The bidirectional transmission of signals from the ER to mitochondria through tethering proteins in MAMs manifests the dynamic exchange of lipids, calcium, and other signaling molecules. 148 The dysregulation of the ER-mitochondrial communications profoundly influences the cellular metabolism of BC cells and other cells in the TME. Further, many of the molecules alter downstream pathways and myriad signaling molecules, like intracellular calcium, that have already been implicated in regulating metastasis, 96 , 149 , 150 drug resistance, 151 and immune function. 152 Therefore, understanding ER-mitochondrial interplay has substantial clinical relevance and promise. There are clearly promising new avenues for developing targeted therapies, offering a unique strategy to combat BC effectively. However, translating this knowledge into clinical applications necessitates further research. Continued investigation into the specific molecular mechanisms that govern this crosstalk is crucial for identifying clinically useful targets. Further, developing novel therapeutic strategies that modulate ER-mitochondrial interplay—either by targeting mitochondrial fusion or fission, promoting controlled ER stress, or disrupting aberrant ER-mitochondria tethering—holds immense potential for suppressing BC progression. Ultimately, this approach will lead to the identification of effective and specific therapies to enhance the outcomes in patients.

Some aspects of the tethering that should be priorities for further research include identifying novel tethering proteins, characterizing regulatory pathways involving post-translational modifications, deciphering the context-dependent roles of specific interactions in mitochondrial dynamics and function, and defining common pathways regulating ER-mitochondrial communications. Each of these refinements will improve off-target or unwanted side effects that lead to suboptimal use of effective therapies or reduced quality of life for patients undergoing treatments with those therapies. Future research should also focus on the design and validation of combinatorial therapies that concurrently target both ER and mitochondrial pathways to overcome single-target treatment limitations and enhance anticancer efficacy in TNBC.

Acknowledgments

The project was supported by GRIG funds, GITAM (Deemed to be University), DST-PURSE [SR/PURSE/2023/169(G)], and DST-ANRF India (CRG/2023/001951). D.R.W. was partially supported by USPHS grant P30-CA168524 from the National Cancer Institute, USA. The authors thank the GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India, for support. The figures were drawn using licensed version of BioRender software.

Author contributions

R.R.M.: conceptualization, funding acquisition, project administration, resources, supervision, visualization, and writing – review and editing; C.P.: writing – original draft; Y.N.: writing – original draft; A.S.S.: writing – original draft; P.B.: writing – original draft; C.M.: writing – original draft; D.R.W.: partial funding acquisition and writing – review and editing; G.P.N.: writing – review and editing.

Declaration of interests

The authors declare no competing interests.

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