The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction.

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma.

Effectively targeting cancer stemness is essential for successful cancer therapy. Recent studies have revealed that SOX2, a pluripotent stem cell factor, significantly contributes to cancer stem cell (CSC)-like characteristics closely associated with cancer malignancy. However, its contradictory impact on patient survival in specific cancer types, including lung adenocarcinoma (LUAD), underscores the need for more comprehensive research to clarify its functional effect on cancer stemness. In this study, we demonstrate that SOX2 is not universally required for the regulation of CSC-like properties in LUAD. We generated SOX2 knockouts in A549, H358, and HCC827 LUAD cells using the CRISPR/Cas9 system. Our results reveal unchanged CSC characteristics, including sustained proliferation, tumor sphere formation, invasion, migration, and therapy resistance, compared to normal cells. Conversely, SOX2 knockdown using conditional shRNA targeting SOX2, significantly reduced CSC traits. However, these loss-of-function effects were not rescued by SOX2 resistant to shRNA, underscoring the potential for SOX2 protein level-independent results in prior siRNA- or shRNA-based research. Ultimately, our findings demonstrate that SOX2 is not absolutely essential in LUAD cancer cells. This emphasizes the necessity of considering cancer subtype-dependent and context-dependent factors when targeting SOX2 overexpression as a potential therapeutic vulnerability in diverse cancers.

Glucose Deprivation Induces Cancer Cell Death through Failure of ROS Regulation.

In previous work, we showed that cancer cells do not depend on glycolysis for ATP production, but they do on fatty acid oxidation. However, we found some cancer cells induced cell death after glucose deprivation along with a decrease of ATP production. We investigated the different response of glucose deprivation with two types of cancer cells including glucose insensitive cancer cells (GIC) which do not change ATP levels, and glucose sensitive cancer cells (GSC) which decrease ATP production in 24 h. Glucose deprivation-induced cell death in GSC by more than twofold after 12 h and by up to tenfold after 24 h accompanied by decreased ATP production to compare to the control (cultured in glucose). Glucose deprivation decreased the levels of metabolic intermediates of the pentose phosphate pathway (PPP) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) in both GSC and GIC. However, glucose deprivation increased reactive oxygen species (ROS) only in GSC, suggesting that GIC have a higher tolerance for decreased NADPH than GSC. The twofold higher ratio of reduced/oxidized glutathione (GSH/GSSG) in GIS than in GSC correlates closely with the twofold lower ROS levels under glucose starvation conditions. Treatment with N-acetylcysteine (NAC) as a precursor to the biologic antioxidant glutathione restored ATP production by 70% and reversed cell death caused by glucose deprivation in GSC. The present findings suggest that glucose deprivation-induced cancer cell death is not caused by decreased ATP levels, but rather triggered by a failure of ROS regulation by the antioxidant system. Conclusion is clear that glucose deprivation-induced cell death is independent from ATP depletion-induced cell death.

BMS794833 inhibits macrophage efferocytosis by directly binding to MERTK and inhibiting its activity.

Myeloid epithelial reproductive proto-oncogene tyrosine kinase (MERTK) plays an essential role in modulating cancer immune tolerance by regulating macrophage efferocytosis. Studies are underway to develop small-molecule chemicals that inhibit MERTK as cancer immunotherapeutic agents, but these efforts are in their early stages. This study identified BMS794833, whose primary targets are MET and VEGFR2, as a potent MERTK inhibitor and developed a real-time efferocytosis monitoring system. The X-ray cocrystal structure revealed that BMS794833 was in contact with the ATP-binding pocket and the allosteric back pocket, rendering MERTK inactive. Homogeneous time-resolved fluorescence kinetic and Western blotting analyses showed that BMS794833 competitively inhibited MERTK activity in vitro and inhibited the autophosphorylation of MERTK in macrophages. We developed a system to monitor MERTK-dependent efferocytosis in real time, and using this system, we confirmed that BMS794833 significantly inhibited the efferocytosis of differentiated macrophages. Finally, BMS794833 significantly inhibited efferocytosis in vivo in a mouse model. These data show that BMS794833 is a type II MERTK inhibitor that regulates macrophage efferocytosis. In addition, the real-time efferocytosis monitoring technology developed in this study has great potential for future applications.

Cholesterol Synthesis Is Important for Breast Cancer Cell Tumor Sphere Formation and Invasion.

Breast cancer has a high risk of recurrence and distant metastasis after remission. Controlling distant metastasis is important for reducing breast cancer mortality, but accomplishing this goal remains elusive. In this study, we investigated the molecular pathways underlying metastasis using cells that mimic the breast cancer distant metastasis process. HCC1143 breast cancer cells were cultured under two-dimensional (2D)-adherent, tumor sphere (TS), and reattached (ReA) culture conditions to mimic primary tumors, circulating tumor cells, and metastasized tumors, respectively. ReA cells demonstrated increased TS formation and enhanced invasion capacity compared to the original 2D-cultured parental cells. In addition, ReA cells had a higher frequency of ESA+CD44+CD24- population, which represents a stem-cell-like cell population. RNA sequencing identified the cholesterol synthesis pathway as one of the most significantly increased pathways in TS and ReA cells compared to parental cells, which was verified by measuring intracellular cholesterol levels. Furthermore, the pharmacological inhibition of the cholesterol synthesis pathway decreased the ability of cancer cells to form TSs and invade. Our results suggest that the cholesterol synthesis pathway plays an important role in the distant metastasis of breast cancer cells by augmenting TS formation and invasion capacity.

Cancer depends on fatty acids for ATP production: A possible link between cancer and obesity.

Several metabolic pathways for the supply of adenosine triphosphate (ATP) have been proposed; however, the major source of reducing power for ADP in cancer remains unclear. Although glycolysis is the source of ATP in tumors according to the Warburg effect, ATP levels do not differ between cancer cells grown in the presence and absence of glucose. Several theories have been proposed to explain the supply of ATP in cancer, including metabolic reprograming in the tumor microenvironment. However, these theories are based on the production of ATP by the TCA-OxPhos pathway, which is inconsistent with the Warburg effect. We found that blocking fatty acid oxidation (FAO) in the presence of glucose significantly decreased ATP production in various cancer cells. This suggests that cancer cells depend on fatty acids to produce ATP through FAO instead of glycolysis. We observed that cancer cell growth mainly relies on metabolic nutrients and oxygen systemically supplied through the bloodstream instead of metabolic reprogramming. In a spontaneous mouse tumor model (KrasG12D; Pdx1-cre), tumor growth was 2-fold higher in mice fed a high-fat diet (low-carbo diet) that caused obesity, whereas a calorie-balanced, low-fat diet (high-carbo diet) inhibited tumor growth by 3-fold compared with that in mice fed a control/normal diet. This 5-fold difference in tumor growth between mice fed low-fat and high-fat diets suggests that fat-induced obesity promotes cancer growth, and tumor growth depends on fatty acids as the primary source of energy.

O-GlcNAcylation of Sox2 at threonine 258 regulates the self-renewal and early cell fate of embryonic stem cells.

Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions.

Reply to Krupenko et al. Comment on "Lee et al. The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer Cancers 2020, 12, 1382".

In the Cancers paper, we observed the increase ALDH1L1 protein expression following oncogenesis, as well as a therapeutic effect, by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC) [...].

Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes.

In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51-VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function.

PGC1α Loss Promotes Lung Cancer Metastasis through Epithelial-Mesenchymal Transition.

PGC1α oppositely regulates cancer metastasis in melanoma, breast, and pancreatic cancer; however, little is known about its impact on lung cancer metastasis. Transcriptome and in vivo xenograft analysis show that a decreased PGC1α correlates with the epithelial-mesenchymal transition (EMT) and lung cancer metastasis. The deletion of a single Pgc1α allele in mice promotes bone metastasis of KrasG12D-driven lung cancer. Mechanistically, PGC1α predominantly activates ID1 expression, which interferes with TCF4-TWIST1 cooperation during EMT. Bioinformatic and clinical studies have shown that PGC1α and ID1 are downregulated in lung cancer, and correlate with a poor survival rate. Our study indicates that TCF4-TWIST1-mediated EMT, which is regulated by the PGC1α-ID1 transcriptional axis, is a potential diagnostic and therapeutic target for metastatic lung cancer.

Overall survival of pancreatic ductal adenocarcinoma is doubled by Aldh7a1 deletion in the KPC mouse.

Rationale: The activity of aldehyde dehydrogenase 7A1 (ALDH7A1), an enzyme that catalyzes the lipid peroxidation of fatty aldehydes was found to be upregulated in pancreatic ductal adenocarcinoma (PDAC). ALDH7A1 knockdown significantly reduced tumor formation in PDAC. We raised a question how ALDH7A1 contributes to cancer progression. Methods: To answer the question, the role of ALDH7A1 in energy metabolism was investigated by knocking down and knockdown gene in mouse model, because the role of ALDH7A1 has been reported as a catabolic enzyme catalyzing fatty aldehyde from lipid peroxidation to fatty acid. Oxygen consumption rate (OCR), ATP production, mitochondrial membrane potential, proliferation assay and immunoblotting were performed. In in vivo study, two human PDAC cell lines were used for pre-clinical xenograft model as well as spontaneous PDAC model of KPC mice was also employed for anti-cancer therapeutic effect. Results:ALDH7A1 knockdown significantly reduced tumor formation with reduction of OCR and ATP production, which was inversely correlated with increase of 4-hydroxynonenal. This implies that ALDH7A1 is critical to process fatty aldehydes from lipid peroxidation. Overall survival of PDAC is doubled by cross breeding of KPC (KrasG12D; Trp53R172H; Pdx1-Cre) and Aldh7a1-/- mice. Conclusion: Inhibitions of ALDH7A1 and oxidative phosphorylation using gossypol and phenformin resulted in a regression of tumor formation in xenograft mice model and KPC mice model.

FAM188B Downregulation Sensitizes Lung Cancer Cells to Anoikis via EGFR Downregulation and Inhibits Tumor Metastasis In Vivo.

Anoikis is a type of apoptosis induced by cell detachment from the extracellular matrix (ECM), which removes mislocalized cells. Acquisition of anoikis resistance is critical for cancer cells to survive during circulation and, thus, metastasize at a secondary site. Although the sensitization of cancer cells to anoikis is a potential strategy to prevent metastasis, the mechanism underlying anoikis resistance is not well defined. Although family with sequence similarity 188 member B (FAM188B) is predicted as a new deubiquitinase (DUB) member, its biological function has not been fully studied. In this study, we demonstrated that FAM188B knockdown sensitized anoikis of lung cancer cell lines expressing WT-EGFR (A549 and H1299) or TKI-resistant EGFR mutant T790M/L858R (H1975). FAM188B knockdown using si-FAM188B inhibited the growth of all three human lung cancer cell lines cultured in both attachment and suspension conditions. FAM188B knockdown resulted in EGFR downregulation and thus decreased its activity. FAM188B knockdown decreased the activities of several oncogenic proteins downstream of EGFR that are involved in anoikis resistance, including pAkt, pSrc, and pSTAT3, with little changes to their protein levels. Intriguingly, si-FAM188B treatment increased EGFR mRNA levels but decreased its protein levels, which was reversed by treatment with the proteasomal inhibitor MG132, indicating that FAM188B regulates EGFR levels via the proteasomal pathway. In addition, cells transfected with si-FAM188B had decreased expression of FOXM1, an oncogenic transcription factor involved in cell growth and survival. Moreover, FAM188B downregulation reduced metastatic characteristics, such as cell adhesion, invasion, and migration, as well as growth in 3D culture conditions. Finally, tail vein injection of si-FAM188B-treated A549 cells resulted in a decrease in lung metastasis and an increase in mice survival in vivo. Taken together, these findings indicate that FAM188B plays an important role in anoikis resistance and metastatic characteristics by maintaining the levels of various oncogenic proteins and/or their activity, leading to tumor malignancy. Our study suggests FAM188B as a potential target for controlling tumor malignancy.

A Quenched Annexin V-Fluorophore for the Real-Time Fluorescence Imaging of Apoptotic Processes In Vitro and In Vivo.

Annexin-based probes have long been used to study apoptotic cell death, which is of key importance to many areas of biological research, drug discovery, and clinical applications. Although apoptosis is a dynamic biological event with cell-to-cell variations, current annexin-based probes are impractical for monitoring apoptosis in real-time. Herein, a quenched annexin V-near-infrared fluorophore conjugate (Q-annexin V) is reported as the first OFF-ON annexin protein-based molecular sensor for real-time near-infrared fluorescence imaging of apoptosis. Q-annexin V is non-fluorescent in the extracellular region, due to photoinduced electron transfer interactions between the conjugated dye and amino acid quenchers (tryptophan and tyrosine). The probe becomes highly fluorescent when bound to phosphatidylserines on the outer layer of cell membranes during apoptosis, thereby enabling apoptosis to be monitored in real-time in 2D and 3D cell structures. In particular, Q-annexin V shows superior utility for in vivo apoptosis fluorescence imaging in animal models of cisplatin-induced acute kidney injury and cancer immune therapy, compared to the conventional polarity-sensitive pSIVA-IANBD or annexin V-Alexa647 conjugates.

Oxoglutarate Carrier Inhibition Reduced Melanoma Growth and Invasion by Reducing ATP Production.

Recent findings indicate that (a) mitochondria in proliferating cancer cells are functional, (b) cancer cells use more oxygen than normal cells for oxidative phosphorylation, and (c) cancer cells critically rely on cytosolic NADH transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. In a spontaneous lung cancer model, tumor growth was reduced by 50% in heterozygous oxoglutarate carrier (OGC) knock-out mice compared with wild-type counterparts. To determine the mechanism through which OGC promotes tumor growth, the effects of the OGC inhibitor N-phenylmaleimide (NPM) on mitochondrial activity, oxygen consumption, and ATP production were evaluated in melanoma cell lines. NPM suppressed oxygen consumption and decreased ATP production in melanoma cells in a dose-dependent manner. NPM also reduced the proliferation of melanoma cells. To test the effects of NPM on tumor growth and metastasis in vivo, NPM was administered in a human melanoma xenograft model. NPM reduced tumor growth by approximately 50% and reduced melanoma invasion by 70% at a dose of 20 mg/kg. Therefore, blocking OGC activity may be a useful approach for cancer therapy.

SEZ6L2 Is an Important Regulator of Drug-Resistant Cells and Tumor Spheroid Cells in Lung Adenocarcinoma.

Many lung cancer deaths result from relapses in distant organs, such as the brain or bones, after standard chemotherapy. For cancer cells to spread to other organs, they must survive as circulating tumor cells (CTCs) in blood vessels. Thus, reducing distant recurrence after chemotherapy requires simultaneously inhibiting drug resistance and CTC survival. Here, we investigated the molecular pathways and genes that are commonly altered in drug-resistant lung cancer cells and lung tumor spheroid (TS) cells. First, RNA sequencing was performed in drug-resistant cells and TS cells originating from H460 and A549 lung cancer cells. Bioinformatic pathway analysis showed that cell cycle-related pathways were downregulated in drug-resistant cells, and cholesterol biosynthesis-related pathways were upregulated in TS cells. Seizure-related 6 homolog-like 2 (SEZ6L2) was selected as a gene that was commonly upregulated in both drug-resistant cells and TS cells, and that showed elevated expression in samples from lung adenocarcinoma patients. Second, the protein expression of SEZ6L2 was analyzed by flow cytometry. The proportions of SEZ6L2 positive cells among both drug-resistant cells and TS cells was increased. Finally, as SEZ6L2 is a transmembrane protein with an extracellular region, the function of SEZ6L2 was disrupted by treatment with an anti-SEZ6L2 antibody. Treatment with the anti-SEZ6L2 antibody reduced drug resistance and TS formation. Overall, our data showed that SEZ6L2 plays an important role in drug resistance and TS formation and may be a therapeutic target for reducing distant recurrence of lung adenocarcinoma.

Crystal Structure of the Kinase Domain of MerTK in Complex with AZD7762 Provides Clues for Structure-Based Drug Development.

Aberrant tyrosine-protein kinase Mer (MerTK) expression triggers prosurvival signaling and contributes to cell survival, invasive motility, and chemoresistance in many kinds of cancers. In addition, recent reports suggested that MerTK could be a primary target for abnormal platelet aggregation. Consequently, MerTK inhibitors may promote cancer cell death, sensitize cells to chemotherapy, and act as new antiplatelet agents. We screened an inhouse chemical library to discover novel small-molecule MerTK inhibitors, and identified AZD7762, which is known as a checkpoint-kinase (Chk) inhibitor. The inhibition of MerTK by AZD7762 was validated using an in vitro homogeneous time-resolved fluorescence (HTRF) assay and through monitoring the decrease in phosphorylated MerTK in two lung cancer cell lines. We also determined the crystal structure of the MerTK:AZD7762 complex and revealed the binding mode of AZD7762 to MerTK. Structural information from the MerTK:AZD7762 complex and its comparison with other MerTK:inhibitor structures gave us new insights for optimizing the development of inhibitors targeting MerTK.

Phosphorylation of OCT4 Serine 236 Inhibits Germ Cell Tumor Growth by Inducing Differentiation.

Octamer-binding transcription factor 4 (Oct4) plays an important role in maintaining pluripotency in embryonic stem cells and is closely related to the malignancies of various cancers. Although posttranslational modifications of Oct4 have been widely studied, most of these have not yet been fully characterized, especially in cancer. In this study, we investigated the role of phosphorylation of serine 236 of OCT4 [OCT4 (S236)] in human germ cell tumors (GCTs). OCT4 was phosphorylated at S236 in a cell cycle-dependent manner in a patient sample and GCT cell lines. The substitution of endogenous OCT4 by a mimic of phosphorylated OCT4 with a serine-to-aspartate mutation at S236 (S236D) resulted in tumor cell differentiation, growth retardation, and inhibition of tumor sphere formation. GCT cells expressing OCT4 S236D instead of endogenous OCT4 were similar to cells with OCT4 depletion at the mRNA transcript level as well as in the phenotype. OCT4 S236D also induced tumor cell differentiation and growth retardation in mouse xenograft experiments. Inhibition of protein phosphatase 1 by chemicals or short hairpin RNAs increased phosphorylation at OCT4 (S236) and resulted in the differentiation of GCTs. These results reveal the role of OCT4 (S236) phosphorylation in GCTs and suggest a new strategy for suppressing OCT4 in cancer.

ATP Production Relies on Fatty Acid Oxidation Rather than Glycolysis in Pancreatic Ductal Adenocarcinoma.

Glycolysis is known as the main pathway for ATP production in cancer cells. However, in cancer cells, glucose deprivation for 24 h does not reduce ATP levels, whereas it does suppress lactate production. In this study, metabolic pathways were blocked to identify the main pathway of ATP production in pancreatic ductal adenocarcinoma (PDAC). Blocking fatty acid oxidation (FAO) decreased ATP production by 40% in cancer cells with no effect on normal cells. The effects of calorie balanced high- or low-fat diets were tested to determine whether cancer growth is modulated by fatty acids instead of calories. A low-fat diet caused a 70% decrease in pancreatic preneoplastic lesions compared with the control, whereas a high-fat diet caused a two-fold increase in preneoplastic lesions accompanied with increase of ATP production in the Kras (G12D)/Pdx1-cre PDAC model. The present results suggest that ATP production in cancer cells is dependent on FAO rather than on glycolysis, which can be a therapeutic approach by targeting cancer energy metabolism.

Targeting Oxidative Phosphorylation Reverses Drug Resistance in Cancer Cells by Blocking Autophagy Recycling.

The greatest challenge in cancer therapy is posed by drug-resistant recurrence following treatment. Anticancer chemotherapy is largely focused on targeting the rapid proliferation and biosynthesis of cancer cells. This strategy has the potential to trigger autophagy, enabling cancer cell survival through the recycling of molecules and energy essential for biosynthesis, leading to drug resistance. Autophagy recycling contributes amino acids and ATP to restore mTOR complex 1 (mTORC1) activity, which leads to cell survival. However, autophagy with mTORC1 activation can be stalled by reducing the ATP level. We have previously shown that cytosolic NADH production supported by aldehyde dehydrogenase (ALDH) is critical for supplying ATP through oxidative phosphorylation (OxPhos) in cancer cell mitochondria. Inhibitors of the mitochondrial complex I of the OxPhos electron transfer chain and ALDH significantly reduce the ATP level selectively in cancer cells, terminating autophagy triggered by anticancer drug treatment. With the aim of overcoming drug resistance, we investigated combining the inhibition of mitochondrial complex I, using phenformin, and ALDH, using gossypol, with anticancer drug treatment. Here, we show that OxPhos targeting combined with anticancer drugs acts synergistically to enhance the anticancer effect in mouse xenograft models of various cancers, which suggests a potential therapeutic approach for drug-resistant cancer.

Cancer cells undergoing epigenetic transition show short-term resistance and are transformed into cells with medium-term resistance by drug treatment.

To elucidate the epigenetic mechanisms of drug resistance, epigenetically reprogrammed H460 cancer cells (R-H460) were established by the transient introduction of reprogramming factors. Then, the R-H460 cells were induced to differentiate by the withdrawal of stem cell media for various durations, which resulted in differentiated R-H460 cells (dR-H460). Notably, dR-H460 cells differentiated for 13 days (13dR-H460 cells) formed a significantly greater number of colonies showing drug resistance to both cisplatin and paclitaxel, whereas the dR-H460 cells differentiated for 40 days (40dR-H460 cells) lost drug resistance; this suggests that 13dR-cancer cells present short-term resistance (less than a month). Similarly, increased drug resistance to both cisplatin and paclitaxel was observed in another R-cancer cell model prepared from N87 cells. The resistant phenotype of the cisplatin-resistant (CR) colonies obtained through cisplatin treatment was maintained for 2-3 months after drug treatment, suggesting that drug treatment transforms cells with short-term resistance into cells with medium-term resistance. In single-cell analyses, heterogeneity was not found to increase in 13dR-H460 cells, suggesting that cancer cells with short-term resistance, rather than heterogeneous cells, may confer epigenetically driven drug resistance in our reprogrammed cancer model. The epigenetically driven short-term and medium-term drug resistance mechanisms could provide new cancer-fighting strategies involving the control of cancer cells during epigenetic transition.