Depletion of proteasome subunit PSMD1 induces cancer cell death via protein ubiquitination and DNA damage, irrespective of p53 status.

Hepatocellular carcinoma (HCC) is characterized by high incidence and fatality rates worldwide. In our exploration of prognostic factors in HCC, the 26s proteasome subunit, non-ATPase 1 (PSMD1) protein emerged as a significant contributor, demonstrating its potential as a therapeutic target in this aggressive cancer. PSMD1 is a subunit of the 19S regulatory particle in the 26S proteasome complex; the 19S particle controls the deubiquitination of ubiquitinated proteins, which are then degraded by the proteolytic activity of the complex. Proteasome-targeting in cancer therapy has received significant attention because of its practical application as an established anticancer agent. We investigated whether PSMD1 plays a critical role in cancer owing to its prognostic significance. PSMD1 depletion induced cell cycle arrest in G2/M phase, DNA damage and apoptosis of cancer cells, irrespective of the p53 status. PSMD1 depletion-mediated cell death was accompanied by an increase in overall protein ubiquitination. These phenotypes occurred exclusively in cancer cells, with no effects observed in normal cells. These findings indicate that PSMD1 depletion-mediated ubiquitination of cellular proteins induces cell cycle arrest and eventual death in cancer cells, emphasizing PSMD1 as a potential therapeutic target in HCC.

Elevated level of PLRG1 is critical for the proliferation and maintenance of genome stability of tumor cells.

Pleiotropic regulator 1 (PLRG1), a highly conserved element in the spliceosome, can form a NineTeen Complex (NTC) with Prp19, SPF27, and CDC5L. This complex plays crucial roles in both pre-mRNA splicing and DNA repair processes. Here, we provide evidence that PLRG1 has a multifaceted impact on cancer cell proliferation. Comparing its expression levels in cancer and normal cells, we observed that PLRG1 was upregulated in various tumor tissues and cell lines. Knockdown of PLRG1 resulted in tumor-specific cell death. Depletion of PLRG1 had notable effects, including mitotic arrest, microtubule instability, endoplasmic reticulum (ER) stress, and accumulation of autophagy, ultimately culminating in apoptosis. Our results also demonstrated that PLRG1 downregulation contributed to DNA damage in cancer cells, which we confirmed through experimental validation as DNA repair impairment. Interestingly, when PLRG1 was decreased in normal cells, it induced G1 arrest as a self-protective mechanism, distinguishing it from effects observed in cancer cells. These results highlight multifaceted impacts of PLRG1 in cancer and underscore its potential as a novel anti-cancer strategy by selectively targeting cancer cells. [BMB Reports 2023; 56(11): 612-617].

NADH elevation during chronic hypoxia leads to VHL-mediated HIF-1α degradation via SIRT1 inhibition.

BACKGROUND: Under conditions of hypoxia, cancer cells with hypoxia inducible factor-1α (HIF-1α) from heterogeneous tumor cells show greater aggression and progression in an effort to compensate for harsh environmental conditions. Extensive study on the stability of HIF-1α under conditions of acute hypoxia in cancer progression has been conducted, however, understanding of its involvement during the chronic phase is limited. METHODS: In this study, we investigated the effect of SIRT1 on HIF1 stability in a typical chronic hypoxic conditon that maintains cells for 24 h under hypoxia using Western blotting, co-IP, measurement of intracellular NAD + and NADH levels, semi-quantitative RT-PCR analysis, invasion assay, gene knockdown. RESULTS: Here we demonstrated that the high concentration of pyruvate in the medium, which can be easily overlooked, has an effect on the stability of HIF-1α. We also demonstrated that NADH functions as a signal for conveyance of HIF-1α degradation via the SIRT1 and VHL signaling pathway under conditions of chronic hypoxia, which in turn leads to attenuation of hypoxically strengthened invasion and angiogenic activities. A steep increase in the level of NADH occurs during chronic hypoxia, leading to upregulation of acetylation and degradation of HIF-1α via inactivation of SIRT1. Of particular interest, p300-mediated acetylation at lysine 709 of HIF-1α is recogonized by VHL, which leads to degradation of HIF-1α via ubiquitin/proteasome machinary under conditions of chronic hypoxia. In addition, we demonstrated that NADH-elevation-induced acetylation and subsequent degradation of HIF-1α was independent of proline hydroxylation. CONCLUSIONS: Our findings suggest a critical role of SIRT1 as a metabolic sensor in coordination of hypoxic status via regulation of HIF-1α stability. These results also demonstrate the involvement of VHL in degradation of HIF-1α through recognition of PHD-mediated hydroxylation in normoxia and p300-mediated HIF-1α acetylation in hypoxia.

NPFFR2 Contributes to the Malignancy of Hepatocellular Carcinoma Development by Activating RhoA/YAP Signaling.

G protein-coupled receptors (GPCRs) are a diverse family of cell surface receptors implicated in various physiological functions, making them common targets for approved drugs. Many GPCRs are abnormally activated in cancers and have emerged as therapeutic targets for cancer. Neuropeptide FF receptor 2 (NPFFR2) is a GPCR that helps regulate pain and modulates the opioid system; however, its function remains unknown in cancers. Here, we found that NPFFR2 is significantly up-regulated in liver cancer and its expression is related to poor prognosis. Silencing of NPFFR2 reduced the malignancy of liver cancer cells by decreasing cell survival, invasion, and migration, while its overexpression increased invasion, migration, and anchorage-independent cell growth. Moreover, we found that the malignant function of NPFFR2 depends on RhoA and YAP signaling. Inhibition of Rho kinase activity completely restored the phenotypes induced by NPFFR2, and RhoA/F-Actin/YAP signaling was controlled by NPFFR2. These findings demonstrate that NPFFR2 may be a potential target for the treatment of hepatocellular carcinoma.

Kinesin light chain 4 as a new target for lung cancer chemoresistance via targeted inhibition of checkpoint kinases in the DNA repair network.

The poor therapeutic efficacy of non-small cell lung cancer (NSCLC) is partly attributed to the acquisition of chemoresistance. To investigate the mechanism underlying this resistance, we examined the potential link between kinesin light chain 4 (KLC4), which we have previously reported to be associated with radioresistance in NSCLC, and sensitivity to chemotherapy in human lung cancer cell lines. KLC4 protein levels in lung cancer cells correlated with the degree of chemoresistance to cisplatin treatment. Furthermore, KLC4 silencing enhanced the cytotoxic effect of cisplatin by promoting DNA double-strand breaks and apoptosis. These effects were mediated by interaction with the checkpoint kinase CHK2, as KLC4 knockdown increased CHK2 activation, which was further enhanced in combination with cisplatin treatment. In addition, KLC4 and CHEK2 expression levels showed negative correlation in lung tumor samples from patients, and KLC4 overexpression correlated negatively with survival. Our results indicate a novel link between the KLC4 and CHK2 pathways regulating DNA damage response in chemoresistance, and highlight KLC4 as a candidate for developing lung cancer-specific drugs and customized targeted molecular therapy.

Ionizing radiation attracts tumor targeting and apoptosis by radiotropic lysyl oxidase traceable nanoparticles.

Lysyl oxidase (LOX) is a cell-secreted amine oxidase that crosslinks collagen and elastin in extracellular microenvironment. LOX-traceable nanoparticles (LOXab-NPs) consisting of LOX antibodies (LOXab) and paclitaxel, can accumulate at high concentrations at radiation-treated target sites, as a tumor-targeting drug carrier for chemotherapy. Tumor-targeting and anticancer effects of PLGA based LOXab-NPs in vitro and in vivo were evaluated at radiation-targeted site. In the in vivo A549 lung carcinoma xenograft model, we showed highly specific tumor targeting (above 7.0 times higher) of LOXab-NPs on irradiated tumors. Notably, systemically administered NPs delayed tumor growth, reducing tumor volumes by more than 2 times compared with non-irradiated groups (222% vs. >500%) over 2 weeks. Radiotropic LOXab-NPs can serve as chemotherapeutic vehicles for combined targeted chemo-radiotherapy in clinical oncology.

G3BP1 Depletion Increases Radiosensitisation by Inducing Oxidative Stress in Response to DNA Damage.

BACKGROUND: RAS GTPase-activating protein-binding protein (G3BP1) is an RNA-binding protein that is essential for assembling stress granules. Many functions related to the survival and progression of cancer have been reported. The current study aimed to investigate the role of G3BP1 in radio-sensitisation of cancer cells. MATERIALS AND METHODS: Radiation sensitivity and chemosensitivity were analysed in A549 and H460 cells transfected with G3BP1 siRNAs, and N-acetyl-L-cysteine (NAC) was used to elucidate the involvement of reactive oxygen species (ROS). RESULTS: G3BP1 depletion sensitised lung cancer cell lines to radiation, and the effect was related to ROS. G3BP1 depletion impaired the intracellular ROS scavenging system and NAC abolished the radiation-sensitive phenotypes caused by G3BP1 depletion. CONCLUSION: The study suggested G3BP1 as a promising target for radio- and chemosensitisation of lung cancer.

Benzylideneacetone Derivatives Inhibit Osteoclastogenesis and Activate Osteoblastogenesis Independently Based on Specific Structure-Activity Relationship.

(E)-3,4-Dihydroxybenzylideneacetone (compound 1) inhibited receptor activator of NF-κB ligand-induced osteoclastogenesis of C57BL/6 bone marrow monocyte/macrophages with IC50 of 7.8 µM (IC50 of alendronate, 3.7 µM) while stimulating the differentiation of MC3T3-E1 osteoblastic cells, accompanied by the induction of Runt-related transcription factor 2, alkaline phosphatase, and osteocalcin. (E)-4-(3-Hydroxy-4-methoxyphenyl)-3-buten-2-one (compound 2c) showed a dramatically increased osteoclast-inhibitory potency with IC50 of 0.11 µM while sustaining osteoblast-stimulatory activity. (E)-4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one (compound 2g) stimulated alkaline phosphatase production 2-fold at 50 µM without changing osteoclast-inhibitory activity, compared with compound 1. Oral administration of compounds 1, 2c, and 2g prevented ovariectomy-induced osteoporosis in ddY mice to a degree proportional to their osteoclastogenesis-inhibitory potencies. The administration of 1 (mg/kg)/d compound 2c ameliorated histomorphometry of osteoporotic bone to a degree comparable with 10 (mg/kg)/d alendronate. Conclusively, the in vitro capacity of a few benzylideneacetone derivatives to inhibit osteoclastogenesis supported by independent osteoblastogenesis activation was convincingly reflected in in vivo management of osteoporosis, suggesting a potential novel therapeutics for osteopenic diseases.

Cluh plays a pivotal role during adipogenesis by regulating the activity of mitochondria.

Cluh is a cytosolic protein that is known to specifically bind the mRNAs of nuclear-encoded mitochondrial proteins and play critical roles in mitochondrial biogenesis. Here, we report the role of Cluh in adipogenesis. Our study shows that mRNA expression of Cluh is stimulated during adipogenesis, and that cAMP/Creb signalling increases its transcription. Cluh depletion impaired proper adipocyte differentiation, with reductions seen in lipid droplets and adipogenic marker gene expression. Interestingly, the inductions of the brown adipocyte-specific genes, Ucp1, Cidea and Cox7a1, are severely blocked by Cluh depletion during brown adipogenesis. Mitochondrial respiration and the stability of mRNAs encoding mitochondrial proteins are reduced by Cluh depletion during brown adipogenesis. These results suggest that Cluh, which is induced during adipogenesis, promotes the post-transcriptional regulation of mitochondrial proteins and supports differentiation.

Inhibition of ß-site amyloid precursor protein cleaving enzyme 1 and cholinesterases by pterosins via a specific structure-activity relationship with a strong BBB permeability.

We extracted 15 pterosin derivatives from Pteridium aquilinum that inhibited ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) and cholinesterases involved in the pathogenesis of Alzheimer's disease (AD). (2R)-Pterosin B inhibited BACE1, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with an IC50 of 29.6, 16.2 and 48.1 µM, respectively. The Ki values and binding energies (kcal/mol) between pterosins and BACE1, AChE, and BChE corresponded to the respective IC50 values. (2R)-Pterosin B was a noncompetitive inhibitor against human BACE1 and BChE as well as a mixed-type inhibitor against AChE, binding to the active sites of the corresponding enzymes. Molecular docking simulation of mixed-type and noncompetitive inhibitors for BACE1, AChE, and BChE indicated novel binding site-directed inhibition of the enzymes by pterosins and the structure-activity relationship. (2R)-Pterosin B exhibited a strong BBB permeability with an effective permeability (Pe) of 60.3×10-6 cm/s on PAMPA-BBB. (2R)-Pterosin B and (2R,3 R)-pteroside C significantly decreased the secretion of Aß peptides from neuroblastoma cells that overexpressed human ß-amyloid precursor protein at 500 µM. Conclusively, our study suggested that several pterosins are potential scaffolds for multitarget-directed ligands (MTDLs) for AD therapeutics.

TMEM165, a Golgi transmembrane protein, is a novel marker for hepatocellular carcinoma and its depletion impairs invasion activity.

Transmembrane protein 165 (TMEM165), a Golgi protein, functions in ion homeostasis and vesicular trafficking in the Golgi apparatus. While mutations in TMEM165 are known to cause human 'congenital disorders of glycosylation', a recessive autosomal metabolic disease, the potential association of this protein with human cancer development has not been explored to date. In the present study, we revealed that TMEM165 is overexpressed in HCC and its depletion weakens the invasive activity of cancer cells through suppression of matrix metalloproteinase­2 (MMP­2) expression. Levels of TMEM165 mRNA and protein were clearly increased in HCC patient tissues and cell cultures. Quantitative real­time RT­PCR analysis of fresh HCC tissues (n=88) revealed association of TMEM165 overexpression with more frequent macroscopic vascular invasion, microscopic serosal invasion and higher α­fetoprotein levels. Notably, depletion of TMEM165 led to a marked decrease in the invasive activity of two different HCC cell types, Huh7 and SNU475, accompanied by downregulation of MMP­2. Our collective findings clearly indicated that TMEM165 contributed to the progression of HCC by promoting invasive activity, supporting its utility as a novel biomarker and therapeutic target for cancer.

MicroRNA-196b enhances the radiosensitivity of SNU-638 gastric cancer cells by targeting RAD23B.

Gastric cancer is characterized by resistance to ionizing radiation. The development of resistance to radiotherapy in gastric cancer patients is one of the obstacles to effective radiotherapy. MicroRNAs are small well-conserved non-coding RNA species that regulate post-transcriptional activation. Our study aimed to investigate the role of miR-196b in radiation-induced gastric cancer. In the present study, we found that miR-196b expression was significantly reduced following radiation. The ectopic miR-196b expression sensitized SNU-638 gastric cancer cells and increased γ-H2AX foci upon radiation treatment. Bioinformatics analysis suggested that the DNA repair protein RAD23B was a putative target gene of miR-196b. Overexpression of miR-196b suppressed RAD23B expression in SNU-638 cells. Reporter assays further showed that miR-196b inhibited RAD23B 3'-UTR luciferase activity. Knockdown of RAD23B by small interfering RNA transfection closely mimicked the outcomes of miR-196b transfection, leading to impaired DNA damage repair in gastric cancer cells. Our results show that miR-196b improved radiosensitivity of SNU-638 cells by targeting RAD23B. Our data indicate that miR-196b is a potential target to enhance the effect of radiation treatment on gastric cancer cells. These findings will provide evidence for a new therapeutic target in radiotherapy.

Protein arginine methyltransferase 5 is implicated in the aggressiveness of human hepatocellular carcinoma and controls the invasive activity of cancer cells.

Protein arginine methyltransferase 5 (PRMT5) is a protein that catalyzes transfer of methyl groups to the arginine residues of proteins and is involved in diverse cellular and biological responses. While the participation of PRMT5 in cancer progression has been increasingly documented, its association with the invasive phenotype currently remains poorly understood. In the present study, we revealed that PRMT5 is overexpressed in human hepatocellular carcinoma (HCC) and in colon cancer and its depletion leads to the suppression of cell invasive activity via the reduction of the expression of MMP-2. Real-time quantitative RT-PCR analysis of 120 HCC patient tissues revealed the overexpression of PRMT5 in HCC and the association of PRMT5 with aggressive clinicopathological parameters, such as poorer differentiation (P=0.004), more frequent hepatic vein invasion (P=0.019), larger tumor size (P=0.011) and higher α-fetoprotein levels (P=0.020). Similarly to the data obtained with HCC, overexpression of PRMT5 was also displayed in colon cancer tissues, compared to matched non-tumor regions. Consistent with the significant association of the overexpression of PRMT5 with hepatic vein invasion in patient specimens, PRMT5 depletion via siRNA transfection led to a marked reduction in the invasion rate in both HCC and colon cancer cells. Reduced invasion associated with PRMT5 depletion was accompanied by a decrease in the expression of MMP-2. Collectively, our results indicated that PRMT5 overexpression in HCC and colon cancer cells contributed to their acquisition of aggressive characteristics, such as invasiveness, thus presenting a promising therapeutic target for the treatment of these diseases.

Promoter methylation of cysteine dioxygenase type 1: gene silencing and tumorigenesis in hepatocellular carcinoma.

BACKGROUNDS/AIMS: Cysteine dioxygenase type 1 (CDO1) acts as a tumor suppressor and is silenced by promoter methylation in various malignancies. The relationship between the CDO1 methylation status and hepatocellular carcinoma (HCC) tumorigenesis was evaluated. METHODS: Using a HCC cell line (SNU423), an in vitro demethylation study was performed to confirm whether promoter methylation causes CDO1 down-regulation. The SNU423 cells transfected with the CDO1 cell function was compared to that of naïve cells. An in vivo study using immunohistochemical staining of HCC specimens that were collected from patients who underwent curative liver resection was also performed. RESULTS: CDO1 was activated after demethylation treatment in the HCC specimens. Moreover, tumor cell proliferation, colony-forming, migration, and invasion activities significantly decreased after CDO1 transfection (p<0.05). The percentage of tumors that were larger than 5 cm was higher in patients who had a lower expression of CDO1 (p=0.030). Vascular invasion and histological grade were independent prognostic factors for poor overall and recurrence-free survival. The degree of CDO1 expression was not an independent prognostic factor in this study's population. CONCLUSIONS: These results suggested that methylation down-regulated CDO1 expression in the HCC cells. CDO1 methylation may be a potentially valuable diagnostic biomarker for HCC.

Tumor stroma with senescence-associated secretory phenotype in steatohepatitic hepatocellular carcinoma.

Senescence secretome was recently reported to promote liver cancer in an obese mouse model. Steatohepatitic hepatocellular carcinoma (SH-HCC), a new variant of HCC, has been found in metabolic syndrome patients, and pericellular fibrosis, a characteristic feature of SH-HCC, suggests that alteration of the tumor stroma might play an important role in SH-HCC development. Clinicopathological characteristics and tumor stroma showing senescence and senescence-associated secretory phenotype (SASP) were investigated in 21 SH-HCCs and 34 conventional HCCs (C-HCCs). The expression of α-smooth muscle actin (α-SMA), p21Waf1/Cif1, γ-H2AX, and IL-6 was investigated by immunohistochemistry or immunofluorescence. SH-HCCs were associated with older age, higher body mass index, and a higher incidence of metabolic syndrome, compared to C-HCC (P <0.05, all). The numbers of α-SMA-positive cancer-associated fibroblasts (CAFs) (P = 0.049) and α-SMA-positive CAFs co-expressing p21Waf1/Cif1 (P = 0.038), γ-H2AX (P = 0.065), and IL-6 (P = 0.048) were greater for SH-HCCs than C-HCCs. Additionally, non-tumoral liver from SH-HCCs showed a higher incidence of non-alcoholic fatty liver disease and a higher number of α-SMA-positive stellate cells expressing γ-H2AX and p21Waf1/Cif1 than that from C-HCCs (P <0.05, all). In conclusion, SH-HCCs are considered to occur more frequently in metabolic syndrome patients. Therein, senescent and damaged CAFs, as well as non-tumoral stellate cells, expressing SASP including IL-6 may contribute to the development of SH-HCC.

The mitochondrial hinge protein, UQCRH, is a novel prognostic factor for hepatocellular carcinoma.

Alterations in mitochondrial respiration contribute to the development and progression of cancer via abnormal biogenesis, including generation of reactive oxygen species. Ubiquinol-cytochrome c reductase hinge protein (UQCRH) consists of the cytochrome bc1 complex serving respiration in mitochondria. In the present study, we analyzed UQCRH abnormalities in hepatocellular carcinoma (HCC) and its association with clinical outcomes of patients. UQCRH expression in HCC was determined via semiquantitative and quantitative real-time reverse transcriptase polymerase chain reaction of 96 surgically resected HCC tissues positive for hepatitis B virus surface antigen. UQCRH was frequently overexpressed in HCC tissues (46.8%, based on 2.1-fold cutoff). UQCRH overexpression was observed in HCCs with larger tumor size, poorer differentiation, or vascular invasion. Kaplan-Meier analysis revealed significantly shorter overall (P = 0.005) and recurrence-free survival (P = 0.027) in patients with tumors overexpressing UQCRH. The prognostic impact of UQCRH was significant in subgroups of patients divided according to the α-fetoprotein (AFP) level. The patient subgroup with higher AFP levels (≥20 ng/mL) exhibited significant differences in 5-year overall (18.5% vs. 67.9%) and recurrence-free survival rates (11.1% vs. 46.4%) between groups with and without UQCRH overexpression. In contrast, no marked survival differences were observed between subgroups with lower AFP levels (<20 ng/mL). Multivariate analysis defined UQCRH as an independent poor prognostic factor. Conclusively, our results indicate that UQCRH overexpression is correlated with poor outcomes of HCC patients. Furthermore, in patients grouped as high risk based on elevated AFP, lack of UQCRH overexpression could be a useful indicator for clinical treatment.

The TLR7 agonist imiquimod induces anti-cancer effects via autophagic cell death and enhances anti-tumoral and systemic immunity during radiotherapy for melanoma.

Toll-like receptor (TLR) ligands are strongly considered immune-adjuvants for cancer immunotherapy and have been shown to exert direct anti-cancer effects. This study was performed to evaluate the synergistic anti-cancer and anti-metastatic effects of the TLR7 agonist imiquimod (IMQ) during radiotherapy for melanoma. The pretreatment of B16F10 or B16F1 cells with IMQ combined with γ-ionizing radiation (IR) led to enhanced cell death via autophagy, as demonstrated by increased expression levels of autophagy-related genes, and an increased number of autophagosomes in both cell lines. The results also confirmed that the autophagy process was accelerated via the reactive oxygen species (ROS)-mediated MAPK and NF-κB signaling pathway in the cells pretreated with IMQ combined with IR. Mice subcutaneously injected with melanoma cells showed a reduced tumor growth rate after treatment with IMQ and IR. Treatment with 3-methyladenine (3-MA), ameliorated the anti-cancer effect of IMQ combined with IR. Additionally, the combination therapy enhanced anti-cancer immunity, as demonstrated by an increased number of CD8+ T cells and decreased numbers of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) in the tumor lesions. Moreover, the combination therapy decreased the number of metastatic nodules in the lungs of mice that were injected with B16F10 cells via the tail vein. In addition, the combination therapy enhanced systemic anti-cancer immunity by increasing the abundances of T cell populations expressing IFN-γ and TNF-α. Therefore, these findings suggest that IMQ could serve as a radiosensitizer and immune booster during radiotherapy for melanoma patients.

Mael is essential for cancer cell survival and tumorigenesis through protection of genetic integrity.

Germ line-specific genes are activated in somatic cells during tumorigenesis, and are accordingly referred to as cancer germline genes. Such genes that act on piRNA (Piwi-interacting RNA) processing play an important role in the progression of cancer cells. Here, we show that the spermatogenic transposon silencer maelstrom (Mael), a piRNA-processing factor, is required for malignant transformation and survival of cancer cells. A specific Mael isoform was distinctively overexpressed in diverse human cancer cell lines and its depletion resulted in cancer-specific cell death, characterized by apoptosis and senescence, accompanied by an increase in reactive oxygen-species and DNA damage. These biochemical changes and death phenotypes induced by Mael depletion were dependent on ATM. Interestingly Mael was essential for Myc/Ras-induced transformation, and its overexpression inhibited Ras-induced senescence. In addition, Mael repressed retrotransposon activity in cancer cells. These results suggest that Mael depletion induces ATM-dependent DNA damage, consequently leading to cell death specifically in cancer cells. Moreover, Mael possesses oncogenic potential that can protect against genetic instability.

p31comet-Induced Cell Death Is Mediated by Binding and Inactivation of Mad2.

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.