Methamphetamine use shortens telomere length in male adults and rats.

BACKGROUND: Methamphetamine (MA) use increases the risk of age-related diseases. However, it remains uncertain whether MA use exhibits accelerated biological aging, as indicated by telomere length (TL), a proposed marker of aging. Here we conducted studies in both humans and rats to investigate the association between MA use and TL. METHODS: We recruited 125 male MA users and 66 healthy controls, aged 30-40 years. MA users were diagnosed using DSM-5 criteria and categorized into two groups: non-severe (n = 78) and severe (n = 47) MA use disorder (MUD). MA-treated conditioned place preference (CPP) rats were utilized to validate our clinical investigations. TL was assessed using real-time polymerase chain reaction. RESULTS: At clinical levels, MA users exhibited significantly shorter leukocyte TL compared to healthy controls. Among MA users, individuals with severe MUD had significantly shorter leukocyte TL than those with non-severe MUD. Importantly, both univariate and multivariate linear regression analyses demonstrated a negative association between the severity of MA use and leukocyte TL. In a rat model of MA-induced CPP, leukocyte TL was also significantly shortened after MA administration, especially in rats with higher CPP expression or reinstatement scores. CONCLUSION: MA use shortened TL, and the severity of MA use was negatively correlated with TL. These findings provide new insights into the pathophysiology of accelerated aging caused by MA use and may have implications for identifying biomarkers and developing novel treatment strategies for MUD.

HPIP is an essential scaffolding protein running through the EGFR-RAS-ERK pathway and drives tumorigenesis.

The EGFR-RAS-ERK pathway plays a key role in cancer development and progression. However, the integral assembly of EGFR-RAS-ERK signaling complexes from the upstream component EGFR to the downstream component ERK is largely unknown. Here, we show that hematopoietic PBX-interacting protein (HPIP) interacts with all classical components of the EGFR-RAS-ERK pathway and forms at least two complexes with overlapping components. Experiments of HPIP knockout or knockdown and chemical inhibition of HPIP expression showed that HPIP is required for EGFR-RAS-ERK signaling complex formation, EGFR-RAS-ERK signaling activation, and EGFR-RAS-ERK signaling-mediated promotion of aerobic glycolysis as well as cancer cell growth in vitro and in vivo. HPIP expression is correlated with EGFR-RAS-ERK signaling activation and predicts worse clinical outcomes in patients with lung cancer. These results provide insights into EGFR-RAS-ERK signaling complex formation and EGFR-RAS-ERK signaling regulation and suggest that HPIP may be a promising therapeutic target for cancer with dysregulated EGFR-RAS-ERK signaling.

CHMP4A stimulates CD8+ T-lymphocyte infiltration and inhibits breast tumor growth via the LSD1/IFNß axis.

CD8+ T lymphocyte-mediated immunity strategies have represented attractive weapons against breast cancer (BC) recently. However, the mechanisms underlying CD8+ T-lymphocyte infiltration still remain obscure. Here, using bioinformatics analysis, we identified four hub prognostic genes related to CD8+ T-lymphocyte infiltration (CHMP4A, CXCL9, GRHL2, and RPS29), among which CHMP4A was the most significant gene. High CHMP4A mRNA expression was significantly associated with longer overall survival (OS) in BC patients. Functional experiments showed that CHMP4A had the ability to promote CD8+ T-lymphocyte recruitment and infiltration and suppressed BC growth in vitro and in vivo. Mechanistically, CHMP4A stimulates CD8+ T-lymphocyte infiltration by downregulating LSD1 expression, leading to HERV dsRNA accumulation, and promoting IFNß and its downstream chemokine production. Collectively, CHMP4A is not only a novel positive predictor for prognosis in BC but also a stimulator of CD8+ T-lymphocyte infiltration regulated by the LSD1/IFNß pathway. This study suggests that CHMP4A may be a novel target for improving the effectiveness of immunotherapy in patients with BC.

HUNK inhibits epithelial-mesenchymal transition of CRC via direct phosphorylation of GEF-H1 and activating RhoA/LIMK-1/CFL-1.

Epithelial-mesenchymal transition (EMT) is associated with the invasive and metastatic phenotypes in colorectal cancer (CRC). However, the mechanisms underlying EMT in CRC are not completely understood. In this study, we find that HUNK inhibits EMT and metastasis of CRC cells via its substrate GEF-H1 in a kinase-dependent manner. Mechanistically, HUNK directly phosphorylates GEF-H1 at serine 645 (S645) site, which activates RhoA and consequently leads to a cascade of phosphorylation of LIMK-1/CFL-1, thereby stabilizing F-actin and inhibiting EMT. Clinically, the levels of both HUNK expression and phosphorylation S645 of GEH-H1 are not only downregulated in CRC tissues with metastasis compared with that without metastasis, but also positively correlated among these tissues. Our findings highlight the importance of HUNK kinase direct phosphorylation of GEF-H1 in regulation of EMT and metastasis of CRC.

Let-7b-5p inhibits breast cancer cell growth and metastasis via repression of hexokinase 2-mediated aerobic glycolysis.

Hexokinase 2 (HK2), a critical rate-limiting enzyme in the glycolytic pathway catalyzing hexose phosphorylation, is overexpressed in multiple human cancers and associated with poor clinicopathological features. Drugs targeting aerobic glycolysis regulators, including HK2, are in development. However, the physiological significance of HK2 inhibitors and mechanisms of HK2 inhibition in cancer cells remain largely unclear. Herein, we show that microRNA-let-7b-5p (let-7b-5p) represses HK2 expression by targeting its 3'-untranslated region. By suppressing HK2-mediated aerobic glycolysis, let-7b-5p restrains breast tumor growth and metastasis both in vitro and in vivo. In patients with breast cancer, let-7b-5p expression is significantly downregulated and is negatively correlated with HK2 expression. Our findings indicate that the let-7b-5p/HK2 axis plays a key role in aerobic glycolysis as well as breast tumor proliferation and metastasis, and targeting this axis is a potential therapeutic strategy for breast cancer.

MiR-3180 inhibits hepatocellular carcinoma growth and metastasis by targeting lipid synthesis and uptake.

PURPOSE: Reprogrammed lipid metabolism is a hallmark of cancer that provides energy, materials, and signaling molecules for rapid cancer cell growth. Cancer cells acquire fatty acids primarily through de novo synthesis and uptake. Targeting altered lipid metabolic pathways is a promising anticancer strategy. However, their regulators have not been fully investigated, especially those targeting both synthesis and uptake. METHODS: Immunohistochemistry was performed on samples from patients with hepatocellular carcinoma (HCC) to establish the correlation between miR-3180, stearoyl-CoA desaturase-1 (SCD1), and CD36 expression, quantified via qRT-PCR and western blotting. The correlation was analyzed using a luciferase reporter assay. Cell proliferation, migration, and invasion were analyzed using CCK-8, wound healing, and transwell assays, respectively. Oil Red O staining and flow cytometry were used to detect lipids. Triglycerides and cholesterol levels were analyzed using a reagent test kit. CY3-labeled oleic acid transport was analyzed using an oleic acid transport assay. Tumor growth and metastasis were detected in vivo in a xenograft mouse model. RESULTS: MiR-3180 suppressed de novo fatty acid synthesis and uptake by targeting the key lipid synthesis enzyme SCD1 and key lipid transporter CD36. MiR-3180 suppressed HCC cell proliferation, migration, and invasion in an SCD1- and CD36-dependent manner in vitro. The mouse model demonstrated that miR-3180 inhibits HCC tumor growth and metastasis by inhibiting SCD1- and CD36-mediated de novo fatty acid synthesis and uptake. MiR-3180 expression was downregulated in HCC tissues and negatively correlated with SCD1 and CD36 levels. Patients with high miR-3180 levels showed better prognosis than those with low levels. CONCLUSIONS: Our investigation indicates that miR-3180 is a critical regulator involved in de novo fatty acid synthesis and uptake, which inhibits HCC tumor growth and metastasis by suppressing SCD1 and CD36. Therefore, miR-3180 is a novel therapeutic target and prognostic indicator for patients with HCC.

Epitranscriptic regulation of HRAS by N6-methyladenosine drives tumor progression.

Overexpression of Ras, in addition to the oncogenic mutations, occurs in various human cancers. However, the mechanisms for epitranscriptic regulation of RAS in tumorigenesis remain unclear. Here, we report that the widespread N6-methyladenosine (m6A) modification of HRAS, but not KRAS and NRAS, is higher in cancer tissues compared with the adjacent tissues, which results in the increased expression of H-Ras protein, thus promoting cancer cell proliferation and metastasis. Mechanistically, three m6A modification sites of HRAS 3' UTR, which is regulated by FTO and bound by YTHDF1, but not YTHDF2 nor YTHDF3, promote its protein expression by the enhanced translational elongation. In addition, targeting HRAS m6A modification decreases cancer proliferation and metastasis. Clinically, up-regulated H-Ras expression correlates with down-regulated FTO and up-regulated YTHDF1 expression in various cancers. Collectively, our study reveals a linking between specific m6A modification sites of HRAS and tumor progression, which provides a new strategy to target oncogenic Ras signaling.

[Eukaryotic expression of human Y-box binding protein 1 (YB-1) promotes the proliferation and migration of human HepG2 cells].

Objective To establish the eukaryotic expression vector of Y-box-binding protein 1 (YB-1) with FLAG-tagged and transfect it into hepatocellular carcinoma HepG2 cells to identify the effects of YB-1 on the proliferation and migration. Methods Human YB-1 gene was amplified from the human ovary library by PCR. YB-1 fraction was double enzyme digested and connected with pcDNA3.0-FLAG vector to construct eukaryotic expression vector pcDNA3.0-FlAG-YB-1, which was transfected into HepG2 cells. The expression of YB-1 was detected by Western blotting, and the effect of YB-1 on the proliferation of HepG2 cells was determined by CCK-8 assay and clone formation. The effect of YB-1 on the migration of HepG2 cells was analyzed by wound healing assays. Results The eukaryotic expression vector pcDNA3.0-FLAG-YB-1 was successfully established. YB-1 protein can be expressed in HepG2 cells, and YB-1 promoted the proliferation and migration of HepG2 cells. Conclusion YB-1 promotes the proliferation and migration of HepG2 cells.

DNA damage contributes to age-associated differences in SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is known to disproportionately affect older individuals. How aging processes affect SARS-CoV-2 infection and disease progression remains largely unknown. Here, we found that DNA damage, one of the hallmarks of aging, promoted SARS-CoV-2 infection in vitro and in vivo. SARS-CoV-2 entry was facilitated by DNA damage caused by extrinsic genotoxic stress or telomere dysfunction and hampered by inhibition of the DNA damage response (DDR). Mechanistic analysis revealed that DDR increased expression of angiotensin-converting enzyme 2 (ACE2), the primary receptor of SARS-CoV-2, by activation of transcription factor c-Jun. Importantly, in vivo experiment using a mouse-adapted viral strain also verified the significant roles of DNA damage in viral entry and severity of infection. Expression of ACE2 was elevated in the older human and mice tissues and positively correlated with γH2AX, a DNA damage biomarker, and phosphorylated c-Jun (p-c-Jun). Finally, nicotinamide mononucleotide (NMN) and MDL-800, which promote DNA repair, alleviated SARS-CoV-2 infection and disease severity in vitro and in vivo. Taken together, our data provide insights into the age-associated differences in SARS-CoV-2 infection and a novel approach for antiviral intervention.

Interaction between BEND5 and RBPJ suppresses breast cancer growth and metastasis via inhibiting Notch signaling.

High frequent metastasis is the major cause of breast cancer (BC) mortality among women. However, the molecular mechanisms underlying BC metastasis remain largely unknown. Here, we identified six hub BC metastasis driver genes (BEND5, HSD11B1, NEDD9, SAA2, SH2D2A and TNFSF4) through bioinformatics analysis, among which BEND5 is the most significant gene. Low BEND5 expression predicted advanced stage and shorter overall survival in BC patients. Functional experiments showed that BEND5 could suppress BC growth and metastasis in vitro and in vivo. Mechanistically, BEND5 inhibits Notch signaling via directly interacting with transcription factor RBPJ/CSL. BEN domain of BEND5 interacts with the N-terminal domain (NTD) domain of RBPJ, thus preventing mastermind like transcriptional coactivator (MAML) from forming a transcription activation complex with RBPJ. Our study provides a novel insight into regulatory mechanisms underlying Notch signaling and suggests that BEND5 may become a promising target for BC therapy.

Transcriptional Repression of Aerobic Glycolysis by OVOL2 in Breast Cancer.

Aerobic glycolysis (Warburg effect), a hallmark of cancer, plays a critical role in cancer cell growth and metastasis; however, direct inhibition of the Warburg effect remains largely unknown. Herein, the transcription factor OVO-like zinc finger 2 (OVOL2) is demonstrated to directly repress the expression of several glycolytic genes, blocking the Warburg effect and breast tumor growth and metastasis in vitro and in vivo. OVOL2 inhibits glycolysis by recruiting the nuclear receptor co-repressor (NCoR) and histone deacetylase 3 (HDAC3). The tumor suppressor p53, a key regulator of cancer metabolism, activates OVOL2 by binding to the oncoprotein mouse double minute 2 homolog (MDM2) and inhibiting MDM2-mediated ubiquitination and degradation of OVOL2. OVOL2 expression is negatively correlated with glycolytic gene expression and can be a good predictor of prognosis in patients with breast cancer. Therefore, targeting the p53/MDM2/OVOL2 axis provides a potential avenue for cancer treatment, especially breast cancer.

Lactate Dehydrogenase B Is Required for Pancreatic Cancer Cell Immortalization Through Activation of Telomerase Activity.

Telomerase activity is elevated in most cancer cells and is required for telomere length maintenance and immortalization of cancer cells. Glucose metabolic reprogramming is a hallmark of cancer and accompanied with increased expression of key metabolic enzymes. Whether these enzymes influence telomerase activity and cell immortalization remains unclear. In the current study, we screened metabolic enzymes using telomerase activity assay and identified lactate dehydrogenase B (LDHB) as a regulator of telomerase activity. Sodium lactate and sodium pyruvate did not influence telomerase activity, indicating LDHB regulates telomerase activity independent of its metabolism regulating function. Further studies revealed that LDHB directly interacted with TERT and regulated the interaction between TERT and TERC. Additionally, long-term knockdown of LDHB inhibited cancer cell growth and induced cell senescence in vitro and in vivo. Higher LDHB expression was detected in pancreatic cancer tissues compared with that in adjacent normal tissues and expression of LDHB correlated negatively with prognosis. Thus, we identified LDHB as the first glucose metabolic enzyme contributing to telomerase activity and pancreatic cancer cell immortalization.

DMDRMR promotes angiogenesis via antagonizing DAB2IP in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) patients are highly angiogenic and treated by targeted therapies against VEGFA/VEGFR signaling pathway. However, tumors with such targeted therapies remain a significant clinic challenge. Understanding the underlying mechanism against angiogenesis is highly desired. Here, we demonstrated that the lncRNA DMDRMR serves as a sponge of miR-378a-5p to increase EZH2 and SMURF1 expression, thus promoting EZH2-mediated transcriptional repression of DAB2IP and SMURF1-mediated degradation of DAB2IP. Consequently, this axis activates VEGFA/VEGFR2 signaling pathway, resulting in angiogenesis and resistance of tumor cells to sunitinib in ccRCC. Moreover, the competing endogenous RNA regulatory axis of DMDRMR is clinically relevant to ccRCC pathogenesis and prognosis of patients with ccRCC. Our results support that the DMDRMR/miR-378a-5p/DAB2IP axis may serve as a novel target for combination diagnosis or therapy of ccRCC patients. Our findings may have highly clinical relevance for future translation to develop the targeted therapies for patients with ccRCC.

Differential comparison of genotoxic effects of aristolochic acid I and II in human cells by the mass spectroscopic quantification of γ-H2AX.

Aristolochic acids (AAs) are known to be the potent genotoxic carcinogens, of which aristolochic acid I (AAI) and aristolochic acid II (AAII) are the two representative compounds. As the carcinogenic risk of herbs containing AAs is a global health issue, quantitative evaluation of genotoxicity is needed for the risk assessment of AAs. γ-H2AX, which is an acknowledged attractive bifunctional biomarker for simultaneously reflect the DNA damage response and repair, was used to quantitatively determine the DNA damage and repair properties of AAI and AAII in human cell lines, based on our previously developed mass spectrometry method. Results indicated that both AAI and AAII could increase the level of γ-H2AX in cells in a concentration-dependent manner, and the increased level of γ-H2AX induced by AAI was relatively higher than that induced by AAII. Time-effect curves showed that the change tendency of the proportion of γ-H2AX was obviously different in the later period, particularly afterwards 8 h post exposure. Additionally, AAI and AAII induced an opposite change of expression levels of DNA damage repair-associated proteins (ERCC1 and p53) in HepG2 cells, revealing their distinct molecular mechanisms. Findings of the present study are helpful for understanding the genotoxicity mechanism of AAI and AAII.

USP18 reduces paclitaxol sensitivity of triple-negative breast cancer via autophagy.

Paclitaxol is a first-line treatment for triple-negative breast cancer (TNBC). The molecular mechanisms underlying paclitaxol resistance in TNBC remain largely unclear. In this study, differential expressed genes (DEGs) between TNBC cells and paclitaxol-resistant (taxol-R) TNBC cells were screened by bioinformatics analysis. Among these DEGs, USP18 mRNA expression was significantly increased in taxol-R TNBC cells. USP18 overexpression reduced paclitaxol sensitivity by decreasing paclitaxol-induced apoptosis and cell cycle arrest in TNBC cells. In contrast, USP18 knockdown increased paclitaxol mediated anticancer activity in taxol-R TNBC cells in vitro and in vivo. Mechanistically, USP18 induced autophagy, an important pathway in chemotherapy resistance. The autophagy inhibitor leupeptin could effectively reverse the effect of USP18 on paclitaxol resistance phenotype. These findings suggested that USP18 may be a promising target for overcoming paclitaxol resistance in TNBC.

MicroRNA-377-3p inhibits hepatocellular carcinoma growth and metastasis through negative regulation of CPT1C-mediated fatty acid oxidation.

BACKGROUND: Altered lipid metabolism is closely related to the occurrence and development of hepatocellular carcinoma (HCC). Carnitine palmitoyltransferase 1C (CPT1C) is a member of CPT1 family and plays a key role in cancer development and progression. However, how microRNAs (miRNAs) regulate CPT1C-mediated fatty acid transport and oxidation remains to be elucidated. METHODS: Oil Red O staining, mitochondrial, and lipid droplets immunofluorescence staining were used to detect the functions of miR-377-3p and CPT1C in fatty acid oxidation. Colocalization of palmitate and mitochondria was performed to investigate the function of miR-377-3p and CPT1C in fatty acid transport into mitochondria. Fatty acid oxidation (FAO) assay was used to detect the function of miR-377-3p and CPT1C in FAO. Cell proliferation, migration and invasion assays and animal experiments were used to evaluate the role of miR-377-3p/CPT1C axis in HCC progression in vitro and in vivo. Immunofluorescence staining was used to identify the clinical significance of miR-377-3p and CPT1C in HCC patients. RESULTS: MiR-377-3p inhibits CPT1C expression by targeting its 3'-untranslated region. Through repression of CPT1C, miR-377-3p suppresses fatty acid oxidation by preventing fatty acid from entering into mitochondria and decreasing ATP production in HCC cells. Inhibiting fatty acid oxidation abolishes the ability of miR-377-3p/CPT1C axis to regulate HCC proliferation, migration, invasion and metastasis in vitro and in vivo. In HCC patients, CPT1C is significantly upregulated, and miR-377-3p expression and lipid droplets are negatively correlated with CPT1C expression. High expression of miR-377-3p and CPT1C predict better and worse clinical outcomes, respectively. CONCLUSIONS: We uncover the key function and the relevant mechanisms of the miR-377-3p/CPT1C axis in HCC, which might provide a potential target for the treatment of HCC.

LCDR regulates the integrity of lysosomal membrane by hnRNP K-stabilized LAPTM5 transcript and promotes cell survival.

Lysosome plays important roles in cellular homeostasis, and its dysregulation contributes to tumor growth and survival. However, the understanding of regulation and the underlying mechanism of lysosome in cancer survival is incomplete. Here, we reveal a role for a histone acetylation-regulated long noncoding RNA termed lysosome cell death regulator (LCDR) in lung cancer cell survival, in which its knockdown promotes apoptosis. Mechanistically, LCDR binds to heterogenous nuclear ribonucleoprotein K (hnRNP K) to regulate the stability of the lysosomal-associated protein transmembrane 5 (LAPTM5) transcript that maintains the integrity of the lysosomal membrane. Knockdown of LCDR, hnRNP K, or LAPTM5 promotes lysosomal membrane permeabilization and lysosomal cell death, thus consequently resulting in apoptosis. LAPTM5 overexpression or cathepsin B inhibitor partially restores the effects of this axis on lysosomal cell death in vitro and in vivo. Similarly, targeting LCDR significantly decreased tumor growth of patient-derived xenografts of lung adenocarcinoma (LUAD) and had significant cell death using nanoparticles (NPs)-mediated systematic short interfering RNA delivery. Moreover, LCDR/hnRNP K/LAPTM5 are up-regulated in LUAD tissues, and coexpression of this axis shows the increased diagnostic value for LUAD. Collectively, we identified a long noncoding RNA that regulates lysosome function at the posttranscriptional level. These findings shed light on LCDR/hnRNP K/LAPTM5 as potential therapeutic targets, and targeting lysosome is a promising strategy in cancer treatment.