NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

Wang, Ya-Chin; Tsai, Ming-Chao; Chen, Yaw‐Sen; Hsieh, Pei‐Min; Hung, Chao‐Ming; Lin, Hung‐Yu; Hsu, Yao-Chun; Yeh, Jen‐Hao; Hsiao, Po‐Jen; Su, Yu-Cheih; Ma, Ching-Hou; Lee, Chih-Yuan; Lin, Chen-Si; Shu, Chih‐Wen; Li, Yuchan; Tsai, Mei-Hsing; Lin, James Yu; Peng, Wei‐Hao; Yu, Ming‐Lung; Lin, Chih‐Wen

doi:10.1002/hep4.2019

Cited by 8 publications

(4 citation statements)

References 34 publications

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“…mTOR activity is inhibited by NPRL2 by interacting and directly binding with Rag GTPase, RagD in particular, and interferes with mTOR activity (29). mTOR was found to be downregulated in NPRL2-restored A549-NPRL2 ++/++ , which was consistent with others (12,32), whereas knocking down NPRL2 increased the Rag GTPases and mTOR activation (24). This mTOR downregulation was found to be associated with decreased expression of downstream mediators including phospho-S6, phospho-4E-BP1, phospho-PRAS40, and phospho-GSK-3β in NPRL2 restored cells.…”

Section: Discussionsupporting

confidence: 90%

“…The TME was significantly altered when NPRL2 was permanently restored, which was characterized by increased pro-immunogenic signatures including increased TILs, CD8 + T, and HLADR + DC, which led to significantly reduced tumor growth in humanized mice. On the contrary, the knockdown of NPRL2 promoted cell growth and proliferation in vitro and in vivo (24). Ingenuity pathway analysis showed the significant downregulation of pro-tumorigenic TGFβ1, TGFβ2, and CD47 signaling in anti-PD1 resistant tumors after NPRL2 gene therapy.…”

Section: Discussionmentioning

confidence: 99%

“…Restoration of NPRL2 is multifunctional including inducing apoptosis in cancer cells to inhibit tumor growth. Conversely, the efficient down-regulation of NPRL2 protein expression by both the shRNA and siRNA systems enhanced proliferation, migration, and colony formation in vitro as well as significantly increased tumor growth in hepatocellular carcinoma (24)…”

Section: Discussionmentioning

confidence: 99%

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NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

Meraz,

Majidi,

Song

et al. 2024

Preprint

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NPRL2/TUSC4 is a tumor suppressor gene whose expression is reduced in many cancers including NSCLC. Restoration of NPRL2 expression in cancer cells induces DNA damage which leads to cell cycle arrest and apoptosis. We investigated the antitumor immune responses to NPRL2 gene therapy in aPD1R/ KRAS/STK11mt NSCLC in a humanized mouse model. Humanized mice were generated by transplanting fresh human cord blood derived CD34 stem cells into sub-lethally irradiated NSG mice. Lung metastases were developed from KRAS/STK11mt/aPD1R A549 cells in humanized mice and treated with NPRL2 gene-loaded cationic lipid nanoparticles (DOTAP-NPRL2) with or without pembrolizumab (aPD1). NPRL2 treatment reduced lung metastases significantly, whereas pembrolizumab was ineffective. The antitumor effect was greater in humanized than non-humanized mice suggesting that an immune response contributed to antitumor activity. NPRL2 combined with pembrolizumab was not synergistic in the KRAS/STK11mt/aPD1R tumors but was synergistic in the KRASwt/aPD1S H1299 tumors. Consistent with the A549 humanized mouse model, NPRL2 showed a significantly strong antitumor effect on KRASmt/aPD1R LLC2 syngeneic tumors, whereas aPD1 was ineffective. The antitumor effect of NPRL2 was correlated with increased infiltration of human cytotoxic immune cells and Ag-presenting HLA-DR+ DC, CD11c DC, and downregulation of myeloid and regulatory T cells in the TME. The antitumor effect of NPRL2 was significantly abolished upon in-vivo depletion of CD8 T, macrophages, and CD4 T cells. However, the antitumor effect remained unaffected upon in-vivo depletion of NK cells. A distinct pattern of gene expression profile was found in lung met after NPRL2 treatment in humanized mice. The expression of genes associated with T cell functions, including IFNγ, CD8b, CD7, TNFSF18, ITGA1, GATA3, and TBX21 was significantly increased, whereas the expression of genes associated with negative regulation of T cell functions, including FOXP3, TGFB1, TGFB2, and IL-10RA were strongly inhibited upon NPRL2 treatment. NPRL2 downregulated the expression of T cell co-inhibitory molecules, including CTLA4, ICOS, LAG3, PDCD1, CD274, IDO1, PDCD1LG2, CD47, and KLRB1. Tumors established from NPRL2 stably expressing cells in humanized mice exhibited significantly slower growth compared to controls. TME analysis showed an increased presence of human CD45+, CD3+ T, CD8+ T cells, and HLA-DR+ dendritic cells and a decreased percentage of Treg, CD3+PD1+T cells, MDSC, and CD163+ TAM in NPRL2-expressing tumors. In-vitro, NPRL2 stably expressing cells showed a substantial increase in colony formation inhibition and heightened sensitivity to carboplatin in colony formation, apoptosis, and PARP cleavage assays. Stable expression of NPRL2 resulted in the downregulation of MAPK and AKT-mTOR growth signaling through inhibition of pAKT, pmTOR, pPRAS40, p4E-BP1, and pS6 expression. Taken together, these data suggest that NPRL2 gene therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen presentation and cytotoxic immune cell activation.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

Meraz,

Majidi,

Song

et al. 2024

Preprint

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“…[ 21 , 22 ] Similarly, TM6SF1, NEU1, NPRL2, and TMEM106B, as key genes for lysosome formation, could enhance lysosomal function and thus further regulate tumor progression. [ 23 , 24 ] Epithelial mesenchymal transition (EMT), a crucial stage in building the tumor microenvironment, plays an integral part in the progression of LUAD. Yuan et al found that Glypican-5, an oncogene, could inhibit the process of EMT in lung cancer, so as to suppress tumor growth and metastasis.…”

Section: Discussionmentioning

confidence: 99%

Lysosome-related genes: A new prognostic marker for lung adenocarcinoma

Hu,

Chen,

et al. 2023

Medicine

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Currently, a reliable early prognostic marker has not been identified for lung adenocarcinoma (LUAD), the most common malignancy. Recent studies demonstrated that lysosomal rupture is involved in cancer migration, progression, and immune microenvironment formation. We performed a bioinformatics analysis of lysosomal rupture to investigate whether lysosome-related genes (LRGs) are key in LUAD. The analysis identified 23 LRGs. Cytoscape visualization identified 10 core genes (CCNA2, DLGAP5, BUB1B, KIF2C, PBK, CDC20, NCAPG, ASPM, KIF4A, ANLN). With the 23 LRGs, we established a new risk scoring rule to classify patients with LUAD into high- and low-risk groups and verified the accuracy of the risk score by receiver operating characteristic curves and established a nomogram to evaluate clinical patients. Immunotherapy effectiveness between the high- and low-risk groups was evaluated based on the tumor mutational burden and analyses of immune cell infiltration and drug sensitivity. Pathway enrichment analysis revealed that lysosomes were closely associated with glucose metabolism, amino acid metabolism, and the immune response in patients with LUAD. Lysosomes are a likely new therapeutic target and provide new directions and ideas for treating and managing patients with LUAD.

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NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

et al. 2022

Self Cite

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Hepatocellular carcinoma (HCC) is a highly invasive malignancy. Recently, GATOR1 (Gap Activity TOward Rags 1) complexes have been shown to play an important role in regulating tumor growth. NPRL2 is a critical component of the GATOR1 complex. Therefore, this study used NPRL2 knockdown to investigate how GATORC1 regulates the prognosis and development of HCC via the mammalian target of rapamycin (mTOR) and autophagy signaling pathways. We established HepG2 cells with NPRL2 knockdown using small interfering RNA (siRNA) and short hairpin RNA (shRNA) systems. The siRNA‐mediated and shRNA‐mediated NPRL2 down‐regulation significantly reduced the expression of NPRL2 and two other GATPOR1 complex components, NPRL3 and DEPDC5, in HepG2 cells; furthermore, the efficient down‐regulation of NPRL2 protein expression by both the shRNA and siRNA systems enhanced the proliferation, migration, and colony formation in vitro. Additionally, the NPRL2 down‐regulation significantly increased HCC growth in the subcutaneous and orthotopic xenograft mouse models. The NPRL2 down‐regulation increased the Rag GTPases and mTOR activation and inhibited autophagy in vitro and in vivo. Moreover, the NPRL2 level in the tumors was significantly associated with mortality, recurrence, the serum alpha fetoprotein level, the tumor size, the American Joint Committee on Cancer stage, and the Barcelona Clinic Liver Cancer stage. Low NPRL2, NPRL3, DEPDC5, and LC3, and high p62 and mTOR protein expression in the tumors was significantly associated with disease‐free survival and overall survival in 300 patients with HCC after surgical resection. Conclusion: The efficient down‐regulation of NPRL2 significantly increased HCC proliferation, migration, and colony formation in vitro, and increased HCC growth in vivo. Low NPRL2 protein expression in the tumors was closely correlated with poorer clinical outcomes in patients with HCC. These results provide a mechanistic understanding of HCC and aid the development of treatments for HCC.

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NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

Cited by 8 publications

References 34 publications

NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

Lysosome-related genes: A new prognostic marker for lung adenocarcinoma

NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

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