Argininosuccinate synthase 1 suppresses tumor progression through activation of PERK/eIF2α/ATF4/CHOP axis in hepatocellular carcinoma

Abstract Background Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide, and liver cancer has increased in mortality due to liver cancer because it was detected at an advanced stages in patients with liver dysfunction, making HCC a lethal cancer. Accordingly, we aim to new targets for HCC drug discovery using HCC tumor spheroids. Methods Our comparative proteomic analysis of HCC cells grown in culture as monolayers (2D) and spheroids (3D) revealed that argininosuccinate synthase 1 (ASS1) expression was higher in 3D cells than in 2D cells due to upregulated endoplasmic reticulum (ER) stress responses. We investigated the clinical value of ASS1 in Korean patients with HCC. The mechanism underlying ASS1-mediated tumor suppression was investigated in HCC spheroids. ASS1-mediated improvement of chemotherapy efficiency was observed using high content screening in an HCC xenograft mouse model. Results Studies of tumor tissue from Korean HCC patients showed that, although ASS1 expression was low in most samples, high levels of ASS1 were associated with favorable overall survival of patients. Here, we found that bidirectional interactions between ASS1 ER stress responses in HCC-derived multicellular tumor spheroids can limit HCC progression. ASS1 overexpression effectively inhibited tumor growth and enhanced the efficacy of in vitro and in vivo anti-HCC combination chemotherapy via activation of the PERK/eIF2α/ATF4/CHOP axis, but was not dependent on the status of p53 and arginine metabolism. Conclusions These results demonstrate the critical functional roles for the arginine metabolism–independent tumor suppressor activity of ASS1 in HCC and suggest that upregulating ASS1 in these tumors is a potential strategy in HCC cells with low ASS1 expression.

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Argininosuccinate Synthase 1 Suppresses Tumor Progression Through Activation of PERK/eIF2α/ATF4/CHOP Axis in Hepatocellular Carcinoma

10.21203/rs.3.rs-125320/v1 ◽

2020 ◽

Author(s):

Sanghwa Kim ◽

Minji Lee ◽

Yeonhwa Song ◽

Su-Yeon Lee ◽

Inhee Choi ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cancer ◽

Er Stress ◽

Stress Responses ◽

Arginine Metabolism ◽

Tumor Spheroids ◽

Clinical Value ◽

Suppressor Activity ◽

Argininosuccinate Synthase ◽

Hcc Cells

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide, and liver cancer has increased in mortality due to liver cancer because it was detected at an advanced stages in patients with liver dysfunction, making HCC a lethal cancer. Accordingly, we aim to new targets for HCC drug discovery using HCC tumor spheroids.Methods: Our comparative proteomic analysis of HCC cells grown in culture as monolayers (2D) and spheroids (3D) revealed that argininosuccinate synthase 1 (ASS1) expression was higher in 3D cells than in 2D cells due to upregulated endoplasmic reticulum (ER) stress responses. We investigated the clinical value of ASS1 in Korean patients with HCC. The mechanism underlying ASS1-mediated tumor suppression was investigated in HCC spheroids. ASS1-mediated improvement of chemotherapy efficiency was observed using high content screening in an HCC xenograft mouse model.Results: Studies of tumor tissue from Korean HCC patients showed that, although ASS1 expression was low in most samples, high levels of ASS1 were associated with favorable overall survival of patients. Here, we found that bidirectional interactions between ASS1 ER stress responses in HCC-derived multicellular tumor spheroids can limit HCC progression. ASS1 overexpression effectively inhibited tumor growth and enhanced the efficacy of in vitro and in vivo anti-HCC combination chemotherapy via activation of the PERK/eIF2α/ATF4/CHOP axis, but was not dependent on the status of p53 and arginine metabolism.Conclusion: These results demonstrate the critical functional roles for the arginine-metabolism–independent tumor suppressor activity of ASS1 in HCC and suggest that upregulating ASS1 in these tumors is a potential strategy in HCC cells with low ASS1 expression.

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Intermedin facilitates hepatocellular carcinoma cell survival and invasion via ERK1/2-EGR1/DDIT3 signaling cascade

Scientific Reports ◽

10.1038/s41598-020-80066-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Fei Xiao ◽

Hongyu Li ◽

Zhongxue Feng ◽

Luping Huang ◽

Lingmiao Kong ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Er Stress ◽

Cell Survival ◽

Vascular Remodeling ◽

Therapeutic Potential ◽

Blood Perfusion ◽

Signaling Cascade ◽

Apoptotic Pathway ◽

High Level ◽

Hcc Cells

AbstractAs one of the most malignant cancer types, hepatocellular carcinoma (HCC) is highly invasive and capable of metastasizing to distant organs. Intermedin (IMD), an endogenous peptide belonging to the calcitonin family, has been suggested playing important roles in cancer cell survival and invasion, including in HCC. However, how IMD affects the behavior of HCC cells and the underlying mechanisms have not been fully elucidated. Here, we show that IMD maintains an important homeostatic state by activating the ERK1/2-EGR1 (early growth response 1) signaling cascade, through which HCC cells acquire a highly invasive ability via significantly enhanced filopodia formation. The inhibition of IMD blocks the phosphorylation of ERK1/2, resulting in EGR1 downregulation and endoplasmic reticulum stress (ER) stress, which is evidenced by the upregulation of ER stress marker DDIT3 (DNA damage-inducible transcript 3). The high level of DDIT3 induces HCC cells into an ER-stress related apoptotic pathway. Along with our previous finding that IMD plays critical roles in the vascular remodeling process that improves tumor blood perfusion, IMD may facilitate the acquisition of increased invasive abilities and a survival benefit by HCC cells, and it is easier for HCC cells to obtain blood supply via the vascular remodeling activities of IMD. According to these results, blockade of IMD activity may have therapeutic potential in the treatment of HCC.

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Highly fluorescent QD probes labeling hepatocellular carcinoma cells

RSC Advances ◽

10.1039/c4ra10873f ◽

2015 ◽

Vol 5 (3) ◽

pp. 1841-1845 ◽

Cited By ~ 5

Author(s):

Baiqi Wang ◽

Hetao Chen ◽

Rui Yang ◽

Fang Wang ◽

Ping Zhou ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cancer ◽

Cancer Cells ◽

Carcinoma Cells ◽

Hepatocellular Carcinoma Cells ◽

Liver Cancer Cells ◽

Hcc Cells

The red signals from the cytoplasm of HCC cells reveal that the QD probes can specifically label liver cancer cells.

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Telocytes Promote the Metastasis of Hepatocellular Carcinoma by Activating ERK Signaling Pathway and Sponging miR-942-3p to Impact MMP9

10.21203/rs.3.rs-364558/v1 ◽

2021 ◽

Author(s):

Ying Xu ◽

Hu Tian ◽

Chao Guang Luan ◽

Kai Sun ◽

peng Jin Bao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cancer ◽

Signaling Pathway ◽

Erk Signaling ◽

Cancer Tissue ◽

Proliferative Potential ◽

Cancer Tissues ◽

Hcc Cells

Abstract Background: Hepatocellular carcinoma(HCC) in China is considered as a familiar malignant tumor with poor prognosis, high metastasis and disease relapse. Telocytes(TCs) have been verified to participate in progresses of tumorigenesis, invasions and migrations by secreting functional proteins and transmitting cell-to-cell information. Extracellular signal-regulared protein kinase(ERK) signal pathway is a vital mechanism driving cell proliferation, metastasis and apoptosis, but whether this molecular signaling mechanism contributes to matrix metalloproteinase-9(MMP) expression of TCs remains unclear. Methods: Telocytes and MMP9 expression in the liver cancer tissues are measured by immunohistochemistry assay, Westen blot assay and RT-PCR technique, meanwhile primary telocytes from liver para-cancer tissues are cultured in vitro. To demonstrate the function of telocytes for hepatocellular carcinoma, the metastatic cancer animal model is established by three typs of liver cancer cell-lines in vivo. Results: In our study, we elucidate that TCs in the para-cancer tissue can promote the metastasis of HCC cells by MMP-9 expression, in vitro and in vivo. PDGF derived from HCC cells has a capacity to activate Ras/ERK signaling pathway of TC as a result of accelerating MMP-9 expression, but it’s no significant for proliferative potential and apoptotic rate of TCs. While tyrosine kinase inhibitors and miR-942-3p suppress MMP-9 expression to make loss functions of TCs. Various mutations of TCs are also tested and single nucleotide polymorphisms of MMP-9 may be the potentially molecular mechanism of increasing protein expression in the invasive process of HCC. Conclusion: Our results demonstrate two potential mechanisms between HCC cells and TCs, suggesting that TC is a novel marker and target on deciphering reasons of cancer metastasis.

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Hepatitis B virus small envelope protein promotes HCC angiogenesis via ER stress signaling to upregulate VEGFA expression

Journal of Virology ◽

10.1128/jvi.01975-21 ◽

2021 ◽

Author(s):

Shu-Xiang Wu ◽

Shuang-Shuang Ye ◽

Yu-Xiang Hong ◽

Yan Chen ◽

Biao Wang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Endoplasmic Reticulum ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Er Stress ◽

Viral Protein ◽

Unfolded Protein ◽

B Virus ◽

Protein Response ◽

Hcc Cells

Hepatocellular carcinoma (HCC) is a hypervascular tumor and accumulating evidence has indicated that stimulation of angiogenesis by HBV may contribute to HCC malignancy. The small protein of hepatitis B virus surface antigen (HBsAg), SHBs, is the most abundant HBV viral protein and has a close clinical association with HCC, however, whether SHBs contributes to HCC angiogenesis remains unknown. This study reports that forced expression of SHBs in HCC cells promoted xenograft tumor growth and increased the microvessel density (MVD) within the tumors. Consistently, HBsAg was also positively correlated with MVD count in HCC patients’ specimens. The conditioned media from the SHBs-transfected HCC cells increased the capillary tube formation and migration of human umbilical vein endothelial cells (HUVECs). Intriguingly, overexpression of SHBs increased VEGFA expression at both mRNA and protein levels. A higher VEGFA expression level was also observed in the xenograft tumors transplanted with SHBs-expressing HCC cells and in HBsAg-positive HCC tumor tissues as compared to their negative controls. As expected, in the culture supernatants, the secretion of VEGFA was also significantly enhanced from HCC cells expressing SHBs, which promoted HUVECs migration and vessel formation. Furthermore, all the three unfolded protein response (UPR) sensors IRE1α, PERK and ATF6 associated with endoplasmic reticulum (ER) stress were found activated in the SHBs-expressing cells and correlated with VEGFA protein expression and secretion. Taken together, these results suggest an important role of SHBs in HCC angiogenesis and may highlight a potential target for preventive and therapeutic intervention of HBV-related HCC and its malignant progression. IMPORTANCE Chronic hepatitis B virus infection is one of the important risk factors for the development and progression of hepatocellular carcinoma (HCC). HCC is characteristic of hypervascularization even at early phases of the disease due to overexpression of angiogenic factors like vascular endothelial growth factor-A (VEGFA). However, a detailed mechanism in the HBV-induced angiogenesis remains to be established. In this study, we demonstrate for the first time that the most abundant HBV viral protein, i.e. small surface antigens (SHBs) can enhance the angiogenic capacity of HCC cells by upregulation of VEGFA expression both in vitro and in vivo . Mechanistically, SHBs induced endoplasmic reticulum (ER) stress which consequently activated unfolded protein response (UPR) signaling to increase VEGFA expression and secretion. This study suggests that SHBs plays an important pro-angiogenic role in HBV-associated HCC and may represent a potential target for anti-angiogenic therapy in the HCC.

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Silencing non-SMC chromosome-associated polypeptide G inhibits proliferation and induces apoptosis in hepatocellular carcinoma cells

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2018-0195 ◽

2018 ◽

Vol 96 (12) ◽

pp. 1246-1254 ◽

Cited By ~ 10

Author(s):

Kaikun Liu ◽

Yumin Li ◽

Bo Yu ◽

Furong Wang ◽

Taiyu Mi ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Liver Cancer ◽

Protein Expression ◽

Mrna Expression ◽

Mrna Level ◽

A Subunit ◽

Underlying Mechanisms ◽

Colony Forming Capacity ◽

Hcc Cells

The present study was designed to investigate the significance of non–structural maintenance of chromosomes (non-SMC) chromosome-associated polypeptide G (NCAPG), a subunit of condensin complex I, in the development of hepatocellular carcinoma (HCC). NCAPG protein expression in human HCC and paracancerous hepatic tissues were examined using immunohistochemistry, and NCAPG mRNA expression in HCC cell lines were quantified using quantitative RT–PCR. Lentivirus-mediated RNA interference was used to silence NCAPG in HCC cells. Cell proliferation was monitored by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Apoptosis was determined by flow cytometry. The results showed that increased protein expression of NCAPG was found in HCC tissues compared with the matched paracancerous hepatic tissues. At the mRNA level, increased expression of NCAPG was found in HCC cells as opposed to the normal hepatocytes. Silencing of NCAPG in BEL-7404 and SMMC-7721 cells led to decreased cell proliferation and increased apoptosis. These changes were associated with increased mRNA expressions of P53, P27, and Bad, but decreased mRNA expression of EGFR, Akt, survivin, and JNK. NCAPG might play an oncogenic role in the development of liver cancer. Further studies to clarify its role and underlying mechanisms in the development of liver cancer are warranted.

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Treatment with a New Barbituric Acid Derivative Exerts Antiproliferative and Antimigratory Effects against Sorafenib Resistance in Hepatocellular Carcinoma

Molecules ◽

10.3390/molecules25122856 ◽

2020 ◽

Vol 25 (12) ◽

pp. 2856

Author(s):

Yi-Jen Liao ◽

Shih-Ming Hsu ◽

Chia-Ying Chien ◽

Yuan-Hsi Wang ◽

Ming-Hua Hsu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cancer ◽

Acid Derivative ◽

Cancer Cell ◽

Barbituric Acid ◽

Dependent Manner ◽

Mesenchymal Transition ◽

Barbituric Acid Derivative ◽

Barbituric Acid Derivatives ◽

Hcc Cells

Hepatocellular carcinoma (HCC) is a common cause of cancer death worldwide. Sorafenib, a multikinase inhibitor, is the first-line drug approved by the Food and Drug Administration (FDA) for the treatment of patients with advanced HCC. However, most patients who continuously receive sorafenib may acquire resistance to this drug. Therefore, it is important to develop a new compound to treat liver cancer and sorafenib-resistant liver cancer. Barbituric acid derivatives have been used as antiasthmatic drugs in the clinic. We previously reported that a novel barbituric acid derivative inhibited carbon tetrachloride-induced liver fibrosis in mice, but its effects on liver cancer remain unknown. Thus, the purpose of this study was to investigate the antitumor effect of barbituric acid derivatives on HCC cells and sorafenib-resistant HCC cells (HCC-SRs). Our findings reveal that one of the barbituric acid derivatives, BA-5, significantly inhibited HCC and HCC-SR cell viability in a dose- and time-dependent manner. Therefore, compound BA-5 was selected for further experiments. Western blot data revealed that BA-5 treatment decreased the phosphorylation of AKT/p70s6k without affecting the MAPK pathway and increased cleaved PARP and cleaved caspase-7 in both HCC and HCC-SR cells. Since epithelial-mesenchymal transition plays a significant role in regulating cancer invasion and migration, we used the wound healing assay to evaluate the antimigratory effect of compound BA-5. The results showed that BA-5 treatment inhibited HCC and HCC-SR cell migration and reduced Vimentin protein expression. These results were confirmed by microarray analysis showing that BA-5 treatment influenced cancer cell motility and growth-related pathways. In the xenograft mouse model experiment, BA-5 administration significantly inhibited HCC cancer cell growth in mice. Furthermore, the combination of BA-5 with a low dose of regorafenib synergistically inhibited HCC-SR cell proliferation. In conclusion, our study showed that the barbituric acid derivative BA-5 is a new candidate for HCC and sorafenib-resistant HCC therapy.

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Inhibition of protein phosphatase 1 stimulates noncanonical ER stress eIF2α activation to enhance fisetin-induced chemosensitivity in HDAC inhibitor-resistant hepatocellular carcinoma cells

Cancers ◽

10.3390/cancers11070918 ◽

2019 ◽

Vol 11 (7) ◽

pp. 918 ◽

Cited By ~ 5

Author(s):

Liu ◽

Yu-Chun ◽

Chang ◽

Kuo ◽

Chen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Protein Phosphatase ◽

Cell Apoptosis ◽

Histone Deacetylase Inhibitors ◽

Molecular Mechanisms ◽

Chemotherapeutic Agents ◽

Protein Phosphatase 1 ◽

Hcc Cells

Hepatocellular carcinoma (HCC) is a common fatal type of malignant tumor that has highly metastatic and recurrent properties. Fisetin is a natural flavonoid found in various vegetables and fruits which exhibits anti-cancer and anti-inflammatory properties, as well as other effects. Thus, we hypothesized that fisetin can act as an adjuvant therapy in cancer or drug-resistant cancer cells, and further investigated the molecular mechanisms underlying the development of drug-resistance in HCC cells. We found that fisetin effectively inhibited the cell viability of not only parental cells but also histone deacetylase inhibitors-resistant (HDACis-R) cells and enhanced the chemosensitivity of HCC cells. Interestingly, fisetin did not induce cell apoptosis through the activation of the endoplasmic reticulum (ER) stress sensor of protein kinase R (PKR)-like endoplasmic reticulum kinase, but rather through the non-canonical pathway of the protein phosphatase 1 (PP1)-mediated suppression of eIF2α phosphorylation. Moreover, fisetin-induced cell apoptosis was reversed by treatment with PP1 activator or eIF2α siRNA in HCC cells. Based on these observations, we suggest that PP1-eIF2α pathways are significantly involved in the effect of fisetin on HCC apoptosis. Thus, fisetin may act as a novel anticancer drug and new chemotherapy adjuvant which can improve the efficacy of chemotherapeutic agents and diminish their side-effects.

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Milk Fat Globule-EGF Factor 8 Contributes to Progression of Hepatocellular Carcinoma

Cancers ◽

10.3390/cancers12020403 ◽

2020 ◽

Vol 12 (2) ◽

pp. 403

Author(s):

Duck Sung Ko ◽

Su Hyun Kim ◽

Ji Young Park ◽

Gyunggyu Lee ◽

Hyo Jin Kim ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cancer ◽

Milk Fat ◽

Neutralizing Antibodies ◽

Wide Spectrum ◽

Loss Of Function ◽

Function Study ◽

Milk Fat Globule ◽

Fat Globule ◽

Hcc Cells

Milk fat globule-EGF factor 8 (MFG-E8) is an anti-inflammatory glycoprotein that mediates a wide spectrum of pathophysiological processes. MFG-E8 has been studied as a key regulator of cancer cell invasion, migration, and proliferation in different tissues and organs. However, potential roles of MFG-E8 in the growth and progression of liver cancer have not been investigated to date. Here, we analyzed 33 human hepatocellular carcinoma (HCC) samples and found that levels of MFG-E8 expression were significantly higher in HCC cells than in normal liver tissues. In addition, our in vitro gain-of-function study in three different HCC cell lines revealed that overexpression of MFG-E8 promoted the proliferation and migration of HCC cells, as determined by RT-qPCR, MTT assays, and wound healing analyses. Conversely, an MFG-E8 loss-of function study showed that proliferation capacity was significantly reduced by MFG-E8 knockdown in HCC cells. Additionally, MFG-E8 activity-neutralizing antibodies profoundly inhibited both migration and proliferation of HCC cells, attenuating their tumorigenic properties. These reductions in migration and proliferation were rescued by treatment of HCC cells with recombinant MFG-E8 protein. Furthermore, an in vivo HCC xenograft study showed that the number of proliferating HCC cells and tumor volume/weight were all significantly increased by MFG-E8 overexpression, compared to control mice. These results clearly show that MFG-E8 plays an important role in HCC progression and may provide a basis for future mechanistic studies and new strategies for the treatment of liver cancer.

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