ABSTRACT
ATP-binding cassette (ABC) transporters mediate multidrug resistance and cancer stem cell properties in various model systems. Yet, their biological significance in cancers, especially in hepatocellular carcinoma (HCC), remains unclear. In this study, we investigated the function of ABCF1 in HCC and explored its potential as a therapeutic target. ABCF1 was highly expressed in fetal mouse livers but not in normal adult livers. ABCF1 expression was upregulated in HCCs. These results demonstrate that ABCF1 functions as a hepatic oncofetal protein. We further demonstrated elevated ABCF1 expression in HCC cells upon acquiring chemoresistance. Suppression of ABCF1 by siRNA sensitized both parental cells and chemoresistant cells to chemotherapeutic agents. Reversely, ABCF1 overexpression promoted chemoresistance and drug efflux. In addition, overexpression of ABCF1 enhanced migration, spheroid and colony formation and epithelial-mesenchymal transition (EMT) induction. RNA sequencing analysis revealed EMT inducers HIF1α/IL8 and Sox4 as potential mediators for the oncogenic effect of ABCF1 in HCC progression. Together, this study illustrates that ABCF1 is a novel potential therapeutic target for HCC treatment.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Carcinoma, Hepatocellular/metabolism , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , Liver Neoplasms/metabolism , Neoplastic Stem Cells/drug effects , ATP-Binding Cassette Transporters/genetics , Animals , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Movement , Gene Expression Regulation, Neoplastic , Hep G2 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice, Inbred ICR , Mice, Nude , Neoplasm Invasiveness , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , SOXC Transcription Factors/genetics , SOXC Transcription Factors/metabolism , Signal Transduction , Xenograft Model Antitumor AssaysABSTRACT
The use of monoclonal antibody (mAb) has become a unique means of targeted therapy for human cancers. mAb-based therapies have shown survival benefits by applying alone or in combination with chemotherapeutics. Being a humanized biomolecule with exquisite target specificity, mAb demonstrated effects in a relatively lower dose range with limited off-target harm to the patients. Nowadays, novel targets involved in tumorigenic mechanisms and biomarkers expressed exclusively on cancer cell surface are being constantly discovered. The potential effects of their specific mAb could be investigated in the preclinical cancer model. In this chapter, we outlined our experimental procedures in determining the feasibility of novel mAb in the preclinical cancer model, with an example of progranulin (PGRN/GEP) mAb against hepatocellular carcinoma (HCC) tumor in mouse model. This chapter included the establishment of subcutaneous and orthotopic HCC tumor in mouse model, the injection of the mouse monoclonal antibody in combination with cytotoxic chemotherapeutics, the assessment of tumor development, and the analyses of the molecular changes of the tumor cells.
Subject(s)
Antibodies, Monoclonal/metabolism , Progranulins/metabolism , Animals , Apoptosis , Cell Line, Tumor , Disease Models, Animal , Flow Cytometry , Male , Mice, Inbred BALB C , Mice, Nude , RNA, Messenger/genetics , RNA, Messenger/metabolism , Spheroids, Cellular/pathology , Tumor Stem Cell AssayABSTRACT
BACKGROUND: Granulin-epithelin precursor (GEP) is a secretory growth factor, which has been demonstrated to control cancer growth, invasion, drug resistance and immune escape. Our previous studies and others also demonstrated its potential in targeted therapy. Comprehensive characterization of GEP partner on cancer cells are warranted. We have previously shown that GEP interacted with heparan sulfate on the surface of liver cancer cells and the interaction is crucial for GEP-mediated signaling transduction. This study aims to characterize GEP protein partner at the cell membrane with the co-immunoprecipitation and mass spectrometry approach. METHODS: The membrane fraction from liver cancer model Hep3B was used for capturing binding partner with the specific monoclonal antibody against GEP. The precipitated proteins were analyzed by mass spectrometry. After identifying the GEP binding partner, this specific interaction was validated in additional liver cancer cell line HepG2 by co-immunoprecipitation using GRP78 and GEP antibodies, respectively, as the bait. GRP78 transcript levels in hepatocellular carcinoma (HCC) clinical samples (n = 77 pairs) were examined by real-time quantitative RT-PCR. GEP and GRP78 protein expressions were investigated by immunohistochemistry on paraffin sections. RESULTS: We identified the GEP-binding protein as 78-kDa glucose-regulated protein (GRP78, also named heat shock 70-kDa protein 5, HSPA5). This interaction was validated in independent HCC cell lines. Increased GRP78 mRNA levels were demonstrated in liver cancer tissues compared with the paralleled liver tissues (t-test, P = 0.002). GRP78 and GEP transcript levels were significantly correlated (Spearman's correlation, P = 0.001), and the proteins were also detectable in the cytoplasm of liver cancer cells by immunohistochemical staining. CONCLUSIONS: GRP78 and GEP are interacting protein partners in liver cancer cells and may play a role in GEP-mediated cancer progression in HCC.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Heat-Shock Proteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Liver Neoplasms/metabolism , Signal Transduction , Cell Line, Tumor , Endoplasmic Reticulum Chaperone BiP , Female , Gene Expression Regulation, Neoplastic , Heat-Shock Proteins/genetics , Hep G2 Cells , Humans , Intercellular Signaling Peptides and Proteins/genetics , Male , Middle Aged , Progranulins , Protein BindingABSTRACT
Granulin-epithelin precursor (GEP) has been demonstrated to confer enhanced cancer stem-like cell properties in hepatocellular carcinoma (HCC) cell line models in our previous studies. Here, we aimed to examine the GEP-expressing cells in relation to the stem cell related molecules and stem-like cell properties in the prospective HCC clinical cohort. GEP protein levels were significantly higher in HCCs than the paralleled non-tumor liver tissues, and associated with venous infiltration. GEPhigh cells isolated from clinical HCC samples exhibited higher levels of stem cell marker CD133, pluripotency-associated signaling molecules ß-catenin, Oct4, SOX2, Nanog, and chemodrug transporter ABCB5. In addition, GEPhigh cells possessed preferential ability to form colonies and spheroids, and enhanced in vivo tumor-initiating ability while their xenografts were able to be serially subpassaged into secondary mouse recipients. Expression levels of GEP and pluripotency-associated genes were further examined in the retrospective HCC cohort and demonstrated significant correlation of GEP with ß-catenin. Notably, HCC patients with high GEP and ß-catenin levels demonstrated poor recurrence-free survival. In summary, GEP-positive HCC cells directly isolated from clinical specimens showed ß-catenin elevation and cancer stem-like cell properties.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Intercellular Signaling Peptides and Proteins/genetics , Liver Neoplasms/genetics , Neoplastic Stem Cells/metabolism , beta Catenin/genetics , Animals , Biomarkers, Tumor/metabolism , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cohort Studies , Hep G2 Cells , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Kaplan-Meier Estimate , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mice, Inbred NOD , Mice, SCID , Middle Aged , Neoplasm Recurrence, Local , Progranulins , RNA Interference , Transplantation, Heterologous , beta Catenin/metabolismABSTRACT
Most tumour cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and evade apoptosis. Intriguingly, the molecular mechanisms that link the Warburg effect with the suppression of apoptosis are not well understood. In this study, using loss-of-function studies in vitro and in vivo, we show that the anti-apoptotic protein poly(ADP-ribose) polymerase (PARP)14 promotes aerobic glycolysis in human hepatocellular carcinoma (HCC) by maintaining low activity of the pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. Notably, PARP14 is highly expressed in HCC primary tumours and associated with poor patient prognosis. Mechanistically, PARP14 inhibits the pro-apoptotic kinase JNK1, which results in the activation of PKM2 through phosphorylation of Thr365. Moreover, targeting PARP14 enhances the sensitization of HCC cells to anti-HCC agents. Our findings indicate that the PARP14-JNK1-PKM2 regulatory axis is an important determinant for the Warburg effect in tumour cells and provide a mechanistic link between apoptosis and metabolism.
