Purpose: To investigate the role of macrophages in tumor progression under sorafenib treatment and to explore whether combination of drugs that deplete macrophages improved the antitumor effect of sorafenib. Experimental Design: Tumor growth, lung metastasis, and tumor angiogenesis were observed in HCCLM3-R and SMMC7721, two human hepatocellular carcinoma xenograft nude mouse models, when treated with sorafenib (30 mg/kg daily, n = 6 per group) or a vehicle as control. Macrophage infiltration was measured in the peripheral blood and in sorafenib-treated tumor by immunohistochemistry and flow cytometry with F4/80 antibody and CD11b antibody. The effect of macrophage depletion on tumor angiogenesis and metastasis after sorafenib treatment, using two drug target macrophages, zoledronic acid (ZA) and clodrolip, was measured in the two models of hepatocellular carcinoma. Results: Although sorafenib significantly inhibited tumor growth and lung metastasis, it induced a significant increase in peripheral recruitment and intratumoral infiltration of F4/80- and CD11b-positive cells, which was accompanied with elevation of colony-stimulating factor-1, stromal-derived factor 1α, and vascular endothelial growth factor in the tumor and elevation of plasma colony-stimulating factor-1 and mouse vascular endothelial growth factor in peripheral blood, suggesting the role of macrophages in tumor progression under sorafenib treatment. Depletion of macrophages by clodrolip or ZA in combination with sorafenib significantly inhibited tumor progression, tumor angiogenesis, and lung metastasis compared with mice treated with sorafenib alone. ZA was more effective than clodrolip. Conclusions: Macrophages may have an important role in tumor progression under sorafenib treatment. ZA is promising when combined with sorafenib to enhance its antitumor effect. Clin Cancer Res; 16(13); 3420–30. ©2010 AACR.

https://clincancerres.aacrjournals.org/content/clincanres/16/13/3420.full.pdf

Depletion of Tumor-Associated Macrophages Enhances the Effect of Sorafenib in Metastatic Liver Cancer Models by Antimetastatic and Antiangiogenic Effects

Cholesterol metabolism produces essential membrane components as well as metabolites with a variety of biological functions. In the tumour microenvironment, cell-intrinsic and cell-extrinsic cues reprogram cholesterol metabolism and consequently promote tumourigenesis. Cholesterol-derived metabolites play complex roles in supporting cancer progression and suppressing immune responses. Preclinical and clinical studies have shown that manipulating cholesterol metabolism inhibits tumour growth, reshapes the immunological landscape and reinvigorates anti-tumour immunity. Here, we review cholesterol metabolism in cancer cells, its role in cancer progression and the mechanisms through which cholesterol metabolites affect immune cells in the tumour microenvironment. We also discuss therapeutic strategies aimed at interfering with cholesterol metabolism, and how the combination of such approaches with existing anti-cancer therapies can have synergistic effects, thus offering new therapeutic opportunities.

Cholesterol metabolism in cancer: mechanisms and therapeutic opportunities

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the subtilases that promotes the internalization and degradation of LDL receptor in liver and thereby controls the level of LDL cholesterol in plasma. Here, we show that the expression of PCSK9 in HepG2 cells is completely dependent on the absence or presence of sterols. The minimal promoter region of the PCSK9 gene contains a sterol-regulatory element (SRE), which makes the transcription of PCSK9 dependent on sterols. Expression of nuclear forms of sterol-regulatory element binding protein-1 (SREBP-1) and SREBP-2 dramatically increased the promoter activity of PCSK9. In vitro-translated nuclear forms of SREBPs showed interactions with SRE, whereas mutations in SRE abolished their binding. In vivo studies in mice showed that Pcsk9 protein and mRNA were decreased significantly by fasting and increased by refeeding. However, supplementation with 2% cholesterol in the diet prevented the increase in Pcsk9. The amounts of Pcsk9 mRNA in livers of refed mice showed correlated regulation by the changes in the nuclear form of Srebp-2. In summary, it is suggested that the expression of PCSK9 is regulated by sterol at the transcriptional level in HepG2 cells and that both SREBP-1 and SREBP-2 can transcriptionally activate PCSK9 via SRE in its proximal promoter region in vitro. However, in vivo, it is suggested that the sterol-dependent regulation of PCSK9 is mediated predominantly by SREBP-2.

https://ir.ymlib.yonsei.ac.kr/bitstream/22282913/106946/1/T200800695.pdf

Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatory element binding protein-2.

Coupled scRNA-Seq and Intracellular Protein Activity Reveal an Immunosuppressive Role of TREM2 in Cancer.

Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient's response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease.

/pdf/dna-methylation-biomarkers-cancer-and-beyond-1uzinlzmgc.pdf

DNA Methylation Biomarkers: Cancer and Beyond

Developing predictive biomarkers that can detect the tipping point before metastasis of hepatocellular carcinoma (HCC), is critical to prevent further irreversible deterioration. To discover such early-warning signals or biomarkers of pulmonary metastasis in HCC, we analyse time-series gene expression data in spontaneous pulmonary metastasis mice HCCLM3-RFP model with our dynamic network biomarker (DNB) method, and identify CALML3 as a core DNB member. All experimental results of gain-of-function and loss-of-function studies show that CALML3 could indicate metastasis initiation and act as a suppressor of metastasis. We also reveal the biological role of CALML3 in metastasis initiation at a network level, including proximal regulation and cascading influences in dysfunctional pathways. Our further experiments and clinical samples show that DNB with CALML3 reduced pulmonary metastasis in liver cancer. Actually, loss of CALML3 predicts shorter overall and relapse-free survival in postoperative HCC patients, thus providing a prognostic biomarker and therapy target in HCC.

/pdf/dynamic-network-biomarker-indicates-pulmonary-metastasis-at-44jp7t89ph.pdf

Dynamic network biomarker indicates pulmonary metastasis at the tipping point of hepatocellular carcinoma

Fibrinogen-like protein 1 (FGL1)—Lymphocyte activating gene 3 (LAG-3) pathway is a promising immunotherapeutic target and has synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1). However, the prognostic significance of FGL1-LAG-3 pathway and the correlation with PD-L1 in hepatocellular carcinoma (HCC) remain unknown. The levels of LAG-3, FGL1, PD-L1 and cytotoxic T (CD8+T) cells in 143 HCC patients were assessed by multiplex immunofluorescence. Associations between the marker’s expression and clinical significances were studied. We found FGL1 and LAG-3 densities were elevated while PD-L1 and CD8 were decreased in HCC tissues compared to adjacent normal liver tissues. High levels of FGL1 were strongly associated with high densities of LAG-3+cells but not PD-L1. CD8+ T cells densities had positive correlation with PD-L1 levels and negative association with FGL1 expression. Elevated densities of LAG-3+cells and low levels of CD8+ T cells were correlated with poor disease outcome. Moreover, LAG-3+cells deteriorated patient stratification based on the abundance of CD8+ T cells. Patients with positive PD-L1 expression on tumor cells (PD-L1 TC+) tended to have an improved survival than that with negative PD-L1 expression on tumor cells (PD-L1 TC−). Furthermore, PD-L1 TC− in combination with high densities of LAG-3+cells showed the worst prognosis, and PD-L1 TC+ patients with low densities of LAG-3+cells had the best prognosis. LAG-3, FGL1, PD-L1 and CD8 have distinct tissue distribution and relationships with each other. High levels of LAG-3+cells and CD8+ T cells represent unfavorable and favorable prognostic biomarkers for HCC respectively.

/pdf/expression-and-clinical-significance-of-lag-3-fgl1-pd-l1-and-1hcxoo3w5w.pdf

Expression and clinical significance of LAG-3, FGL1, PD-L1 and CD8+T cells in hepatocellular carcinoma using multiplex quantitative analysis.

Summary The discovery that mutations in the EGFR gene are detected in up to 50% of lung adenocarcinoma patients, along with the development of highly efficacious epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), has revolutionized the treatment of this frequently occurring lung malignancy Indeed, the clinical success of these TKIs constitutes a critical milestone in targeted cancer therapy Three generations of EGFR-TKIs are currently approved for the treatment of EGFR mutation-positive non-small cell lung cancer (NSCLC) The first-generation TKIs include erlotinib, gefitinib, lapatinib, and icotinib; the second-generation ErbB family blockers include afatinib, neratinib, and dacomitinib; whereas osimertinib, approved by the FDA on 2015, is a third-generation TKI targeting EGFR harboring specific mutations Compared with the first- and second-generation TKIs, third-generation EGFR inhibitors display a significant advantage in terms of patient survival For example, the median overall survival in NSCLC patients receiving osimertinib reached 386 months Unfortunately, however, like other targeted therapies, new EGFR mutations, as well as additional drug-resistance mechanisms emerge rapidly after treatment, posing formidable obstacles to cancer therapeutics aimed at surmounting this chemoresistance In this review, we summarize the molecular mechanisms underlying resistance to third-generation EGFR inhibitors and the ongoing efforts to address and overcome this chemoresistance We also discuss the current status of fourth-generation EGFR inhibitors, which are of great value in overcoming resistance to EGFR inhibitors that appear to have greater therapeutic benefits in the clinic

Acquired resistance to third-generation EGFR-TKIs and emerging next-generation EGFR inhibitors.

Hepatocellular carcinoma (HCC) develops in a complex microenvironment characterized by chronic inflammation. In recent years, cholesterol metabolic abnormalities have been implicated the importance in cancer cell physiology. This study was designed to investigate the relationship between inflammation and cholesterol accumulation in HCC cells. Human HCC cells HepG2 and Huh7 were cultured and stimulated with lipopolysaccharide (LPS) for 24 h. The changes of HCC cells related to cholesterol metabolism including intracellular cholesterol concentrations, cholesterol uptake, and the expression of cholesterol-related genes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), LDL receptor (LDLR), sterol regulatory element-binding transcription factor 2 (SREBF2), and proprotein convertase subtilisin/kexin 9 (PCSK9) were comparatively analyzed. Simultaneously, the effects of nuclear factor-kappa B (NF-κB) signaling pathway on cholesterol metabolism were clarified by knocking-down of nuclear factor kappa-B kinase subunit alpha (IKKα) and TGF-beta-activated kinase 1 and MAP3K7-binding protein 3 (TAB3) via RNAi and microRNA (miR)-195. Subsequently, the roles of cholesterol accumulation in LPS induced pro-inflammatory effects were further investigated. Pro-inflammatory factor LPS significantly increased intracellular cholesterol accumulation by upregulating the expression of HMGCR, LDLR, and SREBF2, while downregulating the expression of PCSK9. These effects were revealed to depend on NF-κB signaling pathway by knocking-down and overexpression of IKKα and TAB3. Additionally, miR-195, a regulator directly targeting IKKα and TAB3, blocked the effects of cholesterol accumulation, further supporting the critical role of pro-inflammation NF-κB signaling in regulating cholesterol accumulation. Intriguingly, the accumulation of cholesterol conversely exerted an augmented pro-inflammation effects by further activating NF-κB signaling pathway. These results indicated that pro-inflammation effects of NF-κB signaling could be augmented by a positive feedback via enhancing the cholesterol accumulation in liver cancer cells.

/pdf/pro-inflammation-nf-kb-signaling-triggers-a-positive-2fgbwaj8v4.pdf

Pro-inflammation NF-κB signaling triggers a positive feedback via enhancing cholesterol accumulation in liver cancer cells

Triggering receptor expressed on myeloid cells 2 (TREM2) is involved in nonmalignant pathological processes. However, TREM2’s function in malignant diseases, especially in hepatocellular carcinoma (HCC) remains unknown. In the present study, we report that TREM2 is a novel tumor suppressor in HCC. TREM2 expression was obviously decreased in hepatoma cells (especially metastatic HCC cells), and in most human HCC tissues (especially extrahepatic metastatic tumors). Reduced tumor TREM2 expression was correlated with poor prognosis of HCC patients, and with aggressive pathological features (BCLC stage, tumor size, tumor encapsulation, vascular invasion, and tumor differentiation). TREM2 knockdown substantially promoted cell growth, migration, and invasion in vitro and in vivo, while TREM2 overexpression produced the opposite effect. TREM2 suppressed HCC metastasis by inhibiting epithelial-mesenchymal transition, accompanied by abnormal expression of epithelial and mesenchymal markers. Further study revealed that downregulation of TREM2 in HCC was regulated by miR-31-5p. Moreover, by directly interacting with β-catenin, TREM2 attenuated oncogenic and metastatic behaviors by inhibiting Akt and GSK3β phosphorylation, and activating β-catenin. TREM2 suppressed carcinogenesis and metastasis in HCC by targeting the PI3K/Akt/β-catenin pathway. Thus, we propose that TREM2 may be a candidate prognostic biomarker in malignant diseases and TREM2 restoration might be a prospective strategy for HCC therapy.

/pdf/trem2-acts-as-a-tumor-suppressor-in-hepatocellular-carcinoma-3qbqetiu7w.pdf

Biwei Yang

Papers

Dynamic network biomarker indicates pulmonary metastasis at the tipping point of hepatocellular carcinoma

Expression and clinical significance of LAG-3, FGL1, PD-L1 and CD8+T cells in hepatocellular carcinoma using multiplex quantitative analysis.

Acquired resistance to third-generation EGFR-TKIs and emerging next-generation EGFR inhibitors.

Pro-inflammation NF-κB signaling triggers a positive feedback via enhancing cholesterol accumulation in liver cancer cells

TREM2 acts as a tumor suppressor in hepatocellular carcinoma by targeting the PI3K/Akt/β-catenin pathway.