Experimental Models Of Hepatocellular Carcinoma Research Articles

Effects of mesenchymal stem cells versus curcumin on sonic hedgehog signaling in experimental model of Hepatocellular Carcinoma.

Sonic Hedgehog (SHH) is a fundamental signaling pathway that controls tissue reconstruction, stem cell biology, and differentiation and has a role in gut tissue homeostasis and development. Dysregulation of SHH leads to the development of HCC. The present study was conducted to compare the effects of mesenchymal stem cells (MSCs) and curcumin on SHH molecular targets in an experimental model of HCC in rats. One hundred rats were divided equally into the following groups: control group, HCC group, HCC group received MSCs, HCC group received curcumin, and HCC group received MSCs and curcumin. Histopathological examinations were performed, and gene expression of SHH signaling target genes (SHH, PTCH1, SMOH, and GLI1) was assessed by real-time PCR in rat liver tissue. Results showed that SHH target genes were significantly upregulated in HCC-untreated rat groups and in MSC-treated groups, with no significant difference between them. Administration of curcumin with or without combined administration of MSCs led to a significant down-regulation of SHH target genes, with no significant differences between both groups. As regards the histopathological examination of liver tissues, both curcumin and MSCs, either through separate use or their combined use, led to a significant restoration of normal liver pathology. In conclusion, SHH signaling is upregulated in the HCC experimental model. MSCs do not inhibit the upregulated SHH target genes in HCC. Curcumin use with or without MSCs administration led to a significant down-regulation of SHH signaling in HCC and a significant restoration of normal liver pathology.

Circular RNA DNAH14 molecular mechanism in an experimental model of hepatocellular carcinoma treated with Cobalt chloride to mimic the hypoxia-like response of transcatheter arterial chemoembolization

Transcatheter arterial chemoembolization (TACE) is the primary local treatment for patients with unresectable hepatocellular carcinoma (HCC). Numerous studies have demonstrated the pivotal role of circular RNAs (circRNAs) in TACE efficacy. This study aimed to investigate the function of circular RNA DNAH14 (circDNAH14) in TACE for HCC and to elucidate its molecular mechanisms. To simulate hypoxia conditions experienced during TACE, HCC cells were treated with cobalt chloride. The expression levels of circDNAH14, microRNA-508-3p (miR-508-3p), and Prothymosin Alpha (PTMA) were modulated via transfection for knockdown or overexpression. Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine assays, flow cytometry, and Transwell assays, along with epithelial-mesenchymal transition (EMT) evaluations, were employed to assess cell proliferation, apoptosis, invasion, migration, and EMT. The results indicated that hypoxia treatment downregulated the expression of circDNAH14 and PTMA while upregulating miR-508-3p. Such treatment suppressed HCC cell proliferation, invasion, migration, and EMT, and induced apoptosis. Knockdown of circDNAH14 or PTMA intensified the suppressive effects of hypoxia on the malignant behaviors of HCC cells. Conversely, upregulation of miR-508-3p or PTMA mitigated the effects of circDNAH14 overexpression and knockdown, respectively. Mechanistically, circDNAH14 was found to competitively bind to miR-508-3p, thereby regulating PTMA expression. In vivo, nude mouse xenograft experiments demonstrated that circDNAH14 knockdown augmented the hypoxia-induced suppression of HCC tumor growth. In conclusion, circDNAH14 mitigates the suppressive effects of hypoxia on HCC, both in vitro and in vivo, by competitively binding to miR-508-3p and regulating PTMA expression.

Hepatoprotective Effect Assessment of C-Phycocyanin on Hepatocellular Carcinoma Rat Model by Using Photoacoustic Spectroscopy.

Hepatocellular carcinoma (HCC) is the most common primary neoplasia of the liver with elevated mortality. Experimental treatment with antioxidants has a beneficial effect on the experimental models of HCC. Arthrospira maxima (spirulina) and its phycocyanin have antitumoral action on different tumoral cells. However, it is unknown whether phycocyanin is the responsible molecule for the antitumoral effect on HCC. Photoacoustic spectroscopy (PAS) stands out among other spectroscopy techniques for its versatility of samples analyzed. This technique makes it possible to obtain the optical absorption spectrum of solid or liquid, dark or transparent samples. Previous studies report that assessing liver damage in rats produced by the modified resistant hepatocyte model (MRHM) is possible by analyzing their blood optical absorption spectrum. This study aimed to investigate, by PAS, the effect of phycocyanin obtained from spirulina on hepatic dysfunction. The optical absorption spectra analysis of the rat blood indicates the damage level induced by the MRHM group, being in concordance with the carried out biological conventional studies results, indicating an increase in the activity of hepatic enzymes, oxidative stress, Bax/Bcl2 ratio, cdk2, and AKT2 expression results, with a reduction in p53 expression. Also, PAS results suggest that phycocyanin decreases induced damage, due to the prevention of the Bax, AKT2, and p53 altered expression and the tumor progression in a HCC rat model.

Pirfenidone regulates antioxidant response via NRF2 in an experimental model of hepatocellular carcinoma

Pirfenidone regulates antioxidant response via NRF2 in an experimental model of hepatocellular carcinoma

Non-invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma.

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1α expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Addition of tyrosine kinase inhibitors (TKIs) in patients (pts) with unresectable hepatocellular carcinoma (HCC) who progress on first-line immunotherapy (IO).

e16193 Background: Atezolizumab plus bevacizumab is now standard first-line therapy for advanced HCC. Early phase trials in HCC and phase III trials in other solid tumors testing IO and TKI have shown greater antitumor activity with the combination than with either agent alone. Cancer and vascular endothelial cells release VEGF, which not only increases angiogenesis, promoting tumor growth, but also induces an immunosuppressive tumor microenvironment (TME). Preclinical studies suggest that TKIs with anti-VEGF activity have immunomodulatory potential, enabling the infiltration and activation of effector T cells to the TME. Experimental models of HCC with combination of IO plus TKI also reverted immune escape mechanisms mediated by β-catenin, Wnt, and TGFβ activation and by Treg or neutrophil infiltration. At our institution, pts who were treated with IO alone in the front line often had a TKI added on progression of disease (PD). We aimed to evaluate whether the addition of a TKI could rescue IO failure. Methods: We conducted an IRB-approved retrospective chart review of pts with unresectable HCC at Mount Sinai Health System who received IO from 1/2017 to 6/2021 and who subsequently received a TKI on PD. We assessed the objective response rate (ORR), median progression-free survival (mPFS) and overall survival (mOS). Follow-up CT/MRI imaging studies were conducted every 2-3 months after addition of TKI. Results: Of 277 pts who received first-line IO during the study period, 46 evaluable pts subsequently had TKIs added to IO upon PD. Pts were predominantly male (37/46), with median age 63 years, and 38 had cirrhosis. Etiologies of HCC were as follows: 9 HBV, 22 HCV, 15 alcohol-related, 7 NASH-associated. All pts received first-line nivolumab. TKIs added were lenvatinib (44/46) and sorafenib (2). At the start of IO, most pts (31/46) had Child Pugh class A liver disease and performance status ECOG 0 (31/46). TKIs were added upon PD and after a median of 5.8 months (range 2.1-28.1 months) from IO initiation. ORR of IO plus TKI was 15% (7/46; 2 complete responses and 5 partial responses). 16 pts had stable disease, and 13 had PD. Among the 11/46 pts who initially responded to first-line IO, 2 achieved a response with TKI. There were 5 responses among the 35/46 non-responders to first-line IO. TKI-related grade 3-4 adverse events, including liver toxicity, rash, and encephalopathy, led to discontinuation in 7/46 pts. mOS after adding TKI was 9.5 months (95% CI: 6.1-13.7) and mPFS was 5.4 months (95% CI: 3.6-9.7). The mOS and mPFS of adding TKI after PD did not differ between IO initial responders and non-responders. Overall, mOS from IO initiation was 17.6 months (95% CI: 14.5-24.4). Conclusions: Adding a TKI after PD on single agent IO in pts with advanced HCC did not add a signal of clinical benefit compared with reported outcomes from second-line TKIs alone.

Cabozantinib Enhances Anti-PD1 Activity and Elicits a Neutrophil-Based Immune Response in Hepatocellular Carcinoma.

Immune checkpoint inhibitors combined with antiangiogenic agents produce benefits in the treatment of advanced hepatocellular carcinoma (HCC). We investigated the efficacy and immunomodulatory activity of cabozantinib alone and combined with anti-PD1 in experimental models of HCC, and explored the potential target population that might benefit from this combination. C57BL/6J mice bearing subcutaneous Hepa1-6 or Hep53.4 tumors received cabozantinib, anti-PD1, their combination, or placebo. Tumor and blood samples were analyzed by flow cytometry, IHC, transcriptome, and cytokine profiling. Cabozantinib-related effects were validated in a colorectal cancer patient-derived xenograft model. Transcriptomic data from three human HCC cohorts (cohort 1: n = 167, cohort 2: n = 57, The Cancer Genome Atlas: n = 319) were used to cluster patients according to neutrophil features, and assess their impact on survival. The combination of cabozantinib and anti-PD1 showed increased antitumor efficacy compared with monotherapy and placebo (P < 0.05). Cabozantinib alone significantly increased neutrophil infiltration and reduced intratumor CD8+PD1+ T-cell proportions, while the combination with anti-PD1 further stimulated both effects and significantly decreased regulatory T cell (Treg) infiltration (all P < 0.05). In blood, cabozantinib and especially combination increased the proportions of overall T cells (P < 0.01) and memory/effector T cells (P < 0.05), while lowering the neutrophil-to-lymphocyte ratio (P < 0.001 for combination). Unsupervised clustering of human HCCs revealed that high tumor enrichment in neutrophil features observed with the treatment combination was linked to less aggressive tumors with more differentiated and less proliferative phenotypes. Cabozantinib in combination with anti-PD1 enhanced antitumor immunity by bringing together innate neutrophil-driven and adaptive immune responses, a mechanism of action which favors this approach for HCC treatment.

A FAK Inhibitor Boosts Anti-PD1 Immunotherapy in a Hepatocellular Carcinoma Mouse Model.

Anti-PD-1/PD-L1 immunotherapy has limited efficacy in hepatocellular carcinoma (HCC) and does not benefit all patients. A FAK inhibitor (VS-4718) has been reported to improve the microenvironment in some tumors. This study aimed to investigate the effect of the combination of the FAK inhibitor VS4718 and anti-PD1 for the treatment of HCC in a mouse model and its possible mechanism of action. The expression of FAK and infiltrated immune cells in human HCC from the data of TCGA were analyzed. A primary murine HCC model was established via protooncogene (c-Met/β-catenin) transfection. The pathological characteristics of tumors were examined after the mice were treated with VS4718 and/or anti-PD1 therapy. This study revealed that FAK is highly expressed in human HCC and is associated with poor prognosis of OS (overall survival) and PFS (progress free survival) in HCC patients. Immune cell infiltration (CD8+ T, Tregs, M0, M2, CAFs and MDSCs) was correlated with FAK expression. In the experimental HCC model, the combination of a FAK inhibitor VS4718 and an anti-PD1 antibody had a better effect than monotherapy against HCC. VS4718 reduced the number of Tregs and macrophages but increased the number of CD8+ T cells in HCC mice. Notably, FAK inhibitor promoted the expression of PD-L1 in HCC. This study suggested that combination of the FAK inhibitor VS4718 and anti-PD1 could be a potential therapy for HCC by improving the immune environment, reducing liver fibrosis and simultaneously preventing PD1 from binding to the increased PD-L1 induced by FAK inhibitor VS4718.

Clinical and Experimental Evaluation of Diagnostic Significance of Alpha-Fetoprotein and Osteopontin at the Early Stage of Hepatocellular Cancer.

We evaluated the possibility of using an experimental model of hepatocellular carcinoma to study oncomarkers of primary liver cancer and compared the diagnostic efficacy of alpha-fetoprotein and osteopontin in the experiment and in clinical practice. Experimental studies were performed on a model of hepatocellular carcinoma induced by administration of diethyl nitrosamine to Fisher-344 rats. In addition, the levels of α-fetoprotein and osteopontin were determined in 35 patients with hepatocellular carcinoma detected at stages I-II according to TNM classification. The proposed model of liver cancer in rats reflects the sequence of stages characteristic of hepatocellular carcinoma in humans: liver fibrosis-cirrhosis-cancer. This model is applicable for the study of tumor markers at the early stage of tumor development. Osteopontin was found to have a more powerful diagnostic potential then alpha-fetoprotein.

The Mutational and Transcriptional Landscapes of Hepatocarcinogenesis in a Rat Model.

SummaryHepatocellular carcinoma (HCC) initiation is characterized by stepwise accumulation of molecular alterations, during which the early events are largely unknown. Here, we presented a comprehensive genomic and transcriptomic landscape at stages of hepatitis, cirrhosis, and HCC by using a diethylnitrosamine-induced rat HCC model. We observed the early occurrence of gene instability and aberrant cancer associated signaling pathways in liver hepatitis. We further characterized the progressive molecular changes during hepatocarcinogenesis, wherein the intense rivalry between tumor-suppressive and oncogenic strengths occurred in cirrhosis stage. Despite the significant pathological difference, mutation signatures and expression landscape are highly similar between hepatitis and cirrhosis stages. Furthermore, we identified PI3K-Akt signaling pathway as a key pathway in the process of hepatocarcinogenesis through integrative analysis, and PIK3CD is a potential biomarker indicating HCC recurrence. The dynamic immune response during hepatocarcinogenesis, such as continuous decline of monocytes, suggests an immunological intervention strategy beyond chemoprevention for liver cancer.

Chemerin Is Induced in Non-Alcoholic Fatty Liver Disease and Hepatitis B-Related Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma (HCC) is a frequent liver cancer and high expression of bioactive chemerin in hepatocytes was protective in experimental HCC models. The main risk factors for HCC are non-alcoholic fatty liver disease (NAFLD), hepatitis B and C infections. The current analysis showed that chemerin protein was induced in HCC tissues of NAFLD and hepatitis B infected patients. This upregulation was modest in patients with unknown disease etiology and not detected in hepatitis C infected patients. Protein levels of the chemerin receptor CMKLR1 strongly declined in the tumors of NAFLD patients and patients with unclear disease etiology but not in patients with viral infections. Our results demonstrate that the expression of chemerin in HCC is related to disease etiology and this could also apply to the role of chemerin in human HCC. In contrast to the present findings, chemerin was shown to be low in the HCC tissues of Asian patients with mostly viral disease etiology and this indicates ancestry-specific regulation of chemerin in HCC.Chemerin is protective in experimental models of hepatocellular carcinoma (HCC). Noteworthy, chemerin mRNA and protein were reduced in HCC tissues of Asian patients with mostly hepatitis B disease etiology. The current study nevertheless showed that chemerin protein was induced in tumor tissues of European HCC patients with non-alcoholic fatty liver disease (NAFLD) and patients with unclear disease etiology. A similar regulation was observed in hepatitis B virus (HBV), but not in hepatitis C virus (HCV), related HCC. The apparent discrepancy between the regulation of chemerin in HBV-HCC obtained from our study and recent reports led us to use the chemerin antibodies applied in the previous assays. These antibodies could not equally detect different chemerin isoforms, which were overexpressed in HepG2 cells. Higher chemerin protein in HCC was nevertheless confirmed by the use of all antibodies. Chemerin protein was low in Huh7 and PLC/PRF/5 cells whereas HepG2 and Hep3B cells had chemerin protein similar as primary human hepatocytes. Besides, the anti-tumor effects of retinoids in hepatocyte cell lines did not enclose upregulation of chemerin, which was initially discovered as a tazarotene induced protein in the skin. Finally, protein levels of the chemerin receptor, chemokine-like receptor 1 (CMKLR1), declined in non-viral, and tended to be lower in HBV-HCC tissues suggesting reduced chemerin activity in the tumors. To sum up, our work showed an opposite regulation of chemerin and CMKLR1 in NAFLD and HBV associated HCC. In HCV-HCC neither chemerin nor its receptor were changed in the tumor tissues. Current findings do not support a critical role of total chemerin protein levels in HCC of non-viral and viral etiology. Accordingly, tumor-localized chemerin protein was not associated with tumor-node-metastasis classification.

Abstract 5312: Imeglimin alone or in combination with sorafenib showed potent anti-tumor effect in human hepatocellular carcinoma: A new kid on the block for HCC treatment

Abstract Background: Diabetes as a standalone or as part of metabolic syndrome is recognized as a major risk factor in hepatocellular carcinoma (HCC). Metformin one of the most widely used first-line drug for the treatment of type II diabetes showed antitumor activity in preclinical models of HCC, and in some clinical trials. Imeglimin, a first-in-class tetrahydrotriazine oral antidiabetic agents, is currently in phase III clinical trial in diabetes with a good safety profile. Imeglimin stands as an AMPK activator and based on historical data with metformin may be a safe potentially interesting novel therapeutic drugs for patients with HCC. Hence, the current study aims to evaluate the effects of imeglimin alone or in combination with sorafenib compared to metformin in an experimental model of HCC. Methods: All in vivo experiments were carried out with ethical committee approval and met the standards required by the UKCCCR guidelines. HepG2 HCC cells were injected subcutaneously (2 × 106cells) into the flank of female athymic nude mice. Two weeks after cell inoculation, 90% mice developed single subcutaneous palpable tumors. Mice were randomized in the groups of treatment. Mice were then treated 5 days a week by oral gavage with 75 mg/kg/day metformin (n=14-metformin75), 75 mg/kg/day imeglimin (n=14-Imeglimin75), 150 mg/kg/day imeglimin (n=14-Imeglimin150), 40 mg/kg/day sorafenib (n=14), metformin75 plus sorafenib (n=14) or imeglimin75 plus sorafenib (n=14). Body weight and tumor volumes were assessed thrice a week. Mice were sacrificed after 63 days of treatment to meet ethical requirements (tumor volume &amp;lt; 2000 mm3). Tumor were weighted, excised and OCT-embedded to prevent tissue degradation from frozen conservation. Results: Our experiments showed a 90% uptake in the engrafted mice. Regardless of the administrated treatment, no toxicity was observed as shown by the body weight follow-up. After 63 days of treatment, each treatment arm showed a slight difference with placebo (n=14). Mean tumor volumes were 1096±208 mm3 (P&amp;gt;0.05), 795±222 mm3 (P&amp;gt;0.05), 563±135 mm3 (P&amp;lt;0.05), and 495±163 mm3(P&amp;lt;0.05) for metformin75 alone, imeglimin75 alone, imeglimin150 alone or sorafenib alone, respectively, versus 1132±156 mm3 in the placebo group. Moreover, the combination of imeglimin75 plus sorafenib (375±121 mm3) displayed an increased antitumor activity compared to metformin75 plus sorafenib (515±112 mm3). In addition, at day 59 of treatment, we reached a tumor growth inhibition of 27%, 49%, and 66% in the imeglimin75 alone, imeglimin150 alone, and combination group imeglimin75 plus sorafenib, respectively. At the time of the meeting, we will be displayed IHC analysis in addition to assess tumor angiogenesis. Conclusion: This study showed the antitumor effects of imeglimin, a novel AMPK activator in human HCC. Furthermore, imeglimin potentiates the antitumor effects of sorafenib, which may represent an interesting therapeutic option for patients with HCC. Citation Format: Annemilaï Tijeras-Raballand, Matthieu Martinet, Valérie Paradis, Jean-Pierre Bizzari, Eric Janin, Eric Raymond. Imeglimin alone or in combination with sorafenib showed potent anti-tumor effect in human hepatocellular carcinoma: A new kid on the block for HCC treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5312.

Androgen Receptor Enhances the Efficacy of Sorafenib Against Hepatocellular Carcinoma Through Enriched EpCAM Stemness.

The role of androgen receptor (AR) in hepatocellular carcinoma (HCC) development is controversial. Therefore, the translational value of targeting AR in HCC is unknown. Sorafenib, a multiple kinase inhibitor, is the standard therapy for patients with unresectable HCC. This study investigated sorafenib effect on AR in experimental models of HCC. AR cDNA was introduced into HCC cells and in vitro cell growth and in vivo tumor growth were measured. Sphere cells, as well as epithelial cell adhesion molecule-positive (EpCAM+) and CD133+ cells were isolated from HCC cells with/without AR expression to observe in vitro/in vivo effects. Liver specific AR knockout in mouse models of spontaneous HCC (carcinogen-induced and hepatitis B virus-related HCC) was also implemented to examine gene expression. HCC cells/tumors were treated with sorafenib in order to determine effects on tumor growth and related gene expression. AR cDNA increased transactivation function, increased colony/sphere-forming activities, and enhanced tumorigenicity in HCC cells compared to their parental cells. Expression of the stemness marker EpCAM was also dramatically increased. In carcinogen-and HBV-induced HCC models, EpCAM+ cells were significantly reduced in AR-knockout mice compared to wild-type HCCs. In addition, AR reduced sorafenib-related signals, e.g. extracellular-regulated kinase, AKT serine/threonine kinase 1, and p38 mitogen-activated protein kinase, compared to that in parental cells. Regarding sorafenib cytotoxicity, AR-expressing cells were vulnerable to treatment. Moreover, the half maximal-inhibitory concentration (IC50) was drastically lowered in AR+/EpCAM+ compared to AR-/EpCAM- sphere cells. Strikingly, the IC50 in AR+/CD133+ vs. AR-/CD133+ cells were similar. Moreover, sorafenib robustly suppressed tumor growth in implanted AR+/EpCAM+ cells but not AR-/EpCAM- ones. Finally, bioinformatics analyses revealed EpCAM to be a prognostic biomarker in Asian and non-alcohol-consuming patients with HCC, suggesting suitability of a sorafenib regimen for such patients. AR+/EpCAM+ may be a marker of responsiveness to sorafenib for patients with HCC. Prospective surveys associating AR/EpCAM expression with therapy outcomes are essential.

Thyroid Hormone Effects on Mesenchymal Stem Cell Biology in the Tumour Microenvironment.

Non-classical thyroid hormone signalling via cell surface receptor integrin αvβ3, expressed on most cancer cells and proliferating endothelial cells, has been shown to drive tumour cell proliferation and survival, as well as angiogenesis. Tumours develop within a complex microenvironment that is composed of many different cell types, including mesenchymal stem cells. These multipotent progenitor cells actively home to growing tumours where they differentiate into cancer-associated fibroblast-like cells and blood vessel-stabilising pericytes and thus support the tumour's fibrovascular network. Integrin αvβ3 expression on mesenchymal stem cells makes them susceptible to thyroid hormone stimulation. Indeed, our studies demonstrated - for the first time - that thyroid hormones stimulate the differentiation of mesenchymal stem cells towards a carcinoma-associated fibroblast-/pericyte-like and hypoxia-responsive, pro-angiogenic phenotype, characterised by the secretion of numerous paracrine pro-angiogenic factors, in addition to driving their migration, invasion, and recruitment to the tumour microenvironment in an experimental hepatocellular carcinoma model. The deaminated thyroid hormone metabolite tetrac, a specific inhibitor of thyroid hormone action at the integrin site, reverses these effects. The modulation of mesenchymal stem cell signalling and recruitment by thyroid hormones via integrin αvβ3 adds a further layer to the multifaceted effects of thyroid hormones on tumour progression, with important implications for the management of cancer patients and suggests a novel mechanism for the anti-tumour activity of tetrac.

Roles and Regulation of Long Noncoding RNAs in Hepatocellular Carcinoma.

Next-generation sequencing has uncovered thousands of long noncoding RNAs (lncRNA). Many are reported to be aberrantly expressed in various cancers, including hepatocellular carcinoma (HCC), and play key roles in tumorigenesis. This review provides an in-depth discussion of the oncogenic mechanisms reported to be associated with deregulated HCC-associated lncRNAs. Transcriptional expression of lncRNAs in HCC is modulated through transcription factors, or epigenetically by aberrant histone acetylation or DNA methylation, and posttranscriptionally by lncRNA transcript stability modulated by miRNAs and RNA-binding proteins. Seventy-four deregulated lncRNAs have been identified in HCC, of which, 52 are upregulated. This review maps the oncogenic roles of these deregulated lncRNAs by integrating diverse datasets including clinicopathologic features, affected cancer phenotypes, associated miRNA and/or protein-interacting partners as well as modulated gene/protein expression. Notably, 63 deregulated lncRNAs are significantly associated with clinicopathologic features of HCC. Twenty-three deregulated lncRNAs associated with both tumor and metastatic clinical features were also tumorigenic and prometastatic in experimental models of HCC, and eight of these mapped to known cancer pathways. Fifty-two upregulated lncRNAs exhibit oncogenic properties and are associated with prominent hallmarks of cancer, whereas 22 downregulated lncRNAs have tumor-suppressive properties. Aberrantly expressed lncRNAs in HCC exert pleiotropic effects on miRNAs, mRNAs, and proteins. They affect multiple cancer phenotypes by altering miRNA and mRNA expression and stability, as well as through effects on protein expression, degradation, structure, or interactions with transcriptional regulators. Hence, these insights reveal novel lncRNAs as potential biomarkers and may enable the design of precision therapy for HCC.

Abstract 3274: LTX-401 as a novel antitumor and immunotherapeutic agent in an experimental liver cancer model

Abstract Liver cancer is estimated to be the third most common cause of cancer-related mortality worldwide in both sexes. Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer in which potential curative surgical options are limited and overall survival poor. Hence, there is an unmet need for new and improved therapies. LTX-401 is a novel oncolytic compound with potent antitumor properties. Owing to its amphiphatic nature, LTX-401 may affect the integrity of cancer cell membranes and induce rapid cell death. Transmission electron microscopy indeed revealed a necrotic cell death phenotype accompanied by massive vacuolization of the cytoplasm and dilated mitochondria. Additionally, LTX-401 induces features of immunogenic cell death (ICD) as seen by the extracellular release of ATP and HMGB1, as well as the mitochondrial DAMP cytochrome C. The antitumor effects of LTX-401 has been explored in an experimental model of hepatocellular carcinoma (JM1 model) using immunocompetent rats. When injected into subcutaneously established lesions, the majority of animals went into remission followed by systemic protective immune responses. Additionally, LTX-401 could affect the growth of distal tumor deposits simulating metastases, hence indicating immune-mediated abscopal responses. Intratumoral treatment of orthotopic liver tumors resulted in cure of approximately half of the animals treated, which later displayed protection against subcutaneous tumor (re)challenge. Splenocytes harvested from immunized animals produced significantly larger amounts of IFN-y when stimulated with tumor cells ex vivo, indicating the presence of tumor-specific (memory) T cells. Histological examinations revealed that LTX-401 caused massive hemorrhagic tumor necrosis, both in subcutaneous and intrahepatic compartments, followed by increased infiltration of T cells. Taken together these results indicate that oncolytic compounds generate an immunogenic tumor environment and may be therapeutically exploited as a novel antitumor and immunotherapeutic approach in HCC. Citation Format: Brynjar Mauseth, Ketil Camilio, Ji-Hua Shi, Øystein Rekdal, Baldur Sveinbjørnsson, Pål-Dag Line. LTX-401 as a novel antitumor and immunotherapeutic agent in an experimental liver cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3274.

Chemosensitizing effect of Alpinia officinarum rhizome extract in cisplatin-treated rats with hepatocellular carcinoma

This study was conducted to estimate the preventing and sensitizing efficiency of Alpinia officinarum rhizome extract (AORE) in an experimental model of hepatocellular carcinoma (HCC) +/− cisplatin. HCC was induced by a single intraperitoneal (i.p) dose of diethylnitrosamine (DENA, 200mg/kg). After 14 days, phenobarbitone (PB, 0.05%) was added to drinking water for 14 weeks to promote hepatocarcinogenesis. Cisplatin (CP) was given in a dose of 1.5 mg/kg (i.p), twice a week, alone or with AORE (400 mg/kg daily, orally) for 21 days. AORE was tried as a protective before the induction of HCC for three weeks as well. Results revealed that DENA/PB elevated hepatic indices as ALT and AST and total bilirubin with declining serum total protein. It increased oxidative stress, as hepatic malondialdehyde (MDA) with depressed hepatic reduced glutathione (GSH) contents, superoxide dismutase (SOD) and catalase activities. This was accompanied by an increase in hepatic expression of antioxidant genes (thioredoxin and glutaredoxin). Hepatocarcinogenesis was detected by histopathological changes in liver sections and the elevation of serum alpha-fetoprotein (AFP) level. Treatment with CP partially restored altered hepatic functions and oxidative stress markers. It also showed a partial decrease in the expression of antioxidant genes, improving histopathological changes in the liver and AFP level in serum. The treatment with AORE alone or AORE+CP enhanced hepatic function and oxidative stress markers. It also caused a decrease in the expression of antioxidant genes and improved histopathological changes in liver and serum AFP level. This effect is more potent than the treatment with CP alone. Our study suggested that AORE can be used as a promising natural chemoprevention or a chemosensitizing agent against hepatocarcinogenesis.

FRI-067 - Combination of 4-Methylumbelliferone and Adenoviral Gene Transfer of Interleukin-12 Reduced the Expression of Cancer Stem Cells Markers, and Showed a Potent Antitumor Effect in an Experimental Hepatocellular Carcinoma Model with Fibrosis

FRI-067 - Combination of 4-Methylumbelliferone and Adenoviral Gene Transfer of Interleukin-12 Reduced the Expression of Cancer Stem Cells Markers, and Showed a Potent Antitumor Effect in an Experimental Hepatocellular Carcinoma Model with Fibrosis

Establishment of a dual-color fluorescence tracing orthotopic transplantation model of hepatocellular carcinoma.

Different experimental models of hepatocellular carcinoma (HCC) have been used to investigate the biological mechanisms of hepatocarcinogenesis and its progression. However, previous studies have highlighted the difficulty of distinguishing between the tumor cells and stroma in experimental models of HCC. Therefore the aim of the present study was to establish a red-green dual-color fluorescence tracing orthotopic transplantation model of HCC, and investigate its practical values. Stable high red fluorescent protein (RFP)-expressing HepG2 human hepatoma cells and Hepa1-6 mice hepatoma cells were injected into the right liver lobe of green fluorescent protein-expressing nude mice. The growth and metastasis of the tumors were visualized using a whole-body in vivo fluorescence imaging system in real time. HCC tissues were extracted from tumor-bearing mice, and cut into 5-µm serial frozen slices. The organizational structure of the transplanted tumors was observed under a microscope. A dual-color fluorescence tracing orthotopic transplantation tumor model of HCC was successfully established with a success rate of 100%. The growth and metastasis of the tumors were visualized at each stage of development in the tumor-bearing mice. Tumor cells with red fluorescence and host cells with green fluorescence were identified to merge in the reconstruction region of tumor tissue. The invasion, migration, and cell fusion between tumor and host cells was observed clearly. The dual-color fluorescence tracing ortho-topic transplantation model of HCC was determined to be a stable and reliable method for tracking tumor progression. Mutual interactions between hepatoma cells and host tissues may be observed directly using this model, further elucidating the development of the tumor microenvironment.

Targeting the insulin-like growth factor 1 (IGF1R) and insulin (IR) receptor with a dual IGF1R/IR inhibitor, OSI-906, to potentiate mTOR inhibitor effects in experimental models of hepatocellular carcinoma (HCC)

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