Human Hepatocellular Carcinoma Xenograft Model Research Articles

Curcumin inhibits proliferation of hepatocellular carcinoma cells by blocking PTPN1 and PTPN11 expression.

The antitumor mechanism of curcumin is unclear, especially in hepatocellular carcinoma (HCC) cells. To clarify the mechanism of action of curcumin in the effective treatment of HCC, the targets of curcumin were screened and validated. Candidate genes of curcumin for HCC were screened using the traditional Chinese medicine systems pharmacology (TCMSP) database and validated using The Cancer Genome Atlas (TCGA) database. The correlation of mRNA expression levels between key candidate genes was identified in the TCGA liver hepatocellular carcinoma (LIHC) dataset. The effects on prognosis were analyzed to identify the target gene of curcumin, which inhibits HCC cell proliferation. Based on the subcutaneous xenograft model of human HCC in nude mice, the expression levels of target proteins were observed using immunohistochemistry. The analysis results of the present study identified the target genes of curcumin, which were obtained by screening the TCSMP database. The protein tyrosine phosphatase non-receptor type 1 (PTPN1) was obtained from TCGA database analysis of the targeted genes. The expression levels of PTPN1 and its homologous sequence genes in TCGA LIHC project was analyzed to identify the potential target gene of curcumin, for use in HCC treatment. Next, xenograft experiments were performed to investigate the therapeutic effects of curcumin in an animal model. Curcumin was demonstrated to inhibit the growth of HCC xenograft tumors in mice. Immunohistochemistry results demonstrated that the protein expression levels of PTPN1 and PTPN11 in the curcumin group were significantly lower compared with those in the control group. In conclusion, these results demonstrated that curcumin inhibits the proliferation of HCC cells by inhibiting the expression of PTPN1 and PTPN11.

Abstract 1862: Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models

Abstract Delta-like 1 homolog (DLK-1), a non-canonical notch ligand, is a membrane protein with 6 tandem EGF-like motifs in extracellular region. While the expression of DLK-1 is restricted in some neuroendocrine tissues such as adrenal gland and hypophysis in normal adult tissues, its over-expression in cancers have been reported in a variety of cancer types, such as hepatocellular carcinoma (HCC), small cell lung carcinoma, ovarian carcinoma, adrenal cortical carcinoma, pancreatic carcinoma, gastrointestinal stromal tumor, etc. Interestingly, it has been suggested that DLK-1 is a marker of cancer stem cells in HCC. CBA-1205 is a humanized antibody (IgG1/κ) targeting DLK-1, which is glycoengineered by GlymaxX® to potentiate antibody-dependent cellular cytotoxicity (ADCC) activity. CBA-1205 showed no toxicology issues in cynomolgus monkey GLP studies and the Phase 1 clinical trial in patients with solid tumors is under way (JapicCTI-205384). Previously, we demonstrated potent anti-tumor efficacy of CBA-1205 as a monotherapy in multiple human DLK-1 positive cancer xenograft mouse models including HCC (AACR 2019 annual meeting). Lenvatinib is an oral multi-kinase inhibitor which has been used for 1st line therapy of unresectable advanced HCC globally. The purpose of present study is to evaluate the combinatorial efficacy of CBA-1205 with lenvatinib in two HCC xenograft models (Hep3B and HepG2). Hep3B model was sensitive to lenvatinib monotherapy; oral daily dosing of lenvatinib at 3 mg/kg treatment showed 50% tumor growth inhibition (TGI) and at 10 mg/kg treatment showed more than 80% TGI. In contrast HepG2 model was less sensitive to Lenvatinib; TGI at 3 mg/kg treatment group was 20 % and that for 10 mg/kg treatment group was 50 %. When CBA-1205 (1 mg/kg, twice a week, total 4 times) was intraperitoneally administered in combination with 3 mg/kg of lenvatinib (daily, total 10 times) in Hep3B xenograft mice, synergistic and long-lasting anti-tumor efficacy was observed compared either with CBA-1205 treatment alone or lenvatinib treatment alone. Tumor growth of all mice (N=8) treated with CBA-1205 and lenvatinib showed almost complete inhibition even 2 weeks after the last dosing. More importantly, 4 out of 8 mice in CBA-1205 and lenvatinib treatment group showed tumor regression. Similar results were obtained with CBA-1205 at 1 mg/kg dosage in combination with lenvatinib at 10 mg/kg dosage in HepG2 xenograft model. In conclusion, we demonstrated synergistic and long-lasting anti-tumor efficacy of CBA-1205 in combination with lenvatinib in two different HCC pre-clinical models suggesting that the combination could be a new treatment option for unresectable advanced HCC. Citation Format: Koji Nakamura, Izumi Sakaguchi, Kota Takahashi. Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1862.

Abstract 2743: Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma

Abstract Background: The telomerase-specific oncolytic adenovirus Telomelysin along with the histone deacetylase inhibitor AR42 have both demonstrated anticancer effects in preclinical models of human hepatocellular carcinoma (HCC). However, the clinical development of Telomelysin may be hindered by human antiviral immunity and tumor resistance. Combining oncolytic and epigenetic therapies has been reported to be a viable approach for treating various cancers. We investigated the potential synergism of Telomelysin and AR42 and the relevant underlying mechanisms. Method: HCC cell lines (PLC5 and Hep3B) were tested and median effect analysis was performed to estimate potential synergistic antiproliferative effects. Apoptosis was assessed by flow cytometry and Western blotting. The expression of coxsackievirus and adenovirus receptor (CAR) was measured through flow cytometry and immunofluorescence staining. Human telomerase reverse transcriptase (hTERT) mRNA expression was assessed by reverse transcription polymerase chain reaction. Western blotting was conducted to examine the activation of pivotal signaling pathways and the interferon (IFN) response. HCC cells constitutively overexpressing Akt were cloned for molecular mechanism verification. MK-2206 was used to inhibit the activation of Akt signaling. In vivo efficacy was determined through xenograft models of human HCC. Results: Telomelysin and AR42 exhibited synergistic antiproliferative effects in human HCC models in vitro and in vivo. Apoptosis induced by Telomelysin was significantly enhanced by AR42 in both PLC5 and Hep3B HCC cells. AR42 treatment unexpectedly attenuated the expression of the CAR and the mRNA levels of hTERT, which may be positively associated with the cytotoxicity of Telomelysin. Meanwhile, the cellular antiviral interferon response was not altered by AR42 treatment. Further, we found that Telomelysin enhanced Akt phosphorylation in HCC cells. AR42 reduced Telomelysin-induced phospho-Akt activation and enhanced Telomelysin-induced apoptosis. The correlation of Akt phosphorylation with drug-induced apoptosis was validated in HCC cells with upregulated or downregulated Akt signaling. Conclusions: Combination therapy with Telomelysin and AR42 demonstrates synergistic anti-HCC efficacy. Clinical trials investigating this new combination regimen are warranted. Keywords: Hepatocellular carcinoma, oncolytic adenovirus, Telomelysin, HDAC inhibitor, Akt Citation Format: Zhong-Zhe Lin, Mickey C-T. Hu, Chiun Hsu, Yao-Ming Wu, Yen-Shen Lu, Ja-An Annie Ho, Shiou-Hwei Yeh, Pei-Jer Chen, Ann-Lii Cheng. Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2743.

Development of human hepatocellular carcinoma in X-linked severe combined immunodeficient pigs: An orthotopic xenograft model.

Hepatocellular carcinoma (HCC) is the fifth most common primary tumor and the third leading cause of cancer-related deaths worldwide. Rodent models of HCC have contributed to the advancement of studies investigating liver carcinogenesis, tumor-host interactions, and drug screening. However, their small size renders them unsuitable for surgical or clinical imaging studies, necessitating the development of larger-size HCC models. Here, we developed a xenograft model of human HCC in X-linked interleukin-2 receptor gamma chain gene (Il2rg)-targeted severe combined immunodeficient (SCID) pigs. HepG2 cell suspension in serum-free medium containing 50% membrane matrix was directly injected into the liver parenchyma of eight X-linked Il2rg-targeted SCID pigs (6.6-15.6 kg) via ultrasonography-guided percutaneous puncture. Tumor engraftment was evaluated weekly using ultrasonography, and cone-beam computed tomography was performed during arterial portography (CTAP) and hepatic arteriography (CTHA) to evaluate the hemodynamics of engrafted tumors. The engrafted tumors were histologically analyzed following necropsy and assessed for pathological similarities to human HCCs. Macroscopic tumor formation was observed in seven of the eight pigs (simple nodular tumors in three and multinodular tumors in four). Engrafted tumors were identified as low-echoic upon ultrasonography and as perfusion-defect nodules on the CTAP images. Meanwhile, CTHA showed that the tumors were hyperattenuating. Further, histopathological findings of the engrafted tumors were consistent with those of human HCC. In conclusion, the porcine model of human HCC, successfully generated herein, might help develop more effective therapeutic strategies for HCC.

LXRα activation and Raf inhibition trigger lethal lipotoxicity in liver cancer.

The success of molecular therapies targeting specific metabolic pathways in cancer is often limited by the plasticity and adaptability of metabolic networks. Here we show that pharmacologically induced lipotoxicity represents a promising therapeutic strategy for the treatment of hepatocellular carcinoma (HCC). LXRα-induced liponeogenesis and Raf-1 inhibition are synthetic lethal in HCC owing to a toxic accumulation of saturated fatty acids. Raf-1 was found to bind and activate SCD1, and conformation-changing DFG-out Raf inhibitors could disrupt this interaction, thereby blocking fatty acid desaturation and inducing lethal lipotoxicity. Studies in genetically engineered and nonalcoholic steatohepatitis-induced HCC mouse models and xenograft models of human HCC revealed that therapies comprising LXR agonists and Raf inhibitors were well tolerated and capable of overcoming therapy resistance in HCC. Conceptually, our study suggests pharmacologically induced lipotoxicity as a new mode for metabolic targeting of liver cancer.

A novel targeted GPC3/CD3 bispecific antibody for the treatment hepatocellular carcinoma

ABSTRACT Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer but has shown limited success to date in the treatment of advanced stage. Recruitment of T cells for cancer treatment is a rapidly growing strategy in immunotherapy such as chimeric antigen receptor T cells and bispecific antibodies. However, unwanted aggregations, structural instability or short serum half-life are major challenges of bispecific antibodies. Here, we developed a new format of T cell-redirecting antibody that is bispecific for membrane proteoglycans GPC3 of HCC and the T-cell-specific antigen CD3, which demonstrated to be favorable stability and productivity. Cross-linking of T cells with GPC3 positive tumor cells by the anti-GPC3/CD3 bispecific antibody-mediated potent GPC3-dependent and concentration-dependent cytotoxicity in vitro. Administration of the bispecific antibody with different concentrations in murine xenograft models of human HCC significantly inhibited tumor growth. In addition, no effects on tumor growth were observed in the absence of human effector cells or the bispecific antibody. Taken together, the anti-GPC3/CD3 bispecific antibody might be a potential therapeutic treatment for HCC.

Kinesin family member 15 promotes cancer stem cell phenotype and malignancy via reactive oxygen species imbalance in hepatocellular carcinoma

The development and progression of hepatocellular carcinoma (HCC) is associated with the presence of cancer stem cells (CSCs). In the present study, kinesin family member 15 (KIF15) expression was shown to be overexpressed in HCC tissues, cell lines, and CSCs. Patients with HCC with high KIF15 expression had shortened overall survival (OS) and high recurrence probability. Downregulation of KIF15 in vitro as well as in HCC organoids resulted in a significant reduction in sphere formation and expression of stemness-related genes. KIF15 downregulation in human HCC xenograft models delayed tumor initiation, growth, and metastasis. KIF15 was also demonstrated to interact with phosphoglycerate dehydrogenase (PHGDH) and inhibit proteasomal degradation of PHGDH, thus promoting CSC phenotype and malignancy via PHGDH-mediated intracellular reactive oxygen species (ROS) imbalance in HCC. Moreover, AAA nuclear coregulator cancer-associated protein (ANCCA) upregulation acts as a key mediator in KIF15 expression upregulation in HCC. Conclusion: In this study, we found that KIF15 promotes the CSC phenotype and malignancy via PHGDH-mediated ROS imbalance in HCC. These findings highlight potential therapeutic targets for HCC.

Baicalein Targets GTPase-Mediated Autophagy to Eliminate Liver Tumor-Initiating Stem Cell-Like Cells Resistant to mTORC1 Inhibition.

Drug resistance is a major problem in the treatment of liver cancer. Mammalian Target of Rapamycin 1 (mTORC1) inhibitors have been tested for the treatment of liver cancer based on hyperactive mTOR in this malignancy. However, their clinical trials showed poor outcome, most likely due to their ability to upregulate CD133 and promote chemoresistance. The CD133+ tumor-initiating stem cell-like cells (TICs) isolated from mouse and human liver tumors are chemoresistant, and identification of an approach to abrogate this resistance is desired. In search of a compound that rescinds resistance of TICs to mTORC1 inhibition and improves chemotherapy, we identified baicalein (BC), which selectively chemosensitizes TICs and the human hepatocellular carcinoma (HCC) cell line Huh7 cells but not mouse and human primary hepatocytes. Nanobead pull-down and mass-spectrometric analysis, biochemical binding assay, and three-dimensional computational modeling studies reveal BC's ability to competitively inhibit guanosine triphosphate binding of SAR1B guanosine triphosphatase, which is essential for autophagy. Indeed, BC suppresses autophagy induced by an mTORC1 inhibitor and synergizes cell death caused by mTORC1 inhibition in TIC and Huh7 spheroid formation and in the patient-derived xenograft model of HCC. The BC-induced chemosensitization is rescued by SAR1B expression and phenocopied by SAR1B knockdown in cancer cells treated with a mTORC1 inhibitor. Conclusion: These results identify SAR1B as a target in liver TICs and HCC cells resistant to mTORC1 inhibition.

Apatinib targets both tumor and endothelial cells in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed malignancies worldwide with poor prognosis and tends to be hypervascular. Aberrant expression of the vascular endothelial growth factor 2 (VEGFR‐2) has been implicated in the progression of HCC and represents a valid target for anticancer therapy. Apatinib, a small molecule inhibitor of VEGFR‐2 tyrosine kinase, shows strong antitumor activity in various tumors. This study is designed to evaluate the activity of apatinib on both human umbilical vein vascular endothelial cells (HUVECs) and HCC cell lines (in vitro and in vivo), and also to investigate the characteristics and possible mechanisms underlying these effects by molecular biology methods. Following the results in our study, apatinib inhibited phosphorylation of VEGFR‐2 in HUVECs and blocked in vitro endothelial cell migration and tube formation. Concentration‐dependent antiproliferative effects of apatinib were also observed in all 6 HCC cell lines including SK‐Hep‐1, HepG2, Hep3B, Huh‐7, PLC/PRF/5, SMMC‐7721. Moreover, response to apatinib of HCC cell lines was significantly correlated with VEGFR‐2 expression level. Additionally, apatinib significantly inhibit VEGF‐triggered VEGFR‐2 phosphorylation and activation of downstream signaling molecules such as Akt and ERK1/2 in HCCs. Apatinib can also induce a cell cycle arrest at G2/M phase and promote HCC apoptosis tested in vitro. In vivo data showed that apatinib can effectively inhibit tumor growth, decreased angiogenesis, as well as induced HCC apoptosis (in some tumors), and thus prolonged animal survival in a mouse xenograft model of human HCC. Our findings suggested that apatinib is a highly potent, oral active anti‐angiogenic, and anti‐HCC agent. The results from current study provide a clear biological rationale to evaluate apatinib as a new agent in HCC in clinical setting, especially for the VEGFR‐2 overexpression ones.

Palbociclib enhances radiosensitivity of hepatocellular carcinoma and cholangiocarcinoma via inhibiting ataxia telangiectasia–mutated kinase–mediated DNA damage response

AimPalbociclib is an oral cyclin-dependent kinase 4/6 inhibitor, which is efficacious in treating breast cancer. Currently, there are numerous active clinical trials testing palbociclib alone or in combination with other medications for treating various types of malignancies. Here, we evaluated the anti-cancer effect of palbociclib in combination with radiation therapy (RT) for treating human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) and addressed the molecular mechanism behind the combination therapy. MethodsImmunofluorescence staining of γH2AX or 53BP1 was used to determine the effect of palbociclib on double-strand break (DSB) repair. Clonogenic assays, sphere formation and cell death ELISA were performed to study the sensitising effect of palbociclib on radiation-induced cytotoxicity. Signal alteration in DSB repair pathways was examined by Western blot analysis. Finally, we evaluated the in vivo anti-cancer activity and the associated molecular events of the combination therapy in a preclinical HCC xenograft model. ResultsPalbociclib affected the kinetics of DNA repair and enhanced the radiation sensitivity of HCC and CCA cells. Importantly, we found that palbociclib inhibits ataxia telangiectasia–mutated (ATM) kinase, the key upstream kinase responding to RT-induced DSBs. Furthermore, we showed that the inhibitory effect of palbociclib on RT-induced ATM kinase activation is mediated by protein phosphatase 5 (PP5). Both in vitro and in vivo investigations revealed that the inhibition of the PP5-ATM axis by palbociclib after DNA damage is responsible for the synergism between palbociclib and RT. ConclusionOur findings provide a novel combination strategy against liver cancer cells. Clinical trials using palbociclib as an adjuvant in RT are warranted.

Abstract 2927: Antitumor and antiangiogenesis activities of E7386, an orally active CBP/β-catenin modulator, as a single agent and in combination with lenvatinib in human HCC xenograft models

Abstract E7386 is a novel orally active CBP/β-catenin modulator that has showed anti-tumor activity in human tumor xenograft models with aberrant activation of Wnt/β-catenin signaling pathway. Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide. In HCC, it has been reported that aberrant activation of the canonical Wnt/β-catenin signaling pathway frequently occurs (20-40%). In this study, we investigated anti-tumor activity of E7386 as monotherapy or in combination with lenvatinib in human HCC xenograft models. Lenvatinib is a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors and oncogenic kinases. A recent Phase III study demonstrated the treatment effect of lenvatinib on OS by statistical confirmation of non-inferiority as compared with sorafenib patients with unresectable HCC. We evaluated the anti-tumor activity of E7386 as a single agent and in combination with lenvatinib in 4 human HCC cell lines (SNU398, HepG2, Hep3B2.1-7 and Huh-7) xenograft models. These cell lines were injected subcutaneously into male nude mice, and tumors were allowed to grow to 150-200 mm3 in size. Then, the mice were given oral administration of E7386 and/or lenvatinib for 14 days. Single treatment of E7386 demonstrated statistically significant anti-tumor effect in HepG2 and SNU398 xenograft models with β-catenin mutation; delta T/C value (dT/C) of 60% at 100 mg/kg qd in HepG2 model, dT/C of 74% and at 50 mg/kg qd in SNU398 model. On the other hand, no tumor growth inhibition was observed in Hep3B2.1-7 and Huh-7 xenograft models with wild type β-catenin. Lenvatinib at 10 mg/kg showed the anti-tumor effect in all 4 xenograft models, dT/C of 68%, 43%, 52% and 70% in SNU398, HepG2, Hep3B2.17 and Huh-7, respectively. E7386 at 50 mg/kg in combination with 10 mg/kg of lenvatinib showed more potent anti-tumor effects compared with each single treatment irrespective of β-catenin mutation status, dT/C of 35%, 14%, 9% and 17% in SNU398, HepG2, Hep3B2.17 and Huh-7, respectively. Similar combined activities were observed at the range of 6.25-50 mg/kg of E7386 with lenvatinib at 10 mg/kg. Next, we examined the effect on tumor angiogenesis. Each 50 mg/kg of E7386 and 10 mg/kg of lenvatinib decreased the microvessel density (MVD) in SNU398, reduction ratio of 37% and 79%, respectively. The combination treatment significantly decreased MVD more than each single treatment (reduction ratio of 91.4-95.1% at 12.5-50 mg/kg of E7386 with lenvatinib at 10 mg/kg). Similar combination effects were also observed in HepG2 and Huh-7 xenograft models.In conclusion, these results indicate that the combination of E7386 with lenvatinib exerted enhanced anti-angiogenic activity against tumor vessels and demonstrated the potent anti-tumor activity in HCC preclinical models. Citation Format: Kazuhiko Yamada, Yusaku Hori, Hiroshi Kamiyama, Takayuki Kimura, Yasuhiro Funahashi, Junji Matsui, Yoichi Ozawa. Antitumor and antiangiogenesis activities of E7386, an orally active CBP/β-catenin modulator, as a single agent and in combination with lenvatinib in human HCC xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2927.

Reducing protein regulator of cytokinesis 1 as a prospective therapy for hepatocellular carcinoma

Proteins that bind to microtubule are important for cell cycle, and some of these proteins show oncogenic characteristics with mechanisms not fully understood. Herein we demonstrate overexpression of protein regulator of cytokinesis 1 (PRC1), a microtubule-associated regulator of mitosis, in human hepatocellular carcinoma (HCC). Moreover, upregulated PRC1 is associated with lower survival rates of HCC patients. Mechanistically, reducing PRC1 blocks mitotic exit of HCC cells at telophase in a spindle assembly checkpoint independent manner, and acts synergistically with microtubule-associated agents (MTAs) to suppress p53-wt or p53-null HCC cells in a p53- or p14ARF-dependent manner; while overexpressing PRC1 increases the resistance of HCC to taxol. A combined treatment of taxol/shPRC1 results in 90% suppression of tumor growth in subcutaneous HCC xenograft models. In orthotopic xenograft mice, reducing PRC1 significantly alleviates HCC development and hepatic injury. Together, our results suggest a dual-mitotic suppression approach against HCC by combining MTAs with cytokinesis inhibition, which blocks mitosis at both metaphase and telophase.

PFKFB3 blockade inhibits hepatocellular carcinoma growth by impairing DNA repair through AKT

Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key molecule of glucose metabolism in cytoplasm, has been found in various tumors. Emerging evidence has suggested that PFKFB3 is also located in the nucleus and apparent in regulatory functions other than glycolysis. In this study, we found that PFKFB3 expression is associated with hepatocellular carcinoma (HCC) growth and located mainly in the nucleus of tumor cells. PFKFB3 overexpression was associated with large tumor size (p = 0.04) and poor survival of patients with HCC (p = 0.027). Knockdown of PFKFB3 inhibited HCC growth, not only by reducing glucose consumption but also by damaging the DNA repair function, leading to G2/M phase arrest and apoptosis. In animal studies, overexpression of PFKFB3 is associated with increased tumor growth. Mechanistically, PFKFB3 silencing decreased AKT phosphorylation and reduced the expression of ERCC1, which is an important DNA repair protein. Moreover, PFK15, a selective PFKFB3 inhibitor, significantly inhibited tumor growth in a xenograft model of human HCC. PFKFB3 is a potential novel target in the treatment of HCC.

CMTM5 is downregulated and suppresses tumour growth in hepatocellular carcinoma through regulating PI3K-AKT signalling

BackgroundHuman chemokine like factor (CKLF)-like MAL and related proteins for vesicle trafficking transmembrane, domain-containing member 5 (CMTM5) has been shown to involved and may function as a tumour suppressor in tumorigenesis. The current study aimed to investigate the expression and function of CMTM5 in human hepatocellular carcinoma (HCC).MethodsCMTM5 expression was examined by immunohistochemistry, and its clinical significance was analysed in 76 HCC specimens. The role and molecular mechanisms of CMTM5 in cell proliferation, apoptosis and invasion were examined in vitro and in vivo.ResultsCMTM5 expression was significantly downregulated in HCC tissues as well as cell lines. The expression of CMTM5 was absent in 77.6% of HCC tissues compared with 3.9% in normal liver tissues. Low CMTM5 expression was significantly correlated with poor overall survival in patients with HCC (P = 0.009). Restoring CMTM5 expression in Huh7 cells significantly inhibited cell growth, promoted cell apoptosis, and reduced cell metastatic and invasion ability compared with mock transfected cells in vitro. Overexpression of CMTM5 also suppressed xenograft tumour growth in vivo in a HCC xenograft model. Reduced cell growth and metastasis ability mediated by CMTM5 overexpression was associated with downregulation of PI3K/AKT and its downstream Bcl2, cyclinD1, cyclinE, MMP2 and MMP9 expressions, and an upregulation of p21, Bax, Bad, cleaved caspase3 expressions.ConclusionsOur data suggest that CMTM5 might function as a tumour suppressor in human HCC, and represent a valuable potential therapeutic target for HCC.

Enhanced NOLC1 promotes cell senescence and represses hepatocellular carcinoma cell proliferation by disturbing the organization of nucleolus.

SummaryThe nucleolus is a key organelle that is responsible for the synthesis of rRNA and assembly of ribosomal subunits, which is also the center of metabolic control because of the critical role of ribosomes in protein synthesis. Perturbations of rRNA biogenesis are closely related to cell senescence and tumor progression; however, the underlying molecular mechanisms are not well understood. Here, we report that cellular senescence‐inhibited gene (CSIG) knockdown up‐regulated NOLC1 by stabilizing the 5′UTR of NOLC1 mRNA, and elevated NOLC1 induced the retention of NOG1 in the nucleolus, which is responsible for rRNA processing. Besides, the expression of NOLC1 was negatively correlated with CSIG in the aged mouse tissue and replicative senescent 2BS cells, and the down‐regulation of NOLC1 could rescue CSIG knockdown‐induced 2BS senescence. Additionally, NOLC1 expression was decreased in human hepatocellular carcinoma (HCC) tissue, and the ectopic expression of NOLC1 repressed the proliferation of HCC cells and tumor growth in a HCC xenograft model.

Anti-GPC3-CAR T Cells Suppress the Growth of Tumor Cells in Patient-Derived Xenografts of Hepatocellular Carcinoma

The lack of a general clinic-relevant model for human cancer is a major impediment to the acceleration of novel therapeutic approaches for clinical use. We propose to establish and characterize primary human hepatocellular carcinoma (HCC) xenografts that can be used to evaluate the cytotoxicity of adoptive chimeric antigen receptor (CAR) T cells and accelerate the clinical translation of CAR T cells used in HCC. Primary HCCs were used to establish the xenografts. The morphology, immunological markers, and gene expression characteristics of xenografts were detected and compared to those of the corresponding primary tumors. CAR T cells were adoptively transplanted into patient-derived xenograft (PDX) models of HCC. The cytotoxicity of CAR T cells in vivo was evaluated. PDX1, PDX2, and PDX3 were established using primary tumors from three individual HCC patients. All three PDXs maintained original tumor characteristics in their morphology, immunological markers, and gene expression. Tumors in PDX1 grew relatively slower than that in PDX2 and PDX3. Glypican 3 (GPC3)-CAR T cells efficiently suppressed tumor growth in PDX3 and impressively eradicated tumor cells from PDX1 and PDX2, in which GPC3 proteins were highly expressed. GPC3-CAR T cells were capable of effectively eliminating tumors in PDX model of HCC. Therefore, GPC3-CAR T cell therapy is a promising candidate for HCC treatment.

New Natural Pigment Fraction Isolated from Saw Palmetto: Potential for Adjuvant Therapy of Hepatocellular Carcinoma.

For the first time, we discovered a small proportion of aqueous fraction from Saw Palmetto apart from the fatty acid-rich fraction exhibited pharmacological activity. Therefore, this study aims to explore the anti-tumor potential of red pigmented aqueous fraction of Saw Palmetto, NYG on human hepatocellular carcinoma and its possible targets. Subcutaneous xenograft and orthotopic implantation models of HCC were used to evaluate the tumor inhibitory effect of NYG. Human hepatocellular carcinoma (HCC) cell lines and human umbilical vein endothelial cells (HUVEC) were used as in vitro model. The mRNA expression was conducted by qPCR. Protein expression was monitored by immunoblotting and immunohistochemistry. Cell migration and blood vessel formation were determined by chamber assay and tube formation assay, respectively. Significant tumor inhibition of NYG in dose-dependent manner was observed on subcutaneous xenograft and orthotopic HCC model. NYG has no direct action on cell viability or VEGF secretion of HCC cells. However, NYG reduced in vitro migration and vessel formation activities of HUVEC cells, as well as in vivo intratumoral neovascularization. NYG attenuated extracellular signal-regulated kinases (ERK) activation in endothelial cells, which may be associated with the suppression of migration and tube formation of HUVEC. NYG suppressed tumor expansion of HCC via inhibiting neovascularization, and may be potential adjuvant treatment for HCC.

Abstract 3256: Highly potent monoclonal and bispecific anti-angiogenic antibodies to VEGF and angiopoietin-2

Abstract The angiogenic factor Vascular Endothelial Growth Factor (VEGF) is a well-established target for cancer therapy. The humanized monoclonal antibody (mAb) bevacizumab (Avastin®), which binds and neutralizes VEGF, is widely prescribed for the treatment of colon, lung and certain other tumors, but typically provides a relatively modest survival benefit of several months. It is unknown whether the limited benefit is due to redundancy in angiogenic pathways or to the inability of bevacizumab at the doses used to fully neutralize VEGF in the tumor microenvironment. However, the exploration of higher doses of bevacizumab in the clinical setting has been very limited due to lack of incentive to perform dose-ranging studies for an already approved drug, the expense of the drug, and concern about toxicity. To determine whether more effective neutralization of VEGF would be possible and useful, we have therefore used an intensive immunization protocol to generate a new murine mAb designated VE1 that binds VEGF with very high affinity. A humanized form of this mAb, HuVE1, retains the full affinity of VE1 and binds VEGF about 5-fold better than bevacizumab. In side-by-side comparisons, HuVE1 also blocked the binding of VEGF to its receptor with an IC50 that is 5 to 10-fold lower than for bevacizumab, and it correspondingly inhibited VEGF-induced proliferation of HUVEC about 5-fold better than bevacizumab. HuVE1 almost completely blocked growth of primary human hepatocellular carcinoma (HCC) xenografts in one model, and showed a trend toward better efficacy than bevacizumab in a second HCC xenograft model. For potentially even greater anti-angiogenic efficacy, we developed a humanized mAb HuA2T that potently neutralizes angiopoietin-2 (Ang-2), a cytokine that acts through the Tie-2 receptor to promote angiogenesis, especially in combination with VEGF. The synergy between VEGF and Ang-2 provides a strong rationale to develop a bispecific mAb that neutralizes both these factors. We therefore developed such a mAb using the IgG-like bispecific antibody format Bs(scFv)4-IgG consisting of a homodimer of two monomers, each monomer having a single chain Fv from HuVE1 linked to a light chain constant region, and a single chain Fv from HuA2T linked to a heavy chain constant region. While, as is common in construction of bispecific antibodies, this bispecific HuA2T/HuVE1 mAb did lose some binding affinity for VEGF and Ang-2 relative to the original mAbs, its VEGF binding activity was still greater than that of bevacizumab, illustrating the importance of starting with an extremely high activity mAb. Based on its potency in vitro and in animal models, we believe that HuVE1, in either natural or bispecific form, has the potential for greater clinical efficacy than bevacizumab and thus merits further investigation. Citation Format: Hangil Park, April Zhang, Yi Ding, Lihong Wang, Zhengran Wang, Maximiliano Vasquez, Cary Queen, Jin Kim. Highly potent monoclonal and bispecific anti-angiogenic antibodies to VEGF and angiopoietin-2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3256.

Structure-based optimization leads to the discovery of NSC765844, a highly potent, less toxic and orally efficacious dual PI3K/mTOR inhibitor

The phosphoinositide 3-kinase (PI3K) family is one of the most frequently activated enzymes in a wide range of human cancers; thus, inhibition of PI3K represents a promising strategy for cancer therapy. Herein, a series of benzylamine substituted arylsulfonamides were designed and synthesized as dual PI3K/mTOR inhibitors using a strategy integrating focused library design and virtual screening, resulting in the discovery of 13b (NSC765844). The compound 13b exhibits highly potent enzyme inhibition with IC50s of 1.3, 1.8, 1.5, 3.8 and 3.8 nM for PI3Kα, β, γ, δ, and mTOR, respectively. 13b was further evaluated in NCI by an in vitro cytotoxic screening program. Broad-spectrum antitumor activities with mean GI50 value of 18.6 nM against approximately 60 human tumor cell lines were found. 13b displayed favorable physicochemical properties and superior pharmacokinetic profiles for animal studies. It significantly inhibited tumor growth when administered orally in an A549 non-small-cell lung carcinoma xenograft and BEL7404 human hepatocellular carcinoma xenograft models. On the basis of its excellent in vivo efficacy and superior pharmacokinetic profiles, 13b has been selected for further preclinical investigation as a promising anticancer drug candidate.

Optical Imaging of Mesenchymal Epithelial Transition Factor (MET) for Enhanced Detection and Characterization of Primary and Metastatic Hepatic Tumors.

Purpose: To assess optical imaging of Mesenchymal-Epithelial Transition factor (MET) for delineation and characterization of intrahepatic models of human hepatocellular carcinoma (HCC) and metastatic colorectal cancer (CRC), and thereby demonstrate its potential use in precision oncology.Materials and Methods: MET expression in human CRC and HCC was assessed in tissue microarrays. We used GE-137, a modified cyanine 5-tagged peptide for MET targeting. HepG2 and Huh-7 (HCC) and HT-29 (CRC) cells with MET overexpression, and LNCaP cells (negative control) with minimal MET expression were incubated with the probe. Correlation between the relative fluorescence signal intensity and cellular MET expression level was assessed. Flow cytometry was used to assess probe specific binding and dissociation constant (Kd). Orthotopic xenograft models of human HCC and metastatic CRC were generated in nu/nu mice by subcapsular implantation of cells. Epifluorescence imaging was performed to capture the changes in deferential probe accumulation at different time points after injection. Target-to-liver background ratio (TBR) was calculated and the probe biodistribution within different organs was assessed. Histopathologic analysis of extracted xenografts was performed to correlate the tumors MET expression with probe uptake by cancer cells.Results: Approximately 91.5% of HCC and 81% of CRC microarray cores showed MET expression. HCC and CRC cells incubated with the probe showed substantial fluorescence compared to control LNCaP, with strong correlation between fluorescence signal and MET expression (R2 = 0.99, p < 0.001). Probe binding affinity to MET (Kd) was measured to be 2.9 ± 0.36 nM. Epifluorescence imaging showed intense uptake in subcapsular tumors with peak TBR of 5.46 ± 0.46 in Huh-7, 3.55 ± 0.38 in HepG2, and 15.93 ± 0.61 in HT-29 orthotopic xenografts at 4 hours post-injection (mean ± standard deviation). We demonstrated that in vivo probe uptake in xenografts is specific and can be blocked when co-injected with unlabeled peptide; for instance the epifluorescence TBR is reduced from 13.5 ± 1.2 to 1.7 ± 0.3 (p < 0.05) in HT-29 and from 5.3 ± 0.8 to 1.4 ± 0.2 (p < 0.05) in Huh-7 xenografts after co-injection with unlabeled peptides. Biodistribution studies showed predominantly renal clearance of the probe.Conclusion: Optical imaging of MET resulted in high TBR in animal models of primary and metastatic hepatic tumors suggesting its utility for procedural guidance.