Modelling the Sorafenib-resistant Liver Cancer Microenvironment by Using 3-D Spheroids.

Liver cancer is the third leading cause of cancer-related mortality, and hepatocellular carcinoma (HCC) is the most common form of liver cancer, and it usually occurs in the setting of chronic liver disease and cirrhosis. For patients with advanced HCC, systemic treatment is the first choice - however, resistance occurs frequently. Sorafenib was the first tyrosine kinase inhibitor approved for advanced HCC, and resistance to the therapy is a serious concern. When sorafenib therapy fails in a patient, it can be challenging to decide whether they can undergo a second-line therapy, and to determine which therapy they will be able to tolerate. Thus, physiologically relevant in vitro preclinical models are crucial for screening potential therapies, and 3-D tumour spheroids permit studies of tumour pathobiology. In this study, a drug-resistant 3-D tumour spheroid model was developed, based on sorafenib-resistant hepatocellular carcinoma cells, LX2 stellate cells and THP-1 monocytes. Model tumour spheroids that were formed with the sorafenib-resistant cells demonstrated lower diffusion of doxorubicin and exhibited increased resistance to regorafenib. Moreover, in the sorafenib-resistant spheroids, there was increased presence of CD68-positive cells and a reduction in inflammatory marker secretion. The sorafenib-resistant cell line-derived spheroids also showed a higher expression of FGF-19, PDGF-AA and GDF-15, which are known to be involved in malignancies. This multi-cell type spheroid model represents a potentially useful system to test drug candidates in a microenvironment that mimics the drug-resistant tumour microenvironment in HCC.

EGFR and Lyn inhibition augments regorafenib induced cell death in sorafenib resistant 3D tumor spheroid model.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and the third most lethal malignancy worldwide. Patients with unresectable HCC receive systemic therapies, traditionally sorafenib or lenvatinib as first line therapy. Despite its poor therapeutic response and high rates of resistance, in most countries, sorafenib still remains the globally used first-line treatment for advanced HCC. Thus, preclinical models demonstrating sorafenib resistance are crucial. 3D tumor spheroid models are becoming extremely important as screening platforms for drug therapies. In this paper, we utilized sorafenib resistant Huh7 cell line and LX2 hepatic stellate cell line to establish a sorafenib resistant 3D tumor spheroid model which can be used to test second-line treatment options. Our analysis demonstrated that sorafenib resistant 3D tumor spheroids are also more resistant to regorafenib and exhibit diverse features compared to parental tumor spheroids. Sorafenib resistant spheroids had higher CD24 and EpCAM positive cancer stem cell populations. In addition, several oncogenic kinases are upregulated in the sorafenib resistant spheroids. Importantly, combined inhibition of EGFR and Lyn kinase in sorafenib resistant tumor spheroids are effective in inducing cell death. Our model proved to be an affordable and useful model to mimic drug resistant tumor microenvironment in HCC and provided novel insights into candidates for new combinational therapies.

Changes in Wnt and TGF-ß Signaling Mediate the Development of Regorafenib Resistance in Hepatocellular Carcinoma Cell Line HuH7.

Hepatocellular carcinoma (HCC) is an aggressive, chemo resistant neoplasm with poor prognosis and limited treatment options. Exploring activated pathways upon drug treatment can be used to discover more effective anticancer agents to overcome therapy resistance and enhance therapeutic outcomes for patients with advanced HCC. Human tumor-derived cell lines recapitulate HCC diversity and are widely used for studying mechanisms that drive drug resistance in HCC. In this study, we show that regorafenib treatment activates Wnt/ß-catenin signaling only in hepatoblast-like HCC cell lines and induces enrichment of markers associated with hepatic stem/progenitor cells. Moreover, activation of Wnt/ß-catenin signaling via Wnt3a/R-Spo1 treatment protects these cells from regorafenib induced apoptosis. On the other hand, regorafenib resistant cells established by long-term regorafenib treatment demonstrate diminished Wnt/ß-catenin signaling activity while TGF-ß signaling activity of these cells is significantly enhanced. Regorafenib resistant cells (RRCs) also show increased expression of several mesenchymal genes along with an induction of CD24 and CD133 cancer stem cell markers. Moreover, regorafenib resistant cells also exhibit significantly augmented in vitro and in vivo migration capacity which could be reversed by TGF-ß type 1 receptor (TGFb -R1) inhibition. When combined with regorafenib treatment, TGFß-R1 inhibition also significantly decreased colony formation ability and augmented cell death in resistant spheroids. Importantly, when we knocked down TGFß-R1 using a lentiviral plasmid, regorafenib resistant cells entered senescence indicating that this pathway is important for their survival. Treatment of RRCs with TGFß-R1 inhibitor and regorafenib significantly abolished pSTAT3, pSMAD2 and pERK (44/42) expression suggesting the involvement of both canonical and non-canonical pathways. In conclusion, our data suggest that HCC tumors with aberrant activation in the Wnt/ß-catenin pathway, might have higher intrinsic regorafenib resistance and the inhibition of this pathway along with regorafenib administration might increase regorafenib-induced cell death in combinational therapies. However, to resolve acquired regorafenib resistance developed in HCC patients, the combined use of TGF-ß pathway inhibitors and Regorafenib constitute a promising approach that can increase regorafenib sensitization and prevent tumor recurrence.

LGR5/R-Spo1/Wnt3a axis promotes stemness and aggressive phenotype in hepatoblast-like hepatocellular carcinoma cell lines.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with "hepatoblast-like" appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and ß-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/ß-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/ß-catenin pathway.

Doxorubicin-induced senescence promotes stemness and tumorigenicity in EpCAM-/CD133- nonstem cell population in hepatocellular carcinoma cell line, HuH-7.

The therapeutic induction of senescence is a potential means to treat cancer, primarily acting through the induction of a persistent growth-arrested state in tumors. However, recent studies have indicated that therapy-induced senescence (TIS) in tumor cells allows for the prolonged survival of a subgroup of cells in a dormant state, with the potential to re-enter the cell cycle along with an increased stemness gene expression. Residual cells after TIS with increased cancer stem cell phenotype may have profound implications for tumor aggressiveness and disease recurrence. Herein, we investigated senescence-associated stemness in EpCAM+/CD133+ liver cancer stem cell and EpCAM-/CD133- nonstem cell populations in HuH7 cell line. We demonstrated that treatment with doxorubicin induces senescence in both cell populations, accompanied by a significant increase in the expression of reprogramming genes SOX2, KLF4, and c-MYC as well as liver stemness-related genes EpCAM, CK19, and ANXA3 and the multidrug resistance-related gene ABCG2. Moreover, doxorubicin treatment significantly increased EpCAM + population in nonstem cells indicating senescence-associated reprogramming of nonstem cell population. Also, Wnt/ß-catenin target genes were increased in these cells, while inhibition of this signaling pathway decreased stem cell gene expression. Importantly, Dox-treated EpCAM-/CD133- nonstem cells had increased in vivo tumor-forming ability. In addition, when SASP-CM from Dox-treated cells were applied onto hIPSC-derived hepatocytes, senescence was induced in hepatocytes along with an increased expression of TGF-ß, KLF4, and AXIN2. Importantly, SASP-CM was not able to induce senescence in Hep3B-TR cells, a derivative line rendered resistant to TGF-ß signaling. Furthermore, ELISA experiments revealed that the SASP-CM of Dox-treated cells contain inflammatory cytokines IL8 and IP10. In summary, our findings further emphasize the importance of carefully dissecting the beneficial and detrimental aspects of prosenescence therapy in HCC and support the potential use of senolytic drugs in HCC treatment in order to eliminate adverse effects of TIS.

Effect of adipocyte-secreted factors on EpCAM+/CD133+ hepatic stem cell population.

Recent epidemiological studies have associated obesity with a variety of cancer types including HCC. However, the tumor initiating role of obesity in hepatocarcinogenesis is still unknown. The objective of this paper is to investigate the effect of adipocyte-secreted factors on EpCAM+/CD133+ cancer stem cells and to identify which factors play a role in modulating hepatic cancer stem cell behavior. Our results demonstrated that adipocyte-secreted factors affect motility and drug resistance of EpCAM+/CD133+ cells. When incubated with adipocyte conditioned media, EpCAM+/CD133+ cells exhibited augmented motility and reduced sorafenib-induced apoptosis. Using array-based system, we identified secretion of several cytokines such as IL6, IL8 and MCP1 by cultured adipocytes and activation of c-Met, STAT3 and ERK1/2 signaling pathways in EpCAM+/CD133+ cells incubated with adipocyte conditioned media. Treating EpCAM+/CD133+ cancer stem cells with IL6 receptor blocking antibody or c-Met inhibitor SU11274 both reduced the increase in motility; however SU11274 had greater effect on relieving protection from sorafenib-induced apoptosis. These results indicate that adipocyte-secreted factors might regulate cancer stem cell behavior through several signaling molecules including c-Met, STAT3 and ERK1/2 and inhibition of these signaling pathways offer novel strategies in targeting the effect of adipose-derived cytokines in cancer.

Elevated hepatocyte growth factor expression as an autocrine c-Met activation mechanism in acquired resistance to sorafenib in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Limitations in HCC treatment result due to poor prognosis and resistance against traditional radiotherapy and chemotherapies. The multikinase inhibitor sorafenib is the only FDA approved drug available for advanced HCC patients, and development of second-line treatment options for patients who cannot tolerate or develop resistance to sorafenib is an urgent medical need. In this study, we established sorafenib-resistant cells from Huh7 and Mahlavu cell lines by long-term sorafenib exposure. Sorafenib-resistant HCC cells acquired spindle-shape morphology, upregulated mesenchymal markers, and showed significant increase in both migration and invasion abilities compared to their parental counterparts. Moreover, after long-term sorafenib treatment, HCC cells showed induction of hepatocyte growth factor (HGF) synthesis and secretion along with increased levels of c-Met kinase and its active phosphorylated form, indicating autocrine activation of HGF/c-Met signaling. Importantly, the combined treatment of the resistant cells with c-Met kinase inhibitor SU11274 and HGF neutralizing antibody significantly reversed the increased invasion ability of the cells. The combined treatment also significantly augmented sorafenib-induced apoptosis, suggesting restoration of sorafenib sensitivity. These results describe, for the first time, compensatory upregulation of HGF synthesis leading to autocrine activation of HGF/c-Met signaling as a novel cellular strategy in the acquisition of sorafenib resistance. Therefore, we suggest that combinatorial therapeutic strategies with HGF and c-Met inhibitors comprise promising candidates for overcoming sorafenib resistance.