Devorah Olam

Multiple adaptive mechanisms to chronic liver disease revealed at early stages of liver carcinogenesis in the Mdr2-knockout mice.

Molecular events preceding the development of hepatocellular carcinoma were studied in the Mdr2-knockout (Mdr2-KO) mice. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by hepatocellular carcinoma development after the age of 1 year. Liver tissue samples of Mdr2-KO mice in the early and late precancerous stages of liver disease were subjected to histologic, biochemical, and gene expression profiling analysis. In an early stage, multiple protective mechanisms were found, including induction of many anti-inflammatory and antioxidant genes and increase of total antioxidant capacity of liver tissue. Despite stimulation of hepatocyte DNA replication, their mitotic activity was blocked at this stage. In the late stage of the disease, although the total antioxidant capacity of liver tissue of Mdr2-KO mice was normal, and inflammation was less prominent, many protective genes remained overexpressed. Increased mitotic activity of hepatocytes resulted in multiple dysplastic nodules, some of them being steatotic. Expression of many genes regulating lipid and phospholipid metabolism was distorted, including up-regulation of choline kinase A, a known oncogene. Many other oncogenes, including cyclin D1, Jun, and some Ras homologues, were up-regulated in Mdr2-KO mice at both stages of liver disease. However, we found no increase of Ras activation. Our data suggest that some of the adaptive mechanisms induced in the early stages of hepatic disease, which protect the liver from injury, could have an effect in hepatocarcinogenesis at later stages of the disease in this hepatocellular carcinoma model.

Molecular Mechanisms of Liver Carcinogenesis in the Mdr2-Knockout Mice

Mouse models of hepatocellular carcinoma (HCC) simulate specific subgroups of human HCC. We investigated hepatocarcinogenesis in Mdr2-knockout (Mdr2-KO) mice, a model of inflammation-associated HCC, using gene expression profiling and immunohistochemical analyses. Gene expression profiling showed that although Mdr2-KO mice differ from other published murine HCC models, they share several important deregulated pathways and many coordinately differentially expressed genes with human HCC data sets. Analysis of genome positions of differentially expressed genes in liver tumors revealed a prolonged region of down-regulated genes on murine chromosome 8 in three of the six analyzed tumor samples. This region is syntenic to human chromosomal regions that are frequently deleted in human HCC and harbor multiple tumor suppressor genes. Real-time reverse transcription-PCR analysis of 16 tumor samples confirmed down-regulation of several tumor suppressors in most tumors. We show that in the aged Mdr2-KO mice, cyclin D1 nuclear level is increased in dysplastic hepatocytes that do not form nodules; however, it is decreased in most dysplastic nodules and in liver tumors. We found that this decrease is mostly at the protein, rather than the mRNA, level. These findings raise the question on the role of cyclin D1 at early stages of hepatocarcinogenesis in the Mdr2-KO HCC model. Furthermore, we show that most liver tumors in Mdr2-KO mice were characterized by the absence of β-catenin activation. In conclusion, the Mdr2-KO mouse may serve as a model for β-catenin–negative subgroup of human HCCs characterized by low nuclear cyclin D1 levels in tumor cells and by down-regulation of multiple tumor suppressor genes. (Mol Cancer Res 2007;5(11):1159–70)

Molecular mechanisms of the chemopreventive effect on hepatocellular carcinoma development in Mdr2 knockout mice

Dietary antioxidants and selenium compounds were shown to have a therapeutic effect against hepatocellular carcinoma in several mouse models. We tested the effects of tannic acid and selenomethionine on hepatocellular carcinoma development in Mdr2 knockout (Mdr2-KO) mice. Mdr2-KO and age-matched Mdr2 heterozygous control mice were fed with tannic acid or selenomethionine during the first 3 months of life. Then, several mice from each group were sacrificed, and liver tissue samples were removed for analysis. The remaining mice were fed a regular diet until the age of 16 months, at which time the number and size of liver tumors were determined. Liver tissue samples of 3-month-old mice were subjected to gene expression profiling analysis using cDNA macroarrays containing probes for 240 genes that regulate responses to oxidative stress and inflammation or lipid metabolism. Both tannic acid and selenomethionine had partial chemopreventive effect on development of hepatocellular carcinoma in Mdr2-KO mice: they reduced the incidence of large tumor nodules (diameter >1 cm) at age 16 months. Both agents inhibited gene expression and reversed up-regulation of many genes that control inflammation or response to oxidative stress in Mdr2-KO livers at age 3 months. This inhibitory effect on gene expression correlated with the ability of agents to reduce incidence of large tumors: selenomethionine was more active than tannic acid in both aspects. Understanding the molecular mechanism of chemoprevention effect could improve our therapeutic modalities while using these agents. [Mol Cancer Ther 2007;6(4):1283–91]

HCV Tumor Promoting Effect Is Dependent on Host Genetic Background

Background The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo. Methodology/Principal Findings We crossed HCV-Tg mice that do not develop HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and investigated the potential contributing factors for the generated phenotype by gene expression and protein analyses. The Mdr2-KO/HCV-Tg females from the N2 generation of this breeding (having 75% of the FVB/N genome and 25% of the C57BL/6 genome) produced significantly larger tumors in comparison with Mdr2-KO mice. In parallel, the Mdr2-KO/HCV-Tg females had an enhanced inflammatory gene expression signature. However, in the N7 generation (having 99.2% of the FVB/N genome and 0.8% of the C57BL/6 genome) there was no difference in tumor development between Mdr2-KO/HCV-Tg and Mdr2-KO animals of both sexes. The HCV transgene was similarly expressed in the livers of Mdr2-KO/HCV-Tg females of both generations, as revealed by detection of the HCV transcript and the core protein. Conclusion These findings suggest that the HCV transgene accelerated inflammation-associated hepatocarcinogenesis in a host genetic background-dependent manner.

The Sphingosine-1-Phosphate Modulator FTY720 Targets Multiple Myeloma via the CXCR4/CXCL12 Pathway

Purpose: To explore the functional consequences of possible cross-talk between the CXCR4/CXCL12 and the sphingosine-1-phosphate (S1P) pathways in multiple myeloma (MM) cells and to evaluate the effect of S1P targeting with the FTY720 modulator as a potential anti-MM therapeutic strategy. Experimental Design and Results: S1P targeting with FTY720 induces MM cell apoptosis. The combination of FTY720 with the SPHK1 inhibitor SKI-II results in synergistic inhibition of MM growth. CXCR4/CXCL12-enhanced expression correlates with reduced MM cell sensitivity to both FTY720 and SKI-II inhibitors, and with SPHK1 coexpression in both cell lines and primary MM bone marrow (BM) samples, suggesting regulative cross-talk between the CXCR4/CXCL12 and SPHK1 pathways in MM cells. FTY720 was found to directly target CXCR4. FTY720 profoundly reduces CXCR4 cell-surface levels and abrogates the CXCR4-mediated functions of migration toward CXCL12 and signaling pathway activation. Moreover, FTY720 cooperates with bortezomib, inducing its cytotoxic activity and abrogating the bortezomib-mediated increase in CXCR4 expression. FTY720 effectively targets bortezomib-resistant cells and increases their sensitivity to bortezomib, promoting DNA damage. Finally, in a recently developed novel xenograft model of CXCR4-dependent systemic MM with BM involvement, FTY720 treatment effectively reduces tumor burden in the BM of MM-bearing mice. FTY720 in combination with bortezomib demonstrates superior tumor growth inhibition and abrogates bortezomib-induced CXCR4 increase on MM cells. Conclusions: Altogether, our work identifies a cross-talk between the S1P and CXCR4 pathways in MM cells and provides a preclinical rationale for the therapeutic application of FTY720 in combination with bortezomib in patients with MM. Clin Cancer Res; 23(7); 1733–47. ©2016 AACR.

Testing transgenic regulatory elements through live mouse imaging

To overcome positional and methylation effects on transgene expression, we developed a universal cloning cassette for in vivo assessment of regulatory elements using the luciferase reporter gene and the CCCD camera. Monitoring luciferase expression pattern in live mice enables screening of large numbers of transgenic founders quickly and inexpensively. We demonstrate that in the engineered transgenic mice, the chicken β‐globin 5′HS4 insulator did not always provide the desirable expression pattern, and the Island Element, responsible for the demethylation of the surrounding DNA region, was not beneficial. Both tested liver‐specific and developmentally regulated promoters exhibited the expected expression pattern in most transgenic founders.

CXCR4 promotes neuroblastoma growth and therapeutic resistance through miR-15a/16-1 mediated ERK and BCL2/cyclin D1 pathways

CXCR4 expression in neuroblastoma tumors correlates with disease severity. In this study, we describe mechanisms by which CXCR4 signaling controls neuroblastoma tumor growth and response to therapy. We found that overexpression of CXCR4 or stimulation with CXCL12 supports neuroblastoma tumorigenesis. Moreover, CXCR4 inhibition with the high-affinity CXCR4 antagonist BL-8040 prevented tumor growth and reduced survival of tumor cells. These effects were mediated by the upregulation of miR-15a/16-1, which resulted in downregulation of their target genes BCL-2 and cyclin D1, as well as inhibition of ERK. Overexpression of miR-15a/16-1 in cells increased cell death, whereas antagomirs to miR-15a/16-1 abolished the proapoptotic effects of BL-8040. CXCR4 overexpression also increased miR-15a/16-1, shifting their oncogenic dependency from the BCL-2 to the ERK signaling pathway. Overall, our results demonstrate the therapeutic potential of CXCR4 inhibition in neuroblastoma treatment and provide a rationale to test combination therapies employing CXCR4 and BCL-2 inhibitors to increase the efficacy of these agents.Significance: These results provide a mechanistic rationale for combination therapy of CXCR4 and BCL-2 inhibitors to treat a common and commonly aggressive pediatric cancer.Cancer Res; 78(6); 1471-83. ©2017 AACR.

Tumor-suppressive effect of S-adenosylmethionine supplementation in a murine model of inflammation-mediated hepatocarcinogenesis is dependent on treatment longevity

Chronic inflammation precedes the majority of hepatocellular carcinoma (HCC) cases. We investigated the chemopreventive potential of S-adenosylmethionine (SAM), an essential donor for all methylation reactions in the cell, at the late precancerous stage of HCC development using the Mdr2-knockout (Mdr2-KO, Abcb4-/-) mice, a model of inflammation-mediated hepatocarcinogenesis. Previously, we revealed down-regulation of the genes regulating SAM metabolism in the liver of these mice at the precancerous stages. Now, we have supplied Mdr2-KO mice at the late precancerous stage with SAM during either a short-term (17 days) or a long-term (51 days) period and explored the effects of such supplementation on tumor development, DNA methylation and gene expression in the liver. The short-term SAM supplementation significantly decreased the number of small tumor nodules, proliferating hepatocytes and the total DNA methylation level, while it increased expression of the tumor suppressor proteins Mat1a and p21. Surprisingly, the long-term SAM supplementation did not affect tumor growth and hepatocyte proliferation, while it increased the total liver DNA methylation. Our results demonstrate that the short-term SAM supplementation in the Mdr2-KO mice inhibited liver tumor development potentially by increasing multiple tumor suppressor mechanisms resulting in cell cycle arrest. The long-term SAM supplementation resulted in a bypass of the cell cycle arrest in this HCC model by a yet unknown mechanism.Chronic inflammation precedes the majority of hepatocellular carcinoma (HCC) cases. We investigated the chemopreventive potential of S-adenosylmethionine (SAM), an essential donor for all methylation reactions in the cell, at the late precancerous stage of HCC development using the Mdr2-knockout (Mdr2-KO, Abcb4−/−) mice, a model of inflammation-mediated hepatocarcinogenesis. Previously, we revealed down-regulation of the genes regulating SAM metabolism in the liver of these mice at the precancerous stages. Now, we have supplied Mdr2-KO mice at the late precancerous stage with SAM during either a short-term (17 days) or a long-term (51 days) period and explored the effects of such supplementation on tumor development, DNA methylation and gene expression in the liver. The short-term SAM supplementation significantly decreased the number of small tumor nodules, proliferating hepatocytes and the total DNA methylation level, while it increased expression of the tumor suppressor proteins Mat1a and p21. Surprisingly, the long-term SAM supplementation did not affect tumor growth and hepatocyte proliferation, while it increased the total liver DNA methylation. Our results demonstrate that the short-term SAM supplementation in the Mdr2-KO mice inhibited liver tumor development potentially by increasing multiple tumor suppressor mechanisms resulting in cell cycle arrest. The long-term SAM supplementation resulted in a bypass of the cell cycle arrest in this HCC model by a yet unknown mechanism.