LIPOTOXICITY, HEPATIC STEATOSIS AND HEPATOCARCINOMA: ROLE OF THE MITOCHONDRIAL FAMILY ARMC10 / ARMCX

Eduardo Soriano Universitat de Barcelona Francesc Villarroya CIBER Fisiopatologia de l’Obesitat i Nutrició Beatriz Mínguez Hospital Universitari Vall d’Hebron

2 1. Project summary

Hepatic steatosis is the most common cause of chronic liver injury in the western world. The mechanism by which hepatic steatosis may contribute to the development of hepatocellular carcinoma (HCC) is not known, although liver tumors are increasingly diagnosed in the fatty liver of patients without cirrhosis. The Armcx / Armc10 belong to a family of recently described mitochondrial proteins, characterized by the presence of β-catenin domains; its expression is reduced in epithelial cancers and in cell lines derived from carcinomas. Currently, 6 Armcx (1-6) and one pseudogene (similar to Armcx6) are known, all of them located in a cluster of the X (Xq21.33-q22.2), which suggests the hypothesis of the existence of genetic duplications. This new family shows that these genes are highly expressed in liver and adipose tissue.

The main objective of this work has been to determine the role of this family of mitochondrial proteins in hepatic steatosis and hepatocarcinoma. Using a variety of techniques, including tumor analysis in knockout mice, histology, biochemistry and molecular biology and expression techniques in tissues, we investigated how the expression of Armcx / Armc10 genes regulate the development of hepatocellular carcinoma.

The most important results show that depletion of the Armcx3 gene protects mice from obesity associated with diet, probably due to the induction of "Browning" in white adipose tissue, which is accompanied by better insulin sensitivity and a reduction of liver damage caused by hepatic steatosis. Secondly, the inactivation of the Armcx3 gene in mice dramatically reduces the appearance of tumors in the liver, induced by DEN treatment, both in normal diet and fat diet. This effect is associated with various alterations in signaling pathways, and with an increase in apoptosis and infiltration of macrophages within tumors. Finally, human tissue expression studies show that the Armcx3 and Armc10 genes are overexpressed in hepatocarcinoma. Studies of genetic silencing in human cell lines show that the inhibition of Armcx3 and Armc10 genes decreases the proliferation, viability and invasiveness of liver tumor cells.

3 We conclude that the inhibition / inactivation of the Armcx3 gene inhibits the appearance of hepatocarcinoma, which may represent a new therapeutic tool for the treatment of these tumors.

2. Results obtained

To determine the role of the Armcx3 gene in liver metabolism, we generated conditional knockout mice for that gene. Mice are inactivated at postnatal ages by tamoxifen injections. Both the control (fA3Cre-) and the Armcx3 deficient mice (KO) were fed with low (LFD) or high (HFD) fat diets for sixteen weeks. KO mice fed with HFD gained less weight than fA3 / Cre- mice under the same diet, despite a higher caloric intake. These KO mice, whether fed with LFD or HFD, also showed a better homeostasis of glucose, as indicated by the results obtained with glucose tolerance tests and insulin, and the HOMA-IR index. In addition, KO mice had lower levels of fat content in the liver, especially when they were fed with HFD. This resistance of KO mice to the development of hepatic steatosis was accompanied by lower levels of alanine amino transferase activity in plasma, indicating a reduction in liver injury. On the other hand, the KO mice experienced an induction in the expression of different gene markers of thermogenic activity, such as UCP1 or Dio2, in the subcutaneous white adipose tissue (scWAT), indicating an activation of the "Browning" phenomenon in this deposit (transformation of white adipose tissue in a brown adipose tissue phenotype). In summary, these results suggest that depletion of Armcx3 protects mice from obesity associated with diet, probably due to the induction of "Browning" in the scWAT, which is accompanied by better insulin sensitivity and a reduction in liver damage produced by hepatic steatosis.

Second, and in order to determine the possible involvement of the Alex3 protein ( gene) in the development of HCC, we used N-nitrosodiethylamine (DEN) as a liver carcinogen reagent for the Alex3-KO mouse model. The postnatal mice (P1-P3) were treated with tamoxifen and 14 days later they were injected with DEN. 4 weeks after the administration of DEN, the control mice (fA3 / Cre-) and KO were separated into two dietary groups and fed both with a low-fat diet (LFD) or a high-fat diet (HFD). According to the data cited above, we observed that KO mice fed HFD obtained less body weight than control mice (fA3 / Cre-). Tumors of the mice were evaluated at 30

4 weeks of age. Mice that did not have the Alex3 protein had decreased the number of tumors, the size of the tumor and the maximum tumor size, compared to the control animals (fA3 / Cre-), with a significant protective effect. This effect was much more marked and significant in mice fed with HFD.

Histological analyses of liver sections revealed that KO mice were resistant to the development of hepatic steatosis. In addition, we observed a lower activity of alanine aminotransferase in plasma, which indicates a reduction in hepatocellular injury in KO mice. Therefore, inactivation of the Armcx3 gene decreases the metabolic phenotype and contributes to a decrease in hepatic tumorigenesis induced by DEN caused by obesity associated with diet.

Hepatocellular proliferation was analyzed in tumors by Ki67 and BrdU staining. Surprisingly, inactivation of Alex3 does not affect cell proliferation. However, we found a significant increase in activated caspase-3 in Alex3-KO mice fed with HFD, compared to controls (fA3 / Cre-), especially in animals fed with HFD. Consistent with this data of apoptosis, immunostaining for F4 / 80 indicated a greater accumulation of macrophages in tumors of Alex3-KO mice fed with HFD compared to control mice. These results demonstrated that Alex3 inhibits hepatocellular apoptosis and macrophage infiltration in tumors of mice treated with DEN, especially in mice fed with HFD.

To analyze the gene and protein expression of Armc10, Armcx3 and CTNNB1 (β- catenin) in human liver samples from patients with HCC and NASH, the gene and protein expression of Armcx3, Armc10 and CTNNB1 (β-Catenin) were analyzed by qRTPCR, WB and IHC in human liver samples from control (LC), NASH, cirrhotic and HCC patients in an exploratory cohort of fresh frozen tissue (FF) and in a cohort of paraffin tissue (FFPE). Armcx3 expression analysis in the FF cohort showed that 14% of the HCC samples and 25% of the NASH samples had overexpression at the gene level. This trend could be corroborated in the cohort of FFPE samples in which 35% of samples with HCC showed an overexpression of Armcx3 (increase> 2.5) and 30% of samples presented a positive IHC. The Armc10 gene expression showed significant differences between the control liver groups and the NASH group (0.9 ± 12:18) vs (4.2 ± 12:38) respectively, as well as between the control liver and hepatocellular carcinoma (0.9 ± 12:18) vs (3.9 ± 12:59) respectively. The expression in the cirrhotic

5 liver group did not present significant differences with the rest of the pathologies studied (2.4 ± 0.68). The expression of CTNNB1 was, as expected, significantly higher in the group of hepatocarcinoma samples than in the control liver group (8.7 ± 2.90) vs (1 ± 12:16). The rest of the groups studied did not present significant differences in their expression compared with the rest: NASH (3.4 ± 12:36) and cirrhotic liver (2 ± 0.65). The statistical study of the correlation between the overexpression of Armc10 and CTNNB1, showed a significant positive relationship with a p value lower than 0.05 and an R2 = 0.3941 reinforcing the hypothesis of the relationship between both genes. Validation of these results in the FFPE tissue cohort corroborated the expression of Armc10 and CTNNB1; 45% of the hepatocarcinoma samples had overexpression of Armc10 and 47% of CTNNB1, showing a statistically significant positive correlation with an R2 = 0.7218.

Finally, we investigated the potential role that these genes could play in the development of hepatocellular carcinoma through its silencing in models of human hepatocellular carcinoma in vitro. We designed interference RNAs (siRNAs) in order to silence the expression of Armc10 and Armcx3 and analyzed how silencing affected variables such as viability, invasiveness and clonality in hepatocellular carcinoma cell lines. The human hepatocarcinoma cell lines with higher basal expression of Armcx3 and Armc10 were chosen to be transiently transfected with an inhibitory RNA. Silencing levels of more than 80% were obtained in the selected cell lines (Hep3B, SNU423, SNU449 and SNU182). Feasibility, cloning and invasiveness tests were performed in the different human hepatocarcinoma cell lines at different incubation times (24, 48 and 72 hours). It was observed that the transient inhibition of the expression of the genes significantly decreased the cell viability, measured by the method of reduction of salts of tetrazolium (MTT), after 48 hours. Effects were also observed in the cloning studies that were carried out in two weeks after transfection, by staining the colonies with crystal violet. The colony count showed differences of up to 75% less in the formation of colonies in the cells to which the expression of the gene had been inhibited with respect to the control cells. The invasive capacity of the cells was determined by the line test (wound healing assay) in which it was possible to observe how the cells that had been inhibited the expression of Armcx3 and Armc10 progressed more slowly than those that retained the expression of these genes intact. We conclude therefore that the inhibition of the Armcx3 and Armc10 genes decreases the proliferation, viability and invasiveness of liver tumor cells.

6 3. Relevance and possible future implications

The first important conclusion is that the presence of these genes is necessary to generate a metabolic phenotype in the liver and other tissues, associated with obesity (including hepatic steatosis). This is especially evident when animals are treated with a fatty diet. Therefore, the inhibition of this gene has a protective effect on these phenotypes associated with liver and metabolic disease.

The second important conclusion is that the inactivation of these genes protects against the appearance and growth of liver tumors, in our case in experimental animals induced by treatment with DEN. Therefore, the pharmacological or genetic inhibition of these genes can be beneficial and protective for the appearance and / or progression of hepatic tumors.

Finally, the data in human tissue show a good correlation between high expression of these genes and liver disease, including tumors, which led us to propose this protein family as new markers of hepatic tumor disease. Second, the inhibition of these genes in human tumor cell lines represents a proof of concept showing that it reduces their tumoral activity.

In summary, we have identified a new gene family, whose action is necessary to develop liver disease associated with metabolism and tumors. This suggests that the development of drugs and other inhibitory therapies of these genes and proteins may be of therapeutic interest in the treatment of these diseases.

4. Generated literature

Mirra S, Ulloa F, Gutierrez-Vallejo I, Martì E and Soriano E. Function of Armcx3 and Arm10/SVH genes in the regulation of progenitor proliferation and neural differentiation in the spinal cord. Frontiers Cellular Neuroscience. (2016), doi: 10.3389/fncel.2016.00047

7 Mirra S, Gavaldà A, Manso Y, Higuera M, Serrat R, Mínguez B, Soriano E* and Villaroya F*. The mitochondrial protein Armcx3 confers susceptibilty to hepatic in response to hepatic lipotoxicity.(2018) (in preparation) (* co-senior authors)

Higuera M, Mirra S, Gavaldà A, Salcedo MT, Ventura M, Balibrea JM, Villarroya F, Soriano E and Mínguez B. Impact of the Armcx1-6/Armc10 family genes in non- alcoholic steatohepatitis and hepatocellular carcinoma. (2018) (in preparation)

Gavaldà A, Mirra S, Serrat R, Manso Y, Villaroya F* and Soriano E*. Downregulation of Armcx3 and Armc10 genes results in metabolic dysfunction in vivo. (2018) (in preparation). (* co-senior authors)

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