p19Arf inhibits aggressive progression of H-ras-driven hepatocellular carcinoma

p19Arf inhibits aggressive progression of H-ras-driven hepatocellular carcinoma Abstract Arf, a well-established tumor suppressor, is either mutated or downregulated in a wide array of cancers. However, its role in hepatocellular carcinoma (HCC) progression is controversial. Conflicting observations have been published regarding its expression in HCC. In this study, we provide clear genetic evidence demonstrating a protective role of p19Arf in hepatocarcinogenesis. Using Ras-induced mouse model, we show that p19Arf deficiency accelerates progression of aggressive HCC in vivo. To investigate the role of p14ARF in human liver cancers, we analyzed its expression in human HCC using immunohistochemistry (IHC). We observe lack of nucleolar p14ARF in 43.02% of human HCC samples and that low expression of p14ARF strongly correlates with the early onset of HCC. Importantly, cirrhotic livers that did not progress to HCC harbor higher expression of the p14ARF protein in hepatocytes compared with that in cirrhotic livers with HCC. These results are significant because they suggest that nucleolar p14ARF can be used as early prognostic marker in chronic liver disease to reliably identify patients with high risk for developing liver cancer. Currently, there is no effective systemic therapy for advanced liver cancer; hence, more efficient patient screening and early detection of HCC would significantly contribute to the eradication of this devastating disease. Introduction Liver cancer is the third leading cause of cancer related death worldwide, accounting for more than 600 000 deaths each year (1). The most common type of liver cancer in adult patients is hepatocellular carcinoma (HCC). Unfortunately, HCC is diagnosed often at a late stage when surgery is no longer an option; hence, patients survive less than 1 year after initial diagnosis, even with intervention. Therefore, there is an urgent need for better understanding of this disease. More than 80% of HCC develops as the last stage of chronic liver disease caused by hepatitis C virus (HCV) or hepatitis B virus (HBV) infection, alcohol abuse, dietary exposure to aflatoxin, obesity or metabolic syndrome (2). They all cause damage and necrosis of hepatocytes, followed by inflammation, proliferation and subsequent liver regeneration (3). If insults are persistent, cycles of damage and repair continue, and disease progresses to chronic hepatitis followed by liver cirrhosis, with severely compromised liver function. In liver cirrhosis, regenerative capacity of hepatocytes is exhausted, and functional tissue has been replaced with fibrous scar, making environment suitable for the HCC nodules to develop (4). Because most of the HCCs develop as the terminal stage of chronic liver disease, more effective screening of the population under high risk would result in early detection and subsequent treatment of early stage cancers. Identification of specific markers that could reliably identify patients with high risk would significantly contribute to the eradication of this disease. The roles of the tumor suppressor protein ARF (alternative reading frame; p14ARF in humans and p19Arf in mice) in suppression of HCC have remained unclear. ARF protein expression is induced in response to oncogenic stimuli in many tumors (5). Once expressed, ARF protein prevents abnormal cellular proliferation by sequestering Mdm2 to nucleolus and stabilizing p53 tumor suppressor protein (6). ARF can also induce p53-independent cell cycle arrest by binding to c-myc, E2F1 and FoxM1 (7–9), translocating them to nucleolus, therefore, preventing their transcriptional activation of cell cycle-promoting target genes. The cell cycle-regulatory function of ARF has also been linked to cellular senescence (10). That is consistent with the observations that in a variety of tumors, expression of ARF is quenched through DNA methylation and silencing, suggesting that suppression of ARF expression is critical for tumorigenesis. However, the role of ARF in HCC progression has been controversial, as opposite results have been reported regarding its expression in human HCC (11–14). Anzola et al. (12) reported inactivation of p14Arf gene in 62.4% of the tumor samples and 26.4% of the corresponding non-tumor samples harvested from HCC patients. Their work demonstrates that p14ARF gene in human HCC can be inactivated by different mechanisms including promoter hypermethylation, homozygous deletions, loss of heterozygosity and less frequently point mutations, with hypermethylation being the main cause of inactivation. Importantly, these results suggest that p14ARF gene silencing plays an important role in the pathogenesis of HCC and that could be one of the early events in HCC development. On the other hand, Tannapfel et al. (13) demonstrated p14Arf promotor methylation in only 9% of patient samples. Moreover, Teruaki et al. (14) failed to detect any hypermethylation of the p14ARF promoter in Japanese HCC samples. In that study, only 1 out of 44 HCC samples exhibited homozygous deletion, and 2 out of 44 HCC samples harbored mutation. Surprisingly, they observed increases in p14Arf mRNA expression in the remaining 41 HCC samples, compared with paired non-tumor tissue. Furthermore, although the association was not statistically significant, the expression of p14ARF mRNA was correlated with poorly differentiated tumor phenotype. One of the most commonly perturbed signaling pathways in HCC is the Ras signaling pathway (15–17). For example, recent studies discovered epigenetic silencing of negative regulators of Ras signaling pathway, GAPs (GTPase-activating proteins) in human HCC samples. Calvisi et al. (16) found silencing of RASAL1, DAB2IP and NF1 in over 70% of analyzed HCC. On the other hand, study with 69 Chinese patients demonstrated mutations in H-Ras codons 40 and 61, in about 71% of the patients (17). Hence, although Ras mutations are less common in HCC, Ras-pathway is ubiquitously activated in human HCC due to the silencing of its negative regulators. In this study, using a Ras-transgenic mouse model, we provide genetic evidence that p19Arf plays a strong suppressive role on HCC development and progression. For the first time, we analyzed p14ARF protein expression in human HCC samples using immunohistochemistry (IHC). We observed lack of nucleolar p14ARF in HCC cells in 43.02% of analyzed human HCC samples. We provide evidence that loss of p14ARF protein expression in cirrhotic liver can be used as prognostic marker for HCC development. Materials and methods Animal studies The University of Illinois at Chicago institutional animal care and use committee preapproved all animal experiments. Previously described H-rasV12 mice (18) were crossed with earlier described p19Arf−/− C57/BL6 animals (19) to obtain p19Arf+/+ H-rasV12 and p19Arf−/− H-rasV12 mice. The male mice were, aged ≤ 10 months, humanely sacrificed and analyzed for tumor development. If the mice reached humane end point earlier, they were humanely sacrificed and analyzed for tumor development. Tissue microarray We obtained tissue microarray (TMA) of human HCC core samples collected at the University of Illinois Health Sciences Center following the guidelines of University of Illinois and NIH policy on human sample studies. All tissue TMA core samples were obtained from liver explants of patients who have undergone liver transplantation procedure, from 1999 to 2007. For most of the patients, three cores were included in the TMA. Pathologist diagnosed each core, and only the ones confirmed to be HCC were studied. We analyzed 86 HCC patient samples for p14ARF expression. HCC cores included tumors grade 1 to grade 3. We had available age information for 59 patients, ranging from 29 to 82 years of age. Average age of patients from the HCC set is 57.27 years (median 58 years). On the same TMA, there were 43 cores with cirrhotic hepatocytes harvested from the livers that developed HCC. They were also analyzed for p14ARF expression. Furthermore, we obtained TMA of human cirrhotic core samples collected at the University of Illinois Health, from 2000 to 2007, from liver explants of patients who have undergone liver transplantation procedure but did not develop HCC. Most of the patients were presented with two cores. Average age of patients in the cirrhosis set is 50.018 years (median 49 years), ranging from 25 to 75 years of age. Immunohistochemistry For IHC, tissues were fixed overnight in 10% neutral buffered formalin, rinsed in phosphate-buffered saline and left in 75% ethanol until processing. Afterwards, tissues were passed through grades of alcohols and embedded in paraffin. Sections(5 μm thick) were prepared, baked overnight at 55°C and stained following standard procedures. Briefly, antigen retrieval was performed using sodium citrate buffer, and primary antibodies were incubated overnight at 4°C. Visualization was done using 3,3’-diaminobenzidine and counterstained using Hematoxylin (Polyscientific). All used reagents are from Vector Labs unless otherwise indicated. Staining was performed using rabbit monoclonal Abcam Ki67 antibody, Santa Cruz mouse monoclonal FoxM1 antibody and Abcam p19Arf ab80 antibody. TMA was stained with Novus Biological p14ARF antibody, NB200-335, at dilution of 1:100 and with p53 mouse monoclonal M7001 from DAKO. Expression studies Total RNA was Trizol (Invitrogen) extracted from harvested tumors, and cDNA was synthesized using Bio-Rad reverse transcriptase. cDNA was amplified using SYBR Green (Bio-Rad) and analyzed via iCycler software. Used primer sequences are following: FoxM1 F 5′- GAGGAAAGAGCACCTTCAGC-3′, R 5′- AGGCAATGTCTCCTTGATGG-3′, GAPDH F 5′-AACTTTGGCATTGTGG AAGG-3′, R 5′- CCATCCACAGTCTTCTGGGT-3′, Bmi1 F 5′- AGAGGGATGGACTAC GAATGC-3′, R 5′- AACAGGAAGAGGTGGAGGGAAC-3′, Nanog F 5′- AGCCCTG ATTCTTCTACCAGTCCC-3′, R 5′- ACAGTCCGCATCTTCTGCTTCC-3′, c-myc F 5′- TAACTCGAGGAGGAGCTGGA-3′, R 5′- GCCAAGGTTGTGAGGTTAGG-3′, Cdc25B F 5′- CCCTTCCCTGTTTTCCTTTC-3′, R 5′- ACACACACTCCTGCCAT AGG-3′, FoxA3 F 5′-TGAATCCTGTGCCCACCAT-3′, R 5′-AGCTGAGTGGGTT CAAGGTCAT-3′, CD44 F 5′- TGCATTTGGTGAACAAGGAA-3′, R 5′- GGAATG ACGTCTCCAATCGT-3′, CD90 F 5′- GCCTGACCCGAGAGAAGAAGAAG-3′, R 5′-TGGTGGTGAAGTTCGCTAGAGTAAG-3′, IL6 F 5′- TGATGCACTTGCAGAAA ACA-3′, R 5′- ACCAGAGGAAATTTTCAATAGGC-3′. Statistical analysis Statistical significance was calculated using Student’s t-test (two-tailed). Statistically significant changes were indicated with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Results Deletion of p19Arf accelerates HCC development in vivo To study role of Arf in HCC progression in vivo, we used oncogenic Ras-driven mouse model of HCC. In our model system, oncogenic form of H-Ras (H-ras12V) is expressed under control of albumin promoter, only in the liver epithelial cells. Male mice of this strain develop liver tumors at age of 8–9 months with penetrance of almost 100% (18). It is an excellent, clinically relevant model system to study liver cancer in vivo. To investigate role of p19Arf in HCC development, we crossed H-ras12V mice with p19Arf knockout strain and obtained p19Arf−/−H-ras12V males. As a control group, we generated p19Arf+/+ H-ras12V male mice. As expected, all control males developed liver tumors by age of 10 months, when the livers were harvested (Figure 1A). In sharp contrast, none of the p19Arf-deficient male mice survived 10 months of age. Figure 1. View largeDownload slide Loss of p19Arf accelerates progression of H-ras12V driven HCCs. Ten months old p19Arf+/+ H-ras12V male mice and moribund p19Arf−/− H-ras12V (from 4- to 10-month old) male mice were sacrificed, livers were harvested and examined for tumors (A). Survival of male p19Arf+/+ Hras12V and p19Arf−/− H-ras12V mice is shown in (B). The difference in survival is statistically significant with the P-value of 0.01076. Graph in (C) shows drastic increase in the liver weight (as percentage of total body weight) of p19Arf−/− H-ras12V male mice. (D) percentage of tumor nodules of indicated sizes. IHC staining for p19Arf protein on liver non-tumor and liver tumor sections harvested from 10-month-old male p19Arf+/+H-ras12V mice is shown in (E) (top). Quantification of p19Arf positive cells is presented in the bottom panel E. Figure 1. View largeDownload slide Loss of p19Arf accelerates progression of H-ras12V driven HCCs. Ten months old p19Arf+/+ H-ras12V male mice and moribund p19Arf−/− H-ras12V (from 4- to 10-month old) male mice were sacrificed, livers were harvested and examined for tumors (A). Survival of male p19Arf+/+ Hras12V and p19Arf−/− H-ras12V mice is shown in (B). The difference in survival is statistically significant with the P-value of 0.01076. Graph in (C) shows drastic increase in the liver weight (as percentage of total body weight) of p19Arf−/− H-ras12V male mice. (D) percentage of tumor nodules of indicated sizes. IHC staining for p19Arf protein on liver non-tumor and liver tumor sections harvested from 10-month-old male p19Arf+/+H-ras12V mice is shown in (E) (top). Quantification of p19Arf positive cells is presented in the bottom panel E. We followed 24 male p19Arf+/+ H-ras12V mice and 15 male p19Arf−/− H-ras12V mice for survival. There was a clear decrease in survival of p19Arf-deficient, H-ras12V-expressing mice (Figure 1B). The shortest surviving knockout male mouse reached humane end point as early as 5 months of age, while the oldest p19Arf-deficient male mice reached the end point by 9 months of age. The control mice (p19Arf+/+H-ras12V) survived through 10 months of age when experiment was terminated (Figure 1B), even though they all developed liver tumor nodules. Of note, we aged four p19Arf+/+H-ras12V male mice to 14 months of age, and although they all had large liver tumor nodules, they were not moribund (data not shown). We harvested livers with tumors from control mice at 10 months of age and livers with tumors from p19Arf-deficient mice when they were moribund. The representative harvested livers with tumors are shown in Figure 1A. To quantify tumor burden, we calculated liver weight as percentage of total body weight (Figure 1C). Tumor burden was significantly higher in Arf-deficient male mice compared with the control mice (Figure 1C). Furthermore, we measured diameter of harvested tumors and found that liver tumors harvested from the Arf-deficient mice were larger than tumors harvested from control mice, with around 20% of liver tumors reaching more than 2 cm in diameter in knockout mice (Figure 1D). We did not observe any macrometastasis. Furthermore, we reasoned that if p19Arf is important for Ras-driven HCC development, its expression should diminish in p19Arf+/+ Hras12V tumors. To explore that, we examined p19Arf expression using IHC in non-tumor and tumor tissue harvested from 10-month-old p19Arf+/+H-ras12V male mice. We detected significant decrease in p19Arf protein in tumor cells compared with non-malignant hepatocytes (Figure 1E), further indicating the importance of p19Arf in suppression of Ras-driven HCC development. These results provide clear genetic evidence that p19Arf protects liver from Ras-driven hepatocarcinogenesis in vivo. p19Arf−/−H-ras12V liver tumors are histologically heterogeneous Tumors harvested from p19Arf+/+H-ras12V and p19Arf−/−H-ras12V mice were further analyzed by IHC. Histological analysis of the p14Arf-expressing tumors showed uniform growth pattern with predominantly fatty changes (95.24%, or 20 out of 21 analyzed tumors displayed fatty changes), some solid growth (6/21) and cytoplasmic inclusions (11/21). The tumors developed in p19Arf-deficient mice, on the other hand, were histologically heterogeneous (Figure 2). Most of the analyzed p19Arf-deficient tumors displayed solid growth pattern (9 out of 11 or 81.82%) or trabecular growth pattern (9 out of 11 or 81.82%). Moreover, we found fatty changes in 6 out of 11 analyzed tumors (54.5%) and cytoplasmic inclusions in 3 out of 11 (27.27%). Additionally, we found 2 out of 11 Arf-deficient tumors to have pseudoglandular growth pattern and one with focal sclerosis. No pseudoglandular growth pattern or focal sclerosis was observed in the Arf-expressing tumors. While 57.14% (12 out of 21) of tumors harvested from p19Arf-expressing animals had fibrotic changes, only 14.28% or 3 out of 11 tumors harvested from p19Arf-deficient animals exhibited fibrotic changes. Necrosis was prominent in 81.82% of analyzed p19Arf knockout tumors (9 out of 11 tumors), while only 4.76% (1 out of 21) of p19Arf+/+ tumors contained necrotic portions. Interestingly, we observed significant immune infiltrates in 72.73% (or 8 out of 11) of analyzed p19Arf-deficient tumors, while only 38.09% (8 out of 21) of the p19Arf-expressing tumors contained immune infiltrates. Representative low magnification images of p19Arf-deficient H-ras-driven HCCs are shown in Supplementary Figure 1A-D, available at Carcinogenesis Online, while tables in Figure 2A and B show observed growth pattern changes in analyzed control tumors (Figure 2A) and p19Arf-deficient tumors (Figure 2B). Every column represents different tumor, while black field describes presence of designated pattern in indicated tumor sample. Figure 2. View largeDownload slide Growth pattern of p19Arf+/+ H-ras12V and p19Arf−/−H-ras12V liver tumors. Liver tumors were harvested from male mice, sectioned and stained with H&E. Table in (A) summarizes histology of tumors harvested from p19Arf+/+ H-ras12V mice. Histology of p19Arf−/− H-ras12V tumors is shown in (B). Black boxes in the tables show presence of indicated growth pattern, while the grey boxes represent presence of indicated growth pattern in low levels in particular tumor. White boxes signify absence of the indicated growth pattern. Figure 2. View largeDownload slide Growth pattern of p19Arf+/+ H-ras12V and p19Arf−/−H-ras12V liver tumors. Liver tumors were harvested from male mice, sectioned and stained with H&E. Table in (A) summarizes histology of tumors harvested from p19Arf+/+ H-ras12V mice. Histology of p19Arf−/− H-ras12V tumors is shown in (B). Black boxes in the tables show presence of indicated growth pattern, while the grey boxes represent presence of indicated growth pattern in low levels in particular tumor. White boxes signify absence of the indicated growth pattern. p19Arf-deficient tumors are highly proliferative and they express stemness signature genes To evaluate proliferation capacity of the tumors, we used Ki67-specific immunohistochemical staining (Figure 3A). We quantified the HCC cells for Ki67 expression. There was significant increase in percentage of Ki67 positive HCC cells in p19Arf-deficient tumors, indicating an increase in proliferation. Quantification of the Ki67 staining is shown in Figure 3A, bottom panel. Figure 3. View largeDownload slide p19Arf-deficient tumors are highly proliferative and highly express stemness signature genes. To analyze proliferation of tumor cells, HCC sections of both genotypes were stained for Ki67. Representative images of Ki67 IHC staining and quantification of Ki67 positive tumor cells are shown in (A). We quantified Ki67 positive tumor cells in 30 fields from three different tumors harvested from p19Arf+/+ H-ras12V mice and in 25 fields from five different tumors harvested from p19Arf−/− H-ras12V mice. Total RNA was isolated from harvested tumors and analyzed for Bmi1, Nanog, c-myc, FoxA3, CD90 and IL6 expression by RT-PCR. The results are shown in (B), (C), (D), (E), (F) and (G), respectively. Each bar represents mRNA isolated from different tumor. Green bars represent p19Arf-expressing tumors, and blue bars represent p19Arf-deficient tumors. Statistical analyses for all genes are shown in (H). Figure 3. View largeDownload slide p19Arf-deficient tumors are highly proliferative and highly express stemness signature genes. To analyze proliferation of tumor cells, HCC sections of both genotypes were stained for Ki67. Representative images of Ki67 IHC staining and quantification of Ki67 positive tumor cells are shown in (A). We quantified Ki67 positive tumor cells in 30 fields from three different tumors harvested from p19Arf+/+ H-ras12V mice and in 25 fields from five different tumors harvested from p19Arf−/− H-ras12V mice. Total RNA was isolated from harvested tumors and analyzed for Bmi1, Nanog, c-myc, FoxA3, CD90 and IL6 expression by RT-PCR. The results are shown in (B), (C), (D), (E), (F) and (G), respectively. Each bar represents mRNA isolated from different tumor. Green bars represent p19Arf-expressing tumors, and blue bars represent p19Arf-deficient tumors. Statistical analyses for all genes are shown in (H). Liver tumors with high expression of stemness genes are considered more aggressive and related to poor prognosis in human HCC patients (20). Because tumor harboring p19Arf knockout male mice had shorter survival and more aggressive tumors compared with control, we analyzed expression of stemness genes in tumors of both genotypes using quantitative real time PCR (Figure 3). Interestingly, we observed higher expression of stemness genes in p19Arf-deficient tumors. Each bar in Figure 3B–G represents normalized amount of indicated mRNA in different tumor. All green bars correspond to tumors harvested from control animals, while blue bars correspond to tumors harvested from p19Arf-deficient animals. We found significantly higher expression of Bmi1, Nanog and c-myc in p19Arf knockout Ras-driven tumors (Figure 3B–D). Interestingly, as shown in Figure 3E, we observed lower expression of FoxA3 in p19Arf-deficient tumors. FoxA3 is one of the liver differentiation genes, and it has been shown previously that FoxA3 expression is low in murine fast growing HCCs (21). Furthermore, we observed increase in CD90 mRNA in knockout tumors (Figure 3F). In human HCC, it has been demonstrated that CD90-expressing HCC cells are stem-like cancer cells (22). Moreover, we observed higher IL6 expression in knockout liver tumors (Figure 3G), previously shown to be indispensable for malignant progression in the liver (23) and necessary for liver cancer progenitor cells in vivo growth (24). Statistical analyses for all genes are shown in Figure 3H. Additionally, we analyzed expression of ARF trans-activators (Dmp1, E2F1, E2F2 and E2F3) and ARF repressors (Twist, Tbx2/3 and Pokemon) in p19Arf+/+H-ras12V and p19Arf−/− H-ras12V tumors using real time PCR. Surprisingly, we found that Twist mRNA was significantly upregulated, while Tbx3 mRNA was significantly downregulated in the p19Arf null tumors (Supplementary Figure 1E, available at Carcinogenesis Online). Also, Dmp1 mRNA and E2F2 mRNA were significantly upregulated in p19Arf null tumors (Supplementary Figure 1E, available at Carcinogenesis Online). High FoxM1 expression in p19Arf-deficient tumors FoxM1 is transcription factor tightly correlated with proliferation and stemnes genes expression in human and mouse tumors (25–29). Ras-driven HCCs are addicted to FoxM1, and following FoxM1 deletion, they exhibit decreased proliferation rate, increase in reactive oxygen species accumulation and apoptosis, loss of stem-like cancer cells (CD44+ and EpCAM positive cells) and decreased expression of stemness genes (28). Because Arf-deficient HCC cells proliferate faster and have high expression of stemness genes, we investigated FoxM1 expression in p19Arf knockout and control tumors. To assay for FoxM1 in Ras-driven tumors with and without p19Arf expression, we utilized IHC and real time PCR assays. As shown in Figure 4A, we observed high levels of the FoxM1 protein in p19Arf knockout liver tumors. We quantified FoxM1 expression in six tumors harvested from p19Arf+/+H-ras12V mice and five tumors harvested from p19Arf−/− H-ras12V mice. FoxM1 positive tumor cells were counted in at least five fields for each tumor. In the bottom panel of the Figure 4A, the graph shows clear increases in FoxM1 expression in p19Arf null tumor cells. Moreover, quantitative difference in FoxM1 expression was also observed in RT-PCR assays. Total RNA has been isolated from control and knockout liver tumors and assayed for FoxM1 expression (Figure 4B). We observed higher FoxM1 mRNA levels in p19Arf-deficient tumors. As expected, increase in expression of FoxM1 target genes in knockout tumors paralleled increase in expression of FoxM1 in them. For example, we observed higher expression of CD44 (Figure 4C) and Cdc25B (Figure 4D) in p19Arf-deficient tumors, both shown to be transcriptionally regulated by FoxM1 (28,30). Statistical analyses for each gene are shown in Figure 4E. Figure 4. View largeDownload slide p19Arf−/− H-ras12V liver tumors express high levels of FoxM1. FoxM1 IHC staining and quantification, as well as FoxM1 mRNA expression in tumors of indicated genotypes are shown in (A) and (B), respectively. CD44 mRNA and Cdc25B mRNA expression in tumors of indicated genotypes are shown in (C) and (D), respectively. For mRNA quantification, total RNA was isolated from tumors of indicated genotype and analyzed by RT-PCR. Each bar represents mRNA isolated from different tumor. Green bars represent p19Arf-expressing tumors, and blue bars represent p19Arf-deficient tumors. Statistical analyses for all genes are shown in (E). Figure 4. View largeDownload slide p19Arf−/− H-ras12V liver tumors express high levels of FoxM1. FoxM1 IHC staining and quantification, as well as FoxM1 mRNA expression in tumors of indicated genotypes are shown in (A) and (B), respectively. CD44 mRNA and Cdc25B mRNA expression in tumors of indicated genotypes are shown in (C) and (D), respectively. For mRNA quantification, total RNA was isolated from tumors of indicated genotype and analyzed by RT-PCR. Each bar represents mRNA isolated from different tumor. Green bars represent p19Arf-expressing tumors, and blue bars represent p19Arf-deficient tumors. Statistical analyses for all genes are shown in (E). p14ARF expression inversely correlates with the age of human HCC onset and development of HCC in liver cirrhosis To investigate whether p14ARF plays similar role in human HCC development as in Ras-induced mouse model of HCC, we analyzed p14ARF protein expression in human HCC samples using IHC. We obtained TMA of human HCC core samples collected at the University of Illinois Health. All tissue TMA core samples were obtained from liver explants of patients who have undergone liver transplantation procedure. Because ARF localizes in nucleolus (6–9), we quantified only tumor cells positive for nucleolar p14ARF. Out of 86 HCC TMA core samples analyzed, 43.02% (37 out of 86) of the samples were negative for the nucleolar p14ARF protein in tumor cells. About 20.93% of samples had low nucleolar p14ARF (up to 25% positive tumor cells within a tissue core), 23.26% had moderate (25–50% of positive tumor cells) and 12.8% of HCC samples had high nucleolar p14ARF (more than 50% positive tumor cells). We did not observe any significant correlation between p14ARF expression in tumor cells and tumor grades (data not shown). Interestingly, we observed that tumors harvested from younger HCC patients exhibited lower nucleolar p14ARF compared with tumors harvested from the older patients (Figure 5A). We had available age information for 59 patients that we divided into two age groups. Thirty-one patients were considered younger, with age range from 29 to 59 years, average 49.1 years (median 53 years). Twenty-eight patients were considered older, with age range from 60 to 82 years and average of 66.3 years (median 65.5 years). We scored p14ARF staining as percentage of positive HCC cells for nucleolar p14ARF multiplied by intensity of the staining. Figure 5A shows clear difference in expression profile of p14ARF in tumor cells of younger patients compared with tumor cells of older patients. The mean of p14ARF score in younger patients was 7.659 ± 2.212, while the mean of p14ARF score in older patients was 26.48 ± 7.283. Together with the data obtained using p19Arf−/− H-rasV12 mouse model, these data suggest that loss of p14ARF/p19ARF expression accelerates HCC development. Figure 5. View largeDownload slide Low p14Arf expression correlates with early onset of HCC and higher chances of liver cirrhosis progressing to HCC. Human HCC tissues microarray was stained with p14ARF antibody. Total of 86 patient samples were analyzed and scored for nucleolar p14ARF in tumor cells. Staining score was calculated by multiplying percentage of p14ARF positive HCC cells by staining intensity (from 0 to 2, where 0 is no staining, 0.5 is weak, 1 is moderate and 2 is strong staining). Fifty-nine samples had available age information, and we divided them into two age groups; younger patients (31 patients total, ranging from 29 to 59 years of age) and older patients (28 patients total, ranging from 60 to 82 years of age). We scored p14ARF staining as percentage of positive HCC cells multiplied by intensity of the staining. p14ARF expression was significantly lower in patients with early onset of the disease (A). TMA of liver biopsies collected from patients with liver cirrhosis was stained for p14ARF. Eighty-nine samples from patients with cirrhosis that did not develop HCC and 43 samples collected from patients with liver cirrhosis that progressed to HCC were analyzed. p14ARF expression was significantly lower in cirrhotic livers that did develop HCC (B). Figure 5. View largeDownload slide Low p14Arf expression correlates with early onset of HCC and higher chances of liver cirrhosis progressing to HCC. Human HCC tissues microarray was stained with p14ARF antibody. Total of 86 patient samples were analyzed and scored for nucleolar p14ARF in tumor cells. Staining score was calculated by multiplying percentage of p14ARF positive HCC cells by staining intensity (from 0 to 2, where 0 is no staining, 0.5 is weak, 1 is moderate and 2 is strong staining). Fifty-nine samples had available age information, and we divided them into two age groups; younger patients (31 patients total, ranging from 29 to 59 years of age) and older patients (28 patients total, ranging from 60 to 82 years of age). We scored p14ARF staining as percentage of positive HCC cells multiplied by intensity of the staining. p14ARF expression was significantly lower in patients with early onset of the disease (A). TMA of liver biopsies collected from patients with liver cirrhosis was stained for p14ARF. Eighty-nine samples from patients with cirrhosis that did not develop HCC and 43 samples collected from patients with liver cirrhosis that progressed to HCC were analyzed. p14ARF expression was significantly lower in cirrhotic livers that did develop HCC (B). To evaluate whether higher p14ARF levels seen in HCC from older patients may reflect generally increased ARF in aging tissues (due to accumulation of damage or senescence), we analyzed p14ARF expression in non-cirrhotic, non-malignant hepatocytes. We had small sample size of eight cores harvested from four different patients ranging from 29 to 58 years of age. In those samples, we did not observe age-dependent increase in nucleolar p14ARF (Supplementary Figure 2, available at Carcinogenesis Online). Though we observed loss of nucleolar p14ARF in 43.02% of analyzed HCC cores, some samples did harbor high p14ARF expression (18.6% or 16 out of 86 samples had scored higher than 50). Because it has been demonstrated previously that p14ARF expression can be very high in cells with p53 mutation/deletion (31–34), we investigated if there is correlation between p53 and p14ARF in our cohort of patients. We utilized method developed by Hsia et al. and assayed for p53 expression by IHC on TMA (35). Using IHC and parallel gene sequencing, Hsia et al. demonstrated that human HCC samples stained negative for p53 protein retained wild-type p53 gene and that HCC samples exhibiting high p53 protein expression (50–95% of HCC cells stained in the sample) exhibited mutated form of p53 (94% of assay sensitivity) (35). Applying that idea, we stained HCC TMA with p53 antibody and observed that out of 67 HCC patient samples analyzed, only 4 had 50% or more tumor cells positive for p53 (potentially harboring mutations in p53). The average p14ARF score in that group was 86.83 (Supplementary Figure 3, available at Carcinogenesis Online). Out of 67, 37 HCC samples had no p53-expressing tumor cells (potentially wt p53 alleles). The average score for p14ARF expression in that group was 32.03. P-value showed significant statistical significance (P = 0.023). If additional samples with p53-positive tumor cells (scores with 10% or more of p53 positive tumor cells) are included in the analysis, in 12 patient samples, average p14ARF score is 60.22. The trend is still there, but P-value is not statistically significant (P = 0.071). These data suggest that corrupted feedback loop caused by mutation in p53 gene could be responsible for high p14ARF expression in human HCC samples; however, the sample size of the patients with high p53 expression is too small for conclusion. The p53 staining and graphs are shown in Supplementary Figure 3, available at Carcinogenesis Online. Moreover, we obtained TMA of cirrhotic, non-malignant liver tissue core samples collected from explants of patients who have undergone liver transplantation procedure. We analyzed 89 patient samples obtained from liver explants of patients with cirrhosis who did not develop HCC and 43 non-malignant liver tissue core samples collected from explants of patients with liver cirrhosis who developed HCC. Interestingly, we observed significant difference in nucleolar p14ARF between non-malignant liver tissue core samples of patients with and without HCC (Figure 5B). Irrespective of etiology, p14ARF expression was significantly higher in non-malignant liver tissue core samples of patients with cirrhosis who did not develop HCC (mean of 27.56 ± 2.67) than in the non-malignant liver tissue core samples of patients with cirrhosis that progressed to HCC (mean of 17 ± 3.034); once again suggesting that p14ARF protects damaged liver from developing HCC. These findings are significant because they suggest that lack of nucleolar p14ARF in non-malignant hepatocytes can be used as a marker for aggressive chronic liver disease with high likelihood of progressing to liver cancer. Discussion In this study, we demonstrated that tumor suppressor p19/p14ARF protects liver from hepatocarcinogenesis. Using Ras-driven HCC mouse model, we provided genetic evidence that p19Arf deficiency accelerates liver tumor development and progression. Moreover, we observed loss of nucleolar p14ARF in 43.02% of analyzed human HCC samples. Interestingly, in the analyzed HCC patient cohort, we found that lower nucleolar p14ARF in younger patients correlates with HCC development. Furthermore, we observed higher nucleolar p14ARF in non-malignant hepatocytes in patients with cirrhosis that did not progress to HCC compared with cirrhotic liver that did progress to HCC, suggesting that p14ARF protects cirrhotic livers from HCC development. In the small sample size of cores with non-cirrhotic, non-malignant hepatocytes, we did not observe age-dependent increase in nucleolar p14ARF. Because of the limited sample number, we could not rule out the possibility of age-dependent increase in p14ARF expression. Importantly, however, the TMA set with cirrhotic samples that did not progress to HCC and expressed p14ARF at higher levels were collected from the younger patients (average age 50.018 years, median age 49 years) compared with the cirrhotic samples that did progress to HCC and low p14ARF (average patient age 57.27 years, median patient age 58 years). These findings are significant in several ways. First, although p14/p19ARF role as tumor suppressor is well established, its role in hepatocarcinogenesis is controversial. Our study using Ras-driven mouse model of HCC clearly demonstrates protective role of p19Arf. Moreover, we provided evidence that p19Arf deletion not only accelerates HCC development but also increases heterogeneity of the HCCs. Liver tumors formed in p19Arf-deficient background are more aggressive, they proliferate faster and express the stemnes signature genes, such as Nanog, Bmi1, c-myc, IL6, CD44 and CD90, at high levels. Human patients with HCCs with high expression of these genes have poorer prognosis and shorter survival, the phenotype observed in Arf null H-ras12V male mice as well. It is noteworthy that many of the analyzed genes (Figure 3 and 4) exhibited high intertumor variation in expression, especially Nanog, IL6 and FoxM1. We failed to correlate those variations to age of sacrifice, size of tumors or tumor growth pattern. It is important to note that p19Arf knockout mice do have shorter survival compared with wild-type littermates (19), due to spontaneous tumor development. It has been demonstrated that p19Arf-deficient mice develop mainly sarcomas and lymphomas (19) and have median survival of 38 weeks. No HCC development has been reported in this strain. However, animals used in this study, p19Arf−/− H-ras12V male mice, have median survival of 30 weeks of age, shorter than p19Arf−/− mice. They all had liver cancers at the time of the death and no other visible tumors, suggesting that the cause of their death is aggressive HCC. In contrast, all control, p19Arf+/+ H-ras12V male mice were alive at 10 months of age when the experiment has been stopped. It has been shown that senescence plays an important role in limiting fibrosis and hepatocarcinogenesis in mice (36,37). ARF is a well-known senescence factor, and it has been demonstrated that p19Arf-dependent Ras-induced senescence of premalignant hepatocytes plays crucial role in inducing tumor surveillance program as a part of tumor suppressive mechanism (38). It is possible that lack of senescence in p19Arf−/−H-ras12V premalignant hepatocytes causes earlier tumor development in that strain. Interestingly, we observed high FoxM1 levels in p19Arf null HCCs. FoxM1 is well-known pro-proliferation transcription factor (29,30), as well as an activator of stemness gene expression (25,26,28). It is highly possible that high FoxM1 levels drive aggressive phenotype of the p19Arf null HCCs. Besides being best known for sequestering Mdm2 in the nucleolus and activating p53 pathway (6), p14/p19ARF also sequesters FoxM1 in the nucleolus, rendering it transcriptionally inactive (9); hence, it is easy to understand having more active FoxM1 transcriptional network in the p19Arf null HCC cells. Interestingly, in knockout tumors, not only that FoxM1 was more transcriptionally active, there was more FoxM1 expressed both on mRNA and protein levels. That could be explained by FoxM1 auto-activation of its own expression; it has been demonstrated that FoxM1 can activate its own transcription by positive feedback loop (39). Importantly, our mouse HCC study can be related to human HCC pathogenesis. With our mouse model, we demonstrated that p19Arf deficiency leads to early onset of HCC in vivo, and we observed that human HCC with early onset express low p14ARF. As noted above, role of ARF in liver cancer progression has been controversial. Opposing reports have been published, and they mainly focused on promoter methylation status or p14Arf mRNA levels in liver tumors (11–14). Using IHC, we examined nucleolar p14ARF protein expression in human HCC cells. We found lack of nucleolar p14ARF in 43.02% of examined HCC samples. In that regard, it is noteworthy that Bmi1 and Pokemon, regulators of p14ARF expression, were shown to be upregulated in human HCCs (40–42). Moreover, Mdm2, a target of p14ARF, was shown to be overexpressed in 52.5% of human HCCs (43). Interestingly, we found that nucleolar p14ARF was lower in tumors harvested from younger HCC patients and that cirrhotic livers that did not progress to HCC had higher nucleolar p14ARF compared with cirrhotic livers that did progress to HCC. Cirrhosis of the liver is late stage of chronic liver disease, and most of the HCCs (around 80%) develop in the context of liver cirrhosis. Our finding that hepatocytes in the cirrhotic livers without HCC development exhibit higher nucleolar p14ARF protein compared with hepatocytes in cirrhotic livers that did develop HCC is very significant because it suggests that p14ARF expression can protect chronically injured liver from developing HCC. We propose that nucleolar p14ARF can be used as a marker for progressive liver disease that harbors higher chances for developing to terminal-phase HCC. 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PLoS One , 7, e51916. Google Scholar CrossRef Search ADS PubMed  43. Zhang, M.F.et al.   ( 2009) Correlation between expression of p53, p21/WAF1, and MDM2 proteins and their prognostic significance in primary hepatocellular carcinoma. J. Transl. Med ., 7, 110. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Carcinogenesis Oxford University Press

p19Arf inhibits aggressive progression of H-ras-driven hepatocellular carcinoma

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Abstract

Abstract Arf, a well-established tumor suppressor, is either mutated or downregulated in a wide array of cancers. However, its role in hepatocellular carcinoma (HCC) progression is controversial. Conflicting observations have been published regarding its expression in HCC. In this study, we provide clear genetic evidence demonstrating a protective role of p19Arf in hepatocarcinogenesis. Using Ras-induced mouse model, we show that p19Arf deficiency accelerates progression of aggressive HCC in vivo. To investigate the role of p14ARF in human liver cancers, we analyzed its expression in human HCC using immunohistochemistry (IHC). We observe lack of nucleolar p14ARF in 43.02% of human HCC samples and that low expression of p14ARF strongly correlates with the early onset of HCC. Importantly, cirrhotic livers that did not progress to HCC harbor higher expression of the p14ARF protein in hepatocytes compared with that in cirrhotic livers with HCC. These results are significant because they suggest that nucleolar p14ARF can be used as early prognostic marker in chronic liver disease to reliably identify patients with high risk for developing liver cancer. Currently, there is no effective systemic therapy for advanced liver cancer; hence, more efficient patient screening and early detection of HCC would significantly contribute to the eradication of this devastating disease. Introduction Liver cancer is the third leading cause of cancer related death worldwide, accounting for more than 600 000 deaths each year (1). The most common type of liver cancer in adult patients is hepatocellular carcinoma (HCC). Unfortunately, HCC is diagnosed often at a late stage when surgery is no longer an option; hence, patients survive less than 1 year after initial diagnosis, even with intervention. Therefore, there is an urgent need for better understanding of this disease. More than 80% of HCC develops as the last stage of chronic liver disease caused by hepatitis C virus (HCV) or hepatitis B virus (HBV) infection, alcohol abuse, dietary exposure to aflatoxin, obesity or metabolic syndrome (2). They all cause damage and necrosis of hepatocytes, followed by inflammation, proliferation and subsequent liver regeneration (3). If insults are persistent, cycles of damage and repair continue, and disease progresses to chronic hepatitis followed by liver cirrhosis, with severely compromised liver function. In liver cirrhosis, regenerative capacity of hepatocytes is exhausted, and functional tissue has been replaced with fibrous scar, making environment suitable for the HCC nodules to develop (4). Because most of the HCCs develop as the terminal stage of chronic liver disease, more effective screening of the population under high risk would result in early detection and subsequent treatment of early stage cancers. Identification of specific markers that could reliably identify patients with high risk would significantly contribute to the eradication of this disease. The roles of the tumor suppressor protein ARF (alternative reading frame; p14ARF in humans and p19Arf in mice) in suppression of HCC have remained unclear. ARF protein expression is induced in response to oncogenic stimuli in many tumors (5). Once expressed, ARF protein prevents abnormal cellular proliferation by sequestering Mdm2 to nucleolus and stabilizing p53 tumor suppressor protein (6). ARF can also induce p53-independent cell cycle arrest by binding to c-myc, E2F1 and FoxM1 (7–9), translocating them to nucleolus, therefore, preventing their transcriptional activation of cell cycle-promoting target genes. The cell cycle-regulatory function of ARF has also been linked to cellular senescence (10). That is consistent with the observations that in a variety of tumors, expression of ARF is quenched through DNA methylation and silencing, suggesting that suppression of ARF expression is critical for tumorigenesis. However, the role of ARF in HCC progression has been controversial, as opposite results have been reported regarding its expression in human HCC (11–14). Anzola et al. (12) reported inactivation of p14Arf gene in 62.4% of the tumor samples and 26.4% of the corresponding non-tumor samples harvested from HCC patients. Their work demonstrates that p14ARF gene in human HCC can be inactivated by different mechanisms including promoter hypermethylation, homozygous deletions, loss of heterozygosity and less frequently point mutations, with hypermethylation being the main cause of inactivation. Importantly, these results suggest that p14ARF gene silencing plays an important role in the pathogenesis of HCC and that could be one of the early events in HCC development. On the other hand, Tannapfel et al. (13) demonstrated p14Arf promotor methylation in only 9% of patient samples. Moreover, Teruaki et al. (14) failed to detect any hypermethylation of the p14ARF promoter in Japanese HCC samples. In that study, only 1 out of 44 HCC samples exhibited homozygous deletion, and 2 out of 44 HCC samples harbored mutation. Surprisingly, they observed increases in p14Arf mRNA expression in the remaining 41 HCC samples, compared with paired non-tumor tissue. Furthermore, although the association was not statistically significant, the expression of p14ARF mRNA was correlated with poorly differentiated tumor phenotype. One of the most commonly perturbed signaling pathways in HCC is the Ras signaling pathway (15–17). For example, recent studies discovered epigenetic silencing of negative regulators of Ras signaling pathway, GAPs (GTPase-activating proteins) in human HCC samples. Calvisi et al. (16) found silencing of RASAL1, DAB2IP and NF1 in over 70% of analyzed HCC. On the other hand, study with 69 Chinese patients demonstrated mutations in H-Ras codons 40 and 61, in about 71% of the patients (17). Hence, although Ras mutations are less common in HCC, Ras-pathway is ubiquitously activated in human HCC due to the silencing of its negative regulators. In this study, using a Ras-transgenic mouse model, we provide genetic evidence that p19Arf plays a strong suppressive role on HCC development and progression. For the first time, we analyzed p14ARF protein expression in human HCC samples using immunohistochemistry (IHC). We observed lack of nucleolar p14ARF in HCC cells in 43.02% of analyzed human HCC samples. We provide evidence that loss of p14ARF protein expression in cirrhotic liver can be used as prognostic marker for HCC development. Materials and methods Animal studies The University of Illinois at Chicago institutional animal care and use committee preapproved all animal experiments. Previously described H-rasV12 mice (18) were crossed with earlier described p19Arf−/− C57/BL6 animals (19) to obtain p19Arf+/+ H-rasV12 and p19Arf−/− H-rasV12 mice. The male mice were, aged ≤ 10 months, humanely sacrificed and analyzed for tumor development. If the mice reached humane end point earlier, they were humanely sacrificed and analyzed for tumor development. Tissue microarray We obtained tissue microarray (TMA) of human HCC core samples collected at the University of Illinois Health Sciences Center following the guidelines of University of Illinois and NIH policy on human sample studies. All tissue TMA core samples were obtained from liver explants of patients who have undergone liver transplantation procedure, from 1999 to 2007. For most of the patients, three cores were included in the TMA. Pathologist diagnosed each core, and only the ones confirmed to be HCC were studied. We analyzed 86 HCC patient samples for p14ARF expression. HCC cores included tumors grade 1 to grade 3. We had available age information for 59 patients, ranging from 29 to 82 years of age. Average age of patients from the HCC set is 57.27 years (median 58 years). On the same TMA, there were 43 cores with cirrhotic hepatocytes harvested from the livers that developed HCC. They were also analyzed for p14ARF expression. Furthermore, we obtained TMA of human cirrhotic core samples collected at the University of Illinois Health, from 2000 to 2007, from liver explants of patients who have undergone liver transplantation procedure but did not develop HCC. Most of the patients were presented with two cores. Average age of patients in the cirrhosis set is 50.018 years (median 49 years), ranging from 25 to 75 years of age. Immunohistochemistry For IHC, tissues were fixed overnight in 10% neutral buffered formalin, rinsed in phosphate-buffered saline and left in 75% ethanol until processing. Afterwards, tissues were passed through grades of alcohols and embedded in paraffin. Sections(5 μm thick) were prepared, baked overnight at 55°C and stained following standard procedures. Briefly, antigen retrieval was performed using sodium citrate buffer, and primary antibodies were incubated overnight at 4°C. Visualization was done using 3,3’-diaminobenzidine and counterstained using Hematoxylin (Polyscientific). All used reagents are from Vector Labs unless otherwise indicated. Staining was performed using rabbit monoclonal Abcam Ki67 antibody, Santa Cruz mouse monoclonal FoxM1 antibody and Abcam p19Arf ab80 antibody. TMA was stained with Novus Biological p14ARF antibody, NB200-335, at dilution of 1:100 and with p53 mouse monoclonal M7001 from DAKO. Expression studies Total RNA was Trizol (Invitrogen) extracted from harvested tumors, and cDNA was synthesized using Bio-Rad reverse transcriptase. cDNA was amplified using SYBR Green (Bio-Rad) and analyzed via iCycler software. Used primer sequences are following: FoxM1 F 5′- GAGGAAAGAGCACCTTCAGC-3′, R 5′- AGGCAATGTCTCCTTGATGG-3′, GAPDH F 5′-AACTTTGGCATTGTGG AAGG-3′, R 5′- CCATCCACAGTCTTCTGGGT-3′, Bmi1 F 5′- AGAGGGATGGACTAC GAATGC-3′, R 5′- AACAGGAAGAGGTGGAGGGAAC-3′, Nanog F 5′- AGCCCTG ATTCTTCTACCAGTCCC-3′, R 5′- ACAGTCCGCATCTTCTGCTTCC-3′, c-myc F 5′- TAACTCGAGGAGGAGCTGGA-3′, R 5′- GCCAAGGTTGTGAGGTTAGG-3′, Cdc25B F 5′- CCCTTCCCTGTTTTCCTTTC-3′, R 5′- ACACACACTCCTGCCAT AGG-3′, FoxA3 F 5′-TGAATCCTGTGCCCACCAT-3′, R 5′-AGCTGAGTGGGTT CAAGGTCAT-3′, CD44 F 5′- TGCATTTGGTGAACAAGGAA-3′, R 5′- GGAATG ACGTCTCCAATCGT-3′, CD90 F 5′- GCCTGACCCGAGAGAAGAAGAAG-3′, R 5′-TGGTGGTGAAGTTCGCTAGAGTAAG-3′, IL6 F 5′- TGATGCACTTGCAGAAA ACA-3′, R 5′- ACCAGAGGAAATTTTCAATAGGC-3′. Statistical analysis Statistical significance was calculated using Student’s t-test (two-tailed). Statistically significant changes were indicated with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Results Deletion of p19Arf accelerates HCC development in vivo To study role of Arf in HCC progression in vivo, we used oncogenic Ras-driven mouse model of HCC. In our model system, oncogenic form of H-Ras (H-ras12V) is expressed under control of albumin promoter, only in the liver epithelial cells. Male mice of this strain develop liver tumors at age of 8–9 months with penetrance of almost 100% (18). It is an excellent, clinically relevant model system to study liver cancer in vivo. To investigate role of p19Arf in HCC development, we crossed H-ras12V mice with p19Arf knockout strain and obtained p19Arf−/−H-ras12V males. As a control group, we generated p19Arf+/+ H-ras12V male mice. As expected, all control males developed liver tumors by age of 10 months, when the livers were harvested (Figure 1A). In sharp contrast, none of the p19Arf-deficient male mice survived 10 months of age. Figure 1. View largeDownload slide Loss of p19Arf accelerates progression of H-ras12V driven HCCs. Ten months old p19Arf+/+ H-ras12V male mice and moribund p19Arf−/− H-ras12V (from 4- to 10-month old) male mice were sacrificed, livers were harvested and examined for tumors (A). Survival of male p19Arf+/+ Hras12V and p19Arf−/− H-ras12V mice is shown in (B). The difference in survival is statistically significant with the P-value of 0.01076. Graph in (C) shows drastic increase in the liver weight (as percentage of total body weight) of p19Arf−/− H-ras12V male mice. (D) percentage of tumor nodules of indicated sizes. IHC staining for p19Arf protein on liver non-tumor and liver tumor sections harvested from 10-month-old male p19Arf+/+H-ras12V mice is shown in (E) (top). Quantification of p19Arf positive cells is presented in the bottom panel E. Figure 1. View largeDownload slide Loss of p19Arf accelerates progression of H-ras12V driven HCCs. Ten months old p19Arf+/+ H-ras12V male mice and moribund p19Arf−/− H-ras12V (from 4- to 10-month old) male mice were sacrificed, livers were harvested and examined for tumors (A). Survival of male p19Arf+/+ Hras12V and p19Arf−/− H-ras12V mice is shown in (B). The difference in survival is statistically significant with the P-value of 0.01076. Graph in (C) shows drastic increase in the liver weight (as percentage of total body weight) of p19Arf−/− H-ras12V male mice. (D) percentage of tumor nodules of indicated sizes. IHC staining for p19Arf protein on liver non-tumor and liver tumor sections harvested from 10-month-old male p19Arf+/+H-ras12V mice is shown in (E) (top). Quantification of p19Arf positive cells is presented in the bottom panel E. We followed 24 male p19Arf+/+ H-ras12V mice and 15 male p19Arf−/− H-ras12V mice for survival. There was a clear decrease in survival of p19Arf-deficient, H-ras12V-expressing mice (Figure 1B). The shortest surviving knockout male mouse reached humane end point as early as 5 months of age, while the oldest p19Arf-deficient male mice reached the end point by 9 months of age. The control mice (p19Arf+/+H-ras12V) survived through 10 months of age when experiment was terminated (Figure 1B), even though they all developed liver tumor nodules. Of note, we aged four p19Arf+/+H-ras12V male mice to 14 months of age, and although they all had large liver tumor nodules, they were not moribund (data not shown). We harvested livers with tumors from control mice at 10 months of age and livers with tumors from p19Arf-deficient mice when they were moribund. The representative harvested livers with tumors are shown in Figure 1A. To quantify tumor burden, we calculated liver weight as percentage of total body weight (Figure 1C). Tumor burden was significantly higher in Arf-deficient male mice compared with the control mice (Figure 1C). Furthermore, we measured diameter of harvested tumors and found that liver tumors harvested from the Arf-deficient mice were larger than tumors harvested from control mice, with around 20% of liver tumors reaching more than 2 cm in diameter in knockout mice (Figure 1D). We did not observe any macrometastasis. Furthermore, we reasoned that if p19Arf is important for Ras-driven HCC development, its expression should diminish in p19Arf+/+ Hras12V tumors. To explore that, we examined p19Arf expression using IHC in non-tumor and tumor tissue harvested from 10-month-old p19Arf+/+H-ras12V male mice. We detected significant decrease in p19Arf protein in tumor cells compared with non-malignant hepatocytes (Figure 1E), further indicating the importance of p19Arf in suppression of Ras-driven HCC development. These results provide clear genetic evidence that p19Arf protects liver from Ras-driven hepatocarcinogenesis in vivo. p19Arf−/−H-ras12V liver tumors are histologically heterogeneous Tumors harvested from p19Arf+/+H-ras12V and p19Arf−/−H-ras12V mice were further analyzed by IHC. Histological analysis of the p14Arf-expressing tumors showed uniform growth pattern with predominantly fatty changes (95.24%, or 20 out of 21 analyzed tumors displayed fatty changes), some solid growth (6/21) and cytoplasmic inclusions (11/21). The tumors developed in p19Arf-deficient mice, on the other hand, were histologically heterogeneous (Figure 2). Most of the analyzed p19Arf-deficient tumors displayed solid growth pattern (9 out of 11 or 81.82%) or trabecular growth pattern (9 out of 11 or 81.82%). Moreover, we found fatty changes in 6 out of 11 analyzed tumors (54.5%) and cytoplasmic inclusions in 3 out of 11 (27.27%). Additionally, we found 2 out of 11 Arf-deficient tumors to have pseudoglandular growth pattern and one with focal sclerosis. No pseudoglandular growth pattern or focal sclerosis was observed in the Arf-expressing tumors. While 57.14% (12 out of 21) of tumors harvested from p19Arf-expressing animals had fibrotic changes, only 14.28% or 3 out of 11 tumors harvested from p19Arf-deficient animals exhibited fibrotic changes. Necrosis was prominent in 81.82% of analyzed p19Arf knockout tumors (9 out of 11 tumors), while only 4.76% (1 out of 21) of p19Arf+/+ tumors contained necrotic portions. Interestingly, we observed significant immune infiltrates in 72.73% (or 8 out of 11) of analyzed p19Arf-deficient tumors, while only 38.09% (8 out of 21) of the p19Arf-expressing tumors contained immune infiltrates. Representative low magnification images of p19Arf-deficient H-ras-driven HCCs are shown in Supplementary Figure 1A-D, available at Carcinogenesis Online, while tables in Figure 2A and B show observed growth pattern changes in analyzed control tumors (Figure 2A) and p19Arf-deficient tumors (Figure 2B). Every column represents different tumor, while black field describes presence of designated pattern in indicated tumor sample. Figure 2. View largeDownload slide Growth pattern of p19Arf+/+ H-ras12V and p19Arf−/−H-ras12V liver tumors. Liver tumors were harvested from male mice, sectioned and stained with H&E. Table in (A) summarizes histology of tumors harvested from p19Arf+/+ H-ras12V mice. Histology of p19Arf−/− H-ras12V tumors is shown in (B). Black boxes in the tables show presence of indicated growth pattern, while the grey boxes represent presence of indicated growth pattern in low levels in particular tumor. White boxes signify absence of the indicated growth pattern. Figure 2. View largeDownload slide Growth pattern of p19Arf+/+ H-ras12V and p19Arf−/−H-ras12V liver tumors. Liver tumors were harvested from male mice, sectioned and stained with H&E. Table in (A) summarizes histology of tumors harvested from p19Arf+/+ H-ras12V mice. Histology of p19Arf−/− H-ras12V tumors is shown in (B). Black boxes in the tables show presence of indicated growth pattern, while the grey boxes represent presence of indicated growth pattern in low levels in particular tumor. White boxes signify absence of the indicated growth pattern. p19Arf-deficient tumors are highly proliferative and they express stemness signature genes To evaluate proliferation capacity of the tumors, we used Ki67-specific immunohistochemical staining (Figure 3A). We quantified the HCC cells for Ki67 expression. There was significant increase in percentage of Ki67 positive HCC cells in p19Arf-deficient tumors, indicating an increase in proliferation. Quantification of the Ki67 staining is shown in Figure 3A, bottom panel. Figure 3. View largeDownload slide p19Arf-deficient tumors are highly proliferative and highly express stemness signature genes. To analyze proliferation of tumor cells, HCC sections of both genotypes were stained for Ki67. Representative images of Ki67 IHC staining and quantification of Ki67 positive tumor cells are shown in (A). We quantified Ki67 positive tumor cells in 30 fields from three different tumors harvested from p19Arf+/+ H-ras12V mice and in 25 fields from five different tumors harvested from p19Arf−/− H-ras12V mice. Total RNA was isolated from harvested tumors and analyzed for Bmi1, Nanog, c-myc, FoxA3, CD90 and IL6 expression by RT-PCR. The results are shown in (B), (C), (D), (E), (F) and (G), respectively. Each bar represents mRNA isolated from different tumor. Green bars represent p19Arf-expressing tumors, and blue bars represent p19Arf-deficient tumors. Statistical analyses for all genes are shown in (H). Figure 3. View largeDownload slide p19Arf-deficient tumors are highly proliferative and highly express stemness signature genes. To analyze proliferation of tumor cells, HCC sections of both genotypes were stained for Ki67. Representative images of Ki67 IHC staining and quantification of Ki67 positive tumor cells are shown in (A). We quantified Ki67 positive tumor cells in 30 fields from three different tumors harvested from p19Arf+/+ H-ras12V mice and in 25 fields from five different tumors harvested from p19Arf−/− H-ras12V mice. Total RNA was isolated from harvested tumors and analyzed for Bmi1, Nanog, c-myc, FoxA3, CD90 and IL6 expression by RT-PCR. The results are shown in (B), (C), (D), (E), (F) and (G), respectively. Each bar represents mRNA isolated from different tumor. Green bars represent p19Arf-expressing tumors, and blue bars represent p19Arf-deficient tumors. Statistical analyses for all genes are shown in (H). Liver tumors with high expression of stemness genes are considered more aggressive and related to poor prognosis in human HCC patients (20). Because tumor harboring p19Arf knockout male mice had shorter survival and more aggressive tumors compared with control, we analyzed expression of stemness genes in tumors of both genotypes using quantitative real time PCR (Figure 3). Interestingly, we observed higher expression of stemness genes in p19Arf-deficient tumors. Each bar in Figure 3B–G represents normalized amount of indicated mRNA in different tumor. All green bars correspond to tumors harvested from control animals, while blue bars correspond to tumors harvested from p19Arf-deficient animals. We found significantly higher expression of Bmi1, Nanog and c-myc in p19Arf knockout Ras-driven tumors (Figure 3B–D). Interestingly, as shown in Figure 3E, we observed lower expression of FoxA3 in p19Arf-deficient tumors. FoxA3 is one of the liver differentiation genes, and it has been shown previously that FoxA3 expression is low in murine fast growing HCCs (21). Furthermore, we observed increase in CD90 mRNA in knockout tumors (Figure 3F). In human HCC, it has been demonstrated that CD90-expressing HCC cells are stem-like cancer cells (22). Moreover, we observed higher IL6 expression in knockout liver tumors (Figure 3G), previously shown to be indispensable for malignant progression in the liver (23) and necessary for liver cancer progenitor cells in vivo growth (24). Statistical analyses for all genes are shown in Figure 3H. Additionally, we analyzed expression of ARF trans-activators (Dmp1, E2F1, E2F2 and E2F3) and ARF repressors (Twist, Tbx2/3 and Pokemon) in p19Arf+/+H-ras12V and p19Arf−/− H-ras12V tumors using real time PCR. Surprisingly, we found that Twist mRNA was significantly upregulated, while Tbx3 mRNA was significantly downregulated in the p19Arf null tumors (Supplementary Figure 1E, available at Carcinogenesis Online). Also, Dmp1 mRNA and E2F2 mRNA were significantly upregulated in p19Arf null tumors (Supplementary Figure 1E, available at Carcinogenesis Online). High FoxM1 expression in p19Arf-deficient tumors FoxM1 is transcription factor tightly correlated with proliferation and stemnes genes expression in human and mouse tumors (25–29). Ras-driven HCCs are addicted to FoxM1, and following FoxM1 deletion, they exhibit decreased proliferation rate, increase in reactive oxygen species accumulation and apoptosis, loss of stem-like cancer cells (CD44+ and EpCAM positive cells) and decreased expression of stemness genes (28). Because Arf-deficient HCC cells proliferate faster and have high expression of stemness genes, we investigated FoxM1 expression in p19Arf knockout and control tumors. To assay for FoxM1 in Ras-driven tumors with and without p19Arf expression, we utilized IHC and real time PCR assays. As shown in Figure 4A, we observed high levels of the FoxM1 protein in p19Arf knockout liver tumors. We quantified FoxM1 expression in six tumors harvested from p19Arf+/+H-ras12V mice and five tumors harvested from p19Arf−/− H-ras12V mice. FoxM1 positive tumor cells were counted in at least five fields for each tumor. In the bottom panel of the Figure 4A, the graph shows clear increases in FoxM1 expression in p19Arf null tumor cells. Moreover, quantitative difference in FoxM1 expression was also observed in RT-PCR assays. Total RNA has been isolated from control and knockout liver tumors and assayed for FoxM1 expression (Figure 4B). We observed higher FoxM1 mRNA levels in p19Arf-deficient tumors. As expected, increase in expression of FoxM1 target genes in knockout tumors paralleled increase in expression of FoxM1 in them. For example, we observed higher expression of CD44 (Figure 4C) and Cdc25B (Figure 4D) in p19Arf-deficient tumors, both shown to be transcriptionally regulated by FoxM1 (28,30). Statistical analyses for each gene are shown in Figure 4E. Figure 4. View largeDownload slide p19Arf−/− H-ras12V liver tumors express high levels of FoxM1. FoxM1 IHC staining and quantification, as well as FoxM1 mRNA expression in tumors of indicated genotypes are shown in (A) and (B), respectively. CD44 mRNA and Cdc25B mRNA expression in tumors of indicated genotypes are shown in (C) and (D), respectively. For mRNA quantification, total RNA was isolated from tumors of indicated genotype and analyzed by RT-PCR. Each bar represents mRNA isolated from different tumor. Green bars represent p19Arf-expressing tumors, and blue bars represent p19Arf-deficient tumors. Statistical analyses for all genes are shown in (E). Figure 4. View largeDownload slide p19Arf−/− H-ras12V liver tumors express high levels of FoxM1. FoxM1 IHC staining and quantification, as well as FoxM1 mRNA expression in tumors of indicated genotypes are shown in (A) and (B), respectively. CD44 mRNA and Cdc25B mRNA expression in tumors of indicated genotypes are shown in (C) and (D), respectively. For mRNA quantification, total RNA was isolated from tumors of indicated genotype and analyzed by RT-PCR. Each bar represents mRNA isolated from different tumor. Green bars represent p19Arf-expressing tumors, and blue bars represent p19Arf-deficient tumors. Statistical analyses for all genes are shown in (E). p14ARF expression inversely correlates with the age of human HCC onset and development of HCC in liver cirrhosis To investigate whether p14ARF plays similar role in human HCC development as in Ras-induced mouse model of HCC, we analyzed p14ARF protein expression in human HCC samples using IHC. We obtained TMA of human HCC core samples collected at the University of Illinois Health. All tissue TMA core samples were obtained from liver explants of patients who have undergone liver transplantation procedure. Because ARF localizes in nucleolus (6–9), we quantified only tumor cells positive for nucleolar p14ARF. Out of 86 HCC TMA core samples analyzed, 43.02% (37 out of 86) of the samples were negative for the nucleolar p14ARF protein in tumor cells. About 20.93% of samples had low nucleolar p14ARF (up to 25% positive tumor cells within a tissue core), 23.26% had moderate (25–50% of positive tumor cells) and 12.8% of HCC samples had high nucleolar p14ARF (more than 50% positive tumor cells). We did not observe any significant correlation between p14ARF expression in tumor cells and tumor grades (data not shown). Interestingly, we observed that tumors harvested from younger HCC patients exhibited lower nucleolar p14ARF compared with tumors harvested from the older patients (Figure 5A). We had available age information for 59 patients that we divided into two age groups. Thirty-one patients were considered younger, with age range from 29 to 59 years, average 49.1 years (median 53 years). Twenty-eight patients were considered older, with age range from 60 to 82 years and average of 66.3 years (median 65.5 years). We scored p14ARF staining as percentage of positive HCC cells for nucleolar p14ARF multiplied by intensity of the staining. Figure 5A shows clear difference in expression profile of p14ARF in tumor cells of younger patients compared with tumor cells of older patients. The mean of p14ARF score in younger patients was 7.659 ± 2.212, while the mean of p14ARF score in older patients was 26.48 ± 7.283. Together with the data obtained using p19Arf−/− H-rasV12 mouse model, these data suggest that loss of p14ARF/p19ARF expression accelerates HCC development. Figure 5. View largeDownload slide Low p14Arf expression correlates with early onset of HCC and higher chances of liver cirrhosis progressing to HCC. Human HCC tissues microarray was stained with p14ARF antibody. Total of 86 patient samples were analyzed and scored for nucleolar p14ARF in tumor cells. Staining score was calculated by multiplying percentage of p14ARF positive HCC cells by staining intensity (from 0 to 2, where 0 is no staining, 0.5 is weak, 1 is moderate and 2 is strong staining). Fifty-nine samples had available age information, and we divided them into two age groups; younger patients (31 patients total, ranging from 29 to 59 years of age) and older patients (28 patients total, ranging from 60 to 82 years of age). We scored p14ARF staining as percentage of positive HCC cells multiplied by intensity of the staining. p14ARF expression was significantly lower in patients with early onset of the disease (A). TMA of liver biopsies collected from patients with liver cirrhosis was stained for p14ARF. Eighty-nine samples from patients with cirrhosis that did not develop HCC and 43 samples collected from patients with liver cirrhosis that progressed to HCC were analyzed. p14ARF expression was significantly lower in cirrhotic livers that did develop HCC (B). Figure 5. View largeDownload slide Low p14Arf expression correlates with early onset of HCC and higher chances of liver cirrhosis progressing to HCC. Human HCC tissues microarray was stained with p14ARF antibody. Total of 86 patient samples were analyzed and scored for nucleolar p14ARF in tumor cells. Staining score was calculated by multiplying percentage of p14ARF positive HCC cells by staining intensity (from 0 to 2, where 0 is no staining, 0.5 is weak, 1 is moderate and 2 is strong staining). Fifty-nine samples had available age information, and we divided them into two age groups; younger patients (31 patients total, ranging from 29 to 59 years of age) and older patients (28 patients total, ranging from 60 to 82 years of age). We scored p14ARF staining as percentage of positive HCC cells multiplied by intensity of the staining. p14ARF expression was significantly lower in patients with early onset of the disease (A). TMA of liver biopsies collected from patients with liver cirrhosis was stained for p14ARF. Eighty-nine samples from patients with cirrhosis that did not develop HCC and 43 samples collected from patients with liver cirrhosis that progressed to HCC were analyzed. p14ARF expression was significantly lower in cirrhotic livers that did develop HCC (B). To evaluate whether higher p14ARF levels seen in HCC from older patients may reflect generally increased ARF in aging tissues (due to accumulation of damage or senescence), we analyzed p14ARF expression in non-cirrhotic, non-malignant hepatocytes. We had small sample size of eight cores harvested from four different patients ranging from 29 to 58 years of age. In those samples, we did not observe age-dependent increase in nucleolar p14ARF (Supplementary Figure 2, available at Carcinogenesis Online). Though we observed loss of nucleolar p14ARF in 43.02% of analyzed HCC cores, some samples did harbor high p14ARF expression (18.6% or 16 out of 86 samples had scored higher than 50). Because it has been demonstrated previously that p14ARF expression can be very high in cells with p53 mutation/deletion (31–34), we investigated if there is correlation between p53 and p14ARF in our cohort of patients. We utilized method developed by Hsia et al. and assayed for p53 expression by IHC on TMA (35). Using IHC and parallel gene sequencing, Hsia et al. demonstrated that human HCC samples stained negative for p53 protein retained wild-type p53 gene and that HCC samples exhibiting high p53 protein expression (50–95% of HCC cells stained in the sample) exhibited mutated form of p53 (94% of assay sensitivity) (35). Applying that idea, we stained HCC TMA with p53 antibody and observed that out of 67 HCC patient samples analyzed, only 4 had 50% or more tumor cells positive for p53 (potentially harboring mutations in p53). The average p14ARF score in that group was 86.83 (Supplementary Figure 3, available at Carcinogenesis Online). Out of 67, 37 HCC samples had no p53-expressing tumor cells (potentially wt p53 alleles). The average score for p14ARF expression in that group was 32.03. P-value showed significant statistical significance (P = 0.023). If additional samples with p53-positive tumor cells (scores with 10% or more of p53 positive tumor cells) are included in the analysis, in 12 patient samples, average p14ARF score is 60.22. The trend is still there, but P-value is not statistically significant (P = 0.071). These data suggest that corrupted feedback loop caused by mutation in p53 gene could be responsible for high p14ARF expression in human HCC samples; however, the sample size of the patients with high p53 expression is too small for conclusion. The p53 staining and graphs are shown in Supplementary Figure 3, available at Carcinogenesis Online. Moreover, we obtained TMA of cirrhotic, non-malignant liver tissue core samples collected from explants of patients who have undergone liver transplantation procedure. We analyzed 89 patient samples obtained from liver explants of patients with cirrhosis who did not develop HCC and 43 non-malignant liver tissue core samples collected from explants of patients with liver cirrhosis who developed HCC. Interestingly, we observed significant difference in nucleolar p14ARF between non-malignant liver tissue core samples of patients with and without HCC (Figure 5B). Irrespective of etiology, p14ARF expression was significantly higher in non-malignant liver tissue core samples of patients with cirrhosis who did not develop HCC (mean of 27.56 ± 2.67) than in the non-malignant liver tissue core samples of patients with cirrhosis that progressed to HCC (mean of 17 ± 3.034); once again suggesting that p14ARF protects damaged liver from developing HCC. These findings are significant because they suggest that lack of nucleolar p14ARF in non-malignant hepatocytes can be used as a marker for aggressive chronic liver disease with high likelihood of progressing to liver cancer. Discussion In this study, we demonstrated that tumor suppressor p19/p14ARF protects liver from hepatocarcinogenesis. Using Ras-driven HCC mouse model, we provided genetic evidence that p19Arf deficiency accelerates liver tumor development and progression. Moreover, we observed loss of nucleolar p14ARF in 43.02% of analyzed human HCC samples. Interestingly, in the analyzed HCC patient cohort, we found that lower nucleolar p14ARF in younger patients correlates with HCC development. Furthermore, we observed higher nucleolar p14ARF in non-malignant hepatocytes in patients with cirrhosis that did not progress to HCC compared with cirrhotic liver that did progress to HCC, suggesting that p14ARF protects cirrhotic livers from HCC development. In the small sample size of cores with non-cirrhotic, non-malignant hepatocytes, we did not observe age-dependent increase in nucleolar p14ARF. Because of the limited sample number, we could not rule out the possibility of age-dependent increase in p14ARF expression. Importantly, however, the TMA set with cirrhotic samples that did not progress to HCC and expressed p14ARF at higher levels were collected from the younger patients (average age 50.018 years, median age 49 years) compared with the cirrhotic samples that did progress to HCC and low p14ARF (average patient age 57.27 years, median patient age 58 years). These findings are significant in several ways. First, although p14/p19ARF role as tumor suppressor is well established, its role in hepatocarcinogenesis is controversial. Our study using Ras-driven mouse model of HCC clearly demonstrates protective role of p19Arf. Moreover, we provided evidence that p19Arf deletion not only accelerates HCC development but also increases heterogeneity of the HCCs. Liver tumors formed in p19Arf-deficient background are more aggressive, they proliferate faster and express the stemnes signature genes, such as Nanog, Bmi1, c-myc, IL6, CD44 and CD90, at high levels. Human patients with HCCs with high expression of these genes have poorer prognosis and shorter survival, the phenotype observed in Arf null H-ras12V male mice as well. It is noteworthy that many of the analyzed genes (Figure 3 and 4) exhibited high intertumor variation in expression, especially Nanog, IL6 and FoxM1. We failed to correlate those variations to age of sacrifice, size of tumors or tumor growth pattern. It is important to note that p19Arf knockout mice do have shorter survival compared with wild-type littermates (19), due to spontaneous tumor development. It has been demonstrated that p19Arf-deficient mice develop mainly sarcomas and lymphomas (19) and have median survival of 38 weeks. No HCC development has been reported in this strain. However, animals used in this study, p19Arf−/− H-ras12V male mice, have median survival of 30 weeks of age, shorter than p19Arf−/− mice. They all had liver cancers at the time of the death and no other visible tumors, suggesting that the cause of their death is aggressive HCC. In contrast, all control, p19Arf+/+ H-ras12V male mice were alive at 10 months of age when the experiment has been stopped. It has been shown that senescence plays an important role in limiting fibrosis and hepatocarcinogenesis in mice (36,37). ARF is a well-known senescence factor, and it has been demonstrated that p19Arf-dependent Ras-induced senescence of premalignant hepatocytes plays crucial role in inducing tumor surveillance program as a part of tumor suppressive mechanism (38). It is possible that lack of senescence in p19Arf−/−H-ras12V premalignant hepatocytes causes earlier tumor development in that strain. Interestingly, we observed high FoxM1 levels in p19Arf null HCCs. FoxM1 is well-known pro-proliferation transcription factor (29,30), as well as an activator of stemness gene expression (25,26,28). It is highly possible that high FoxM1 levels drive aggressive phenotype of the p19Arf null HCCs. Besides being best known for sequestering Mdm2 in the nucleolus and activating p53 pathway (6), p14/p19ARF also sequesters FoxM1 in the nucleolus, rendering it transcriptionally inactive (9); hence, it is easy to understand having more active FoxM1 transcriptional network in the p19Arf null HCC cells. Interestingly, in knockout tumors, not only that FoxM1 was more transcriptionally active, there was more FoxM1 expressed both on mRNA and protein levels. That could be explained by FoxM1 auto-activation of its own expression; it has been demonstrated that FoxM1 can activate its own transcription by positive feedback loop (39). Importantly, our mouse HCC study can be related to human HCC pathogenesis. With our mouse model, we demonstrated that p19Arf deficiency leads to early onset of HCC in vivo, and we observed that human HCC with early onset express low p14ARF. As noted above, role of ARF in liver cancer progression has been controversial. Opposing reports have been published, and they mainly focused on promoter methylation status or p14Arf mRNA levels in liver tumors (11–14). Using IHC, we examined nucleolar p14ARF protein expression in human HCC cells. We found lack of nucleolar p14ARF in 43.02% of examined HCC samples. In that regard, it is noteworthy that Bmi1 and Pokemon, regulators of p14ARF expression, were shown to be upregulated in human HCCs (40–42). Moreover, Mdm2, a target of p14ARF, was shown to be overexpressed in 52.5% of human HCCs (43). Interestingly, we found that nucleolar p14ARF was lower in tumors harvested from younger HCC patients and that cirrhotic livers that did not progress to HCC had higher nucleolar p14ARF compared with cirrhotic livers that did progress to HCC. Cirrhosis of the liver is late stage of chronic liver disease, and most of the HCCs (around 80%) develop in the context of liver cirrhosis. Our finding that hepatocytes in the cirrhotic livers without HCC development exhibit higher nucleolar p14ARF protein compared with hepatocytes in cirrhotic livers that did develop HCC is very significant because it suggests that p14ARF expression can protect chronically injured liver from developing HCC. We propose that nucleolar p14ARF can be used as a marker for progressive liver disease that harbors higher chances for developing to terminal-phase HCC. 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Journal

CarcinogenesisOxford University Press

Published: Mar 1, 2018

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