Defective DNA strand break repair causes chromosomal instability and accelerates liver carcinogenesis in mice

Teoh, Narcissus; Dan, Yock Young; Swisshelm, Karen; Lehman, Stacey L.; Wright, Jocelyn H.; Haque, Jamil; Gu, Yansong; Fausto, Nelson

doi:10.1002/hep.22194

Cited by 77 publications

(70 citation statements)

References 47 publications

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“…In the present study, we demonstrated that obese, diabetic foz/foz mice exhibit early onset (6 mths) DEN-induced HCC, whereas lean Wt mice do not develop HCC until 9 mths, a time course consistent with previous studies [18,32]. The earlier onset and more aggressive nature of DEN-induced HCC with obesity and diabetes were supported by the high rate of pulmonary metastases at 9 mths, 60% in obese vs. 10% in lean Wt mice.…”

Section: Discussionsupporting

confidence: 90%

Obesity and diabetes accelerate hepatocarcinogenesis via hepatocyte proliferation independent of NF-kB or Akt/mTORC1

et al. 2016

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Background: There are strong links between obesity, diabetes and hepatocellular carcinoma (HCC), but molecular mechanisms remain unclear. Aim: We tested the proposed involvement of NF-κB, IL-6/STAT3 and Akt/mTORC1 before onset (at 3 months) and at onset (6 months) of accelerated hepatocarcinogenesis in DEN-injected obese and diabetic foz/foz compared to lean wildtype (Wt) mice, and also studied the hepatocyte proliferative response to DNA damage between the obese and lean lines. Methods:Male foz/foz and Wt littermates fed normal chow were DEN-injected (10mg/kg i.p.) at age 12-15 days. To test the effect of mTOR inhibitor on growth of dysplastic hepatocytes, a separate cohort of DEN-injected foz/foz mice was administered rapamycin (4 mg/kg body weight/day). Results: foz/foz mice developed obesity, hyperinsulinemia, diabetes, adipokine dysregulation and fatty liver, without increased serum or liver TNF-α or serum IL-6. All DEN-injected foz/foz mice developed HCC by 6 mths vs. 0/10 lean Wt. At 3 mths, there were more dysplastic hepatocytes in DEN-injected foz/foz than Wt, with increased liver injury (serum ALT), hepatocyte apoptosis (M30-positive cells) and proliferation (cyclin D1, cyclin E, PCNA), but neither NF-κB nor STAT3 activation. foz/foz livers exhibited upregulation of DNA damage sensors ATM and ATR, with inadequate cell cycle checkpoint controls (CHK1, CHK2, p53, p21). Akt and mTORC1 were highly activated in livers from foz/foz vs. Wt mice. Despite such activation, rapamycin failed to reduce growth of dysplastic hepatocytes. Conclusions: Accelerated DEN-induced HCC in obese/diabetic mice is linked to enhanced growth of dysplastic hepatocytes that cannot be attributed to NF-κB or IL-6/STAT3 activation, nor to sustained mTORC1 activation. The critical mechanism for obesity-enhanced hepatocarcinogenesis lies in the disconnection between hepatocellular injury with DNA damage, and an unrestrained proliferative response. Relevance for patients: This study supports the epidemiological data linking obesity, diabetes and fatty liver disease with increased risk for developing HCC. The findings also suggest that mTORC1 inhibition may not be beneficial in the prevention of obesity-related hepatocarcinogenesis.

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Section: Discussionsupporting

confidence: 90%

Obesity and diabetes accelerate hepatocarcinogenesis via hepatocyte proliferation independent of NF-kB or Akt/mTORC1

et al. 2016

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“…Chromosomal abnormalities occur in 75% of blood cancers and in more than 90% of solid tumors including hepatocellular carcinoma (HCC) (25,26). The overexpression of the receptor tyrosine kinase Axl along with the release of soluble Axl (sAxl) was detected in HCC and correlated with poor survival of HCC patients (27,28).…”

Section: Introductionmentioning

confidence: 99%

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

et al. 2017

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Abstract. Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl --1, HLF-Axl --2, SNU449-Axl --1, SNU449-Axl --2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl -clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region.However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl --1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl --1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

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“…The data on the effect of Ku70 or Ku80 disruption in mice or cell lines on susceptibility to DNA damaging agents are conflicting and unclear. Several studies have claimed an increased rate of mutation, chromosomal instability, and increased carcinogenesis (7,12,20,26,41), while other investigators observed a converse decrease in mutation frequency and chromosomal rearrangements (32,44). It appears that the particular mouse strain or cell line used may play a role in this discrepancy.…”

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confidence: 98%

Tagging of Endogenous Genes in aToxoplasma gondiiStrain Lacking Ku80

2009

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As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a ⌬ku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductasethymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the ⌬ku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the ⌬ku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.Toxoplasma gondii is an obligate intracellular protozoan in the phylum Apicomplexa that has garnered more intense study in recent decades. This is due in part to the genetic tractability and ease of growth of T. gondii and also because knowledge obtained for this parasite is often relevant to its kin, including other pathogens of medical and veterinary importance, such as Plasmodium falciparum, the parasite that causes malaria (11). The genome sequence of Toxoplasma was recently completed (10), opening the door to identifying novel genes involved in a variety of events, such as cell invasion, replication, gliding motility, metabolism, stage conversion, and virulence. With a large number of new genes of interest, it is essential to have tools that enable investigators to perform studies on a larger scale. One bottleneck for the analysis of novel genes is protein localization, since this generally necessitates time-consuming production of antibodies that often require additional affinity purification. Protein localization by ectopic expression of a tagged construct can also be problematic due to overexpression or mistiming of expression from a heterologous promoter. If genes could be tagged directly on the chromosome, this would better mimic natural expression and allow the use of fluorescent protein tags or standardized antibodies to monitor localization.DNA double-strand breaks...

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Defective DNA strand break repair causes chromosomal instability and accelerates liver carcinogenesis in mice

Cited by 77 publications

References 47 publications

Obesity and diabetes accelerate hepatocarcinogenesis via hepatocyte proliferation independent of NF-kB or Akt/mTORC1

Obesity and diabetes accelerate hepatocarcinogenesis via hepatocyte proliferation independent of NF-kB or Akt/mTORC1

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

Tagging of Endogenous Genes in aToxoplasma gondiiStrain Lacking Ku80

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