The Stress-Regulated Transcription Factor CHOP Promotes Hepatic Inflammatory Gene Expression, Fibrosis, and Oncogenesis

<div><p>Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma (HCC). Although these conditions also lead to integrated stress response (ISR) or unfolded protein response (UPR) activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is up-regulated in liver tumors in human HCC and two mouse models thereof. <i>Chop</i>-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. <i>Chop</i>-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases.</p></div

CHOP is induced by fibrotic insult and promotes fibrosis.

<p>(A) CCl₄ injection promotes collagen deposition. Masson's trichrome staining was used to assess collagen deposition (blue) in formalin-fixed paraffin-embedded liver sections of mice injected i.p. with 10% CCl₄ twice a week for 12 weeks, or with mineral oil as a control. (B–C) CHOP expression is associated with CCl₄-challenged livers. (B) CHOP was detected by IHC in mouse livers from (A) and samples were counterstained with hematoxylin as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003937#pgen-1003937-g001" target="_blank">Figure 1C</a>. Representative data are shown. (C) The number of CHOP-positive nuclei per microscopic field from (B) was quantitated in multiple fields from 3 mice per group, and is expressed here as mean +/− S.E.M. per field. (D–E) <i>Chop</i> deletion attenuates DEN-induced fibrosis. Mild-to-moderate fibrosis is seen in wild-type but not <i>Chop</i>−/− livers by trichrome staining after DEN treatment as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003937#pgen-1003937-g002" target="_blank">Figure 2</a>. Sections from two separate DEN-treated animals of each genotype are shown. (E) A METAVIR score was blindly determined from trichrome stains as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003937#s4" target="_blank">Materials and Methods</a>, and the p-value was calculated by Asymptotic Wilcoxon Rank Sum Test. 0 = no fibrosis; 1 = portal fibrosis lacking septa; 2 = portal fibrosis with some septa; 3 = abundant septa; 4 = cirrhosis.</p

CHOP promotes hepatocellular apoptosis and proliferation.

<p>(A) <i>Chop</i>−/− mice show reduced cell death upon DEN challenge. Apoptosis in vehicle- or DEN-treated livers of wild-type or <i>Chop</i>−/− mice was detected by IHC staining for the cleaved form of Caspase-3. Liver nodule borders are indicated by a dashed outline. Hematoxylin counterstain is not shown because it interfered with Caspase-3 staining. In (A–C), representative images are shown. (B–D) Reduced proliferation in <i>Chop</i>−/− DEN-challenged animals. Hepatocellular proliferation was detected by IHC staining for PCNA (B) or Ki-67 (C). (D) The extent of PCNA and Ki-67 staining was quantitated blindly as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003937#s4" target="_blank">Materials and Methods</a>. P-values were determined by Asymptotic Wilcoxon Rank Sum Test.</p

Model for the role of CHOP in HCC.

<p>The simplest interpretation of the data presented is a linear pathway whereby CHOP promotes apoptosis, which in turn activates inflammatory signaling, leading to fibrosis, compensatory proliferation, and ultimately tumorigenesis. Fibrosis and cellular transformation might in turn exacerbate cellular stress and CHOP expression, amplifying the tumor-promoting function of CHOP.</p

Microarray profiling reveals CHOP-dependent differences in inflammation and ribosomal biogenesis genes.

<p>(A) Basal expression differences in <i>Chop</i>−/− mice. 18,138 significantly expressed genes were analyzed using Illumina MouseRef-8 v2.0 BeadChips, with total RNA from 9 month old mouse liver homogenate as input. The number and percentage of genes upregulated (upper left) and downregulated (lower right) in wild-type versus <i>Chop</i>−/− tissue is shown, showing only genes differing by 1.5-fold or more, p<0.05. Expression is given on a log₂ scale. In (A), (D), and (E), the position of <i>Chop</i> is shown as a yellow-filled circle. (B–C) Suppressed basal expression of genes involved in immune and inflammatory pathways in <i>Chop</i>−/− mice. (B, top) GO pathways enriched among all regulated genes in PBS-treated wild-type versus <i>Chop</i>−/− animals were determined using DAVID, with all pathways that were significantly enriched after Bonferroni correction shown. (B, bottom) Same as (top), but considering only genes downregulated in <i>Chop</i>−/− animals. (C) Average array-determined expression of genotype-dependent immune- and inflammation-related genes from PBS-treated animals is shown on a log₂ scale +/− S.D.M. All genes shown are differentially expressed >1.5-fold (i.e., >0.585 on the y-axis), p<0.05. (D) DEN treatment alters gene expression in wild-type mice. Scatter plot of all genes differentially expressed (>1.5-fold; p<0.05) in tumors from 9 month-old DEN-treated wild-type animals versus normal liver tissue in age-matched PBS-treated wild-type animals. The positions of <i>Nfe2</i>, <i>Cyclin D1</i> (<i>Ccnd1</i>), and <i>β-catenin</i> are indicated by red diamonds. (E–F) Uniform upregulation of genes involved in ribosome biogenesis in tumors from <i>Chop</i>−/− mice. (E) Scatter plot of gene expression from tumor tissue of DEN-treated wild-type animals compared to <i>Chop</i>−/− animals is presented, showing all genes differing by >1.5-fold, p<0.05. (F) Average expression of all ribosomal subunit genes that were significantly different (p<0.05, fold-change >1.5) in <i>Chop</i>−/− versus wild-type large tumors is shown on a log₂ scale +/− S.D.M. as in (C).</p

CHOP is upregulated in a genetic mouse model of HCC.

<p>(A) eIF2α-dependent genes are upregulated in tumors induced by <i>SB</i> mutagenesis. Relative expression of the indicated UPR genes in tumors versus normal mouse liver tissue, as quantified by transcriptome sequencing. Sequencing was performed on total RNA from mouse liver tumors generated by T2/Onc3 transposition into the <i>Rtl1</i> locus (n = 8) and age-matched normal liver tissue (n = 7). The branch of UPR signaling to which each gene is most responsive is indicated. Here and in (B), error bars represent means +/− S.D.M. Here and elsewhere: *, p<0.05; **, p<0.01; ***, p<0.001. (B) qRT-PCR of <i>SB</i>-induced tumors confirms upregulation of <i>Chop</i>. Relative expression of the indicated genes quantitated by qRT-PCR from tumors arising from T2/Onc3 <i>Rtl1</i> locus integrants (n = 7), compared to expression in normal tissue (n = 5). Relative expression of total (tot) and spliced (spl) <i>Xbp1</i> mRNA is also shown. mRNA levels were normalized against average expression of 2 housekeeping genes (<i>Btf3</i> and <i>Ppia</i>). (C and D) CHOP and BiP expression, respectively, in tumors from <i>Rtl1</i> locus integrants and normal liver tissue from age-matched control animals using IHC. Also shown for CHOP staining are hematoxylin-counterstained samples, with exemplar CHOP-positive nuclei indicated by arrowheads. All scale bars throughout this work represent 50 µm unless indicated otherwise. The same anti-mouse immunoglobulin secondary antibody was used for both CHOP and BiP immunostains.</p

CHOP is expressed in human HCC tumors.

<p>(A) Histology of tumor-proximal liver (uninvolved HCC) and tumor (two regions) tissue from the same patient. H&E images are representative of 28 tumor samples and 5 uninvolved matched controls. For (A) only, scale bar = 100 µm (B–C) CHOP immunostaining is associated with human HCC but not uninvolved regions. (B) Samples from uninvolved tumor-proximal tissue and HCC tumors were probed for CHOP expression by immunohistochemistry. CHOP-positive nuclei are indicated by arrowheads. Cellular and nuclear architecture in some HCC samples was distorted as seen in the non-counterstained image, accounting for the large nuclei. Scale bar = 50 µm. (C) The average number of CHOP-positive nuclei per microscopic field per patient was quantitated blindly from 5 HCC tumors and their uninvolved matched controls, as well as from 4 non-HCC specimens, and these were aggregated by Boxplot. P-values were calculated by Asymptotic Wilcoxon Rank Sum Test. (D) HCC-associated CHOP is expressed in hepatocytes. Expression of CHOP (brown) and cell type-specific markers (blue) for hepatocytes (cytokeratin-18), macrophages (CD68), bile ducts (cytokeratin-7), or activated stellate cells (smooth muscle actin) is shown by immunohistochemistry, with a hematoxylin counterstain applied. Representative data are shown. Note that only hepatocytes show CHOP-positive nuclei; arrowheads are used to show CHOP-positive cells in other samples. Scale bar = 50 µm. Insets show 25 µm×25 µm regions with distinct CHOP-positive nuclei.</p

Identification of <i>Rtl1</i>, a Retrotransposon-Derived Imprinted Gene, as a Novel Driver of Hepatocarcinogenesis

<div><p>We previously utilized a Sleeping Beauty (SB) transposon mutagenesis screen to discover novel drivers of HCC. This approach identified recurrent mutations within the <i>Dlk1-Dio3</i> imprinted domain, indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis. For the current study, we performed transcriptome and small RNA sequencing to profile gene expression in SB–induced HCCs in an attempt to clarify the genetic element(s) contributing to tumorigenesis. We identified strong induction of <i>Retrotransposon-like 1</i> (<i>Rtl1</i>) expression as the only consistent alteration detected in all SB–induced tumors with <i>Dlk1-Dio3</i> integrations, suggesting that <i>Rtl1</i> activation serves as a driver of HCC. While previous studies have identified correlations between disrupted expression of multiple <i>Dlk1-Dio3</i> domain members and HCC, we show here that direct modulation of a single domain member, <i>Rtl1</i>, can promote hepatocarcinogenesis <i>in vivo</i>. Overexpression of <i>Rtl1</i> in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant (86%) tumor formation. Additionally, we detected overexpression of <i>RTL1</i> in 30% of analyzed human HCC samples, indicating the potential relevance of this locus as a therapeutic target for patients. The <i>Rtl1</i> locus is evolutionarily derived from the domestication of a retrotransposon. In addition to identifying <i>Rtl1</i> as a novel driver of HCC, our study represents one of the first direct <i>in vivo</i> demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis.</p> </div

<i>Rtl1</i>-expressing mouse HCCs resemble human S1 subclass.

<p>Expression levels for the gene sets defining human HCC subclasses S1, S2, and S3 were analyzed in SB-induced HCCs and normal livers. Gene Set Enrichment Analysis (GSEA) was conducted for each subclass independently to assess the significance of differential expression between tumor and normal samples. Heat maps generated by GSEA are shown. This analysis revealed a significant (p = 0.039) overexpression of the genes defining human subclass S1 in SB-induced HCCs, as compared to normal liver.</p

<i>Dlk1-Dio3</i> domain transposon integration sites in SB–induced HCC and effects on domain expression.

<p>(A) The <i>Dlk1-Dio3</i> imprinted domain spans ∼800 kilobases at the distal end of mouse chromosome 12 (human chr14q32). Three protein-coding genes are expressed from the paternal allele (<i>Dlk1</i>, <i>Rtl1</i>, and <i>Dio3</i>). The maternal allele encodes four lncRNAs (<i>Meg3</i>, <i>Rtl1as</i>, <i>Rian</i>, and <i>Mirg</i>), as well as several miRNAs and snoRNAs. SB transposon and AAV integration sites found to be associated with HCC development in mice are depicted. (B) Intensity plot showing normalized expression levels of long transcripts within and surrounding the <i>Dlk1-Dio3</i> domain in SB-induced HCCs and normal livers. (C) Intensity plot showing normalized expression of <i>Dlk1-Dio3</i> domain miRNAs. miRNAs contained within lncRNAs are indicated. miRNAs with no detected expression across all samples were omitted.</p