Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (gt;99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMOdependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness

Mutated in colorectal cancer (MCC): a putative tumour suppressor gene in colorectal cancer

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC.MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis.MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur

A very minimal population of human vimentin⁺ HSCs/myofibroblasts express a primary cilium, with none detected on CD31⁺ endothelial cells.

<p>Human ALD liver tissue was examined for the expression of primary cilia (α-acetylated tubulin, green; γ-tubulin, red) by vimentin⁺ (grey) HSCs/myofibroblasts <b>(A)</b> or CD31+ (grey) ECs <b>(C)</b>. <b>(A)</b> The majority of vimentin⁺ cells were Pc^-ve in the tissues examined. Representative image shown, displaying absence of Pc on vimentin⁺ cells. To confirm this result, ciliary protein Arl13b (green) was co-stained with vimentin (grey). Rare Arl13b ciliary structures (arrow) co-localised with vimentin⁺ cells. Final panel in A illustrates rare Pc⁺ (α-acetylated tubulin, green; γ-tubulin, red) vimentin⁺ (grey) HSCs/myofibroblasts, at the cirrhotic interface. <b>(B)</b> Number of vimentin⁺ Pc⁺ cells or vimentin⁺ Pc^neg cells per FOV (<i>n</i> = 3 ALD samples, 8 FOV/sample). <b>(C)</b> No Pc were detected on CD31⁺ cells in the tissues examined (ALD <i>n</i> = 3, 8 FOV/sample). Representative image shown. All images obtained using confocal microscopy, 63x objective. DAPI, blue. White arrows illustrate Pc. * Non-specific liver autofluorescence.</p

Gene changes in BMOL1.2 cells following GANT61 treatment.

<p>Gene changes in BMOL1.2 cells following GANT61 treatment.</p

Patient sample, age, sex, fibrosis staging (Scheuer score) and SHH expression.

<p>Patient sample, age, sex, fibrosis staging (Scheuer score) and SHH expression.</p

SHH expression in human donor and cirrhotic liver.

<p><b>(A)</b> Frozen (4 μm) human donor (<i>n</i> = 5), and cirrhotic liver sections [ALD (<i>n</i> = 6), NASH (<i>n</i> = 3), PBC (<i>n</i> = 1)] were screened for SHH (C-terminus) by immunofluorescence. 5x objective. Representative images shown. <b>(B)</b> qRT-PCR for <i>SHH</i> in human donor and ALD samples. Mean±S.E.M. Significant (*) difference between means (One-sided student t-test, *<i>p</i><0.05). <b>(C)</b> SHH expression (red) by EpCAM⁺ LPCs (green) in donor liver. 63x objective. <b>(D, E)</b> Comprehensive characterisation of SHH expression by liver cell populations in ALD. Images obtained using 63x objective. The majority of SHH is produced by hepatocytes. <b>(D)</b> SHH (red) is expressed by EpCAM⁺ LPCs (green). Co-localisation in merged images indicated by yellow. A subset of CD31⁺ ECs (green) at the cirrhotic interface express SHH. <b>(E)</b> SHH (red) is not expressed by CD45⁺ leukocytes (green). Minimal SHH is expressed by vimentin⁺ ECs (solid arrows), with negligible SHH expressed by vimentin⁺ myofibroblasts (dashed arrows). DAPI, blue.</p

Inhibition of GLI signaling reduces the viability of liver progenitor cell lines.

<p>LPC lines BMOL1.2 <b>(A, C, E, G)</b> and BMOL-TAT <b>(B, D, F, H)</b> were grown over 72 h in the presence of signaling inhibitors for GLI (GANT61; <b>A, B</b>), Notch (DAPT; <b>C,D</b>) c-Met (SGX523; <b>E, F</b>), Wnt (XAV939; <b>G, H</b>) signaling and matching [DMSO]. Viability was determined by ALAMAR Blue assays, and an interaction between time and dose was assessed using two-way ANOVA followed by Tukey’s multiple comparisons test (α = 0.001) (Prism, GraphPad). **** <i>p</i><0.001.</p

Changes to transcript expression in two liver progenitor cell lines following GLI inhibition.

<p>Heat map representing changes to gene transcript levels in BMOL1.2 (<i>n</i> = 3) or BMOL-TAT (<i>n</i> = 3) lines following GANT61 (10 μM) 8 h treatment. Log₂ intensity scale shown. Downregulated genes (blue) are shown above the yellow dashed line, with upregulated genes (red) below. Grey indicates no data.</p

Common gene changes in BMOL1.2 and BMOL-TAT cells following GANT61 treatment.

<p>Common gene changes in BMOL1.2 and BMOL-TAT cells following GANT61 treatment.</p