Luc Gailhouste

Fibrillar collagen scoring by second harmonic microscopy: a new tool in the assessment of liver fibrosis.

BACKGROUND & AIMS Imaging of supramolecular structures by multiphoton microscopy offers significant advantages for studying specific fibrillar compounds in biological tissues. In this study, we aimed to demonstrate the relevance of Second Harmonic Generation (SHG) for assessing and quantifying, without staining, fibrillar collagen in liver fibrosis. METHODS We first showed the relationship between SHG signal and collagen forms over-produced and accumulated during fibrosis progression. Taking this property into consideration, we developed an innovative method to precisely quantify the fibrosis area in histological slices by scoring of fibrillar collagen deposits (Fibrosis-SHG index). RESULTS The scoring method was routinely applied to 119 biopsies from patients with chronic liver disease allowing a fast and accurate measurement of fibrosis correlated with the Fibrosis-Metavir score (rho=0.75, p<0.0001). The technique allowed discriminating patients with advanced (moderate to severe) fibrosis (AUROC=0.88, p<0.0001) and cirrhosis (AUROC=0.89, p<0.0001). Taking advantage of its continuous gradation, the Fibrosis-SHG index also allowed the discrimination of several levels of fibrosis within the same F-Metavir stage. The SHG process presented several advantages such as a high reliability and sensitivity that lead to a standardized evaluation of hepatic fibrosis in liver biopsies without staining and pathological examination. CONCLUSIONS Second harmonic microscopy emerges as an original and powerful tool in the assessment of liver fibrosis and offers new possibilities for the evaluation of experimental protocols. We expect that this technology could easily be applicable in the study of other fibro-proliferative pathologies.

miR-148a plays a pivotal role in the liver by promoting the hepatospecific phenotype and suppressing the invasiveness of transformed cells.

MicroRNAs (miRNAs) are evolutionary conserved small RNAs that post‐transcriptionally regulate the expression of target genes. To date, the role of miRNAs in liver development is not fully understood. By using an experimental model that allows the induced and controlled differentiation of mouse fetal hepatoblasts (MFHs) into mature hepatocytes, we identified miR‐148a as a hepatospecific miRNA highly expressed in adult liver. The main finding of this study revealed that miR‐148a was critical for hepatic differentiation through the direct targeting of DNA methyltransferase (DNMT) 1, a major enzyme responsible for epigenetic silencing, thereby allowing the promotion of the “adult liver” phenotype. It was also confirmed that the reduction of DNMT1 by RNA interference significantly promoted the expression of the major hepatic biomarkers. In addition to the essential role of miR‐148a in hepatocyte maturation, we identified its beneficial effect through the repression of hepatocellular carcinoma (HCC) cell malignancy. miR‐148a expression was frequently down‐regulated in biopsies of HCC patients as well as in mouse and human HCC cell lines. Overexpressing miR‐148a led to an enhancement of albumin production and a drastic inhibition of the invasive properties of HCC cells, whereas miR‐148a silencing had the opposite consequences. Finally, we showed that miR‐148a exerted its tumor‐suppressive effect by regulating the c‐Met oncogene, regardless of the DNMT1 expression level. Conclusion: miR‐148a is essential for the physiology of the liver because it promotes the hepatospecific phenotype and acts as a tumor suppressor. Most important, this report is the first to demonstrate a functional role for a specific miRNA in liver development through regulation of the DNMT1 enzyme. (Hepatology 2013;53:1153–1165)

RNAi-mediated ERK2 knockdown inhibits growth of tumor cells in vitro and in vivo.

The MAPK MEK/ERK pathway is often upregulated in cancer cells and represents an attractive target for development of anticancer drugs. Only few data concerning the specific functions of ERK1 and 2 are reported in the literature. In this report, we investigated the specific role of ERK1 and 2 in liver tumor growth both in vitro and in vivo. DNA synthesis and cells in S phase analysed by flow cytometry, correlated with strong inhibition of Cdk1 and cyclin E levels, are strongly reduced after exposure to the MEK inhibitor, U0126. We obtained a significant reduction of colony formation in soft agar assays and a reduction in the size of tumor xenografts in nude mice treated with U0126. Then, we could specifically abolished ERK1 or 2 expression by small-interfering RNA (siRNA) and demonstrated that ERK2 knockdown but not ERK1 interferes with the process of replication. Moreover, we found that colony formation and tumor growth in vivo were significantly inhibited by targeting ERK2 using stable chemically modified siRNA. Taken together, our results emphasize the importance of the MEK/ERK pathway in liver cancer cell growth in vitro and in vivo and argue for a crucial role of ERK2 in this regulation.

Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

We investigated the specific role of the mitogen‐activated protein kinase (MAPK) extracellular signal‐regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long‐term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)‐dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF‐dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2‐dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Conclusion: Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long‐term survival and cell cycle regulation of hepatocytes. (HEPATOLOGY 2009.)

RNAi-mediated MEK1 knock-down prevents ERK1/2 activation and abolishes human hepatocarcinoma growth in vitro and in vivo

The mitogen‐activated protein kinases MEK/ERK pathway regulates fundamental processes in malignant cells and represents an attractive target in the development of new cancer treatments especially for human hepatocarcinoma highly resistant to chemotherapy. Although gene extinction experiments have suggested distinct roles for these proteins, the MEK/ERK cascade remains widely considered as exhibiting an overlap of functions. To investigate the functionality of each kinase in tumorigenesis, we have generated stably knock‐down clones for MEK1/2 and ERK1/2 isoforms in the human hepatocellular carcinoma line HuH7. Our results have shown that RNAi strategy allows a specific disruption of the targeted kinases and argued for the critical function of MEK1 in liver tumor growth. Transient and stable extinction experiments demonstrated that MEK1 isoform acts as a major element in the signal transduction by phosphorylating ERK1 and ERK2 after growth factors stimulation, whereas oncogenic level of ERK1/2 phosphorylation appears to be MEK1 and MEK2 dependent in basal condition. In addition, silencing of MEK1 or ERK2 abolished cell proliferation and DNA replication in vitro as well as tumor growth in vivo after injection in rodent. In contrast, targeting MEK2 or ERK1 had no effect on hepatocarcinoma progression. These results strongly corroborate the relevance of targeting the MEK cascade as attested by pharmacologic drugs and support the potential application of RNAi in future development of more effective cancer therapies. Our study emphasizes the importance of the MEK/ERK pathway in human hepatocarcinoma cell growth and argues for a crucial role of MEK1 and ERK2 in this regulation.

Cancer-related microRNAs and their role as tumor suppressors and oncogenes in hepatocellular carcinoma

MicroRNAs (miRNAs) have emerged as key factors involved in several biological processes, including development, differentiation, cell proliferation, and tumorigenesis. In hepatocellular carcinoma (HCC), miRNAs frequently present aberrant expression profiles, which make them potentially attractive for diagnostic or prognostic applications. Currently, accumulating evidence is indicating the role of miRNAs as tumor suppressors or oncogenes in hepatic malignancies. In particular, comprehensive studies have made possible a better understanding of HCC behavior, such as tumor growth, response to therapies, metastatic potential, or recurrence, regarding the altered expression of cancer-related miRNAs. Based on these findings, efforts are under way to define new markers for liver cancer in both invasive (hepatic biopsy or tumor resection) and non-invasive (circulating miRNAs in blood serum) ways. Due to their implication in the control of various cell processes altered in HCC, cancer-related miRNAs also offer encouraging perspectives for the development of innovative cancer therapies. In this article, we review the importance of miRNA deregulation in HCC progression and the role of these small non-coding RNAs as tumor suppressors and oncogenes. The significance of miRNAs in HCC diagnosis and miRNA-based therapeutic strategies is then discussed.

A robust collagen scoring method for human liver fibrosis by second harmonic microscopy

Second Harmonic Generation (SHG) microscopy offers the opportunity to image collagen of type I without staining. We recently showed that a simple scoring method, based on SHG images of histological human liver biopsies, correlates well with the Metavir assessment of fibrosis level (Gailhouste et al., J. Hepatol., 2010). In this article, we present a detailed study of this new scoring method with two different objective lenses. By using measurements of the objectives point spread functions and of the photomultiplier gain, and a simple model of the SHG intensity, we show that our scoring method, applied to human liver biopsies, is robust to the objectives numerical aperture (NA) for low NA, the choice of the reference sample and laser power, and the spatial sampling rate. The simplicity and robustness of our collagen scoring method may open new opportunities in the quantification of collagen content in different organs, which is of main importance in providing diagnostic information and evaluation of therapeutic efficiency.

Commitment of Annexin A2 in recruitment of microRNAs into extracellular vesicles.

Extracellular vesicles (EVs) contain microRNAs (miRNAs). However, the exact molecular mechanisms of the recruitment of miRNAs in EVs are not well characterized. Based on proteomic analysis, we identified that silencing of Annexin A2 (ANXA2) significantly decreased the amount of miRNAs in EVs. In addition, microarray analysis revealed that ANXA2 regulated the loading of miRNAs into EVs in a sequence independent manner. Lastly, immunoprecipitation analysis confirmed that ANXA2 could bind miRNAs in EVs in the presence of Ca2+. These observations demonstrate that ANXA2 plays an important role in the packaging process of miRNAs into EVs.

Potential applications of miRNAs as diagnostic and prognostic markers in liver cancer.

Primary liver tumors are mainly represented by hepatocellular hepatocarcinoma (HCC), one of the most aggressive and resistant forms of cancer. Numerous studies have reported the key role of microRNAs (miRNAs) in development, cell proliferation, apoptosis, and tumor biology. The alteration of cancer-related miRNA expression can be associated with tumorigenesis. In HCC, deregulated miRNAs frequently act as oncogenes or altered tumor suppressors. Distinct subtypes of hepatic cancer can also be related to an aberrant expression of particular miRNAs, arguing for the significance of using miRNAs as tumor biomarkers in order to refine the HCC grading assessment. In this article, we review the latest reports regarding miRNA profiling and the potential of small RNAs in HCC diagnosis. The relevance of cancer-related miRNA signatures for the prognosis and better understanding of liver cancer outcome is then considered.

The complexity of ERK1 and ERK2 MAPKs in multiple hepatocyte fate responses

Recent reports suggest that extracellular signal‐regulated kinase (ERK1) and ERK2 mitogen‐activated protein kinases (MAPK) may direct specific biological functions under certain contexts. In this study, we investigated the role of early and sustained epidermal growth factor (EGF) stimulation on long‐term hepatocyte differentiation and the possible role of ERK1 and ERK2 in this process. We demonstrate a long‐term survival and an elevated level of differentiation up to 3 weeks. The differentiation state of hepatocytes is supported by sustained expression of aldolase B, albumin, and the detoxifying enzymes CYP1A2, 2B2, and 3A23. Similarly to freshly isolated cells, cultured hepatocytes also retain the ability to respond to 3‐methylcholanthrene (3MC) and phenobarbital (PB), two known CYP inducers. In addition, we show evidence that continuous MAPK/ERK kinase (MEK) inhibition enhances the level of differentiation. Using RNA interference approaches against ERK1 and ERK2, we demonstrate that this effect requires both ERK1 and ERK2 activity, whereas the specific ERK1 knockdown promotes cell survival and the specific ERK2 knockdown regulates cell proliferation. In conclusion, we demonstrate that early and sustained EGF stimulation greatly extends long‐term hepatocyte survival and differentiation, and that inhibition of the ERK1/2 MAPK pathway potentiates these pro‐survival/pro‐differentiation phenotypes. We clearly attest that specific ERK1 and ERK2 MAPKs determine hepatocyte survival and proliferation, respectively, whereas dual inhibition is required to stabilize a highly differentiated state. J. Cell. Physiol. 227: 59–69, 2012.