Lack of Matrilin-2 Favors Liver Tumor Development via Erk1/2 and GSK-3 beta Pathways In Vivo

Matrilin-2 (Matn2) is a multidomain adaptor protein which plays a role in the assembly of extracellular matrix (ECM). It is produced by oval cells during stem cell-driven liver regeneration. In our study, the impact of Matn2 on hepatocarcinogenesis was investigated in Matn2(-/-) mice comparing them with wild-type (WT) mice in a diethylnitrosamine (DEN) model. The liver tissue was analyzed macroscopically, histologically and immunohistochemically, at protein level by Proteome Profiler Arrays and Western blot analysis. Matn2(-/-) mice exhibited higher susceptibility to hepatocarcinogenesis compared to wild-type mice. In the liver of Matn2(-/-) mice, spontaneous microscopic tumor foci were detected without DEN treatment. After 15 mu g/g body weight DEN treatment, the liver of Matn2(-/-) mice contained macroscopic tumors of both larger number and size than the WT liver. In contrast with the WT liver, spontaneous phosphorylation of EGFR, Erk1/2 GSK-3 alpha/beta and retinoblastoma protein (p-Rb), decrease in p21/CIP1 level, and increase in beta-Catenin protein expression were detected in Matn2(-/-) livers. Focal Ki-67 positivity of these samples provided additional support to our presumption that the lack of Matn2 drives the liver into a pro-proliferatory state, making it prone to tumor development. This enhanced proliferative capacity was further increased in the tumor nodules of DEN-treated Matn2(-/-) livers. Our study suggests that Matn2 functions as a tumor suppressor in hepatocarcinogenesis, and in this process activation of EGFR together with that of Erk1/2, as well as inactivation of GSK-3 beta, play strategic roles

Representative immunofluorescence and immunohistochemical stains of WT control, WT DEN-treated, <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN-treated mouse livers.

<p>Scale bars represent 0.1(insets) for paraffin-embedded samples and 0.05 mm for frozen tissue sections.</p

Differences between <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN group compared to <i>Matn2^-/-</i> control.

<p>Differences between <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN group compared to <i>Matn2^-/-</i> control.</p

Differences between WT DEN and <i>Matn2^-/-</i> DEN group compared to WT DEN.

<p>Differences between WT DEN and <i>Matn2^-/-</i> DEN group compared to WT DEN.</p

Representative Western blots of intracellular regulatory proteins

<p>in WT control, WT DEN-treated, <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN-treated mouse livers (<b>A/1, A/2</b>). Results of densitometrical analysis of band intensities expressed as values normalized to β-Actin loading control (<b>B</b>). Data are expressed as mean ± SD, n = 3.</p

Representative immunohistochemical stains of WT control, WT DEN-treated, <i>Matn2^-/-</i> control and <i>Matn2^-/-</i> DEN-treated mouse livers (A).

<p>Scale bars represent 0.1(insets) for paraffin-embedded samples. Changes in cell cycle regulation. Ki-67 proliferation index (B). In knockout samples an average of 4.1 Ki-67 positive cells were counted per field of view compared to 0.7 in WT (p<0.001) (B). Results are expressed as mean ± SD. <b>Representative Western blots of cell cycle regulatory proteins (C) in WT control, WT DEN-treated, </b><b><i>Matn2^-/-</i></b><b> control and </b><b><i>Matn2^-/-</i></b><b> DEN-treated mouse livers.</b> Diagrams of band intensities expressed as values normalized to β-Actin loading control (D). Data are expressed as mean ± SD, n = 3.</p

Differences between WT control and WT DEN group compared to WT control.

<p>Differences between WT control and WT DEN group compared to WT control.</p

Results of Phospho-RTK antibody array.

<p>Picture of RTK array membrane (<b>A</b>). Densitometry of phosphorylation signals in WT control (dark gray bars) and WT DEN-treated (white bars) samples, compared to control (black bars) and <i>Matn2^-/-</i> DEN-treated (light gray bars) samples (<b>B</b>). Data are expressed as mean ± SD, n = 3.</p

Differences between WT control and <i>Matn2^-/-</i> control group compared to WT control.

<p>Differences between WT control and <i>Matn2^-/-</i> control group compared to WT control.</p

Results of DEN treatment in WT and <i>Matn2^-/-</i> animals.

<p>Representative pictures of the macroscopic appearance of WT and <i>Matn2^-/-</i> DEN-treated mouse livers. 10 months after DEN exposure the number and size of macroscopic tumors were greater in <i>Matn2^-/-</i> than in WT livers (<b>A</b>). The number of tumors/animal (<b>B</b>) and the tumor volume (<b>C</b>) were significantly higher in <i>Matn2^-/-</i> mice compared to WT mice after DEN-treatment (n = 9 for WT DEN, n = 13 for <i>Matn2^-/-</i> DEN, **p<0.01).</p