WNT5A Inhibits Hepatocyte Proliferation and Concludes β-Catenin Signaling in Liver Regeneration

Activation of Wnt/β-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased β-catenin–T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in β-catenin–T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses β-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.Other than its roles in hepatic development, metabolic zonation, and hepatocarcinogenesis, β-catenin signaling plays an important role in liver regeneration (LR).¹ Liver is a unique organ in its ability to respond physiologically through non–stem cell–dependent regeneration after surgical or toxin-induced loss of hepatic mass.² After partial hepatectomy (PH), several signaling pathways get activated in tandem to ensure proper initiation and progression of the regenerative process, which entails orderly proliferation of all resident cells of the liver.² One pathway that is activated early during LR is the Wnt/β-catenin signaling pathway. This pathway has been shown to be highly relevant in ensuring proper G₁ to S phase transition of hepatocytes through regulation of its key target, cyclin-D1.^3,4 In fact, several studies have shown that disruption of Wnt/β-catenin signaling in various genetic mouse models leads to blunted LR, although, as many other signaling molecules, β-catenin too has been deemed redundant because its loss delays, but does not completely prevent, regeneration.An important attribute of LR is the ability of this process to spontaneously terminate when the normal hepatic mass consistent with the prehepatectomy mass is re-established. Because hepatocytes have seemingly unlimited proliferative capacity,^5,6 there must exist some regulatory mechanisms to stop the LR process. It has been suggested that pathways such as transforming growth factor-β signaling may play a role in the termination of LR.⁷ Furthermore, the role of extracellular matrix in regulating hepatocyte proliferation, especially during LR, has also been recently suggested.⁸We took an alternative approach to address termination mechanisms of LR. We hypothesized that because a series of proproliferative signaling pathways are turned on to initiate LR soon after PH, identification of negative regulators of such signaling mechanisms may yield clues to the overall LR termination processes. Because β-catenin is a proproliferative signal in LR, identification of what terminates β-catenin signaling might eventually be contributing to cessation of LR. Interestingly, examination of β-catenin signaling, cyclin-D1 expression, and markers of S phase in albumin-cre–driven Wntless knockout (KO) or liver-specific KOs of Wntless (Wls-LKO) mice revealed temporal prolongation of the hepatocyte proliferative program. This suggested that hepatocytes might be a source of Wnt that negatively regulates β-catenin signaling, because Wntless protein is specific for only Wnt secretion. Further analysis revealed Wnt5a to be differentially regulated during LR and contributed to inhibiting β-catenin activity in primary hepatocytes and liver tumor cells. Intriguingly, liver-specific Wnt5a KO lacked any spontaneous phenotype and displayed normal initiation and termination of LR. This is likely because of adaptive changes such as reduced expression of Frizzled-2 in the Wnt5a-LKO hepatocytes. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation in vitro and in vivo. Furthermore, we provide evidence that Wnt5a may be one of the effectors that is secreted by hepatocytes to suppress β-catenin signaling in hepatocytes in an autocrine manner, which, through Frizzled-2, terminates β-catenin signaling, thereby contributing to timely conclusion of the process of LR.     

Sarcoidosis en nasofaringe,una extraña localización

Sarcoidosis is a systemic granulomatous disease that usually has a pulmonary presentation. The extrapulmonary organs most frequently affected are lymph nodes, eyes and skin. Rhinopharyngeal involvement is extremely rare. We describe a case of sarcoidosis which was diagnosed through its location in the nasopharynx.     

Efficient treatment of breast cancer xenografts with multifunctionalized iron oxide nanoparticles combining magnetic hyperthermia and anti-cancer drug delivery

Introduction

Tumor cells can effectively be killed by heat, e.g. by using magnetic hyperthermia. The main challenge in the field, however, is the generation of therapeutic temperatures selectively in the whole tumor region. We aimed to improve magnetic hyperthermia of breast cancer by using innovative nanoparticles which display a high heating potential and are functionalized with a cell internalization and a chemotherapeutic agent to increase cell death.

Methods

The superparamagnetic iron oxide nanoparticles (MF66) were electrostatically functionalized with either Nucant multivalent pseudopeptide (N6L; MF66-N6L), doxorubicin (DOX; MF66-DOX) or both (MF66-N6LDOX). Their cytotoxic potential was assessed in a breast adenocarcinoma cell line MDA-MB-231. Therapeutic efficacy was analyzed on subcutaneous MDA-MB-231 tumor bearing female athymic nude mice.

Results

All nanoparticle variants showed an excellent heating potential around 500 W/g Fe in the alternating magnetic field (AMF, conditions: H = 15.4 kA/m, f = 435 kHz). We could show a gradual inter- and intracellular release of the ligands, and nanoparticle uptake in cells was increased by the N6L functionalization. MF66-DOX and MF66-N6LDOX in combination with hyperthermia were more cytotoxic to breast cancer cells than the respective free ligands. We observed a substantial tumor growth inhibition (to 40% of the initial tumor volume, complete tumor regression in many cases) after intratumoral injection of the nanoparticles in vivo. The proliferative activity of the remaining tumor tissue was distinctly reduced.

Conclusion

The therapeutic effects of breast cancer magnetic hyperthermia could be strongly enhanced by the combination of MF66 functionalized with N6L and DOX and magnetic hyperthermia. Our approach combines two ways of tumor cell killing (magnetic hyperthermia and chemotherapy) and represents a straightforward strategy for translation into the clinical practice when injecting nanoparticles intratumorally.

Electronic supplementary material

The online version of this article (doi:10.1186/s13058-015-0576-1) contains supplementary material, which is available to authorized users.