Mechanisms regulating CXCR4 intracellular traffic and polarization in human hepatocellular carcinoma cells: cross-talk with the TGF-β pathway

CXCR4 is a chemokine receptor originally discovered and cloned in leukocytes that functions by controling the migration to the inflammatory foci. The CXCR4 chemokine receptor and its ligand, SDF-1α (also named CXCL12), are two important molecules involved in the crosstalk between cancer cells and the microenvironment, which has a relevant role in tumour progression, and consequently are considered as promising targets for cancer therapy. The CXCR4/SDF-1α axis is implicated in the expansion and progression of a wide variety of tumors, including prostate, oesophageal, and liver cancer, among others. Moreover, the CXCR4/SDF-1α axis has been also related to other processes of tumor development such as initiation, angiogenesis, metastasis, invasion and cell survival. Thus tumors expressing high levels of CXCR4 are correlated with a poor prognosis of the patient. Intracellular trafficking pathways that control CXCR4 export to the plasma membrane are a key regulatory point of CXCR4 function, especially those that regulate trafficking through the endosomal bodies, whose role as a cell signaling platforms could be determinant for the polarized expression of CXCR4. Our group has previously reported that TGF-β regulates CXCR4 expression and its asymmetrical distribution within the cell in the alleged migration front. The general aim of this project is to analyze the mechanisms regulating the asymmetric export of CXCR4 to the plasma membrane of HCC cells and their possible correlation with the mechanisms that control cell polarity. CXCR4 is expressed in the cell surface of PLC/PRF/5 and Hep3B human HCC cell lines but significant higher levels are found in intracellular compartments. TGF-β pathway blockade reduces cell surface localization of CXCR4 in Hep3B cells, which display mesenchymal features for which an overactivation of TGF-β pathway has been previously described. CXCR4 reaches plasma membrane through the Golgi-AP1-exocyst axis and requires the functionality of the exocyst complex for tethering of the exocytic vesicle to the plasma membrane. CXCR4 is internalized through different endocytic pathways depending on the epithelial or mesenchymal profile of the PLC/PRF/5 and Hep3B HCC cells. While epithelial-like HCC cells internalize CXCR4 through dynamin-dependent pathways, concretely clathrin- and caveolin-dependent pathways, HCC cells displaying some mesenchymal characteristics use dynamin-independent endocytosis mechanisms, particularly macropinocytosis. SDF-1α induces the internalization of CXCR4 from plasma membrane to recycling endosomes. A common Rab11+ recycling pathway is used by HCC cells while a rapid Rab4+/Rab5+ recycling pathway is only used by HCC cells with an epithelial-like phenotype. Otherwise, EMT can be induced by affecting the expression and function of Par3 and Par6 proteins, but mechanisms regulating Par proteins remain obscure. Here we found out that the loss of Par3 in PLC/PRF/5 cells implies a disruption of tight junctions, as could be expected, but also a disruption of adherens junctions. Furthermore, none of the analyzed EMT transcription factors were up-regulated after Par3-silencing while vimentin expression behaved as expected after TGF-β treatment, indicating that the loss of Par3 may trigger an EMT process independently of classical EMT transcription factors. Finally, the loss of Par3 facilitates migration of epithelial PLC/PRF/5 cells. However, TGF-β- induced cell motility was found to triplicate the migratory ability of PLC/PRF/5 cells when compared to the Par3-silenced ones. This data fits with the fact that the down-regulation of Par3 is able to disrupt cell-cell junctions and decrease epithelial marker levels but it is unable to induce mesenchymal features, contrary to TGF-β-treated cells, which actually increased vimentin mRNA levels. Taken together, it is suggested that additional mechanisms needed for a fully efficient migration that are triggered by TGF-β are not regulated by Par3.CXCR4 es un receptor de quimiocinas implicado en procesos fisiológicos, como la homeostasis del sistema inmune y la hematopoyesis, así como en procesos patológicos, destacando su papel como inductor de la migración celular en respuesta a gradientes de SDF-1alfa, su ligando específico, en células tumorales. El tráfico intracelular de los receptores de membrana constituye uno de los mecanismos de regulación de su actividad. Asimismo, los mecanismos que controlan la polaridad celular son determinantes en la localización y, consecuentemente, en la actividad de los receptores. Se ha descrito que TGF-β juega un papel determinante en la regulación tanto del tráfico intracelular como de la polaridad celular. En este trabajo se analizan los mecanismos que regulan la exportación asimétrica de CXCR4 en la membrana plasmática de células de carcinoma hepatocelular celular (CHC) humano y su correlación con los mecanismos que controlan la polaridad celular. Describimos que CXCR4 se encuentra en la superficie celular de células de CHC aunque se localiza mayoritariamente en compartimentos intracelulares. La sobreactivación de la vía del TGF-β promueve la localización de CXCR4 en la superficie celular. Asimismo, se describe la vía exocítica de CXCR4 en células de CHC, viajando a través del eje Golgi-AP1- exocisto. Además, se demuestra la necesidad de la funcionalidad del exocisto en el anclaje de la vesícula exocítica en la membrana plasmática para la exportación de CXCR4. También se analizan las vías de internalización de CXCR4, observándose que las células PLC/PRF/5, con un perfil epitelial, utilizan la vía dinamina-dependiente; mientras que las Hep3B, con características mesenquimales, utilizan la macropinocitosis como vía endocítica. SDF-1alfa provoca la internalización de CXCR4 y su posterior reciclaje a través de vesículas Rab11-positivas en ambas líneas celulares, pero sólo en las PLC/PRF/5 se observa reciclaje (rápido) por la vía de Rab4. Otro aspecto relevante es la regulación de la localización subcelular de PAR3 por TGF-β dependiendo de las características epiteliales/mesenquimales de las células de CHC. La inhibición de la vía del TGF-β en células mesenquimales promueve la localización de PAR3 en la membrana plasmática coincidiendo con la formación de uniones célula-célula. Finalmente, definimos que PAR3 juega un papel crucial en el mantenimiento del fenotipo epitelial en células de CHC estabilizando las uniones célula-célula (tanto las "tight" como las "adherens junctions") independientemente de la expresión de los factores de transcripción clásicos de transición epitelio- mesénquima. Además, la localización de PAR3 en la membrana plasmática previene la distribución asimétrica de CXCR4 en el citoplasma de las células PLC/PRF/5, inhibiendo así la migración celular