G Protein-Coupled Receptors in Cancer.

Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of "cancer driver" GPCRs. Emerging data on GPCR biology point to functional selectivity and "biased agonism"; hence, there is a diminishing enthusiasm for the concept of "one drug per GPCR target" and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.

Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer.

Mammalian protease-activated-receptor-1 and -2 (PAR1 and PAR2) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR1 and PAR2 act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR2 devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR1 signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR1-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR1 pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR1 ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR2 was utilized in a xenograft mouse model, it inhibited PAR1-PAR2-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR1-C-tail in vitro and decreased markedly selective PAR1-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR1 and PAR2 in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR1-PAR2 complex formation but no PAR1-CXCR4 complex was formed. Taken together, our observations show that PAR1 and PAR2 act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome.

The GTPase domain of gamma-tubulin is required for normal mitochondrial function and spatial organization.

In the cell, γ-tubulin establishes a cellular network of threads named the γ-string meshwork. However, the functions of this meshwork remain to be determined. We investigated the traits of the meshwork and show that γ-strings have the ability to connect the cytoplasm and the mitochondrial DNA together. We also show that γ-tubulin has a role in the maintenance of the mitochondrial network and functions as reduced levels of γ-tubulin or impairment of its GTPase domain disrupts the mitochondrial network and alters both their respiratory capacity and the expression of mitochondrial-related genes. By contrast, reduced mitochondrial number or increased protein levels of γ-tubulin DNA-binding domain enhanced the association of γ-tubulin with mitochondria. Our results demonstrate that γ-tubulin is an important mitochondrial structural component that maintains the mitochondrial network, providing mitochondria with a cellular infrastructure. We propose that γ-tubulin provides a cytoskeletal element that gives form to the mitochondrial network.

Low-density lipoprotein receptor-related protein 6 is a novel coreceptor of protease-activated receptor-2 in the dynamics of cancer-associated ß-catenin stabilization.

Protease-activated receptor-2 (PAR2) plays a central role in cancer; however, the molecular machinery of PAR2-instigated tumors remains to be elucidated. We show that PAR2 is a potent inducer of ß-catenin stabilization, a core process in cancer biology, leading to its transcriptional activity. Novel association of low-density lipoprotein-related protein 6 (LRP6), a known coreceptor of Frizzleds (Fz), with PAR2 takes place following PAR2 activation. The association between PAR2 and LRP6 was demonstrated employing co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), and confocal microscopy analysis. The association was further supported by ZDOCK protein-protein server. PAR2-LRP6 interaction promotes rapid phosphorylation of LRP6, which results in the recruitment of Axin. Confocal microscopy of PAR2-driven mammary gland tumors in vivo, as well as in vitro confirms the association between PAR2 and LRP6. Indeed, shRNA silencing of LRP6 potently inhibits PAR2-induced ß-catenin stabilization, demonstrating its critical role in the induced path. We have previously shown a novel link between protease-activated receptor-1 (PAR1) and ß-catenin stabilization, both in a transgenic (tg) mouse model with overexpression of human PAR1 (hPar1) in the mammary glands, and in cancer epithelial cell lines. Unlike in PAR1-Gα13 axis, both Gα12 and Gα13 are equally involved in PAR2-induced ß-catenin stabilization. Disheveled (DVL) is translocated to the cell nucleus through the DVL-PDZ domain. Collectively, our data demonstrate a novel PAR2-LRP6-Axin interaction as a key axis of PAR2-induced ß-catenin stabilization in cancer. This newly described axis enhances our understanding of cancer biology, and opens new avenues for future development of anti-cancer therapies.