PI3K-Akt signaling is critical for the development, progression, and metastasis of malignant tumors, but its role in the tumor microenvironment has been relatively little studied. Here, we report that the Akt substrate Girdin, an actin-binding protein that regulates cell migration, is expressed and activated by Akt phosphorylation in cancer-associated fibroblasts (CAF) and blood vessels within the tumor microenvironment. Lewis lung tumors grafted into mice defective in Akt-mediated Girdin phosphorylation (SA transgenic mice) exhibited a decrease in both CAF infiltration and tumor growth, compared with wild-type (WT) host control animals. Contrasting with the findings of other studies, we found that Akt-dependent phosphorylation of Girdin was not a rate-limiting step in the growth of endothelial cells. In addition, Lewis lung tumors displayed limited outgrowth when cotransplanted with CAF derived from tumor-bearing SA transgenic mice, compared with CAF derived from tumor-bearing WT mice. Collectively, our results revealed a role for Akt-mediated Girdin phosphorylation in CAF during tumor progression, highlighting the need to inhibit Akt function in both tumor cells and cells that comprise the tumor microenvironment. Cancer Res; 75(5); 813-23. Ó2015 AACR.

No abstract

RET is a tyrosine kinase receptor that plays an important role in the development of the enteric nervous system and the kidney (1, 2). It has been demonstrated that members of the glial cell line-derived neurotrophic factor (GDNF) 1 family including GDNF, neurturin, artemin, and persephin represent the RET ligands. The RET activation by these neurotrophic factors are mediated by their binding to glycosylphosphatidylinositol-anchored co-receptors termed GDNF family receptor ␣ 1-4 (GFR␣ 1-4) (1, 2). GDNF, neurturin, artemin, and persephin use GFR␣ 1, GFR␣ 2, GFR␣ 3, and GFR␣ 4 as the preferred receptors, respectively, and play specific roles in vivo through these preferred ligand-receptor complex formation. For example, Gdnf-or Gfr␣1-deficient mice had the phenotype quite similar to that of Ret-deficient mice, exhibiting the defects of enteric neurons as well as renal agenesis or dysgenesis (3-8). In Nrtn-or Gfra2-deficient mice, the parasympathetic innervation was markedly reduced in the lachrymal and submandibular glands and the intestine (9, 10). Gfr␣3-deficient mice showed severe defect of the superior cervical ganglion (11).RET mutations are responsible for development of several human diseases including Hirschsprung's disease, multiple endocrine neoplasia (MEN) type 2A and 2B, familial medullary thyroid carcinoma (FMTC), and papillary thyroid carcinoma (PTC) (1, 2, 12). Loss-of-function mutations of RET lead to the development of Hirschsprung's disease (13, 14), a malformation characterized by the absence of autonomous enteric neurons. On the other hand, gain-of-function mutations of RET contribute to the development of human neoplastic diseases including MEN 2A, MEN 2B, FMTC, and PTC (12,15,16). MEN 2A, MEN 2B, and FMTC are caused by germ-line point mutations of RET, and PTC is caused by its somatic rearrangement. MEN 2A and MEN 2B share the clinical feature of medullary thyroid carcinoma (MTC) and pheochromocytoma, and FMTC is characterized by the development of MTC alone. In addition, ϳ10 -30% of MEN 2A patients develop parathyroid hyperplasia, whereas MEN 2B patients show a more complex phenotype including ganglioneuromatosis of the gastrointestinal tract, mucosal neuroma, and marfanoid habitus. MTCs developed in MEN 2B appear more aggressive than those in MEN 2A and FMTC.Activated RET recruits a variety of signaling molecules including Grb2, Grb7, Grb10, Shc, Enigma, SNT/Frs2, Dok, insulin receptor substrate-1 (IRS-1), and phospholipase C␥ (17-29). As is the case for other receptor tyrosine kinases, phosphorylated tyrosine residues in the kinase domain and carboxyl-terminal tail of RET represent docking sites for these molecules. For example, phosphorylated tyrosines 905, 1015, and 1096 were identified as docking sites for Grb7/Grb10, phospholipase C␥, and Grb2, respectively (17,20,28,29). Intriguingly, several signaling molecules including Shc, Enigma, SNT/ Frs2, Dok, and IRS-1 bind to phosphorylated tyrosine 1062 (18, 19, 21-23, 25, 26) through which the Ras/Erk, PI3-K/Akt, p38MAPK, c-Jun amino-term...

Rationale: It is well established that the migration and proliferation of vascular smooth muscle cells (VSMCs) have major roles in the vascular remodeling process. Our previous study showed that the Akt substrate Girdin, which is expressed in VSMCs and endothelial cells, is essential for postnatal angiogenesis. However, the function of Girdin and its Akt-mediated phosphorylation in VSMCs and their in vivo roles in vascular remodeling remain to be elucidated. Objective:We investigated the function of Girdin and its Akt-mediated phosphorylation using cultured VSMCs and animal models of vascular remodeling. Methods and Results:The depletion of Girdin by RNA interference disrupted the rearrangement of the actin cytoskeleton in VSMCs, resulting in impaired cell migration. The depletion of Girdin also inhibited VSMC proliferation. Girdin expression was highly upregulated and its serine at position 1416 was phosphorylated in the neointima of carotid arteries after balloon injury in a rat model. The introduction of an adenovirus harboring short hairpin RNA against Girdin attenuated the proliferation of VSMCs and neointima formation without affecting reendothelialization. Furthermore, we found that neointima formation after femoral wire injury was significantly attenuated in Girdin S1416A knock-in mice, in which the Akt phosphorylation site of Girdin was mutated, thus indicating a major role for Girdin phosphorylation in vascular remodeling. Conclusions: These findings indicate that Girdin and its

Angiogenic cell therapy represents a novel strategy for ischemic diseases, but some patients show poor responses. We investigated the therapeutic potential of an induced pluripotent stem (iPS) cell sheet created by a novel magnetite tissue engineering technology (Mag-TE) for reparative angiogenesis. Mouse iPS cell-derived Flk-1+ cells were incubated with magnetic nanoparticle-containing liposomes (MCLs). MCL-labeled Flk-1+ cells were mixed with diluted extracellular matrix (ECM) precursor and a magnet was placed on the reverse side. Magnetized Flk-1+ cells formed multi-layered cell sheets according to magnetic force. Implantation of the Flk-1+ cell sheet accelerated revascularization of ischemic hindlimbs relative to the contralateral limbs in nude mice as measured by laser Doppler blood flow and capillary density analyses. The Flk-1+ cell sheet also increased the expressions of VEGF and bFGF in ischemic tissue. iPS cell-derived Flk-1+ cell sheets created by this novel Mag-TE method represent a promising new modality for therapeutic angiogenesis.