Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

Abstract

Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4β1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4β1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis.

Dual-activity PI3K–BRD4 inhibitor for the orthogonal inhibition of MYC to block tumor growth and metastasis

“…The human neuroblastoma cell line SKNBE2 was obtained from ATCC. The Panc02 cell line from C57BL/6 mice has been previously described (34). All cell lines were tested for mycoplasma contamination and grown in DMEM (Invitrogen) supplemented with 10% FBS, 2 mM glutamine, and 1% penicillin-streptomycin at 37°C in a 5% CO 2 atmosphere.…”

“…For spontaneous metastasis, orthotopic pancreatic tumors were initiated by implanting 1 × 10 6 Panc02 into the pancreas of syngeneic mice as described before (34). After 20 d of tumor implantation, mice were treated with either (i ) 30 mg/kg of SF2523 formulated in 15% DMA + 30% captisol, (ii ) 30 mg/kg of JQ1 formulated in 30% captisol in combination with 30 mg/kg of BKM120 formulated in 15% ethanol + 15% cremaphore, (iii) vehicle (15% ethanol + 15% cremaphore, as control), or (iv) another vehicle (15% DMA + 30% captisol, as control) five times a week, until tumors were removed on day 35.…”

A Macrophage-Dominant PI3K Isoform Controls Hypoxia-Induced HIF1α and HIF2α Stability and Tumor Growth, Angiogenesis, and Metastasis

“…Isolation of bone marrow-derived macrophages, hypoxia and in vitro inhibitor experiments Bone marrow-derived macrophages (BMDM) were isolated as described previously and cultured under MCSF (75 ng/mL) þ 20% FBS conditions (31). On day 7, most of the adherent cells were macrophages as confirmed by FACS analysis (>90% Mac1-and F4/80-positive cells).…”

“…cDNA (2 mL) was amplified by RT-PCR reactions with 1Â SYBR green supermix (Bio-) in 96-well plates on an CFX96 Real time system (Bio-Rad), using different primers for mouse gene. Sequence of primers used were reported before (31). Relative expression levels were normalized to GAPDH expression according to the formula: 2…”

“…Lewis lung carcinoma (LLC) and B16 F10 melanoma obtained from the ATCC were cultured as described previously (31). LLC cells (1 Â 10 5 ) were injected subcutaneously into syngeneic 4-to 6-week-old mice.…”