Scientists say that they have discovered that a single protein may control tumor inflammation and progression. They claim that tumor- derived chemoattractants that stimulate myeloid cell receptor tyrosine kinases (RTKs), toll-like/IL-1 receptors (TLR/IL1Rs), and GPCRs unexpectedly activate a single PI3-kinase isoform known as p110γ and a single integrin called α4β1 to promote myeloid cell recruitment to tumors and tumor progression.
The team, based at the University of California, San Diego (UCSD) and University of Torino in Italy, reports their findings in Cancer Cell. The paper is titled “Receptor Tyrosine Kinases and TLR/IL1Rs Unexpectedly Activate Myeloid Cell PI3Kg, A Single Convergent Point Promoting Tumor Inflammation and Progression.” Their work overturns the widely held tenet that p110γ is only activated by GPCRs, says USCD’s Judith A. Varner, Ph.D., and colleagues.
The team first confirmed that CD11b+ myeloid cells are recruited to and extensively populate a range of human and murine tumors, including murine and human breast, pancreatic, and lung carcinomas but not corresponding normal tissues. These cells persistently invaded growing tumors over time until as much as 25% of a tumor’s mass comprised myeloid cells.
Furthermore, tumor inflammation was directly proportional to angiogenesis throughout the growth of the tumor. Additional studies showed that inflammatory factors in the tumor microenvironment were derived from both tumor and inflammatory cells.
As most tumors produce multiple chemoattractants, the researchers then looked to see whether a common mechanism was involved in regulating myeloid cell recruitment to tumors. They tested the ability of chemoattractants to promote myeloid cell adhesion to endothelial cells (ECs) in vivo and in vitro.
They took this approach because in order to exit the blood stream in response to signals released from diseased tissues, immune cells transiently adhere to and transmigrate through vascular endothelium, a process that depends on adhesion to EC receptors. A diverse range of tumor-derived chemoattractants was able to tigger EC adhesion by myeloid cells.
Previous studies have shown that the myeloid cell receptors α4β1 and αMβ2 are involved in recognizing the EC surface adhesion proteins VCAM-1 and ICAM-1 and that blocking α4β1 but not αMβ2 suppresses murine and human myeloid cell adhesion to ECs. Dr. Varner’s team confirmed that murine myeloid cells in which integrin α4 expression was ablated by siRNA also failed to adhere to EC and the EC surface adhesion protein VCAM-1.
“As chemoattractants had no effect on EC expression of α4 ligands during these assays, these results indicate that diverse chemoattractants stimulate myeloid cell adhesion by selectively increasing integrin α4β1 but not αMβ2,” the authors note.
Indeed, further investigation indicated that chemokines, cytokines, and growth factors that activate RTKs, TLR/IL1Rs, and GPCRs all activate integrin α4β1, thereby promoting murine and human myeloid cell adhesion to ECs. Importantly, knocking out integrin α4 in tumors implanted in mice provide evidence that integrin α4 activation is required for trafficking and infiltration of myeloid cells into tumors in vivo.
The observation that the chemoattractants stimulating structurally diverse GPCRs, RTKs, TLR/IL1Rs, and type I cytokine receptors all activate myeloid cell integrin α4β1 indicates that a common downstream signaling pathway might link these receptors, the authors write. When they evaluated inhibitors of various signaling pathways using myeloid cell adhesion assays, they found that selective inhibitors of PI3Kγ (p110γ) and Ras GTPases blocked myeloid cell adhesion to endothelium, while inhibitors of other PI3K isoforms and signaling proteins had no effect on adhesion.
The class IB PI3K p110γ has been well-characterized as a GPCR-activated lipid kinase that promotes chemokine-stimulated chemotaxis and polarization of neutrophils, lymphocytes, and thymocytes in vitro and in vivo. p110γ-knockout mice and animals that express an inactivated form of p110γ have been shown to exhibit defects in granulocyte responses to chemokines.
Previous studies have in addition shown that p110γ is essential for GPCR- but not RTK-mediated activation of PI3K activity, which makes the new observation that p110γ inhibitors blocked adhesion in response to GPCR as well as RTK ligands particularly unexpected, the team stresses.
Using p110γ-specific knockout or inhibition methods, the researchers went on to demonstrate that growth factors, interleukins, and chemokines, which are ligands for RTKs, TLR/IL-1Rs, and GPCRs, promoted adhesion of wild-type, but not of P110γ-knockout myeloid cells or those with inactive P110γ to ECs or VCAM-1. Given that the knockdown cells still expressed normal levels of α4 integrin, these results indicated that p110γ is necessary for integrin adhesive activity. siRNA knockdown of p110γ but not other PI3K catalytic subunits also suppressed adhesion to ECs or VCAM-1, regardless of the stimulus, the authors add.
“Although prior studies have indicated that only GPCR ligands activate p110γ, our results indicate that ligands for RTKs and TLR/IL1Rs promote p110γ activity and p110γ-dependent integrin α4β1 activation,” they claim. “Importantly, p110γ is also sufficient for integrin α4β1 activation as cells from p110γCAAX mice, which express membrane-targeted, constitutively activated p110γ adhered strongly to ECs even in the absence of stimulation.”
In a further set of studies the team went on to confirm the pivotal role of p110γ but not other PI3K isoforms in promoting α4β1-mediated myeloid cell trafficking to tumors in vivo. They surprisingly found that p110γ was the major PI3K catalytic isoform expressed in primary myeloid cells, whereas lymphocytes express large amounts of p110δ, while LLC cells express large amounts of p110α and β and little γ or δ. Additional in vitro studies demonstrated that growth factors and cytokines activate p110γ directly, rather than indirectly through GPCRs.
The combined collection of findings made particular sense when viewed in a clinical context. The researchers tested the efficacy of a selective p110γ inhibitor or a pan-PI3K inhibitor, in tumor-bearing mice. While both inhibitors suppressed lung cancer inflammation angiogenesis and tumor growth in vivo, activity of the P110γ inhibitor was related to the inhibition of tumor inflammation and angiogenesis, without directly affecting the tumor cells themselves.
While a number of clinical trials are already under way to test pan-PI3K inhibitors in cancer patients, “our studies indicate that p110γ is an excellent target for a relatively nontoxic cancer therapeutic, as this isoform is primarily expressed by myeloid cells and is a convergence point of diverse chemoattractant signaling pathways that are required for tumor inflammation and tumor progression,” the authors conclude.