SummaryThe mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood. Here, we show that the growth of tumors formed by mutant BrafV600E mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation. Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in BrafV600E mouse melanoma cells, as well as in NrasG12D melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways. This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species. Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.

Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (PKC), the major cellular receptor for this class of agents. We demonstrate here that this proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogenactivated protein kinase (ERK/MAPK) cascade. It is shown that dominant-negative PKC-␣ inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (␣, ␤ 1 , ␦, , , and ) were employed. Transient transfection of these PKC mutants into Cos-7 cells showed that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes ␣ and ) are potent activators of c-Raf1, atypical PKC-cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-␣ and PKC-was abrogated for RafCAAX, which is a membrane-localized, partially active form of c-Raf1. We further established that activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, we describe a novel Raf desensitization induced by PKC-␣, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in 12-Otetradecanoylphorbol-13-acetate induced mitogenesis and provide evidence for multiple PKC controls acting on this MAPK cascade.To date, 11 members of the protein kinase C (PKC) superfamily have been identified (for reviews, see references 13, 28, 45, and 52). On the basis of their biochemical properties and sequence homologies, they have been divided into three groups: the conventional PKCs (cPKC-␣, -␤ 1 , -␤ 2 , and -␥), which are activated in a diacylglycerol (DAG)-and calciumdependent manner; the calcium-independent but DAG-dependent novel PKCs (nPKC-␦, -ε, -, -, and -, also termed PKD); and a third group consisting of atypical PKCs (aPKCand -/). The members of this last group of isotypes are unresponsive to DAG and calcium and, in contrast to c-and nPKCs, do not respond to phorbol esters. The existence of this large family of PKC isotypes suggests that individual PKC isotypes likely have specific roles in signal transduction. We have been interested in determining if such specificity exists in the case of the extracellular signal-regulated kinase/mitogenactivated protein kinase (ERK/MAPK) cascade, by which PKC may mediate some of its effects on cell growth and differentiation.The MAPK cascade, which involves the kinases Raf, MAPK/ ERK kinase (MEK), and ERK/MAPK, is ubiquitously expressed in mammalian cells and serves to couple various cell surface ...

SummaryIntravital imaging of BRAF-mutant melanoma cells containing an ERK/MAPK biosensor reveals how the tumor microenvironment affects response to BRAF inhibition by PLX4720. Initially, melanoma cells respond to PLX4720, but rapid reactivation of ERK/MAPK is observed in areas of high stromal density. This is linked to “paradoxical” activation of melanoma-associated fibroblasts by PLX4720 and the promotion of matrix production and remodeling leading to elevated integrin β1/FAK/Src signaling in melanoma cells. Fibronectin-rich matrices with 3–12 kPa elastic modulus are sufficient to provide PLX4720 tolerance. Co-inhibition of BRAF and FAK abolished ERK reactivation and led to more effective control of BRAF-mutant melanoma. We propose that paradoxically activated MAFs provide a “safe haven” for melanoma cells to tolerate BRAF inhibition.

A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras‐dependent activation of a protein kinase cascade consisting of Raf‐1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf‐1, we have examined the properties of p74Raf‐1 and oncogenic Src that are necessary for activation of p74Raf‐1. We show that in mammalian cells activation of p74Raf‐1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf‐1 to interact with p21Ras‐GTP. The Ras/Raf interaction is required for p21Ras‐GTP to bring p74Raf‐1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane‐bound pp60Src. When oncogenic Src is expressed with Raf‐1, p74Raf‐1 is activated 5‐fold; however, when co‐expressed with oncogenic Ras and Src, Raf‐1 is activated 25‐fold and this is associated with a further 3‐fold increase in tyrosine phosphorylation. Thus, p21Ras‐GTP is the limiting component in bringing p74Raf‐1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf‐1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras‐GTP recruiting p74Raf‐1 to the plasma membrane. Thus, the role of Ras in Raf‐1 activation is to bring p74Raf‐1 to the plasma membrane for at least two different activation steps.