PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
 
Cell. 2017 November 30; 171(6): 1301–1315.e14.
PMCID: PMC5720393

Myc Cooperates with Ras by Programming Inflammation and Immune Suppression

Summary

The two oncogenes KRas and Myc cooperate to drive tumorigenesis, but the mechanism underlying this remains unclear. In a mouse lung model of KRasG12D-driven adenomas, we find that co-activation of Myc drives the immediate transition to highly proliferative and invasive adenocarcinomas marked by highly inflammatory, angiogenic, and immune-suppressed stroma. We identify epithelial-derived signaling molecules CCL9 and IL-23 as the principal instructing signals for stromal reprogramming. CCL9 mediates recruitment of macrophages, angiogenesis, and PD-L1-dependent expulsion of T and B cells. IL-23 orchestrates exclusion of adaptive T and B cells and innate immune NK cells. Co-blockade of both CCL9 and IL-23 abrogates Myc-induced tumor progression. Subsequent deactivation of Myc in established adenocarcinomas triggers immediate reversal of all stromal changes and tumor regression, which are independent of CD4+CD8+ T cells but substantially dependent on returning NK cells. We show that Myc extensively programs an immune suppressive stroma that is obligatory for tumor progression.

Keywords: Myc, oncogene cooperation, lung cancer, tumor microenvironment, IL-23, CCL9, immune suppression, Ras, NK cells, inflammation

Graphical Abstract

An external file that holds a picture, illustration, etc.
Object name is fx1.jpg

Introduction

The 1983 discovery of oncogenic cooperation between activated Ras and Myc (Land et al., 1983) demonstrated the obligate interdependence of individual oncogenic mutations. However, 30 years on, the mechanisms underlying Ras/Myc cooperation remain elusive. Early studies focused solely on Ras and Myc’s complementary cell-intrinsic outputs, such as their co-induction and/or stabilization of cell cycle proteins (Born et al., 1994, Leone et al., 1997, Wang et al., 2011) and Myc itself (Sears et al., 2000), on the reciprocal abrogation of Ras-induced senescence by Myc (Evan and Littlewood, 1998, Vaqué et al., 2005) and Ras-dependent suppression of Myc-induced apoptosis (Evan and Littlewood, 1998, Tsuneoka and Mekada, 2000), or on the capacity of Myc to overcome blockade to self-renewal in Ras-driven cells (Dong et al., 2011, Ischenko et al., 2013). However, co-transgenic in vivo studies in mice demonstrated that oncogenic cooperation between Ras and Myc in diverse tissues (Alexander et al., 1989, Andres et al., 1988, Boxer et al., 2004, Compere et al., 1989, Podsypanina et al., 2008, Tran et al., 2008, Yaari et al., 2005) necessarily involves modulation of interactions between the tumor cells and their stroma.

Both Ras and Myc are implicated in human non-small cell lung cancer (NSCLC). Oncogenic KRas mutations are thought to be causal drivers in ~30% of NSCLC, especially in smokers (Rodenhuis et al., 1988), and correlate with poor prognosis (Graziano et al., 1999). Mutant KRas is principally thought to drive precocious mitogenic signaling but is also credited with reprogramming tumor cell metabolism (Kimmelman, 2015), suppressing apoptosis (Cox and Der, 2003, Kauffmann-Zeh et al., 1997) and promoting migration, metastasis (Campbell and Der, 2004), angiogenesis (Kranenburg et al., 2004, Sparmann and Bar-Sagi, 2004), and inflammation (Karin, 2005)—the latter two presumably by indirect signaling, since oncogenic Ras is confined to the epithelial tumor cell compartment. Indeed, oncogenic KRas is a potent inducer of various cytokines in many tumor types, including lung, where IL-8 (CXCL8) and IL-6 both contribute to lung cancer’s signature inflammatory phenotype (Ancrile et al., 2008, Campbell and Der, 2004, Ji et al., 2006, Kranenburg et al., 2004, Sparmann and Bar-Sagi, 2004, Sunaga et al., 2012). Aberrant Myc expression is also implicated in lung cancer. It is demonstrably overexpressed in >70% of NSCLC (Richardson and Johnson, 1993), with overt Myc gene amplification in the ~20% of tumors with poorest prognosis (Iwakawa et al., 2011, Seo et al., 2014, Wolfer et al., 2010). Precocious Myc activity is causally implicated in cancers principally through its capacity to drive tumor cell proliferation; engage biosynthetic cell metabolism; and promote angiogenesis, invasion, and metastasis (Dang, 2013, Rapp et al., 2009, Shchors et al., 2006, Sodir et al., 2011, Wolfer et al., 2010). Even in NSCLC not overtly driven by mutations in Ras or Myc themselves, endogenous Ras and Myc both play prominent, even obligate, roles as downstream conduits for diverse upstream oncogenic drivers.

Here, we specifically explore the cooperative contribution made by Myc deregulation to the evolution and progression of KRasG12D-driven lung tumors in vivo. Using a rapidly and reversibly switchable genetic system (Murphy et al., 2008), we define the causal sequence of events by which Myc drives conversion of indolent adenomas to aggressive, inflammatory, and immune-suppressed adenocarcinomas and by which subsequent Myc de-activation triggers tumor regression. Our data show a profound role for Myc in re-programming the tumor microenvironment—especially the inflammatory and immune components of tumor stroma.

Results

Myc Deregulation, without Elevated Expression, Cooperates with KRasG12D to Accelerate Lung Tumorigenesis In Vivo

To investigate Myc cooperation with KRasG12D in vivo, we used mice heterozygous for the LSL-KRasG12D allele (Jackson et al., 2001) and homozygous for Rosa26-lox-STOP-lox MycERT2 (R26LSLMycERT2) (Murphy et al., 2008). In these LSL-KRasG12D;Rosa26-lox-STOP-lox MycERT2 mice (hereafter referred to as KM), inhalation of Cre-recombinase-expressing adenovirus (AdV) triggers sporadic coincident expression in lung of oncogenic KRasG12D from its endogenous promoter and reversibly activatable 4-OHT-dependent MycERT2 driven from the constitutively active Rosa26 promoter at low, quasi-physiological levels (Murphy et al., 2008).

As reported (Jackson et al., 2001), activation of endogenous KRasG12D alone in lung epithelium elicits slow outgrowth of multiple independent lesions. Multiple small foci of atypical epithelial and adenomatous hyperplasia are evident by 6 weeks after AdV-Cre inhalation, progressing to indolent and non-invasive adenomas by 12–18 weeks. Aggressive and invasive adenocarcinomas emerge sporadically much later, presumably through additional oncogenic lesions. Activation of MycERT2 (for 6 weeks) in 12-week-old indolent KRasG12D-driven lesions dramatically accelerated tumorigenesis over that in KRasG12D-only controls (Figures 1A, A,S1A,S1A, A,S2A,S2A, and S2B), resulting in highly proliferative, invasive tumors heavily infiltrated with leukocytes and with inchoate, nascent vasculature indicative of ongoing angiogenesis (Figures 1B–1D). Myc activation profoundly accelerated lung tumor progression at every stage of adenoma evolution (Figures S2A and S2B), triggering a precipitous drop in survival (Figure S2C). Activation of MycERT2 in the absence of KRasG12D elicited no discernible lung phenotype (Figure S2D), while tamoxifen treatment alone had no effect on KRasG12D-only lung tumors (not shown). The aggressive phenotypes of KRasG12D tumors following MycERT2 activation were indistinguishable from those of KRasG12D tumors driven by constitutive Rosa26-driven Myc (not shown).

Figure 1
Deregulated Myc Cooperates Oncogenically with KRasG12D in Lung
Figure S1
Schematic Representations of Animal Experiments, Related to Figures 1, ,2,2, ,3,3, ,4,4, ,5,5, ,6,6, and and77
Figure S2
Deregulated Myc Cooperates Oncogenically with KRasG12D at All Stages of Lung Adenoma Evolution, Related to Figure 1

Myc Deregulation in Tumor Cells Rapidly Induces Diverse Stromal Changes

The lung tumors resulting from long-term combined KRasG12D and Myc activity are more advanced and aggressive than the indolent tumors driven by KRasG12D alone, being markedly more proliferative, invasive, inflammatory, and angiogenic. Such dramatic stromal changes could either be indirect, passive consequences of the disruption of normal lung architecture caused by Myc-driven tumor growth or, alternatively, direct proximal consequences of Myc activation within the adenoma epithelial compartment. Hence, to determine how Myc activation causally elicits its diverse phenotypic changes, we used the rapid, synchronous, and reversible in vivo activation possible with MycERT2 (Pelengaris et al., 2002, Shchors et al., 2006, Sodir et al., 2011). Matched cohorts of adult KM mice were infected with AdV-Cre, and 12 weeks later, tamoxifen was systemically administered to activate Myc in the epithelial cells of the incipient lung adenomas (Figure S1B). Myc activation triggered proliferation broadly across the whole adenoma mass (Figure 2A), as well as immediate and widespread changes in the tumor-associated stroma. Within 24 hr of Myc activation, we saw a dramatic influx of CD206+ macrophages (Figure 2B) into tumor masses and concurrent loss of CD3+ T (Figure 2B) and B220+ B cells (Figure S3A). We also saw a rapid decline of NKp46+ natural killer (NK) cells (Figure 2B) from the juxta-tumoral blood vessels and tertiary lymphoid structures (TLS) to which they principally localize (Fridman et al., 2012, Goc et al., 2013, Joshi et al., 2015) and a fall in total NK cell numbers in the entire lungs (Figure S3B). This rapid expulsion of NK cells is especially intriguing given that Myc activation also triggered a profound upregulation of Rae-1 NKG2D ligands and downregulation of major histocompatibility complex (MHC) class I (Figure S3C)—both potent activating signals for NK-like cells (Morvan and Lanier, 2016)—in lung adenoma cells. Finally, Myc activation triggered the abrupt onset of angiogenesis, marked by loss of vessel integrity, increased vessel leakiness, and relief of the widespread hypoxia characteristic of the original indolent adenomas (Figures 2C and andS3D).S3D). All of these diverse changes in tumor and stroma preceded overt Myc-induced tumor expansion, and all were sustained so long as Myc activity was maintained (see below).

Figure 2
Deregulation of Myc in Epithelial Adenoma Cells Immediately Re-programs the Tumor Stroma
Figure S3
Deregulated Myc Rapidly Re-programs Tumor Immunity, Related to Figure 2

Myc-Induced Stromal Changes in Lung Tumors Are Instructed by CCL9 and IL-23

To exclude the possibility that some of the phenotype we observe might be a consequence of Cre induction of MycERT2 in long-lived resident alveolar macrophages we directly assessed whether MycERT2 is expressed in the lung tumor-associated macrophages using two independent macrophage markers co-expressed on lung tumor-associated macrophages (Figure S3E)—the mannose receptor CD206 and EMR1, a GPCR family adhesion molecule recognized by the F4/80 antibody. First, we showed that abundant nuclear in situ MycERT2 RNA signals, while evident across the entire adenoma epithelial compartment of KM mice, are entirely absent from all CD206+ macrophages (Figure S3F). Second, analysis of lung macrophages harvested from disaggregated KM mouse lungs and purified by F4/80 antibody affinity showed complete absence of MycERT2 mRNA expression, all of which was confined to the F4/80 negative lung cell fraction (Figure S3G). Hence, none of the stromal changes we observe are the result of direct activation of MycERT2 in macrophages.

As MycERT2 is activated only in the epithelial compartment of the KM lung adenomas, the rapid stromal changes that Myc elicits must be mediated by signals released from those epithelial cells. To identify them, we activated MycERT2 in adenoma-bearing KM mice, harvested their lungs after 8 hr, and used whole-lung protein extracts to interrogate an inflammation antibody array. Of 40 potential candidates, only two inflammatory signals were detectably upregulated by Myc: the chemokine CCL9 (a.k.a. MIP-1γ) and the p40 subunit common to IL-12 and IL-23 (Figures 3A and 3B). This upregulation was confirmed immunohistochemically: by 3 days post Myc activation, intense cytoplasmic CCL9 immunostaining and cell surface immunostaining for the p19 subunit of IL-23 were evident (Figure S4A) in TTF1+ epithelial cells (Stenhouse et al., 2004) while absent from both non-tamoxifen-treated controls (Figure S4B) and tamoxifen-treated KRasG12D-only-driven adenomas (not shown). The extremely rapid induction of both CCL9 and IL-23 following epithelial Myc activation, plus their histological co-localization, confirm the adenoma epithelial compartment as the source of CCL9 and IL-23 production and secretion.

Figure 3
IL-23 and CCL9 Mediate Myc-Induced Remodeling of Lung Tumor Stroma
Figure S4
Myc Induces IL-23 and CCL9 in the Epithelial Compartment of Lung Adenomas and Their Continued Expression Is Required to Maintain an Immune-Suppressed Phenotype, Related to Figure 3

To determine whether CCL9 and IL-23 release is the proximal trigger for the stromal changes that Myc activation elicits, adenoma-bearing KM mice were pre-treated with CCL9- and/or IL-23-blocking antibodies and Myc then activated by tamoxifen administration (Figures S1C and S1D). Co-blockade of both CCL9 and IL-23 sharply reduced Myc-induced macrophage influx; blocked the Myc-induced angiogenic switch; and forestalled the loss of T, B, and NK cells (Figures 3C and andS4C).S4C). And while Myc-induced tumor cell proliferation was only modestly suppressed, it was now accompanied by significant tumor cell apoptosis (Figure 3C), resulting in significant reduction in tumor burden by 7 days’ blockade (Figures 3D and 3E). To define the individual roles of CCL9 and IL-23 in mediating Myc-induced stromal changes, we used antibodies to inhibit each signal individually. Blockade of IL-23 had no discernible impact on either Myc-induced macrophage influx or angiogenesis but profoundly blocked Myc-induced loss of T, B, and NK cells; weakly suppressed tumor cell proliferation; and potently exacerbated tumor cell apoptosis (Figure 3C). By contrast, blockade of CCL9 alone profoundly inhibited Myc-induced macrophage influx, angiogenesis, and T cell loss but had no impact on Myc-driven NK cell exclusion, weakly inhibited B cell loss, and only modestly inhibited tumor cell proliferation or exacerbated apoptosis (Figure 3C).

To dissect further the causal sequence by which CCL9 and IL-23 modify lung tumor stroma, we next examined the mechanism behind Myc-induced angiogenesis. The rapid remodeling of tumor vasculature that Myc triggers is a prototypical effect of VEGF, and indeed, immunohistochemistry using the GVM39 monoclonal antibody that specifically recognizes VEGF bound to its cognate VEGFR2 receptor (Brekken et al., 1998) indicated rapid VEGF engagement of endothelial cell VEGFR2 following Myc activation (Figure 4A). To identify the source of VEGF, we assayed VEGFA expression in F4/80-affinity-isolated lung macrophages, since macrophages are established drivers of angiogenesis in diverse tumors (Pollard, 2004). VEGFA mRNA expression was evident in the F4/80+ cell fraction of tamoxifen-treated KM lungs (Figure 4B), but only after MycERT2 activation. By contrast, expression of PD-L1, constitutive in F4/80+ KM lung macrophages (Igarashi et al., 2016), was unaffected by Myc activation status (Figure 4B). Together with our observation that Myc-driven KM lung tumor angiogenesis is completely absent when macrophage influx is abrogated by CCL9 blockade, our data identify macrophage-derived VEGF as the principal instigator of Myc-induced lung adenoma angiogenesis.

Figure 4
Macrophages Mediate Myc-Induced Angiogenesis and T Cell Exclusion

Since CCL9 blockade prevented Myc-induced loss of T cells from KM adenomas, we hypothesized that incoming macrophages might mediate T cell exclusion. Immunostaining showed PD-L1 expression on lung macrophages but absent from the adenoma epithelium, irrespective of Myc status (Figure 4C). After Myc activation, F4/80+ PD-L1+ macrophages rapidly accumulated in the tumors (Figure 4D), coinciding temporally with loss of CD3+ T cells (see Figure 2B). To test directly whether accumulation of PD-L1-positive macrophages was causally responsible for the T cell loss, we activated MycERT2 in adenoma-bearing KM mice while concurrently administering PD-L1-blocking immunoglobulin (Figure S1E). PD-L1 blockade completely abolished Myc-induced T cell loss and partially suppressed B cell loss but had no discernible impact on Myc-induced macrophage influx, angiogenesis, or the rapid decline in juxta-tumoral NK cells (Figure 4E). Moreover, PD-L1 blockade—and consequent persistence of T cells—had no measurable inhibitory impact on tumor cell proliferation (Figure 4E), tumor growth, or tumor burden (Figure 4F).

Taken together, our data delineate a causal sequence whereby Myc activation in lung epithelial adenoma cells triggers rapid release of IL-23 and CCL9. IL-23 drives the rapid exit of T, B, and NK cells, and since IL-23 blockade triggers tumor cell apoptosis, we infer its activity is required for outgrowth of Myc-driven lung adenocarcinomas. CCL9 is principally responsible for the rapid recruitment of PD-L1+ macrophages into the tumor environment whose rapid induction and release of VEGF drives the CCL9-dependent angiogenic switch while the PD-L1 they express is, along with IL-23, required for the rapid loss of T, but not of NK, cells.

Lung Tumors, and Their Associated Stroma, Rapidly Acquire Dependency upon Myc Activity for Their Maintenance

Previous transgenic studies of tumors co-driven by oncogenic Ras and Myc concur that such tumors are dependent on both oncogenes for their maintenance. Acquisition of such dependence, sometimes dubbed “oncogene addiction,” is a widely reported phenomenon, although its underlying mechanistic basis remains unknown.

To address whether KRasG12D-driven lung tumors acquire dependency on Myc, we activated MycERT2 in adenoma-bearing KM mice for 6 weeks and then de-activated it by tamoxifen withdrawal (Figure S1F) (Wilson et al., 2014). Myc de-activation triggered an immediate drop in tumor cell proliferation, rapid exit of macrophages and re-entry of CD3+ T cells, an influx of NKp46+ NK cells, and induction of tumor apoptosis (Figure 5A) and tumor regression, evident by the appearance of voids within the tumor masses and profound drop in overall tumor burden (Figure 5B). Concurrently, tumor blood vessels “normalized” (Figure 5C) and hypoxia rose (Figure S5).

Figure 5
Myc De-activation Triggers Immediate Collapse of Myc-Induced Stroma Together with Tumor Regression
Figure S5
Myc De-activation Immediately Reverses Normoxia in KM Lung Tumors, Related to Figure 5

To ascertain how rapidly Myc dependency arises, Myc was activated in adenoma-bearing KM mice for 7 days—sufficient time to induce all stromal changes and for significant increase in tumor load (Figures 6A and andS1G).S1G). De-activating Myc, even after this short period of Myc activity, triggered an immediate drop in tumor cell proliferation, rapid efflux of macrophages, normalization of vasculature, and repopulation by T and NK cells (Figures 6B and 6C). Hence, the dependency of KRasG12D-driven lung adenocarcinomas on deregulated Myc activity arises immediately across the whole tumor mass and across all tumors. Of note, however, Myc de-activation failed to drive complete tumor regression; rather, the tumors regressed back to their previously indolent KRasG12D-only adenoma state and thereafter persisted indefinitely (Figures 6D and andS1S1H).

Figure 6
Dependency on Myc is Rapidly Acquired by Lung Adenocarcinomas

The tight temporal coincidence between tumor cell apoptosis and re-entry of T and NK cells (Figure 6B) prompted us to address whether T or NK cells play a causal role in Myc-de-activation-induced tumor regression. To investigate this, we depleted KM mice of either CD4+ helper and CD8+ effector T cells or NK cells using appropriate antibodies (Figure S1I). Systemic anti-CD4/CD8 treatment efficiently ablated all detectable circulating and splenic T cells (Figure 7A) and completely abrogated the re-population of tumors by CD3+ T cells following Myc de-activation (Figure 7B). However, absence of CD4+/CD8+ T cells had no measurable inhibitory effect on induction of tumor apoptosis (Figure 7C), nor did it inhibit macrophage efflux or influx of NK cells (Figure S6A). By contrast, systemic depletion of NKp46+ NK cells using asiolo-GM1 antibody (Figures 7A and 7B), while having no impact on Myc deactivation-induced T cell influx (Figure S6B), profoundly inhibited both efflux of macrophages (Figure S6B) and induction of the apoptosis responsible for tumor regression (Figure 7C).

Figure 7
Depletion of NKp46+ Cells, but Not of CD4+ and CD8+ T Cells, Retards Tumor Regression following Myc De-activation
Figure S6
Depletion of NKp46+ Cells, but Not of CD4+ and CD8+ T Cells, Retards Reversion of Myc-Driven Tumor Stromal Changes following Myc De-activation, Related to Figure 7

Discussion

To address the mechanism and dynamics of cooperation between oncogenic KRasG12D and Myc in discrete tissues in vivo, we used KM mice that combine the well-characterized LSL-KRasG12D mouse lung tumor model (Jackson et al., 2001) with our Rosa26-LSL-MycERT2 mouse (Murphy et al., 2008). Inhalation by KM mice of AdV-Cre sporadically co-induces expression of both oncogenic KRasG12D and tamoxifen-switchable MycERT2 in bronchioalveolar epithelium; in the resulting adenomas, oncogenic KRasG12D activity is expressed constitutively while Myc activity may be reversibly superimposed at will. Adeno-Cre activation of KRasG12D alone initiates formation of sporadic dysplasias that slowly progress to indolent adenomas and only rarely and sporadically evolve into aggressive adenocarcinomas. Activation of Myc alone had no observable effect on lung tissues. By contrast, sustained co-activation of Myc drove dramatic progression into highly proliferative, invasive, angiogenic, inflammatory, and lethal adenocarcinomas.

Long-term Myc activation studies provide little mechanistic cause-and-effect information as to how Myc deregulation drives progression of indolent KRasG12D-driven adenomas into aggressive adenocarcinomas. In particular, whether the profound stromal changes that Myc elicits are direct consequences of Myc programming or chronic host tissue responses to intrusion by the tumor is unclear. To distinguish between these two possibilities, we used the remarkably synchronous and rapid in vivo Myc activation afforded by the MycERT2 switchable system (Christophorou et al., 2005, Littlewood et al., 1995, Pelengaris et al., 2002, Shchors et al., 2006, Wilson et al., 2014) to delineate the temporal sequence of events by which Myc activation drives transition to adenocarcinoma.

Acute MycERT2 activation in indolent KRasG12D-driven lung adenomas rapidly reprogrammed both hematopoietic and endothelial stromal compartments, driving rapid influx of CD206+ macrophages prototypically associated with tissue remodeling and immune regulation in diverse tissues (Martinez and Gordon, 2014, Rőszer, 2015) including lung (Aggarwal et al., 2014); rapid loss of T, B, and NK cells; and vasculature remodeling coincident with transition from localized hypoxia to normoxia. These diverse stromal changes preceded any significant expansion of the tumor masses. Hence, the signature “stromatype” of the aggressive adenocarcinomas induced by sustained co-activity of KRasG12D and Myc is an immediate, instructed consequence of Myc activation, not an indirect or generic tissue reaction to disruption by the expanding tumor in its midst.

Since MycERT2 is activated only in the epithelial compartment of KM adenomas, the consequent stromal changes must be mediated by signals originating in that epithelium. A broad search identified only two candidates, CCL9 and IL-23, as induced in lung adenomas with sufficient rapidity (by 8 hr) to be proximal causes of the Myc-induced stromal changes we observe. Both immunohistochemically localized to the TTF1+ epithelial compartment. CCL9, a.k.a. MIP-1γ, is a murine ligand for the CCR1/CD191 receptor present on monocytes, T cells, and some immature CD34+ myeloid-derived suppressor cells (MDSCs). It is a potent chemo-attractant for monocytes, macrophages, and MDSCs (White et al., 2013), while CCR1 ligation is implicated in progression of adenomas to carcinomas (Kitamura et al., 2007, Koizumi et al., 2007) and inflammatory lung pathologies (Kitamura et al., 2015). IL-23 is a pro-inflammatory cytokine that suppresses wound resolution and is pro-tumorigenic in many tissues (Langowski et al., 2006), including lung (Baird et al., 2013). IL-23 is the principal trigger of TH17 lymphocytes, an ROR1γ-dependent T cell subset whose consequent secretion of IL-17 and IL-22 induces, in turn, production of diverse inflammatory cytokines, chemokines, and prostaglandins by many stromal cell types (Gaffen et al., 2014). TH17 cells are also highly immunosuppressive (Bailey et al., 2014, Chang, 2015, Chang et al., 2014). IL-23 is also a potent suppressor of innate immunity, most notably NK cells (Teng et al., 2010). IL-23 shares a common p40 subunit with IL-12 that, in many ways, antagonizes IL-23 in cancers by boosting anti-tumor immunity (Ngiow et al., 2013, Teng et al., 2012) and promoting injury resolution and subsequent tissue re-normalization (Ngiow et al., 2013).

Co-blockade of both CCL9 and IL-23 profoundly inhibited all the diverse stromal changes that Myc elicits, suppressed Myc-induced tumor cell proliferation, and triggered abrupt onset of apoptosis. Blockade of CCL9 alone had little impact on tumor cell proliferation or apoptosis but completely inhibited Myc-induced macrophage influx, angiogenesis, and T cell loss. It also reduced loss of B cells yet had no inhibitory impact on Myc-driven loss of juxta-tumoral NK cells. By contrast, blockade of IL-23 alone had no inhibitory impact on macrophage influx or angiogenesis but strongly suppressed T cell loss, completely blocked the loss of NK and B cells, mildly suppressed tumor cell proliferation, and unlike blockade of CCL9 alone, triggered widespread tumor cell apoptosis. From these studies, we draw several conclusions (Figure S7). First, CCL9 and IL-23 are the principal epithelial signals that instruct the immediate and diverse stromal changes that Myc elicits. Second, while both CCL9 and IL-23 are crucial for the rapid loss of T and B cells following Myc activation, CCL9 alone is required to drive macrophage recruitment and angiogenesis, while IL-23 alone is required for rapid exclusion of NK cells. Third, since blockade of IL-23, but not CCL9, triggers substantial tumor cell apoptosis, IL-23 plays a unique role in survival and maintenance of Myc-driven lung tumors.

Figure S7
Myc Instructs Stromal Changes in Lung Adenomas, Related to Figures 2, ,3,3, ,4,4, and and77

Myc activation rapidly induced a profound angiogenic switch, evident by blood vessel permeability and loss of integrity that coincided with VEGF activity. VEGF was potently induced in the macrophage compartment following Myc activation, implicating macrophages as the principal angiogenic mediators, consistent with the inhibition of Myc-induced angiogenesis by CCL9 blockade and the widely reported angiogenic role of macrophages (Chanmee et al., 2014, Riabov et al., 2014) (Figure S7). In exploring the mechanism underlying the peculiar obligate role of CCL9 in Myc-driven exclusion of T and B cells, but not NK cells, we noted the rapid rise in the lung tumors of cells expressing the immunosuppressive ligand PD-L1 (a.k.a. B7-H1 or CD274) following Myc activation. However, PD-L1 expression was not directly induced by Myc, as recently reported (Casey et al., 2016), but entirely confined to incoming F4/80+ macrophage-like cells. These are known to express PD-L1 (Igarashi et al., 2016), whose increase is a simple consequence of CCL9-dependent macrophage influx. Systemic blockade of PD-L1 completely abrogated Myc-induced loss of CD3+ T cells—confirming macrophage-derived PD-L1 as necessary for Myc-induced T cell depletion—but had no effect on NK exclusion or macrophage influx. The extremely rapid Myc-induced, CCL9-dependent T cell depletion occurred without detectable T cell apoptosis, and this, together with the equally rapid re-appearance of T cells in adenomas upon Myc de-activation, suggests that migration of T cells out and in to adenoma tissue is the likely mechanism for the rapid T cell dynamics that Myc elicits.

We next assessed Myc’s role in maintaining lung adenocarcinomas. Acute Myc de-activation in established KM adenocarcinomas triggered rapid and synchronous regression of all tumors, characterized by a direct reversal of all Myc-induced stromal changes: macrophages rapidly exited the tumor masses; tumor vasculature reformed into discrete vessels (“vascular re-normalization”) and widespread hypoxia reappeared; T, B, and NK cells rapidly repopulated the tumors and their environs; and tumor cell apoptosis and tumor regression rapidly ensued. Remarkably, even very short-term (7 days) activation of ectopic Myc was sufficient to establish such Myc dependency in tumors. Acquired dependency on driver oncogenes (oncogene addiction) is a pervasive phenomenon that provides the underpinning rationale for most current targeted therapies. Its mechanism remains unclear, although co-dependency of oncogenic mutations, the abrupt re-instatement of cell intrinsic or immune checkpoints, asynchrony in attenuation rates of mitogenic versus survival signals, and collapse of tumor stroma have all been implicated (Restifo, 2010, Sharma et al., 2006, Sodir et al., 2011, Tran et al., 2011). Intriguingly, once the Myc-driven KM tumors had regressed back to their previous KRasG12D-only adenoma state and size, tumor regression stalled indefinitely. Similarly incomplete regression upon Myc de-activation has been seen in other Myc transgenic models (Boxer et al., 2004, Podsypanina et al., 2008, Tran et al., 2008). It contrasts with the complete regression—indeed, eradication—seen upon inhibition of Myc in KRasG12D-driven lung tumors via the dominant negative Omomyc mutant (Soucek et al., 2008, Soucek et al., 2013)—an apparent discrepancy that likely arises because Omomyc inhibits not only Myc transgenes, but also endogenous Myc. The rapidity with which Myc dependency arises is incompatible with the idea that Myc addiction reflects acquired co-dependency upon other oncogenic mutations. Rather, our data favor a tight temporal and spatial co-dependence between Myc driven tumor cell expansion and Myc-driven tumor stroma. However, precisely which attributes of the Myc-instructed tumor stroma are so essential for tumor maintenance are less clear. T cells do not appear to be involved, since abrogation of Myc-driven T cell expulsion by PD-L1 blockade has no inhibitory impact on Myc-driven lung tumor growth, progression, or burden. Moreover, while systemic depletion of CD4+ and CD8+ T cells prevented T cell re-colonization of tumors following Myc de-activation, it does not measurably retard either stromal collapse or tumor regression. By contrast, our data do suggest a possible role for NK cells in limiting KM tumor growth. Myc rapidly induced expression of Rae-1, the principal activating ligands for the NK cell NKG2D receptor, in adenoma epithelial cells. It also downregulated MHC class I, a potent trigger of NK cell activation as well as a means of adaptive T cell evasion (Töpfer et al., 2011). Given two such potent NK cell activators, it is difficult to see how KM tumors could survive without the rapid clearance of NK cells that IL-23 mediates. Indeed, blockade of IL-23 in Myc-stimulated KM tumors triggered profound tumor apoptosis that was not seen when expulsion of T cells alone was blocked by PD-L1 blockade. We also note that the transient wave of apoptosis (Figure 6C) responsible for regression of KM tumors induced by Myc deactivation precisely coincides temporally with transient influx of NK cells and is significantly suppressed when NK cells, but not CD4+CD8+ T cells, are systemically ablated.

Advantageous though it may be for tumors, the question remains as to why Myc is bestowed with the capacity to drive rapid clearance of adaptive and innate lymphocytes cells, a property of Myc we see across diverse tissues. We guess the answer lies in the physiological role of Myc as the transcriptional coordinator of the diverse array of intra- and extracellular processes needed for regeneration in lung (Dong et al., 2011) and other tissues after injury. Such regeneration requires complex, tissue-specific interplay between epithelial, myeloid, mesenchymal, and endothelial stromal elements needed to clear debris, seal the wound, and regenerate the tissue. In sterile situations (where pathogens are no complication), this rapid outgrowth phase is typically associated with rapid influx of immunosuppressive monocytic and myeloid-derived suppressor cells (Wynn and Vannella, 2016) followed by a resolution phase marked by return of lymphoid cells (D’Alessio et al., 2009, Wu et al., 2013) during which epithelial regeneration attenuates and extensive tissue and vascular remodeling reconstruct the original tissue architecture. The similarities between this biphasic physiological process, regeneration followed by resolution, and Myc-driven tumorigenesis followed by Myc de-activation-induced tumor regression, are provocative.

STAR[large star]Methods

Key Resources Table

Table thumbnail

Contact for Reagent and Resource Sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Gerard I. Evan (gie20@cam.ac.uk).

Experimental Model and Subject Details

Mice and in vivo procedures

All treatments and procedures of mice were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee at UCSF and Home Office UK guidelines under project licenses to G.I.E., (70/7586, 80/2396) at the University of Cambridge. Mice were maintained on regular diet in a pathogen-free facility on a 12 hr light/dark cycle with continuous access to food and water. Lsl-KrasG12D (Jackson et al., 2001) and LSL-Rosa26MIE/MIE (Murphy et al., 2008) mice have been described previously. For activation of MycERT2, Tamoxifen (Sigma; T5648) dissolved in peanut oil was administered daily by intraperitoneal (IP) injection for a maximum of 6 weeks at a dose of 1 mg/20 g body mass. To deliver adenovirus-Cre recombinase (AdV-Cre), mice were anesthetized with 2.5% Avertin (250 ml/20 g body mass) or isoflurane (Zoetis, IsoFlo 250 ml) and 5x107 plaque-forming units of AdV-Cre were administered as described previously (Fasbender et al., 1998). Mice were between 8 and 12 weeks old at time of AdV-Cre infection, and between 20-28 weeks of age at time of euthanasia and analysis. Equivalent female and male age-matched littermates were divided over experimental and control mice. Lectins were administered by retro-orbital injection prior to sacrifice and Hypoxyprobe and blocking antibodies for PD-L1, IL23p19, CCL9, CD4, CD8 and asialo-GM1 were administered by IP injection at doses and frequency described in method details and Figure S1.

Method Details

Immunohistochemistry and Immunofluorescence

Mice were euthanized and cardiac-perfused with PBS followed by 10% neutral-buffered formalin (Sigma-Aldrich, 501320). Lungs were removed, fixed overnight in neutral-buffered formalin and processed for paraffin embedding. Tissue sections were stained with hematoxylin and eosin (H&E) using standard reagents and protocols. For frozen sections, lungs were directly embedded in OCT (VWR Chemicals, 361603E) and either frozen on dry ice or snap frozen in liquid Nitrogen, embedded in OCT, and stored at −80°C. For immunofluorescence analysis of VEGF bound to its receptor (VEGF:VEGFR2), OCT frozen cryostat-cut 10 μm sections were air-dried, serum blocked and incubated with the GV39M antibody overnight. For immunohistochemical and immunofluorescence analysis, sections were de-paraffinized, rehydrated, and boiled in a microwave for 10 minutes in 10 mM citrate buffer (pH 6.0) or treated with 20 μg/ml Proteinase K for antigen retrieval. Antibodies were incubated overnight at 4°C except for anti-CD3, which was incubated for 20 minutes at room temperature. Antibodies used (see key resources table): F4/80 (Cl:A3-1); MMR/CD206; CD45 (30-F11); CD3 (SP7); CD274/PD-L1; Ki67 (SP6); c-Myc (Y69); CD31/PECAM (MEC13.3); Pan-endothelial cell antigen/PVLAP (MECA32); IL23p19; CCL9/10/MIP1γ; TTF1 (EP1584Y); TTF1 (8G7G3/1); Hypoxyprobe (4.3.11.3); Pan-Rae-1; MHC1 (ER-HR52); CD45R/B220 (RA3-6B2); CD335/NKp46 (29A1.4). Commercial antibodies were purchased from Cedarlane, Merck, Bio-Rad, R&D Systems, BD, ThermoFisher, Abcam, East Coast Biologics, Novus, Hypoxyprobe and BioLegend. HRP- conjugated secondary antibodies (Vectastain Elite ABC Kits: PK-6200; Universal, PK-6101; Rabbit, PK-6104; Rat, PK-6105; Goat) were applied for 30 min and visualized with DAB (Vector Laboratories; SK-4100), or secondary Alexa Fluor 488 or −455 dye-conjugated antibodies (Life Technologies) applied for 30 minutes at room temperature. Fluorescence antibody-labeled slides were mounted in fluorescent mounting medium (Prolong Molecular Probes; P36934) post-treatment with 0.5 μg/ml Hoechst counter-stain. TUNEL analysis was performed using the Apoptag Fluorescein In Situ Apoptosis Detection Kit (Millipore; S7110) according to manufacturer’s instructions. Briefly, tissue sections were pre-treated in Proteinase K (20 μg/ml) for 15 min at room temperature, washed in deionized water twice for 2 min each, and returned to PBS. Sections were then covered with equilibration buffer for a minimum of 2 min followed by incubation at 37°C for 1 hr with a 1:5 dilution of TdT enzyme in reaction buffer, followed by fluorescein anti-digoxigenin conjugated secondary antibody, washed, and mounted. To stain tissues systemically for hypoxia, 60 mg/kg hypoxyprobe-1 (1-{[2-hydroxy-3-piperdinyl] propyl}-2-nitroimidazole hydrochloride) (Hypoxyprobe; HP1-100 kit) was administered through IP injection in saline 15 min prior to euthanasia. Protein adducts of reductively activated pimonidazole were identified through immunohistochemistry in fixed tissues with a monoclonal antibody against hypoxyprobe-1.

RNA-ISH for MycERT2 was performed with a custom-designed probe targeting nucleotides 2110-3619 of Myc-ER-ires-EGFP (Murphy et al., 2008), amplified with RNAscope 2.5 HD Reagent Kit (Advanced Cell Diagnostics; 322300) and developed with the TSA Plus kit (PerkinElmer; NEL760001KT) according to manufacturer’s instructions. Images were collected with an Axiovert 5100 TV inverted fluorescence microscope (Zeiss) and Open Lab 3.5.1 software, or with an Axiovert 100 inverted microscope (Zeiss) equipped with a Hamamatsu Orca digital camera (University of California, San Francisco), and a Zeiss Axio Imager M2 microscope and Axiovision Rel 4.8 software (University of Cambridge).

Isolation of mouse lung tumor-associated macrophage RNA

After heart-perfusion with PBS, mouse lungs were harvested and macrophages isolated following the MACS cell-separation protocol according to manufacturer’s instructions (Miltenyi Biotec). Briefly: lung tissue was minced, incubated 15 minutes at 37°C in Ca2+/Mg2+-free PBS with 0.5% EDTA, passed through 40 μm Falcon cell strainers (352340) and re-suspended in erythrocyte lysis buffer (RBC Tris-buffered ammonium chloride pH7.2) followed by dissociation buffer (PBS pH7.2 with 2mM EDTA). The resulting homogenate was incubated with anti-F4/80 biotin-conjugated antibody (Miltenyi Biotec, 130-101-893) followed by streptavidin-conjugated microbeads (Miltenyi Biotec, 130-101-893), or incubated with anti-F4/80 microbead-conjugated antibody (Miltenyi Biotec, 130-110-443), passed through magnetic columns and the macrophages then transferred to TRizol (Ambion, 15596018) for RNA isolation with the RNeasy Plus Mini Kit (QIAGEN, 74134), according to manufacturer’s instructions.

Immune Array and determination of MycERT2, PD-L1, and VEGFA mRNA

14 weeks post AdV-Cre KM mouse lungs were isolated 8 hours after IP injection with either oil or tamoxifen and then snap frozen in liquid nitrogen. Whole (tumor-laden) lung protein samples were isolated and incubated on a mouse inflammation antibody array (Abcam, ab133999) according to manufacturer instructions. The intensity of the signals was analyzed using ImageJ.

Total RNA (0.5-1 mg) isolated from F4/80- lung- and tumor tissue or F4/80+ macrophages was reverse transcribed using the High Capacity cDNA RT kit (Applied Biosystems, 4374966). Real-time quantitative RT–PCR (Fast Sybr Green, Applied Biosystems, 4385612) was used to quantify mRNA levels. The TBP and β-actin genes were used as an internal amplification control. TBP forward primer: 5′-ACTTCGTGCAAGAAATGCTGAAT-3′, TBP reverse primer: 5′- CAGTTGTCCGTGGCTCTCTTATT-3′. β-actin forward primer: 5′-GACGATATCGCTGCGCTGG −3′, β-actin reverse primer: 5′-CCACGATGGAGGGGAATA-3′. MycERT2 primers; forward: 5′-ATTTCTGAAGACTTGTTGCGGAAA-3′, reverse: 5′- GCTGTTCTTAGAGCGTTTGATCATGA-3′ (Murphy et al., Cancer Cell 14 (6), 2008). PD-L1 primers; forward: 5′-GACCAGCTTTTGAAGGGAAATG-3′, reverse: 5′-CTGGTTGATTTTGCGGTATGG-3′. VEGFA primers were from Bio-Rad (10025636, qMmuCED0040260). Real-time PCR reactions were performed on an Eppendorf Mastercycler Realpex 2 and analyzed with accompanying software.

Blocking antibodies and Flow Cytometric analysis

For antibody blocking experiments, mice were IP injected with either anti-IL23p19 or anti-CCL9 individually or together (see Figures 3 and andS4)S4) or with anti-PD-L1 every two days, starting one day before Myc activation by tamoxifen injection (see Figures 4 and andS1).S1). For tumor regression studies, antibody ablation of CD4+ and CD8+ T cells or NKp46+ cells was initiated four days before tamoxifen withdrawal (see Figures 7, ,S1,S1, and andS6).S6). Concentrations of antibodies and their isotype controls were adjusted in PBS as follows (see key resources table): IL23p19 (MMp19B2) and Mouse IgG2b κ Isotype control (MPC-11) 150 μg/mouse/IP; CCL9/10/MIP1γ (AF463) and goat IgG control (AB-108-C) 50 μg/mouse/IP; CD274/PD-L1 (10F.9G2) and Rat IgG2b κ Isotype control (RTK4530) 150 μg/mouse/IP; CD4 (GK1.5) and CD8 (2.43) and Rat IgG control (LTF2) 200 μg/mouse/IP; asialo-GM1 and Rabbit IgG control 100 μl/mouse/IP and 100 μg/mouse/IP, respectively. For Flow Cytometric identification of CD4 and CD8 positive T cells in spleen and blood of anti-CD4 and anti-CD8 treated mice, CD3ε (145-2C11), CD8α (53-6.7), CD4 (RM4-5, GK1.5) and CD45 (30-F11)-specific antibodies were used. For Flow Cytometric identification of NKp46 positive NK cells in blood and spleen of anti-asialo-GM1 treated mice NKp46 (29A1.4)-specific antibody was used. For isolation and analysis of lung-specific NKp46+ cells whole lung tissue was minced, incubated 15 minutes at 37°C in Ca2+/Mg2+-free PBS with 0.5% EDTA, passed through 40 μm Falcon cell strainers (352340) and re-suspended in erythrocyte lysis buffer (RBC Tris-buffered ammonium chloride pH7.2) followed by dissociation buffer (PBS pH7.2 with 2mM EDTA). The cell suspension was incubated with FITC-NKp46-specific antibody (eBioscience, 11-3351). Flow Cytometry was performed on an Accuri C6 Flow cytometer (BD Biosciences) with accompanying software. Commercial antibodies were purchased from R&D Systems, BioLegend, BioXcell and Wako Chemicals.

Lectins

Lectin dyes Fluorescein-Lycopersicon esculentum (FL-1171) and Rhodamine-Ricinus communis agglutinin I (RL-1082) were obtained from Vector Labs. Three minutes before sacrifice 100 μL of a 1:2 Fluorescein:Rhodamine solution was administered by retro-orbital injection. Lungs were then harvested and fluorescence imaged on de-paraffinized tissue sections counterstained with Hoechst dye.

Quantification and Statistical Analysis

Scanning H&E slides, ImageJ, and GraphPad.

H&E sections were scanned with an Aperio AT2 microscope (Leica Biosystems) at 20X magnification (resolution 0.5 microns per pixel) and analyzed with Aperio Software. Quantifications were performed using ImageJ. Statistical analysis was performed in GraphPad. Data points on the scatter dot graphs that portray quantification per Field of View represent one tumor (small data points) and averages per mouse (large data points) simultaneously. A minimum of five tumors per mouse were analyzed. For comparison, quantification of histological markers was only performed on tumor sections stained at the same time. For endothelial cell marker CD31 the immunohistochemistry staining intensity was calculated as percentage CD31-positive area per Field of View using ImageJ. For proliferation marker Ki67 the immunohistochemistry staining was quantified as percentage of total cells per Field of View using ImageJ. For quantification of NK cell marker NKp46 a complete section of the lung of a mouse was analyzed. Scoring (none, ≤ 10 cells, > 10 cells) was based on the amount of juxta-tumoral NKp46+ cells visible. Only tumors directly lining and associated with clearly distinguishable vasculature were taken into consideration. P values are derived from mouse-average comparisons between groups and determined via Student’s t test or two-way ANOVA (see figure legends). Bar graphs are represented as mean with standard deviation. Quantifications in scatter dot-plot graphs show median with interquartile range.

Author Contributions

R.M.K. and G.I.E. conceived the project. R.M.K., N.M.S., L.B.S., T.D.L., and G.I.E. designed experiments. R.M.K. performed all experiments. N.M.S., C.H.W., D.L.B., and L.P. assisted with some experiments. R.M.K., T.D.L., and G.I.E. wrote the manuscript. G.I.E. supervised the study. All authors discussed results and revised the manuscript.

Acknowledgments

We thank the members of the Evan laboratory for discussion and advice and Alessandra Perfetto and Irina Pshenichnaya at the University of Cambridge and Fanya Rostker at UCSF Helen Diller Family Cancer Center for expert mouse work assistance and outstanding technical support. The study was supported by NWO-Rubicon 825.08.022 grant from the Netherlands Organisation for Scientific Research to R.M.K. and by program grants to G.I.E. (Cancer Research UK C4750/A12077 and C4750/A19013, NIH/NCI 2RO1 CA98018, European Research Council 294851, and the SU2C/CR UK/Lustgarten/CR UK/Lustgarten Transatlantic Pancreatic Cancer Dream Team C4750/A22585).

Notes

Published: November 30, 2017

Footnotes

Supplemental Information includes seven figures and can be found with this article online at https://doi.org/10.1016/j.cell.2017.11.013.

References

Aggarwal N.R., King L.S., D’Alessio F.R. Diverse macrophage populations mediate acute lung inflammation and resolution. Am. J. Physiol. Lung Cell. Mol. Physiol. 2014;306:L709–L725. [PubMed]
Alexander W.S., Adams J.M., Cory S. Oncogene cooperation in lymphocyte transformation: malignant conversion of E mu-myc transgenic pre-B cells in vitro is enhanced by v-H-ras or v-raf but not v-abl. Mol. Cell. Biol. 1989;9:67–73. [PubMed]
Ancrile B.B., O’Hayer K.M., Counter C.M. Oncogenic ras-induced expression of cytokines: a new target of anti-cancer therapeutics. Mol. Interv. 2008;8:22–27. [PubMed]
Andres A.C., van der Valk M.A., Schönenberger C.A., Flückiger F., LeMeur M., Gerlinger P., Groner B. Ha-ras and c-myc oncogene expression interferes with morphological and functional differentiation of mammary epithelial cells in single and double transgenic mice. Genes Dev. 1988;2:1486–1495. [PubMed]
Bailey S.R., Nelson M.H., Himes R.A., Li Z., Mehrotra S., Paulos C.M. Th17 cells in cancer: the ultimate identity crisis. Front. Immunol. 2014;5:276. [PubMed]
Baird A.-M., Leonard J., Naicker K.M., Kilmartin L., O’Byrne K.J., Gray S.G. IL-23 is pro-proliferative, epigenetically regulated and modulated by chemotherapy in non-small cell lung cancer. Lung Cancer. 2013;79:83–90. [PubMed]
Born T.L., Frost J.A., Schönthal A., Prendergast G.C., Feramisco J.R. c-Myc cooperates with activated Ras to induce the cdc2 promoter. Mol. Cell. Biol. 1994;14:5710–5718. [PubMed]
Boxer R.B., Jang J.W., Sintasath L., Chodosh L.A. Lack of sustained regression of c-MYC-induced mammary adenocarcinomas following brief or prolonged MYC inactivation. Cancer Cell. 2004;6:577–586. [PubMed]
Brekken R.A., Huang X., King S.W., Thorpe P.E. Vascular endothelial growth factor as a marker of tumor endothelium. Cancer Res. 1998;58:1952–1959. [PubMed]
Campbell P.M., Der C.J. Oncogenic Ras and its role in tumor cell invasion and metastasis. Semin. Cancer Biol. 2004;14:105–114. [PubMed]
Casey S.C., Tong L., Li Y., Do R., Walz S., Fitzgerald K.N., Gouw A.M., Baylot V., Gütgemann I., Eilers M., Felsher D.W. MYC regulates the antitumor immune response through CD47 and PD-L1. Science. 2016;352:227–231. [PubMed]
Chang S.H. Tumorigenic Th17 cells in oncogenic Kras-driven and inflammation-accelerated lung cancer. OncoImmunology. 2015;4:e955704. [PubMed]
Chang S.H., Mirabolfathinejad S.G., Katta H., Cumpian A.M., Gong L., Caetano M.S., Moghaddam S.J., Dong C. T helper 17 cells play a critical pathogenic role in lung cancer. Proc. Natl. Acad. Sci. USA. 2014;111:5664–5669. [PubMed]
Chanmee T., Ontong P., Konno K., Itano N. Tumor-associated macrophages as major players in the tumor microenvironment. Cancers (Basel) 2014;6:1670–1690. [PubMed]
Christophorou M.A., Martin-Zanca D., Soucek L., Lawlor E.R., Brown-Swigart L., Verschuren E.W., Evan G.I. Temporal dissection of p53 function in vitro and in vivo. Nat. Genet. 2005;37:718–726. [PubMed]
Compere S.J., Baldacci P., Sharpe A.H., Thompson T., Land H., Jaenisch R. The ras and myc oncogenes cooperate in tumor induction in many tissues when introduced into midgestation mouse embryos by retroviral vectors. Proc. Natl. Acad. Sci. USA. 1989;86:2224–2228. [PubMed]
Cox A.D., Der C.J. The dark side of Ras: regulation of apoptosis. Oncogene. 2003;22:8999–9006. [PubMed]
D’Alessio F.R., Tsushima K., Aggarwal N.R., West E.E., Willett M.H., Britos M.F., Pipeling M.R., Brower R.G., Tuder R.M., McDyer J.F., King L.S. CD4+CD25+Foxp3+ Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury. J. Clin. Invest. 2009;119:2898–2913. [PubMed]
Dang C.V. MYC, metabolism, cell growth, and tumorigenesis. Cold Spring Harb. Perspect. Med. 2013;3 [PMC free article] [PubMed]
Dong J., Sutor S., Jiang G., Cao Y., Asmann Y.W., Wigle D.A. c-Myc regulates self-renewal in bronchoalveolar stem cells. PLoS ONE. 2011;6:e23707. [PubMed]
Evan G., Littlewood T. A matter of life and cell death. Science. 1998;281:1317–1322. [PubMed]
Fasbender A., Lee J.H., Walters R.W., Moninger T.O., Zabner J., Welsh M.J. Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo. J. Clin. Invest. 1998;102:184–193. [PubMed]
Fridman W.H., Pagès F., Sautès-Fridman C., Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat. Rev. Cancer. 2012;12:298–306. [PubMed]
Gaffen S.L., Jain R., Garg A.V., Cua D.J. The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing. Nat. Rev. Immunol. 2014;14:585–600. [PubMed]
Goc J., Fridman W.H., Sautès-Fridman C., Dieu-Nosjean M.-C. Characteristics of tertiary lymphoid structures in primary cancers. OncoImmunology. 2013;2:e26836. [PubMed]
Graziano S.L., Gamble G.P., Newman N.B., Abbott L.Z., Rooney M., Mookherjee S., Lamb M.L., Kohman L.J., Poiesz B.J. Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer. J. Clin. Oncol. 1999;17:668–675. [PubMed]
Igarashi T., Teramoto K., Ishida M., Hanaoka J., Daigo Y. Scoring of PD-L1 expression intensity on pulmonary adenocarcinomas and the correlations with clinicopathological factors. ESMO Open. 2016;1:e000083. [PubMed]
Ischenko I., Zhi J., Moll U.M., Nemajerova A., Petrenko O. Direct reprogramming by oncogenic Ras and Myc. Proc. Natl. Acad. Sci. USA. 2013;110:3937–3942. [PubMed]
Iwakawa R., Kohno T., Kato M., Shiraishi K., Tsuta K., Noguchi M., Ogawa S., Yokota J. MYC amplification as a prognostic marker of early-stage lung adenocarcinoma identified by whole genome copy number analysis. Clin. Cancer Res. 2011;17:1481–1489. [PubMed]
Jackson E.L., Willis N., Mercer K., Bronson R.T., Crowley D., Montoya R., Jacks T., Tuveson D.A. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 2001;15:3243–3248. [PubMed]
Ji H., Houghton A.M., Mariani T.J., Perera S., Kim C.B., Padera R., Tonon G., McNamara K., Marconcini L.A., Hezel A. K-ras activation generates an inflammatory response in lung tumors. Oncogene. 2006;25:2105–2112. [PubMed]
Joshi N.S., Akama-Garren E.H., Lu Y., Lee D.-Y., Chang G.P., Li A., DuPage M., Tammela T., Kerper N.R., Farago A.F. Regulatory T Cells in Tumor-Associated Tertiary Lymphoid Structures Suppress Anti-tumor T Cell Responses. Immunity. 2015;43:579–590. [PubMed]
Karin M. Inflammation and cancer: the long reach of Ras. Nat. Med. 2005;11:20–21. [PubMed]
Kauffmann-Zeh A., Rodriguez-Viciana P., Ulrich E., Gilbert C., Coffer P., Downward J., Evan G. Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature. 1997;385:544–548. [PubMed]
Kimmelman A.C. Metabolic Dependencies in RAS-Driven Cancers. Clin. Cancer Res. 2015;21:1828–1834. [PubMed]
Kitamura T., Kometani K., Hashida H., Matsunaga A., Miyoshi H., Hosogi H., Aoki M., Oshima M., Hattori M., Takabayashi A. SMAD4-deficient intestinal tumors recruit CCR1+ myeloid cells that promote invasion. Nat. Genet. 2007;39:467–475. [PubMed]
Kitamura T., Qian B.-Z., Soong D., Cassetta L., Noy R., Sugano G., Kato Y., Li J., Pollard J.W. CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages. J. Exp. Med. 2015;212:1043–1059. [PubMed]
Koizumi K., Hojo S., Akashi T., Yasumoto K., Saiki I. Chemokine receptors in cancer metastasis and cancer cell-derived chemokines in host immune response. Cancer Sci. 2007;98:1652–1658. [PubMed]
Kranenburg O., Gebbink M.F.B.G., Voest E.E. Stimulation of angiogenesis by Ras proteins. Biochim. Biophys. Acta. 2004;1654:23–37. [PubMed]
Land H., Parada L.F., Weinberg R.A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 1983;304:596–602. [PubMed]
Langowski J.L., Zhang X., Wu L., Mattson J.D., Chen T., Smith K., Basham B., McClanahan T., Kastelein R.A., Oft M. IL-23 promotes tumour incidence and growth. Nature. 2006;442:461–465. [PubMed]
Leone G., DeGregori J., Sears R., Jakoi L., Nevins J.R. Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature. 1997;387:422–426. [PubMed]
Littlewood T.D., Hancock D.C., Danielian P.S., Parker M.G., Evan G.I. A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res. 1995;23:1686–1690. [PubMed]
Martinez F.O., Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. 2014;6:13. [PubMed]
Morvan M.G., Lanier L.L. NK cells and cancer: you can teach innate cells new tricks. Nat. Rev. Cancer. 2016;16:7–19. [PubMed]
Murphy D.J., Junttila M.R., Pouyet L., Karnezis A., Shchors K., Bui D.A., Brown-Swigart L., Johnson L., Evan G.I. Distinct thresholds govern Myc’s biological output in vivo. Cancer Cell. 2008;14:447–457. [PubMed]
Ngiow S.F., Teng M.W.L., Smyth M.J. A balance of interleukin-12 and -23 in cancer. Trends Immunol. 2013;34:548–555. [PubMed]
Pelengaris S., Khan M., Evan G.I. Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell. 2002;109:321–334. [PubMed]
Podsypanina K., Politi K., Beverly L.J., Varmus H.E. Oncogene cooperation in tumor maintenance and tumor recurrence in mouse mammary tumors induced by Myc and mutant Kras. Proc. Natl. Acad. Sci. USA. 2008;105:5242–5247. [PubMed]
Pollard J.W. Tumour-educated macrophages promote tumour progression and metastasis. Nat. Rev. Cancer. 2004;4:71–78. [PubMed]
Rapp U.R., Korn C., Ceteci F., Karreman C., Luetkenhaus K., Serafin V., Zanucco E., Castro I., Potapenko T. MYC is a metastasis gene for non-small-cell lung cancer. PLoS ONE. 2009;4:e6029. [PubMed]
Restifo N.P. Can antitumor immunity help to explain “oncogene addiction”? Cancer Cell. 2010;18:403–405. [PubMed]
Riabov V., Gudima A., Wang N., Mickley A., Orekhov A., Kzhyshkowska J. Role of tumor associated macrophages in tumor angiogenesis and lymphangiogenesis. Front. Physiol. 2014;5:75. [PubMed]
Richardson G.E., Johnson B.E. The biology of lung cancer. Semin. Oncol. 1993;20:105–127. [PubMed]
Rodenhuis S., Slebos R.J., Boot A.J., Evers S.G., Mooi W.J., Wagenaar S.S., van Bodegom P.C., Bos J.L. Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung. Cancer Res. 1988;48:5738–5741. [PubMed]
Rőszer T. Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms. Mediators Inflamm. 2015;2015:816460. [PubMed]
Sears R., Nuckolls F., Haura E., Taya Y., Tamai K., Nevins J.R. Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev. 2000;14:2501–2514. [PubMed]
Seo A.N., Yang J.M., Kim H., Jheon S., Kim K., Lee C.T., Jin Y., Yun S., Chung J.H., Paik J.H. Clinicopathologic and prognostic significance of c-MYC copy number gain in lung adenocarcinomas. Br. J. Cancer. 2014;110:2688–2699. [PubMed]
Sharma S.V., Gajowniczek P., Way I.P., Lee D.Y., Jiang J., Yuza Y., Classon M., Haber D.A., Settleman J. A common signaling cascade may underlie “addiction” to the Src, BCR-ABL, and EGF receptor oncogenes. Cancer Cell. 2006;10:425–435. [PubMed]
Shchors K., Shchors E., Rostker F., Lawlor E.R., Brown-Swigart L., Evan G.I. The Myc-dependent angiogenic switch in tumors is mediated by interleukin 1beta. Genes Dev. 2006;20:2527–2538. [PubMed]
Sodir N.M., Swigart L.B., Karnezis A.N., Hanahan D., Evan G.I., Soucek L. Endogenous Myc maintains the tumor microenvironment. Genes Dev. 2011;25:907–916. [PubMed]
Soucek L., Whitfield J., Martins C.P., Finch A.J., Murphy D.J., Sodir N.M., Karnezis A.N., Swigart L.B., Nasi S., Evan G.I. Modelling Myc inhibition as a cancer therapy. Nature. 2008;455:679–683. [PubMed]
Soucek L., Whitfield J.R., Sodir N.M., Massó-Vallés D., Serrano E., Karnezis A.N., Swigart L.B., Evan G.I. Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice. Genes Dev. 2013;27:504–513. [PubMed]
Sparmann A., Bar-Sagi D. Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis. Cancer Cell. 2004;6:447–458. [PubMed]
Stenhouse G., Fyfe N., King G., Chapman A., Kerr K.M. Thyroid transcription factor 1 in pulmonary adenocarcinoma. J. Clin. Pathol. 2004;57:383–387. [PubMed]
Sunaga N., Imai H., Shimizu K., Shames D.S., Kakegawa S., Girard L., Sato M., Kaira K., Ishizuka T., Gazdar A.F. Oncogenic KRAS-induced interleukin-8 overexpression promotes cell growth and migration and contributes to aggressive phenotypes of non-small cell lung cancer. Int. J. Cancer. 2012;130:1733–1744. [PubMed]
Teng M.W.L., Andrews D.M., McLaughlin N., von Scheidt B., Ngiow S.F., Möller A., Hill G.R., Iwakura Y., Oft M., Smyth M.J. IL-23 suppresses innate immune response independently of IL-17A during carcinogenesis and metastasis. Proc. Natl. Acad. Sci. USA. 2010;107:8328–8333. [PubMed]
Teng M.W.L., Vesely M.D., Duret H., McLaughlin N., Towne J.E., Schreiber R.D., Smyth M.J. Opposing roles for IL-23 and IL-12 in maintaining occult cancer in an equilibrium state. Cancer Res. 2012;72:3987–3996. [PubMed]
Töpfer K., Kempe S., Müller N., Schmitz M., Bachmann M., Cartellieri M., Schackert G., Temme A. Tumor evasion from T cell surveillance. J. Biomed. Biotechnol. 2011;2011:918471. [PubMed]
Tran P.T., Fan A.C., Bendapudi P.K., Koh S., Komatsubara K., Chen J., Horng G., Bellovin D.I., Giuriato S., Wang C.S. Combined Inactivation of MYC and K-Ras oncogenes reverses tumorigenesis in lung adenocarcinomas and lymphomas. PLoS ONE. 2008;3:e2125. [PubMed]
Tran P.T., Bendapudi P.K., Lin H.J., Choi P., Koh S., Chen J., Horng G., Hughes N.P., Schwartz L.H., Miller V.A. Survival and death signals can predict tumor response to therapy after oncogene inactivation. Sci. Transl. Med. 2011;3:103ra199. [PMC free article] [PubMed]
Tsuneoka M., Mekada E. Ras/MEK signaling suppresses Myc-dependent apoptosis in cells transformed by c-myc and activated ras. Oncogene. 2000;19:115–123. [PubMed]
Vaqué J.P., Navascues J., Shiio Y., Laiho M., Ajenjo N., Mauleon I., Matallanas D., Crespo P., León J. Myc antagonizes Ras-mediated growth arrest in leukemia cells through the inhibition of the Ras-ERK-p21Cip1 pathway. J. Biol. Chem. 2005;280:1112–1122. [PubMed]
Wang C., Lisanti M.P., Liao D.J. Reviewing once more the c-myc and Ras collaboration: converging at the cyclin D1-CDK4 complex and challenging basic concepts of cancer biology. Cell Cycle. 2011;10:57–67. [PubMed]
White G.E., Iqbal A.J., Greaves D.R. CC chemokine receptors and chronic inflammation--therapeutic opportunities and pharmacological challenges. Pharmacol. Rev. 2013;65:47–89. [PubMed]
Wilson C.H., Gamper I., Perfetto A., Auw J., Littlewood T.D., Evan G.I. The kinetics of ER fusion protein activation in vivo. Oncogene. 2014;33:4877–4880. [PubMed]
Wolfer A., Wittner B.S., Irimia D., Flavin R.J., Lupien M., Gunawardane R.N., Meyer C.A., Lightcap E.S., Tamayo P., Mesirov J.P. MYC regulation of a “poor-prognosis” metastatic cancer cell state. Proc. Natl. Acad. Sci. USA. 2010;107:3698–3703. [PubMed]
Wu Q., Gardiner G.J., Berry E., Wagner S.R., Lu T., Clay B.S., Moore T.V., Ferreira C.M., Williams J.W., Luster A.D. ICOS-expressing lymphocytes promote resolution of CD8-mediated lung injury in a mouse model of lung rejection. PLoS ONE. 2013;8:e72955. [PubMed]
Wynn T.A., Vannella K.M. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity. 2016;44:450–462. [PubMed]
Yaari S., Jacob-Hirsch J., Amariglio N., Haklai R., Rechavi G., Kloog Y. Disruption of cooperation between Ras and MycN in human neuroblastoma cells promotes growth arrest. Clin. Cancer Res. 2005;11:4321–4330. [PubMed]