Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer.

Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8(+) cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology.

Loss of P53 Function Activates JAK2-STAT3 Signaling to Promote Pancreatic Tumor Growth, Stroma Modification, and Gemcitabine Resistance in Mice and Is Associated With Patient Survival.

BACKGROUND & AIMS: One treatment strategy for pancreatic ductal adenocarcinoma is to modify, rather than deplete, the tumor stroma. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) is associated with progression of pancreatic and other solid tumors. We investigated whether loss of P53 function contributes to persistent activation of STAT3 and modification of the pancreatic tumor stroma in patients and mice. METHODS: Stat3, Il6st (encodes gp130), or Trp53 were disrupted, or a mutant form of P53 (P53R172H) or transgenic sgp130 were expressed, in mice that developed pancreatic tumors resulting from expression of activated KRAS (KrasG12D, KC mice). Pancreata were collected and analyzed by immunohistochemistry, in situ hybridization, quantitative reverse-transcription polymerase chain reaction (qPCR), or immunoblot assays; fluorescence-activated cell sorting was performed to identify immune cells. We obtained frozen pancreatic tumor specimens from patients and measured levels of phosphorylated STAT3 and P53 by immunohistochemistry; protein levels were associated with survival using Kaplan-Meier analyses. We measured levels of STAT3, P53, ligands for gp130, interleukin 6, cytokines, sonic hedgehog signaling, STAT3 phosphorylation (activation), and accumulation of reactive oxygen species in primary pancreatic cells from mice. Mice with pancreatic tumors were given gemcitabine and a Janus kinase 2 (JAK2) inhibitor; tumor growth was monitored by 3-dimensional ultrasound. RESULTS: STAT3 was phosphorylated constitutively in pancreatic tumor cells from KC mice with loss or mutation of P53. Tumor cells of these mice accumulated reactive oxygen species and had lower activity of the phosphatase SHP2 and prolonged phosphorylation of JAK2 compared with tumors from KC mice with functional P53. These processes did not require the gp130 receptor. Genetic disruption of Stat3 in mice, or pharmacologic inhibitors of JAK2 or STAT3 activation, reduced fibrosis and the numbers of pancreatic stellate cells in the tumor stroma and altered the types of immune cells that infiltrated tumors. Mice given a combination of gemcitabine and a JAK2 inhibitor formed smaller tumors and survived longer than mice given control agents; the tumor stroma had fewer activated pancreatic stellate cells, lower levels of periostin, and alterations in collagen production and organization. Phosphorylation of STAT3 correlated with P53 mutation and features of infiltrating immune cells in human pancreatic tumors. Patients whose tumors had lower levels of phosphorylated STAT3 and functional P53 had significantly longer survival times than patients with high levels of phosphorylated STAT3 and P53 mutation. CONCLUSIONS: In pancreatic tumors of mice, loss of P53 function activates JAK2-STAT3 signaling, which promotes modification of the tumor stroma and tumor growth and resistance to gemcitabine. In human pancreatic tumors, STAT3 phosphorylation correlated with P53 mutation and patient survival time. Inhibitors of this pathway slow tumor growth and stroma formation, alter immune cell infiltration, and prolong survival of mice. Transcript profiling: ArrayExpress accession number: E-MTAB-3278.

Macrophage scavenger receptor a promotes tumor progression in murine models of ovarian and pancreatic cancer.

Alternatively activated macrophages express the pattern recognition receptor scavenger receptor A (SR-A). We demonstrated previously that coculture of macrophages with tumor cells upregulates macrophage SR-A expression. We show in this study that macrophage SR-A deficiency inhibits tumor cell migration in a coculture assay. We further demonstrate that coculture of tumor-associated macrophages and tumor cells induces secretion of factors that are recognized by SR-A on tumor-associated macrophages. We tentatively identified several potential ligands for the SR-A receptor in tumor cell-macrophage cocultures by mass spectrometry. Competing with the coculture-induced ligand in our invasion assay recapitulates SR-A deficiency and leads to similar inhibition of tumor cell invasion. In line with our in vitro findings, tumor progression and metastasis are inhibited in SR-A(-/-) mice in two in vivo models of ovarian and pancreatic cancer. Finally, treatment of tumor-bearing mice with 4F, a small peptide SR-A ligand able to compete with physiological SR-A ligands in vitro, recapitulates the inhibition of tumor progression and metastasis observed in SR-A(-/-) mice. Our observations suggest that SR-A may be a potential drug target in the prevention of metastatic cancer progression.

Inflammation and cancer: a double-edged sword.

Recent literature has highlighted an important role of inflammation in promoting cancer. However, the immune system can also play a central role in protecting the body against cancer as well as infection, although its role in cancer is not well understood. A study published in the September issue of Nature Medicine adds a new twist to the role of inflammation in cancer. Apetoh et al. describe how activation of innate immunity after conventional radiation or chemotherapy can trigger protective antitumor immunity.

Cancer-related inflammation.

Solid tumors consist of neoplastic cells, non-malignant stromal cells, and migratory hematopoietic cells. Complex interactions between the cell types in this microenvironment regulate tumor growth, progression, metastasis, and angiogenesis. The cells and mediators of inflammation form a major part of the epithelial tumor microenvironment. In some cancers, inflammatory conditions precede development of malignancy; in others, oncogenic change drives a tumor-promoting inflammatory milieu. Whatever its origin, this "smoldering" inflammation aids proliferation and survival of malignant cells, stimulates angiogenesis and metastasis, subverts adaptive immunity, and alters response to hormones and chemotherapy. Cytokines are major mediators of communication between cells in the inflammatory tumor microenvironment. It is known that neoplastic cells often over-express proinflammatory mediators including proteases, eicosanoids, cytokines, and chemokines. Several cytokines such as macrophage migratory inhibitory factor (MIF), TNF-α, IL-6, IL-17, IL-12, IL-23, IL-10, and TGF-ß have been linked with both experimental and human cancers and can either promote or inhibit tumor development. MIF is a major cytokine in many cancers and there is evidence that the cytokine is produced by both malignant cells and infiltrating leukocytes. In this article we will discuss the role of cancer-associated inflammation and the particular role of MIF in malignant disease.

A distinct DNA methylation profile associated with microsatellite and chromosomal stable sporadic colorectal cancers.

Aberrant DNA methylation, microsatellite instability (MSI) and chromosomal instability (CIN) are well-characterised molecular features of sporadic colorectal cancers (CRCs). In addition to CpG island methylator phenotype (CIMP) associated with MSI, an intermediate methylation subgroup is also a feature of non-MSI cancers. A large proportion of CRCs have no evidence of either MSI or CIN, here called Microsatellite and Chromosomal Stable (MACS), and require their methylation profile to be established. The clinical and molecular features of 170 sporadic CRC patients were investigated and stratified into MSI, CIN and MACS groups. MACS were most often found in the left colon and had a significantly lower BRAF mutation frequency (p < 0.001) compared with MSI. MACS had better survival [hazard ratio (HR) = 0.244, p = 0.017] compared with CIN, but were similar to MSI. The methylation status of 1,505 CpG loci from cancer-related genes was analysed in a subset of CRCs (n = 44 normal-tumour pairs) and compared with CIN, MSI and MACS status. Using two-way hierarchical clustering, three subgroups were identified, which associated with CIN, MSI and MACS status. Using significance analysis of microarray, 16 CpG loci demonstrating methylation changes associated with MACS were identified. A combination of six loci identified MACS with 81% sensitivity and 93% specificity. This result now requires independent validation. Hypomethylation of a CpG locus within the sonic hedgehog (SHH) promoter correlated with increased gene expression and was associated significantly with MACS cancers. In conclusion, we propose that MACS have distinct clinicopathological features and can be distinguished from other CRCs by a specific set of methylation loci.

Resistance--the true face of biological defiance.

Biological therapeutics are widely used in chronic inflammatory and malignant disease. The underlying mechanisms of treatment failure for these drugs are poorly understood. Resistance to these biological agents and the further subdivision into intrinsic and acquired resistance are not clearly defined. In this review, we explore the current understanding of the mechanisms of action of several biological agents as well as the complex biological processes that underlie resistance. A better understanding of why biologicals fail might help to improve their single or combinational use and will ultimately help to alleviate disease burden more efficiently.

Regulation of macrophage function in tumors: the multifaceted role of NF-kappaB.

The pivotal role of tumor-associated macrophages (TAMs) in tumor progression is now well established. TAMs have been shown to influence multiple steps in tumor development including the growth, survival, invasion, and metastasis of tumor cells as well as angiogenesis and lymphangiogenesis in tumors. The molecular circuits that polarize TAMs toward such a protumoral phenotype are now the focus of intense investigation. The transcription factor, nuclear factor-kappaB (NF-kappaB), is a master regulator of many cellular processes and been shown to regulate various pathways that impact on the function of TAMs. Much evidence for this has come from the use of elegant transgenic murine tumor models in which modification of single components of the NF-kappaB signaling pathway has been shown to regulate the pro-tumor repertoire of TAMs. Here, we outline this evidence and attempt to reconcile the various views that have emerged recently over the exact role of NF-kappaB in this phenomenon.

Activated macrophages in the tumour microenvironment-dancing to the tune of TLR and NF-kappaB.

A large number of variables have been identified which appear to influence macrophage phenotype within the tumour microenvironment. These include reciprocal chemical and physical interactions with tumour cells and with non-malignant cells of the tumour microenvironment, tissue oxygen tension, and the origin and prior experience of the particular macrophage population. In this review we outline the key evidence for these influences and consider how macrophage phenotype is acquired and the relevance of the TLR-NF-kappaB pathway.

Investigating macrophage and malignant cell interactions in vitro.

Within most human and murine cancers there is an abundant macrophage population, attracted to the tumor microenvironment by cytokines and chemokines such as CSF-1 (M-CSF) and CCL2 (MCP-1) (Cell 124:263-266, 2006). Despite their intrinsic antitumor activity there is usually, but not always, a positive association between the extent of the macrophage infiltrate in tumors and a bad prognosis (Cell 124:263-266, 2006; Nat Rev Cancer 4:71-78, 2004). According to Condeelis and Pollard (Nat Rev Cancer 4:71-78, 2004), tumor-associated macrophages are obligate partners for malignant cell migration, invasion, and metastasis. These conclusions are based not only on association studies, but also on experiments demonstrating that ablation of macrophage function, or their infiltration into experimental tumors, inhibits growth and metastasis (J Exp Med 193:727-740, 2001). While it has become well appreciated that the extensive macrophage infiltrate of tumors can correlate with tumor progression, there is little understanding of the precise nature of interactions between malignant cells and macro-phages and the mechanisms by which these promote cancer. There are several experimental approaches to study the interactions between macrophages and tumor cells in vitro, which we will describe here.

The inflammatory cytokine tumor necrosis factor-alpha generates an autocrine tumor-promoting network in epithelial ovarian cancer cells.

Constitutive expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is characteristic of malignant ovarian surface epithelium. We investigated the hypothesis that this autocrine action of TNF-alpha generates and sustains a network of other mediators that promote peritoneal cancer growth and spread. When compared with two ovarian cancer cell lines that did not make TNF-alpha, constitutive production of TNF-alpha was associated with greater release of the chemokines CCL2 and CXCL12, the cytokines interleukin-6 (IL-6) and macrophage migration-inhibitory factor (MIF), and the angiogenic factor vascular endothelial growth factor (VEGF). TNF-alpha production was associated also with increased peritoneal dissemination when the ovarian cancer cells were xenografted. We next used RNA interference to generate stable knockdown of TNF-alpha in ovarian cancer cells. Production of CCL2, CXCL12, VEGF, IL-6, and MIF was decreased significantly in these cells compared with wild-type or mock-transfected cells, but in vitro growth rates were unaltered. Tumor growth and dissemination in vivo were significantly reduced when stable knockdown of TNF-alpha was achieved. Tumors derived from TNF-alpha knockdown cells were noninvasive and well circumscribed and showed high levels of apoptosis, even in the smallest deposits. This was reflected in reduced vascularization of TNF-alpha knockdown tumors. Furthermore, culture supernatants from such cells failed to stimulate endothelial cell growth in vitro. We conclude that autocrine production of TNF-alpha by ovarian cancer cells stimulates a constitutive network of other cytokines, angiogenic factors, and chemokines that may act in an autocrine/paracrine manner to promote colonization of the peritoneum and neovascularization of developing tumor deposits.

Ovarian cancer cell-derived migration inhibitory factor enhances tumor growth, progression, and angiogenesis.

In view of our previous findings that tumor cell-derived macrophage migration inhibitory factor (MIF) increased macrophage-mediated ovarian cancer cell invasiveness in vitro, we investigated the wider significance of ovarian cancer cell-derived MIF for tumor growth, metastasis, and angiogenesis. We found that MIF is expressed in borderline and malignant ovarian tumors, and active MIF is found in malignant ascitic fluid. We next investigated the expression and function of MIF in a syngeneic ovarian cancer model. Stable knockdown of MIF in the murine ovarian cancer cell line ID8 decreased in vivo tumor burden and overall survival. Tumors arising from MIF knockdown cells had decreased proliferation and significantly increased apoptosis. This was associated with an increased phosphorylation of p53 and reduced Akt phosphorylation. MIF knockdown led to a changed cytokine profile in the ascitic microenvironment; tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-10 expression were all significantly decreased. Accompanying this decrease in cytokine expression was a significant decrease in macrophage infiltration into ascites. Additionally, MIF knockdown reduced the expression of proangiogenic cytokines vascular endothelial growth factor and keratinocyte chemoattractant (KC) and reduced the amount of endothelial cells in the malignant ascites. We conclude that autocrine production of MIF by ovarian cancer cells stimulates other cytokines, chemokines, and angiogenic factors that may promote colonization of the peritoneum and neovascularization of tumor deposits.

The inflammatory cytokine tumor necrosis factor-alpha regulates chemokine receptor expression on ovarian cancer cells.

Epithelial ovarian cancer cells express the chemokine receptor, CXCR4, which may be associated with increased survival and metastatic potential, but the regulation of this receptor is not understood. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is found in ovarian cancer biopsies and is associated with increased tumor grade. In this report, we show that CXCR4 expression on human epithelial ovarian cancer cells is associated with, and can be modulated by, TNF-alpha. Ovarian cancer cells with high endogenous expression of TNF-alpha expressed higher levels of CXCR4 mRNA and protein than cells with low TNF-alpha expression. Stimulation of ovarian cancer cell lines and primary epithelial cancer cells with TNF-alpha resulted in increased CXCR4 mRNA and protein. The TNF-alpha-stimulated increase in CXCR4 mRNA was due partly to de novo synthesis, and up-regulation of CXCR4 cell surface protein increased migration to the CXCR4 ligand CXCL12. CXCR4 mRNA and protein was down-regulated by anti-TNF-alpha antibody or by targeting TNF-alpha mRNA using RNAi. TNF-alpha stimulation activated components of the nuclear factor kappaB pathway, and overexpression of the inhibitor of kappaB also reduced CXCR4 expression. Coculture of macrophages with ovarian cancer cells also resulted in cancer cell up-regulation of CXCR4 mRNA in a TNF-alpha-dependent manner. Finally, there was a correlation between the levels of TNF-alpha and CXCR4 mRNA in clinical biopsies of ovarian cancer, and TNF-alpha protein was expressed in CXCR4-positive tumor cells. TNF-alpha is a critical mediator of tumor promotion in a number of experimental cancers. Our data suggest that one mechanism may be through nuclear factor kappaB-dependent induction of CXCR4.

Utility of procalcitonin concentration in the evaluation of patients with malignant diseases and elevated C-reactive protein plasma concentrations.

BACKGROUND: Elevated plasma concentrations of the C-reactive protein (CRP) are frequently found in patients with malignant diseases. Discrimination between infection and noninfectious acute-phase reactions is essential for therapeutic decisions. METHODS: Because increased procalcitonin (PCT) concentrations have been described predominantly in patients with a systemic infection, PCT plasma concentrations were measured prospectively in 111 patients with a hemato-oncological condition with a CRP concentration >8 mg/L. RESULTS: Documented cases of infection were identified in 42 patients, 39 patients had unexplained fever, and 30 patients had no signs of infection. Twenty patients in the latter group were classified as having an elevated CRP concentration caused by a high tumor load (tumor group), and 8 had elevated concentrations that were drug related (drug group). Median CRP concentrations did not differ significantly between groups of patients with and without infection. PCT concentrations were higher in patients with an infection than in patients without an infection and were within the normal range in all patients in the drug and tumor groups. As shown by receiver operating characteristic analysis, PCT concentration was a significant discriminator between having and not having infection, having infection and being in the tumor group, and having infection and being in the drug group. In contrast, CRP concentration was only a predictor of being in the drug group, when the cut-off point was set at 85.1 mg/L, which limited its clinical applicability. CONCLUSIONS: PCT concentration contributes significantly to the differential diagnosis for elevated CRP concentrations in patients with hemato-oncological conditions and facilitates therapeutic decisions.

A role for endothelin-2 and its receptors in breast tumor cell invasion.

We have studied the role of endothelins (ET-1, ET-2 and ET-3) and ET receptors (ET-RA and ET-RB) in the invasive capacity of breast tumor cells, which express ET-1 and ET-2 as well as ET-RA and ET-RB. Of five human breast tumor cell lines tested, all expressed mRNAs for ET-1, ET-2, and ET-RB. ET-RA mRNA was expressed by four of five tumor cell lines. Breast tumor cells migrated toward ET-1 and ET-2 but not toward ET-3. Chemotaxis involved signaling via both receptors, and a pertussis toxin-sensitive p42/p44 mitogen-activated protein kinase (MAPK)-mediated pathway that could be inhibited by MAPK kinase (MEK)1/2 antagonists. Chemotaxis toward ETs did not involve p38 or stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) and was not inhibited by hypoxia. Incubation of tumor cells with ET-2 also increased chemotaxis toward the chemokines CXCL12 and CCL21. As well as inducing chemotaxis of tumor cells, ET-1 and ET-2 increased tumor cell invasion through Matrigel. Furthermore, stimulation of macrophage/tumor cell cocultures with ETs led to increased matrix metalloproteinase (MMP)-2 and -9 production by macrophages and a marked increase in invasion of tumor cells. Antagonism of either ET-RA or ET-RB decreased the invasion seen in ET-stimulated cocultures, as did a broad-spectrum MMP inhibitor. Immunohistochemical staining of human breast tumor sections showed increased ET and ET receptor protein expression by tumor cells in invasive ductal carcinoma compared with normal breast tissue or ductal carcinoma in situ. Furthermore, tumor cell ET and receptor expression was stronger at the invasive margin of invasive ductal carcinomas, in the lymphovascular space, and in lymph node metastases. ET expression often colocalized with ET-RB expression in all neoplastic tissue indicating a possible autocrine action of ETs. We suggest that expression of ETs and their receptors by human breast tumors, particularly in conjunction with a high macrophage infiltrate, may have a role in the progression of breast cancer and the invasion of tumor cells.

Have lessons from past failures brought us closer to the success of immunotherapy in metastatic pancreatic cancer?

Pancreatic cancer is extremely resistant to chemo- and radiation-therapies due to its inherent genetic instability, the local immunosuppressive microenvironment and the remarkable desmoplastic stromal changes which characterize this cancer. Therefore, there is an urgent need for improvement on standard current therapeutic options. Immunotherapies aimed at harnessing endogenous antitumor immunity have shown promise in multiple tumor types. In this review, we give an overview of new immune-related therapeutic strategies currently being tested in clinical trials in pancreatic cancer. We propose that immunotherapeutic strategies in combination with current therapies may offer new hopes in this most deadly disease.

Expression of endothelins and their receptors promotes an invasive phenotype of breast tumor cells but is insufficient to induce invasion in benign cells.

There is increased staining of endothelins (ET-1, -2, and -3) and receptors (ET-RA and -RB) in invasive breast tumors compared to nonneoplastic tissue, and ETs stimulate MCF-7 cell invasion in vitro. We analyzed ETstimulation of benign and transformed mammary epithelial cells, and whether expression of ETs is sufficient to induce invasiveness. In breast cancer patient serum, ET-1 was increased in those patients with lymph node metastases compared to those with no lymph node involvement; ETs, however, had no mitogenic effect on breast tumor cell lines in vitro. The benign mammary epithelial cell line, hTERT-HME1, and the poorly invasive breast tumor cell line MCF-7 secreted low levels of ET-1, while the invasive cell lines SKBR3 and MDAMB231 secreted high levels. Expression of the ETs and receptors by the cell lines broadly correlated with their in vitro invasiveness; overexpression of ETs in MCF-7 cells increased basal invasion. ET-mediated invasion involved both receptors and a calcium influx to induce a pertussis toxin-sensitive MAPK pathway. MMP-14 activity was induced via ET-RA in an autocrine manner. In contrast to transformed cells, ET stimulation or overexpression did not induce an invasive phenotype in benign cells. Benign cells do not respond to ETs, and ET expression is not sufficient to induce invasion; however, the level of ET production by tumor cells correlates with their invasiveness, and increasing expression of the ET axis promotes breast tumor cell invasion via both receptors, while MMP-14 is induced via ET-RA.

Tumour Microenvironment: Overview with an Emphasis on the Colorectal Liver Metastasis Pathway.

The tumour microenvironment (TME) represents a dynamic network that plays an important role in tumour initiation, proliferation, growth, and metastasis. Cell behaviour may be regulated by interplay of molecular interactions involving positive and negative reinforcement as well as a high level of cross-talk, which determines this system. Additionally, cancer involves cell proliferation, its malignancy defined by the tumour's ability to break down normal tissue architecture and by a dynamic process of invasion and metastasis. The metastatic cascade is regulated by a chain of molecular steps which triggers the progression of the developing cancer cell in the primary tumour into a number of transformations, leading to invasion and proceeding to metastases. Tumour-associated macrophages (TAMs) play a key-role in the progression from inflammatory conditions to cancer; TAMs are also capable of infiltrating the tumour microenvironment. Furthermore, myeloid-derived suppressor cells (MDSCs), a population of inhibitory immune cells, have been reported to increase in various cancer types, although characterising human MDSCs remains difficult, as their phenotype is quite variable. The future of cancer treatment is likely to involve creating more drugs that target these elements as well as others. An overview of the tumour's microenvironment is, therefore, presented in this paper, focusing on the metastatic pathways of primary colorectal cancer to the liver.