Non-hematopoietic expression of IDO is integrally required for inflammatory tumor promotion

Indoleamine 2,3-dioxygenase (IDO) is generally considered to be immunosuppressive but recent findings suggest this characterization oversimplifies its role in disease pathogenesis. Recently, we showed that IDO is essential for tumor outgrowth in the classical two-stage model of inflammatory skin carcinogenesis. Here, we report that IDO loss did not exacerbate classical inflammatory responses. Rather, IDO induction could be elicited by environmental signals and tumor promoters as an integral component of the inflammatory tissue microenvironment even in the absence of cancer. IDO loss had limited impact on tumor outgrowth in carcinogenesis models that lacked an explicit inflammatory tumor promoter. In the context of inflammatory carcinogenesis where IDO was critical to tumor development, the most important source of IDO was radiation-resistant non-hematopoietic cells, consistent with evidence that loss of the IDO regulatory tumor suppressor gene Bin1 in transformed skin cells facilitates IDO-mediated immune escape by a cell autonomous mechanism. Taken together, our results identify IDO as an integral component of ‘cancer-associated’ inflammation that tilts the immune system toward tumor support. More generally, they promote the concept that mediators of immune escape and cancer-associated inflammation may be genetically synonymous.     

The resolution of inflammation and cancer

Inflammation has long been thought to contribute to the development of cancer; however there is also clear evidence that the immune system can recognize and eliminate cancer cells. Current research suggests that cancer-associated inflammation has a dual role in tumor progression; inflammatory mediators promote the malignant activity of cancer cells by acting as growth factors and also stimulate angiogenesis, however, cancer-associated inflammation is also linked with immune-suppression that allows cancer cells to evade detection by the immune system. In this review we will discuss the dual role of inflammation in cancer and how endogenous anti-inflammatory mechanisms may equally be important in carcinogenesis.     

Immunoepigenetics: the unseen side of cancer immunoediting

Cancer immunosurveillance representing, till recently, the explanatory framework relating cancer and the immune system, does not convincingly explain tumor escape. At the beginning of the decade, a new theory emerged, namely the immunoediting theory, and it comprehensively defines the role of the immune system in carcinogenesis. The core of this theory embraces the concept that the immune system on the one hand protects the body from cancer and on the other it shapes the immunogenicity of these cancers, thus presents a persuasive rationalization of the resistance of tumors against the immune response. With the immune system playing, in this context, such a pivotal role in shaping the tumor immune profile and in subsequent oncogenesis, it seems rather paradoxical to accept the immunocompetent host's immune system as a constant moiety. While DNA mutations of immune genes create a rather polymorphic condition, their frequency is much lower than that of other genetic events. Of these, epigenetic alterations give rise to new epialleles, which can reach up to 100% per locus. Bearing in mind that cancer is characterized by a tremendous amount of epigenetic aberrations, in both gene and global level, it is reasonable to postulate that, for the same unknown causes, analogous aberrations could affect the immune genes. Should this be the case, the relation between oncogenesis and the immune system appears much more dynamic and complex. Such an immunoepigenetic approach to carcinogenesis could improve our understanding of a series of common cancer-related aspects, such as environmental risk factors, effectiveness of demethylating agents, failure of current immunotherapies, etc. Moreover, this immunoepigenetic paradigm will take the current perception of the immune system and cancer interrelation further and beyond, constituting that the immunoresistant cancer cell phenotype is not shaped by the immune system acting as a steady and rigid evolutionary pressure, but rather as an extremely dynamic variable.     

Stimulator of interferon genes (STING): A “new chapter” in virus-associated cancer research. Lessons from wild-derived mouse models of innate immunity

Thanks to the numerous studies that have been carried out recently in the field of cytosolic DNA sensing, STING (Stimulator of Interferon Genes) is now recognized as a key mediator of innate immune signaling. A substantial body of evidence derived from in vivo mouse models demonstrates that STING-regulated pathways underlie the pathogenesis of many diseases including infectious diseases and cancers. It has also become evident from these studies that STING is a promising therapeutic target for the treatment of cancer. However, mouse strains commonly used for modelling innate immune response against infections or tumors do not allow investigators to accurately reproduce certain specific characteristics of immune response observed in human cells. In this review, we will discuss recent data demonstrating that the use of wild-derived genetically distinct inbred mice as a model for investigation into the innate immunity signaling networks may provide valuable insight into the STING-regulated pathways specific for human cells. The maximum complexity of STING-mediated mechanisms can probably be seen in case of DNA virus-induced carcinogenesis in which STING may perform unexpected biological activities. Therefore, in another part of this review we will summarize emerging data on the role of STING in human DNA virus-related oncopathologies, with particular attention to HPV-associated cervical cancer, aiming to demonstrate that STING indeed “starts a new chapter” in research on this issue and that wild-derived mouse models of STING-mediated response to infections will probably be helpful in finding out molecular basis for clinical observations.     

Arsenic possibly influences carcinogenesis by affecting arginine and zinc metabolism

The role of arsenic in carcinogenesis is controversial. There is no doubt arsenic can influence carcinogenesis under certain conditions. However, a review of the findings relating arsenic to cancer indicates that arsenic mainly affects carcinogenesis indirectly by influencing other metabolic systems (i.e., immune system) or nutrients (i.e., arginine, zinc) that may have a more direct role in the carcinogenic process. Depending upon the level of exposure, arsenic can either inhibit or activate interferon, an inhibitor of virus replication. Furthermore, arsenic can apparently inhibit some virusinduced tumorigenesis. However, once a tumor is initiated, arsenic enhances tumor growth, possibly by affecting the immune response. Recent experiments in our laboratory demonstrated that arsenic metabolically interacts with arginine and zinc, both of which apparently influence the immune response. Arsenic evidently has a role that strongly influences the metabolism of arginine, which is an immunostimulatory amino acid. Furthermore, the effect of arsenic on arginine metabolism is apparently modified by the zinc status of the animal. Because arsenic can apparently affect cancer development through several indirect or direct mechanisms, probably the only general conclusion that can be made about arsenic and cancer is that arsenic, depending upon dosage, route of administration, and chemical form, modifies the induction or development of some tumors.     

MicroRNAs as biomarkers of cervical cancer development: a literature review on miR-125b and miR-34a

MicroRNAs are non-coding RNAs with important functions in several biological processes, such as, regulation of cell cycle, immune response, inflammation, and apoptosis. In fact, deregulation and abnormal expression of these molecules is associated with human pathologies including cancer and several have already emerged as potential prognostic biomarkers in different neoplasias. miR-34a is directly regulated by p53 and acts as tumor suppressor while miR-125b plays a significant role in immune response and apoptosis. In cervical carcinogenesis, HPV proteins seem to interact with both miR-34a and miR-125b changing its expression and promoting persistent infection and cervical cancer development. In this review we describe the potential role of miR-125b and miR-34a in cervical carcinogenesis, including interaction with HPV and mechanism of deregulation. Additionally, their clinical applications in cervical cancer as prognostic/predictive biomarkers are also briefly discussed.     

Dual roles of immune cells and their factors in cancer development and progression

Traditional wisdom holds that intact immune responses, such as immune surveillance or immunoediting, are required for preventing and inhibiting tumor development; but recent evidence has also indicated that unresolved immune responses, such as chronic inflammation, can promote the growth and progression of cancer. Within the immune system, cytotoxic CD8(+) and CD4(+) Th1 T cells, along with their characteristically produced cytokine IFN-γ, function as the major anti-tumor immune effector cells, whereas tumor associated macrophages (TAM) or myeloid-derived suppressive cells (MDSC) and their derived cytokines IL-6, TNF, IL-1β and IL-23 are generally recognized as dominant tumor-promoting forces. However, the roles played by Th17 cells, CD4(+) CD25(+) Foxp3(+) regulatory T lymphocytes and immunoregulatory cytokines such as TGF-β in tumor development and survival remain elusive. These immune cells and the cellular factors produced from them, including both immunosuppressive and inflammatory cytokines, play dual roles in promoting or discouraging cancer development, and their ultimate role in cancer progression may rely heavily on the tumor microenvironment and the events leading to initial propagation of carcinogenesis.     

A novel combination immunotherapy for cancer by IL-13Rα2-targeted DNA vaccine and immunotoxin in murine tumor models

Optimum efficacy of therapeutic cancer vaccines may require combinations that generate effective antitumor immune responses, as well as overcome immune evasion and tolerance mechanisms mediated by progressing tumor. Previous studies showed that IL-13Rα2, a unique tumor-associated Ag, is a promising target for cancer immunotherapy. A targeted cytotoxin composed of IL-13 and mutated Pseudomonas exotoxin induced specific killing of IL-13Rα2(+) tumor cells. When combined with IL-13Rα2 DNA cancer vaccine, surprisingly, it mediated synergistic antitumor effects on tumor growth and metastasis in established murine breast carcinoma and sarcoma tumor models. The mechanism of synergistic activity involved direct killing of tumor cells and cell-mediated immune responses, as well as elimination of myeloid-derived suppressor cells and, consequently, regulatory T cells. These novel results provide a strong rationale for combining immunotoxins with cancer vaccines for the treatment of patients with advanced cancer.     

The CXCR4 Antagonist AMD3465 Regulates Oncogenic Signaling and Invasiveness In Vitro and Prevents Breast Cancer Growth and Metastasis In Vivo

CXCR4, the receptor for stromal-derived factor-1, is reportedly involved in breast carcinogenesis. However, the mechanisms through which CXCR4 contributes to breast cancer cell growth and metastases are poorly understood. In this study, we examined the putative in vitro and in vivo anti-cancer effects of the specific CXCR4 inhibitor AMD3465. Here, we report that AMD3465 triggers a reduction in breast cancer cell invasiveness in vitro, and promotes marked changes in oncogenic signaling proteins including a reduction in STAT3, JAK2, AKT, and CXCR4 phosphorylation and the reduced expression of GSK3 and cMYC. Using three breast cancer cell lines as murine syngeneic immunocompetent breast cancer models, we found that AMD3465 inhibited breast tumor formation and reduced tumor cell metastases to the lung and liver. Furthermore, treatment with AMD3465 significantly reduced the infiltration of myeloid CD11b positive cells at the aforementioned metastatic sites as well as the spleen implying this agent could regulate the formation of the tumor microenvironment and conceivably the premetastatic niche. In conclusion, our studies suggest that AMD3465 inhibits breast cancer growth and metastases by acting on tumor cells as well as immune cells that constitute the tumor microenvironment. This process appears to be regulated, at least in part, through the modulation of oncogenic signaling that includes the STAT3 pathway. Thus, CXCR4 could be a novel target for breast cancer therapy.     

Chronic Mycobacterium marinum infection acts as a tumor promoter in Japanese Medaka (Oryzias latipes)

An accumulating body of research indicates there is an increased cancer risk associated with chronic infections. The genus Mycobacterium contains a number of species, including M. tuberculosis, which mount chronic infections and have been implicated in higher cancer risk. Several non-tuberculosis mycobacterial species, including M. marinum, are known to cause chronic infections in fish and like human tuberculosis, often go undetected. The elevated carcinogenic potential for fish colonies infected with Mycobacterium spp. could have far reaching implications because fish models are widely used to study human diseases. Japanese medaka (Oryzias latipes) is an established laboratory fish model for toxicology, mutagenesis, and carcinogenesis; and produces a chronic tuberculosis-like disease when infected by M. marinum. We examined the role that chronic mycobacterial infections play in cancer risk for medaka. Experimental M. marinum infections of medaka alone did not increase the mutational loads or proliferative lesion incidence in all tissues examined. However, we showed that chronic M. marinum infections increased hepatocellular proliferative lesions in fish also exposed to low doses of the mutagen benzo[a]pyrene. These results indicate that chronic mycobacterial infections of medaka are acting as tumor promoters and thereby suggest increased human risks for cancer promotion in human populations burdened with chronic tuberculosis infections.     

Detection of mycoplasma infection in circulating tumor cells in patients with hepatocellular carcinoma

Many studies have shown that persistent infections of bacteria promote carcinogenesis and metastasis. Infectious agents and their products can modulate cancer progression through the induction of host inflammatory and immune responses. The presence of circulating tumor cells (CTCs) is considered as an important indicator in the metastatic cascade. We unintentionally produced a monoclonal antibody (MAb) CA27 against the mycoplasmal p37 protein in mycoplasma-infected cancer cells during the searching process of novel surface markers of CTCs. Mycoplasma-infected cells were enriched by CA27-conjugated magnetic beads in the peripheral blood mononuclear cells in patients with hepatocellular carcinoma (HCC) and analyzed by confocal microscopy with anti-CD45 and CA27 antibodies. CD45-negative and CA27-positive cells were readily detected in three out of seven patients (range 12–30/8.5 ml blood), indicating that they are mycoplasma-infected circulating epithelial cells. CA27-positive cells had larger size than CD45-positive hematological lineage cells, high nuclear to cytoplasmic ratios and irregular nuclear morphology, which identified them as CTCs. The results show for the first time the existence of mycoplasma-infected CTCs in patients with HCC and suggest a possible correlation between mycoplasma infection and the development of cancer metastasis.     

Cancer immunotherapy with CpG-ODN

Bacterial DNA and synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODN) are the ligands for the Toll-like receptor 9 (TLR9), which is expressed by B-lymphocytes and a subset of dendritic cells. CpG-ODN are strong activators of both innate and specific immunity, and drive the immune response towards the Th1 phenotype. Given the promising results obtained in several experimental models of allergies or infections, CpG-ODN are now entering clinical trials for these diseases. In cancer, promising approaches combined CpG-ODN with tumor antigens, monoclonal antibodies or dendritic cells. When no relevant tumor antigen is known, CpG-ODN can be used alone to activate locally the innate immunity and trigger a tumor-specific immune response, overcoming the need for the identification of a tumoral antigen. Preclinical models have shown impressive results and several clinical trials are on-going worldwide in melanoma, lymphoma, renal carcinoma, breast cancer and glioblastoma.     

Perspective on the dynamics of cancer

Background

The genetic diversity of cancer and the dynamic interactions between heterogeneous tumor cells, the stroma and immune cells present daunting challenges to the development of effective cancer therapies. Although cancer biology is more understood than ever, this has not translated into therapies that overcome drug resistance, cancer recurrence and metastasis. The future development of effective therapies will require more understanding of the dynamics of homeostatic dysregulation that drives cancer growth and progression.

Results

Cancer dynamics are explored using a model involving genes mediating the regulatory interactions between the signaling and metabolic pathways. The exploration is informed by a proposed genetic dysregulation measure of cellular processes. The analysis of the interaction dynamics between cancer cells, cancer associated fibroblasts, and tumor associate macrophages suggests that the mutual dependence of these cells promotes cancer growth and proliferation. In particular, MTOR and AMPK are hypothesized to be concurrently activated in cancer cells by amino acids recycled from the stroma. This leads to a proliferative growth supported by an upregulated glycolysis and a tricarboxylic acid cycle driven by glutamine sourced from the stroma. In other words, while genetic aberrations ignite carcinogenesis and lead to the dysregulation of key cellular processes, it is postulated that the dysregulation of metabolism locks cancer cells in a state of mutual dependence with the tumor microenvironment and deepens the tumor’s inflammation and immunosuppressive state which perpetuates as a result the growth and proliferation dynamics of cancer.

Conclusions

Cancer therapies should aim for a progressive disruption of the dynamics of interactions between cancer cells and the tumor microenvironment by targeting metabolic dysregulation and inflammation to partially restore tissue homeostasis and turn on the immune cancer kill switch. One potentially effective cancer therapeutic strategy is to induce the reduction of lactate and steer the tumor microenvironment to a state of reduced inflammation so as to enable an effective intervention of the immune system. The translation of this therapeutic approach into treatment regimens would however require more understanding of the adaptive complexity of cancer resulting from the interactions of cancer cells with the tumor microenvironment and the immune system.

     

Application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum; may exist in greater concentrations than their cognate antigens; and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.