NSAID activated gene (NAG-1), a modulator of tumorigenesis

The NSAID activated gene (NAG-1), a member of the TGF-beta superfamily, is involved in tumor progression and development. The over-expression of NAG-1 in cancer cells results in growth arrest and increase in apoptosis, suggesting that NAG-1 has anti-tumorigenic activity. This conclusion is further supported by results of experiments with transgenic mice that ubiquitously express human NAG-1. These transgenic mice are resistant to the development of intestinal tumors following treatment with azoxymethane or by introduction of a mutant APC gene. In contrast, other data suggest a pro-tumorigenic role for NAG-1, for example, high expression of NAG-1 is frequently observed in tumors. NAG-1 may be like other members of the TGF-beta superfamily, acting as a tumor suppressor in the early stages, but acting pro-tumorigenic at the later stages of tumor progression. The expression of NAG-1 can be increased by treatment with drugs and chemicals documented to prevent tumor formation and development. Most notable is the increase in NAG-1 expression by the inhibitors of cyclooxygenases that prevent human colorectal cancer development. The regulation of NAG-1 is complex, but these agents act through either p53 or EGR-1 related pathways. In addition, an increase in NAG-1 is observed in inhibition of the AKT/GSK-3beta pathway, suggesting NAG-1 alters cell survival. Thus, NAG-1 expression is regulated by tumor suppressor pathways and appears to modulate tumor progression.     

A critical role for autophagy in pancreatic cancer

Autophagy is a regulated catabolic process that leads to the lysosomal degradation of damaged proteins, organelles and other macromolecules, with subsequent recycling of bioenergetic intermediates. The role of autophagy in cancer is undoubtedly complex and likely dependent on tumor type and on the cellular and developmental context. While it has been well demonstrated that autophagy may function as a tumor suppressor, there is mounting evidence that autophagy may have pro-tumorigenic roles, e.g., promoting therapeutic resistance as well as survival under stresses such as hypoxia and nutrient deprivation. These two, seemingly disparate functions can be reconciled by a possible temporal role of autophagy during tumor development, initially suppressing tumor initiation yet supporting tumor growth at later stages.     

A critical role for autophagy in pancreatic cancer

Autophagy is a regulated catabolic process that leads to the lysosomal degradation of damaged proteins, organelles and other macromolecules, with subsequent recycling of bioenergetic intermediates. The role of autophagy in cancer is undoubtedly complex and likely dependent on tumor type and on the cellular and developmental context. While it has been well demonstrated that autophagy may function as a tumor suppressor, there is mounting evidence that autophagy may have pro-tumorigenic roles, e.g., promoting therapeutic resistance as well as survival under stresses such as hypoxia and nutrient deprivation. These two, seemingly disparate functions can be reconciled by a possible temporal role of autophagy during tumor development, initially suppressing tumor initiation yet supporting tumor growth at later stages.     

Dynamics of chemiluminescence response of Syrian hamster blood neutrophils to the growth of 10 different strains of tumor cells]

The production of H2O2 by blood neutrophils of Syrian hamsters, bearing 10 different transplanted tumors was studied at different stages of tumor growth by the luminol-dependent chemiluminescence (CL) test. It was demonstrated that during the tumors growth, two types of the blood neutrophils CL reaction can be registered. The first type of reaction represents very early and significant decrease of spontaneous CL of blood neutrophils, already evident before the appearance of palpable tumor nodules in animals, bearing in vivo selected cell strains. Subsequent subcutaneous tumor appearance in the animals was followed by increased CL of neutrophils. The second type of reaction was characteristic for in vitro transformed cells never selected in vivo. In this case the increase of CL of blood neutrophils at early stages of tumor growth was followed with the decrease of this activity at the period of active tumor growth. Possible relation of this reactions to the survival and growth of different tumor cells in vivo is discussed.     

Heterogeneous Distribution of Fetal Microchimerism in Local Breast Cancer Environment

Fetal cells enter maternal circulation during pregnancy and persist in the woman’s body for decades, achieving a form of physiological microchimerism. These cells were also evidenced in tumors. We investigated the frequency and concentration of fetal microchimerism in the local breast cancer environment. From 19 patients with confirmed breast neoplasia, after breast surgical resection, we collected three fresh specimens from the tumor core, breast tissue at tumor periphery, and adjacent normal breast tissue. The presence of male DNA was analyzed with a quantitative PCR assay for the sex determining region gene (SRY) gene. In the group of women who had given birth to at least one son, we detected fetal microchimerism in 100% of samples from tumors and their periphery and in 64% (9 of 14) of those from normal breast tissue. The tissues from the tumor and its periphery carry a significantly increased number of SRY copies compared to its neighboring common breast tissue (p = 0.005). The median of the normalized SRY-signal was about 77 (range, 3.2–21467) and 14-fold (range, 1.3–2690) greater in the tumor and respectively in the periphery than in the normal breast tissue. In addition, the relative expression of the SRY gene had a median 5.5 times larger in the tumor than in its periphery (range, 1.1–389.4). We found a heterogeneous distribution of fetal microchimerism in breast cancer environment. In women with sons, breast neoplasia harbors male cells at significantly higher levels than in peripheral and normal breast tissue.     

Prognostic utility of circulating transforming growth factor beta 1 in breast cancer patients

Transforming growth factor betas (TGF-?s) are multifunctional cytokines with a biphasic role in breast tumorigenesis, acting as tumor suppressors at early stages while stimulating tumor progression at later stages (TGF-? switch). Among the 3 human isoforms, TGF-?1 is known to be overexpressed in several tumor types including breast tumors. TGF-? signaling and "crosstalk" in the tumor microenvironment presents a unique challenge and an opportunity to develop novel therapies. We assessed circulating TGF-?1 levels by ELISA in blood samples from 117 previously untreated breast cancer patients in this prospective study to explore the TGF-? switch at the forefront. The levels were correlated with clinicopathological prognosticators like age, menopausal status, nodal status, histological type, histological grade, necrosis, stromal involvement, and survival. Higher mean preoperative serum TGF-?1 was observed in early-stage patients than controls (p=0.05) as revealed by receiver operating characteristic (ROC) analysis. Elevation of TGF-?1 was evident in patients with advanced-stage breast cancer compared with those having early-stage disease (p=0.0001). Prognosticators of an aggressive phenotype were associated with higher TGF-?1 levels, and higher levels thus announced the likelihood of relapse, marking the role of TGF-?1 as a tumor promoter and evidencing the existence of a TGF-? switch. Moreover, higher levels of TGF-?1 shortened the overall survival in breast cancer patients (p=0.010). The results indicate that circulating TGF-?1 may be used as a predictive and prognostic marker in breast carcinoma.     

CXCL7-induced macrophage infiltration in lung tumor is independent of CXCR2 expression: CXCL7-induced macrophage chemotaxis in LLC tumors

Chemokines play diverse roles in modulating the immune response during tumor development. Levels of CXC chemokine ligand 7 (CXCL7) protein vary during tumorigenesis, and the evidence suggests that this chemokine serves as a novel biomarker of early-stage lung cancer. We investigated the effect of CXCL7 gene expression on the infiltration of myeloid cells into the tumor microenvironment in Lewis lung carcinoma (LLC). Tumors established from LLC cells overexpressing CXCL7 (CXCL7-LLC tumors) increased the infiltration of CD206⁺ M2 macrophages at the early stages of tumorigenesis. This infiltration was independent of CXCR2 expression on either tumor cells or macrophages. CXCL7-LLC tumors developed faster than control-LLC tumors (IRES-LLC tumor) did. The extent of CD4⁺ T cell, CD8⁺ T cell, and natural killer T cell infiltration was similar between the two tumor groups. Our findings suggest that CXCL7 attracts macrophages especially at the tumor site and may accelerate lung tumor development in the early stages.     

In situ detection and distribution of inflammatory cytokines during the course of infection with Nocardia brasiliensis

Actinomycetoma, caused by the intracellular bacterium Nocardia brasiliensis, is characterized by an infiltration of several inflammatory cell populations. To explore aspects of the immune response in the pathogenesis of these bacteria we injected 10(6) CFU in footpads of BALB/c mice. After 1, 2, 3, 4, 7, 30 and 90 days immunohistochemistry was performed to compare presence and distribution of the inflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IFN-gamma, IL-4, IL-10, and TGF-beta. Analysis of serial paraffin tissue sections showed strong participation and differences in distribution of cytokine-producing cells during the course of infection. Several TNF-alpha immunoreactive lymphocytes of the dermis were present during the course of the infection, but absent in the site of inflammation. During the first 4 days, IL-1 beta immunoreactivity was observed in dendritic epidermal cells and in cells surrounding the neutrophils around the grain. In later stages of infection, immunoreactive cells to this cytokine were mainly in the periphery of the microabscesses. Strong immunoreactivity was observed with IL-6 during the course of infection. Some cells in the epidermis and dermis, as well as muscle cells and several cells at the periphery of the microabscesses, showed strong IL-6 immunoreactivity. Cells immunoreactive to IL-4, IL-10, IFN-gamma and TGF-beta were present at the site of infection and, in later stages, in cells at the periphery of the microabscesses. In conclusion a mix of proinflammatory and antiinflammatory cytokines are produced at the same time by host cells. According to their distribution, inflammatory cytokines seems to have different functions during the course of infection with the intracellular bacterium N. brasiliensis.     

Stage-Stratified Analysis of Prognostic Significance of Tumor Size in Patients with Gastric Cancer

Background

The prognostic significance of tumor size in gastric cancer is not well defined. The objective of this study was to identify the prognostic value of tumor size in patients with gastric cancer.

Methods

We retrospectively reviewed a total of 1800 patients with gastric cancer admitted to our hospital between 1997 and 2007. These patients were divided into two groups according to tumor size: small size group (SSG, tumor ≤5 cm) and large size group (LSG, tumor >5 cm). We compared clinico-pathologic features of the two groups and investigated the prognostic factors by performing univariate, multivariate, and stage- stratified analyses according to tumor size.

Results

LSG had more aggressive clinico-pathologic features than SSG. Tumor size was an independent prognostic indicator in patients with gastric cancer. In a stratified-pT, pN, and pTNM analysis, survival of patients with LSG was significantly worse than that of patients with SSG and advanced stage. Tumor size was not a significant predictor of survival in patients with early stage tumors. Large tumor size was associated with shorter survival in patients with stages N0, N1, N2, and N3, and stages I, II, III, and IV.

Conclusions

Tumor size is a simple and practical prognostic factor in patients with gastric cancer. Tumor size could supplement clinical staging in the future.     

Depletion of regulatory T cells facilitates growth of established tumors: a mechanism involving the regulation of myeloid-derived suppressor cells by lipoxin A4

Regulatory T cells (Tregs) are thought to facilitate tumor development by suppressing protective antitumor immune responses. However, recent clinical and laboratory studies show that Tregs are a favorable element against cancer. In this study, we provide evidence that Tregs have both promoting and inhibiting effects on tumors, depending on the stage of tumor development. By using 0.5 mg cyclophosphamide, we constructed a murine liver cancer model in which Tregs were continuously and selectively depleted. Under such conditions, we found that tumor growth was inhibited at early stages but accelerated later on. Analysis of the tumor microenvironment disclosed that long-term Treg depletion by 0.5 mg cyclophosphamide treatment induced Gr-1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs). Ablation of MDSCs by anti-Gr-1 Ab blocked Treg depletion-induced promotion of tumor growth. Furthermore, lipoxygenases 5 and 12, two enzymes participating in the biosynthesis of the lipid anti-inflammatory mediator lipoxin A(4), were upregulated or downregulated by Treg depletion or adoptive transfer. Correspondingly, the levels of lipoxin A(4) were increased or decreased. Lipoxin A(4) thus regulated the induction of MDSCs in response to Treg depletion. These findings suggest that Tregs may play different roles at different stages of tumor growth: promoting early and inhibiting late tumor growth. Our study also suggests that the interplay among Tregs, MDSCs, and lipoxin A(4) tunes the regulation of tumor-associated inflammation.     

Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression

Prostate cancer (PCa) is the second-leading cause of cancer-related mortality, after lung cancer, in men from developed countries. In its early stages, primary tumor growth is dependent on androgens, thus generally can be controlled by androgen deprivation therapy (ADT). Eventually however, the disease progresses to castration-resistant prostate cancer (CRPC), a lethal form in need of more effective treatments. G-protein coupled receptors (GPCRs) comprise a large clan of cell surface proteins that have been implicated as therapeutic targets in PCa growth and progression. The findings reported here provide intriguing evidence of a role for the newly characterized glutamate family member GPR158 in PCa growth and progression. We found that GPR158 promotes PCa cell proliferation independent of androgen receptor (AR) functionality and that this requires its localization in the nucleus of the cell. This suggests that GPR158 acts by mechanisms different from other GPCRs. GPR158 expression is stimulated by androgens and GPR158 stimulates AR expression, implying a potential to sensitize tumors to low androgen conditions during ADT via a positive feedback loop. Further, we found GPR158 expression correlates with a neuroendocrine (NE) differentiation phenotype and promotes anchorage-independent colony formation implying a role for GPR158 in therapeutic progression and tumor formation. GPR158 expression was increased at the invading front of prostate tumors that formed in the genetically defined conditional Pten knockout mouse model, and co-localized with elevated AR expression in the cell nucleus. Kaplan-Meier analysis on a dataset from the Memorial Sloan Kettering cancer genome portal showed that increased GPR158 expression in tumors is associated with lower disease-free survival. Our findings strongly suggest that pharmaceuticals targeting GPR158 activities could represent a novel and innovative approach to the prevention and management of CRPC.     

Significance of host heparanase in promoting tumor growth and metastasis

Heparanase, the sole heparan sulfate degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, angiogenesis and metastasis. Much of the impact of heparanase on tumor progression is related to its function in mediating tumor-host crosstalk, priming the tumor microenvironment to better support tumor growth and metastasis. We have utilized mice over-expressing (Hpa-tg) heparanase to reveal the role of host heparanase in tumor initiation, growth and metastasis. While in wild type mice tumor development in response to DMBA carcinogenesis was restricted to the mammary gland, Hpa-tg mice developed tumors also in their lungs and liver, associating with reduced survival of the tumor-bearing mice. Consistently, xenograft tumors (lymphoma, melanoma, lung carcinoma, pancreatic carcinoma) transplanted in Hpa-tg mice exhibited accelerated tumor growth and shorter survival of the tumor-bearing mice compared with wild type mice. Hpa-tg mice were also more prone to the development of metastases following intravenous or subcutaneous injection of tumor cells. In some models, the growth advantage was associated with infiltration of heparanase-high host cells into the tumors. However, in other models, heparanase-high host cells were not detected in the primary tumor, implying that the growth advantage in Hpa-tg mice is due to systemic factors. Indeed, we found that plasma from Hpa-tg mice enhanced tumor cell migration and invasion attributed to increased levels of pro-tumorigenic factors (i.e., RANKL, SPARC, MIP-2) in the plasma of Hpa-Tg vs. wild type mice. Furthermore, tumor aggressiveness and short survival time were demonstrated in wild type mice transplanted with bone marrow derived from Hpa-tg but not wild type mice. These results were attributed, among other factors, to upregulation of pro-tumorigenic (i.e., IL35⁺) and downregulation of anti-tumorigenic (i.e., IFN-γ⁺) T-cell subpopulations in the spleen, lymph nodes and blood of Hpa-tg vs. wild type mice and their increased infiltration into the primary tumor. Collectively, our results emphasize the significance of host heparanase in mediating the pro-tumorigenic and pro-metastatic interactions between the tumor cells and the host tumor microenvironment, immune cells and systemic factors.     

SP/NK1R system regulates carcinogenesis in prostate cancer: Shedding light on the antitumoral function of aprepitant

AimsProstate cancer continues to be one of the main global health issues in men. Neuropeptide substance P (SP) acting via neurokinin-1receptor (NK1R) promotes tumorigenicity in many human malignant tumors. However, its pro-tumorigenic functions and the therapeutic effects of its inhibition in prostate cancer remain unclear.MethodsMTT assay was employed for measuring cellular proliferation and cytotoxicity. mRNAs and proteins expression levels were evaluated by qRT-PCR and western blot assay, respectively. Gelatinase activity was assessed by zymography. The migration ability was defined using wound-healing assay. Flow cytometry was employed to evaluate the cell cycle distribution. We also performed an in vivo experiment in a mouse model of prostate cancer to confirm the in vitro therapeutic effect of targeting the SP/NK1R system.ResultsWe found a noticeable increase in the expression of the truncated isoform of NK1R as an oncogenic NK1R splice variant in tumor cells. We also demonstrated that SP promotes both proliferative and migrative phenotypes of prostate cancer through modifying cell cycle-related proteins (c-Myc, cyclin D1, cyclin B1, p21), and apoptosis-related genes (Bcl-2 and Bax), promoting cell migration and increasing MMP-2 and MMP-9 expression and activity, while aprepitant administration could remarkably reverse these effects. SP also stimulated tumor growth in vivo, which was correlated with shorter survival times, while aprepitant reversed this effect and led to significantly longer survival time.SignificanceOur findings suggest that SP/NK1R system may serve as a novel therapeutic target in prostate cancer and support the possible candidacy of aprepitant in future prostate cancer therapy.     

Unilateral multicentric breast cancer

Clinical characteristics of unilateral multicentric breast cancer (UMBC) were explored depending on aggressiveness, survival rate, disease-free period and local recurrence. The study included 296 women with breast cancer, surgically treated between 1990 and 2001. UMBC was histologically proved in 29 (9.8%) patients. Multicentricity was defined by following criteria: a) tumor with minimum one satellite node in the same or other quadrant of the breast; b) minimum one cut through the breast without tumor cells; c) histopathologically, discontinued tumors with intra-ductal invasion. The average age of patients was 63.4 (range 36-85). There were 9 (31.0%) women with one satellite node, 7 (24.1%) women with two satellite nodes, and 13 (44.8%) women with three or more satellite nodes. At the operation, axilla was positive in 20 (68.9%) women. Steroid receptors were highly positive in 12 (41.4%) patients. Primary and secondary tumors were of the same histological type in 26 (89.6%) patients. Local recurrence was found in only 3 (10.3%) patients. A five-year period without disease was achieved in 24 (82.7%) women. Kaplan-Meier analysis showed a significantly higher survival rate at lower tumor stages (I or II) unlike in advanced stages with predominantly N2 grade. The results of this study showed a slightly lower five-year disease-free period than in the case of patients with monocentric breast cancer (MOBC). The survival rate was significantly lower at all advanced stages, especially determined by N2 axilla. Therefore, the conclusion is that multicentricity doesn't increase the risk of poor prognosis, especially at lower tumor stages.     

Expression of mast cell proteases correlates with mast cell maturation and angiogenesis during tumor progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.     

Complexities of TGF-β targeted cancer therapy

Many advanced tumors produce excessive amounts of Transforming Growth Factor-β (TGF-β) which, in normal epithelial cells, is a potent growth inhibitor. However, in oncogenically activated cells, the homeostatic action of TGF-β is often diverted along alternative pathways. Hence, TGF-β signaling elicits protective or tumor suppressive effects during the early growth-sensitive stages of tumorigenesis. However, later in tumor development when carcinoma cells become refractory to TGF-β-mediated growth inhibition, the tumor cell responds by stimulating pathways with tumor progressing effects. At late stages of malignancy, tumor progression is driven by TGF-β overload. The tumor microenvironment is a target of TGF-β action that stimulates tumor progression via pro-tumorigenic effects on vascular, immune, and fibroblastic cells. Bone is one of the richest sources of TGF-β in the body and a common site for dissemination of breast cancer metastases. Osteoclastic degradation of bone matrix, which accompanies establishment and growth of metastases, triggers further release of bone-derived TGF-β. This leads to a vicious positive feedback of tumor progression, driven by ever increasing levels of TGF-β released from both the tumor and bone matrix. It is for this reason, that pharmaceutical companies have developed therapeutic agents that block TGF-β signaling. Nonetheless, the choice of drug design and dosing strategy can affect the efficacy of TGF-β therapeutics. This review will describe pre-clinical and clinical data of four major classes of TGF-β inhibitor, namely i) ligand traps, ii) antisense oligonucleotides, iii) receptor kinase inhibitors and iv) peptide aptamers. Long term dosing strategies with TGF-β inhibitors may be ill-advised, since this class of drug has potentially highly pleiotropic activity, and development of drug resistance might potentiate tumor progression. Current paradigms for the use of TGF-β inhibitors in oncology have therefore moved towards the use of combinatorial therapies and short term dosing, with considerable promise for the clinic.     

Pro-Tumorigenic Phosphorylation of p120 Catenin in Renal and Breast Cancer

Altered protein expression and phosphorylation are common events during malignant transformation. These perturbations have been widely explored in the context of E-cadherin cell-cell adhesion complexes, which are central in the maintenance of the normal epithelial phenotype. A major component of these complexes is p120 catenin (p120), which binds and stabilizes E-cadherin to promote its adhesive and tumor suppressing function. However, p120 is also an essential mediator of pro-tumorigenic signals driven by oncogenes, such as Src, and can be phosphorylated at multiple sites. Although alterations in p120 expression have been extensively studied by immunohistochemistry (IHC) in the context of tumor progression, little is known about the status and role of p120 phosphorylation in cancer. Here we show that tyrosine and threonine phosphorylation of p120 in two sites, Y228 and T916, is elevated in renal and breast tumor tissue samples. We also show that tyrosine phosphorylation of p120 at its N-terminus, including at the Y228 site is required for its pro-tumorigenic potential. In contrast, phosphorylation of p120 at T916 does not affect this p120 function. However, phosphorylation of p120 at T916 interferes with epitope recognition of the most commonly used p120 antibody, namely pp120. As a result, this antibody selectively underrepresents p120 levels in tumor tissues, where p120 is phosphorylated. Overall, our data support a role of p120 phosphorylation as a marker and mediator of tumor transformation. Importantly, they also argue that the level and localization of p120 in human cancer tissues immunostained with pp120 needs to be re-evaluated.