The relationship between angiogenesis and the immune response in carcinogenesis and the progression of malignant disease

Recent studies have demonstrated that angiogenesis and suppressed cell- mediated immunity (CMI) play a central role in the pathogenesis of malignant disease facilitating tumour growth, invasion and metastasis. In the majority of tumours, the malignant process is preceded by a pathological condition or exposure to an irritant which itself is associated with the induction of angiogenesis and/or suppressed CMI. These include: cigarette smoking, chronic bronchitis and lung cancer; chronic oesophagitis and oesophageal cancer; chronic viral infections such as human papilloma virus and ano-genital cancers, chronic hepatitis B and C and hepatocellular carcinoma, and Epstein- Barr virus (EBV) and lymphomas; chronic inflammatory conditions such as Crohn's disease and ulcerative colitis and colorectal cancer; asbestos exposure and mesothelioma and excessive sunlight exposure/sunburn and malignant melanoma. Chronic exposure to growth factors (insulin-like growth factor-I in acromegaly), mutations in tumour suppressor genes (TP53 in Li Fraumeni syndrome) and long-term exposure to immunosuppressive agents (cyclosporin A) may also give rise to similar environments and are associated with the development of a range of solid tumours. The increased blood supply would facilitate the development and proliferation of an abnormal clone or clones of cells arising as the result of: (a) an inherited genetic abnormality; and/or (b) acquired somatic mutations, the latter due to local production and/or enhanced delivery of carcinogens and mutagenic growth factors. With progressive detrimental mutations and growth-induced tumour hypoxia, the transformed cell, to a lesser or greater extent, may amplify the angiogenic process and CMI suppression, thereby facilitating further tumour growth and metastasis. There is accumulating evidence that long-term treatment with cyclo-oxygenase inhibitors (aspirin and indomethacin), cytokines such as interferon-α, anti-oestrogens (tamoxifen and raloxifene) and captopril significantly reduces the incidence of solid tumours such as breast and colorectal cancer. These agents are anti-angiogenic and, in the case of aspirin, indomethacin and interferon-α have proven immunomodulatory effects. Collectively these observations indicate that angiogenesis and suppressed CMI play a central role in the development and progression of malignant disease. (C) 2000 Elsevier Science Ltd.

Inflammation in lung carcinogenesis

It was Dvorak in 1986 that postulated 'tumours are wounds that do not heal' as they share common cellular and molecular mechanisms, which are active in both wounds and in cancer tissue. Inflammation is a crucial part of the innate immune system that protects against pathogens and initiates adaptive immunity. Acute inflammation is usually a rapid and self-limiting process, however it does not always resolve. This leads to the establishment of a chronic inflammatory state and provides the perfect environment for carcinogenesis. Inflammation and cancer have long had an association, going back as far as Virchow in 1863, when leucocytes were noted in neoplastic tissue. It has been estimated that approximately 25% of all malignancies are initiated or exacerbated by inflammation caused by infectious agents. Furthermore, inflammation is linked to all of the six hallmarks of cancer (evasion of apoptosis, insensitivity to anti-growth signals, unlimited replicative potential, angiogenesis, increase in survival factors and invasion and metastasis). It is thought that inflammation may play a critical role in lung carcinogenesis given that individuals with inflammatory lung conditions have an increased risk of lung cancer development. Cigarette smoking can also induce inflammation in the lung and smokers are at a higher risk of developing lung cancer than non-smokers. However, exposure to a number of environmental agents such as radon, have also been demonstrated as a causative factor in this disease. This chapter will focus on inflammation as a contributory factor in non small cell lung cancer (NSCLC), concentrating primarily on the pathological involvement of the pro-inflammatory cytokines, TNF-α, IL-1β, and the CXC (ELR+) chemokine family. Targeting of inflammatory mediators will also be discussed as a therapeutic strategy in this disease. © 2013 by Nova Science Publishers, Inc. All rights reserved.

Hormonal carcinogenesis in breast cancer : cellular and molecular studies of malignant progression

We have established and characterized a series of variant cell lines in which to identify the critical factors associated with E2-induced malignant progression, and the acquisition to tamoxifen resistance in human breast cancer. Sublines of the hormone-dependent MCF-7 cell line (MCF7/MIII and MCF7/LCC1) form stable, invasive, estrogen independent tumors in the mammary fat pads of ovariectomized athymic nude mice. These cells retain expression of both estrogen (ER) and progesterone receptors (PGR), but retain sensitivity to each of the major structural classes of antiestrogens. The tamoxifen-resistant MCF7/LCC2 cells retain sensitivity to the inhibitory effects of the steroidal antiestrogen ICI 182780. By comparing the parental hormone-dependent and variant hormone-independent cells, we have demonstrated an altered expression of some estrogen regulated genes (PGR, pS2, cathepsin D) in the hormone-independent variants. Other genes remain normally estrogen regulated (ER, laminin receptor, EGF-receptor). These data strongly implicate the altered regulation of a specific subset or network of estrogen regulated genes in the malignant progression of human breast cancer. Some of the primary response genes in this network may exhibit dose-response and induction kinetics similar to pS2, which is constitutively upregulated in the MCF7/MIII, MCF7/LCC1 and MCF7/LCC2 cells.

Glutathione transferase activities in renal carcinomas and adjacent normal renal tissues : factors influencing renal carcinogenesis induced by xenobiotics

In general, the biological activation of nephrocarcinogenic chlorinated hydrocarbons proceeds via conjugatiton with glutathione. It has mostly been assamed that the main site of initial conjugation is the liver, followed by a mandatory transfer of intermediates to the kidney. It was therefore of interest to study the enzyme activities of subgroups of glutathione transferases (GSTs) in renal cancers and the surrounding normal renal tissues of the same individuals (n = 21). For genotyping the individuals with respect to known polymorphic GST isozymes the following substrates with differential specificity were used: 1-chloro-2,4-dinitrobenzene for overall GST activity (except GST θ); 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for GST α; 1,2-dichloro-4-nitro-benzene for GST μ; ethacrynic acid and 4-vinylpyridine for GST π; and methyl chloride for GST θ. In general, the normal tissues were able to metabolize the test substrates. A general decrease in individual GST enzyme activities was apparent in the course of cancerization, and in some (exceptional) cases individual activities, expressed in the normal renal tissue, were lost in the tumour tissue. The GST enzyme activities in tumours were independent of tumour stage, or the age and gender of the patients. There was little influence of known polymorphisms of GSTM1, GSTM3 and GSTP1 upon the activities towards the test substrates, whereas the influence of GSTT1 polymorphism on the activity towads methyl chloride was straightforward. In general, the present findings support the concept that the initial GST-dependent bioactivation step of nephrocarcinogenic chlorinated hydrocarbons may take place in the kidney itself. This should be a consideration in toxicokinetic modelling.

Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.

Cooperation of Notch and Ras/MAPK signaling pathways in human breast carcinogenesis

Background: Recent studies have implicated aberrant Notch signaling in breast cancers. Yet, relatively little is known about the pattern of expression of various components of the Notch pathway, or its mechanism of action. To better understand the role of the Notch pathway in breast cancer, we have undertaken a detailed expression analysis of various Notch receptors, their ligands, and downstream targets at different stages of breast cancer progression. Results: We report here that there is a general increase in the expression levels of Notch 1, 2, 4, Jagged1, Jagged2, and Delta-like 4 proteins in breast cancers, with simultaneous upregulation of multiple Notch receptors and ligands in a given cancer tissue. While Notch3 and Delta-like1 were undetectable in normal tissues, moderate to high expression was detected in several cancers. We detected the presence of active, cleaved Notch1, along with downstream targets of the Notch pathway, Hes1/Hes5, in similar to 75% of breast cancers, clearly indicating that in a large proportion of breast cancers Notch signaling is aberrantly activated. Furthermore, we detected cleaved Notch1 and Hes1/5 in early precursors of breast cancers - hyperplasia and ductal carcinoma in situ suggesting that aberrant Notch activation may be an early event in breast cancer progression. Mechanistically, while constitutively active Notch1 alone failed to transform immortalized breast cells, it synergized with the Ras/MAPK pathway to mediate transformation. This cooperation is reflected in vivo, as a subset of cleaved Notch positive tumors additionally expressed phopsho-Erk1/2 in the nuclei. Such cases exhibited high node positivity, suggesting that Notch-Ras cooperation may lead to poor prognosis. Conclusions: High level expression of Notch receptors and ligands, and its increased activation in several breast cancers and early precursors, places Notch signaling as a key player in breast cancer pathogenesis. Its cooperation with the Ras/MAPK pathway in transformation offers combined inhibition of the two pathways as a new modality for breast cancer treatment.

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

mRNA-binding protein HuR in breast carcinogenesis

Breast cancer is the most common cancer among women. Although its prognosis has improved nowadays, methods to predict the progression of the disease or to treat it are not comprehensive. This thesis work was initiated to elucidate in breast carcinogenesis the role of HuR, a ubiquitously expressed mRNA-binding protein that regulates gene expression posttranscriptionally. HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm, and this nucleocytoplasmic translocation is important for its function as a RNA-stabilizing and translational regulator. HuR has been associated with diverse cellular processes, for example carcinogenesis. The specific aims of my thesis work were to study the prognostic value of HuR in breast cancer and to clarify the mechanisms by which HuR contributes to breast carcinogenesis. My ultimate goal is, by better understanding the role of HuR in breast carcinogenesis, to aid in the discovery of novel targets for cancer therapies. HuR expression and localization was studied in paraffin-embedded preinvasive (atypical ductal hyperplasia, ADH, and ductal carcinoma in situ, DCIS) specimens as well in sporadic and familial breast cancer specimens. Our results show that cytoplasmic HuR expression was already elevated in ADH and remained elevated in DCIS as well as in cancer specimens. Clinicopathological analysis showed that cytoplasmic HuR expression associated with the more aggressive form of the disease in DCIS, and in cancer specimens it proved an independent marker for poor prognosis. Importantly, cytoplasmic HuR expression was significantly associated with poor outcome in the subgroups of small (2 cm) and axillary lymph node-negative breast cancers. HuR proved to be the first mRNA stability protein the expression of which is associated in breast cancer with poor outcome. To explore the mechanisms of HuR in breast carcinogenesis, lentiviral constructs were developed to inhibit and to overexpress the HuR expression in a breast epithelial cell line (184B5Me). Our results suggest that HuR mediates breast carcinogenesis by participating in processes important in cell transformation, in programmed cell death, and in cell invasion. Global gene expression analysis shows that HuR regulates genes participating in diverse cellular processes, and affects several pathways important in cancer development. In addition, we identified two novel target transcripts (connective tissue growth factor, CTGF, and Ras oncogene family member 31, RAB31) for HuR. In conclusion, because cytoplasmic HuR expression in breast cancer can predict the outcome of the disease it could serve in clinics as a prognostic marker. HuR accumulates in the cytoplasm even at its non-invasive stage (ADH and DCIS) of the carcinogenic process and supports functions essential in cell alteration. These data suggest that HuR contributes to carcinogenesis of the breast epithelium.

Role of CIP2A in carcinogenesis

Worldwide and notably in the developed countries, cancer is an increasing cause of morbidity and mortality, being the second most common cause of death after ischemic heart disease. Now and in the future new cancer cases need to be diagnosed earlier. Prognostic factors may be helpful in recognizing and handling those patients who need more aggressive therapy, and it is also desirable to predict treatment response accurately. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein predominantly expressed in malignant tissues and inhibiting protein phosphatase 2A (PP2A) activity; it is a promising target for cancer therapy. The aim of this thesis was to evaluate the prognostic role of CIP2A in solid cancers, and for this purpose to explore expression of CIP2A, and investigating regulation of CIP2A in order to gain insight into signalling pathways leading to alteration in prognosis. Patients diagnosed with gastric, serous ovarian, tongue, or colorectal cancer at Helsinki University Central Hospital were included. Tumour tissue microarrays assembled from specimens from these patients were prepared and stained immunohistochemically for CIP2A protein expression. Associations with clinicopathologic parameters and other biomarkers were explored, and survival analyses were done according to the Kaplan-Meier method. Study of the role of CIP2A in intracellular signalling in vitro involved gastric, ovarian, and tongue cancer cell lines. We found CIP2A to be highly expressed in gastric, ovarian, tongue, and colorectal cancer specimens. CIP2A was associated with clinicopathologic parameters characterizing an aggressive disease, namely advanced stage, high grade, p53 immunopositivity, and high proliferation index. CIP2A led to recognition of gastric, ovarian, and tongue cancer patients with poor prognosis, however, with a cancer type-specific cut-off level for prognostic significance. In tongue cancer, it served as an independent prognostic marker. In contrast, in colorectal cancer, CIP2A provided no prognostic value. In cancer cell lines, CIP2A was highly expressed at both protein and mRNA levels, and promoted cell proliferation and anchorage-independent growth. In gastric cancer, we demonstrated with a MYCER construct in mouse embryo fibroblasts that activation of MYC led to increased CIP2A mRNA expression, and hence we suggested that a positive feedback mechanism between CIP2A and MYC may potentiate and prolong the oncogenic activity of these proteins. We demonstrated in ovarian cancer an association between CIP2A and EGFR protein overexpression and EGFR gene amplification. In ovarian and tongue cancer cells we showed that depletion of EGFR downregulates CIP2A expression. In conclusion, high CIP2A expression occurred frequently among patients with aggressive disease. CIP2A may serve as a prognostic marker in gastric, ovarian, and tongue cancer and thus may help in tailoring therapy for cancer patients. The positive feedback mechanism between CIP2A and MYC, as well as the positive regulation of CIP2A by EGFR, are a few signalling pathways regulating and regulated by CIP2A. These and other mechanisms need to be studied further, however. CIP2A is a potential target for therapy, and its potential role as predictive marker and as a tumour marker in serum requires exploration.

HPV infection, a described cause for human cancer: HPV detection and molecular mechanisms of HPV carcinogenesis (Review).

[en]Human papillomavirus (HPV) belongs to the Papillomaviridae virus family and it is one of the most common sexual transmission infections. HPV genome is composed of eight genes, including two early genes and six late genes. Among these late genes, E6 and E7 code for proteins that trigger cell-cycle re-entry in infected cells, which can lead to cervical cancer development. The IARC (International Agency for Research Cancer) proposed a guideline based on Hill’s criteria to determine whether the relation between HPV infection and cervical cancer is causal or not. Epidemiological studies have demonstrated that HPV infection is a necessary but non-sufficient cause for cervical cancer. Furthermore, HPV infection is considered the first necessary cause described of a human cancer, being HPV16 and 18 carcinogenic to humans and the most studied types. Cervical cancer is the second leading cause of cancer death among women worldwide. Different screening programs are carried out with the aim of preventing cervical cancer; such as cytologies and HPV tests. There are two main methods which are equally usable to detect HPV: the real-time PCR assays and the array assays. Regarding the molecular mechanisms of HPV mediated malignancies, E2, E6 and E7 proteins of HPV16 lead to immune response evasion, inducing IL-10 and TGF-β1 gene expression. Besides, E6 and E7 proteins allow cell-cycle reentry, phosphorylating RB and ubiquitinating p53 respectively. HPV genome integration in host genome leads to the alteration of host and viral genes expression, including oncogenes and tumor suppressor genes. However, the differences of E6 and E7 oncoproteins in different HPV types is poorly known due to the fact that almost the most studied HPV type has been HPV16.

Molecular pathology of RUNX3 in human carcinogenesis

A major goal of molecular biology is to elucidate the mechanisms underlying cancer development and progression in order to achieve early detection, better diagnosis and staging and novel preventive and therapeutic strategies. We feel that an understanding of Runt-related transcription factor 3 (RUNX3)-regulated biological pathways will directly impact our knowledge of these areas of human carcinogenesis. The RUNX3 transcription factor is a downstream effector of the transforming growth factor-beta (TGF-beta) signaling pathway, and has a critical role in the regulation of cell proliferation and cell death by apoptosis, and in angiogenesis, cell adhesion and invasion. We previously identified RUNX3 as a major gastric tumor suppressor by establishing a causal relationship between loss of function and gastric carcinogenesis. More recently, we showed that RUNX3 functions as a bona fide initiator of colonic carcinogenesis by linking the Wnt oncogenic and TGF-beta tumor suppressive pathways. Apart from gastric and colorectal cancers. a multitude of epithelial cancers exhibit inactivation of RUNX3, thereby making it a putative tumor suppressor in human neoplasia. This review highlights our current understanding of the molecular mechanisms of RUNX3 inactivation in the context of cancer development and progression. (C) 2009 Elsevier B.V. All rights reserved.

RUNX3 Inactivation in Colorectal Polyps Arising Through Different Pathways of Colonic Carcinogenesis

OBJECTIVES: We hypothesized that RUNX3 inactivation by promoter hypermethylation in colorectal polyps is an early molecular event in colorectal carcinogenesis.
METHODS: RUNX3 protein expression was analyzed immunohistochemically in 50 sporadic colorectal polyps comprising 19 hyperplastic polyps (HPs), 14 traditional serrated adenomas (TSAs), and 17 sporadic traditional adenomas (sTAs) as well as in 19 familial adenomatous polyposis (FAP) samples from 10 patients showing aberrant crypt foci (ACF) (n=91), small adenomas (SmAds) (n=40), and large adenomas (LAds) (n=13). In addition, we assessed the frequency of promoter hypermethylation of RUNX3 by methylation-specific PCR (MSP) in all the 50 sporadic polyps as well as 38 microdissected FAP polyps comprising ACF, SmAds, and LAds obtained from 7 FAP samples. A total of 12 normal colon samples were also included for RUNX3 MSP analysis.
RESULTS: Compared to normal colon (2 of 12, 16%) and sTAs (3 of 17, 18%), HPs (15 of 19, 79%) and TSAs (8 of 14, 57%) displayed significant inactivation of RUNX3 (P<0.05). In FAP, RUNX3 inactivation was more frequently seen in ACF (78 of 91, 86%), SmAds (25 of 40, 62%), and LAds (6 of 13, 46%) compared to normal mucosa (0 of 19, 0%) in the same samples (all P<0.05). Promoter hypermethylation of RUNX3 was significantly higher in colorectal polyps (64 of 87, 74%) compared to normal colon (2 of 12, 16%) (P=0.001). Serrated polyps such as HPs (17 of 19, 89%) and TSAs (12 of 14, 86%) were significantly more methylated than sTAs (7 of 17, 44%) (P=0.004). RUNX3 hypermethylation was observed in 28 of the total 38 (74%) FAP polyps. Overall, RUNX3 promoter methylation correlated with inactivation of RUNX3 expression in sporadic (27 of 36, 75%) (P=0.022) and FAP (21 of 28, 75%) (P=0.021) polyps.
CONCLUSIONS: Our data suggest that RUNX3 inactivation due to promoter hypermethylation in colorectal polyps represents an early event in colorectal cancer (CRC) progression. In addition, epigenetic RUNX3 inactivation is a frequent event in the serrated colonic polyps as well as in the ACF of FAP polyps.