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ROMANIAN ACADEMY
PhD THESIS
INTRACELLULAR SIGNALING IN MALIGNANT PROLIFERATION
ABSTRACT
PhD student: Laura Georgiana Necula
PhD thesis advisor: Gabriela Anton, PhD, CS I
The purpose of the experimental studies was to analyze the changes that occur in the
signaling pathways in gastric tumor tissue in an attempt to open up new perspectives in
understanding the molecular mechanisms involved in neoplastic transformation in the
progression of gastric cancer and, not least, to identify new opportunities for therapeutic
approach by highlighting new gastric cancer biomarkers.
Identifying alterations that occur in the signaling pathways and processes of
interaction and compensation of signaling in gastric carcinogenesis may be useful for
developing new targeted cancer therapies. It is also necessary to identify biomarkers for early
detection and prognosis in patients with gastric adenocarcinoma.
During the process of malignant transformation in tumor cells, there are a number of
changes that include the ability to proliferate independently of exogenous growth factors or
growth-inhibitory signals. Tumoral cells are also able to avoid the mechanisms of the
limitation of cell proliferation (apoptosis), cause the angiogenic response, may invade
surrounding tissues and generate distant metastases. Most of these tumor processes are due to
changes in the signaling pathways that in normal cell control cell proliferation and survival.
Currently there are many studies focused on identifying changes that occur in the
signaling pathways in malignant transformation processes and future anti-tumor therapy seeks
to replace the current chemotherapeutic drugs with agents targeting specific signaling
molecules or cascades.
According to GLOBOCAN data in Romania, breast cancer, lung cancer, cervical
cancer, colorectal cancer, prostate cancer, stomach cancer and ovarian cancer represent
malignancies with high incidence and mortality rate. In the literature there are numerous
studies that support the involvement of the signaling pathways alterations in each of these
types of cancers. Gastric cancer is a leading cause of cancer death worldwide since the early
stages of carcinogenesis are asymptomatic and most patients are diagnosed with advanced
tumor stage. Also, there are currently no biomarkers for early detection, prognosis and
chemotherapy resistance in patients with gastric cancer. The most effective treatment is
surgical resection of the tumor due to the fact that gastric cancer cells have low response to
chemotherapy and radiotherapy.
In this regard, it is necessary to conduct further studies focused on identifying the
genetic alterations involved in neoplastic transformation that can facilitate the development of
new methods of prevention, diagnosis and treatment of gastric adenocarcinoma.
The aim of the PhD thesis was the identification of the molecular etiology of gastric
adenocarcinoma by exploring the signaling pathways involved in carcinogenesis, in order to
identify gene alterations that lead to neoplastic transformation.
The main objectives were: to investigate the activation of the gp130 receptor subunit,
the analysis of the signaling pathways involved in gastric carcinogenesis and to identify new
biomarkers that can be correlated with prognosis in patients with gastric adenocarcinoma. In
this respect sequencing analysis was performed to identify possible mutations in gp130 gene
in gastric cancer tissues, which may lead to constitutive activation of proliferative signaling
pathways. Plasma and tissular levels of the major cytokines (IL-6 family) that signaling
through gp130 was also analyzed. The results were correlated with the presence or absence of
Helicobacter pylori infection which is currently considered the main active factor in gastric
carcinogenesis.
To identify new biomarkers for early detection and prognosis in patients with gastric
cancer, genomic studies have been carried out. Thus, the study aimed to analyze gene
expression in gastric cancer and to identify genes whose expression are significantly altered in
gastric tumor tissue compared to normal adjacent tissue. The differences in gene expression
between primary tumor stages and advanced tumor stages with metastasis were analyzed.
Gene expression and proteomics data were used for the analysis of the signaling pathways
involved in the process of carcinogenesis.
The results obtained from analyzing the level of cytokines that signal through gp130
receptor subunit (IL-6, IL-11, LIF) in plasma and tumor/normal tissues, collected from
patients with adenocarcinoma gastric reveal a positive correlation between the level of IL-6
and IL-11 and tumor progression. The expression of IL-6 plasma level also increases in a
manner dependent on the stage of the gastric tumor. The tissular and plasma levels of IL-6
were statistically significant associated with a poor prognosis. Thus, plasma and tissue levels
of IL-6 may be considered markers of the aggressiveness of the tumor, having prognostic
value in patients with gastric adenocarcinoma.
The results obtained from the evaluation of H. pylori infection suggest that H. pylori
presence represents a risk factor for gastric carcinogenesis but it requires other alterations in
gastric tissue that facilitate malignant transformation. The obtained results showed that IL-6
plasma expression is increased but it is not induced by the presence of H. pylori.
Therefore, IL-6 could be considered good marker of aggressiveness in gastric
adenocarcinoma.
The sequence analysis of exon 17 of the gene IL6ST encoding gp130 did not reveal
any mutation in the major phosphorylation sites of the receptor subunit gp130 (Y759, Y767,
Y814, Y905, Y915) in patients with gastric adenocarcinoma. However, activation of gp130
signaling may take place because of a high level of IL-6 and IL-11 in gastric cancer tissue that
has been positively correlated with the tumor stage, emphasizing the role of these cytokines in
gastric carcinogenesis.
The results of the gene expression analysis in primary lesions showed intense
metabolic processes of triglycerides, collagen, glycerol and an increase in the level of
expression of proteases and genes related to the biosynthesis and morphogenesis processes. In
advanced gastric adenocarcinoma the overexpressed genes are involved in cytoskeletal
rearrangement, cell migration, and regulation of the phosphorylation and anti-apoptosis
processes.
Downregulated genes in tumor tissue are involved in normal physiological processes
of the stomach such as gastric motility and synthesis, appetite regulation and also genes
associated with metabolic processes, chemical homeostasis and apoptosis process.
The results of protein and gene expression analysis showed the activation of STAT3
signaling by IL-6 and IL-11 in gastric tumor tissue simultaneously with activation of JNK /
p38 MAPK, Wnt/β-catenin and PI3K/AKT cascades. All of these signaling pathways are
involved in proliferation and invasiveness. The findings suggest the need for targeting
multiple signaling pathways in anti-tumor therapy of gastric adenocarcinoma.
The novelty, the most important result of this study was the identification of a panel of
seven genes with significantly increased expression in gastric tumor tissue and associated
with tumor progression: KRT17, COL10A1, KIAA1199, SPP1, IL11, S100A2 and MMP3.
Moreover, COL10A1, KRT17 and SALL4 could be potential biomarkers for early detection
of gastric carcinogenesis, having increased expression in primary tumor stages. Importantly,
increased COL10A1 gene expression was positively correlated with short-term survival in
gastric carcinogenesis.
These biomarkers could be particularly useful in the early detection and assessment of
the evolution of patients with gastric adenocarcinoma.
The results of these studies were disseminated through publication of scientific papers
in journals with international impact (ISI journals) such as:
- Necula LG, Chivu-Economescu M, et. al. IL-6 and IL-11 as markers for tumor
aggressiveness and prognosis in gastric adenocarcinoma patients without mutations in Gp130
subunits. Journal of Gastrointestinal and Liver Diseases 2012, 21(1), 23-29 (ISSN 18418724). 25 citations, IF 1.891.
- Chivu Economescu M, Necula LG, et al. Identification of potential biomarkers for early
and advanced gastric adenocarcinoma detection, Hepato-Gastroenterology 2010; 58(104):
1453-1464 (ISSN: 0172-6390). 15 citations, IF 0.792.