Introduction all, as diagnostic blood and/or tissue biomarkers

Introduction
Epithelial invasive ovarian cancer (EOC) is the second most common gynaecological
malignancy 1 and one of the ten types of cancers with the highest incidence and mortality
rates 2. It is considered to be a challenge of 21st-century medicine because approximately
77-78% of the cases are still diagnosed in advanced stages III or IV 3. In 2011, the results
of PLCO (Prostate, Lung, Colorectal, Ovarian Cancer Screening) trial had concluded that all
screening tests have no contribution in a decrease of mortality due to ovarian cancer. In 2017
the results of a collaborative re-analysis from Ovarian Cancer Cohort Consortium suggested
that IGF-1 concentration is inversely correlated with EOC risk independent of histological
phenotype 4-5.
The role of IGF system components in carcinogenesis of ovarian cancer was based on
investigative epidemiological results, experiments in vivo and in vitro and on attempts of
application of drugs affecting the IGF axis 6. Investigative hypotheses in original papers
were based on physiological functions manifested by the entire family of IGF (ligands,
receptors, binding proteins, adaptor molecules) 6. In the context of carcinogenesis, the
essential functions of IGF family involve intensification of proliferation, inhibition of cell
apoptosis as well influence on cell transformation through the synthesis of several regulatory
proteins 7. Components of IGF axis control survival and influence metastases of cells.
Interactions of IGF axis components may be of a direct or indirect nature. The immediate
effects are linked to activation of PI3K/Akt signaling pathway, in which the initiating role is
played by, first of all, IGF-1 and IGF-1R. The activity of this signaling pathway leads to an
increased mutagenesis, cell cycle progression, and protection against different apoptotic
stresses. Indirect effects of IGF axis depend on interactions between IGFs and other molecules
essential for cancer aetiology (e.g. sex hormones, products if suppressor genes, viruses, other
growth factors) and the style of life (nutrition, physical activity). In oncogenesis of several
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cancers also the age of the patient remains of crucial importance, which is also reflected by
dysregulation in IGF axis 8-9.
By clinicians, components of IGF system are considered, first of all, as diagnostic
blood and/or tissue biomarkers of cancer, prognostic factors and attractive target of current
anti-tumour therapies. Several mechanisms in which IGF system components act in the
process of carcinogenesis await clarification, mainly due to multifactorial aetiology of the
diseases (lung, skin, breast, prostate, cervix, colorectal, gastric, pancreatic, liver cancers).
Pinpointing of the role played in carcinogenesis by any single signaling pathway remains to
be particularly difficult 6, 10.
Gene coding the human protein IGF-1, placed in the long arm of chromosome 12
(12q22-24.1), covers an area of nearly 90kbp and contains six exons separated by very long
(1.9-50kbp) introns. The sequence of the IGF-1 gene is most conservative, and its
transcription is under the control of two promoters: P1 and P2. It is considered that almost
90% of IGF-I transcripts remain under the control of P1. The P1 human promoter region
consists of 322 nucleotides located in the region of 5’UTR and exon 1 of the regulatory region
in 1630. The most conservative is a 322-nucleotide stretch of 5’UTR. The P1 promoter
sequence requires typical sequences of other genes, such as TATA or CCAAT elements, and
the rest of the area is rich in GC. The P1 promoter has 5 sections protected from DNase
digestion: HS3A, HS3B, HS3C, HS3D, HS3E. The HS3D place is thought to be responsible
for the regulation of IGF-I expression by estrogens 11-12. 5′(CA)n repeats in the P1
promoter region of the IGF-I gene, 1 kb upstream from the transcription site, are a highly
polymorphic microsatellite, comprising a variable length of a cytosine-adenosine (CA) repeat
sequences. The number of CA repeats ranges between 10 to 24 with the most common allele
containing 19 (CA)(192 bp) repeats, characteristic for Caucasians 11-12. Many studies
suggest that the number of CA repeats in the promoter region is inversely correlated with the
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transcriptional activity. In the literature, the involvement of the polymorphism of CA
promoter dinucleotide repeats is still controversial in clinical disorders such as cancer,
diabetes, cardiovascular diseases, birth weight and body height and the IGF-1 serum level
13-15.
Our study aimed the analysis of CA repeat polymorphism in the P1 promoter region of
the IGF-1 gene in blood serum and cells obtained from Caucasian females with epithelial
ovarian cancer (EOC) and women with benign ovarian cysts (the reference group). We
identified (CA)n repeats in the P1 region of IGF-1, the blood serum levels of IGF-1, IGFBP1,
IGFBP-3 and IGF-1 expression in tissues and analysed the relationship between these
factors in different types of microsatellite polymorphism in the P1 promoter region of the
IGF-1 gene and epithelial invasive ovarian cancer development. It is worth noting that,
hitherto no studies related to ovarian cancer stages and the microsatellite polymorphism
profile of the IGF-1 gene P1 promoter region were reported.
Methods
Ethics statements
The research was accepted by the Ethics Committee of the Medical University of
Lublin (Lublin, Poland; Resolution of the Bioethics Committee no. 0254/263/2011). Written
informed consent was received from all patients included, and the investigation was
performed under the principles of the Helsinki Declaration.
Study participants
Biological material used for the estimation of IGF 1 level and (CA)n repeats of the P1
promoter region of the IGF 1 gene included peripheral blood and tissue fragments. Peripheral
blood was collected from the antecubital vein before surgery from patients recruited in the
present study. Tissue sections were embedded in paraffin (Sigma-Aldrich, St. Louis, MO,
USA) from patients who underwent surgery at the Department of Gynecological Oncology
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and Gynecology, Medical University of Lublin (Lublin, Poland) between November 2010 and
December 2016. A total of 33 tissue samples were obtained from postmenopausal women
with epithelial ovarian cancer (EOC). All patients with EOC underwent radical surgery. For
staging of ovarian cancer, the FIGO (International Federation of Gynecology and Obstetrics)
standards from 2010 (7th edition) were applied. The number of samples classified as FIGO
stages I, II, III C and IV were 4 (12.1%), 6 (18.2%), 18 (54.5%) and 5 (15.2%), respectively.
Diagnosis and reclassification were performed histologically by two independent
pathologists. The reference group consisted of 27 tissue samples from patients referred to the
department for diagnostic procedures of tumour ovaries in which histopathological
examination found cystic simplex ovarii or cystic follicularis ovarii. Patients with hormone
replacement therapy, other types of cancer, systemic diseases, ischemic heart disease,
peripheral vascular diseases, thyroid diseases and/or other endocrine diseases as well as liver
and bile duct diseases were rejected from the research. The average age of the patients with
OC was 52?8 range: 27-76 years. The mean age in the reference group was 48?