Dissertation committee:
Φραγκούλης Γ. Εμμανουήλ, Ομότιμος Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Μουτσάτσου-Λαδίκου Παρασκευή, Καθηγήτρια Ιατρικής Σχολής, ΕΚΠΑ
Σκορίλας Ανδρέας, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Τρουγκάκος Ιωάννης, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Κλετσας Δημήτριος, Ερευνητής Α', Ε.Κ.ΕΦ.Ε. "Δημόκριτος"
Σίδερης Διαμάντης,Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Κοντός Χρήστος, Επίκουρος Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Summary:
Both breast and prostate cancer are among the most common types of cancer and are the second cause of cancer-related deaths in the female and male population respectively. However the progress in understanding molecular aspects of breast and
prostate carcinogenesis and the advances in clinical management of these patients
denote the paradox of the above-mentioned high rates. Much of this paradox is
attributed to the clinical and molecular heterogeneity of breast and prostate cancer,
which renders them a collection of diseases with variable clinical behaviors, and to the
inadequacy of established clinical/pathological markers to predict the outcome of most
tumors and to guide optimal treatment decisions. Therefore the recognition of novel,
sensitive and reliable molecular markers, capable of improving disease diagnosis,
prognosis and treatment monitoring is of great importance.
BCL2L12, as a member of the BCL-2 family, is an apoptosis-related gene, which is
subjected to excessive alternative splicing, producing a total of 63 alternative splice
variants, eighteen of which code for different isoforms of the BCL2L12 protein.
Currently, the most studied isoform is BCL2L12 is.1, but still its apoptotic role remains
ambiguous, since it seems that the pre-apoptotic or anti-apoptotic behavior of this
protein depends on the cellular environment. Several lines of evidence support the
significance of BCL2L12 in cancer from a clinical point of view as well.
Human RNase κ is an endoribonuclease, coding by a gene that consists of three
exons and two intervening introns. Recently, our group demonstrated that RNase κ is
subjected to alternative splicing, within the first intron, producing two transcript
variants, RNase κ-01 and RNase κ-02, which code for two structurally different protein
isoforms. The high percentage of amino acid conservation of human RNase κ along with its expression in a wide spectrum of tissues and developmental stages, denote its
significant, but yet largely obscure, biological function. Moreover, RNase κ exhibits
increased or decreased expression levels in several cancer types, suggesting its possible
clinical utility as molecular cancer marker.
A rapidly developing scientific field in cancer-related research is the study of
microRNAs (miRNAs). MiRNAs constitute a large family of small molecules that
negatively regulate, at the post-transcriptional level, the expression of genes involved in
key-cellular processes. Recent data describe the marked deregulation of miRNA levels in
human malignancies and suggest that they might represent novel promising cancer
biomarkers. The miR-96-182-183 cluster targets the BCL2L12, whereas the miR-423-5p
is suggested by several algorithms to target RNase κ. These miRNAs are associated with
the progression of several neoplasms.
The aim of the present thesis was the expression analysis and study of BCL2L12
variants, RNase κ, the miR-96-182-183 cluster and miR-423-5p in breast and prostate
tumor, as well as their clinical evaluation as biomarkers helpful for the differential
diagnosis and prognosis of these patients. An additional aim was to experimentally
validate that miR-423-5p targets RNase κ gene.
In order to fulfill these objectives, a statistically significant sample size of breast
cancer tissues and matched non-malignant breast tissues as well as of cancerous and
benign prostate tissues was initially processed via homogenization. Total RNA was
isolated from the abovementioned tissue specimens and was tested in terms of quality
and quantity via agarose gel electrophoresis and spectrophotometric analysis,
respectively. Subsequently, total RNA was reversed transcribed into cDNA. The total
RNA that was used for miRNA expression analysis, was subjected to polyadenylation,
prior to reverse transcription that was conducted in the presence of a modified poly(T)
adapter. The expression of BCL2L12 transcript variants and RNase κ was, firstly, studied
in breast and prostate tissues, via regular RT-PCR and agarose gel electrophoresis. The
mRNA transcripts, as well as miRNAs expression levels, were further quantified via RealTime PCR, using SYBR Green chemistry and the relative quantification method.
Furthermore, we examined the miR-423-5p/ RNase κ interaction using bioinformatic
tools for miRNA target prediction and molecular biology techniques. The clinical evaluation of the prognostic and the differential diagnostic potential of the biomolecules under study was accomplished by using extensive biostatistical analyses. More precisely, non-parametric statistical tests were used to identify any associations between the expression levels of the genes under study and the patients’ clinicopathological characteristics. ROC curve analysis was used in order to evaluate the
differential diagnostic value of the examined biomolecules. Kaplan-Meier curves and
univariate logistic regression analyses were used to study the effect of each factor in the
disease-free survival of the breast and prostate cancer patients. The independence of the
prognostic information provided by the biomolecules under study was tested via
multivariate Cox logistic regression analysis.
Expression analyses showed that BCL2L12 v.1 ,v.2 και v.4 are overexpressed in
breast cancer tissues compared to the non-cancerous component (v.1,p < 0.001; v.2, p =
0.009; v.4, p = 0.004). Increased BCL2L12 v.4 mRNA expression was associated with
markers of unfavorable prognosis namely, advanced tumor grade (p = 0.002), ER- (p =
0.015)/PR- (p < 0.001) negativity, Ki-67-positivity (p = 0.007) and high NPI score (p =
0.033). Survival analysis disclosed that BCL2L12 v.2 overexpression (HR= 0.45, p =
0.010), is a strong and independent marker of favorable prognosis for breast cancer
patients. Regarding prostate tissues, it was found that BCL2L12 v.1 and v.5 are
overexpressed in prostate cancer patients compared to BPH ones (v.1, p=0.016; v.5,
p=0.011). BCL2L12 v.5 mRNA levels were also elevated in patients with tumors of
advanced stage (pT2c and pT3a).
Regarding RNase κ the statistical analysis revealed a significant downregulation
of RNase κ in CaP patients compared to BPH ones (p=0.002). RNase κ overexpression
was associated with decreased risk of CaP development and can discriminate between
CaP and BPH independently of serum PSA levels (p=0.001). RNase κ upregulation was
also associated with less advanced (P=0.018) and less aggressive (p=0.001) tumors as
well as with longer progression-free survival (PFS) (p=0.003). Finally univariate
bootstrap Cox regression confirmed that RNase κ was associated with favorable
prognosis (HR=0.85, p=0.002).
Quantitative expression analysis showed that the miR-96-182-183 cluster is
significantly upregulated in breast cancer tissues compared to the non-cancerous
component (miR-96, p<0.001; miR-182, p<0.001; miR-183, p<0.001), but it is
downregulated in women with triple negative breast cancer (miR-96, p=0.013; miR-182,
p=0.021; miR-183, p=0.04). Indeed ROC analysis disclosed that these miRNAs exhibited
a significant potential to distinguish tumors characterized as triple negative from those
belonging to other molecular subtypes (miR-96, AUC=0.652, p=0.013; miR-182,
AUC=0.642, p=0.021; miR-183, AUC=0.620, p<0.05). Survival analysis disclosed that
miR-183 overexpression is a strong and independent marker of unfavorable prognosis
in breast cancer (HR= 0.13, p=0.004). In prostate cancer the miR-96-182-183 cluster is also upregulated and as ROC analysis showed these miRNAs can differensuffering from BPH and patients with CaP (miR-96, AUC= 0.611, p = 0.00AUC= 0.617, p = 0.004; miR-183, AUC= 0.643, p = 0.001). miR-182 anupregulation were also associated with more advanced (miR-182: p=0.00183: p<0.001)and more aggressive (miR-182: p=0.03 and miR-183: p=0whereas miR-96 overexpression is a strong and independent marker prognosis in prostate cancer.
Conversely, miR-423-5p is downregulated in breast cancer tissueMoreover, increased miR-423-5p expression was associated with markersprognosis namely, initial tumor grade (p = 0.001), ER-positivity (p = 0.negativity (p = 0.035) and luminal phenotype (p=0.004). Similarly mupregulated in patients suffering from BPH (p<0.001) and according to miR-423-5p expression levels can discriminate between prostate cancer and0.713, p < 0.001).
Finally we developed the appropriate methodologies for the validation of the miR-423-5p/RNase κ interaction. By performing these mwe concluded to the already experimentally confirmed conclusion that targets Ogt, and we provided evidence for the firtst time that miR-423-5p taκ.
In conclusion, the biomolecules that were studied representmolecular indicators that could aid in the differential diagnosis of and provprognostic information for breast and prostate cancer patients.
