Summary:
Colon cancer is the most frequent malignant disease of the digestive system in
USA and Europe. In our country, colon cancer ranked fourth among causes of
death from neoplasms. The biomarkers used in modern clinical practice are the
Carcinoembryonic Antigen (CEA), the Carbohydrate Antigen 19-9 (CA 19-9), the
72-4 Cancer Antigen (CA 72-4) and the Tissue plasminogen activator (PLAT).
Although several prognostic and predictive markers for colon cancer have been
recognized, very few of them are currently used in clinical practice.
The human tissue kallikrein gene family is the largest contiguous gene family
of proteases in the human genome, with no intervention from other genes. It is
a very attractive field of study, as one of its members, the famous PSA/KLK3,
is the most accepted and widely used cancer biomarker, until today. Studies
have suggested other family members as potential biomarkers for prognosis
or/and prediction of colon cancer, including KLK4 and KLK7, at mRNA level, and
KLK4, KLK7 and KLK11, at protein level.
KLK10 gene is a new member of the kallikrein tissue family. Several studies
have been conducted to assess the potential role of KLK10 as a tumor marker.
Due to its higher levels in tumor tissues and serum of patients with ovarian
cancer, KLK10 has been proposed as diagnostic and prognostic marker for ovarian
cancer, in combination with the already used marker CA125, for increased
diagnostic sensitivity.
In this thesis, we performed clinical evaluation of the KLK10 gene for its
possible application as a new prognostic marker for colorectal cancer. The mRNA
expression study was performed with conventional RT-PCR and quantitative PCR
methodology in real-time (real-time PCR), and the protein was detected and
measured by Enzyme-Linked Immunosorbent Assay (ELISA). Analytical protocols
were developed to determine the mRNA expression of KLK10 gene in colorectal
cancer based on reverse transcription (RT), polymerase chain reaction (PCR) and
quantitative real-time PCR (real-time PCR) methodology. Detection and
quantification of protein KLK10 performed by Enzyme-Linked Immunosorbent Assay
(ELISA). There weren’t any ready kit to be used for the abovementioned
experiments, all the assays were developed in our lab, so we also performed
quality control in order to ensure the reliability of the results. We collected
a statistically significant size from cancerous and non- cancerous colon tissue
samples (194 tissues), from patients with complete clinical history. For the
analysis of data, an extensive biostatistical analysis was performed. We used
parametric and nonparametric methodologies, ROC analysis, Kaplan-Meier survival
analysis, as well as univariate and multivariate Cox regression analysis.
The results obtained showed that the mRNA expression of the KLK10 gene is
statistically associated with several clinicopathological parameters, including
Duke’s Stage (P=0.036), Grade (P=0.025), lymph nodes status (P=0.046) and
distant organs metastasis (P=0.027). The Kaplan Meier survival analysis showed
statistically significantly shorter possibility of disease-free (DFS) and
overall survival (OS) of patients with positive KLK10 mRNA expression (P=0.014
and P=0.020, respectively). The expression of KLK10 protein was statistically
significant higher in tumor samples than in normal counterparts (P<0.001). In
conclusion, the cancerous colon tissues examined showed a higher expression of
KLK10 gene. The over-expression of this appears to be associated with poor
prognosis in patients with colon cancer and may constitute a useful novel
molecular biomarker.
We then cultured colorectal adenocarcinoma cell line HT-29, for which we
constructed viability curves, followed by treatment with chemotherapy drugs
used in systematic treatment of colon cancer (fluorouracil, oxaliplatin), as
well as with other widely used cancer drugs (taxotere, cisplatin, gemcitabine).
For the first time, after the treament with the administered agent, we removed
the agent and we continued the cell culture, in order to evaluate whether the
cells continue to die or if they revert to normal cell division, when the drug
is removed. Then, we evaluated the mRNA expression profiles of KLK10 gene, as
well as other genes involved in apoptosis: BCL2, BCLX, MCL1, BAX, BAK1, BIM,
BID, PUMA (BBC3), NΟΧΑ (PMAIP1), BIRC5 (Survivin), and P21 (CDKN1A).
KLK10 mRNA expression levels were elevated, after the treatment with
fluorouracil and cisplatin, while smaller scale changes were observed with the
treatment with other drugs. Finally, what was very interesting, was the fact
that KLK10 mRNA pattern of expression followed the mRNA pattern of expression
of the proapoptotic NOXA gene.
Regarding the BCL2 family members, depending on the chemotherapeutic agent
used, other members expression levels increased, while others decreased and
others remained the same. The transcriptional activation of some members in
each treatment with cytotoxic agent, however, suggests that, depending on the
stimulus, the proapoptotic members begin their path to apoptosis. The position
of each molecule may be of strategic importance in order to ensure the pathway
of apoptosis. This could reveal a unified role in proapoptotic BH3-only family
members. Perhaps such a mechanism is established by the transcriptional
activation of different members of the family, during the treatment with
different chemotherapeutic agents. It is worth noting, finally, that the mRNA
expression of the antiapoptotic BCL2 gene was downregulated in the presence of
all the abovementioned cytotoxic agents, which is in agreement with previous
studies where overexpression of the gene was combined with tolerance to
chemotherapy. A second important point that emerges from this study, is the use
of taxotere and cisplatin in colon cancer chemotherapy. From treatment with
both agents, the cells continued to die, even after their removal from the
flask.
Finally, the expression of the KLK10 gene, at both mRNA and protein level, has
features of an unfavorable prognostic marker for colon cancer, as it is
associated with shorter disease free and overall survival, and this is the most
important result of this thesis. Its transcriptional activation after treatment
with chemotherapeutic agents, perhaps assigns it a possible role as a biomarker
for response to chemotherapy. The understanding of regulation mechanisms and
the discovery of the physiological substrate of this enzyme, will lead to its
evaluation as a potential target for colon cancer treatment.
