Despite new methods and combined strategies, conventional cancer chemotherapy still lacks
specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in
the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor
genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or
specifically silencing any given target gene. Concerning gene silencing, attention has recently
shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi
machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs
(miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced,
respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the
function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target
gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On
the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the
research community new important perspectives for the comprehension of the physiological and,
above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs
expression have been identified in several types of neoplasia and it has also been proposed that the
overexpression of genes in cancer cells may be due to the disruption of a control network in which
relevant miRNA are implicated. For these reasons, I focused my research on a possible link
between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC),
since it has been established that the transition adenoma-adenocarcinoma and the progression of
CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is
involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell
invasion, tumour growth and metastatization.
On the basis of data reported in the literature, the first aim of my research was to develop an
innovative and effective tool, based on the RNAi mechanism, able to silence strongly and
specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show
that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2
gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced
angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a
specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2
previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon
cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2
deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus,
results reported here indicate an easy-to-use, powerful and high selective virus-based method to
knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore,
they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as
therapeutic agent for human cancers overexpressing COX-2.
In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2
expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by
COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic
pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and
with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These
promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was
effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and
pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of
Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but
also the most specific system to downregulate COX-2 in colon cancer cells.
Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of
CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for
this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in
mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes,
which encode for two bacterial factors needed for successful transfer of shRNA in mammalian
cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for
the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and
could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing
mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29
and HCA-7 cell lines were in high agreement with data previously collected after the transfection of
pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system
for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool
for the treatment of colorectal cancer.
Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I
proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer.
In particular, it is known that components of the miRNA/RNAi pathway may be altered during the
progressive development of colorectal cancer (CRC), and it has been already demonstrated that
some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was
that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or
more tumor suppressor miRNAs.
In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR-
101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro
modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in
COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and
COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic
environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is
known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I
observed that COX-2 overexpression induced by hypoxia is always coupled to a significant
decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation
could be involved in the adaption of cancer cells to the hypoxic environment that strongly
characterize CRC tissues
