The tumor-suppressive functions of the human INK4A locus
Tumor suppressors are genes found
inactivated by mutations or deletions in a subset of
human cancers. However, each individual tumor
contains many genetic alterations, complicating the
study of the contribution of each variation to
tumorigenesis. We studied the tumor-suppressive
functions of the human INK4A locus, which encodes
for the p16INK4A and p14ARF genes and is mutated in
many cancers. In mouse model systems, p19ARF plays
an important protective role from oncogene induced
transformation whereas p16INK4A loss causes only
limited induction of tumors. Using pSUPER technology
we showed that p16INK4A, but not p14ARF, is the
major tumor suppressor of the human INK4A locus in
response to oncogenic stress. We are currently
engaged in the elucidation of the p14ARF independent
pathways that are activated by oncogenic
stress.
The pSUPER system is sensitive to
mutations to the extent that a single nucleotide
mismatch in the targeting sequence abrogates its
ability to suppress gene expression. This feature
opened opportunities for developing new therapy
approaches by targeting disease-derived transcripts
(e.g oncogenes) with dominant activating mutations.
One such example is the occurrence of mutated RAS
alleles in many human tumors. To examine the role of
RAS mutants in tumor maintenance we have developed a
pSUPER tool to specifically target oncogenic
K-RASV12 allele without affecting its wild type
counterpart. We showed that this tool powerfully
inhibits the tumorigenicity of cancer cell lines
that harbor this exact type of genetic alteration.
As K-RASV12 is a frequent event in human cancer, the
pSUPER-K-RASV12 and similar expression vectors can
be used now to identify essential genetic events in
human cancers and also may possibly serve as genetic
tools for cancer therapy.
Identification and characterization of novel tumor suppressor genes
In a complementary approach, we are
using the pSUPER RNAi library to target putative
tumor suppressor in order to identify and define
combinations of genetic events that are capable of
converting primary human cells to cancerous cells.
To uncover novel tumour-suppressor genes affecting
the RAS pathway, we screened an RNA-interference
library for knockdown constructs that transform
human primary cells in the absence of oncogenic RAS.
We report the identification of PITX1, whose
inhibition induces the RAS pathway and
tumorgenicity. Interestingly, we observed low
expression of PITX1 in prostate and bladder tumours
and in colon cancer cell lines containing wild-type
RAS. Restoration of PITX1 in the colon cancer cells
inhibited tumorigenicity, in a wild-type RAS
dependent manner. Finally, we identified the
mechanism through which PITX1 affect RAS activity.
PITX1 activates RASAL1, a member of the
RAS-GTPase-activating protein family, by direct
binding to its promoter. Altogether, our results
outline an approach to uncover tumour suppressor
genes that function as inhibitors of the RAS
pathway, which can also be used for other cancer
causing pathways, such as the p53 and pRb. The data
extracted from such study enables us to understand
the function of various tumor suppressors in tumor
progression and to identify new critical genes
required for cancer development in humans.
