Biology Reference
In-Depth Information
these data demonstrated that the MMR-c-Abl-p73
a
/
MMR-c-Abl-GADD45
a
signal transduction pathway
plays an important role in anticancer therapies. Under-
standing these signaling pathways is an important step
for improving therapy of cancers with defects in these
pathways due to altered MMR.
Based on our own and other studies, we noticed that
inhibition, or knockdown, of c-Abl kinase activity pre-
vented MMR-dependent G
2
cell cycle checkpoint arrest
and apoptotic responses, as well as lethality and long-
term survival. However, we were able to segregate roles
for p73
a
and GADD45
a
in MMR-dependent apoptosis
and G
2
arrest responses, respectively. These data highly
suggest that there may be further downstream signal
transduction responses beginning with hMLH1-c-Abl
sensory detection of damage that may exist in the
cellular response to MNNG, cisplatin, TMZ or other
DNA damaging agents that create MMR-dependent
responses. Numerous molecules, such as BRCA1,
Rad51, p300, Pin1, and p21, have been proposed to be
involved in c-Abl-dependent cellular responses to
various cell stresses.
232
e
235
One potential candidate
protein that bridges the gap between MMR and c-Abl
signaling to the downstream induction of GADD45
a
,
is BRCA1. BRCA1, the breast cancer susceptibility
protein-1, has a duel role in both cell cycle G
2
arrest
responses and homologous DSB repair. Similar to p53,
BRCA1 can be phosphorylated and thereby activated
in response to DNA damage, allowing it to act as a tran-
scription factor for several downstream genes, including
GADD45
a
.
197
Since our laboratory observed enhanced
MMR-dependent GADD45
a
induction in response to
fluorinated pyrimidines
94
and MNNG exposures,
90,91
BRCA1 makes an attractive candidate for linkage of c-
Abl to the MMR-dependent regulation of GADD45
a
expression after MNNG or other DNA damaging agents
initially detected by the MMR-c-Abl damage sensory
recognition complex. Interestingly, BRCA1 can also be
activated by ATM kinase after recognition of DSBs, dis-
rupting its interaction from a stable inactive complex
with c-Abl.
233
This disruption allows both proteins to
separate and become fully active, possibly taking part
in separate or mutually synergistic intracellular
signaling. Indeed, a direct interaction between MMR
and BRCA1 complexes has been observed. Wang
et al.
236
demonstrated that BRCA1 physically interacted
with hMSH2 and two of its binding partners, hMSH3
and hMSH6. BRCA1 and hMSH2 are both parts of
a proposed multiprotein complex involved in DNA
damage recognition and repair, known as the BASC
complex.
166
Although our data demonstrated that
induction of cell death by O
6
-meG lesions required
direct signaling by the MMR-c-Abl-p73
a
signaling
cascade, DSBs are simultaneously formed and most
likely contribute to MMR-mediated cell death if not
repaired. Cells defective in homologous recombination
(XRCC2 and BRCA2 mutants) are extremely sensitive
to cell death by apoptosis and chromosomal aberration
formation and less sensitive to sister-chromatid
exchange induction than corresponding wild-type
cells.
237
NOVEL THERAPEUTIC STRATEGIES
FOR THE TREATMENT OF
M
MR-DEFICIENT CANCER
S
Since resistance to chemotherapy due to MMR defi-
ciencies is well established (e.g., for 5-FU, see
Table
9.3
), overcoming this resistance in order to obtain
improved therapeutic efficacy is a major goal of research
in this area. Therefore, it is important in modern cancer
drug discovery to design therapeutic approaches that
identify and exploit the underlying genetic weaknesses
or “Achilles heel” of specific cancer cells. To address
this issue, several strategies have been proposed for
the specific treatment of MMR-deficient cancers.
Restoring MMR Function
The functional cause of a majority of sporadic MMR-
deficient cancers was due to loss of hMLH1 expression
caused by promoter hypermethylation. In these cases,
MMR function can be restored through the use of deme-
thylating agents, such as prolonged azacytidine expo-
sures, to cause demethylation of the hMLH1 promoter,
allowing re-expression of hMLH1 protein and reemer-
gence of its function. As a result, re-expression of both
hMLH1 and hPMS2 can be seen in cells exposed to
azacytidine. Early studies from our laboratory demon-
strated that restoring hMLH1 expression in HCT116
cells caused significantly prolonged G
2
arrest in
response to 6-TG or FdUrd.
94,106
One fluoropyrimidine
derivative, 5-fluoro-2-deoxycytidine (FdCyd), can be
a potent hypomethylating agent when incorporated
into the DNA of exposed cells.
238
The fluorine group
in the 5-position of FdCyd prevents methylation due
to its unbreakable carbon-fluorine bond. Indeed,
exposing cells to FdCyd alters the methylation pattern
of exposed cells.
238
Furthermore, we demonstrated that
exposure of RKO6 cells, which have epigenetic loss of
hMLH1 protein due to promoter hypermethylation,
can re-express hMLH1 in response to prolonged FdCyd
treatments. As a result, cells exposed to FdCyd are
hypersensitive to this drug compared to the resistance
noted to FdUrd or 5-FU. Thus, we proposed that FdCyd
exposures can be used for the re-expression of hMLH1,
thereby converting resistant epigenetically silenced
hMLH1-deficient colon or ovarian cancer cells to sensi-
tive cells due to the restoration of functional MMR.
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