10 research outputs found
RASAL2 Confers Collateral MEK/EGFR Dependency in Chemoresistant Triple-Negative Breast Cancer
Re-Engineered p53 Chimera with Enhanced Homo-Oligomerization That Maintains Tumor Suppressor Activity
The use of the tumor suppressor p53 for gene therapy of cancer
is limited by the dominant negative inactivating effect of mutant
endogenous p53 in cancer cells. We have shown previously that swapping
the tetramerization domain (TD) of p53 with the coiled-coil (CC) from
Bcr allows for our chimeric p53 (p53-CC) to evade hetero-oligomerization
with endogenous mutant p53. This enhances the utility of this construct,
p53-CC, for cancer gene therapy. Because domain swapping to create
p53-CC could result in p53-CC interacting with endogenous Bcr, which
is ubiquitous in cells, modifications on the CC domain are necessary
to minimize potential interactions with Bcr. Hence, we investigated
the possible design of mutations that will improve homodimerization
of CC mutants and disfavor hetero-oligomerization with wild-type CC
(CCwt), with the goal of minimizing potential interactions with endogenous
Bcr in cells. This involved integrated computational and experimental
approaches to rationally design an enhanced version of our chimeric
p53-CC tumor suppressor. Indeed, the resulting lead candidate p53-CCmutE34K-R55E
avoids binding to endogenous Bcr and retains p53 tumor suppressor
activity. Specifically, p53-CCmutE34K-R55E exhibits potent apoptotic
activity in a variety of cancer cell lines, regardless of p53 status
(in cells with mutant p53, wild-type p53, or p53-null cells). This
construct overcomes the dominant negative effect limitation of wt
p53 and has high significance for future gene therapy for treatment
of cancers characterized by p53 dysfunction, which represent over
half of all human cancers
The DNA Binding Domain of p53 Is Sufficient To Trigger a Potent Apoptotic Response at the Mitochondria
The
tumor suppressor p53 is one of the most studied proteins in
human cancer.− While nuclear p53 has been utilized for cancer gene
therapy, mitochondrial targeting of p53 has not been fully exploited
to date., In response to cellular stress, p53 translocates
to the mitochondria and directly interacts with Bcl-2 family proteins
including antiapoptotic Bcl-XL and Bcl-2 and proapoptotic Bak and
Bax. Antiapoptotic Bcl-XL forms inhibitory
complexes with proapoptotic Bak and Bax preventing their homo-oligomerization. Upon translocation to the mitochondria, p53 binds
to Bcl-XL, releases Bak and Bax from the inhibitory complex and enhances
their homo-oligomerization. Bak and Bax
homotetramer formation disrupts the mitochondrial outer membrane,
releases antiapoptotic factors such as cytochrome <i>c</i> and triggers a rapid apoptotic response mediated by caspase induction. It is still unclear if the MDM2 binding domain
(MBD), the proline-rich domain (PRD) and/or DNA binding domain (DBD)
of p53 are the domains responsible for interaction with Bcl-XL.− The purpose of this work is to determine if a smaller functional
domain of p53 is capable of inducing apoptosis similarly to full length
p53. To explore this question, different domains of p53 (MBD, PRD,
DBD) were fused to the mitochondrial targeting signal (MTS) from Bcl-XL
to ensure Bcl-XL specific targeting. The
designed constructs were tested for apoptotic activity (TUNEL, Annexin-V,
and 7-AAD) in 3 different breast cancer cell lines (T47D, MCF-7, MDA-MB-231),
in a cervical cancer cell line (HeLa) and in non-small cell lung adenocarcinoma
cells H1373. Our results indicate that DBD-XL (p53 DBD fused to the
Bcl-XL MTS) reproduces (in T47D cells) or demonstrates increased apoptotic
activity (in MCF-7, MDA-MB-231, and HeLa cells) compared to p53-XL
(full length p53 fused to Bcl-XL MTS). Additionally, mitochondrial
dependent apoptosis assays (TMRE, caspase-9), co-IP and overexpression
of Bcl-XL in T47D cells suggest that DBD fused to XL MTS may bind
to and inhibit Bcl-XL. Taken together, our data demonstrates for the
first time that the DBD of p53 may be the minimally necessary domain
for achieving apoptosis at the mitochondria in multiple cell lines.
This work highlights the role of small functional domains of p53 as
a novel cancer biologic therapy
Enhanced and Selective Killing of Chronic Myelogenous Leukemia Cells with an Engineered BCR-ABL Binding Protein and Imatinib
The oncoprotein Bcr-Abl stimulates prosurvival pathways
and suppresses apoptosis from its exclusively cytoplasmic locale,
but when targeted to the mitochondrial compartment of leukemia cells,
Bcr-Abl was potently cytotoxic. Therefore, we designed a protein construct
to act as a mitochondrial chaperone to move Bcr-Abl to the mitochondria.
The chaperone (i.e., the 43.6 kDa intracellular cryptic escort (iCE))
contains an EGFP tag and two previously characterized motifs: (1)
an optimized Bcr-Abl binding motif that interacts with the coiled-coil
domain of Bcr (ccmut3; 72 residues), and (2) a cryptic mitochondrial
targeting signal (cMTS; 51 residues) that selectively targets the
mitochondria in oxidatively stressed cells (i.e., Bcr-Abl positive
leukemic cells) via phosphorylation at a key residue (T193) by protein
kinase C. While the iCE colocalized with Bcr-Abl, it did not relocalize
to the mitochondria. However, the iCE was selectively toxic to Bcr-Abl
positive K562 cells as compared to Bcr-Abl negative Cos-7 fibroblasts
and 1471.1 murine breast cancer cells. The toxicity of the iCE to
leukemic cells was equivalent to 10 μM imatinib at 48 h and
the iCE combined with imatinib potentiated cell death beyond imatinib
or the iCE alone. Substitution of either the ccmut3 or the cMTS with
another Bcr-Abl binding domain (derived from Ras/Rab interaction protein
1 (RIN1; 295 residues)) or MTS (i.e., the canonical IMS derived from
Smac/Diablo; 49 residues) did not match the cytotoxicity of the iCE.
Additionally, a phosphorylation null mutant of the iCE also abolished
the killing effect. The mitochondrial toxicity of Bcr-Abl and the
iCE in Bcr-Abl positive K562 leukemia cells was confirmed by flow
cytometric analysis of 7-AAD, TUNEL, and annexin-V staining. DNA segmentation
and cell viability were assessed by microscopy. Subcellular localization
of constructs was determined using confocal microscopy (including
statistical colocalization analysis). Overall, the iCE was highly
active against K562 leukemia cells and the killing effect was dependent
upon both the ccmut3 and functional cMTS domains
A Chimeric p53 Evades Mutant p53 Transdominant Inhibition in Cancer Cells
Because
of the dominant negative effect of mutant p53, there has
been limited success with wild-type (wt) p53 cancer gene therapy.
Therefore, an alternative oligomerization domain for p53 was investigated
to enhance the utility of p53 for gene therapy. The tetramerization
domain of p53 was substituted with the coiled-coil (CC) domain from
Bcr (breakpoint cluster region). Our p53 variant (p53-CC) maintains
proper nuclear localization in breast cancer cells detected via fluorescence
microscopy and shows a similar expression profile of p53 target genes
as wt-p53. Additionally, similar tumor suppressor activities of p53-CC
and wt-p53 were detected by terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL), annexin-V, 7-aminoactinomycin D (7-AAD),
and colony-forming assays. Furthermore, p53-CC was found to cause
apoptosis in four different cancer cell lines, regardless of endogenous
p53 status. Interestingly, the transcriptional activity of p53-CC
was higher than wt-p53 in 3 different reporter gene assays. We hypothesized
that the higher transcriptional activity of p53-CC over wt-p53 was
due to the sequestration of wt-p53 by endogenous mutant p53 found
in cancer cells. Co-immunoprecipitation revealed that wt-p53 does
indeed interact with endogenous mutant p53 via its tetramerization
domain, while p53-CC escapes this interaction. Therefore, we investigated
the impact of the presence of a transdominant mutant p53 on tumor
suppressor activities of wt-p53 and p53-CC. Overexpression of a potent
mutant p53 along with wt-p53 or p53-CC revealed that, unlike wt-p53,
p53-CC retains the same level of tumor suppressor activity. Finally,
viral transduction of wt-p53 and p53-CC into a breast cancer cell
line that harbors a tumor derived transdominant mutant p53 validated
that p53-CC indeed evades sequestration and consequent transdominant
inhibition by endogenous mutant p53