Page 157 - ebook

HIPK2 phosphorylates Notch1-IC T2512 via FBW7 in breast cancer
School of Biological Sciences and Technology, Chonnam National University, Yongbong-dong, Buk-ku, Gwangju, 500-757, Republic of Korea
*Corresponding author : Hee-Sae Park, e-mail : proteome@jnu.ac.kr
Abstract
The receptor Notch1 plays an important role in malignant pro- gression of many
cancers, but its regulation is not fully under- stood. In this study, we report that the
kinase HIPK2 is responsible for facilitating the Fbw7-dependent proteasomal
degradation of Notch1 by phosphorylating its intracellular domain (Notch1-IC) within
the Cdc4 phosphodegron motif. Notch1-IC expression was higher in cancer cells
than normal cells. Under genotoxic stress, Notch1-IC was phosphorylated
constitutively by HIPK2 and was maintained at a low level through proteasomal
degradation. HIPK2 phosphorylated the residue T2512 in Notch1-IC. Somatic muta-
tions near this residue rendered Notch1-IC resistant to degradation, as induced
either by HIPK2 overexpression or adriamycin treat- ment. In revealing an important
mechanism of Notch1 stability, the results of this study could offer a therapeutic
strategy to block Notch1-dependent progression in many types of cancer.
Figure 1.
Adriamycin (ADR) affects Notch1 signaling and cell
viability of breast cancer cells.
A, Western blotting of Notch1-IC and HIPK2 in MDA-MB-231
cells treated with the specified dose of adriamycin for 12 hours.
B, Western blotting of Notch1-IC and HIPK2 in MDA-MB-231 cells
treated with 2 mmol/L adriamycin for the indicated time. C,
Western blotting of Notch1-IC, HIPK2, Hes1, and Hes5 in
HEK293 cells treated with 2 mmol/L adriamycin for 12 hours. D,
immunohistochemical staining of Notch1-IC and HIPK2 in serial
tissue arrays of 61 breast cancer samples. Notch1-IC and HIPK2
levels in each sample were semiquantified as high or low in a
double-blind manner, according to the standards presented.
Figure 3
HIPK2 decreases the stability of
through proteasome-dependent degradation.
A, endogenous coimmunoprecipitation of RBP-Jk with
Notch1-IC in HIPK2þ/þ and HIPK2/ MEFs. B, endogenous
coimmunoprecipitation of RBP-Jk with Notch1-IC in HEK293
cells expressing shCon and shHIPK2, and treated
with 2 mmol/L adriamycin for 12 hours. C, levels of Notch1-
IC in HEK293 cells transfected with shCon and shHIPK2 and
treated with 2 mmol/L adriamycin for 12 hours and 100
mmol/L cycloheximide (CHX) for the indicated time were
determined by Western blotting. D, levels of Notch1-IC in
HIPK2þ/þ and HIPK2/ MEFs treatedwith 2 mmol/L adriamycin
for 12 hours and 100 mmol/L cycloheximide for the indicated
time were determined by Western blotting. C and D, we
quantified the intensity of each band using a densitometer
and plotted relative intensities. Data are expressed as means
SD from three independent experiments. E, luciferase
reporter analysis of the transcriptional activity of Notch1-IC
in HIPK2þ/þ and HIPK2/ MEFs treatedwith 5 mmol/L MG132
for 6 hours. Error bars correspond to pooled data from three
independent experiments. F, lysates of HIPK2þ/þ and HIPK2/
MEFs treated with MG132 for 6 hours were analyzed by
Western blotting with antibodies against Notch1-IC and b-
actin. G, endogenous coimmunoprecipitation of Fbw7 with
Notch1-IC in HIPK2þ/þ and HIPK2/ MEFs.
Conclusion
• HIPK2 phosphorylated the T2512 residue of Notch1-IC and facilitated its proteasomal
degradation through Fbw7.
• The levels of phosphorylated Notch1-IC T2512, HIPK2, and Fbw7 were significantly higher in
normal breast tissue than in the tumor tissue.
• After adriamycin treatment, HIPK2 significantly decreased cancer growth and invasion by
increasing the apoptosis of cancer cells expressing Notch1- IC
• Notch1-IC P2513L and Notch1-IC P2515*fs mutants were resistant to HIPK2-induced
phosphorylation
• Phosphorylation of T2512 in Notch1-IC reduces migration, tumor growth, and metastasis.
Introduction
Notch1 is a highly conserved transmembrane protein that plays a crucial role in cancer development, cancer cell survival, prolif- eration, and differentiation, and fate
determination of cancer cells (1). Notch1 signaling is aberrantly activated in breast cancer, and increased expression of Notch1 intracellular domain (Notch1-IC) is associated
with poor survival in patients with various cancers, including breast cancer (2–7). Moreover, proliferation of cells derived from these cancers can be suppressed by
pharmacologic inhibition of Notch1. Therefore, preventing the generation of Notch1-IC is a potential strategy for treating various cancers (8, 9). Fbw7 binds to Notch1-IC via its
WD40 domains and mediates its ubiquitination and degradation by the proteasome system, which promotes proline, glutamic acid, serine, and threonine rich region (PEST)
domain-dependent Notch1-IC degradation (10, 11). Phosphorylation of the T2512 residue of Notch1-IC is important for its recognition by Fbw7, which in turn facilitates its
degrada-tion through the proteasome (12–14). However, the kinase that phosphorylates the T2512 residue has not been conclusively identified.
In this study, we evaluated the crosstalk between HIPK2 and Notch1 signaling during tumorigenesis. We identified that che- motherapeutic drug–induced HIPK2 phosphorylated
the T2512 residue in the Cdc4 phosphodegron (CPD) motif of Notch1-IC, thus facilitating its degradation by Fbw7 ubiquitin ligase. We also found that tissues of patients with
breast cancer showed increased level of Notch1-IC and decreased levels of phosphorylated Notch1-IC T2512, HIPK2, and Fbw7. The chemotherapeutic agent significantly
decreased the growth, invasion, and tumori- genic activity of Notch1-IC–expressing cells. However, this was not observed in cells expressing Notch1-IC T2512A and the somatic
mutants Notch1-IC P2513L and Notch1-IC P2515 frame- shift (P2515 fs) because of the decreased phosphorylation of Notch1-IC. These data suggested that HIPK2-induced
*
phosphor- ylation of Notch1-IC at the T2512 residue plays an important role in cancer prevention and could be a potential biomarker for diagnosing breast cancer treatment.
Figure 2. Figure 5 Figure 6
Adriamycin (ADR) suppresses the transcriptional HIPK2-mediated downregulation of Notch1-IC Somatic mutations in the CPD motif of Notch1-IC
activity of Notch1 by inducing HIPK2 suppresses the tumorigenesis of breast cancer make it resistant to HIPK2
A, luciferase reporter analysis of the transcriptional activity
of Notch1-IC in the presence or absence of HIPK2 in MDA-
MB-231 cells at 12 hours after Adriamycin treatment. Error
bars correspond to pooled data from three independent
experiments. B, Western blotting of Hes1 in HEK293 cells
transiently transfected with the indicated plasmids and
treated with 2 mmol/L adriamycin for 12 hours. C, Western
blotting of Hes1, Hes5, and HIPK2 in HIPK2þ/þ and HIPK2/ A, pie chart representing the distribution of mutated Notch1 in
MEFs treated with 2 mmol/L adriamycin for 12 hours. D, various tissues (data from COSMIC). B, mutations in the CPD
luciferase reporter analysis of the transcriptional activity of motif of Notch1 in leukemia cells. C, Western blotting of
Notch1-IC in HEK293 cells transfected with Notch1-IC, phosphorylated Notch1-IC T2512 in HEK293 cells transiently
shCon, and shHIPK2. Error bars correspond to pooled data transfected with the indicated plasmids and then treated with 5
from three independent experiments. E, luciferase reporter mmol/L MG132 for 6 hours. D, coimmunoprecipitation of anti-
analysis of the transcriptional activity of Notch1-IC in Flag with anti-Myc in HEK293 cells transiently transfected with
HIPK2þ/þ and HIPK2/ MEFs transfected with Notch1-IC. Error the indicated plasmids and then treated with 5 mmol/L MG132
bars correspond to pooled data from three independent
for 6 hours. E, Western blotting of Myc-Notch1-IC, Myc-Notch1-
Notch1-IC experiments. RLU, relative luciferase units. IC P2513L, Myc-Notch1-IC P2515fs, and GFP-HIPK2 in HEK293
cells transiently transfected with the individually indicated
plasmids. F, in vivo Notch1-IC ubiquitination assay in HEK293
cells transiently transfected with the indicated plasmids and
Figure 4 then treated with DMSO or MG132 for 6 hours.
HIPK2 regulates Notch1-IC by phosphorylating its Figure 7.1
T2512 residue.
(AA, HEK293 cells transfected with shNotch1, GFP, Myc- Physiologic effects of somatic mutations in the CPD
Notch1-IC, and Myc-Notch1-IC T2512A, and treated with 2 motif of Notch1-IC.
mmol/L adriamycin for 12 hours were fixed and stained with
DAPI. Apoptotic nuclei of GFP-positive cells were quantified
using a fluorescence microscope. Data are expressed as
mean SD of values from three independent experiments. B,
photomicrographs of wound healing assay of MDA-MB-231
cells expressing either Notch1-IC or shHIPK2. C,
photomicrographs of transwell motility assay of MDA-MB-
231 cells expressing shCon and shHIPK2, and treated with
adriamycin for 12 hours. Bar graph shows the mean number
of cells per filter. Data are expressed as mean SD of values
from three independent experiments. Representative
colonies are shown. D, photomicrographs from Matrigel
invasion assay of MDA-MB-231 cells expressing shCon and
shHIPK2, and treated with adriamycin for 12 hours. Bar
graph shows the mean number of cells per filter. Data are
expressed asmean SD of values from three independent
experiments. Representative colonies are shown. E and F,
colony-forming assay of Notch1-silenced MCF7 (E) and
MDA-MB-231 (F) cells expressing Myc-Notch1-IC WT and
Myc-Notch1-IC T2512A and treated with adriamycin for 12
hours. Cells were further incubated for 14 days, fixed with
4% paraformaldehyde, and stained with 0.5% crystal violet. A, xenograft growth ofMDA-MB-231-SQ cells expressing the
Data are expressed as meanSD of values from three indicated constructs and treated with adriamycin. Tumors
independent experiments. Representative colonies are excised from nude mice, and statistical analysis of mean tumor
shown. G, Western blotting of Notch1-IC, phosphorylated volume and tumor weight. Tumor volumes (mm3) were
Notch1-IC T2512, HIPK2, and Fbw7 in breast cancer tissues measured on indicated days (n ¼ 5 per group). Error bars, mean
(T) and adjacent normal tissues (N). b-Actin was used as the SD; , P < 0.001. Inhibition of tumor growth by adriamycin was
loading control. H, comparison of the relative levels of measured by calculating tumor volume (B) and tumor weight (C).
A, endogenous coimmunoprecipitation of Notch1-IC with HIPK2 Notch1-IC, phosphorylated Notch1-IC T2512, HIPK2, and Representative images of the dissected tumors are shown in A.
in HEK293 cells transiently transfected with the indicated Fbw7 in breast cancer tissues and normal tissues by using D, cell lysates of random tumors selected from eight groups
plasmids and then treated with 2 mmol/L adriamycin (ADR) for paired t test (data are expressed as mean SD; n ¼ 54, , P were analyzed by immunoblotting with antibodies against Myc,
12 hours. B, endogenous coimmunoprecipitation of HIPK2 with phosphorylated Notch1-IC T2512, HIPK2, Fbw7, and b-actin..
Notch1-IC in HIPK2þ/þ and HIPK2/ MEFs. C, immunocomplex < 0.001).
kinase analysis to determine HIPK2-induced phosphorylation of
recombinant GST-Notch1-IC or GST-Fbw7 in HEK293 cells
transfected with the indicated plasmids. Cell lysates were
treated with anti-Myc antibody. Results of 32P-autoradiography Figure 7.2
after SDS-PAGE and corresponding protein loading are shown
at the bottom. D, CLUSTALW alignment of mammalian CPDs. E, Physiologic effects of somatic mutations in the CPD
CLUSTALW alignment of the CPD consensus motif and HIPK2- motif of Notch1-IC.
binding motif of Notch1 sequences from various species;
human Notch1 CPD and HIPK2-binding site (T2512) are shown
in red. F, endogenous Western blotting of phosphorylated
Notch1-IC T2512 in HIPK2/ MEFs transiently transfected with the E, HIPK2 suppresses Notch1 signaling by phosphorylating its
indicated plasmids. G,Western blotting of phosphorylated T2512 residue, which in turn initiates the proteasome-mediated
Notch1-IC T2512 in HEK293 cells transiently transfected with degradation of Notch1-IC by Fbw7 in the nucleus. Notch1-IC forms
the indicated plasmids and then treated with 5 mmol/L MG132 a trimeric complex with Fbw7 and HIPK2. HIPK2 enhances the
for 6 hours. H, luciferase reporter analysis of the transcriptional degradation of Notch1-IC via the Fbw7- dependent proteasome
activity of Notch1-IC in HIPK2/ MEFs transfected with Notch1-IC, pathway.
Notch1-IC T2512A, and HIPK2. Error bars correspond to pooled
data from three independent experiments. I, in vivo Notch1-IC
ubiquitination assay in HEK293 cells transiently transfected
with the indicated plasmids and then treated with DMSO or
MG132 for 6 hours.
Reference
1. Artavanis-Tsakonas, S., M. D. Rand, and R. J. Lake. 1999. Notch signaling: cell fate control and signal integration in development.
Science 284:770-6.
2. Pece S, Serresi M, Santolini E, Capra M, Hulleman E, Galimberti V, et al. Loss of negative regulation by Numb over Notch is relevant
to human breast carcinogenesis. J Cell Biol 2004;167:215–21.
3. Stylianou S, Clarke RB, Brennan K. Aberrant activation of notch signaling in human breast cancer. Cancer Res 2006;66:1517–25.
4. Rizzo P, Miao H, D'Souza G, Osipo C, Song LL, Yun J, et al. Cross-talk between notch and the estrogen receptor in breast cancer
suggests novel therapeutic approaches. Cancer Res 2008;68:5226–35
5. Gupta-Rossi N, Le Bail O, Gonen H, Brou C, Logeat F, Six E, et al. Functional interaction between SEL-10, an F-box protein, and the
nuclear form of activated Notch1 receptor. J Biol Chem 2001;276:34371–8.
6. Stylianou S, Clarke RB, Brennan K. Aberrant activation of notch signaling in human breast cancer. Cancer Res 2006;66:1517–25.
7. Demarest RM, Ratti F, Capobianco AJ. It's T-ALL about Notch. Oncogene 2008;27:5082–91