NCI Mammary Mouse Models Collective (MMMC)

Minutes of Meeting---November 19, 2003

Building 41, Conference Room 507

1:15 p.m.--2:30 p.m.

**Next Meeting: December 8, 2003**

**Mouse Models of Mammary Cancer Retreat**

Marriott Washingtonian Center, Gaithersburg.

8:30 a.m. -- 5:30 p.m.

##### ACTION ITEMS:

### MMMC Members

-   Notify Jeff Green (<jg155c@nih.gov>) of the names of potential MMMC
    seminar speakers.

-   Send abstracts for the MMMC Retreat to Valerie Sanders
    (sanderva@mail.nih.gov) as soon as possible.

##### UPDATES AND NEW BUSINESS:

### Overview of MMMC-Interfaculty Retreat

Dr. Green presented a brief overview of the upcoming retreat at the
Marriott Washingtonian Center in Gaithersburg on December 8. The agenda,
details on submitting abstracts, and other general information about the
meeting, can be found at the following Web site:
<http://www.palladianpartners.com/MMMammary/default.asp>

### Upcoming Seminar

Dr. Andras Nagy will be the guest speaker at the January 14, 2004,
meeting of the MMMC at 1 p.m. in the NIH Building 41 conference room.
The seminar will be broadcast to the Lombardi Cancer Center,
NCI-Frederick, and the Key West Center.

##### SEMINAR:

## Novel Signaling in Breast Cancer---Dr. Mien-Chie Hung, Chairman, Department of Molecular and Cellular Oncology, M. D. Anderson Cancer Center, University of Texas.

Dr. Hung began by explaining that the first part of his talk would focus
on "traditional" cell signaling, while the second part would be on more
novel concepts in membrane signaling. He presented data from recent
studies in his lab examining the relationship between the human
epidermal growth factor receptor-2 (*Her2/neu*; erbB2) and the tumor
suppressor p53.

*Her2/neu* is an oncogene encoding a transmembrane receptor, tyrosine
kinase. Overexpression of *Her2/neu* confers resistance to apoptosis,
promotes cell growth, and is associated with poor clinical outcome in
many human cancers. Regarding its mechanism of activity, earlier work
had demonstrated that *Her2/neu* activates the phosphatidylinositol-3
kinase (PI(3)K)-Akt pathway, thus preventing cells from undergoing
apoptosis. NIH3T3 cells overexpressing *Her2/neu* (*Her2/neu* 3T3 cells)
are resistant to the DNA-damaging agent etoposide, while cells
overexpressing *Her2/neu* and an inactive Akt protein (DN-Akt 3T3 cells)
undergo etoposide-induced apoptosis. The protein p53 induces apoptosis
in response to DNA damage of this sort. Blockage of the Akt pathway with
PI(3)K inhibitors does not affect survival in either wild type or
p53^-^/^-^ cells, but it dramatically increases the sensitization to
etoposide in wild type cells---measured as lower cell activity and
greater apoptosis---demonstrating that p53 is essential for
etoposide-induced apoptosis. In conclusion, an intact Akt pathway seems
to be required for *Her2/neu*-mediated chemoresistance, and there
appears to be a relationship between Akt and p53 in etoposide-induced
apoptosis. Additional evidence for this relationship is that DN-Akt 3T3
cells, unlike *Her2/neu* 3T3 cells, express elevated levels of p53 and
that these levels increase following exposure to etoposide

Dr. Hung reminded the audience that p53 levels are regulated by the
interaction of several proteins, including MDM2, p300, and p19^ARF^. In
unstressed cells, MDM2 binds p53, resulting in p53 ubiquitination,
transport to the cytoplasm, and degradation. Following DNA damage, MDM2
is unable to bind p53 to facilitate nuclear export; consequently, levels
of p53 rise. Nuclear accumulation of p53 was seen in DN-Akt 3T3 cells
treated with etoposide---but not in *Her2/neu* 3T3 cells---implicating
Akt in p53-MDM2 interaction. To allow p53 accumulation in the nucleus of
*Her2/neu* 3T3 cells, nuclear export was inhibited by Leptomycin B. The
p53 protein obtained from *Her2/neu* 3T3 cells, but not from DN-Akt 3T3
cells, was ubiquitinated. Further experiments demonstrated that Akt
binds and phosphorylates MDM2 at two sites, Ser166 and Ser188, close to
sequences crucial for the cellular location of MDM2; immunofluorescence
staining showed that MDM2 is almost exclusively nuclear in *Her2/neu*
3T3 cells, while it is both nuclear and cytoplasmic in the DN-Akt 3T3
cells. Dr. Hung explained that this would suggest that Akt must
phosphorylate MDM2 for it to be translocated to the nucleus. The
phosphorylation state of MDM2 also affects its ability to bind p300 or
p19^ARF^. Phosphorylated MDM2 binds p300, resulting in increased p53
degradation, while nonphosphorylated MDM2 binds p19^ARF^, inhibiting
MDM2's ubiquitin-ligase activity and, thus, stabilizing p53. Dr. Hung
emphasized how blocking the *Her2/neu*-Akt pathway to prevent the
nuclear translocation of MDM2 has clinical implications for increasing
the cytotoxic effects of DNA-damaging drugs.

Dr. Hung concluded the first part of his presentation by describing how
*Her2/neu* overexpression and subsequent activation of the Akt pathway
promotes cell growth. Akt-mediated phosphorylation of the cell-cycle
inhibitor p21^Cip1/WAF1^ results in cytoplasmic localization and allows
interaction with caspase 3 and apoptosis-signal-regulating kinase 1
(ASK1), thus inducing resistance to apoptosis. Nuclear p21^Cip1/WAF1^,
however, inhibits cell growth. The cellular localization of
p21^Cip1/WAF1^ is clinically relevant, as Dr. Hung presented evidence
correlating significantly greater survival in cancer patients with
nuclear rather than cytoplasmic p21^Cip1/WAF1^ expression.

In the second part of his talk, Dr. Hung discussed the nuclear
localization of the epidermal growth factor receptor (EGFR) and its
potential role as a transcription factor. A paper published by his group
in 1994 showed that the *Her2/neu*-encoded protein p185 is localized in
the nucleus and that its C-terminal domain possesses transcriptional
activity when used as "bait" in the yeast two-hybrid system (Xie and
Hung, *BBRC* 203; 1589-98). This was the first published report that a
membrane-receptor tyrosine kinase had transcriptional activity. Work has
since begun to address the function of nuclear EGFR. Immunohistochemical
studies have demonstrated that EGFR is found in the nuclei of highly
proliferating cells as well as those of tumor cells and that the
C-terminal domain of EGFR is associated with strong transcriptional
activity (see Lin *et al.*, Nat. Cell Biol. (2001) 3:802-808).

Dr. Hung explained that the cyclic amplification and selection of
targets (CASTing) technique was employed to determine whether the EGFR
bound directly to DNA and to identify its DNA-binding site. An AT-rich
minimal consensus sequence (ATRS) was identified and subsequently
confirmed as an EGFR-binding site using gel-shift assays.

Because EGFR was found in the nucleus of highly proliferative cells, Dr.
Hung explained that its targets might be genes involved in cell growth;
two ATRS consensus sequences were subsequently identified in the cyclin
D1 promoter. A reporter gene containing the two ATRS sequences derived
from the cyclin D1 promoter was activated *in vitro* by EGF treatment.
Dr. Hung also presented chromatin immunoprecipitation results showing
that EGFR was physically associated with the cyclin D1 promoter,
confirming that cyclin D1 expression is regulated by EGFR.

Dr. Hung concluded that further studies are required to explain how EGFR
is translocated into the nucleus and to define the involvement of its
ligand in regulating DNA-binding and transcriptional activation. He
indicated that the mechanism involved may be common to many other
transmembrane receptors that have also been found in the nucleus, such
as FGFR, IL-1R, and c-erbB-4. He closed his presentation by speculating
on how transmembrane receptors physically translocate to the nucleus.

**ATTENDANCE:**

Dalit Barkan

Xavier Coumoul

Chuxia Deng

Lionel Feigenbaum

Jeff Green

Morihisa Hirota

Jung-Im Huh

Kent Hunter

Sonia Jakowlew

Mindy Lancaster

Wenmei Li

Luanne Lukes

Cristina Montagna

Tri Nguyen

Thomas Ried

Gertraud Robinson

David Salomon

Valerie Sanders

Youping Sun

Binwu Tang

Lalage Wakefield

Weihan Wang

Yumei Ye

######## NOVA Research Company

Mel Simpson