NCI Mammary Mouse Models Collective (MMMC)

Minutes of Meeting---October 8, 2003

NIH Building 41, Conference Room 507

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

**Next Meeting: November 19, 2003**

**Dr. Mien-Chie Hung, M.D. Anderson Cancer Center,**

**University of Texas: *Novel Signaling in Breast Cancer***

NIH Building 41, Conference Room 507

Frederick, Building 549, Executive Board Room

Rockville, Key West Center, Room 300

Lombardi Cancer Center, Room E501

##### 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.

-   Notify Jeff Green if interested in joining the MMMC Steering
    Committee.

-   Notify Natasha Caplen (<ncaplen2@mail.nih.gov>) about siRNA genes
    that should be added to the list available on the MMMC Web site.

##### UPDATES AND NEW BUSINESS:

### Overview of RFA renewal grant

Dr. Green summarized the organizational structure, scientific
accomplishments, and scientific projects of the RFA recompetition grant
proposal submitted in March. The proposal will be reviewed this month,
and results will be announced at the beginning of 2004. Tom Ried and
Jeff Green are the Co-PIs for the grant proposal.

Dr. Green presented an outline of the interactions documented in the
proposal among the Co-PIs, CCR Director, MMMC Steering Committee (Drs.
Anver, Bedigian, Deng,\
Green, Hunter, Ried and Shih), and Faculties (Breast Cancer:
Dr. Vonderhaar; Metastasis: Dr. Hunter; Prevention: Dr. Colburn;
Molecular Targets: Drs. Barrett and Steeg). Dr. Green would like to
enlarge the Steering Committee and he invited MMMC members to notify him
of their interest, or to recommend another member, in serving in this
capacity.

**Update on CCR initiatives for animal models/siRNA:**

Dr. Green explained that Natasha Caplen is negotiating a cooperative
research and development agreement (CRADA) with a company to produce
siRNA for genes of interest to the NCI. A list of the proposed siRNA
genes is available on the MMMC Web site; MMMC members should contact Dr.
Caplen (<ncaplen2@mail.nih.gov>) with any additional genes they feel
should be added to the list.

Dr. Green also reminded the Collective that Xenogen will be presenting a
seminar on the company's *in vivo* biophotonic imaging system on October
10 in Building 31.

Dr. Green introduced Dr. Lyuba Varticovski to provide an update on the
Mouse Mammary Tumor Project.

### Preclinical mouse models

Dr. Varticovski presented preliminary negotiations with Regeneron that
will provide the NCI with access to a high-throughput technology
system--Velocigene--to introduce genetic mutations into mice. This
innovative technology uses BAC for homologous recombination. Depending
on the availability of funding, Regeneron could produce up to 115 mouse
strains for NCI next year. This project is an NCI-wide initiative; Dr.
Varticovski is also exploring a CRADA between Regeneron and CCR for
introducing conditional mutants and RNAi. Dr. Varticovski invited MMMC
members to provide suggestions and participate in the project.

Dr. Varticovski explained why the development of appropriate preclinical
mouse models is necessary for testing and improving new drug therapies.
Current tumor xenograft models are heterogeneous, and the responses are
poorly characterized. Tumor-bearing genetically engineered mice offer "a
fresh start," and a variety of genetically modified mouse strains are
available for testing. If successful, these would provide homogenous
cohorts of mice for drug therapy testing. Both tumor explants and tumor
cell suspensions could be transplanted. Dr. Varticovski explained that
cell suspensions are preferable because the tumor cells would be more
uniformly distributed. A cell suspension can also be frozen and
used/analyzed later; the cells can be implanted both orthotopically and
orthopically.

Dr. Varticovski summarized a pilot project she drafted in collaboration
with MMMC members to study the feasibility of transplanting cell
suspensions. In collaboration with Melinda Hollingshead at
NCI--Frederick, several representative tumor types have been implanted
in the mammary glands of both syngeneic and nude mouse strains.
Preliminary results indicate that cell suspensions do generate tumors in
the implanted mice after approximately 3 weeks post implantation. These
tumors will be analyzed with a comprehensive array of molecular
techniques.

Dr. Varticovski concluded by inviting MMMC members to provide
suggestions and take an active part in this exciting project.

### Overview of MMMC-Interfaculty Retreat.

Dr. Green explained that the MMMC Steering Committee is planning a
one-day retreat to be held December 8 at the Washingtonian Marriott
Hotel in Gaithersburg, MD. The proposed format will include abstracts,
short presentations, posters, and a discussion session.

Dr. Green has begun soliciting one-page abstracts from all members of
the MMMC on work related to the MMMC mission (i.e., mouse models,
mammary cancer, genomics, metastases, therapeutics, etc.). Members who
received supplemental funding during the first phase of the MMMC are
required to submit an abstract of their work; members who have submitted
a proposal as part of the second phase (renewal grant) are required to
submit an abstract outlining their proposed projects. Abstracts should
be sent to Valerie Sanders (sanderva@mail.nih.gov) as soon as possible.

Based on abstracts received, participants will be selected to give short
oral presentations. Each Core (Imaging, Proteomics, Transgenic/Knockout,
Histopathology, Chromosome Aberrations, Microarray, Informatics, and
Technology Transfer) and Faculty (Breast Cancer, Metastases, Prevention,
and Molecular Targets) will present a poster. The retreat will end with
a discussion forum to develop new ideas and direct the future activities
of the Collective.

Audience response to the retreat was overwhelmingly positive. Barbara
Vonderhaar cautioned that many investigators are "meetinged out," and
effort will need to be made to ensure a high level of attendance.
Faculty leaders should encourage their staffs to participate. JoAnne
Zujewski volunteered to assist by persuading clinical colleagues to
attend the retreat. Lalage Wakefield suggested holding the discussion
session in the forum, as this will introduce participants' work and
facilitate interactions at subsequent sessions. She also suggested using
the retreat to focus on what was "unique" about the MMMC, as well as to
articulate what the Collective can do both independently and in
conjunction with the Faculties.

##### SEMINAR:

## Can Dysregulated Expression of Estrogen-Receptor Alpha Initiate the Development of Breast Cancer?---Priscilla Furth, Professor of Oncology, Lombardi Cancer Center, Georgetown University.

Dr. Green introduced the speaker, Priscilla Furth. Dr. Furth is
currently working at the Lombardi Cancer Center at Georgetown
University. She has studied breast cancer for several years and has made
important contributions to the field. She is developing models of breast
cancer and investigating the temporal control of oncogene expression
using inducible systems.

Dr. Furth explained that while high levels of estrogen-receptor alpha
(ERα) represent one of several factors shown to increase the risk of
breast cancer, it is not known whether dysregulated expression of ERα
directly contributes to the development of breast cancer or whether
ERα requires an oncogene or coactivator to do so. To explore this
question, Dr. Furth developed several transgenic mouse models using the
tetracycline-dependent transactivator protein (tTA) and the mouse
mammary virus long-term repeat (MMTV-LTR) to conditionally induce and
deinduce the overexpression of ERα in the mammary epithelium. Phenotypes
were assessed at 4 and 12 months, and the mice were given exogenous
17β-estradiol to increase levels of circulating estrogen and
downregulate endogenous ERα.

Dr. Furth briefly described three tTA-MMTV transgenic models and their
phenotypes. One provides homogeneous, inducible expression of ERα in
mammary epithelial cells of transgenic mice. Dr. Furth indicated that at
4 months, the level of ERα expression in female transgenic mice was
increased about twofold; exogenous 17β-estradiol administration had no
effect on the expression of transgenic ERαIncreased levels of
ERαresulted in i downstream genes, such as
the progesterone receptors A and B, as well as a dramatic increase in
mammary epithelial cell proliferation. The mammary glands of
ERα−e transgenic mice developed double-layer epithelia and
lobular structures resembling human mammary glands. Ductal development
was abnormal, with incomplete filling of the fat pad and aberrant ductal
branching. The window of developmental vulnerability to ERα lasts just
through puberty, because no phenotype develops when ERα is turned off at
3 weeks of age after exposure through *in utero* development and through
the time of development up to weaning at 3 weeks of age.

Dr. Furth explained that the most interesting phenotype seen in the
transgenic mice is ductal hyperplasia because of its relevance to human
breast cancer. More than four layers of mammary epithelia have been
shown to increase the risk of developing carcinoma in humans. The
lesions in the 4-month-old transgenic mice are pathologically similar to
human ductal hyperplasia, with heterogenous, highly proliferative cells
and fenestrations. Cyclin D1 is increased in the ductal hyperplasia
sites. Turning off ERα expression did not cause the ductal hyperplasia
to regress; this result is consistent with human breast cancer data
suggesting that an alteration in estrogen signaling leaves a "footprint"
that may be sufficient to confer risk of developing cancer.

Preliminary results indicate that although 12-month-old transgenic mice
had no palpable tumors, they had hyperplastic alveolar nodules. In
addition, histopathological examination identified mitotic figures
within the epithelial cell structures that were not present at 4 months
of age.

Dr. Furth explained her approach to studying ERα dysregulation in
conjunction with oncogenes. Her laboratory has available a transgenic
MMTV T-Antigen mouse model that targets T-Antigen and ERα to the same
cells. These transgenic mice developed mammary gland tumors that
mimicked some of the characteristics of human breast cancer, including
invasion of the surrounding stroma by the tumor cells and desmoplasia.

Dr. Furth concluded her presentation by summarizing a collaborative
effort with Dr. Anna Reigel at the Lombardi Cancer Center on the
interaction between ERα and the steroid receptor coactivator AIB1
(*Amplified in Breast Cancer 1*). Preliminary results indicate that
dominant gain experiments utilizing transgenic AIB1 mice demonstrated a
phenotype reminiscent of ERα mice, as would be expected if both models
acted through the same pathway.

**ATTENDANCE:**

Miriam Anver

Hendrick Bedigian

Chuxia Deng

Kartiki Desai

Bob Dickson

Lionel Feigenbaum

Jeff Green

Jung-Im Huh

Kent Hunter

Steven Hursting

Sonia Jakowlew

Aleksandra Michalowska

Cristina Montagna

Tri Nguyen

Tinghu Qiu

Sushil Rane

Thomas Ried

Valerie Sanders

Holly Simons Clark

Lyuba Varticovski

Barbara Vonderhaar

Lalake Wakefield

Ling Zhao

JoAnne Zujewski

######## NOVA Research Company

Petra Delgado

Mel Simpson