Summary
Single transgenic TGFa and c-Myc mice were mated to yield double transgenic TGFa/c-Myc
mice. All of these developed synchronous mammary tumors at a mean age of 66 days. An
unexpected finding was that tumor latency and frequency in males and virgin females was
identical. Although approximately one half of single transgenic c-Myc virgin females also
developed mammary gland tumors, these were stochastic and arose after a long latency
period of 9-12 months. Single transgenic virgin TGFa females and males, c-Myc males and
transgene negative littermates did not develop tumors (ages up to 15 months). In addition,
the salivary glands of double transgenic animals developed pathological abnormalities
ranging from hyperplasias to adenocarcinomas. Taken together, TGFa and c-Myc synergize in
an extremely powerful way to cause mammary gland tumorigenesis in males and virgin females
without a requirement for pregnancies.
Citation
Amundadottir et al., 1995. Cell Growth and Differentiation, 6:737-748.
Background
TGFa and c-Myc are implicated in human breast cancer by either gene amplification and/or
overexpression. The c-myc protooncogene is amplified in about one third of breast cancer
cases and overexpressed (without gene amplification) in many more. Although the TGFa gene
is not found amplified in human breast cancer, its expression (and that of other EGF
family members) is frequently increased compared to the normal gland. In addition, in
vitro studies have shown that c-Myc overexpression results in an increased responsiveness
to the effects of mitogenic growth factors including TGFa. High levels of Myc may
therefore permit a tumorigenic transformation by a TGFa/EGFR autocrine growth mechanism,
or it may sensitize cells to such a mechanism. Based on the in vitro collaboration between
TGFa and c-Myc and their importance in human breast cancer we decided to test if the two
collaborated in vivo in mouse mammary gland tumorigenesis. .
Transgene
A human TGFa cDNA driven by the mouse metallothionein 1 promoter and a mouse c-myc cDNA
driven by the MMTV LTR promoter/enhancer
mouse strain
FVB/N (TGFa, Jhappan et al., 1990. Cell, 61:1137-1146) and CD-1xC57BL/6J (c-Myc, Stewart
et al., 1984. Cell, 38:627-637)
All (20/20) double transgenic females and males developed palpable mammary gland tumors a mean age of 66 +/- 12 days. In addition, pathological diagnoses of haematoxylin/eosin stained sections revealed the presence of adenocarcinomas in glands without frank palpable tumors, thus showing that every mammary gland from double transgenic animals was cancerous. Surprisingly, no normal tissue was found adjacent to mammary gland tumors in double transgenic animals, so that the whole glands could be characterized as malignant. Younger TGFa/c-Myc animals (3-5 week old) were also diagnosed as having mammary gland adenocarcinoma by histopathology and tissue transplantation into nude mice. Together, the data suggest that in our model, overexpression of TGFa and c-Myc is sufficient to cause a complete tumorigenic transformation of the mouse mammary gland. A cooperation was also noted in the salivary glands of double transgenic animals although tumor formation was stochastic in this organ.
Histology
Hematoxylin/eosine stained sections of inguinal mammary glands from 3 month old animals.
The normal virgin gland is representative of TGFa and c-Myc single transgenic animals and
of nontransgenic littermates at this age. Note that the gland consists mostly of adipose
cells with a few scattered epithelial ducts. The transformed virgin mammary gland is from
a tumor arising in a double transgenic TGFa/c-Myc animal at three months of age. Observe
that tumor cells fill the whole gland and the absence of adipose tissue. Mammary gland
tumors in TGFa/c-Myc animals were classified as Adenocarcinomas of types A and B.
Mammary development
Mammary gland whole mounts from a wild type and double transgenic TGFa/c-Myc animals
reveal the epithelial network and the lymph node (round structure in middle of gland) in
these developing mammary glands.
Gene Expression
In situ hybridization was used to assess transgene expression in tumors arising in double
transgenic animals (image). Panel (a) shows a brightfield photograph of a control section
hybridized to a sense riboprobe (image). Panel
(b) shows a brightfield photograph of a section hybridized to a c-myc antisense riboprobe
(image). Panel (c) shows a brightfield photograph
of a tumor section hybridized to a tgfa antisense riboprobe (image). Note uniform expression of both transgenes in
a mammary gland tumor from this 3 week old double transgenic animal. In situ hybridization
performed on salivary glands revealed that transgene expression was "patchy" and
that hyperplastic and tumorous areas colocalized with regions that overexpressed both the
tgfa and c-myc transgenes (not shown). This indicates that the presence of both proteins
is required for transformation.
Mechanistic Implications
TGFa and c-Myc are powerful collaborating genes in mouse mammary and salivary gland
tumorigenesis. It appears that additional events are not required for tumorigenesis in the
mammary gland since the entire glands of double trangenic TGFa/c-Myc animals look
transformed by histological appearance and can readily grow when transplanted into nude
mice.
key words
c-myc, TGF-alpha, double transgenic mice
Submitted by: Laufey T. Amundadottir and Robert B. Dickson on April 30, 1996
Laufey Amundadottir Department of Genetics Harvard Medical School 200 Longwood Avenue Boston, MA 02115 Tel: (617) 432-7553 Fax: (617) 432-7663 Jeff Torri and Robert Dickson Georgetown University Medical Center Room W-17B,TRB Georgetown University 3970 Reservoir Road Washington, D.C 20007 Tel: (202) 687-3770 Fax: (202) 687-7505
For further information Contact:
Laufey Amundadottir
e-mail: amundadottir@rascal.med.harvard.edu
contributed May 1996
last update: December 1998
