Akt1 and ERB2 Activity Is Associated with Tumor Growth Inhibition Camille Alfonso, Department of Hum

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Akt1 and ERB2 Activity Is Associated with Tumor
Growth Inhibition Camille Alfonso, Department of
Human Science, Adriana Stoica, Ph.D., Department
of Human Science, School of Nursing and Health
Studies and Department of Oncology ,Georgetown
University.
Introduction Breast cancer is the second
leading cause of cancer death in American women,
and the rate of incidence is increasing rather
rapidly. While there are some known risk factors
for breast cancer, varying from family history to
hormone replacement therapy, the vast majority of
women with breast cancer have no known risks.
Estrogen is the single most important etiological
factor in breast cancer. The anti-estrogen
tamoxifen (Tam) can repress or activate
transcription of estrogen target genes. However,
its agonistic effects can lead to breast tumor
growth.
Results
Effect of ErbB2 and Akt In Vivo Growth of MCF-7
Cells
Abstract MCF-7 has been used for many years as
a model cell line in order to investigate
hormonally-responsive breast cancer.
Estrogen-dependent breast tumors have shown the
tendency to regress following the withdrawal of
estrogen. (1) We inoculated MCF-7 cells into
ovariectomized female athymic nude mice, allowing
the formation of tumors after estradiol
supplementation. The mice tissue samples were
then analyzed for tumor growth and Akt, ER-a, PR
expression/ activity through western blots and
immunohistochemistry and the following
conclusions were discovered. Inoculation of
parental MCF-7 cells into ovariectomized female
athymic nude mice led to tumor appearance 4 weeks
after estradiol supplementation and the selective
ErbB2 inhibitor, AG825 blocked this effect.
Tumor volumes were 80 smaller upon inoculation
of MCF- 7/DN-Akt1 cells K179M-Akt1 or R25C-Akt1.
AG825 inhibited the growth of established tumors
by 60 for MCF-7 or MCF-7/myr-Akt1 cells. In
tumors from MCF-7 and MCF-7/myr-Akt1 xenografts,
decreased ER- a expression and increased PR was
observed, effects that were blocked by the ErbB2
inhibitor. In contrast, in tumors from MCF-7/
K179M-Akt1 or MCF-7/ R25C-Akt1 xenografts, ER-a
expression and activity were inhibited. Tumor
extracts from animals inoculated with MCF-7 cells
with an estradiol pellet or myr-Akt1 cells
without estradiol, showed high p-Akt, that was
blocked by AG825, suggesting that loss of Akt1
activity was associated with tumor growth
inhibition. These results suggest that loss of
Akt1 activity and inhibition of ErbB2 was
associated with tumor growth inhibition.
MCF-7cells (parental (A, E, F) or stably
transfected with empty vector, CMV
((MCF-7/CMV-B), K179M-Akt (C, E), R25C-Akt (D,
E), or myr-Akt (F) were injected subcutaneously
into the flanks of 4-5 weeks old female,
ovariectomized nude BALB/c-nu/nu mice. A-E.
Animals were randomly placed into six groups,
receiving an estradiol pellet (E2) in the
presence or absence of tamoxifen or AG825,
tamoxifen, or AG825 alone. A-D. Tumor volumes
were measured twice per week. E. Animals were
sacrificed after 100 days and tumor weight was
measured. F. Tumors were allowed to grow for
further 3 weeks after tumor formation upon E2
supplementation. At this point, animals were
randomly placed in different groups. In one group
E2 was removed, another group was kept with an E2
pellet, and the 3rd group received AG825. Animals
were sacrificed after 100 days and tumor weight
was measured. Representative experiments of three
/- S.D.
Effect of ErbB2 and Akt1 on ER-a Expression and
Activity in Xenograft Tumors
MCF-7 cells (parental or stably transfected with
CMV, myr-Akt or R25C-Akt) were injected
subcutaneously into the flanks of 4-5 weeks old
female, ovariectomized nude BALB/c-nu/nu mice.
Animals were randomly placed into six groups,
receiving an estradiol pellet in the presence or
absence of tamoxifen or AG825, tamoxifen, or
AG825 alone. Mice were sacrificed after 70-100
days. Tumors were kept in 10 formalin until
paraffin sections from each tumor were prepared.
A, B, D. Immunohistochemical (IHC) staining was
performed using anti-ER-a, PR, Akt, or p-Akt
antibodies. The IHC staining intensity was
categorized into - negative, 1 weak, 2
moderate, and 3 strongly positive. The final
score resulted from the product of staining
intensity and percentage of stained cells. C.
Western blot analysis of parental MCF-7 cell
extract from non-treated cells (C) or treated
with E2 or HRG-b1 or tumor extracts from
ovariectomized female nude mice inoculated with
either CMV or myr-Akt1 cells and not receiving
(C) or receiving an E2 pellet (E2) in the
presence or absence of AG825. Representative of 3
immunoblots.
Methods We inoculated MCF-7 cells into
ovariectomized female athymic nude mice, allowing
the formation of tumors after estradiol
supplementation The mice tissue samples were then
analyzed for tumor growth and Akt, ER-a, PR
expression/ activity through western blots and
immunohistochemistry.
Conclusions

• Inoculation of parental MCF-7 cells into
  ovariectomized female athymic nude mice led to
  tumor appearance 4 weeks after estradiol
  supplementation and the selective ErbB2
  inhibitor, AG825 blocked this effect
• Tumor volumes were 80 smaller upon inoculation
  of MCF-7/DN-Akt1 cells K179M-Akt1 or R25C-Akt1
• Ag825 inhibited the growth of established tumors
  by 60 for MCF-7 or MCF-7/myr-Akt1 cells
• In tumors from MCF-7/myr-Akt1 xenografts,
  decreased ER-a expression and increased PR was
  observed, effects that were blocked by the ErbB2
  inhibitor
• In contrast, in tumors from MCF-7/ K179M-Akt1 or
  MCF-7/ R25C-Akt1 xenografts, ER-a expression and
  activity were inhibited
• Tumor extracts from animals inoculated with MCF-7
  cells with an estradiol pellet or myr-Akt1 cells
  without estradiol, showed high p-Akt, that was
  blocked by AG825, suggesting that loss of Akt1
  activity was associated with tumor growth
  inhibition

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