ErbB2 signaling in breast cancer : the role of ErbB, Akt and ShcA phosphorylation

Breast cancer is the most common malignancy in women and is estimated to account for
more than 200,000 new cancer cases in the United States in the year 2002. It now
represents the second leading cause of death (40,000) from cancer in women. Although
the number of new breast cancer cases has been increasing, the death rate has been
steadily decreasing. This trend may be due to earlier diagnosis, and/or increased survival
resulting from the use of adjuvant therapy.
Clinical outcome is affected by prognostic predictive factors. Prognostic factors are
associated with either the metastatic or the growth potential of the primary tumor, while
predictive factors are associated with the relative sensitivity and/or resistance to specific
therapies. Routinely available prognostic indicators include tumor size, type, and grade,
axillary lymph node status, estrogen and progesterone receptor status. Estrogen receptor
status and progesterone receptor status also serve as predictive factors for expected
response to hormone therapy. Many other molecular markers are being investigated for
their clinical usefulness. One of the major molecular prognostic and predictive markers in
breast cancer is the amplification status of the proto-oncogene ErbB2 (HER-2/ neu).
The ErbB2 proto-oncogene is a component of a four-member family of closely related
growth factor receptors that includes the epidermal growth factor receptor
(ErbB1/HER1), ErbB3 (HER3), and ErbB4 (HER4). The human gene is located on
chromosome 17q21 and encodes a 185-kDa protein with tyrosine kinase activity that is
also known by the designation p185. Structurally, the protein has extracellular,
transmembrane, and a cytoplasmic domain, the latter of which contains the tyrosine
kinase domain and shares significant homology, although is distinct, from EGFR. Under
normal circumstances, low levels of ErbB2 expression are detectable
immunohistochemically in a variety of fetal and adult epithelial cells throughout the
gastrointestinal, respiratory, and genitourinary tracts. Amplification of the ErbB2 protooncogene
or overexpression of the p185 protein, which generally correlate with each
other, has been identified in 10% to 34% of breast cancers as well as in gastrointestinal,
pulmonary, and genitourinary tumors. The mechanism by which overexpressed ErbB2
leads to a neoplastic phenotype occurs by activation of several different signaling
pathways that lead to gene activation, ultimately resulting in cell proliferation. Although
the mechanism of activation of ErbB2 has not been completely elucidated, it is thought to
involve the formation of heterodimers with other members of the epidermal growth factor
family of receptors or spontaneous homodimerization.
This study was designed to compare the prognostic value of phosphorylated ErbB2 in
well characterized primary breast cancer samples. Seventy primary breast cancers with a
median of 45 months of follow-up were analyzed for quantitative levels of
phosphorylated ErbB2 using new sensitive chemiluminescence-linked immunoassay
(CLISA). Phosphorylated ErbB2 data were compared with clinical, histological and
outcome variables as well as quantitative mRNA and protein expression levels of ErbB
family members. ErbB2 – overexpressing tumors contained significantly more
phosphorylated ErbB2, however PY1248 could be detected in some of low ErbB2
expression tumors. ErbB2 phosphorylation was correlated with disease free and overall
survival and reduced estrogen receptor and progesterone receptor contents. Comparison
of ErbB family expression on mRNA level with ErbB2 phosphorylation revealed
significant correlation with ErbB2 and EGFR but inverse correlation with ErbB3 and
ErbB4. Similar correlations were found also with respect to protein expression levels of
these factors.
We have also investigated total (pan) tyrosine, serine and threonine phosphorylated
ErbB2 in 153 breast cancer samples by two-site CLISA assays. Serine and threonine
phosphorylated ErbB2 could be detected only in low ErbB2 – expressing tumors, no
serine and threonine phosphorylation was detectable in ErbB2 overexpressing tumors. As
in case of PY1248, ErbB2 – overexpressing tumors contained significantly more tyrosine
phosphorylated ErbB2, but ErbB2 tyrosine phosphorylation was detectable in some of
low ErbB2 expression tumors as well. Due to the fact that tumors we selected for this
study were mostly aggressive tumors, it was impossible to analyze the prognostic value
of ErbB2 phosphorylations.
Akt1, Akt2 and Akt3 kinases are involved in the signal transduction pathway downstream
of receptor tyrosine kinases via phosphoinosytol-3-kinase, influencing cell growth,
proliferation and survival. Akt2 overexpression and amplification have been described in
breast, ovarian and pancreatic cancers. In this study we measured the quantitative
expression levels of total phosphorylated (P-S473) Akt (Akt1/2/3) by means of a two-site
CLISA on cytosol extracts obtained from 156 primary breast cancer tissue samples. We
aimed to clarify the prognostic significance of activated Akt in primary breast cancer in
association with other tumor biomarkers. Akt phosphorylation was not associated with
the nodal status and the ErbB2 expression. Only very high expression levels of P-Akt
correlated with poor prognosis. More importantly, the prognostic value of P-Akt
expression increased in ErbB2 overexpressing subset of patients. In addition, P-Akt was
found to be associated with mRNA expression levels of several proliferation markers,
such as thymidylate synthase, thymidine kinase 1, survivin, topoisomerase II alpha and
transcription factor E2F, measured by quantitative real-time PCR (Q-RT-PCR).
Shc adapter/docking proteins are an important component of receptor tyrosine kinase
signaling pathways because they are involved in transducing the activation signals from
receptor or cytoplasmic tyrosine kinases to downstream signaling cascades. At least three
genes, shcA, shcB, and shcC, are known to encode Shc proteins. ShcA has been found to
be phosphorylated rapidly and efficiently by all tyrosine kinases tested to date. These
phosphorylation sites have been mapped to Y339, Y240, and Y317. In addition to
tyrosine phosphorylation, ShcA can also be phosphorylated at serine/threonine residues.
We have investigated pan- tyrosine, serine and threonine phosphorylated ShcA in 153
breast cancer samples by two-site CLISA assays. P-ShcA was found to be weekly
associated with PT ErbB2 levels and weekly inversely correlated with P-Akt levels. A
very good correlation was found between PS ShcA and PY SchA.
Since it was the same collective of tumors, as the one used for ErbB2 pan-S, T and Y
phosphorylation assessment, it was also impossible to analyze the prognostic value of
phospho-SchA.