Introduction
BRCA1- and
BRCA2-associated tumors have many morphologic features in common. These include ductal histology, high histologic grade, pushing tumor margins and a notable host lymphocytic response [
1,
2]. Despite these similarities they have distinct molecular signatures;
BRCA1-associated tumors have been shown to be predominantly estrogen receptor negative and to have a basal phenotype [
3‐
5], whereas we have shown previously that
BRCA2-associated tumors have a luminal phenotype characterized by the expression of estrogen receptor and luminal-type cytokeratins [
1]. These two molecular signatures reflect in part the two cell types, basal/myoepithelial and luminal found in the terminal duct lobular unit of the normal breast.
Basal-like human breast cancers have been shown to exhibit aberrations in the epidermal growth factor receptor (EGFR) signaling pathway [
6]. With the exception of Cyclin D1, genes responsible for promoting the growth and survival of
BRCA2-associated cancers have yet to be identified [
7]. Our objective in performing global gene expression analysis on
BRCA1- and
BRCA2-associated tumors was to distinguish these distinct pathways of carcinogenesis and to elucidate novel genes necessary for the transformation and survival of
BRCA2-associated tumors.
Discussion
In recent years, gene expression profiling of breast cancers has improved our understanding of the heterogeneity of the disease and generated hypotheses concerning the development and progression of these cancers. Molecular signatures for
BRCA1-associated tumors have been delineated, but
BRCA2-associated tumors have been less well studied [
5,
7]. To elucidate novel genes involved in the development and progression of
BRCA2-associated tumors and to distinguish distinct pathways of carcinogenesis in
BRCA1 and
BRCA2-associated tumors, we compared gene expression microarray patterns in tumors from
BRCA1 and
BRCA2 carriers.
We found that FGFR2 and FGF1 were more highly expressed in
BRCA2-associated cancers as compared to
BRCA1-associated breast cancers, suggesting the presence of an autocrine growth stimulatory loop. FGF1 and FGFR2 belong to a large family of ligands and receptor tyrosine kinases [
24]. FGF1 is a mitogen that signals through FGFRs and subsequently activates the MAPK signaling cascade. Overexpression of FGF1 in breast cancer cell lines has been shown to result in increased anchorage independent growth and reduced requirement for estrogen in vitro and to increased tumorigenicity, angiogenesis and metastatic behaviour in vivo [
25,
26].
FGF1 is located at 5q31 a region of the genome often subject to loss in
BRCA1-associated cancers [
27] and basal-like cancers [
28].
FGFR2, located at 10q26, encodes at least two receptor isoforms FGFR2-IIIb and FGFR2-IIIc. FGFR2-IIIc is mainly expressed in tissues of mesenchymal origin, whereas FGFR2-IIIb is expressed in the epithelium of many organs including the mammary gland [
29,
30]. The FGFRs in general and FGFR2 specifically have roles in embryogenesis, development and carcinogenesis. FGFR2 mRNA is expressed in many carcinoma cell lines [
31‐
33] and in the breast cancer cell lines examined, FGFR2 expression contributes to their invasive phenotype. Similarly, overexpression of the receptor in normal human mammary epithelial (HME) cells leads to their transformation [
34]. These properties are attributable to the activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling cascades [
34]. Moreover, the gene locus (10q26) is amplified in 2–10% of breast cancers and its expression has been reported in 50–100% of human breast cancers, with high-level expression confined to 4%–12% of cases [
35,
36]. It has also been associated with estrogen (ER) and progesterone (PR) receptor expression and improved overall and disease free survival [
35]. Moreover in two recent independent genome wide association studies (GWAS) single nucleotide polymorphisms (SNPs) within intron 2 of FGFR2 have been causally associated with increased risk of both familial and sporadic breast cancer [
37,
38]. Furthermore this susceptibility locus was associated with younger age of onset and bilateral disease [
37]. The authors speculate that the SNPs have functional effects and that the association with breast cancer risk is mediated through regulation of FGFR2 expression possibly through interaction with the ER. In our study we have shown that FGFR2 expression is positively associated with the
BRCA2 genotype, a tumor group that we have previously shown to be predominantly ER-positive luminal-type tumors [
1]. In addition, we found that FGFR2 expression was positively associated with PR expression, a weak prognostic and predictive factor in breast cancer and often regarded as indicative of a functional ER pathway [
39]. Conversely, FGFR2 expression was negatively correlated with basal-like breast cancers which are known to be ER negative and to carry an adverse prognosis [
5,
6].
Our expression studies support previous reports indicating that
BRCA1-associated breast cancers have a basal-like profile [
3,
5]. In the
BRCA1-associated tumor group, we detected elevated expression of a number of genes, including keratin 17 [
5,
6], vimentin, and caveolin 1 [
20], that have previously been linked to the basal-like cancers. Our results also suggest that Y-box binding protein-1 is more highly expressed in
BRCA1-associated breast cancers (
P = 0.001). This protein has recently been demonstrated to transcriptionally induce EGFR which is commonly overexpressed in basal-like cancers [
40]. In addition we found that stathmin/oncoprotein 18 and osteopontin were overexpressed in
BRCA1-associated tumors relative to
BRCA2-associated tumors. The gene for stathmin, located at 1p36, is one of the 70 genes that compose the ‘70 gene classifier’ that predicts poor prognosis in sporadic breast cancer [
41] a classifier that correlates closely with the poor performing subgroups from the intrinsic gene set of which basal-like cancers are one [
42]. Stathmin is a microtubule depolymerizing protein involved in cell cycle progression and cell motility. It is highly expressed in a number of human malignancies, including breast cancers where it is negatively correlated with ER expression and positively correlated with grade, aneuploidy, proliferation and mutant p53 [
43], all characteristic features of
BRCA1-associated and basal-like cancers [
3,
5,
6].
Osteopontin, a secreted phosphoprotein, has been shown to interact with a diverse range of factors including integrins, CD44, TGFα, EGFR, Met and VEGF leading to the enhancement of cellular migration, invasion, survival and angiogenesis. [
44‐
46]. Osteopontin is expressed in a wide variety of human malignancies and its expression has been reported to be correlated with poor prognosis in breast cancer [
47].
Novel results from our expression study include the identification of members of the Notch and TGFβ signaling pathways, Jagged 1 and TGFβ2 respectively, as being more highly expressed in
BRCA1-associated tumors. Both of these pathways are highly conserved through evolution and play important roles in development, differentiation and tumorigenesis [
48,
49]. Moreover, both pathways are known to play a role in mammary stem cell maintenance or renewal [
50,
51]. Dontu et al. have shown that Notch signaling can act on mammary stem cells to promote self-renewal, on early progenitor cells to promote proliferation, and on multipotent progenitor cells, facilitating myoepithelial cell lineage specific commitment and proliferation [
50]. Furthermore, patients whose breast cancers express Jagged 1 have been found to have a poorer prognosis [
17]. Shipitsin and colleagues showed that TGFβ signaling is upregulated in normal breast stem cells and their malignant counterparts and has prognostic effects [
51].
In conclusion, our molecular profiling demonstrates that BRCA1-associated and BRCA2-associated tumors have distinct molecular profiles. Our results confirm the known link between BRCA1-associated tumors and the basal-like signature and highlight a link between the Notch and TGFβ pathways and BRCA1-associated tumorigenesis. In contrast the data suggest that BRCA2-associated tumors express higher levels of FGFR2 and FGF1, suggesting the existence of an autocrine loop leading to downstream pleiotrophic cellular effects.