The estrogen receptor alpha (ERa) plays a critical role in the pathogenesis and clinical behavior of breast cancer. To obtain further insights into the molecular basis of estrogen-dependent forms of this malignancy, we used real-time quantitative reverse transcription (RT)-PCR to compare the mRNA expression of 560 selected genes in ERa-positive and ERa-negative breast tumors. Fifty-one (9.1%) of the 560 genes were significantly upregulated in ERa-positive breast tumors compared with ERa-negative breast tumors. In addition to well-known ERa-induced genes (PGR, TFF1/PS2, BCL2, ERBB4, CCND1, etc.) and genes recently identified by cDNA microarray-based approaches (GATA3, TFF3, MYB, STC2, HPN/HEPSIN, FOXA1, XBP1, SLC39A6/LIV-1, etc.), an appreciable number of novel genes were identified, many of, which were weakly expressed. This validates the use of large-scale real-time RT-PCR as a method complementary to cDNA microarrays for molecular tumor profiling. Most of the new genes identified here encoded secreted proteins (SEMA3B and CLU), growth factors (BDNF, FGF2 and EGF), growth factor receptors (IL6ST, PTPRT, RET, VEGFR1 and FGFR2) or metabolic enzymes (CYP2B6, CA12, ACADSB, NAT1, LRBA, SLC7A2 and SULT2B1). Importantly, we also identified a large number of genes encoding proteins with either pro-apoptotic (PUMA, NOXA and TATP73) or anti-apoptotic properties (BCL2, DNTP73 and TRAILR3). Surprisingly, only a small proportion of the 51 genes identified in breast tumor biopsy specimens were confirmed to be ERa-regulated and/or E2-regulated in vitro (cultured cell lines). Therefore, this study identified a limited number of genes and signaling pathways, which better delineate the role of ERa in breast cancer. Some of the genes identified here could be useful for diagnosis or for predicting endocrine responsiveness, and could form the basis for novel therapeutic strategies.

Recent data suggest that chemokines could be essential players in breast carcinogenesis. We previously showed that the CXC chemokine CXCL8 (interleukin-8) was overexpressed in estrogen receptor a (ERa)-negative breast cell lines. Analysis of CXCL8 chromosomal location showed that several CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL4, CXCL4V1, CXCL5, CXCL6, CXCL7, and CXCL8) were localized in the same narrow region (360 kb in size) of chromosome 4. We thus hypothesized that they could belong to the same cluster. Quantification of these chemokines in breast tumors showed that samples expressing high CXCL8 also produced elevated levels of CXCL1, CXCL3, and CXCL5, and displayed low content of ERa. CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8 were co-regulated both in tumors and in breast cancer cell lines. CXCL5 and CXCL8 were mainly produced by epithelial cells, whereas CXCL1, CXCL2, and CXCL3 had a high expression in blood cells. The overexpression of these chemokines in tumor cells was not the result of gene amplification, but rather of an enhanced gene transcription. Our data suggest that high CXCL8 expression in tumors is mainly correlated to activating protein-1 (AP-1) pathway and to a minor extent to NF-kB pathway. Interestingly, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 chemokines were present at higher levels in metastases when compared with grade I and III biopsies. High levels of CXCL8, CXCL1, and CXCL3 accounted for a shorter relapse-free survival of ERa-positive patients treated with tamoxifen. In summary, we present evidences that multiple CXC chemokines are co-expressed in CXCL8-positive breast tumors. In addition, these chemokines could account for the higher aggressiveness of these types of tumors.

IntroductionPIK3CA is the oncogene showing the highest frequency of gain-of-function mutations in breast cancer, but the prognostic value of PIK3CA mutation status is controversial.MethodsWe investigated the prognostic significance of PIK3CA mutation status in a series of 452 patients with unilateral invasive primary breast cancer and known long-term outcome (median follow-up 10 years).ResultsPIK3CA mutations were identified in 151 tumors (33.4%). The frequency of PIK3CA mutations differed markedly according to hormone receptor (estrogen receptor alpha [ERα] and progesterone receptor [PR]) and ERBB2 status, ranging from 12.5% in the triple-negative subgroup (ER-/PR-/ERBB2-) to 41.1% in the HR+/ERBB2- subgroup. PIK3CA mutation was associated with significantly longer metastasis-free survival in the overall population (P = 0.0056), and especially in the PR-positive and ERBB2-positive subgroups. In Cox multivariate regression analysis, the prognostic significance of PIK3CA mutation status persisted only in the ERBB2-positive subgroup.ConclusionsThis study confirms the high prevalence of PIK3CA mutations in breast cancer. PIK3CA mutation is an emerging tumor marker which might become used in treatment-choosing process. The independent prognostic value of PIK3CA mutation status in ERBB2-positive breast cancer patients should be now confirmed in larger series of patients included in randomized prospective ERBB2-based clinical trials.

Background: IBC (Inflammatory Breast cancer) is a rare form of breast cancer with a particular phenotype. New molecular targets are needed to improve the treatment of this rapidly fatal disease. Given the role of NF-κB-related genes in cell proliferation, invasiveness, angiogenesis and inflammation, we postulated that they might be deregulated in IBC.

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Our retrospective analysis has identified protease expression and tumor cell proliferation rate as important biological prognostic factors in breast cancer. Prospective clinical trials should be undertaken to confirm these results.

BackgroundThe PI3K/AKT pathway plays a pivotal role in breast cancer development and maintenance. PIK3CA, encoding the PI3K catalytic subunit, is the oncogene exhibiting a high frequency of gain-of-function mutations leading to PI3K/AKT pathway activation in breast cancer. PIK3CA mutations have been observed in 30% to 40% of ERα-positive breast tumors. However the physiopathological role of PIK3CA mutations in breast tumorigenesis remains largely unclear.Methodology/Principal FindingsTo identify relevant downstream target genes and signaling activated by aberrant PI3K/AKT pathway in breast tumors, we first analyzed gene expression with a pangenomic oligonucleotide microarray in a series of 43 ERα-positive tumors with and without PIK3CA mutations. Genes of interest were then investigated in 249 ERα-positive breast tumors by real-time quantitative RT-PCR. A robust collection of 19 genes was found to be differently expressed in PIK3CA-mutated tumors. PIK3CA mutations were associated with over-expression of several genes involved in the Wnt signaling pathway (WNT5A, TCF7L2, MSX2, TNFRSF11B), regulation of gene transcription (SEC14L2, MSX2, TFAP2B, NRIP3) and metal ion binding (CYP4Z1, CYP4Z2P, SLC40A1, LTF, LIMCH1).Conclusion/SignificanceThis new gene set should help to understand the behavior of PIK3CA-mutated cancers and detailed knowledge of Wnt signaling activation could lead to novel therapeutic strategies.

Dok1 is an abundant Ras-GTPase-activating protein-associated tyrosine kinase substrate that negatively regulates cell growth and promotes migration. We now find that I B kinase ␤ (IKK␤) associated with and phosphorylated Dok1 in human epithelial cells and B lymphocytes. IKK␤ phosphorylation of Dok1 depended on Dok1 S439, S443, S446, and S450. Recombinant IKK␤ also phosphorylated Dok1 or Dok1 amino acids 430 -481 in vitro. TNF-␣, IL-1, ␥ radiation, or IKK␤ overexpression phosphorylated Dok1 S443, S446, and S450 in vivo, as detected with Dok1 phospho-S site-specific antisera. Moreover, Dok1 with S 439, S443, S446, and S450 mutated to A was not phosphorylated by IKK␤ in vivo. Surprisingly, mutant Dok1 A439, A 443, A446, and A450 differed from wild-type Dok1 in not inhibiting platelet-derived growth factor-induced extracellular signal-regulated kinase 1͞2 phosphorylation or cell growth. Mutant Dok1 A 439, A443, A446, and A450 also did not promote cell motility, whereas wild-type Dok1 promoted cell motility, and Dok1 E439, E443, E446, and E450 further enhanced cell motility. These data indicate that IKK␤ phosphorylates Dok1 S439S443 and S446S450 after TNF-␣, IL-1, or ␥-radiation and implicate the critical Dok1 serines in Dok1 effects after tyrosine kinase activation.NF-B ͉ serine phosphorylation ͉ cell migration D ok1 or p62 dok is an abundant Ras-GTPase-activating protein-associated adaptor protein that is downstream of growth factor receptor and nonreceptor tyrosine k inases (1, 2). Related proteins include Dok2 (also known as FRIP or Dok-R), Dok3 (also known as Dok-L), Dok4, Dok5, and insulin receptor substrates (3-8). DOK proteins have an N-terminal pleckstrin homology domain, a phosphotyrosine-binding (PTB) domain, and a C terminus rich in proline, serine, and tyrosine (1, 2). The pleckstrin homology domain mediates association with membrane phospholipids, whereas the PTB domain mediates homodimerization and association with phosphotyrosine signaling molecules (9, 10). When it is tyrosine-phosphorylated, Dok1 interacts with other signaling molecules containing Src homology 2 domains, such as Ras-GTPase-activating protein, SHIP1, Nck, Csk, and SH2D1A (11-15).DOK proteins down-modulate tyrosine kinase signaling effects. Dok1 can inhibit mitogen-activated protein kinase activation, cell proliferation, cell transformation, and leukemogenesis (9,10,(16)(17)(18). Dok1 can also affect cell adhesion, spreading, migration, and apoptosis (13,19,20), and modulate T or B cell receptor signaling (18,(21)(22)(23)(24). Pleckstrin homology domainmediated plasma membrane translocation and tyrosine phosphorylation are critical for these Dok1 effects (10, 25).After TNF-␣, IL-1, or Toll receptor signaling, ␥ radiation, or tyrosine kinase signaling, I B kinase ␤ (IKK␤) phosphorylates I B␣ S 32 S 36 , resulting in I B␣ degradation, NF-B activation, and increased transcription of genes important for costimulatory and survival effects (for reviews see refs. 15 and 26-37). Dok1 could have a role in the physiologic integration of tyrosin...