Metabotropic glutamate receptor 1 expression and its polymorphic variants associate with breast cancer phenotypes.

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.

Differentially expressed genes regulating the progression of ductal carcinoma in situ to invasive breast cancer.

Molecular mechanisms mediating the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer remain largely unknown. We used gene expression profiling of human DCIS (n = 53) and invasive breast cancer (n = 51) to discover uniquely expressed genes that may also regulate progression. There were 470 total differentially expressed genes (≥2-fold; P < 0.05). Elevated expression of genes involved in synthesis and organization of extracellular matrix was particularly prominent in the epithelium of invasive breast cancer. The degree of overlap of the genes with nine similar studies in the literature was determined to help prioritize their potential importance, resulting in 74 showing overlap in ≥2 studies (average 3.6 studies/gene; range 2-8 studies). Using hierarchical clustering, the 74-gene profile correctly categorized 96% of samples in this study and 94% of samples from 3 similar independent studies. To study the progression of DCIS to invasive breast cancer in vivo, we introduced human DCIS cell lines engineered to express specific genes into a "mammary intraductal DCIS" xenograft model. Progression of xenografts to invasive breast cancer was dramatically increased by suppressing four genes that were usually elevated in clinical samples of DCIS, including a protease inhibitor (CSTA) and genes involved in cell adhesion and signaling (FAT1, DST, and TMEM45A), strongly suggesting that they normally function to suppress progression. In summary, we have identified unique gene expression profiles of human DCIS and invasive breast cancer, which include novel genes regulating tumor progression. Targeting some of these genes may improve the detection, diagnosis, and therapy of DCIS.

Parity-induced decrease in systemic growth hormone alters mammary gland signaling: a potential role in pregnancy protection from breast cancer.

Early full-term pregnancy is an effective natural protection against breast cancer in both humans and experimental rodents. The protective effect of an early pregnancy is, in part, linked to changes in circulating hormones that are involved in both normal breast development and breast cancer. For example, a reduction in circulating growth hormone (GH) has been shown to protect rats from carcinogen-induced mammary tumors. We examined the ability of a full-term pregnancy to alter the endocrine GH/insulin-like growth factor-I (IGF-I) axis and how this change affected normal mammary gland function in two commonly used rat models (Sprague-Dawley and Wistar Furth). Circulating GH and IGF-I were measured in blood drawn every 30 minutes from parous and age-matched virgin female rats. Mean serum GH levels were significantly decreased (P < 0.01) in parous compared with age-matched virgin rats for both strains. Changes in GH levels were independent of estrous cycle, indicated by a significant (P < 0.05) reduction in circulating levels of GH during estrus and diestrus in both parous strains. Despite the decrease in circulating GH, pituitary GH mRNA levels were unaltered in parous rats. Circulating IGF-I and hepatic IGF-I mRNA were also unaltered by parity in either rat strain. Immunoblot analysis of mammary glands showed decreases in phosphorylation of signal transducer and activator of transcription 5A and Janus-activated kinase 2, suggesting reduced action of GH in the mammary gland. Therefore, although the parity reduction in circulating GH does not affect circulating IGF-I levels, it is possible that reduced GH acts directly at the mammary gland and may play a role in pregnancy protection from breast cancer.

Ductal carcinoma in situ and the emergence of diversity during breast cancer evolution.

PURPOSE: Human invasive breast cancers (IBC) show enormous histologic and biological diversity. This study comprehensively evaluated diversity in ductal carcinoma in situ (DCIS), the immediate precursors of IBCs. EXPERIMENTAL DESIGN: The extent of diversity for conventional histologic grade and standard prognostic biomarkers assessed by immunohistochemistry was evaluated in a series of pure DCIS (n = 200) compared with a contemporaneous series of IBCs (n = 200). A subset of the DCIS (n = 25) was evaluated by DNA microarrays for the presence of luminal, basal, and erbB2 intrinsic subtypes. The extent of diversity within individual cases of DCIS (n = 120) was determined by assessing multiple regions independently for histologic (nuclear) grade and several biomarkers by immunohistochemistry, which approximate microarrays in determining intrinsic subtypes. RESULTS: DCIS showed a broad distribution of conventional histologic grades and standard biomarkers ranging from well to poorly differentiated, nearly identical to IBCs. Microarrays showed the same intrinsic subtypes in DCIS as in IBCs. However, higher resolution analysis showed that multiple histologic grades, biomarker phenotypes, and intrinsic subtypes often coexist within the same DCIS, and these diverse regions probably compete for dominance. Diversity within cases of DCIS was highly correlated with mutated p53 (P = 0.0007). CONCLUSIONS: These results support the hypothesis that poorly differentiated DCIS gradually evolve from well-differentiated DCIS by randomly acquiring genetic defects resulting in increasingly abnormal cellular features. This diversity is amplified by defects resulting in genetic instability (e.g., p53 mutation), and the alterations are propagated to IBC in a manner independent of progression to invasion.

Alterations of gene expression in the development of early hyperplastic precursors of breast cancer.

Enlargement of normal terminal duct lobular units (TDLUs) by hyperplastic columnar epithelial cells is one of the most common abnormalities of growth in the adult female human breast. These hyperplastic enlarged lobular units (HELUs) are important clinically as the earliest histologically identifiable potential precursor of breast cancer. The causes of the hyperplasia are unknown but may include estrogen-simulated growth mediated by estrogen receptor-alpha, which is highly elevated in HELUs and may be fundamental to their development. The present study used DNA microarray technology and RNA from microdissected pure epithelial cells to examine changes in gene expression and molecular pathways associated with the development of HELUs from TDLUs. The results suggest that HELUs evolve from TDLUs primarily by reactivation of pathways involved in embryonic development and suppression of terminal differentiation. Changes in ERBB genes were particularly prominent, including a uniform switch in ligands for the ERBB1 receptor (14-fold decrease in epidermal growth factor and 10-fold increase in amphiregulin, respectively) in HELUs compared with TDLUs. Epidermal growth factor regulates terminal differentiation in adult breast and amphiregulin is critical to normal embryonic breast development. Because HELUs are such early potential precursors of breast cancer, targeting some of these alterations may be especially promising strategies for breast cancer prevention.

Hormones, receptors, and growth in hyperplastic enlarged lobular units: early potential precursors of breast cancer.

INTRODUCTION: The hyperplastic enlarged lobular unit (HELU) is a common alteration in adult female human breast and is the earliest histologically identifiable lesion with premalignant potential. Growth and differentiation in normal epithelium are regulated by estrogen and progesterone, whose effects are mediated through estrogen receptor (ER)-alpha and progesterone receptor (PR). We assessed correlations between growth (proliferation and apoptosis), endogenous hormone levels (using age as a surrogate for menopausal/estrogen status), and ER-alpha/PR expression in HELUs versus adjacent normal terminal duct lobular units (TDLUs) to gain insight into potentially premalignant hyperplasia. METHODS: Proliferation (Ki67 antigen), ER-alpha, and PR were assessed by immunohistochemistry, apoptosis using the TUNEL (terminal transferase-mediated dUTP nick end-labeling) assay, and nuclear colocalization of ER-alpha and Ki67 by dual-labeled immunofluorescence in HELUs and adjacent TDLUs (n = 100-584, depending on the factor) from 324 breasts. All factors were quantified under direct microscopic visualization. ER-alpha/PR expression was semiquantified by estimating the proportion of positive cells (0 = none, 1 = or <1/100, 2 = 1/100 to 1/10, 3 = 1/10 to 1/3, 4 = 1/3 to 2/3, and 5 = or >2/3). Ki67, TUNEL, and colocalization of ER-alpha and Ki67 were scored by absolute counting (%positive). RESULTS: ER-alpha and PR expression were significantly elevated in HELUs versus adjacent TLDUs (average score: 4.5 versus 3.1 and 3.5 versus 2.1; P < 0.0001). Proliferation was also significantly higher in HELUs versus TDLUs (average 6.3% versus 2.0%; P < 0.0001). In contrast, apoptosis was significantly lower in HELUs versus TDLUs (average 0.61% versus 0.22%; P < 0.0001). Changes in proliferation and receptor expression were similar between premenopausal and postmenopausal TDLUs and HELUs, suggesting that hyperplastic cells remain responsive to regulation by estrogen. The proportion of ER-positive/proliferating cells was much higher in HELUs than TDLUs (27.6% vs. 4.9%; P < .0001). CONCLUSION: Development of HELUs is associated with increased proliferation and decreased cell death relative to normal cells. ER-alpha and PR are highly elevated in HELUs, which may contribute to the hyperplasia because they mediate hormonal regulation of growth. An understanding of the fundamental causes of increased levels of receptors and growth may lead to new strategies to prevent breast cancer.