A prospective study of serum insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding protein-3 and breast cancer risk.

The associations between serum concentrations of insulin-like growth factor-I (IGF-I), IGF-II and IGF-binding proteins (IGFBP)-3 and risk of breast cancer were investigated in a nested case-control study involving 117 cases (70 premenopausal and 47 postmenopausal at blood collection) and 350 matched controls within a cohort of women from the island of Guernsey, UK. Women using exogenous hormones at the time of blood collection were excluded. Premenopausal women in the top vs bottom third of serum IGF-I concentration had a nonsignificantly increased risk for breast cancer after adjustment for IGFBP-3 (odds ratio (OR) 1.71; 95% confidence interval (CI): 0.74-3.95; test for linear trend, P=0.21). Serum IGFBP-3 was associated with a reduction in risk in premenopausal women after adjustment for IGF-I (top third vs the bottom third: OR 0.49; 95% CI: 0.21-1.12, P for trend=0.07). Neither IGF-I nor IGFBP-3 was associated with risk in postmenopausal women and serum IGF-II concentration was not associated with risk in pre- or postmenopausal women. These data are compatible with the hypothesis that premenopausal women with a relatively high circulating concentration of IGF-I and low IGFBP-3 are at an increased risk of developing breast cancer

Physical Confirmation and Mapping of Overlapping Rat Mammary Carcinoma Susceptibility QTLs, Mcs2 and Mcs6

Only a portion of the estimated heritability of breast cancer susceptibility has been explained by individual loci. Comparative genetic approaches that first use an experimental organism to map susceptibility QTLs are unbiased methods to identify human orthologs to target in human population-based genetic association studies. Here, overlapping rat mammary carcinoma susceptibility (Mcs) predicted QTLs, Mcs6 and Mcs2, were physically confirmed and mapped to identify the human orthologous region. To physically confirm Mcs6 and Mcs2, congenic lines were established using the Wistar-Furth (WF) rat strain, which is susceptible to developing mammary carcinomas, as the recipient (genetic background) and either Wistar-Kyoto (WKy, Mcs6) or Copenhagen (COP, Mcs2), which are resistant, as donor strains. By comparing Mcs phenotypes of WF.WKy congenic lines with distinct segments of WKy chromosome 7 we physically confirmed and mapped Mcs6 to ∼33 Mb between markers D7Rat171 and gUwm64-3. The predicted Mcs2 QTL was also physically confirmed using segments of COP chromosome 7 introgressed into a susceptible WF background. The Mcs6 and Mcs2 overlapping genomic regions contain multiple annotated genes, but none have a clear or well established link to breast cancer susceptibility. Igf1 and Socs2 are two of multiple potential candidate genes in Mcs6. The human genomic region orthologous to rat Mcs6 is on chromosome 12 from base positions 71,270,266 to 105,502,699. This region has not shown a genome-wide significant association to breast cancer risk in pun studies of breast cancer susceptibility

Absence of the common Insulin-like growth factor-1 19-repeat allele is associated with early age at breast cancer diagnosis in multiparous women

Multiparity decreases the risk of breast cancer in white women, whereas it is a risk factor in black women <50 years. Early-onset breast cancer (<50 years) has been associated with high insulin-like growth factor-1 (IGF-1) levels. Absence of the common IGF1 19 cytosine-adenine (CA)-repeat allele (IGF1-19/-19) inverts the effect of several non-genetic factors on breast cancer risk but the interaction between IGF1-19/-19 and multiparity on breast cancer risk is unknown. As IGF1-19/-19, multiparity and early-onset breast cancer are more common in black than in white women, we aimed to study whether multiparity combined with IGF1-19/-19 increases the risk of early-onset breast cancer. Four hundred and three breast cancer patients diagnosed in Lund, Sweden, at age 25–99 years were genotyped for the IGF1 CA-repeat length using fragment analysis. Overall, 12.9% carried the IGF1-19/-19 genotype. There was a highly significant interaction between multiparity and IGF1-19/-19 on age at breast cancer diagnosis (P=0.007). Among IGF1-19/-19 patients, multiparity was associated with a 9.2 year earlier age at diagnosis compared with uniparity or nulliparity (P=0.006). Multiparity combined with IGF1-19/-19 was associated with an early age at breast cancer diagnosis. If confirmed, IGF1-19/-19 may help identify a subgroup of women for earlier breast cancer screening

Decreased expression of the mannose 6- phosphate/insulin-like growth factor-II receptor promotes growth of human breast cancer cells

BACKGROUND: Loss or mutation of the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) has been found in breast cancer. However, whether or not decreased levels of functional M6P/IGF2R directly contribute to the process of carcinogenesis needs to be further verified by functional studies. METHODS: In this study, using viral and ribozyme strategies we reduced the expression of M6P/IGF2R in human breast cancer cells and then examined the effect on growth and apoptosis of these cells. RESULTS: Our results showed that infection of MCF-7 cells with the adenovirus carrying a ribozyme targeted against the M6P/IGF2R mRNA dramatically reduced the level of transcripts and the functional activity of M6P/IGF2R in these cells. Accordingly, cells treated with the ribozyme exhibited a higher growth rate and a lower apoptotic index than control cells (infected with a control vector). Furthermore, decreased expression of M6P/IGF2R enhanced IGF-II-induced proliferation and reduced cell susceptibility to TNF-induced apoptosis. CONCLUSIONS: These results suggest that M6P/IGF2R functions as a growth suppressor and its loss or mutation may contribute to development and progression of cancer. This study also demonstrates that adenoviral delivery of the ribozyme provides a useful tool for investigating the role of M6P/IGF2R in regulation of cell growth

Mutation analysis of aryl hydrocarbon receptor interacting protein (AIP) gene in colorectal, breast, and prostate cancers

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently identified in individuals with pituitary adenoma predisposition (PAP). These patients have prolactin (PRL) or growth hormone (GH) oversecreting pituitary adenomas, the latter exhibiting acromegaly or gigantism. Loss-of-heterozygosity (LOH) analysis revealed that AIP is lost in PAP tumours, suggesting that it acts as a tumour-suppressor gene. Aryl hydrocarbon receptor interacting protein is involved in several pathways, but it is best characterised as a cytoplasmic partner of the aryl hydrocarbon receptor (AHR). To examine the possible role of AIP in the genesis of common cancers, we performed somatic mutation screening in a series of 373 colorectal cancers (CRCs), 82 breast cancers, and 44 prostate tumour samples. A missense R16H (47G>A) change was identified in two CRC samples, as well as in the respective normal tissues, but was absent in 209 healthy controls. The remaining findings were silent, previously unreported, changes of the coding, non-coding, or untranslated regions of AIP. These results suggest that somatic AIP mutations are not common in CRC, breast, and prostate cancers

Inferring predominant pathways in cellular models of breast cancer using limited sample proteomic profiling

<p>Abstract</p> <p>Background</p> <p>Molecularly targeted drugs inhibit aberrant signaling within oncogenic pathways. Identifying the predominant pathways at work within a tumor is a key step towards tailoring therapies to the patient. Clinical samples pose significant challenges for proteomic profiling, an attractive approach for identifying predominant pathways. The objective of this study was to determine if information obtained from a limited sample (i.e., a single gel replicate) can provide insight into the predominant pathways in two well-characterized breast cancer models.</p> <p>Methods</p> <p>A comparative proteomic analysis of total cell lysates was obtained from two cellular models of breast cancer, BT474 (HER2+/ER+) and SKBR3 (HER2+/ER-), using two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein interaction networks and canonical pathways were extracted from the Ingenuity Pathway Knowledgebase (IPK) based on association with the observed pattern of differentially expressed proteins.</p> <p>Results</p> <p>Of the 304 spots that were picked, 167 protein spots were identified. A threshold of 1.5-fold was used to select 62 proteins used in the analysis. IPK analysis suggested that metabolic pathways were highly associated with protein expression in SKBR3 cells while cell motility pathways were highly associated with BT474 cells. Inferred protein networks were confirmed by observing an up-regulation of IGF-1R and profilin in BT474 and up-regulation of Ras and enolase in SKBR3 using western blot.</p> <p>Conclusion</p> <p>When interpreted in the context of prior information, our results suggest that the overall patterns of differential protein expression obtained from limited samples can still aid in clinical decision making by providing an estimate of the predominant pathways that underpin cellular phenotype.</p

Over-expression of lysophosphatidic acid receptor-2 in human invasive ductal carcinoma

INTRODUCTION: Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse effects on various cells. It interacts with at least three G-protein-coupled transmembrane receptors, namely LPA1, LPA2 and LPA3, whose expression in various tumours has not been fully characterized. In the present study we characterized the expression profile of LPA receptors in human breast cancer tissue and assessed the possible roles of each receptor. METHODS: The relative expression levels of each receptor's mRNA against β-actin mRNA was examined in surgically resected invasive ductal carcinomas and normal gland tissue using real-time RT-PCR. LPA2 expression was also examined immunohistochemically using a rat anti-LPA2 monoclonal antibody. RESULTS: In 25 cases normal and cancer tissue contained LPA1 mRNA at similar levels, whereas the expression level of LPA2 mRNA was significantly increased in cancer tissue as compared with its normal counterpart (3479.0 ± 426.6 versus 1287.3 ± 466.8; P < 0.05). LPA3 was weakly expressed in both cancer and normal gland tissue. In 48 (57%) out of 84 cases, enhanced expression of LPA2 protein was confirmed in carcinoma cells as compared with normal mammary epithelium by immunohistochemistry. Over-expression of LPA2 was detected in 17 (45%) out of 38 premenopausal women, as compared with 31 (67%) out of 46 postmenopausal women, and the difference was statistically significant (P < 0.05). CONCLUSION: These findings suggest that upregulation of LPA2 may play a role in carcinogenesis, particularly in postmenopausal breast cancer

The aromatase inhibitor letrozole and inhibitors of insulin-like growth factor I receptor synergistically induce apoptosis in in vitro models of estrogen-dependent breast cancer

INTRODUCTION: Endocrine-dependent, estrogen receptor positive breast cancer cells proliferate in response to estrogens, synthesized by the cytochrome p450 aromatase enzyme. Letrozole is a potent nonsteroidal aromatase inhibitor that is registered for the treatment of postmenopausal women with advanced metastatic breast cancers and in the neoadjuvant, early, and extended adjuvant indications. Because crosstalk exists between estrogen receptor and insulin-like growth factor I receptor (IGF-IR), the effect of combining a selective IGF-IR inhibitor (NVP-AEW541) with letrozole was assessed in two independent in vitro models of estrogen-dependent breast cancer. METHODS: MCF7 and T47D cells stably expressing aromatase (MCF7/Aro and T47D/Aro) were used as in vitro models of aromatase-driven breast cancer. The role of the IGF-IR pathway in breast cancer cells stimulated only by 17ß-estradiol or androstenedione was assessed by proliferation assays. The combination of letrozole and NVP-AEW541 was assessed for synergy in inhibiting cell proliferation using Chou-Talalay derived equations. Finally, combination or single agent effects on proliferation and apoptosis were assessed using proliferation assays, flow cytometry, and immunoblotting. RESULTS: Both MCF7 and T47D cells, as well as MCF7/Aro and T47D/Aro, exhibited sensitivity to inhibition of 17ß-estradiol dependent proliferation by NVP-AEW541. Letrozole combined with NVP-AEW541 synergistically inhibited androstenedione-dependent proliferation in aromatase-expressing cells with combination index values of 0.6 or less. Synergistic combination effects correlated with higher levels of apoptosis as compared with cells treated with the single agent alone. Treatment with either agent also appeared to inhibit IGF-IR signalling via phosphoinositide 3-kinase. Notably, IGF-IR inhibition had limited effect on estrogen-dependent proliferation in the cell lines, but was clearly required for survival, suggesting that the combination of letrozole and IGF-IR inhibition sensitizes cells to apoptosis. CONCLUSION: Inhibition of the IGF-IR pathway and aromatase was synergistic in two independent estrogen-dependent in vitro models of breast cancer. Moreover, synergism of NVP-AEW541 and letrozole correlated with induction of apoptosis, but not cell cycle arrest, in the cell lines tested. Combination of IGF-IR inhibitors and letrozole may hold promise for the treatment of patients with estrogen-dependent breast cancers

Serum antibodies against p53 in relation to cancer risk and prognosis in breast cancer: a population-based epidemiological study

To perform an epidemiological evaluation of the predictive value of p53 autoantibodies in breast cancer, we measured antibodies against p53 in serum samples from 165 breast cancer patients in comparison with serum samples from 330 healthy controls, selected from the same population as the cases and matched for age, sex and specimen storage time. Median age of patients was 51 years (range 25–64 years). Presence of serum p53 autoantibodies was analysed by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blotting. The lower ELISA reactivities were similar for cases and controls, but presence of high-level reactivity was more common among cases than among controls [odds ratio (OR) 9.03, 95% confidence interval (CI) 2.40–50.43]. Presence of Western blot-detected p53 autoantibodies had a very similar association (OR 10.8, CI 3.0–59.4). Among the cases, we also studied whether there was any correlation between level of anti-p53 antibodies and stage of the disease or survival. There was no significant correlation between presence of antibodies and stage of the disease. There was a significant negative correlation between presence of p53 antibodies and survival (P= 0.003). A stepwise multivariate Cox regression analysis showed that T-stage, age and presence of anti-p53 antibodies were significant independent prognostic variables, with a dose-dependent negative effect on survival for all three variables. We conclude that presence of anti-p53 antibodies are of significance both for the risk of having breast cancer and the risk of dying from breast cancer. 1999 Cancer Research Campaig

Expression of a protease-resistant insulin-like growth factor-binding protein-4 inhibits tumour growth in a murine model of breast cancer

BACKGROUND: Insulin-like growth factor 1 (IGF1) promotes breast cancer and disease progression. Bioavailability of IGF1 is modulated by IGF-binding proteins (IGFBPs). IGFBP4 inhibits IGF1 activity but cleavage by pregnancy-associated plasma protein-A (PAPP-A) protease releases active IGF1. METHODS: Expression of IGF pathway components and PAPP-A was assessed by western blot or RT-PCR. IGFBP4 (dBP4) resistant to PAPP-A cleavage, but retaining IGF-binding capacity, was used to block IGF activity in vivo. 4T1.2 mouse mammary adenocarcinoma cells transfected with empty vector, vector expressing wild-type IGFBP4 or vector expressing dBP4 were implanted in the mammary fat pad of BALB/c mice and tumour growth was assessed. Tumour angiogenesis and endothelial cell apoptosis were assessed by immunohistochemistry. RESULTS: 4T1.2 cells expressed the IGF1R receptor and IGFBP4. PAPP-A was expressed within mammary tumours but not by 4T1.2 cells. Proliferation and vascular endothelial growth factor (VEGF) production by 4T1.2 cells was increased by IGF1(E3R) (recombinant IGF1 resistant to binding by IGFBPs) but not by wild-type IGF1. IGF1-stimulated microvascular endothelial cell proliferation was blocked by recombinant IGFBP4. 4T1.2 tumours expressing dBP4 grew significantly more slowly than controls or tumours expressing wild-type IGFBP4. Inhibition of tumour growth by dBP4 was accompanied by the increased endothelial cell apoptosis. CONCLUSION: Protease-resistant IGFBP4 blocks IGF activity, tumour growth and angiogenesis