bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved methods of detection, and prognostic significance, of vascular invasion in breast cancer.
Authors: Mohammed RA , Ellis IO , Green AR , Paish EC , Gill MS , Martin SG
Published: December 2010

Abstract #3010

Vascular invasion, encompassing both lymphovascular (LVI) and blood vascular invasion (BVI), influences breast cancer patient prognosis. We have previously shown, using an immunohistochemical approach to distinguish between LVI and BVI in a well characterized group of 177 consecutive paraffin-embedded specimens breast cancer patients (52 LN+ and 125 LN-), that VI was detected in 32% of specimens and that 96% of such VI were invasion of lymphatic vessels. The presence of LVI was significantly associated with the presence of metastasis, recurrence and worse disease free interval (DFI) and overall survival (OS). In multivariate analysis, LVI was an independent poor prognostic factor. This original study has been expanded by examining LVI, assessed by podoplanin staining, in 1000 LN-, early stage, breast cancers. In addition to verifying initial results showing the prognostic power of detection of LVI in early stage tumours, it also allowed an examination of LVI in histological and basal phenotypes to determine if the frequency of LVI influences prognosis in different breast cancer phenotypes.  LVI was detected in 212 (21% of specimens). The lowest LVI positivity was observed in tubular carcinoma (5.6%), followed by mucinous (7.1%) and atypical medullary (18.5%) types. The highest LVI positivity was detected in invasive carcinoma of no special type (26%). Tumours with basal phenotype were significantly associated with higher LVI (29%) compared with 19% in non-basal tumours P =0.002.  In the LVI-positive specimens; 203 (76%) specimens had peri-tumoural LVIs and 58 (24%) had an LVI in intra-tumoural vessels (9 specimens had LVIs in the intra-tumoural vessels only and 49 specimens had an LVI in both intra- and peri-tumoural vessels). The frequency of LVI ranged from 1 to 79: 167specimens (79%) had <5 LVIs, 23 (11%) had 6-10 LVIs and 22 (10%) had >10LVIs. There was no significant difference between the frequency of LVI and development of recurrence or death from the disease.  Patients with LVI had a significantly worse prognosis. The 20-year OS rate was 85% in patients without LVI compared with 68% in patients with LVI. In multivariate analysis for OS, tumour size (P =0.004), tumour grade (P< 0.0001), and LVI (P <0.0001) retained significance. However, for DFI, only LVI (P<0.0001) remained significant.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3010.
Are there preoperative predictors for invasive disease in DCIS?.
Authors: McArdle K , Shokuhi S , Chan Y , Vishwanath L , Brown H
Published: December 2010

Abstract #3014

Introduction: Ductal carcinoma in situ (DCIS) of the breast is defined as a proliferation of malignant epithelial cells within breast ducts without evidence of invasion through the basement membrane. Core needle biopsy (CNB) is used to sample both symptomatic and screen detected breast lesions preoperatively. However in a proportion of patients invasive disease is identified following surgical excision and as a result an upstaged patient may need to undergo additional surgery for axillary nodal evaluation such as sentinel lymph node biopsy (SLNB). Our aim was to evaluate predictive factors for invasive disease in this group.  Method: Data from all patients with preoperative diagnosis of DCIS on core biopsy between February 2006 and December 2007 were analysed. Demographic data, screening /symptomatic presentation, palpability, size and histological features of DCIS on core biopsy including presence/absence of comedo necrosis, size of post operative invasion when present and result of axillary staging in this group was recorded. All patients with invasive disease had axillary staging.  Results: A total of 75 patients were identified (all female), median age 63 (range 38-77). 72% (54/75) were screen detected. All palpable lesions were in the symptomatic group (13/19). Invasive disease was identified in 18 (24%) on post operative histology (2 micro-invasions). Only one patient with invasive disease had axillary involvement.    Invasive disease was significantly higher in symptomatic and palpable lesions (P=0.04). Size of the lesion was significantly larger in the invasive group (P=0.04). High grade histology or presence of comedo necrosis was not a predictor of invasive disease in our series.  Conclusion: A quarter of patients were upstaged and required axillary staging. 50% of symptomatic, palpable lesions larger than 20mm were found to be invasive on final histology, this group accounted for the only positive axilla. Symptomatic patients presenting with large, palpable areas of DCIS should be considered for axillary ultrasound scan and/or SLNB.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3014.
Akt-dependent phosphorylation on AIB1 serine 967 contributes to breast cancer tamoxifen resistance.
Authors: Shou J , Osborne KC , Gee J , Nicholson R , Schiff R
Published: December 2010

Abstract #3021

AIB1, a coactivator for nuclear receptors and other transcription factors, is involved in breast cancer development and progression. Overexpression of both AIB1 and HER2/neu (HER2) results in tamoxifen (Tam) resistance in clinical and experimental breast cancer. We hypothesized that HER2 signaling, via its downstream kinase Akt, leads to phosphorylation and activation of AIB1, which may then contribute to development of Tam resistance by reducing its antagonist activity.  We have previously shown that Akt synergizes with AIB1 to enhance estrogen- and Tam-induced estrogen receptor (ER)-dependent transcriptional activity. This synergy is mainly mediated by Akt phosphorylation of AIB1 on serine 967, which is located between the nuclear receptor and the CBP/p300 interacting domains. We showed that this serine residue was phosphorylated specifically by Akt both in vitro and in vivo, including in both acquired and de novo Tam-resistant xenograft models that are associated with increased Akt signaling. More recent data showed that serine 967 of AIB1 is phosphorylated in Tam-resistant MCF7 tumors, and that this phosphorylation is stable in the resistant cells. By using immunoprecipitation and Western blot analysis, in ER positive breast tumors from patients, 90% of tumors were positive for total AIB, while AIB1 phosphorylation was detectable in 60% of tumors. Phosphorylated AIB1 was positively correlated with total and phosphorylated HER2 (Tyrosine 1248) (p=0.004 and p=0.0008 respectively, Fisher's exact test), implying that phophorylation of serine 967 of AIB1 may be stimulated by HER2 in these AIB1 positive tumors. Serine 967 of AIB1 was also phosphorylated in breast cancer cell lines, including both basal (HCC1954, BT20 and SUM190PT) and luminal (T47D, ZR7530, MDAMB361, and UACC812) subtypes, in which HER2 (except BT20) and Akt were activated .  Taken together, Akt phosphorylation of AIB1 serine 967 appears important for AIB1 to function as an ER coactivator and regulator of breast cancer proliferation, and for Tam agonistic activity. Thus phosphorylation at serine 967 of AIB1 may be an essential factor required for Tam resistance as well as for tumor growth independent of ER. Additional regulatory roles of serine 967 in AIB1 function are currently under investigation.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3021.
Overexpression of CD44 in acquired endocrine resistant breast cancer modulates erbB activity and promotes an invasive phenotype.
Authors: Baruah BP , Smith C , Jordan N , Nicholson RI , Robertson J , Barrett-Lee P , Hiscox S
Published: December 2010

Abstract #3027

Background: Acquired endocrine resistance in breast cancer cells is accompanied by altered growth factor receptor signalling and a highly migratory cell phenotype. In tamoxifen-resistant ('TamR') MCF7 cells, our microarray analysis has demonstrated elevated levels of CD44, a transmembrane glycoprotein, known to interact with and modulate the function of a number of growth factor receptors and plays a major role in tumour metastasis. Here, we have explored the role of CD44 as a modulator of erbB family activity in TamR cells and evaluated the expression of CD44 in a series of breast cancer patients on endocrine therapy.  Methods: RT-PCR was performed to confirm overexpression of CD44 at the gene level in TamR cells. Western blotting and immunocytochemistry were used to investigate expression of CD44 at the protein level together with the activity of erbB signalling pathways following stimulation of CD44 by its ligand, hyaluronan (HA), or following stimulation with erbB ligands (TGFα, heregulin β1) after knockdown of CD44 expression using siRNA. Immunofluorescence microscopy was used to determine association between CD44 and erbB family members. Cell migration was assessed by seeding cells onto fibronectin-coated microporous inserts for 24 hours. CD44 expression levels were evaluated using paraffin-embedded tissue sections in a series of primary breast cancer tissues (n=103) from patients receiving endocrine therapy by immunohistochemical staining.  Results: CD44 was overexpressed at the gene and protein level in TamR cells versus their endocrine sensitive counterparts. siRNA-mediated suppression of CD44 expression reduced basal EGFR/Her2 activity in TamR cells together with suppressing their migratory phenotype. siRNA-mediated inhibition of CD44 expression greatly attenuated the ability of erbB ligands to activate erbB receptors and inhibited TGFα and heregulin β1-induced migration. Conversely, stimulation of TamR, but not MCF7 cells, with HA promoted activation of erbB family members together with their downstream signalling intermediates ERK1/2, Src and AKT. Analysis of CD44 expression in clinical tissue revealed an association between elevated CD44 expression and a shortened response to endocrine therapy.  Conclusions: These data demonstrate that overexpression of CD44 accompanies endocrine resistance where it enhances the sensitivity of these cells to factors (erbB ligands, HA) found within the tumour microenvironment. This may implicate CD44 in loss of endocrine response clinically and, therefore, provide a novel therapeutic target in endocrine resistant breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3027.
Overexpression of L1CAM accompanies acquired endocrine resistance and is associated with the development of an aggressive cell phenotype.
Authors: Hiscox S , Jordan N , Crandon-Lewis A , Jiang W , Nicholson RI , Gee J
Published: December 2010

Abstract #3028

Background: Acquired resistance to endocrine therapies is associated with a gain in aggressive features in vitro which may impact on tumour spread in vivo. To identify potential candidate molecules which may promote this adverse cell behaviour, we performed microarray analysis on MCF-7 cell models of fulvestrant resistance ('FasR' cells) applying a commercial algorithm for signalling network discovery. We have subsequently investigated the significance of overexpression of L1CAM, a cell-surface adhesion molecule implicated in a MAPK signalling network and recently associated with tumour metastasis, in FasR cells and in clinical tissue.  Materials and Methods: A network analysis algorithm was applied to t-test significant Affymetrix gene lists derived from FasR vs. MCF-7 cells (Almac Diagnostics). Subsequent to revealing a prominent L1CAM network, L1CAM gene and protein expression were assessed using RT-PCR, Western blotting and immunofluorescence. Inhibition of L1CAM was achieved using siRNA transfection. Cell invasion and migration were measured by seeding onto Matrigel- or fibronectin-coated microporous membranes respectively. After 48 hours, invasive/migratory cells were fixed, stained and counted. Analysis of cell signalling molecules was determined using Western blotting with phospho-specific antibodies. Clinical significance of L1CAM expression was determined by analysis of L1CAM expression in tumour (n=101) vs. normal (n=23) breast tissue using Q-PCR.  Results: Development of fulvestrant resistance in MCF7 cells was associated with ER loss and a gain in migratory and invasive capacity in vitro. L1CAM was overexpressed at both gene (p<0.05) and protein (p<0.01) level in FasR cells whereas little or no L1CAM was detectable in MCF7 cells confirming microarray findings. Network and ontological analysis predicted L1CAM lies upstream of an ERK2 network (26 genes; p<0.0001) impinging on transcription factors/regulators which may promote aggressive cell behaviour. In agreement, siRNA-mediated inhibition of L1CAM suppressed the activity of nuclear MAP kinase and reduced the intrinsic migratory and invasive nature of FasR cells in vitro. Preliminary analysis indicated L1CAM expression was inversely associated with ER status (p<0.01) and reduced overall survival (p=0.01).  Conclusions: These data suggest that L1CAM overexpression associates with emergence of ER-negative fulvestrant resistance in breast cancer, where it may confer a highly motile and invasive phenotype. Additionally, our clinical studies suggest L1CAM may also contribute to inherently aggressive, ER-negative breast cancers. L1CAM signalling may thus provide a new therapeutic target to subvert such aggressive states in vivo.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3028.
Control of luminal type A intrinsic subtype enriched transcription factor network by insulin: implications of diabetes on breast cancer classification.
Authors: McCune KR , Bhat-Nakshatri P , Thorat M , Badve S , Nakshatri H
Published: December 2010

Abstract #3029

Background: Luminal type A and type B represent estrogen receptor alpha (ERalpha)-positive breast cancers with luminal type A expressing higher levels of ERalpha and is associated with better prognosis. Recent studies have identified a specific functional transcription factor network comprising GATA-3, FOXA1 and ERalpha in normal luminal cells as well as in luminal type A breast cancer that dictates their hormone dependence. Signaling molecules that may disrupt this network and force these cells to acquire hormone-independence are not known. T-bet (Tbx21) has been described as a major negative regulator of GATA-3 activity. As the expression and/or activity of some of the above factors are controlled by insulin, the objective of this study was to investigate whether elevated level of insulin, as evidenced in type II diabetes, alters gene expression pattern in luminal type A breast cancers by interrupting GATA-3:FOXA1:ERalpha network and thus forcing these cancers to acquire non-luminal phenotype and/or hormone-independence.  Methodologies: The effect of insulin on the expression of ERalpha, FOXA1, GATA-3 and T-bet was measured in ERalpha-positive MCF-7 cells by Western blot analysis. The effect of T-bet on estrogen-regulated gene expression was measured by stable overexpression of T-bet in MCF-7 cells and subsequent qRT-PCR analysis. Publicly available Oncomine database was used to determine the expression pattern of T-bet and its relation to ERalpha status in primary breast cancers. A proliferation assay was used to determine sensitivity of T-bet overexpressing cells to tamoxifen in the presence and absence of insulin.  Results: Insulin induced the expression of T-bet, which was partially reversed by estrogen. ERalpha and GATA-3 levels were reduced in MCF-7 cells stably overexpressing T-bet suggesting that T-bet reduces GATA-3-dependent ERalpha expression. Estrogen-inducible expression of estrogen target genes GREB-1 and Myb was lower in T-bet overexpressing cells compared to parental cells, although basal expression was elevated in T-bet overexpressing cells. Treatment of T-bet overexpressing cells with insulin decreased tamoxifen sensitivity. Although T-bet expression was generally higher in ERalpha-negative breast cancers compared to ERalpha-positive breast cancers, a subpopulation of ER-positive breast cancers express elevated levels of T-bet.  Conclusions: Insulin may change the gene expression pattern through T-bet-mediated disruption of master cell-type-specific transcriptional network including GATA-3, ERalpha and FOXA1 that dictates the phenotype of hormone-dependent luminal type A breast cancer. T-bet may serve as a marker to identify ERalpha-positive breast cancers that express low levels of GATA-3 and have progressed to hormone-independence.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3029.
Expression of ER-a36, a novel variant of estrogen receptor a, and resistance to tamoxifen treatment in breast cancer.
Authors: Shi L , Dong B , Li Z , Lu Y , Ouyang T , Li J , Wang T , Fan Z , Fan T , Lin B , Wang Z , Xie Y
Published: December 2010

Abstract #3037

Background: Recently, a 36 kDa variant of estrogen receptor a (ER-a66), ER-a36, has been identified and cloned. ER-a36 predominantly localizes on the plasma membrane and in the cytoplasm and mediates a membrane-initiated “nongenomic” signaling pathway. In this study, we investigated the association between ER-a36 expression and tamoxifen resistance in breast cancer patients.  Methods: ER-a36 protein expression in tumors from 710 breast cancer patients with a median follow-up of 7.9 years was assessed using immunohistochemistry (IHC) assay. Survival curves were compared using the log-rank test and multivariate analysis was performed using Cox model. All statistical tests were two-sided.  Results: Among the patients with ER-a66 positive tumors who received tamoxifen treatment (n=307), overexpression of ER-a36 was associated with poorer disease-free survival (DFS) and disease-specific survival (DSS) and remained as an unfavorable independent factor of survival in multivariate analyses (DFS: HR=2.27; 95% CI= 1.40 to 3.68; P=. 001; DSS: HR=2.42; 95% CI= 1.37 to 4.28; P= .002). In contrast, among patients with ER-a66 positive tumors who did not receive tamoxifen (n=129), ER-a36 expression was not associated with survival, indicating a correlation between ER-a36 expression and tamoxifen resistance. Furthermore, ER-a36 expression was not associated with survival in ER-a66 negative tumors whether the patients received tamoxifen (n=73) or not (n=149). Our in vitro experiments with MCF7/ER36 cells also confirmed that high ER-a36 expression resulted in tamoxifen resistance.  Conclusions: Patients with ER-a66 positive tumors that also express high levels of ER-a36 are less likely to benefit from tamoxifen treatment. ER-a36 is an important predictive marker for tamoxifen therapy in ER-a66 positive breast cancer patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3037.
Intermittent treatment of hormone-dependent breast cancer using exemestane: translational research from lab bench to bedside.
Authors: Masri S , Phung S , Chung C , Somlo G , Chen S
Published: December 2010

Abstract #3040

Background and Rationale: Endocrine therapy agents such as aromatase inhibitors (AIs) have shown good clinical efficacy in the treatment of post-menopausal breast cancer patients. Currently, the major clinical hindrance with endocrine therapy is disease progression or acquired resistance. Intermittent treatment of endocrine therapy agents has been proposed in several clinical trials to address whether this treatment strategy can delay the onset of acquired resistance in hormone-dependent breast cancers. To support these clinical trials, we have performed a pre-clinical study using the steroidal AI exemestane (EXE) to understand potential molecular features that differentiate continuous treatment of EXE from intermittent administration.  Methods: The ER+ and aromatase-overexpressing cell culture model system (MCF-7aro) was used in this pre-clinical study. The cells were treated either continuously (T+EXE Cont) or intermittently (T+EXE Interm) for 2 weeks with EXE and 1 week without EXE, in the presence of testosterone (T). In addition, a non-steroidal AI letrozole, was used in an identical intermittent manner.  Results: Based on cell culture studies, acquired resistance was reached at 14 weeks in the T+EXE Cont treated cells, while T+EXE Interm cells were still responsive to EXE. At 34 weeks, acquired resistance developed in the T+EXE Interm cell lines, which demonstrates a delay in time to progression. The non-steroidal AI letrozole did not demonstrate a delay in acquired resistance using this intermittent model. Based on microarray analysis, we observed that gene expression of certain estrogen-responsive genes was elevated in the T+EXE Cont cells, while select genes showed lower levels of up-regulation in T+EXE Interm lines.  Discussion: Based on our previous studies, up-regulation of a key estrogen-responsive gene, amphiregulin (AREG), is responsible for activating EGFR and mediating resistance to EXE. Expression of genes such as AREG are decreased in the T+EXE Interm cell lines, suggesting the suppression of growth factor signaling implicated in driving resistance. In addition, we have reported that EXE acts as an aromatase destabilizer at the protein level. Unlike letrozole or anastrozole, EXE-mediated protein degradation of aromatase may play a key role in reducing estrogen levels that are responsible for driving breast cancer survival and proliferation. Since these pre-clinical studies provide an important mechanistic basis for intermittent endocrine therapy, a clinical trial to evaluate the intermittent use of exemestane has been discussed and is planned to initiate in the near future.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3040.
Co-targeting leptin and insulin-like growth factor I signaling: dramatic effects of epidermal growth factor receptor inhibitors on triple negative breast cancer cells.
Authors: Saxena NK , Smith LT , Knight BB , Sharma M , Sharma D
Published: December 2010

Abstract #3044

Introduction: Obesity is an independent risk factor for breast cancer and obese breast cancer patients exhibit a higher risk for larger tumor burden, increased metastasis and poor response to endocrine therapy. Obesity affects breast carcinogenesis by autocrine and paracrine effects of adipocytokine leptin and IGF-1. We have previously shown that leptin induces growth stimulation of breast cancer cells by recruiting histone acetyltransferases and mediator complex to cyclin D1 promoter via activation of Stat3. In the present study, we found a novel bidirectional crosstalk between IGF-1 and leptin signaling that promotes invasion and migration of triple-negative (ER, PR, HER2 negative) breast cancer cells; MDA-MB-231, MDA-MB-468, MDA-MB-435 and HCC-1806.  Methods and Results: IGF-1 induced significant tyrosine phosphorylation of leptin receptor (Ob-Rb) and leptin induced tyrosine phosphorylation of IGF-1 Receptor (IGF-1R). Combined treatment of leptin and IGF-1 induced synergistic activation of both Ob-Rb and IGF-1R along with activation of Akt and ERK. Furthermore, IGF-1 and leptin synergistically transactivated epidermal growth factor receptor (EGFR) and induced proliferation of triple negative breast cancer cells. We also found that tyrosine phosphorylation of EGFR induced by combined treatment of leptin and IGF-1 was significantly inhibited not only by EGFR tyrosine kinase inhibitor, AG1478 but also with a broad-spectrum matrix metalloproteinase inhibitor, GM6001 indicating that leptin and IGF-1 induced EGFR transactivation is dependent on proteolytic release of EGFR ligands. Intriguingly, we also found that combined treatment of leptin and IGF-1 potently induced invasion of triple negative breast cancer cells in Matrigel invasion and quantitative Electric Cell-Substrate Impedance Sensing invasion assays. Inhibition of leptin and IGF-1 mediated EGFR activation using a highly potent, reversible inhibitor of HER1/EGFR tyrosine kinase, erlotinib or dual epidermal growth factor receptor (EGFR)/human EGFR-2 (HER2) kinase inhibitor, lapatinib effectively inhibited leptin and IGF-1 induced invasion. Also, inhibition of EGFR activation using erlotinib and lapatinib reduced leptin- and IGF-1-induced migration of triple negative breast cancer cells.  Discussion: Taken together these data indicate a novel bidirectional crosstalk between leptin and IGF-1 signaling that transactivates EGFR and promotes metastatic properties, invasion and migration of triple negative breast carcinoma cells. Our novel findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the pro-cancerous effects of leptin and IGF-1 signaling in triple negative breast cancers.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3044.
Interactive effects of low-dose estradiol and tamoxifen on the postmenopausal primate endometrium and mammary gland.
Authors: Wood CE , Cline M
Published: December 2010

Abstract #3047

Background: Long-term use of combined estrogen+progestin hormone therapies increases breast cancer risk in postmenopausal women. Selective estrogen receptor modulators (SERMs) are promising alternatives to progestins, particularly for breast cancer survivors and other women at high-risk for breast cancer considering hormone therapy. The purpose of this study was to evaluate interactive effects of low-dose estradiol (E2) and the SERM tamoxifen on the postmenopausal endometrium and mammary gland.  Materials and Methods: Ovariectomized adult female cynomolgus monkeys were given placebo (n=6), oral estradiol (E2, 0.25 mg/1800 kcal) (n=6), tamoxifen (TAM, 20 mg/1800 kcal) (n=5), or E2+TAM (n=6) for 4 months. Doses of E2 and TAM were scaled to approximate those given to women.  Results: Endometrial thickness and uterine weight were significantly higher in E2, TAM, and E2+TAM groups compared to placebo (P < 0.01 for all). In contrast, endometrial gland and stromal proliferation determined by Ki67 immunolabeling was significantly greater only in the E2 group (P < 0.01 for both). Endometrial Ki67 gene expression was also higher only in the E2 group (P < 0.01). Gene markers of estrogen receptor alpha (ER) activity such as TFF1 and STC2 were significantly increased in E2, TAM, and E2+TAM groups (P < 0.01 for all). On endometrial histology TAM and E2+TAM induced stromal fibrosis and occasional simple glandular hyperplasia with cystic change. In the mammary gland, TFF1 gene expression was increased by all treatments (P < 0.05 for all), while no statistically significant treatment effects were seen for epithelial proliferation or progesterone receptor expression.  Discussion: Previous studies have suggested that the weak estrogen-like effects of tamoxifen in the uterus contribute to increased endometrial cancer risk. Our findings reveal a more complex pattern of tamoxifen action involving increased ER activity, antagonism of E2-induced proliferation, and distinctive stromal remodeling. These results suggest that tamoxifen does not exhibit simple weak estrogen agonist / antagonist effects in the primate endometrium but instead acts through pathways independent of classical ER signaling to alter cancer risk. Future gene profiling should aid in the identification of these alternative pathways.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3047.
Normal breast tissue cells from breast cancer patients express epidermal growth factor receptor HER1-4 protein concentrations characteristic for cancer cells.
Authors: Olsen DA , Oestergaard B , Bokmand S , Jakobsen EH , Brandslund I
Published: December 2010

Abstract #3052

Background: The epidermal growth factor receptor HER2 is expressed to higher than normal concentrations in 25%-30% of human breast cancers and in approximately 90% of these cases the overexpression is a result of gene amplification. The mechanism behind HER2 amplification is unknown but may be caused by continuous stimulation and activation of the HER1 system. Consequently we have mapped the concentrations of all four epidermal growth factor receptors HER1-4 in breast cancer tissue, in autologous non-cancer breast tissue and normal breast tissue from healthy women.  Material and Methods: Tissue samples of malignant and adjacent normal breast tissue were collected from 118 consecutive women admitted for surgical treatment of breast cancer. In addition 26 samples of normal breast tissue from healthy women were collected. The tissue samples weighing between 10 and 40 mg were homogenized and the proteins were extracted. The total protein amount in the samples were measured and the protein concentrations of HER1-4 were determined quantitatively using ELISA expressed as pg/mg total protein.  Results: HER1 demonstrated lower than normal concentrations both in autologous non-cancer tissue and cancer tissue. HER2 showed 4 times higher than normal concentrations in autologous reference tissue and 24 times higher in cancer tissue, while the corresponding figures for HER3 was factor 8 and 13 and for HER4 factor 10 and 8. These differences were significant at p<10-4. Further it was observed that correlations between the receptors were maintained from normal breast tissue to autologous reference breast tissue but were lost in cancer tissue. Adjusting the concentrations of receptors by differences in cell number content did not change differently in the cancer patients.  Discussion: We propose these findings to indicate that breast cancer is a systemic disease where also non-cancer tissue cells in the cancer patients are continuously activated thus favouring the subsequent development of cancer. This may explain the observation that occurrence of new cancers are increased in patients previously treated for cancer. Measurement of decreased HER1 and increased HER2-4 in breast cells from healthy women may in the future be used as an indicator of increased risk for cancer development.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3052.
Comparison between HER-2/neu, estrogen and progesterone receptors in primary breast carcinomas and matched lymph node metastases.
Authors: Desouki MM , Self SE , Wolff DJ
Published: December 2010

Abstract #3053

Background: HER2, estrogen (ER) and progesterone receptors (PR) are important markers for prognostic and therapeutic implications in breast cancer. Fluorescence in situ hybridization (FISH) and IHC for HER2 gene amplification and evaluation of ER and PR in primary tumor are commonly used methods. However, evaluation of these markers in lymph nodes or distant metastases, which may be more biologically significant, is not that applicable in regular clinical basis. In the current work we compared HER2 (by FISH), ER and PR (by IHC) in matched primary breast carcinomas and lymph node metastases.  Design: Formalin fixed, paraffin embedded tissue sections from 39 cases of primary and lymph node metastases were assessed for Her2 (by FISH). Primary tumors of the cases selected were known to be HER2 un-amplified. Also, ER and PR IHC were performed on 36 cases from the same cohort of cases to assess any discrepancy between primary and lymph node metastases of these cases.  Result: Out of 39 cases, one case was HER2 amplified in lymph node metastasis compared to un-amplified primary tumor. Eight percent of cases (3/36) were ER negative in LN mets and 6% (2/36) were less strongly positive compared to the positive primary tumors. Nineteen percent (7/36) were PR negative in LN mets in contrast to the matched positive primary tumors. Regarding PR immunoreactivity, 6% (2/36) were PR-positive in LN met compared to their corresponding negative primary tumors.  Conclusion: While most matched primary breast tumors and lymph node metastases show concordance in HER2, ER, PR status, we found discordance in a minority of cases. These results suggest the possibility that extension of studies in metastatic breast tumors in parallel with that of primary lesions is informative and can be used to determine further treatment options and predict prognosis in breast cancer patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3053.
Reciprocal regulation of ZEB1 and AR in triple negative breast cancer cells.
Authors: Graham TR , Yacoub R , Liu T , Odero-Marah V , Kimbro K , O'Regan RM
Published: December 2010

Abstract #3054

Introduction: Approximately 60-80% of breast tumors are positive for androgen receptor (AR) and negative for estrogen receptor (ER) and progesterone (PR) (Isola et al. J Pathol. 1993). This suggests that AR may function independently of ER and PR to cross-talk with other factors such as Zinc-finger enhancer binding protein (ZEB1). ZEB1 is a transcription factor involved in the progression of breast cancer primarily through promoting epithelial to mesenchymal transition (EMT). ZEB1 represses the expression of E-cadherin by binding to E-box sequences in the promoter, thus decreasing epithelial differentiation and promotes cell migration.  Methods: We established a stable control and ZEB1 knockdown using shRNA in MDA-MB-231 cells, a highly metastatic triple negative breast cancer cell line with mesechymal morphology. The stable cell lines were characterized by immunoblot and RT-PCR analysis and were stimulated with synthetic androgens or an androgen antagonist and cell growth and migration were monitored.  Results: ZEB1 regulated the expression of AR in triple breast cancer cell lines. AR promoter contains two E-boxes, which may be critical to the transcriptional regulation of this gene. ZEB1 suppression by stably transfecting shRNA in MDA-MB-231 cells resulted in a decrease of AR mRNA, protein and AR downstream targets. In addition, ZEB1 suppression reduced the protein expression of Cyclin D1 and decreased cell proliferation. Conversely, blockade of AR signaling with bicalutamide resulted in suppression of ZEB1 protein expression in two triple negative breast cancer cell lines. ZEB1 knockdown in MDA-MB-231 cells sensitized the cells to bicalutamide by inducing a greater amount of cell death and a reduction in migration compared to the control cells.  Conclusions: Taken together, these results indicate that ZEB1 and AR regulate each other to promote cell growth and cell migration in triple negative breast cancer cells. Inhibition of AR signaling could decrease cell proliferation via ZEB1 in a subset of triple negative breast cancers. The growth of triple negative breast cancers that express ZEB1 and AR could be inhibited by antiandrogen treatment.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3054.
Targeting EGFRs and Src signaling with a modified ectodomain of human EGFR (EBIP) and dasatinib in breast cancer.
Authors: Nautiyal J , Yu Y , Patel BB , Majumdar AP
Published: December 2010

Abstract #3069

It is becoming increasingly evident that many solid tumors including breast cancer show increased activation of several growth factor receptors, specifically EGFR and its family members (EGFRs) as well as c-Src, a non-receptor tyrosine kinase that promote proliferation, inhibit apoptosis and induce metastasis. The EGF-receptor (EGFR) consists of an extracellular ligand-binding domain, a hydrophobic transmembrane region, and an intracellular tyrosine kinase domain. The extracellular or ectodomain of EGFR is essential for ligand-binding and subsequent homo/heterodimerization of the receptor. This raises the possibility that the ectodomain of EGFR could be utilized to inhibit EGFR functions and in turn cellular growth. Recently, we generated an ectodomain of human EGFR (hEGFR), composed of 1-448 amino acids fragment of hEGFR and the 30 amino acid epitope (known as “U” region) of rat EGFR-Related Protein (ERRP) fused at the carboxy-terminal end. The new protein, referred to as EBIP (ErbB Inhibitory Protein), as has been observed for ERRP (Xu et al. Mol. Cancer Ther. 2005 (4): 435-42), inhibits growth of colon and breast cancer cells that express varying levels of EGFR and/or its family members. We hypothesize that inhibition of c-Src and EGFRs will be an effective therapeutic strategy for breast cancer. To test our hypothesis we used Dasatinib (BMS-354825; Bristol-Myers Squibb), a newly developed highly potent, ATP-competitive Src and Abl kinase inhibitor and ErbB Inhibitory Protein (EBIP). Four different breast cancer cell lines (MDA-MB-468, SKBr-3, MDA-MB-453 and MDA-MB-231) expressing different levels of EGFR and/or its family members were used. Both EBIP and dasatinib caused a dose dependent inhibition of growth in all breast cancer cells, but dasatinib was less effective compared to EBIP. However, the combination of Dasatinib and EBIP synergistically inhibited cell growth. In EGFR overexpressing MDA-MB-468 cells, the combination therapy also inhibited invasion through extracellular matrix and decreased colony formation. Induction of apoptosis could be attributed to activation of caspases -9 and -8 and by modulating phosphorylation of anti- and pro-apoptotic proteins, Bcl-2 and Bad, respectively. The combination of Dasatinib and EBIP produced a greater inhibition of EGFR and Src activation and their downstream signaling pathways involving Akt and p44/42 MAPK. Additionally, we observed that EGFR promoter activities as well as transactivation of NFκB in MDA-MB-468 cells were greatly reduced. In conclusion, our data suggest that combination treatment of Dasatinib and EBIP that target EGFRs and Src could be a highly effective therapy for breast cancer than mono therapy, as they are likely to impact several aspects of tumor progression.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3069.
Bone marrow disseminated tumor cells, loco-regional and adjuvant treatment are prognostic factors for loco-regional relapses in early breast cancers.
Authors: Bidard F , Kirova Y , Vincent-Salomon A , Alran S , De Rycke Y , Sigal-Zafrani B , Sastre-Garau X , Mignot L , Fourquet A , Pierga J
Published: December 2010

Abstract #307

Purpose: To study in early breast cancer (BC) patients (pts) if bone marrow (BM) disseminated tumor cells (DTC) are associated with a specific loco-regional dissemination of cancer cells, according to the radiotherapy fields and to the pattern of loco-regional relapses.  Patient and methods: BM aspirates were prospectively screened for cytokeratin positive (CK+) cells at primary treatment in stage I-III BC pts. Local recurrence (LR) was defined as recurrence of the breast or chest wall; Regional recurrence (RR) was defined as lymph node recurrence after loco-regional treatment. Irradiation fields, BM DTC status, clinical and pathological variables of the pts were correlated with loco-regional relapse-free survival (LRRFS).  Results: BM DTC were detected in 94 out of 621 pts (15%) and were not associated with the axillary lymph node status and other prognostic factors. After a median follow-up of 56 months, 18 pts (2,9%) experienced loco-regional relapses: 15 LR occurred in the RT fields whereas 2 LR and 1 RR were out. 8 of the 18 relapsing pts (44%) were DTC positive (8 LR in the RT fields). BM DTC detection was the only prognostic factor for LRRFS (p=0.0005, OR=5.2 [2.0-13.1]) in multivariate analysis. In BM DTC positive patients, longer LRRFS were observed in those who were given adjuvant hormonal therapy (p=0.03) and RT on supra-clavicular (SCN) and internal mammary (IMN) nodes (p=0.055) (multivariate analysis).  Conclusions: BM DTC strongly influence loco-regional relapse in early BC patients. Our data support the hypothesis of a different lymphatic spread of BM DTC positive BC and of a possible re-seeding of BM DTC to the breast. Survival analyses suggest that BM DTC positive patients take advantage of adjuvant hormone therapy but also of adjuvant SCN and IMN irradiation. This observation could be one explanation of the influence of systemic treatment on locoregional relapse and of radiotherapy on distant metastatic dissemination.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 307.
Netrin-1: a promising therapeutic target in metastatic breast cancer. Expression analysis in localized and metastatic breast tumors and preclinical validation.
Authors: Fitamant J , Guenebeaud C , Treilleux I , Tabone E , Chabot S , Bernet A , Mehlen P , Bachelot T
Published: December 2010

Abstract #3075

Netrin-1, an axon navigation cue was proposed to play a crucial role during colorectal tumorigenesis by regulating apoptosis. The netrin-1 receptors DCC and UNC5H belong to the family of dependence receptors that share the ability to induce apoptosis in the absence of their ligand. Cells expressing one of these dependence receptors are thus dependent on ligand availability to survive. Such a trait confers on these receptors a tumor suppressor activity by preventing uncontrolled cell proliferation.  We have previously shown in breast cancer that gaining autocrine expression of the ligand netrin-1 would represent for cancer cells a selective advantage. Human and murine netrin-1-expressing mammary metastatic cell lines undergo apoptosis when netrin-1 expression is experimentally decreased or when decoy soluble receptor ectodomains are used. Such treatments prevent metastasis formation in a syngenic mouse model of lung colonization by a mammary cancer cell line (Fitamant et al., PNAS 2008, vol 105).  In order to assess the clinical importance of netrin-1 expression in human breast cancer, we quantified by Q-RT-PCR the mRNA levels of netrin-1 in primary breast cancer biopsies and studied their correlation to pathological and clinical data. We used frozen tumor samples from 120 breast cancer patients treated in our institution between 1993 and 2000. At diagnosis, median age was 50, median tumor size was 40 mm. 11% of the patients had SBR1 tumor, 46% had SBR2 tumor and 43% had SBR3 tumor. Estrogen receptor was positive for 62% of the patients. 17% of the patients were pN0, M0; 71% were pN+, M0 and 12% already had distant metastasis (M+) at time of diagnosis. Although there is no correlation between netrin-1 expression and tumor size, histological SBR grade or hormone receptor status, there is a strong correlation between high netrin-1 expression and the presence of nodal involvement (p=0,007) or distant metastasis at the time of diagnosis (p<0,0001). We hypothesize that high netrin-1 expression allows an early and rapid metastatic dissemination during tumor development. We are currently exploring netrin-1 expression levels at different metastatic sites and are completing survival analysis of our cohort of patients.  In parallel, preclinical mice models of xenografts either of human breast cancer cell lines or fresh human breast tumors were used to evaluate netrin-1 targeting strategy. The preliminary results show that disrupting netrin-1 activity appears to be more efficient than a conventional Adryamicin-Cyclophosphamide treatment in decreasing tumor sizes and suppressing lung metastasis.  These results support netrin-1 as a marker of metastatic dissemination and as a promising therapeutic target in breast cancer progression.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3075.
Nano-gossypin, a novel cdk2/VEGF inhibitor formulation against breast cancer.
Authors: Nair HB , Veerapaneni P , Sophie DM , Kirma NB , Perla RP , Tekmal RR
Published: December 2010

Abstract #3080

Cyclin-dependent kinases (CDKs) play essential roles in the intracellular control of the cell cycle. The encapsulated therapeutic drug molecules as nanoparticles will offer a sustained delivery of chemotherapeutics in to the target tissue. Nanoparticles enhance the therapeutic efficacy of an encapsulated drug by increased and sustained delivery of the drug inside the cell. Most anticancer drugs are taken up non-specifically by all types of cells, resulting in serious side-effects. Therefore, an ideal delivery carrier for an anticancer drug should be able to transport the drug specifically to cancer cells and release the drug molecules inside the cells to the site of their pharmacological activities. Most of the activity proven natural products were not entered in to the clinical practice due to lack of active formulation and/ or effective targeted delivery systems. In our study we found that gossypin (3,5,7,3',4'-Pentahydroxy-8-O-glucosylflavone) is inhibiting cell cycle progression by inhibiting cyclin dependent kinase-2 (cdk2) when compared with roscovitine, a known cdk2 inhibitor. Gossypin has been shown to induce the accumulation of the tumor suppressor p53, cell cycle arrest in G1 and G2/M phases of the cell cycle, and to induce apoptosis in MCF-7, SKBR-3, and MDA-MB-231 cells with a mean IC 50 of 40uM. Gossypin also found to inhibit vascular endothelial growth factor (VEGF) in vitro. Nanoparticle formulation of gossypin effectively inhibited the growth and colony formation in vitro when compared to parent compound. Gossypin might exert inhibition of tumor cell growth and invasion by cell cycle arrest as well as VEGF induced vascular permeability and tumor neovascularization. The dual mode of action of gossypin may results in highly efficacious inhibition of tumor growth and management of breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3080.
The antineoplasic effect of molecular iodine on human mammary cancer involves the activation of apoptotic pathways and the inhibition of angiogenesis.
Authors: Vega-Riveroll L , Mondragón P , Rojas-Aguirre J , Romero-Romo J , Delgado G , Aceves C
Published: December 2010

Abstract #3082

Breast cancer is the most frequent malignant neoplasia in women. One factor thought to influence the incidence of breast cancer is diet. Japanese women, who have a high dietary intake of iodine (5280 vs 209 µg/day in western population) due to ingestion of seaweed, have the lowest incidence of breast cancer in the world. In normal mammary gland, iodine deficiency is involved in dysplasias, which are reversible with I2 but not with iodide administration. Recent data generated in our laboratory showed that continuous treatment with I2, but not iodide, has a potent antineoplastic effect on tumoral progression in N-methyl-N-nitrosourea-treated virgin rats (50-70%). Also I2 treatment in MCF-7 human breast cancer cells, but not in normal mammary cells (MCF-12F), shows a significant apoptotic effect induced by activation of Bax-caspases and AIF-PARP-1 pathways. Based on these findings, a study of women diagnosed with cancer (mammography and tru-cut biopsy) was initiated. Patients were divided into placebo or I2 (5 mg/day) groups for 2 to 5 weeks before surgery. One day before beginning the I2 treatment and the day of the surgery, urine and blood samples were collected. The day before surgery, a second mammography was obtained. In the tumor samples proliferation (PCNA), apoptosis (TUNEL), and vasculature area (CD-31) were analyzed by immunohistochemistry. Bax, Bcl2, p53, and VEGF proteins were analyzed by Western blot. Circulating TSH and T3 levels were measured by RIA and urinary iodide by HPLC. The results showed that although tumor size did not differ between groups, a significantly lower proliferation and increased apoptotic rate were observed in tumors from women supplemented with iodine. In these tumors the ratio Bax/Bcl2 increased, p53 levels were unchanged, and VEGF levels and vasculature area diminished significantly. Serum TSH and T3 showed no changes in any group, indicating that I2 treatment had not compromised the thyroid status. We propose that I2 supplements should be considered for use in clinical trials of breast cancer therapies. We thank Dr. Jorge Álvarez-Aguirre2,3, Dr. Alonso Gallegos-Corona2, Lic. Concepción Correa-Tinajero3, Alejandro Núñez-Nolasco2,3, and Silvia Serrato-Águila2. This work was partially supported by: PAPIIT-UNAM IN201207, and CONACyT 44976-M and 85952.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3082.