bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P5-01-13: Flat Epithelial Atypia: Management and outcome in three Dutch teaching hospitals
Authors: Ghuijs PM , de Vries B , Strobbe LJA , van Deurzen CHM , Heuts EM , Keymeulen KBMI , Lobbes MBI , Wauters CAP , Van de Vijver KKBT , Smidt ML
Published: August 2014

Background: Flat Epithelial Atypia (FEA) is a presumably neoplastic alteration of terminal duct-lobular units, characterized by the replacement of native luminal epithelium by ductal cells demonstrating low-grade cytologic atypia. The architecture shows stratification of epithelial cells. FEA is often accompanied by microcalcifications and therefore discovered in biopsies following screening mammography. FEA is frequently seen in association with ADH (atypical ductal hyperplasia), DCIS (ductal carcinoma in situ), lobular neoplasia and invasive tubular carcinomas. There is emerging evidence suggesting FEA may represent a precursor to DCIS. The risk of subsequent breast carcinoma remains to be defined. The aim of this study is therefore to inventorise the management and outcome of solitary FEA in histological biopsies in three Dutch teaching hospitals.

Materials and Methods: Data of this retrospective multicentre study were collected in a database. Local pathology databases were screened with the terms: ‘FEA’, ‘Flat Epithelial Atypia’, ‘columnar atypia’ and Dutch equivalents. Results were manually screened, only including solitary FEA.

Patient files were viewed for information on presentation, mammography, ultrasound and management: surgery vs follow-up. In case of excision, definitive pathology was added.

Results: We included 103 patients showing only solitary FEA in the primary biopsy. Management of these patients consisted of follow-up for 60 patients (58,3%) and surgery for 43 patients (41,7%, 49 excisions): lumpectomy (42) or mastectomy (7). Reason for choosing mastectomy was preventive in case of contralateral breast cancer or increased familial or genetic risk.

Definitive pathology of lumpectomy or mastectomy showed no abnormalities or solitary FEA in 31 patients; other findings were ADH in 7, LCIS in 4 and DCIS in 7 patients. Some patients showed more than one finding. Invasive breast cancer (IBC) was detected in 3 patients. Only one mastectomy showed invasive disease, located in a different lobe, however.

No incidents occurred in the follow-up group.

Conclusions: No consistent management exists concerning solitary FEA in these three hospitals. Also, one hospital used the diagnosis of FEA inconsistently and interchangingly with other terms. Lack of this study is the retrospective gathering of data, making it difficult to detect the reasons for the chosen management. DCIS or IBC was discovered in 20,4% of all surgical specimens. It was concluded that FEA should be seen as a red flag, indicating the possible presence of a more malignant lesion. Additional research is warranted concerning long term follow-up for this patient group.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-01-13.
Abstract P5-01-14: Discrepancy between routine and expert pathologists in histological assessment of early stage non palpable breast cancer and its impact on loco regional and systemic treatment
Authors: Postma EL , Verkooijen HM , van Diest PJ , Willems SM , van den Bosch MA , van Hillegersberg R
Published: August 2014

Background: Histopathological parameters are essential for deciding on further adjuvant treatment in patients with breast cancer after surgery. Here, we assessed the impact of inter observer variability on further treatment strategy in patients with clinically node negative, non palpable breast carcinomas.

Methods: Clinical and histopathological data of 314 patients with clinically node negative non palpable invasive breast cancer were analysed. Histological assessment of the primary tumour and sentinel nodes was first performed in a routine setting, subsequently central review took place. In cases of discordance between local en central assessment, we determined the impact on locoregional and systemic treatment strategy.

Results: Discordance between local and central review was seen in 13% of the patients (kappa 0.60 95% CI 0.50–0.71) for type, in 12% (k = 0.796 95% CI 0.73–0.86) for grade, in 1% for ER status (k = 0.898 95% CI 0.80–1.0), in 2% for PR status(0.940 95% CI 0.89–0.99). Discrepancy in the assessment of the sentinel node(s) was seen in 2% of the patients. (k = 0.954 95% CI 0.92–0.98). Practising current Dutch Guidelines, loco regional treatment would be affected in 2% (7/310), systemic treatment in 5% (16/310) and both in 1% (2/310) of the patients.

For patients in whom central review would have led to additional systemic treatment (3%), the 10 years mortality and recurrence rate would decrease with a median of 4.6% and 15% respectively.

Conclusion: Inter observer variation in the histopathological assessment of non-palpable breast carcinoma specimens and sentinel nodes results in a different loco regional or systemic treatment advice in 8% of the patients.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-01-14.
Abstract P5-02-03: Large-scale genomic instability consistently identifies BRCA1/2 inactivation in breast cancers
Authors: Stern M-H , Popova T , Manié E , Dubois T , Sigal-Zafrani B , Bollet M , Sastre-Garau X , Vincent-Salomon A , Houdayer C , Stoppa-Lyonnet D
Published: August 2014

The goal of this study was to identify genomic markers predicting actual BRCA1/2 inactivation in breast cancers. By comparing genomic profiles of BRCA1/2 mutated and wildtype basal-like carcinomas (BLC) we developed a signature of BRCAness. The signature accounts for the level of the large scale genomic instability and was demonstrated to predict BRCA1/2 impairment with 100% sensitivity and 90% specificity in the large series of BLCs. We introduced a bioinformatics procedure evaluating the number of large scale rearrangements (denoted as Large Scale Transition or LST) in the genome and showed that the number of LSTs discriminate homologous recombination deficient and proficient tumors (Popova et al, Cancer Research 2012 Aug).

The efficiency of the signature of BRCAness represented by the number of LSTs was then evaluated for the luminal breast tumors and ovarian carcinomas. Luminal breast tumors and ovarian carcinomas could be stratified into two groups according to the number of LSTs. The performance of the LST signature in all types of breast cancers will be presented.

Measurement of LST is a highly reliable, simple and cost effective method to identify patients who should benefit of a BRCA1/2 testing, and to select tumors which may respond to treatment targeting DNA repair deficiencies (platinium salts, PARP inhibitors).

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-02-03.
Abstract P5-03-08: A fluorescence STAT3 reporter preferentially expressed in human breast cancer tumor-initiating cells
Authors: Wei W , Tweardy D , Zhang M , Roarty K , Rosen J , Lewis M
Published: August 2014

Cancer initiating/stem cells are thought to be responsible for treatment-resistance, disease-recurrence and metastasis. Current breast cancer stem cell markers lack direct correlation with stem cell function, which makes the targeted therapeutic design difficult. The STAT3 pathway is a critical regulator of the function of normal stem cells and evidence is accumulating for its important role in breast cancer stem cells, as well as associated “niche” cells. However, due to lack of method for pathway-activity-based live cell separation, whether STAT3 functions in the cancer stem cells themselves or in surrounding niche cells, or both remains unknown. To answer this question, we have constructed lentiviral fluorescent reporters, which enables FACS-sorting of cells differ in STAT3 activity as well as in vivo/in situ localization of STAT3 responsive cells. Our study for the first time separated the STAT3 positive and STAT3 negative cells from the same breast tumor in live, and for the first time provided functional evidences that the STAT3 positive cells are enriched for breast cancer stem cells compared to STAT3 negative cells, as tumor cells positive for reporter activity perform better in both mammosphere formation and dilution transplantation assays compared to the negatives.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-08.
Abstract P5-03-09: Receptor Activator of Nuclear Factor Kappa B (RANK) as a potential therapeutic target in triple-negative breast cancer
Authors: Reyes ME , Masuda H , Zhang D , Reuben JM , Woodward W , Darnay BG , Hortobagyi GN , Ueno NT
Published: August 2014

Background: Triple-negative breast cancer (TNBC) has a poorer prognosis in comparison to other breast cancer subtypes because of its high metastatic rate. There is evidence that cancer stem cells (CSCs), known to have an epithelial-to-mesenchymal (EMT) phenotype, may contribute to the metastasis of breast cancer. Denosumab (an anti-RANK ligand antibody) is currently being explored as a treatment for breast cancer in phase II and III clinical trials. RANK is thought to regulate mammary stem cells. Therefore, we hypothesized that RANK promotes tumorigenicity and metastasis of TNBC via the regulation of EMT and enrichment of CSCs. We sought to determine the effects of RANK expression on the tumorigenic and metastatic activities of TNBC cells and whether RANK is a potential therapeutic target in TNBC.

Methods: Primary breast tumor specimens from the MD Anderson Cancer Center Tumor Bank were stratified by ER/HER2 status. We analyzed RANK mRNA expression levels, obtained by Affymetrix gene chip array, in ER+/HER2-negative breast tumors (n = 22) and ER−/HER2-negative breast tumors including TNBC tumors (n = 18). To study the effects of RANK suppression in TNBC cells, we generated two stable RANK shRNA MDA-MB-231 TNBC cell lines and a negative shRNA cell line as a control and examined whether knockdown of RANK expression correlated with a decrease in mesenchymal markers, vimentin and snail, and stem cell marker, ALDH1, invasion, migration, and tumorigenicity in TNBC cells.

Results: An analysis of RANK mRNA expression levels among human primary breast cancer specimens revealed increased RANK expression in ER−/HER2-negative primary breast tumors including TNBC tumors, compared with ER+/HER2-negative primary breast tumors (p = 0.034). Knocking down RANK in MDA-MB-231 cells significantly inhibited migration (p = 0.0005) and invasion (p = 0.0006) and reduced the stem cell subpopulation, as shown by a decrease in mammospheres (p = 0.0188) and ALDH1 activity. Further, in a three-dimensional (3D) cell culture model, we observed significant inhibition of projections in MDA-MB-231 cells with knockdown of RANK (p < 0.05), which correlated with downregulation of the mesenchymal markers vimentin and snail shown by qRT PCR analysis.

Conclusion: Our data demonstrate that RANK may promote the tumorigenesis of TNBC by enriching cancer stem cells. In future studies, we will investigate the effect of RANK inhibition on metastasis in vivo using a TNBC xenograft model. Our long-term goal is to develop RANK-targeted therapy for TNBC.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-09.
Abstract P5-03-12: Targeting breast cancer stem cells using the autopahgy inhibitor N-Acetyl cysteine
Authors: Dave B , Granados S , Mitra S , Chang JC
Published: August 2014

Introduction: One in eight women is diagnosed with breast cancer in the United States. The most aggressive form of breast cancer is the “triple negative breast cancer” (TNBCs), where there is a lack of expression of all three receptors, namely ER, PR and HER2 and a lack of targeted therapies lead to the highest relapse rate in breast cancer. This makes it imperative to identify and target the mechanism of relapse of this cancer. We have recently identified autophagy as a mechanism of tumor resistant cell survival in breast cancer. We have demonstrated an increase in autophagy in chemotherapy treated patients. Further, we have shown that addition of a stem cell inhibitor against NOTCH reduces autophagy and stem cells population. In order to eliminate these tumor resistant cells from surviving chemotherapy, we plan to target the cell survival pathway of autophagy using N-acetyl csyteine as a novel inhibitor.

Materials and Methods: We treated three triple negative cell lines (SUM159, BT549 and MDA-MB231) with varying concentrations of N-acetyl cysteine and determined its impact on tumor initiating cells via mammosphere formation and FACS sorting of CD44hi/CD24low cells. N-acetyl cysteine affects mitochondrial metabolism so we tested its impact on mitochondrial DNA mass.

Results: N-acetyl cysteine significantly decreased TIC population as evidenced by the remarkable reduction in mammosphere formation efficiency and levels of CD44hi/24low cells at 1 and 10uM in all three cell lines. In two cells lines we have demonstrated that there is a significant increase in mitochondrial mass upon treatment of NAC.

Conclusion: We have currently determined that N-acetyl cysteine works via autophagy and eliminates tumor initiating cell population. We have also demonstrated that this involves changing the mitochondrial mass and overall changes in the metabolism of these cells. This novel interlink between mitochondrial metabolism and autophagy provided a new insight into the role of tumor initiating cells in breast cancer and possible new approaches to treat therapy resistance in triple negative breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-12.
Abstract P5-03-13: Detection of tumor initiating cells (TIC) among the peripherally circulating epithelial tumor cells from patients with breast cancer
Authors: Pachmann K , Zimon D , Pizon M , Carl S , Rabenstein C , Camara O , Pachmann U
Published: August 2014

Background: The detection of tumor cells circulating in the peripheral blood of patients with breast cancer is a sign that cells have been able to leave the primary tumor and survive in the circulation. However, in order to form metastases they require additional properties such as the ability to adhere, self renew and grow. Here we present data that a variable fraction among the circulating tumor cells detected by the maintrac® approach express mRNA of the stem cell gene nanog and of the adhesion molecule vimentin and are capable of forming tumor spheres a property ascribed to TIC.

Patients and methods: Between 10 to 50 circulating epithelial antigen positive cells detected by the maintrac® approach were selected randomly from each of 20 patients with breast cancer before and after surgery and isolated using automated capillary aspiration and deposited individually onto slides for expression profiling. In addition, the circulating tumor cells were cultured without isolation among the white blood cells from 5 patients with breast cancer in different stages of disease using culture methods favoring growth of epithelial cells.

Results: Although no EpCAM mRNA positive cells expressing stem cell genes or the adhesion molecule vimentin were detected before surgery, 10 to 20% of the cells were found to be positive for mRNA of these genes after surgery. Most surprisingly, it was possible to grow tumor spheres (Fig 1) from these circulating cells without previous isolation in all patients in a fraction comprising between 1% of the epithelial antigen positive cells in patients with newly diagnosed tumor up to 10% in a patient with advanced breast cancer.

Conclusion: We here show, that among the peripherally circulating tumor cells a variable fraction is able to express stem cell and adhesion properties and can be grown into tumor spheres, a property ascribed to cells capable of initiating tumors and metastases.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-13.
Abstract P5-03-14: Expression of ALDH1 in metastasizing axillary lymphnodes in breast cancer
Authors: Shi A , Dong Y , Bi L , Xu N , Fan Z , Li S , Yang H , Li Y
Published: August 2014

Background: There is increasing evidences that a wide variety of malignancies, including breast cancer, may be driven by a small subset of ‘tumor-initiating cells’ or ‘cancer stem cells’ (CSC) which are able to form tumors in immunocompromised mice as well as to generate the phenotypic heterogeneity of the initial tumor. Enzyme aldehyde dehydrogenase (ALDH1) has been reported as a possible marker for mammary CSC. These cells are a source of tumor recurrence and metastasis, and are resistant to chemotherapy, radiotherapy and hormone therapy.

Objective: Assuming that the detection of CSC in axillary lymph nodes is more effective to predicting cancer outcome than the widely used detection of cancer cells in axillary lymph nodes, we measure ALDH1 levels to predict their presence into axillary lymph nodes on development of cancer and anticipate outcomes.

Methods: ALDH1 protein was detected by an immunohistochemical technique in 229 cases of breast cancer diagnosed from 2002 to 2011 Follow-up ranged from 11.5 months to 96.9 months, with a mean of 73.9 months. A survival assay was used to determine the relationship between distant metastatic rate and survival rate.

Results: ALDHl expression was detected in 79cases and the Positive rate in metastatic axillary lymph nodes was 34.5%. Negative ER, PR status were related to the ALDH1 positive cases(P = 0.012). See Table 1. Mortality rate between ALDH1 positive cases (50.8%) and negative cases (28.8%) were significantly different (P = 0.001). See Table 2. Further, survival analysis of recurrence-free survivals (RFS) and survival rate decreased significantly between ALDHl positive and negative cases (P = 0.001) (see table 2) and COX analysis shows that ALDH1 expression is an independent predictor of poor outcome in breast cancer(P = 0.011).

Discussion: What cancer stem cells migrate to the axillary nodes have more important prediction than that the matastesis of normal cancar cells in axillary node. It might be a role resulting in dying in breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-14.
Abstract P5-04-01: Expression of epithelial-mesenchymal transition (EMT)-related markers in primary tumors and matched lymph node metastases in breast cancer patients
Authors: Zaczek AJ , Ahrends T , Markiewicz A , Seroczynska B , Szade J , Welnicka-Jaskiewicz M , Jassem J
Published: August 2014

Background. Malignant tumors may include multiple cancer cell populations with various metastatic potential. More aggressive subpopulations might easier be captured in lymph nodes metastases (LNM) than in primary tumors (PT). We evaluated mRNA and protein levels of master EMT regulators: TWIST1, SNAIL and SLUG, protein levels of EMT-related markers: E-cadherin, vimentin, and expression of classical breast cancer receptors: HER2, ER and PR in PT and corresponding LNM. The results were correlated with clinicopathological data and patient outcomes.

Methods. Formalin-fixed paraffin-embedded (FFPE) samples from PT and matched LNM from 42 stage II-III breast cancer patients were examined. Expression of TWIST1, SNAIL and SLUG was measured by RT-qPCR, with median as cut off for positive result. Protein expression was examined by immunohistochemistry on tissue microarrays. For TWIST1, SNAIL, SLUG, E-cadherin and vimentin >10% of positively stained cells defined positive result. ER and PR were scored according to Allred system, and HER2 - according to HercepTest criteria. Results were considered concordant if PT and LNM were both positive or both negative. Concordance was measured by estimating Cohen's kappa coefficient (κ), with κ value equal 1 indicating perfect agreement. Disease-free survival (DFS) and overall survival (OS) were compared using F-Cox test. Hazard ratios (HRs) with 95% confidence intervals (95% CI) were computed using Cox regression analysis.

Results. On average, expression of TWIST1, SNAIL and SLUG was significantly higher in LNM compared to PT (P < 0.00001 for all). Gene and protein levels of TWIST1, SNAIL and SLUG were highly discordant between PT and matched LNM (Tab 1). Receptor conversion, particularly loss of ER and PgR, occurred in 18% and 29% of cases, respectively. Increased expression of TWIST1 and SNAIL in LNM was associated with shorter OS (p = 0.045 and p = 0.022, respectively) and DFS (p = 0.02 and p = 0.01, respectively), whereas their expression in PT had no prognostic impact. Negative-to-positive switch of TWIST, SNAIL and vimentin correlated with shorter OS and DFS. Conversion of ER and PR had no significant impact on survival.

Conclusions. LNM are enriched in cells with more aggressive phenotype, marked by elevated levels of EMT regulators. High expression of TWIST1 and SNAIL in LNM, as well as negative-to-positive conversion of both TWIST1 and SNAIL confer worse prognosis, confirming the correlation of EMT with aggressive disease behavior.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-01.
Abstract P5-04-02: DYRK2 regulates breast cancer invasion via Snail/E-cadherin pathway
Authors: Mimoto R , Imawari Y , Kamio M , Kato K , Nogi H , Toriumi Y , Takeyama H , Yoshida K , Uchida K
Published: August 2014

Introduction: Epithelial-Mesenchymal Transition (EMT) is characterized by the loss of cellular adhesion molecule E-cadherin, and plays a fundamental role during early steps of breast cancer invasion. An important regulator of E-cadherin expression is Snail, a zinc finger transcriptional repressor. Snail is posphorylated by GSK3β and then degraded by βTrCP mediated ubiquitination. Here we found another kinase, DYRK2, regulates the stability of Snail. Knockdown of DYRK2 promoted the EMT and cancer invasion both in vitro and in vivo experiments.

Methods: Cell lines: MCF-7 cells were transfected with pSuper vector (pSuper control) and pSuper shRNA DYRK2 (shRNA-DYRK2) with G418 to isolate stable cell lines.

Immunoblotting: Expression of pertinent molecular markers was determined by western blotting.

RT-PCR: RNA was extracted using TRIsure and 500 ng of total RNA was amplified using Primer Script One Step RT-PCR Kit Ver.2.

Invasion assay: Various cancer cell lines were seeded on the top of the upper chamber with serum free medium while the bottom chambers were filled with medium containing 10% FBS.

In vivo metastasis assay: Cells in PBS were injected into the left ventricle of 7-week-old female nude mice. Metastases to distant organs were confirmed by IVIS2000.

Immunohistochemistry: We acquired paraffin-embedded tissue sections from primary breast tumor cores and from Jikei University of Medicine. Immunohistochemistry for DYRK2 was performed.

Results: In MCF-7 cells, knockdown of DYRK2 increased Snail in the protein level but not in mRNA level. Upon DYRK2 knockdown, MG132 treatment had no effect on additional increase in Snail. DYRK2 knockdown decreased Snail ubiquitination. Stable DYRK2 depletion led to Snail accumulation and E-cadherin abrogation. Fibroblast marker, Vimentin, emerged in DYRK2 depleted cells. For further analysis, we carried out invasion assays. The invasion potential in DYRK2-depleted cells was substantially higher than that in control cells. In xenograft model, we used nude mice received intracardial injections of pSuper control or shRNA-DYRK2 cells. 6 weeks after injections, a significant increase in bone and lung metastasis was observed in shRNA-DYRK2 group. The patients with tumors expressed low DYRK2 showed significantly poor prognosis.

Conclusion: In breast cancer cells, Snail is phosphorylated by DYRK2 and then acquires the ability to be ubiquitinated. In the absence of DYRK2, Snail is unable to be degraded by ubiquitin-proteasome machinery. Accumulation of Snail promotes EMT and cancer invasion, so low expression of DYRK2 leads to poor prognosis in breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-02.
Abstract P5-04-03: Epithelial-mesenchymal transition is associated with in situ to invasive transition of basal-like breast cancer
Authors: Park SY , Choi Y , Kim EJ , Lee HE , Lee HJ , Kang E , Kim S-W
Published: August 2014

BACKGROUND: Epithelial-mesenchymal transition (EMT) is known to play an important role in breast cancer invasion and metastatic progression and to be associated with cancer stem cells and basal-like subtype. However, its role in the progression of in situ to invasive breast carcinoma is uncertain. To investigate this question, we evaluated the difference in the expression of EMT-related markers between pure ductal carcinoma in situ (DCIS) and invasive carcinomas according to molecular subtype.

METHODS: We performed immunohistochemical analyses of EMT-related markers [expression of vimentin, smooth muscle actin (SMA), osteonectin and N-cadherin, translocation of β-catenin and loss of E-cadherin] and breast cancer stem cell markers (CD44+/CD24−, ALDH1) in 320 invasive carcinomas and 179 pure DCIS of breast using tissue microarrays. We also analyzed 39 basal-like invasive cancers with adjacent DCIS component to determine the difference in the expression of EMT-related markers in the invasive and DICS component within individual cases.

RESULTS: In invasive carcinomas, vimentin, SMA and osteonectin were highly expressed in basal-like subtype. In addition, loss of E-cadherin and translocation of β-catenin were most frequently found in basal-like subtype. Furthermore, there were positive correlations between the expression of EMT-related markers and stem cell markers (CD44+/CD24- and ALDH1). However, there was no significant difference in the expression of EMT-related markers according to molecular subtype in pure DCIS. When comparing invasive carcinoma with pure DCIS, expression of EMT-related markers was significantly higher in invasive carcinoma than in pure DCIS. Subgroup analysis revealed higher expression of EMT-related markers in invasive carcinoma than in DCIS in basal-like subtype, but not in non-basal-like subtypes. Moreover, in 39 basal-like invasive cancers with adjacent DCIS, expression of mesenchymal markers was increased in invasive component compared to DCIS component.

CONCLUSIONS: Our study confirmed that EMT is an intrinsic characteristic of basal-like subtype and is associated with breast cancer with stem cell phenotype. However, increased expression of EMT-related markers in invasive carcinoma compared to pure DCIS, especially in basal-like subtype, and in the invasive component of basal-like invasive carcinoma with DCIS component also suggests a role of EMT in the transition of in situ- to invasive carcinoma in basal-like breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-03.
Abstract P5-04-09: Squamous Cell Carcinoma Antigen 1 (SCCA1) Upregulation Causes Epithelial to Mesenchymal Transition (EMT) and Promotes Mammary Tumorigenesis
Authors: Sheshadri N , Ullman E , Catanzaro J , Chen E , Zong W-X
Published: August 2014

SCCA1, a member of the family of serine protease inhibitors, was first identified as a serological marker for cervical cancer but has since been shown to be upregulated in several other cancers including lung, head and neck, liver, skin, and breast. SCCA1 is believed to protect cells from unscheduled cathepsin release and activation upon lysosomal injury. In order to understand the molecular role of SCCA in cancer, we ectopically expressed it in the immortalized yet non-tumorigenic mammary epithelial cell line MCF10A. Expression of SCCA1 impairs the efficiency of lysosomal as well as proteasomal protein degradation pathways. Transcriptional profiling of SCCA expressing cells by microarray demonstrates an altered gene expression pattern that resembles EMT. Phenotypically, SCCA1 expression leads to spindle shaped cell morphology, increased migratory potential, resistance to anoikis, and the ability to form colonies in soft agar. SCCA1-expressing MCF10A cells can form tumors when injected into the mammary fat pad of immune-compromised mice. Correlative to the transforming activity, SCCA1 expressing cells have enhanced Erk and Akt signaling pathways, and have bypassed the requirement of growth factors and hormones that are essential for proliferation of MCF10A cells. In a transgenic mouse model, where SCCA1 is conditionally expressed in mammary epithelial cells under the control of MMTV promoter, increased mammary gland ductal branching and hyperplasia are observed. These findings indicate a previously unknown role of SCCA1 in promoting EMT and tumorigenesis, possibly by inducing chronic misfolded protein stress that contributes to enhance tumor-promoting signaling.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-09.
Abstract P5-05-03: The 5p12 breast cancer susceptibility locus is associated with MRPS30 expression in estrogen receptor - positive tumors
Authors: Quigley DA , Van Loo P , Alnæs GG , Tost J , Zelenika D , Balmain A , Børresen-Dale A-L , Kristensen VN
Published: August 2014

Genome-wide association studies (GWAS) have identified numerous loci linked to breast cancer susceptibility, but few of these loci have been convincingly linked to causal variants. We identified genes whose mRNA expression is linked to germline variation in normal mammary tissue and breast tumors by performing a genome-wide expression Quantitative Trait Locus (eQTL) analysis in 97 samples of normal mammary tissue and 286 breast adenocarcinomas. In the tumors we found 164 cis-acting loci, which affect expression of nearby genes. Twenty nine of these loci are previously unreported in other eQTL studies, including a cis-acting locus at 5p12 affecting expression of the mitochondrial ribosomal 28S subunit protein MRPS30 in estrogen receptor-positive but not estrogen receptor-negative tumors. This locus has been associated with susceptibility to estrogen receptor-positive breast cancer in several GWAS, with MRPS30 being the suggested candidate gene. One of these studies found that the rs771660 variant was most significantly associated with breast cancer in the 5p12 region, and we found that rs771660 was most strongly associated with MRPS30 gene expression. We provide the first evidence that this breast cancer susceptibility locus affects MRPS30 expression specifically in estrogen receptor-positive tumors and suggest a mechanism for estrogen activation of MRPS30 via proteins in the AP-1 complex.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-05-03.
Abstract P5-05-04: Intraductal delivery of RNAi-based therapeutics to gene targets identified through computational systems modeling
Authors: Brock A , Krause S , Li H , Kowalski M , Collins J , Ingber D
Published: August 2014

We utilize a computational systems biology approach to identify gene targets with the potential to serve as targets for therapeutic RNAi. Inferred gene regulatory network models were constructed using whole transcriptome expression profiling for progressive stages of mammary tumorigenesis in FVB C3(1)-SV40Tag mice. These computational models predicted key mediator transcription factors that are critical for hyperplasia and ductal filling in the early stages of disease progression. Putative mediator transcription factors were targeted by RNAi first in a mammary spheroid assay. Based on their ability to restore lumen formation and normal apical-basal polarization to mammary tumor cells, two transcription factors, Hoxa1 and Hoxb13, were selected for validation in vivo. siRNA targeting each of these transcription factors was formulated with lipidoid nanoparticles and delivered to the mammary gland via intraductal injection. Silencing Hoxa1 resulted in a significant reduction in tumor incidence in FVB C3(1)-SV40Tag. This represents a novel strategy to achieve localized silencing of a target gene in the mammary epithelium.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-05-04.
Abstract P5-05-05: Computational modeling of breast cancer growth and spread: the role of matrix degrading enzymes
Authors: Deakin NE , Thompson AM , Chaplain MAJ
Published: August 2014

Background: A “hallmark” of cancer growth and metastatic spread is the process of local invasion of the surrounding tissue. Cancer cells achieve this by the secretion of enzymes involved in proteolysis (tissue degradation) including matrix metalloproteinases (MMPs). These over-expressed proteolytic enzymes then proceed to degrade the host tissue allowing the cancer cells to disseminate throughout the microenvironment by active migration and interaction with components of the extracellular matrix such as collagen. The implications of MMP-2 activation by MMP-14 and the tissue inhibitor of metalloproteinases-2 (TIMP2) can be considered alongside the suitability of the matrix to be invaded. The suitability of the matrix incorporates pore size, since in some highly dense collagen structures such as breast tissue, the cancer cells are unable to physically fit through a porous region and rely on membrane-bound MMPs to forge a path through which degradation by other MMPs and movement of cancer cells can occur.

Method: We have developed a multi-scale mathematical model of breast cancer cell invasion of host tissue by combining both micro- (cell) and macro- (tissue) scale dynamics. The model considers cancer cells and two matrix-degrading enzymes from the MMP family of differing mechanistic actions, namely MMP-2 and the membrane bound MMP-14 (also called MT1-MMP), and their interaction with, and effect on, the extracellular matrix (ECM) in a 2-D spatial domain.

For the macro-scale we use a system of reaction-diffusion-taxis partial differential equations to capture the qualitative dynamics of the migratory response of the cancer cells. The micro-scale operates over a smaller spatial and temporal scale, and as such allows us to explicitly examine the stochastic role which is inherent within the region of enzyme operation. Computational simulations of the equations predict the spatio-temporal evolution of the cancer cell density, the concentration levels of the various enzymes and the density of the extracellular matrix.

Results: The model exhibits a rich range of dynamic and heterogeneous spatio-temporal solutions, which qualitatively match experimental and clinically observed results for aggressive breast carcinoma invasion and can be used to determine the speed of invasion through a variety of structured mediums such as the highly dense collagen constitution of some peritumoral stroma. Using the “worst case” scenario, an estimate for the maximum region of invasion over time from the initial data can be obtained suggesting the likely extent of breast cancer cell invasion into the surrounding tissue.

Conclusion: This modeling is most relevant to and informs the biological processes occurring in early stage breast cancer, in which local invasion is a crucial aspect of tumor behavior.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-05-05.
Abstract P5-06-02: The Role of Breast Tumor Kinase (Brk) in Cell Cycle Control
Authors: Nimnual AS , Chan E
Published: August 2014

Brk promotes cell proliferation and has been associated with multiple mitogenic pathways. However, the role of Brk in the cell cycle is still elusive. Here, we present a mechanism of Brk-induced cell cycle progression. Brk downregulates the cell cycle inhibitor p27 by altering its transcription factor FoxO3a subcellular localization. p27 is a key component of the G1/S cell cycle checkpoint and downregulation of p27 promotes S phase entry. The progression of the cell from G1 to S phase, induced by Brk, is independent of growth factors suggesting a role for Brk in malignant transformation. FoxO3a appears to be a major component in p27 down-regulation by Brk as the effect of Brk on p27 expression is significantly abrogated in cells transfected with siRNA targeting FoxO3a. The induction of FoxO3a nuclear exclusion by Brk is inhibited by LY294002 indicating that the mechanism is mediated by the PI3-kinase/Akt pathway. The mechanism underlying PI3-kinase mediated Brk function is still under investigation with evidence suggesting the involvement of the translocation of epidermal growth factor receptor. These data suggest that perturbation of p27 expression induced by Brk causes S phase entrance. Deregulation of the cell cycle is a key event in neoplasia, and thus, the mechanism presented here likely contributes to breast cancer development.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-06-02.
Abstract P5-07-02: Analysis of a novel synergistic relationship between the FK506 binding protein FKBP52 and beta catenin in androgen receptor signaling pathways
Authors: Storer CL , Olivares K , Fletterick RJ , Webb P , Cox MB
Published: August 2014

Hormone-dependent cancers depend largely on folding and regulation by a chaperone-steroid receptor complex. The final stage of the receptor-chaperone complex consists of a receptor, a heat shock protein 90 (Hsp90) dimer, p23, and an immunophilin. Hormone-dependent prostate cancer progresses due to key interactions between the androgen receptor complex and the ligand dihydrotestosterone. While current treatments block androgen receptor-ligand interactions, these therapies are no longer effective in advanced stage, hormone independent prostate cancer (HRPC), when the receptor is activated even in miniscule levels of hormone. Therefore, we are interested in targeting other members of the androgen receptor complex and signaling cascade, namely the immunophilin FK506 binding protein (FKBP52) and the Wnt cell signaling protein β-catenin. FKBP52 has the ability to potentiate activity of the androgen, progesterone, and glucocorticoid receptors and is a prostate cancer biomarker. β-catenin is a member of the cellular adhesion complex with E-cadherin in healthy cells, but in aberrant situations, β-catenin actively promotes progression and invasion in a number of cancers. Our data from FKBP52 knockout mouse embryonic fibroblast cell lines suggests that FKBP52 and β-catenin work in tandem to synergize androgen receptor (AR) transcriptional activity with the probasin promoter. When AR, FKBP52, and β-catenin are co-transfected, a synergistic up-regulation of AR signaling is observed, as assessed by luciferase reporter assays. According to pull down assays, FKBP52 and β-catenin can interact in the absence of other proteins. Luciferase assays of mutant FKBP52 proteins suggest that FKBP52's binding to β-catenin involves the FKBP52 proline-rich loop and may act independently of Hsp90 binding. Interestingly, it appears that this synergistic effect is promoter-specific, suggesting that regulation by these proteins occurs at the transcriptional level. We have successfully abrogated the synergism of these proteins using the FKBP52-specific inhibitor MJC13, a small molecule developed for use in our laboratory. According to surface plasmon resonance studies, this molecule binds the androgen receptor at the BF-3 regulatory surface, the interaction surface for FKBP52 regulation of receptor. According to recent findings, AR, Wnt, and Human Epidermal Growth Factor Receptor 2 signaling pathways are linked in molecular apocrine breast cancer progression. We therefore hypothesize that our findings could be relevant to breast cancer as well as prostate cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-07-02.
Abstract P5-07-05: Insulin-like growth factor binding protein-3 is a key component of the breast cancer cell response to DNA-damaging therapy
Authors: Baxter RC , Lin MZ , Marzec KA , Martin JL
Published: August 2014

Introduction. Breast cancer cells frequently develop resistance to DNA-damaging therapies, such as radiation and the cytotoxic drugs doxorubicin and etoposide, and activation of the epidermal growth factor receptor (EGFR) may play an important role in this process. However many estrogen receptor (ER)-negative breast tumors respond poorly to DNA-damaging drugs even when combined with an EGFR inhibitor. EGFR can modulate the repair of DNA double strand breaks (DSB) by non-homologous end-joining (NHEJ), by forming protein complexes that include the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). In previous studies we have shown that the growth-regulatory protein, insulin-like growth factor binding protein-3 (IGFBP-3), can translocate to the nucleus, is a substrate for DNA-PKcs, and can transactivate EGFR. The aim of this study was to delineate the role of IGFBP-3 in the response of breast cancer cells to DSB-inducing chemotherapeutic agents.

Results. In the triple-negative breast cancer cell lines MDA-MB-468 and Hs578T, which express IGFBP-3 highly, nuclear localization of EGFR and IGFBP-3, and of their complex as measured by coimmunoprecipitation (coIP), was enhanced by exposure to etoposide or doxorubicin, peaking 2 h after etoposide treatment. This effect was blocked by the EGFR kinase inhibitor gefitinib. Concomitantly, the co-location of EGFR-IGFBP-3 complexes with lipid rafts, visualized by proximity ligation assay (PLA) and confocal microscopy, decreased in response to drug treatment, consistent with the loss of this complex from the plasma membrane. Similar to the nuclear DNA-PKcs-EGFR complex, the nuclear DNA-PKcs-IGFBP-3 complex (seen by both coIP and PLA) was greatest 4 h after treatment. DNA-PKcs activation at serine-2056, required for the repair of DNA DSB and stimulated by etoposide treatment, was decreased by IGFBP-3 downregulation using two separate siRNAs, and this treatment also attenuated the enhanced DNA-PKcs-EGFR complexes seen in response to etoposide. Collectively these data suggest that IGFBP-3 has an obligatory role in the DNA repair response to DNA-damaging therapy through its interactions with EGFR and DNA-PKcs.

Conclusion. IGFBP-3 co-translocation to the nucleus of breast cancer cells and its complex formation with DNA-PKcs and EGFR in response to DNA damage demonstrate its previously unrecognized involvement in the regulation of DNA DSB repair by NHEJ. These novel findings suggests the possibility of a therapeutic approach for sensitizing ER-negative breast cancers to chemo- or radiotherapy by targeting the DNA repair function of IGFBP-3. Supported by the Australian Research Council.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-07-05.
Abstract P5-09-01: Evaluation of BCL2 sequence variant in Iranian women patients with Breast cancer
Authors: Motahari B , Ghaffarpour M , Javadi GH , Houshmand M
Published: August 2014

Introduction: Despite recent advancements in the treatment and management of cancers, Breast cancer still remains the most common malignancy and second leading cause of cancer-related deaths among women in Iran. Apoptosis and cellular proliferation play role an important role during normal mammary development and carcinogenesis of the mammary gland. In normal tissues, the homeostasis between apoptosis and cell proliferation is regulated by Bcl-2 family proteins. Tumor cells tend to interfere with the mechanism of Apoptosis by activating anti apoptotic genes such as bcl-2. BCL-2 gene, located at 18q21.3 and consists of three exons and two promoters (P1 and P2), which have different functions. The second promoter, P2, is located 1,400 bp upstream of the translation initiation site and functions as a negative regulatory element to the P1 promoter. Bcl-2 is the one of the most important anti apoptotic genes. It has been shown that the −938AA genotype s associated with higher Bcl-2 expression levels in B-CLL and poor prognosis. Bcl2 gene has been also demonstrated with breast cancer development and a single nucleotide polymorphism (SNP-938C >A) has been identified recently and can provide a susceptible factor for causing breast cancer.

The main objective of this study was to evaluation of the frequency of specific genetic alterations in the BCL-2 gene (−938,-758 and 21) and the risk of breast cancer in Iranian women patients with Breast cancer.

Materials and Methods: Patients; 27 paired Fresh tumor and adjacent normal samples were obtained from consecutive patients with BC who underwent surgery for mama mastroctomy from IRANIAN National Tumor Bank, National Cancer Institute, Imam Khomeini Hospital Complexes, Medical Tehran University, Tehran, Iran. Histopathological examinations were performed, and all tumors were confirmed as adenocarcinoma.

Mutational analysis: Fresh tumors and their adjacent were extracted for genomic DNA using the QIA amp Mini Kit according to the manufacturer's instructions. We searched DNA samples of Iranian patients for identification of SNP; −938C>A, −758T insertion in the inhibitory P2 promoter and 21A/G in exon 2 of the BCL-2 gene by means of PCR sequencing.

Results and conclusions: In All cases, the frequency of genetic alterations in the BCL-2 gene (−938, −758 and 21) was 16 of 27 (59.29%) heterozygous in −938(C, A) and 5 of 27(18.51%) SNP-938 C>A, 0 of 27(0%)and 8 of 23(34.78%)heterozygous in +21(A, G)and 2 of 23(8.69%)SNP 21A>G respectively. According to our findings, point mutation in −938C and 21A alleles displayed a positive relation with increasing tumorgenesis in breast cancer. Our result indicated that the regulatory BCL2 promoter polymorphism (−938C, A) and coding BCl2 gene (21A, G) may be useful as a tumor marker for increasing tumorgenesis in Iranian women breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-09-01.
Abstract P5-10-01: MicroRNAs −181 and −135a modulate tumour infiltrating immune cell programs in distinct molecular breast cancer subtypes
Authors: Paladini L , Truglia M , Arcangeli A , De Mattos Aruda L , Bianchini G , Iwamoto T , Bottai G , Pusztai L , Calin GA , Di Leo A , Santarpia L
Published: August 2014

Background and Aims: MicroRNAs (miRNAs) are short non-coding RNA whose expression levels have diagnostic and prognostic implications in cancer. However, the relevance of aberrantly expressed miRNAs to breast tumour-immune response has not been established. We aimed to assess the association between deregulated miRNAs and tumour immune response in breast molecular cancer subtypes.

Methods: One hundred and seventy ductal infiltrating breast carcinomas (BC) (ER+/HER2− n=85, HER2+ n=40, ER−/HER2− n=45) were evaluated for the presence of cellular immune components by IHC assay. CD3, CD20 were used as markers for T lymphocytes infiltration (TIL), and B lymphocytes infiltration (BIL), respectively, whereas CD68 for the presence of tumour-associated macrophage (TAM). Total RNA (including miRNA) was extracted from all tumours and 32 highly specific miRNAs and 276 immune genes were evaluated by array and qRT-PCR assays. We derived an integrated immune miRNA-regulatory gene profile to tease out relationships of these genes as potential targets of specific miRNAs. In vitro experiments on BC cell lines were performed to confirm potential links between deregulated miRNAs and immune genes.

Results: Lymphocytes infiltration (LI) was most common in HER2+ and ER−/HER2− BC samples (P < .004 and < .001) although B and T cells components were differently distributed in these 2 BC groups. TAM was a frequent event in ER−/HER2− BC subtype (P < .005). By gene and integration analyses we identified important miRNA immune-modulated target genes associated with prognostic factors and with molecular BC subtypes. We focused our studies on miR-181 and miR-135a that were significantly associated with ER−/HER2− (P < .001) and HER2+ BC (P < .002), respectively. BCs with abnormal expression of miR-181 and miR-135a showed an important association with distinct TIL and TAM components and specific immune genes. Importantly, preclinical data on BC cell lines demonstrated that miR-181 and miR-135a were able to regulate important cell immune-modulators such as SEMA3B and STAT6 signalling pathways, respectively. These 2 cellular pathways in turn were capable to regulate specific cascades of immune gene networks.

Conclusion: We identified specific miRNAs associated with immune cellular components and immune-related tumour response in the 3 main molecular BC subtypes (ER+/HER2−, HER2+ and ER−/HER2). Aberrant expression of miR-181 and miR-135a can contribute to BC tumour immune response programs by regulating important gene targets such as SEMA3B and STAT6 signalling pathways in ER−/HER2− and HER2+ tumours, respectively. Modulation of miRNAs activating tumour immune response programs may have important implications for novel therapeutic strategies in BC.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-01.