Aggressive Behavior Of Breast Cancer Research Articles

Impact of immunohistochemical expression of kinesin family member 18A (Kif18A) and β-catenin in infiltrating breast carcinoma of no special type

BackgroundKIF18A is a regulator of the cell cycle that stimulates the proliferation of cancer cells. The Wnt/β-catenin pathway is involved in different issues’ carcinogenesis and is being examined as a therapeutic target. The relationship between KIF18A and β-catenin in breast cancer was not previously investigated. Therefore, this work aims to study the immunohistochemical expression and correlation of KIF18A and β-catenin in breast-infiltrating duct carcinoma (IDC) and their relation to prognosis.Material and methodsSlides cut from paraffin blocks of 135 IDC and 40 normal breast tissues were stained by KIF18A and β-catenin antibodies. KIF18A cytoplasmic or nucleocytoplasmic staining and β-catenin aberrant expression either nucleo-cytoplasmic or cytoplasmic staining were considered.ResultsNormal breast tissue and IDC showed a significant difference regarding KIF18A and aberrant β-catenin expression. High KIF18A and β-catenin H score values were associated with poor prognostic factors such as high grade, advanced stage, distant metastasis, high Ki67 status, and Her2neu-enriched subtype. There was a significant direct correlation between KIF18A and β-catenin as regards percent and H score values. Prolonged overall survival (OS) was significantly associated with mild intensity and low H score of KIF18A, and low β-catenin H score.ConclusionsKIF18A could be involved in breast carcinogenesis by activating β-catenin. Overexpression of KIF18A and aberrant expression of β-catenin are considered proto-oncogenes of breast cancer development. KIF18A and β-catenin could be poor prognostic markers and predictors of aggressive behavior of breast cancer.

(Thyroid) Hormonal regulation of breast cancer cells.

Thyroid hormone as L-thyroxine (T4) acts nongenomically at physiological concentrations at its cancer cell surface receptor on integrin αvβ3 ('thyrointegrin') to cause cancer cell proliferation. In the case of estrogen receptor (ERα)-positive breast cancer cells, T4 via the integrin promotes ERα-dependent cancer growth in the absence of estrogen. Thus, tumor growth in the post-menopausal patient with ERα-positive cancer may again be ER-dependent because of T4. Additional mechanisms by which T4 may contribute uniquely to aggressive breast cancer behavior-independently of ER-are stimulation of immune checkpoint inhibitor gene expression and of several anti-apoptosis mechanisms. These observations may call for consideration of elimination of host T4 production in breast cancer patients whose response is suboptimal to standard chemotherapy regimens. Euthyroidism in such a setting may be maintained with exogenous 3,3',5-triiodo-L-thyronine (T3).

Concurrent manifestations of Horner\u2019s syndrome and esophageal metastasis of breast cancer: case report of a young woman after a period of non-adherence to treatment: a case report

BackgroundEsophageal involvement and Horner’s syndrome are rare manifestations of breast cancer distant metastases that can pose a significant challenge in diagnosis and treatment. In addition to the more aggressive behavior of breast cancer diagnosed in young women, non-adherence to treatment is associated with increased risk of distant metastasis.Case presentationA 36-year-old Javanese woman presented to our institution with dysphagia, hoarseness, and frequent hiccups. In the 6 weeks prior to the current admission, the patient also reported tingling in the neck and shoulder, anhidrosis in the left hemifacial region, and drooping of the upper left eyelid. She was previously managed as tuberculoid laryngitis. Plain X-rays showed burst fractures of the cervical vertebrae and slight pleural effusion. Laryngoscopy revealed bowing of the vocal cords and liquid residue in the vallecula that was reduced upon chin tuck. Esophageal metastasis was confirmed with endoscopy showing thickening of the wall and positive cytology swab with ductal malignant cells. The patient had a history of breast cancer with a period of loss to follow-up of 4 years.ConclusionsPhysicians should consider potential distant metastasis of breast cancer to the esophagus and sympathetic nervous system of the neck particularly in a high-risk woman with presentation of dysphagia and manifestations of Horner’s syndrome.

Evaluation of breast cancer stem cells in human primary breast carcinoma and their role in aggressive behavior of the disease

To delineate the underlying molecular mechanisms responsible for the intratumoral enrichment of breast cancer stem cells (BCSCs) in aggressive breast tumors, we evaluated the frequency and characteristics of BCSCs within the tumor tissue in primary human breast carcinomas. We assessed the expression profiles of various genes in cancer cells (CC) and stromal cells (SC) from these tumors to delineate the role played by the cellular niche in de novo origin or expansion of intra-tumoral cancer stem cells (CSC). The study included primary tumor and adjacent normal breast tissue specimens from chemotherapy-naïve breast carcinoma patients. The BCSCs, identified as Lin-CD44+CD24- and aldehyde dehydrogenase 1 A1 positive, were enumerated. The flow-cytometrically sorted stromal, and CC were processed for gene expression profiling using a custom-designed polymerase chain reaction array of genes known to facilitate disease progression. The frequency of BCSCs within the tumor mass correlated significantly with histopathological and molecular grades of tumors, indicating a direct relationship of BCSC with the aggressive behavior of breast cancer. Further, a significantly increased expression of the genes associated with growth factors, cytokines and matricellular proteins in tumors were found in high BCSCs compared to Lo-BCSC tumors, suggesting the possible contribution of stromal and CC in an intratumoral expansion of CSCs. Similarly, a significant upregulation of genes associated with hypoxia and angiogenesis in Hi-BCSCs tumors further supported the role of a hypoxic environment. Overall, the findings suggest the molecular crosstalk between SC and CC potentially (directly or indirectly) contributes to the expansion of CSC. The current study highlights the importance of CSC as a potential future predictive/prognostic marker for aggressive breast cancer. The present study predicts the potential risk stratification based on the frequency of BCSCs in primary breast tumors and existing prognostic factors.

Clinical significance of serum 25 hydroxyvitamin D in breast cancer: An Indian scenario

Recent evidences suggest a protective mechanism of vitamin D signaling against breast cancer by the autocrine/paracrine manner and may modestly reduce the risk of breast cancer. Despite lots of sunshine, vitamin D deficiency is widespread in India. Moreover, there are limited studies from Indian population regarding circulatory 25(OH) D and breast cancer risk. Thus, the aim of the present study is to investigate circulatory 25(OH) D in relation to breast cancer risk and its association with various clinico-pathological parameters from Indian population. Total 297 subjects, comprising of 157 controls and 140 breast cancer patients were enrolled for the study. Circulatory 25(OH) D was analyzed by HPLC. Statistical analysis was carried out by SPSS software version 15. Further, subjects were categorized into severe, moderate, mild vitamin D deficiency and sufficiency. The prevalence of severe and moderate 25(OH) D deficiency was higher in breast cancer patients as compared to controls. Mean values of 25(OH) D were lower in breast cancer patients as compared to controls in mild, moderate and severe deficient groups (p = 0.07, p = 0.003 and p = 0.001). Moreover, 25(OH) D was significantly lower in postmenopausal breast cancer patients as compared to premenopausal breast cancer patients, particularly in severe deficient group. The levels of 25(OH) D were lower in ER and PR negative receptor status as compared to the positive receptor in severe deficient category (p = 0.06 and p = 0.09 respectively). Whereas, the mean values of 25(OH) D were lower in HER 2 negative receptor status as compared to positive receptor status in the moderate deficient category (p = 0.09). Further, severe deficient group showed significantly lower levels of 25(OH) Din TNBC as compared to luminal A subtype (p = 0.01). Thus, Results indicate that 25(OH) D deficiency might be associated with increased risk of breast cancer. Moreover, severe 25(OH) D deficiency is associated with aggressive behavior of breast cancer.

Citron kinase (CIT-K) promotes aggressiveness and tumorigenesis of breast cancer cells in vitro and in vivo: preliminary study of the underlying mechanism

ObjectivesCitron kinase (CIT-K), as a key Rho effector, functions to maintain proper structure of the midbody during cell mitosis. This study assessed CIT-K expression and its role in breast cancer cells.MethodsParaffin-embedded breast cancer and para-tumor tissues from 43 invasive breast cancer patients and 33 normal mammary glands were collected for immunohistochemistry. CIT-K expression knockdown was achieved using lentivirus carrying CIT-K shRNA in a wide range of breast cancer cell lines. Cells were then subjected to Western blot, qRT-PCR, cell proliferation, colony formation, transwell, and flow cytometric assays. The tumorigenicity of CIT-K knocked-down breast cancer cells was assessed using the nude mouse xenograft assay. Microarray analysis was performed to elucidate the underlying gene regulation after CIT-K silencing.ResultsCIT-K protein was overexpressed in breast cancer tissues, which is associated with advanced tumor stage, HER-2 expression and Ki-67 expression, whereas knockdown of CIT-K expression reduced breast cancer cell proliferation and colony formation, but promoted tumor cell apoptosis and cell-cycle arrest. Knockdown of CIT-K expression also inhibited breast cancer cell migration and invasion capacity. Moreover, CIT-K knockdown suppressed the tumorigenicity of breast cancer cells in nude mice. Molecularly, the expression of a variety of signaling genes, such as cyclin D1, EGFR, JAK1, TGF-α, PTK2, RAF1, RALB, SOS1, mTOR, and PTGS2, were altered after CIT-K knockdown.ConclusionsThis study demonstrated that CIT-K is associated with aggressive breast cancer behavior and targeting CIT-K may be a novel strategy for the future control of breast cancer.

Discovery of a Glucocorticoid Receptor (GR) Activity Signature Using Selective GR Antagonism in ER-Negative Breast Cancer.

Purpose: Although high glucocorticoid receptor (GR) expression in early-stage estrogen receptor (ER)-negative breast cancer is associated with shortened relapse-free survival (RFS), how associated GR transcriptional activity contributes to aggressive breast cancer behavior is not well understood. Using potent GR antagonists and primary tumor gene expression data, we sought to identify a tumor-relevant gene signature based on GR activity that would be more predictive than GR expression alone.Experimental Design: Global gene expression and GR ChIP-sequencing were performed to identify GR-regulated genes inhibited by two chemically distinct GR antagonists, mifepristone and CORT108297. Differentially expressed genes from MDA-MB-231 cells were cross-evaluated with significantly expressed genes in GR-high versus GR-low ER-negative primary breast cancers. The resulting subset of GR-targeted genes was analyzed in two independent ER-negative breast cancer cohorts to derive and then validate the GR activity signature (GRsig).Results: Gene expression pathway analysis of glucocorticoid-regulated genes (inhibited by GR antagonism) revealed cell survival and invasion functions. GR ChIP-seq analysis demonstrated that GR antagonists decreased GR chromatin association for a subset of genes. A GRsig that comprised n = 74 GR activation-associated genes (also reversed by GR antagonists) was derived from an adjuvant chemotherapy-treated Discovery cohort and found to predict probability of relapse in a separate Validation cohort (HR = 1.9; P = 0.012).Conclusions: The GRsig discovered herein identifies high-risk ER-negative/GR-positive breast cancers most likely to relapse despite administration of adjuvant chemotherapy. Because GR antagonism can reverse expression of these genes, we propose that addition of a GR antagonist to chemotherapy may improve outcome for these high-risk patients. Clin Cancer Res; 24(14); 3433-46. ©2018 AACR.

Ror2 Signaling and Its Relevance in Breast Cancer Progression.

Breast cancer is a heterogeneous disease and has been classified into five molecular subtypes based on gene expression profiles. Signaling processes linked to different breast cancer molecular subtypes and different clinical outcomes are still poorly understood. Aberrant regulation of Wnt signaling has been implicated in breast cancer progression. In particular Ror1/2 receptors and several other members of the non-canonical Wnt signaling pathway were associated with aggressive breast cancer behavior. However, Wnt signals are mediated via multiple complex pathways, and it is clinically important to determine which particular Wnt cascades, including their domains and targets, are deregulated in poor prognosis breast cancer. To investigate activation and outcome of the Ror2-dependent non-canonical Wnt signaling pathway, we overexpressed the Ror2 receptor in MCF-7 and MDA-MB231 breast cancer cells, stimulated the cells with its ligand Wnt5a, and we knocked-down Ror1 in MDA-MB231 cells. We measured the invasive capacity of perturbed cells to assess phenotypic changes, and mRNA was profiled to quantify gene expression changes. Differentially expressed genes were integrated into a literature-based non-canonical Wnt signaling network. The results were further used in the analysis of an independent dataset of breast cancer patients with metastasis-free survival annotation. Overexpression of the Ror2 receptor, stimulation with Wnt5a, as well as the combination of both perturbations enhanced invasiveness of MCF-7 cells. The expression–responsive targets of Ror2 overexpression in MCF-7 induced a Ror2/Wnt module of the non-canonical Wnt signaling pathway. These targets alter regulation of other pathways involved in cell remodeling processing and cell metabolism. Furthermore, the genes of the Ror2/Wnt module were assessed as a gene signature in patient gene expression data and showed an association with clinical outcome. In summary, results of this study indicate a role of a newly defined Ror2/Wnt module in breast cancer progression and present a link between Ror2 expression and increased cell invasiveness.

Pengaruh Indeks Massa Tubuh terhadap Disease-Free Survival Lima Tahun Pasien Kanker Payudara di Rumah Sakit Kanker �Dharmais� Jakarta

ABSTRACTThere are some evidences that the metabolic changes in breast cancer patients with high body mass index (BMI) associated with insulin resistance and, in particular, the related alteration in cytokine production by adipose tissue which are major contributors to the aggressive behavior of breast cancer that develop through their effects in angiogenesis and stimulation of invasive capasity of cancer cells. Retrospective cohort study conducted at the Dharmais National Cancer Hospital aimed to determine the effect of BMI on five-year disease-free survival (DFS) breast cancer patients. This study was conducted from August to November 2014. The samples in this study were collected consecutively as many as 127 patients. From this study, it is known that the five-year DFS of breast cancerpatients was 70.0%. Based on the category of BMI, breast cancer patients with high BMI (>22.9 kg/m2) had the biggest DFS, followed by low BMI (<18.5 kg/m2) and normal BMI (18.5 22.9 kg/m2) that the precentages successively were 75.5%, 68.6%, and 60.4%. Multivariate analysis showed that BMI was not associated with the events of recurrence or metastases (HR 1.055; 95% CI 0.413-2.678) after being controlled by other variables, such as education, sosioeconomic, staging, lymph node involvement, histopathology, occupation, and biological subtypes.ABSTRAKBeberapa bukti menunjukkan perubahan metabolik pada pasien kanker payudara dengan indeks massa tubuh (IMT) tinggi berhubungan resistansi insulin dan khususnya perubahan terkait produksi sitokin oleh jaringan adiposa yang merupakan kontributor utama terhadap sifat agresif dari kanker payudara yang berkembang melalui pengaruhnya terhadap angiogenesis dan stimulasi kemampuan invasif dari sel kanker. Studi kohort retrospektif yang dilakukan di Rumah Sakit Kanker Dharmais ini bertujuan untuk mengetahui pengaruh IMT terhadap disease-free survival (DFS) lima tahun pasien kanker payudara. Penelitian ini dilakukan dari Agustus sampai November 2014. Sampel yangdigunakan pada studi ini diambil secara konsekutif sebanyak 127 pasien. Dari studi ini, diketahui bahwa DFS lima tahun pasien kanker payudara adalah 70,0%. Berdasarkan kategori IMT, pasien kanker payudara dengan IMT tinggi (>22,9 kg/m2) memiliki DFS lima tahun yang paling besar, yaitu 75,5% ; diikuti pasien dengan IMT rendah (<18,5 kg/m2) sebesar 68,6%; dan 60,4% untuk pasien dengan IMT normal (18,522,9 kg/m2). Hasil analisis multivariat menunjukkan bahwa IMT tidak memiliki asosiasi dengan kejadian kekambuhan atau metastase (HR=1,052, 95% CI 0,413-2,678) setelah dikontrol oleh variabel pendidikan, sosioekonomi, stadium, keterlibatan kelenjar getah bening, histopatologi, pekerjaan, dan subtipe biologis.

Functional Studies of CtBP in Breast Cancer Using the Lentiviral pINDUCER System

As a transcriptional corepressor, CtBP plays a significant role in tumorigenesis and tumor progression through epigenetic regulation. Interacting with transcription factors and chromatin modifying enzymes, CtBP mediates repression of tumor suppressor genes that maintain genome stability, repress cellular proliferation and inhibit EMT. This contributes to the evolution of more aggressive forms of multiple epithelial malignancies, including breast cancer. CtBP is also considered a metabolic sensor due to its ability to bind to NADH, the high energy intermediate generated during carbohydrate metabolism. However, despite its potential role in tumor progression and its link to metabolic imbalance, a detailed mechanism explaining how CtBP promotes aggressive breast cancer behavior is unclear. Thus, we are employing an inducible gene expression approach that utilizes the lentiviral pINDUCER system to inducibly manipulate the levels of CtBPs using Doxycycline. The fidelity of this system allows us to characterize CtBP‐regulated pathways including EMT, invasion, metastasis, genome instability, and uncontrolled self‐renewal. To explore the role of CtBP in increasing cancer malignancy, we are implementing a broad array of biological functional assays including wound healing migration assays, Boyden chamber invasion assays, and 3D gland formation assays. We are also conducting clonogenic assays, comet assays, gamma‐H2AX foci assays to further understand how CtBP drives genome instability and activates self‐renewal pathways. Eventually, we will develop orthotopic xenograft mouse models to study breast cancers in vivo. Together, the functional assays manipulated by the pINDUCER system will shed light on the role of CtBP in breast tumorigenesis and progression.

Low spinophilin expression enhances aggressive biological behavior of breast cancer.

Spinophilin, a putative tumor suppressor gene, has been shown to be involved in the pathogenesis of certain types of cancer, but its role has never been systematically explored in breast cancer. In this study, we determined for the first time the expression pattern of spinophilin in human breast cancer molecular subtypes (n = 489) and correlated it with survival (n = 921). We stably reduced spinophilin expression in breast cancer cells and measured effects on cellular growth, apoptosis, anchorage-independent growth, migration, invasion and self-renewal capacity in vitro and metastases formation in vivo. Microarray profiling was used to determine the most abundantly expressed genes in spinophilin-silenced breast cancer cells. Spinophilin expression was significantly lower in basal-like breast cancer (p<0.001) and an independent poor prognostic factor in breast cancer patients (hazard ratio = 1.93, 95% confidence interval: 1.24 -3.03; p = 0.004) A reduction of spinophilin levels increased cellular growth in breast cancer cells (p<0.05), without influencing activation of apoptosis. Anchorage-independent growth, migration and self-renewal capacity in vitro and metastatic potential in vivo were also significantly increased in spinophilin-silenced cells (p<0.05). Finally, we identified several differentially expressed genes in spinophilin-silenced cells. According to our data, low levels of spinophilin are associated with aggressive behavior of breast cancer.

Abstract 1987: The role of lipolysis-stimulated lipoprotein receptor in directing breast cancer cell behavior and subtype

Abstract The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by low gene expression of cell junction and adhesion proteins, and de-regulation of tight junction proteins is often been implicated in tumorigenesis. Herein, we sought to define the role of tricellular tight junction (tTJ) proteins in breast cancer and cancer cell behavior with a focus on lipolysis-stimulated lipoprotein receptor (LSR). LSR was expressed in epithelium, endothelium, adipose and stromal cells within the healthy breast tissue, as well as in tumor epithelium. LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, and ERα positive tumors and breast cancer cell lines. Consistent with the role of LSR in tTJs, LSR levels were significantly reduced in claudin-low breast cancer cell lines. Functional studies illustrated that that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may direct collective cell migration. Moreover, re-introduction of LSR in claudin-low breast cancer cell lines reestablished a family of TJ expression, thereby reverting claudin-low lines to other molecular subtypes. Overexpression of LSR enhanced proliferation and survival in anchorage independent conditions, suggesting that reestablishment of tight junctions is not sufficient to inhibit aggressive cell behaviors in vitro. Collectively, these data highlight an unanticipated role for the tTJ protein, LSR in directing aggressive breast cancer behavior. Citation Format: Jodie M. Fleming, Denise K. Reaves, Katerina D. Fagan-Solis, Karen Dunphy, Shannon D. Oliver. The role of lipolysis-stimulated lipoprotein receptor in directing breast cancer cell behavior and subtype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1987. doi:10.1158/1538-7445.AM2014-1987

AlphaB-crystallin is a marker of aggressive breast cancer behavior but does not independently predict for patient outcome: a combined analysis of two randomized studies.

BackgroundalphaB-crystallin is a small heat shock protein that has recently been characterized as an oncoprotein correlating with the basal core phenotype and with negative prognostic factors in breast carcinomas. The purpose of this study was to evaluate alphaB-crystallin with respect to clinicopathological parameters and the outcome of patients with operable high-risk breast cancer.MethodsA total of 940 tumors were examined, derived from an equal number of patients who had participated in two randomized clinical trials (paclitaxel-containing regimen in 793 cases). Immunohistochemistry for ER, PgR, HER2, Ki67, CK5, CK14, CK17, EGFR, alphaB-crystallin, BRCA1 and p53 was performed. BRCA1 mutation data were available in 89 cases.Resultsalphaβ-crystallin was expressed in 170 cases (18.1%) and more frequently in triple-negative breast carcinomas (TNBC) (45% vs. 14.5% non-TNBC, p < 0.001). alphaB-crystallin protein expression was significantly associated with high Ki67 (Pearson chi-square test, p < 0.001), p53 (p = 0.002) and basal cytokeratin protein expression (p < 0.001), BRCA1 mutations (p = 0.045) and negative ER (p < 0.001) and PgR (p < 0.001). Its overexpression, defined as >30% positive neoplastic cells, was associated with adverse overall survival (Wald’s p = 0.046). However, alphaB-crystallin was not an independent prognostic factor upon multivariate analysis. No interaction between taxane-based therapy and aβ-crystallin expression was observed.ConclusionsIn operable high-risk breast cancer, alphaB-crystallin protein expression is associated with poor prognostic features indicating aggressive tumor behavior, but it does not seem to have an independent impact on patient survival or to interfere with taxane-based therapy.Trial registrationsACTRN12611000506998 (HE10/97 trial) and ACTRN12609001036202 (HE10/00 trial).

The Role of Lipolysis Stimulated Lipoprotein Receptor in Breast Cancer and Directing Breast Cancer Cell Behavior

The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by cancer stem cell-like features and low expression of cell junction and adhesion proteins. Herein, we sought to define the role of lipolysis stimulated lipoprotein receptor (LSR) in breast cancer and cancer cell behavior as LSR was recently correlated with tumor-initiating features. We show that LSR was expressed in epithelium, endothelium, and stromal cells within the healthy breast tissue, as well as in tumor epithelium. In primary breast tumor bioposies, LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, as well as ERα positive tumors and breast cancer cell lines. LSR levels were significantly reduced in claudin-low breast cancer cell lines and functional studies illustrated that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may promote collective cell migration. Re-introduction of LSR in claudin-low breast cancer cell lines reestablished tight junction protein expression and correlated with transepithelial electrical resistance, thereby reverting claudin-low lines to other intrinsic molecular subtypes. Moreover, overexpression of LSR altered gene expression of pathways involved in transformation and tumorigenesis as well as enhanced proliferation and survival in anchorage independent conditions, highlighting that reestablishment of LSR signaling promotes aggressive/tumor initiating cell behaviors. Collectively, these data highlight a direct role for LSR in driving aggressive breast cancer behavior.

Bioinformatics analysis of aggressive behavior of breast cancer via an integrated gene regulatory network.

Breast cancer is one of the most frequently diagnosed cancers in women. Though death from this disease is mainly caused by the metastases of the aggressive cancer cells, few studies have expounded the aggressive behavior of breast cancer. We downloaded the gene expression profiles of GSE40057, including four aggressive and six less-aggressive breast cancer cell lines, from Gene Expression Omnibus and identified the differentially expressed genes (DEGs) between the aggressive and less-aggressive samples. An integrated gene regulatory network was built including DEGs, microRNAs (miRNAs), and transcription factors. Then, motifs and modules of the network were identified. Modules were further analyzed at a functional level using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway to study the aggressive behavior of breast cancer. A total of 764 DEGs were found and two modules were filtered from the integrated gene regulatory network. Totally two motifs and modules for DEGs were identified. Significant GO terms associated with cell proliferation and hormone stimulus of the modules were found and the target genes identified were CAV1, CD44, and TGFβR2. The KEGG pathway analysis discovered that CAV1 and FN1 were significantly enriched in focal adhesion, extracellular matrix (ECM)-receptor interaction, and pathways in cancer. Aggressive behavior of breast cancer was proved to be related to cell proliferation and hormone stimulus. Genes such as CAV1, CD44, TGFβR2, and FN1 might be potential targets to diagnose the aggressive behavior of breast cancer cells.

Expression of tissue levels of matrix metalloproteinases and their inhibitors in breast cancer

BackgroundWe examined mRNA expression for MMP-2, MMP-7, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 in human breast cancer tissues, and the association between their expression and clinicopathological variables. Patients and methodsBreast tissue samples from 120 patients with breast cancer were available for this study. To determine mRNA expression for MMP-2, MMP-7, MMP-9, MT1-MMP, TIMP-1, and TIMP-2, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out on tumor and normal tissues, respectively. ResultsMean MMP-2, MMP-7, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 mRNA expression in the breast cancer was significantly higher than in the normal tissue. In terms of tumor size and lymph node metastasis of breast cancer, the differences in MMP-2, MMP-7, MMP-9, and MT1-MMP mRNA expression levels were significant. ConclusionThe association between the increased expression of MMP-2, MMP-7, MMP-9, and MTI-MMP and clinicopathological parameters reflects a role in predicting the aggressive behavior of breast cancer.

Decreased microRNA-214 levels in breast cancer cells coincides with increased cell proliferation, invasion and accumulation of the Polycomb Ezh2 methyltransferase

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression by inhibiting translation or promoting degradation of specific target messenger RNAs (mRNAs). Alteration of the levels of a number of miRNAs is common in solid and hematological tumors. We have shown previously that miR-214 regulates Ezh2 in skeletal muscle and embryonic stem cells. The current study was aimed at examining the role of miR-214 in breast cancer where miR-214 levels are reduced but whether this phenomenon bears a functional relevance is unknown. MiR-214 expression was inversely correlated with Ezh2 mRNA and protein levels in breast cancer cell lines and at least one copy of the miR-214 alleles was found to be deleted in 24% (6/25) of primary breast tumors. Experimental increase of miR-214 in breast cancer cell lines correlated with reduction of Ezh2 protein levels, a known marker of invasion and aggressive breast cancer behavior. Supporting a direct targeting mechanism, miR-214 decreased luciferase activity from a construct containing the Ezh2 3' untranslated region. Expression of miR-214 specifically reduced cell proliferation of breast cancer cells and inhibited the invasive potential of a highly metastatic breast cancer cell line. These findings indicate that reduced miR-214 levels may contribute to breast tumorigenesis by allowing abnormally elevated Ezh2 accumulation and subsequent unchecked cell proliferation and invasion.

Risk of aggressive breast cancer in women of Han nationality carrying TGFB1 rs1982073 C allele and FGFR2 rs1219648 G allele in North China

The high morbidity and aggressive behavior of breast cancer are associated with genetic variations. Single nucleotide polymorphisms (SNPs) in many genes have been demonstrated as a risk factor for breast cancer. In this study, we evaluated the association of the SNP rs1982073 (Leu10Pro) in transforming growth factor-beta 1 (TGFB1) gene and the SNP rs1219648 in fibroblast growth factor receptor 2 (FGFR2) gene with the risk and aggressiveness of breast cancer among women of Han nationality in North China. TaqMan allelic discrimination assay was performed to genotype rs1982073 and rs1219648 in 447 breast cancer cases and 406 age-matched healthy controls. The results imply that genotype frequencies of rs1982073 and rs1219648 were not significantly different between breast cancer patients and healthy controls. However, for TGFB1 SNP rs1982073, the C-carriers (T/C+C/C genotype) were more likely to bear tumors of greater aggressiveness (histological grade III) than T/T-carriers (grade I-II) (OR = 3.45, 95% CI: 1.48-8.00, P = 0.004). For FGFR2 SNP rs1219648, G-carriers (A/G+G/G genotype) were more significantly linked to tumors with lymph node metastasis than those with A/A genotype (lymph node negative) (OR = 1.69, 95% CI: 1.11-2.58, P = 0.016). Therefore, the SNPs of TGFB1 rs1982073 and FGFR2 rs1219648 may contribute to the identification of patients with more aggressive breast cancers among Han women in North China.

Abstract 4647: Clinical Implications of Rabphillin-3A-Like (RPH3AL/NOC2) Gene Alterations in Breast Cancer

Abstract Background: The Rabphillin-3A-like (RPH3AL) gene, located at the 17p13.3 locus, has been reported to be involved in progression and metastasis of colorectal cancer. Since there have been no studies of this gene in breast cancer (BC), mutations and loss of heterozygosity (LOH) of RPH3AL in BCs were evaluated. Methods: BCs (n=104) and matching benign tissues were evaluated for LOH by use of two locus-specific polymorphic markers (D17S1866 and D17S643) and ABI-3100 sequencing technology. Of 104 cases, 16 had corresponding snap-frozen BCs and benign epithelial tissues. The cDNA samples of these frozen cases were analyzed for RPH3AL mutations by RT-PCR and direct sequencing. Since most of the missense mutations were located in the exon-6 region, the genomic DNA samples of 68 (of 104) BCs and their corresponding benign tissues were assessed only for exon 6 by the use of exon-6 specific primers. Additionally, 96 (of 104) BCs and the corresponding control tissues were analyzed for germ line mutations at 43 base pairs downstream from exon-6. Mutations and LOH status were correlated with clinicopathologic features of BCs and patient overall survival by chi-square and Kaplan-Meier analyses, respectively. Results: LOH analysis demonstrated that 69 of 99 (70%) informative cases exhibited LOH in at least one marker. In total, 19% (13 of 68) of BCs exhibited missense point mutations in the coding region (exon-6) of RPH3AL. Six BCs exhibited mutations at codon 175, two BCs at codon 200, one BC had double mutations at codon 178 and 189, and four BCs had single mutations at codons 165, 174, 181 and 196. There was a germ-line mutation (G&amp;gt;T) at 43 base pairs downstream from exon-6. Genotyping of this alteration showed that 31% (30 of 96) of BCs were homozygous for the G/G allele, 56% (54 of 96) were heterozygous for G/T, and 13% (12 of 96) were homozygous for T/T. In our correlative analysis, most BCs with mutations were associated with lymph-node metastases (9 of 13, 69%) and with an allelic loss (8 of 13, 62%). The G/G alleles were more commonly found in African-Americans than Caucasians (χ2, p=0.003), in ER/PR positive BCs than in ER/PR negative BCs (χ2, p=0.03), and in BCs with advanced stage (χ2, p=0.02). The incidence of LOH increased with nodal metastasis (χ2, p=0.04) and tumor size (χ2, p=0.05) of the BCs. Furthermore, LOH was significantly associated with poor patient survival (log rank, p=0.002). Conclusions: The results suggest that genetic alterations in the RPH3AL gene are associated with aggressive behavior of breast cancer. (Grant Support: UAB-Breast SPORE-Pilot and Susan G. Komen Breast Cancer Foundation, POP138306). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4647.

RCP is a human breast cancer–promoting gene with Ras-activating function

Aggressive forms of cancer are often defined by recurrent chromosomal alterations, yet in most cases, the causal or contributing genetic components remain poorly understood. Here, we utilized microarray informatics to identify candidate oncogenes potentially contributing to aggressive breast cancer behavior. We identified the Rab-coupling protein RCP (also known as RAB11FIP1), which is located at a chromosomal region frequently amplified in breast cancer (8p11-12) as a potential candidate. Overexpression of RCP in MCF10A normal human mammary epithelial cells resulted in acquisition of tumorigenic properties such as loss of contact inhibition, growth-factor independence, and anchorage-independent growth. Conversely, knockdown of RCP in human breast cancer cell lines inhibited colony formation, invasion, and migration in vitro and markedly reduced tumor formation and metastasis in mouse xenograft models. Overexpression of RCP enhanced ERK phosphorylation and increased Ras activation in vitro. As these results indicate that RCP is a multifunctional gene frequently amplified in breast cancer that encodes a protein with Ras-activating function, we suggest it has potential importance as a therapeutic target. Furthermore, these studies provide new insight into the emerging role of the Rab family of small G proteins and their interacting partners in carcinogenesis.