Early Event In Breast Cancer Research Articles

Immune infiltration, aggressive pathology, and poor survival outcomes in RECQL helicase deficient breast cancers

RECQL is essential for genomic stability. Here, we evaluated RECQL in 449 pure ductal carcinomas in situ (DCIS), 152 DCIS components of mixed DCIS/invasive breast cancer (IBC) tumors, 157 IBC components of mixed DCIS/IBC and 50 normal epithelial terminal ductal lobular units (TDLUs). In 726 IBCs, CD8+, FOXP3+, IL17+, PDL1+, PD1+ T-cell infiltration (TILs) were investigated in RECQL deficient and proficient cancers. Tumor mutation burden (TMB) was evaluated in five RECQL germ-line mutation carriers with IBC by genome sequencing. Compared with normal epithelial cells, a striking reduction in nuclear RECQL in DCIS was evident with aggressive pathology and poor survival. In RECQL deficient IBCs, CD8+, FOXP3+, IL17+ or PDL1+ TILs were linked with aggressive pathology and shorter survival. In germline RECQL mutation carriers, increased TMB was observed in 4/5 tumors. We conclude that RECQL loss is an early event in breast cancer and promote immune cell infiltration.

Microenvironmental IL1\u03b2 promotes breast cancer metastatic colonisation in the bone via activation of Wnt signalling

Dissemination of tumour cells to the bone marrow is an early event in breast cancer, however cells may lie dormant for many years before bone metastases develop. Treatment for bone metastases is not curative, therefore new adjuvant therapies which prevent the colonisation of disseminated cells into metastatic lesions are required. There is evidence that cancer stem cells (CSCs) within breast tumours are capable of metastasis, but the mechanism by which these colonise bone is unknown. Here, we establish that bone marrow-derived IL1β stimulates breast cancer cell colonisation in the bone by inducing intracellular NFkB and CREB signalling in breast cancer cells, leading to autocrine Wnt signalling and CSC colony formation. Importantly, we show that inhibition of this pathway prevents both CSC colony formation in the bone environment, and bone metastasis. These findings establish that targeting IL1β-NFKB/CREB-Wnt signalling should be considered for adjuvant therapy to prevent breast cancer bone metastasis.

Abstract LB-313: Upregulation of lactate dehydrogenase A by 14-3-3ζ leads to increased glycolysis critical for breast cancer initiation and progression

Abstract Metabolic reprogramming is a hallmark of cancer. Elevated glycolysis in cancer cells switches the cellular metabolic flux to produce more biological building blocks, thereby facilitating cellular adaptation to the adverse environment and sustaining rapid proliferation. Recently, new evidence has emerged that abnormal metabolic reprogramming may occur at the early-stages of neoplasia and critically contribute to early transformation and cancer initiation. Previously, we reported that 14-3-3ζ overexpression is an early event in human breast cancer, taking place as early as in the atypical ductal hyperplasia (ADH) stage. We therefore hypothesized that 14-3-3ζ overexpression in breast epithelial cells may trigger metabolic reprogramming by elevating glycolysis that contributes to cancer initiation. In the present study, our bioinfomatics analysis of microarray profiling data from the early-stages breast neoplastic lesions revealed that 14-3-3ζ expression is strongly correlated with the expression of genes of the canonical glycolytic pathway, particularly lactate dehydrogenase A (LDHA). Experimentally, increasing 14-3-3ζ expression in human non-transformed mammary epithelial cells (hMECs), MCF10A and MCF12A, transcriptionally up-regulated LDHA expression, elevated glycolytic activity, and promoted early transformation of hMECs. Conversely, knockdown of LDHA in the 14-3-3ζ-overexpressing hMECs significantly reduced glycolytic activity and inhibited transformation. Together, our data demonstrate that overexpression of 14-3-3ζ in early stage pre-cancerous breast epithelial cells may trigger an elevated glycolysis by activating the MEK-ERK-CREB signaling axis and transcriptionally up-regulating LDHA, thereby critically contributes to human breast cancer initiation. Citation Format: Chia-Chi Chang, Chenyu Zhang, Ozgur Sahin, Qingling Zhang, Hai Wang, Jian Zhang, Sumaiyah Rehman, Ping Li, Dihua Yu. Upregulation of lactate dehydrogenase A by 14-3-3ζ leads to increased glycolysis critical for breast cancer initiation and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-313.

A Novel Model of Dormancy for Bone Metastatic Breast Cancer Cells

Mortality of patients with breast cancer is due overwhelmingly to metastatic spread of the disease. Although dissemination is an early event in breast cancer, extended periods of cancer cell dormancy can result in long latency of metastasis development. Deciphering the mechanisms underlying cancer cell dormancy and subsequent growth at the metastatic site would facilitate development of strategies to interfere with these processes. A challenge in this undertaking has been the lack of models for cancer cell dormancy. We have established novel experimental systems that model the bone microenvironment of the breast cancer metastatic niche. These systems are based on 3D cocultures of breast cancer cells with cell types predominant in bone marrow. We identified conditions in which cancer cells are dormant and conditions in which they proliferate. Dormant cancer cells were able to proliferate upon transfer into supportive microenvironment or upon manipulation of signaling pathways that control dormancy. These experimental systems will be instrumental for metastasis studies, particularly the study of cellular dormancy.

Lactate Dehydrogenase-B Is Silenced by Promoter Methylation in a High Frequency of Human Breast Cancers

ObjectiveUnder normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer.Experimental designLactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA.ResultsAbsent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/ 25 cases of breast cancer tissues, but not in 5/ 5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/ 26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O2), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells.ConclusionsLoss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia.

Silencing of tumor suppressor genes RASSF1A, SLIT2, and WIF1 by promoter hypermethylation in hereditary breast cancer

Promoter hypermethylation is gaining strength as one of the main mechanisms through which tumor suppressor genes are silenced during tumor progression. Three tumor suppressor genes are frequently found methylated in their promoter, in concordance with absence of expression, RASSF1A, SLIT2, and WIF1. In addition, a previous array-CGH analysis from our group showed that these genes are found in deleted genomic regions observed in hereditary breast cancer tumors. In the present work we analyzed the methylation status of these three tumor suppressor gene promoters in 47 hereditary breast cancer tumors. Promoter methylation status analysis of hereditary breast tumors revealed high methylation frequencies for the three genes (67% RASSF1A, 80% SLIT2, and 72% WIF1). Additionally, the presence of methylated PCR products was associated with absence of protein expression for the three genes and statistically significant for RASSF1A and WIF1. Interestingly, methylation of all the three genes was found in 4 out of 6 grade I invasive ductal carcinoma tumors. Association between RASSF1A methylation and DCIS tumors was found. These results suggest that silencing of these tumor suppressor genes is an early event in hereditary breast cancer, and could be a marker for pre-malignant phenotypes.

Initiating breast cancer by PIK3CA mutation

PIK3CA mutations confer constitutive activation of PI3K, which initiates intracellular kinase signaling cascades that promote cell proliferation and survival. Recent studies by Meyer and colleagues, and Liu and colleagues demonstrate that expression of the H1047R exon 20 mutant of PIK3CA in luminal mammary epithelial cells induces tumorigenesis, implying that PIK3CA mutation is an early event in breast cancer. PIK3CA-H1047R-initiated tumors exhibit variable dependence on the oncogene and variable sensitivity to PI3K inhibition. Amplification of the oncogenes MYC and MET was observed in tumors that recurred following silencing of PIK3CA-H1047R, suggesting that these pathways represent mechanisms of escape from PI3K inhibition.

Frequent promoter hypermethylation of BRCA2, CDH13, MSH6, PAX5, PAX6 and WT1 in ductal carcinoma in situ and invasive breast cancer

Epigenetic changes are considered to be a frequent event during tumour development. Hypermethylation of promoter CpG islands represents an alternative mechanism to inactivate tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. In search of epigenetic events related to progression, we used MS-MLPA (ME-0002-B1, MRC-Holland, Amsterdam, The Netherlands) to compare the methylation status of 25 breast cancer-related genes between laser-microdissected ductal carcinoma in situ (DCIS) and adjacent invasive ductal cancer (IDC) lesions in 33 breast cancer patients. Using absolute methylation percentages or, alternatively, a 15% cut-off for methylation, promoter methylation in DCIS and IDC was not significantly different for any of the genes studied. Aberrant methylation in at least 50% of both the DCIS and adjacent IDC lesions was observed for PAX6, BRCA2, PAX5, WT1, CDH13 and MSH6. Methylation of MSH6, however, was also frequent in normal breast tissue. In contrast, CDKN2A, CHFR, PYCARD and one of the two analysed RB1 CpG loci were rarely (<5%) methylated in both lesions. CDKN2A and GSTP1 showed significantly (p < 0.002) higher mean methylation levels in increasing grades (I, II, III) of DCIS (1% versus 4% versus 7% for CDKN2A and 6% versus 26% versus 28% for GSTP1). The mean number of methylated genes per sample increased with increasing grades of DCIS (p = 0.014) and IDC (p = 0.109). In contrast to the observations in DCIS, none of the analysed genes showed significantly higher methylation levels with increasing grades of IDC. In conclusion, there were no differences in promoter methylation between DCIS and IDC in the 25 analysed genes, suggesting that DCIS, at the epigenetic level, is as advanced as IDC. Promoter hypermethylation of PAX6, BRCA2, PAX5, WT1, CDH13 and MSH6 seems to be a frequent early event in breast cancer and methylation levels of GSTP1 (and CDKN2A, although still low) seem to increase with increasing DCIS grade.

Abstract 1188: BRCA1 regulates EGFR expression through microRNAs

Abstract Germline mutations in the tumor suppressor BRCA1 (breast cancer 1, early onset) are associated with a substantially higher risk of developing basal-like breast cancer (BLBC). Many sporadic breast cancers express decreased levels of BRCA1 and show a strong resemblance to BRCA1-hereditary tumors. BRCA1-associated tumors are triple-negative (TN), basal-like high grade ductal carcinomas that display an undifferentiated phenotype and frequently overexpress epidermal growth factor receptor (EGFR/ERBB1/HER1) and basal cytokeratins (CK5, CK6, CK14 and CK17) and confer a poor clinical prognosis. Although upregulation of EGFR is commonly observed in BRCA1-associated breast cancer, the mechanism of EGFR regulation by BRCA1 is not understood. EGFR upregulation could occur at multiple transcriptional and/or post-transcriptional levels. MicroRNAs (miRNAs) are an evolutionarily conserved class of small non-coding RNAs that regulate gene expression post-transcriptionally and/or translationally. Several miRNAs are found to be differentially expressed in breast cancer and studies show their involvement in stem cell differentiation, epithelial-to-mesenchymal transition (EMT), invasion and metastasis. Identifying the functional relationship between loss of BRCA1 and miRNAs will help us understand the post-transcriptional mechanism by which BRCA1 deficiency leads to aberrant EGFR expression. In the current study, we show for the first time, a direct inverse correlation between BRCA1 and EGFR protein expression levels in breast cancer cell lines representing the different intrinsic subtypes and in primary mammary epithelial cells. By overexpression and knockdown of BRCA1, we demonstrate that BRCA1 loss results in increased stability of EGFR protein. Using our in vitro system of immortalized primary mammary epithelial cells, we evaluated the effect of functional loss of BRCA1 on the expression of miRNAs in secondary mammospheres which are enriched for stem-like cells and in cells differentiated in vitro. We observed that BRCA1 deficiency leads to dysregulation of miRNAs linked to stem cell renewal, EGFR signaling and EMT. miRNA gain-of-function experiments using Pre-miR™ miRNA precursors and loss-of-function experiments using miRCURY LNA™ miRNA inhibitors were performed with the selected miRNAs to further confirm their effect on EGFR expression. Results suggest that miRNA mediated EGFR deregulation could be a critical step in tumor initiation and progression in BRCA1 deficiency. Since EGFR upregulation is an early event in breast cancer, additional functional studies will provide a novel molecular mechanism by which BRCA1 deficiency leads to the development of BLBC with aberrant EGFR expression mediated by miRNAs. The study will offer new therapeutic approaches for BLBC, a highly aggressive disease with limited therapeutic options. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1188. doi:10.1158/1538-7445.AM2011-1188

PIK3CA Mutations in In situ and Invasive Breast Carcinomas

The PIK3 signaling pathway has been identified as one of the most important and most frequently mutated pathways in breast cancer. Somatic mutations in the catalytic subunit of PIK3CA have been found in a significant fraction of breast carcinomas, and it has been proposed that mutant PIK3CA plays a role in tumor initiation. However, the majority of primary human tumors analyzed for genetic alterations in PIK3CA have been invasive breast carcinomas and the frequency of PIK3CA mutations in preinvasive lesions has not been explored. To investigate this, we sequenced exons 9 and 20 of PIK3CA in pure ductal carcinoma in situ (DCIS), DCIS adjacent to invasive carcinoma, and invasive ductal breast carcinomas. In a subset of cases, both in situ and invasive areas were analyzed from the same tumor. We found that the frequency of PIK3CA mutations was essentially the same ( approximately 30%) in all three histologic groups. In some cases, in situ and invasive areas of the same tumor were discordant for PIK3CA status, and in two cases in which multiple invasive and adjacent in situ areas within the same tumor were analyzed independently, we detected intratumor heterogeneity for PIK3CA mutations. Our results suggest that mutation of PIK3CA is an early event in breast cancer that is more likely to play a role in breast tumor initiation than in invasive progression, although a potential role for exon 9 mutations in the progression of a subset of DCIS cases cannot be excluded.

Disseminated tumor cells in primary breast cancer: Evaluation of the percentage of breast cancer stem cells in bone marrow aspirates of patients receiving neoadjuvant chemotherapy

505 Background: Disseminated tumor cells (DTC) in the bone marrow (BM) are related to poor prognosis of primary breast cancer (PBC). Cancer-initiating stem cells (CSC) have been detected in BM of PBC patients. Downregulation of the Notch pathway leads to murine mammary stem cell expansion and its expression is an early event in breast cancer. We enumerated CSC in BM mononuclear cells (BMC) and assessed the expression of Notch-1, ER, and HER-2 to study the differences between untreated patients and those treated with neoadjuvant chemotherapy. Methods: BM was collected from 90 PBC patients at time of surgery to assess DTC (CD326+CD45-) and CSC (CD326+CD45-CD44+CD24-) by FACS analysis. BM samples were also interrogated for the presence of CSC with ALDH activity using the Aldefluor® detection kit. RNA extracted from 61 BMC was analyzed for Notch-1, ER, and HER-2 transcripts by RT-PCR; the level of each transcript was normalized to that of the housekeeping gene, GAPDH. Results: The median age of patients is 51 years old (range, 27–77 years). A total of 29 patients (32%) received neoadjuvant chemotherapy, including 8 patients with anti-HER-2 targeted therapy. BMC of neoadjuvant-treated patients had a significantly higher median percentage of CSC in BMC (P= 0.046) and a significantly higher proportion of CSC cells within the Aldefluor+ population (P= 0.013) than those of untreated patients. Median level of Notch-1 transcripts in BMC was lower in neoadjuvant-treated patients compared with that of untreated patients (P= 0.07). There was an inverse correlation between the level of Notch-1 transcripts and the percentage of CSC in BMC of untreated (Spearman's rho= -0.519, P &lt; 0.001), but not in neoadjuvant treated (Spearman's rho= -0.483, P= 0.058) patients. Conclusions: Neoadjuvant chemotherapy is associated with expansion of CSC in the BMC of patients with PBC. Untreated patients with higher level of Notch-1 transcripts in the BMC tend to have lower percentages of CSC and suggest that Notch signaling may be crucial for differentiation of CSC. The prognostic value of this finding is under investigation and gene expression profile of CSC may provide indications for novel neoadjuvant therapies. No significant financial relationships to disclose.

Methylation of homeobox genes is a frequent and early epigenetic event in breast cancer

IntroductionAberrant methylation of CpG islands is a hallmark of cancer and occurs at an early stage in breast tumorigenesis. However, its impact on tumor development is not fully determined, and its potential as a diagnostic biomarker remains to be validated. Methylation profiling of invasive breast carcinoma has been largely explored. Conversely, very little and sparse information is available on early-stage breast cancer. To gain insight into the epigenetic switches that may promote and/or contribute to the initial neoplastic events during breast carcinogenesis, we have analyzed the DNA methylation profile of ductal carcinoma in situ, a premalignant breast lesion with a great potential to progress toward invasive carcinoma.MethodsWe have utilized a comprehensive and sensitive array-based DNA mapping technique, the methylated-CpG island recovery assay, to profile the DNA methylation pattern in ductal carcinoma in situ. Differential methylation of CpG islands was compared genome-wide in tumor DNA versus normal DNA utilizing a statistical linear model in the LIMMA software package.ResultsUsing this approach, we have identified 108 significant CpG islands that undergo aberrant DNA methylation in ductal carcinoma in situ and stage I breast tumors, with methylation frequencies greater than or comparable with those of more advanced invasive carcinoma (50% to 93%). A substantial fraction of these hypermethylated CpG islands (32% of the annotated CpG islands) is associated with several homeobox genes, such as the TLX1, HOXB13, and HNF1B genes. Fifty-three percent of the genes hypermethylated in early-stage breast cancer overlap with known Polycomb targets and include homeobox genes and other developmental transcription factors.ConclusionsWe have identified a series of new potential methylation biomarkers that may help elucidate the underlying mechanisms of breast tumorigenesis. More specifically, our results are suggestive of a critical role of homeobox gene methylation in the insurgence and/or progression of breast cancer.

Two-Dimensional Differential Gel Electrophoresis of a Cell Line Derived from a Breast Cancer Micrometastasis Revealed a Stem/Progenitor Cell Protein Profile

Dissemination of primary cancer cells to distant sites is an early event in breast cancer. These cells can invade the bone marrow, rest there, and many years later disseminated tumor cells (DTC) can grow out to form overt metastases. Epithelium specific cytokeratins are commonly used as marker proteins for sensitive detection of metastatic lesions. However, due to difficulties in the detection of DTC, the question arises if DTC necessarily have the same protein expression profile as advanced tumors. On that account, we analyzed the previously uncharacterized breast cancer DTC cell line BC-M1 by 2-D DIGE. Special protein concentration and purification protocols for 2-DE were developed which resulted in high recovery rates and increased display of alkaline proteins. A broad range reference map of metastasis relevant proteins was compiled including the cytokeratins 5, 7, 8, 17, 18, and 19 and several classes of cytoskeleton proteins involved in metastasis like ezrin, gelsolin, vinculin, or vimentin. BC-M1 shows the rare and highly metastatic vimentin/cytokeratin 5 positive and cytokeratin 8/18 negative breast cancer phenotype and expresses Her-2, which is also found in stem cells/progenitor cells of primary tumors. Supported by the detection of several other epithelium-derived proteins, the example BC-M1 indicates that the protein expression profile of DTC might be reminiscent of the expression profile of the early tumor, which differs from the advanced tumor. Hence, DTC from breast cancer patients' bone marrow expressed cytokeratin 5, which further supports our hypothesis.

TP53 mutation is an early event in breast cancer progression.

Abstract Abstract #1047 Background: In breast cancer, previous studies have shown that somatic TP53 mutations cause a more aggressive disease with poor clinical outcome and may impact treatment response. Although TP53 mutation is considered to be an early event in breast cancer, the timing of TP53 mutations is not known, and there are controversies regarding the cellular origin and linear model of breast cancer. The purpose of this study was to investigate whether TP53 mutations are early events in breast cancer progression.&amp;#x2028; Methods: From a population-based cohort of women diagnosed between 1986 and 2004 either with a pure ductal carcinoma in situ (DCIS), a pure invasive cancer (&amp;lt;15mm) or a mixed lesion (i.e. invasive cancer with a DCIS component), we included 118 women with stored frozen tissue. Mixed lesions were microdissected using LCM (laser capture microdissection) on a PALM slide to separate in situ and invasive tumor cells. DNA was isolated using phenol-chloroform extraction. The entire coding sequence of TP53 was analyzed for mutations by direct sequencing on a 3730 DNA analyzer.&amp;#x2028; Results: Of 118 tumor samples, 19 were detected with a TP53 mutation; five 5 of 32 (15.6%) pure DCIS, 4 of 38 (10.5%) pure invasive cancers and 10 of 48 (20.8%) mixed lesions. In the mixed lesions, both the invasive and the DCIS component showed the same mutation in all 5 cases where we successfully could microdissect the two components separately. Pure DCIS demonstrated missense mutations (4/5, 80%) more frequently than pure invasive cancers (2/4, 50%) and mixed lesions (4/10, 40%), although this difference was not statistically significant (p=0.3). Also, the frequency of missense mutations in the DNA binding domain was not statistically different between the three groups.&amp;#x2028; Conclusion: TP53 mutation is likely an early event in breast cancer, occurring previous to or in the in situ stage. Presence of the same mutation in both DCIS and invasive component from the same tumor indicates same cellular origin. The role of mutant TP53 in the progression into invasive cancer is less clear and may vary between subtypes of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1047.

Notch-1 expression is not related to the percentage of CD326+ cells in bone marrow aspirates of patients with primary breast cancer.

Abstract Abstract #4030 Background: The presence of epithelial cells (CD326+CD45dim) in the bone marrow (BM), i.e. disseminated tumor cells (DTC), are related to poor prognosis of primary breast cancer (PBC). Notch signaling pathway is involved in the development of the breast and aberrant activation of Notch signaling is an early event in breast cancer. Others have shown that breast cancer stem cells (BCSC) with the putative phenotype of CD44+CD24- can be identified within cell lines and primary tumor and have upregulated genes including Notch-1. However, it is unclear if Notch-1 is highly expressed by DTC and BCSC in BM of patients with PBC. We hypothesized that the Notch-1 expression is correlated with the level of BCSC within DTC. In this study, we enumerated BCSC in DTC-enriched populations in patients with PBC and assessed the expression of Notch-1 by RT-PCR.&amp;#x2028; Methods and Patients: As part of an ongoing prospective study, from September 2006 to May 2008, 121 BM aspirates were collected from as many patients with PBC. Thirty-seven patients, median age of 52 years (range, 30-76 years), provided BM aspirates under anesthesia at time of surgery. At the time of BM collection, 9 patients had received neoadjuvant therapy. BM mononuclear cells (BMC) were enriched for DTC using anti-CD326 antibody-coated magnetic beads, and isolated using the AutoMACS-Pro cell separator system. DTC and BCSC were identified by multi-color FACS analysis. Thereafter, DTC-enriched cell preparations were analyzed for expression of ER, PR, HER-2, Notch-1, and Numb by RT-PCR; level of each transcript was normalized to a ratio to the house keeping gene GAPDH.&amp;#x2028; Results: The mean ± SEM DTC in BMC was 1.05 % ± 0.09% and the majority was BCSC (79.7% ± 1.87%). The proportion of DTC-enriched fractions that expressed Notch-1, Numb, ER, PR, and HER-2 transcripts was 27/37 (73.0%), 33/37 (89.2%), 24/37 (64.9%), 15/37 (40.5%), and 20/37 (54.1%), respectively. The expression of Notch-1, Numb, ER, PR, and HER-2 transcripts did not correlate with the percentage of DTC. Contrary to our hypothesis, Notch-1 and Numb mRNA expression in DTC-enriched cells negatively correlated with the percentage of BCSC (Spearman's rho = -0.384, p= 0.027; and rho= -0.347, p= 0.048, respectively).&amp;#x2028; Conclusions: Our results show no correlation between the level of Notch-1 expression and the percentage of BCSC in DTC-enriched populations in BM of patients with PBC. Since these studies were conducted in CD326-enriched populations, it underscores the necessity to further purify the CD44+CD24- cells and conduct studies in isolated single cells to definitively delineate the gene expression of BCSC from patients with PBC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4030.

14-3-3ζ Down-regulates p53 in Mammary Epithelial Cells and Confers Luminal Filling

Recent progress in diagnostic tools allows many breast cancers to be detected at an early preinvasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. Previously, we discovered that 14-3-3 zeta is overexpressed in >40% of advanced breast cancers, and this overexpression predicts poor patient survival. Here, we examined at what stage of breast disease 14-3-3 zeta overexpression occurs, and we found that increased expression of 14-3-3 zeta begins at atypical ductal hyperplasia, an early stage of breast disease. To determine whether 14-3-3 zeta overexpression is a decisive early event in breast cancer, we overexpressed 14-3-3 zeta in MCF10A cells and examined its effect in a three-dimensional culture model. We discovered that 14-3-3 zeta overexpression severely disrupted the acini architecture resulting in luminal filling. Proper lumen formation is a result of anoikis, apoptosis due to detachment from the basement membrane. We found that 14-3-3 zeta overexpression conferred resistance to anoikis. Additionally, 14-3-3 zeta overexpression in MCF10A cells and in mammary epithelial cells (MEC) from 14-3-3 zeta transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3 zeta induced hyperactivation of the phosphoinositide 3-kinase/Akt pathway which led to phosphorylation and translocation of the MDM2 E3 ligase resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3 zeta-overexpressing MCF10A acini in three-dimensional cultures. These data suggest that 14-3-3 zeta overexpression is a critical event in early breast disease, and down-regulation of p53 is one of the mechanisms by which 14-3-3 zeta alters MEC acini structure and increases the risk of breast cancer.

Mammary Stem Cells and Breast Cancer—Role of Notch Signalling

Adult stem cells are found in numerous tissues of the body and play a role in tissue development, replacement and repair. Evidence shows that breast stem cells are multipotent and can self renew, which are key characteristics of stem cells, and a single cell enriched with cell surface markers has the ability to grow a fully functional mammary gland in vivo. Many groups have extrapolated the cancer stem cell hypothesis from the haematopoietic system to solid cancers, where using in vitro culture techniques and in vivo transplant models have established evidence of cancer stem cells in colon, pancreas, prostate, brain and breast cancers. In the report we describe the evidence for breast cancer stem cells; studies consistently show that stem cell like and breast cancer initiating populations can be enriched using cell surface makers CD44+/CD24- and have upregulated genes which include Notch. Notch signalling has been highlighted as a pathway involved in the development of the breast and is frequently dysregulated in invasive breast cancer. We have investigated the role of Notch in a pre-invasive breast lesion, ductal carcinoma in situ (DCIS), and have found that aberrant activation of Notch signalling is an early event in breast cancer. High expression of Notch 1 intracellular domain (NICD) in DCIS also predicted a reduced time to recurrence 5 years after surgery. Using a non-adherent sphere culture technique we have grown DCIS mammospheres from primary DCIS tissue, where self-renewal capacity, measured by the number of mammosphere initiating cells, were increased from normal breast tissue. A gamma-secretase inhibitor, DAPT, which inhibits all four Notch receptors and a Notch 4 neutralising antibody were shown to reduce DCIS mammosphere formation, indicating that Notch signalling and other stem cell self-renewal pathways may represent novel therapeutic targets to prevent recurrence of pre-invasive and invasive breast cancer.

SATR-1 hypomethylation is a common and early event in breast cancer

Genome stability and normal gene expression are maintained by a fixed and predetermined DNA methylation pattern, which becomes abnormal in malignant cells. Hypomethylation of satellite DNA sequences is frequently found in tumors and has been associated with an increased frequency of DNA rearrangements and chromosome instability. In this work, we used methylation-sensitive arbitrarily primed polymerase chain reaction (MSAP-PCR) to identify differentially methylated DNA fragments in normal and tumor breast samples. We identified a novel differentially methylated fragment located on chromosome 5 with high similarity to a SATR-1 satellite sequence. This fragment was found to be hypomethylated in 63% of breast tumor cell lines and in 86% of breast tumors relative to normal breast tissue. We found that normal tissue adjacent to breast tumors displayed a variable decrease in methylation and that the decrease observed for most of these adjacent samples was higher than observed for normal breast tissue derived from reduction mammoplasty. The methylation decrease was, however, significantly higher in tumor samples than in adjacent tissue (χ 2 = 154, 1 df, P < 10 −4), suggesting that SATR-1 hypomethylation frequently occurs in the early stages of tumor development. Our results highlight the importance of global DNA hypomethylation as a contributing factor in breast tumorigenesis.

DNA methylation markers - an opportunity to further individualize therapy in breast cancer?

Over the last few decades, a wealth of treatment options have become available for breast cancer. To specifically direct those therapies to patients with the highest need who will receive the greatest benefit, biomarkers are urgently needed. Two specific needs seem to be most pressing: first is the need for prognostic markers, which would determine which group of patients may recover without adjuvant chemotherapy. Second, predictive markers for specific treatments, such as different endocrine treatments, chemotherapies or targeted drugs, are expected to play a major role in the near future. Ideally, such markers should be strong single markers, or low-complexity marker panels containing only a few markers, to allow for easier assay development and improved reproducibility. The possibility to measure the marker(s) in formalin-fixed specimens would greatly facilitate integration into routine clinical practice. A common and early event in breast cancer is aberrant DNA methylation within gene regulatory regions, affecting a variety of genes with different functions. Data from recently published studies indicate that altered DNA methylation carries prognostic as well as predictive information in breast cancer. Together with the technical advantages of a DNA-based marker, DNA methylation may well constitute the ideal biomarker to further individualize breast cancer treatment. Here the recent literature is reviewed and the most interesting markers, which have the potential to significantly change breast cancer treatment and, therefore, warrant further systematic clinical validation, are highlighted.

Aberrant methylation of the maspin promoter is an early event in human breast cancer.

The maspin gene functions as a tumor suppressor in human breasts, and its expression is frequently lost during breast cancer progression. In vitro models of human breast cancer indicate that the loss of maspin expression is closely linked to aberrant methylation of the maspin promoter. We conducted a study on 30 archival ductal carcinoma in situ (DCIS) specimens to determine if aberrant methylation of the maspin promoter occurred in vivo, and whether it occurred early in breast cancer evolution. Healthy tissue obtained from reduction mammoplasty was used as normal control. Results from immunohistochemical analysis indicate that maspin expression is lost in a substantial fraction of DCIS specimens (57%). Bisulfite sequencing of DNA isolated from laser capture-microdissected normal and neoplastic ducts showed that loss of maspin expression was often, but not always, linked to aberrant methylation of the maspin promoter, suggesting that other mechanisms, in addition to aberrant methylation, participate and/or cooperate to silence maspin gene expression. Taken together, these results indicate that aberrant methylation of the maspin promoter is an early event in human breast cancer.