Event In Human Breast Cancer Research Articles

Chromothripsis in Human Breast Cancer.

Chromothripsis is a form of genome instability by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosomes. Widely assumed to be an early event in tumor development, this phenomenon plays a prominent role in tumor onset. In this study, an analysis of chromothripsis in 252 human breast cancers from two patient cohorts (149 metastatic breast cancers, 63 untreated primary tumors, 29 local relapses, and 11 longitudinal pairs) using whole-genome and whole-exome sequencing reveals that chromothripsis affects a substantial proportion of human breast cancers, with a prevalence over 60% in a cohort of metastatic cases and 25% in a cohort comprising predominantly luminal breast cancers. In the vast majority of cases, multiple chromosomes per tumor were affected, with most chromothriptic events on chromosomes 11 and 17 including, among other significantly altered drivers, CCND1, ERBB2, CDK12, and BRCA1. Importantly, chromothripsis generated recurrent fusions that drove tumor development. Chromothripsis-related rearrangements were linked with univocal mutational signatures, with clusters of point mutations due to kataegis in close proximity to the genomic breakpoints and with the activation of specific signaling pathways. Analyzing the temporal order of events in tumors with and without chromothripsis as well as longitudinal analysis of chromothriptic patterns in tumor pairs offered important insights into the role of chromothriptic chromosomes in tumor evolution. SIGNIFICANCE: These findings identify chromothripsis as a major driving event in human breast cancer.

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.

Abstract 1746: Discovery and evaluation of a small molecule KIFC1 inhibitor for breast cancer treatment

Abstract Historically, drugs used in the treatment of certain cancers may cause damage to healthy cells while affecting cancer cells. Therefore, finding compounds that are specific toward cancer cells only is still a high priority in the search for new therapies. In contrast with normal cells, most cancer cells contain multiple centrosomes which are associated with genome instability and tumorigenesis. Cancer cells can avoid multipolar mitosis which can cause cell death by clustering the extra centrosomes into two spindle poles, thereby enabling bipolar division. Kinesin-like protein KIFC1 plays a critical role in centrosome clustering in cancer cells, but is not essential for normal cell survival. Therefore, targeting KIFC1 may give some insight into how a novel therapy can selectively target only cancer cells. Here, we report that KIFC1 up-regulation is a frequent event in human breast cancer and that KIFC1 is highly expressed in all 8 tested human breast cancer cell lines, but is absent in normal human mammary epithelial cells and weakly expressed in 2 human lung fibroblast lines. We also found that depletion of KIFC1 in breast cancer cells induced cell death. From a high throughput screen of 30,000 compounds, we identified a small molecule KIFC1 inhibitor, SRH06, which has an enzymatic IC50 value of 6.5 μM versus KIFC1 and binds directly to KIFC1 without interacting with the microtubule. Results from our computer modeling studies suggested that SRH06 binds to a novel allosteric site on KIFC1 that appears suitable for the development of selective KIFC1 inhibitors. Importantly, SRH06 prevented bipolar clustering of extra-centrosomes in breast cancer cells and significantly reduced colony formation and cell viability, but was less toxic to normal LL47 fibroblasts. Therefore, SRH06 provides a very valuable tool to study the biological function of KIFC1 and serves as a potential lead for the development of a novel therapeutic agent for the treatment of breast cancer. Citation Format: Wei Zhang, Ling Zhai, Wenyan Lu, Yimin Wang, Vandana V. Gupta, Indira Padmalayam, Robert J. Bostwick, Lucile White, Ross Larry, Joseph Maddry, Sam Ananthan, Mark Suto, Bo Xu, Rongbao Li, Yonghe Li. Discovery and evaluation of a small molecule KIFC1 inhibitor for breast cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1746. doi:10.1158/1538-7445.AM2015-1746

Triple negative tumors accumulate significantly less methylglyoxal specific adducts than other human breast cancer subtypes.

Metabolic syndrome and type 2 diabetes are associated with increased risk of breast cancer development and progression. Methylglyoxal (MG), a glycolysis by-product, is generated through a non-enzymatic reaction from triose-phosphate intermediates. This dicarbonyl compound is highly reactive and contributes to the accumulation of advanced glycation end products. In this study, we analyzed the accumulation of Arg-pyrimidine, a MG-arginine adduct, in human breast adenocarcinoma and we observed a consistent increase of Arg-pyrimidine in cancer cells when compared with the non-tumoral counterpart. Further immunohistochemical comparative analysis of breast cancer subtypes revealed that triple negative lesions exhibited low accumulation of Arg-pyrimidine compared with other subtypes. Interestingly, the activity of glyoxalase 1 (Glo-1), an enzyme that detoxifies MG, was significantly higher in triple negative than in other subtype lesions, suggesting that these aggressive tumors are able to develop an efficient response against dicarbonyl stress. Using breast cancer cell lines, we substantiated these clinical observations by showing that, in contrast to triple positive, triple negative cells induced Glo-1 expression and activity in response to MG treatment. This is the first report that Arg-pyrimidine adduct accumulation is a consistent event in human breast cancer with a differential detection between triple negative and other breast cancer subtypes.

Triple negative human breast cancers accumulate significantly less Arg‐pyrimidine moieties, than other subtype lesions (58.3)

Metabolic disorders influence breast cancer development and progression.Methylglyoxal (MGX), is a highly reactive glycolytic byproduct able to inflict carbonyl stress to proteins, lipids and DNA. The possible role of MGX in breast cancer development has not been yet extensively explored. We analyzed the accumulation of Arg‐pyrimidine adducts, in a series of more than 100 human breast adenocarcinoma and we observed a consistent increased of MGX adducts in cancer cells compared to the non‐tumoral counterpart. Most triple negative lesions examined exhibited low accumulation of Arg‐pyrimidine residues compared other subtypes. Intriguingly, the level of expression of glyoxalase 1 (Glo‐1), an enzyme that detoxifies MGX, was similar in both triple negative and other subtype lesions, suggesting a potential difference in the Glo‐1 activity. This hypothesis is supported by our preliminary results on breast cancer cell lines, showing that triple negative cells are more efficient to respond to treatment with MGX by increasing the activity of Glo‐1. Our study represent the first observation that Arg‐pyrimidine adducts accumulation is a consistent event in human breast cancer with a differential detection between triple negative and other breast cancer subtypes. Our preliminary data suggest that methylglyoxal production and detoxification could be a molecular link between metabolic diseases and breast cancer.Grant Funding Source: This study is supported by Founds for National Research (FNRS), Belgium

Aberrantly Methylated DNA as a Biomarker in Breast Cancer

Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients occur early during tumorigenesis. This may open up for effective screening, and analysis of blood or nipple aspirate may later help in diagnosing breast cancer. As a more detailed molecular characterization of different types of breast cancer becomes available, the ability to divide patients into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential as a versatile biomarker tool for screening, diagnosis, prognosis and monitoring of breast cancer. Standardization of methods and biomarker panels will be required to fully exploit this clinical potential.

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.

MicroRNA-335 inhibits tumor reinitiation and is silenced through genetic and epigenetic mechanisms in human breast cancer

Post-transcriptional regulators have emerged as robust effectors of metastasis and display deregulated expression through unknown mechanisms. Here, we reveal that the human microRNA-335 locus undergoes genetic deletion and epigenetic promoter hypermethylation in every metastatic derivative obtained from independent patients' malignant cell populations. Genetic deletion of miR-335 is a common event in human breast cancer, is enriched for in breast cancer metastases, and also correlates with ovarian cancer recurrence. We furthermore identify miR-335 as a robust inhibitor of tumor reinitiation. We thus implicate the miR-335 locus on 7q32.2 as the first selective metastasis suppressor and tumor initiation suppressor locus in human breast cancer.

Agglomerative Epigenetic Aberrations Are a Common Event in Human Breast Cancer

Changes in DNA methylation patterns are a common characteristic of cancer cells. Recent studies suggest that DNA methylation affects not only discrete genes, but it can also affect large chromosomal regions, potentially leading to LRES. It is unclear whether such long-range epigenetic events are relatively rare or frequent occurrences in cancer. Here, we use a high-resolution promoter tiling array approach to analyze DNA methylation in breast cancer specimens and normal breast tissue to address this question. We identified 3,506 cancer-specific differentially methylated regions (DMR) in human breast cancer with 2,033 being hypermethylation events and 1,473 hypomethylation events. Most of these DMRs are recurrent in breast cancer; 90% of the identified DMRs occurred in at least 33% of the samples. Interestingly, we found a nonrandom spatial distribution of aberrantly methylated regions across the genome that showed a tendency to concentrate in relatively small genomic regions. Such agglomerates of hypermethylated and hypomethylated DMRs spanned up to several hundred kilobases and were frequently found at gene family clusters. The hypermethylation events usually occurred in the proximity of the transcription start site in CpG island promoters, whereas hypomethylation events were frequently found in regions of segmental duplication. One example of a newly discovered agglomerate of hypermethylated DMRs associated with gene silencing in breast cancer that we examined in greater detail involved the protocadherin gene family clusters on chromosome 5 (PCDHA, PCDHB, and PCDHG). Taken together, our results suggest that agglomerative epigenetic aberrations are frequent events in human breast cancer.

Inactivation of Wnt inhibitory factor-1 (WIF1) expression by epigenetic silencing is a common event in breast cancer

The Wnt signaling pathway is a powerful and prominent oncogenic mechanism dysregulated in numerous cancer types. While evidence from transgenic mouse models and studies of human tumors clearly indicate that this pathway is of likely importance in human breast cancer, few clues as to the exact molecular nature of Wnt dysregulation have been uncovered in this tumor type. Here, we show that the Wnt inhibitory factor-1 (WIF1) gene, which encodes a secreted protein antagonistic to Wnt-dependent signaling, is targeted for epigenetic silencing in human breast cancer. We show that cultured human breast tumor cell lines display absent or low levels of WIF1 expression that are increased when cells are cultured with the DNA demethylating agent 5-aza-2'-deoxycytidine. Furthermore, the WIF1 promoter is aberrantly hypermethylated in these cells as judged by both methylation-specific PCR and bisulfite genomic sequencing. Using a panel of patient-matched breast tumors and normal breast tissue, we show that WIF1 expression is commonly diminished in breast tumors when compared with normal tissue and that this correlates with WIF1 promoter hypermethylation. Analysis of a panel of 24 primary breast tumors determined that the WIF1 promoter is aberrantly methylated in 67% of these tumors, indicating that epigenetic silencing of this gene is a frequent event in human breast cancer. Using an isogenic panel of cell lines proficient or deficient in the DNA methyltransferases (DNMTs) DNMT1 and/or DNMT3B, we show that hypermethylation of the WIF1 promoter is attributable to the cooperative activity of both DNMT1 and DNMT3B. Our findings establish the WIF1 gene as a target for epigenetic silencing in breast cancer and provide a mechanistic link between the dysregulation of Wnt signaling and breast tumorigenesis.

Epigenetic silencing of DSC3 is a common event in human breast cancer

IntroductionDesmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens.MethodsWe used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines.ResultsDSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure.ConclusionThese results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.

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.

Regulation of fatty acid synthase expression in breast cancer by sterol regulatory element binding protein-1c

Activation of fatty acid synthase (FAS) expression and fatty acid synthesis is a common event in human breast cancer. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate genes involved in lipid metabolism, including FAS. SREBP-1c expression is induced in liver and adipose tissue by insulin and by fasting/refeeding and is critical for nutritional regulation of lipogenic gene expression. In contrast, upregulation of fatty acid metabolism during in vitro transformation of human mammary epithelial cells and in breast cancer cells was driven by increased MAP kinase and PI 3-kinase signaling, which increased SREBP-1 levels. SREBP-1a was more abundant than SREBP-1c in many proliferative tissues and cultured cells and was thus a candidate to regulate lipogenesis for support of membrane synthesis during cell growth. We now show that SREBP-1c and FAS mRNA were both increased by H- ras transformation of MCF-10a breast epithelial cells and were both reduced by exposure of MCF-7 breast cancer cells to the MAP kinase inhibitor, PD98059, or the PI 3-kinase inhibitor, wortmannin, while SREBP-1a and SREBP-2 showed less variation. Similarly, the mRNA levels for FAS and SREBP-1c in a panel of primary human breast cancer samples showed much greater increases than did those for SREBP-1a and SREBP-2 and were significantly correlated with each other, suggesting coordinate regulation of SREBP-1c and FAS in clinical breast cancer. We conclude that regulation of FAS expression in breast cancer is achieved through modulation of SREBP-1c, similar to the regulation in liver and adipose tissue, although the upstream regulation of liopgenesis differs in these tissues.

P53 gain-of-function mutation in codon 175 is a rare event in human breast cancer.

p53 gain-of-function mutation in codon 175 is a rare event in human breast cancer.

P53 protein expression in human breast carcinoma: relationship to expression of epidermal growth factor receptor, c-erbB-2 protein overexpression, and oestrogen receptor.

The expression of p53 protein, oestrogen receptor protein, epidermal growth factor receptor (EGFR) and overexpression of the c-erbB-2 oncoprotein was examined in a series of 149 primary symptomatic breast carcinomas. Expression of p53 was present in 62 of 146 cases (42.5%) of the invasive carcinoma and one of three cases (33.3%) of ductal carcinoma in situ (DCIS) examined. Statistical associations of tumour oestrogen receptor positivity and lack of p53 protein expression, chi 2 = 19.78 (d.f. = 1), P less than 0.001, positive tumour p53 status and poor tumour grade; chi 2 = 14.1 (d.f. = 2), P less than 0.001, EGFR expression chi 2 = 7.07, (d.f. = 1), P less than 0.01 and tumour c-erbB-2 protein overexpression; chi 2 = 4.61 (d.f. = 1), P = 0.032 were identified. Expression of p53 is rare in invasive lobular carcinoma of classical type (8.3% of cases examined) in contrast to other common types of mammary carcinoma. Non-significant trends of p53 protein expression and increased regional tumour recurrence; chi 2 = 3.20 (d.f. = 1), P = 0.074 and also poorer patient survival; chi 2 = 3.76 (d.f. = 1), P = 0.053 were identified. p53 protein expression is a common event in human breast cancer and is present in both DCIS and invasive mammary carcinoma. Abnormal expression of p53 protein is a feature of both in situ and invasive breast carcinoma, implying that the abnormal p53 protein expression may be implicated in the early stages of mammary carcinoma progression.