Understanding Of Breast Cancer Progression Research Articles

Germline variants and breast cancer survival in patients with distant metastases at primary breast cancer diagnosis

Breast cancer metastasis accounts for most of the deaths from breast cancer. Identification of germline variants associated with survival in aggressive types of breast cancer may inform understanding of breast cancer progression and assist treatment. In this analysis, we studied the associations between germline variants and breast cancer survival for patients with distant metastases at primary breast cancer diagnosis. We used data from the Breast Cancer Association Consortium (BCAC) including 1062 women of European ancestry with metastatic breast cancer, 606 of whom died of breast cancer. We identified two germline variants on chromosome 1, rs138569520 and rs146023652, significantly associated with breast cancer-specific survival (P = 3.19 × 10−8 and 4.42 × 10−8). In silico analysis suggested a potential regulatory effect of the variants on the nearby target genes SDE2 and H3F3A. However, the variants showed no evidence of association in a smaller replication dataset. The validation dataset was obtained from the SNPs to Risk of Metastasis (StoRM) study and included 293 patients with metastatic primary breast cancer at diagnosis. Ultimately, larger replication studies are needed to confirm the identified associations.

Expression of Annexin A2 Promotes Cancer Progression in Estrogen Receptor Negative Breast Cancers.

When breast cancer progresses to a metastatic stage, survival rates decline rapidly and it is considered incurable. Thus, deciphering the critical mechanisms of metastasis is of vital importance to develop new treatment options. We hypothesize that studying the proteins that are newly synthesized during the metastatic processes of migration and invasion will greatly enhance our understanding of breast cancer progression. We conducted a mass spectrometry screen following bioorthogonal noncanonical amino acid tagging to elucidate changes in the nascent proteome that occur during epidermal growth factor stimulation in migrating and invading cells. Annexin A2 was identified in this screen and subsequent examination of breast cancer cell lines revealed that Annexin A2 is specifically upregulated in estrogen receptor negative (ER-) cell lines. Furthermore, siRNA knockdown showed that Annexin A2 expression promotes the proliferation, wound healing and directional migration of breast cancer cells. In patients, Annexin A2 expression is increased in ER- breast cancer subtypes. Additionally, high Annexin A2 expression confers a higher probability of distant metastasis specifically for ER- patients. This work establishes a pivotal role of Annexin A2 in breast cancer progression and identifies Annexin A2 as a potential therapeutic target for the more aggressive and harder to treat ER- subtype.

Atypical ductal hyperplasia is a multipotent precursor of breast carcinoma.

The current model for breast cancer progression proposes independent 'low grade (LG)-like' and 'high grade (HG)-like' pathways but lacks a known precursor to HG cancer. We applied low-coverage whole-genome sequencing to atypical ductal hyperplasia (ADH) with and without carcinoma to shed light on breast cancer progression. Fourteen out of twenty isolated ADH cases harboured at least one copy number alteration (CNA), but had fewer aberrations than LG or HG ductal carcinoma in situ (DCIS). ADH carried more HG-like CNA than LG DCIS (e.g. 8q gain). Correspondingly, 64% (7/11) of ADH cases with synchronous HG carcinoma were clonally related, similar to LG carcinoma (67%, 6/9). This study represents a significant shift in our understanding of breast cancer progression, with ADH as a common precursor lesion to the independent 'low grade-like' and 'high grade-like' pathways. These data suggest that ADH can be a precursor of HG breast cancer and that LG and HG carcinomas can evolve from a similar ancestor lesion. We propose that although LG DCIS may be committed to a LG molecular pathway, ADH may remain multipotent, progressing to either LG or HG carcinoma. This multipotent nature suggests that some ADH cases could be more clinically significant than LG DCIS, requiring biomarkers for personalising management. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PO-345 The role of the deubiquitinase USP11 in endocrine-driven breast cancer

IntroductionApproximately 80% of breast cancers overexpress the oestrogen receptor α (ERα) and depend on this key transcriptional regulator for growth. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which remove ubiquitin moieties from proteins, in regulating ERα activity in breast cancer.Material and methodsTo identify DUBs involved in ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all 108 known or putative human DUB genes. We found that suppression of the BRCA-associated DUB, USP11, downregulated ERα transcriptional activity in ZR-75–1 cells. Dual luciferase reporter assays and qRT-PCR were used to determine ERα activity in stable ZR-75–1 USP11 knockdown cell lines. PTMScan technology, which allows for enrichment of ubiquitinated proteins and quantitative profiling by mass spectrometry, was used to reveal the ubiquitinome in these cells and results are pending. RNA sequencing (RNA-seq) technology was carried out in estrogen-independent LCC1 USP11 knockdown cells, were USP11 was hypothesised to play a vital role. Finally, immunohistochemistry (IHC) was used to investigate the prognostic relevance of USP11 in breast cancer patients.Results and discussionsKnockdown of USP11 in ZR-75–1 cells decreased ERα transcriptional activity and mRNA expression of ERα target genes. These results were further validated in a HEK293T USP11 CRISPR knockout model with ectopic ERα expression. Interestingly, USP11 expression was upregulated in LCC1 cells, an isogenic, estrogen-independent model derived from MCF-7 cells. Knockdown of USP11 in LCC1 cells decreased the expression of multiple ERα target genes and cell cycle-associated genes, as determined by RNA-seq. IHC staining of a breast cancer tissue microarray (103 ER +patients) and subsequent Kaplan-Meier analysis of this cohort revealed a significant association between high USP11 expression and poor overall (p=0.030) and breast cancer-specific survival (p=0.041). In silico analysis of publically available breast cancer gene expression datasets further supported an association between high USP11 expression and poor prognosis.ConclusionThese results suggest a role for USP11 in ERα transcriptional activity and reveal a novel mechanism as to how this receptor is regulated in breast cancer.

Abstract P2-05-02: Functional genomic screening identifies ubiquitin-specific protease 11 (USP11) as a novel regulator of ER-alpha transcription in breast cancer

Abstract Approximately 70% of breast cancers overexpress the estrogen receptor α (ERα) and depend on this key transcriptional regulator for growth and differentiation. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer attractive new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which act to remove ubiquitin moieties from proteins, in regulating transcriptional activity of ERα in breast cancer. To identify DUBs involved in the regulation of ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all human DUB genes. The DUB library consisted of pools of four non-overlapping shRNAs targeting all 108 known or putative DUBs (432 shRNAs in total). We found that suppression of a number of DUBs markedly repressed or enhanced the activity of an estrogen-response-element (ERE) luciferase reporter following estradiol (E2) stimulation. Of particular interest, suppression of the BRCA2-associated DUB, USP11, was found to down-regulate ERα transcriptional activity. Subsequent validation using two individual siRNAs targeted to USP11 revealed a notable reduction in expression of endogenous ERα target genes in the ZR-75-1 cell line, as quantified using qRT-PCR. Further validation was carried out in a HEK293T USP11 knockout cell line, where reduced activity of an ERE-luciferase reporter was detected when compared to wild-type cells. This phenotype was rescued with a USP11 overexpression vector, both in the presence and absence of E2. Furthermore, USP11 expression was found to be upregulated in the estrogen-independent cell line LCC1 when compared to their parental MCF7 cells. Knockdown of USP11 in LCC1 cells resulted in decreased mRNA expression of a panel of ERα target genes, while RNA-seq revealed a downregulation of several putative ERα target genes and a downregulation of many cell cycle-associated proteins. To support the prognostic relevance of USP11, immunohistochemical staining of a breast cancer tissue microarray (103 ER+ patients available for final analysis) was performed. Kaplan-Meier analysis of this cohort revealed a highly significant association between high USP11 expression and poor overall (p=0.030) and breast cancer-specific survival (p=0.041). In silico analysis of publically available breast cancer gene expression datasets further supported an association between high USP11 mRNA levels and poor prognosis. We observed a significant correlation between high expression of USP11 mRNA in ER-positive patients and poor distant metastasis-free survival (HR 2, CI 1.37-2.91, p=0.00023). This correlation was also significant in ER-positive patients who had received tamoxifen only (HR 2.9, CI 1.63-5.15, p=0.00015). These results suggest a role for USP11 in driving cellular growth and identify USP11 as novel therapeutic target in breast cancer. Citation Format: Dwane L, Das S, Moran B, O'Connor AE, Mulrane L, Dirac AM, Jirstrom K, Crown JP, Bernards R, Gallagher WM, Ní Chonghaile T, O'Connor DP. Functional genomic screening identifies ubiquitin-specific protease 11 (USP11) as a novel regulator of ER-alpha transcription in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-02.

Abstract 3040: Functional genomic screening identifies USP11 as a novel regulator of ERα transcription in breast cancer

Abstract Approximately 70% of breast cancers overexpress the estrogen receptor α (ERα) and depend on this key transcriptional regulator for growth and differentiation. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer attractive new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which act to remove ubiquitin moieties from proteins, in regulating transcriptional activity of ERα in breast cancer. To identify DUBs involved in the regulation of ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all human DUB genes. The DUB library consisted of pools of four non-overlapping shRNAs targeting all 108 known or putative DUBs (432 shRNAs in total). We found that suppression of a number of DUBs markedly repressed or enhanced the activity of an estrogen-response-element (ERE) luciferase reporter following estradiol (E2) stimulation. Of particular interest, suppression of the BRCA2-associated DUB, USP11, was found to down-regulate ERα transcriptional activity. Subsequent validation using two individual siRNAs targeted to USP11 revealed a notable reduction in expression of endogenous ERα target genes in the ZR-75-1 cell line, as quantified using qRT-PCR. Immunoprecipitation of ERα revealed no physical interaction with USP11, however E2 stimulation resulted in translocation of USP11 to the nucleus, suggesting a potential role in E2-induced transcription. Furthermore, USP11 expression was found to be upregulated in the estrogen-independent cell line LCC1 when compared to their parental MCF7 cells. Knockdown of USP11 in LCC1 cells resulted in decreased mRNA expression of a panel of ERα target genes, suggesting a role for USP11 in an estrogen independent setting. To support the prognostic relevance of USP11, immunohistochemical staining of a breast cancer tissue microarray (n=144) was performed. Kaplan-Meier analysis of this cohort revealed a highly significant association between poor overall survival (OS) (p=0.030) and breast cancer-specific survival (BCSS) (p=0.041). In silico analysis of publically available breast cancer gene expression datasets further supported an association between high USP11 mRNA levels and poor prognosis. We observed a significant correlation between high expression of USP11 mRNA in ER-positive patients and poor distant metastasis-free survival (DMFS) (HR 2, CI 1.37-2.91, p=0.00023). This correlation was also significant in ER-positive patients who had received endocrine therapy only (HR 2.9, CI 1.63-5.15, p=0.00015). These results suggest a role for USP11 in driving cellular growth and identify USP11 as novel therapeutic target in breast cancer. Citation Format: Lisa Dwane, Aisling E. O'Connor, Laoighse Mulrane, Annette M. Dirac, Karin Jirstrom, John P. Crown, Rene Bernards, William M. Gallagher, Tríona Ní Chonghaile, Darran P. O'Connor. Functional genomic screening identifies USP11 as a novel regulator of ERα transcription in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3040. doi:10.1158/1538-7445.AM2017-3040

Breast cancer metastasis - insight into selected molecular mechanisms of the phenomenon.

Metastasis is a complex, multistep biological process, involving a multitude of genes and biomolecules. Despite the successful therapeutic management of breast cancer, including surgery, chemotherapy and radiation therapy, that can control primary tumor growth, metastatic disease remains the greatest clinical challenge in oncology, as these methods are still not very effective in preventing relapses or in the management of breast cancer metastases. The knowledge of its mechanisms is still fragmentary and needs to be broadened in order to improve our therapeutic approach and influence on the long-term control of breast cancer progression. Despite the constant progress in understanding of breast cancer progression, it remains a major health problem around the world. Novel therapeutic modalities are being tested and developed, but the incidence and mortality rates are still frightening. In this paper, we review selected aspects of breast cancer metastasis, including the metastatic cascade and models of dissemination, tumor angiogenesis, disaggregation and migration of cells from the primary tumor, breaking the vascular wall, adaptation to a new environment, organotropism and dormant cells. The interactions between cancer cells and normal host cells contributing significantly to the metastatic cascade are highlighted, and a wide range of signaling and stimulating biomolecules and genes involved in the process are introduced.

Epigenetic and metabolic regulation of breast cancer stem cells.

Breast cancer has a relatively high mortality rate in women due to recurrence and metastasis. Increasing evidence has identified a rare population of cells with stem cell-like properties in breast cancer. These cells, termed cancer stem cells (CSCs), which have the capacity for self-renewal and differentiation, contribute significantly to tumor progression, recurrence, drug resistance and metastasis. Clarifying the mechanisms regulating breast CSCs has important implications for our understanding of breast cancer progression and therapeutics. A strong connection has been found between breast CSCs and epithelial mesenchymal transition (EMT). In addition, recent studies suggest that the maintenance of the breast CSC phenotype is associated with epigenetic and metabolic regulation. In this review, we focus on recent discoveries about the connection between EMT and CSC, and advances made in understanding the roles and mechanisms of epigenetic and metabolic reprogramming in controlling breast CSC properties.

Mining genome sequencing data to identify the genomic features linked to breast cancer histopathology

Background: Genetics and genomics have radically altered our understanding of breast cancer progression. However, the genomic basis of various histopathologic features of breast cancer is not yet well-defined. Materials and Methods: The Cancer Genome Atlas (TCGA) is an international database containing a large collection of human cancer genome sequencing data. cBioPortal is a web tool developed for mining these sequencing data. We performed mining of TCGA sequencing data in an attempt to characterize the genomic features correlated with breast cancer histopathology. We first assessed the quality of the TCGA data using a group of genes with known alterations in various cancers. Both genome-wide gene mutation and copy number changes as well as a group of genes with a high frequency of genetic changes were then correlated with various histopathologic features of invasive breast cancer. Results: Validation of TCGA data using a group of genes with known alterations in breast cancer suggests that the TCGA has accurately documented the genomic abnormalities of multiple malignancies. Further analysis of TCGA breast cancer sequencing data shows that accumulation of specific genomic defects is associated with higher tumor grade, larger tumor size and receptor negativity. Distinct groups of genomic changes were found to be associated with the different grades of invasive ductal carcinoma. The mutator role of the TP53 gene was validated by genomic sequencing data of invasive breast cancer and TP53 mutation was found to play a critical role in defining high tumor grade. Conclusions: Data mining of the TCGA genome sequencing data is an innovative and reliable method to help characterize the genomic abnormalities associated with histopathologic features of invasive breast cancer.

Deregulated Expression of ANXA1 in Human High-Grade Breast Cancers

Background: Breast cancer is a heterogeneous disease comprising a diversity of tumor subtypes. All of them differ from each other resulting in a wide array of clinical pictures, risks of recurrence and response to treatment. In recent years, tumor biomarkers have changed the way breast cancers are diagnosed and treated. In this paper we have sought to investigate the differential expression of ANXA1, a multifunctional calcium binding protein, among various molecular subtypes of breast cancers and in particular triple negative tumors. Methods: ANXA1 was first studied using in-silico analysis on available DNA microarray and RNA sequencing data of human breast tissues. Next we ascertained ANXA1 expression on cell lines and breast carcinoma tissue microarrays along with cognate normal breast tissue. Results: Whereas ANXA1 expression is normally restricted to the normal myoepithelial cell layer it becomes ectopically and aberrantly expressed in tumor cells of a significant minority of aggressive breast cancers. Specifically, we found that ANXA1 expression is severely deregulated in high-grade breast cancers that comprise clinically aggressive tumors such as triple-negative and, to some extent, HER2-positive breast cancers. Conclusion: Our results indicate that ANXA1 is a valuable breast cancer biomarker that can help to segregate and dissect out subsets of high histological grade breast cancers paving the way to a better understanding of breast cancer progression and metastasis.

Abstract 1053: DNMT3B7 alters E-cadherin expression and cell proliferation in breast cancer cells

Abstract Breast cancer is the second-leading cause of cancer death among women in the United States and the progression from non-invasive to invasive/metastatic disease correlates with an increased risk of death. Changes in DNA methylation have been shown to be important in breast cancers. However, the mechanism by which cancer cells acquire and maintain abnormal methylation is not well understood. DNA methyltransferases (DNMTs) are responsible for monitoring and altering DNA methylation patterns in cells. Interestingly, we have identified many aberrant DNMT3B transcripts in cancer cells compared to normal cells. Therefore, we hypothesize that aberrant DNMT3B isoforms alter gene and protein expression to promote tumor progression in breast cancer cells. Our studies indicate that the breast cancer cell lines MCF-7 and MDA-MB-231 express many aberrant DNMT3B isoforms including DNMT3B7 and ΔDNMT3B6. Interestingly, the invasive MDA-MB-231 cells express increased levels of DNMT3B7 and ΔDNMT3B6 compared to the poorly invasive MCF-7 cells. To further our studies, poorly invasive MCF-7 cells were stably transfected with a DNMT3B7 expression construct to determine the role of this aberrant transcript in tumor progression. Preliminary data indicate that the increased expression of DNMT3B7 in these cells correlates with a significant increase in growth. Specifically, cells expressing more DNMT3B7 grow faster than those with lower levels of the aberrant protein. Furthermore, E-cadherin expression is inversely correlated with DNMT3B7 expression indicating that the expression of this aberrant protein may lead to a more aggressive phenotype. Finally, soft agar assays have been performed to determine the functional significance of these findings. The novel results presented in this study suggest the need for additional studies to determine the role of aberrant DNMT3B transcripts in cancer. Ultimately, this work may lead to a better understanding of breast cancer progression and the development of novel therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1053. doi:1538-7445.AM2012-1053

PD03-07: Breast Cancer Heterogeneity and Treatment Resistance: Clues from Metaplastic Tumors.

Abstract At late stage, nearly all breast cancers are heterogeneous and refractory to treatment, like metaplastic breast cancer is at an early stage. These rare carcinomas are highly aggressive and de-differentiated. They are enriched for mesenchymal and stem cell features and essentially fail current therapies. As metaplastic tumors provide a time-compressed picture of breast cancer progression early on, understanding these tumors will yield insight into mechanisms that drive breast cancer into advanced stages and treatment resistance. To investigate a genetic basis for heterogeneity in metaplastic breast cancer, we established a progression model comprising three cell lines. The cell lines were derived from a primary tumor, a local recurrence and a pleural effusion of a 40-year old patient. The primary tumor was a stage III invasive metaplastic, triple negative, inflammatory breast cancer, resected after neoadjuvant chemotherapy (capecitabine and taxotere, then adriamycin and one cycle of bevacizumab). The local recurrence, biopsied seven months post mastectomy, developed after the patient received adjuvant carboplatin and gemcitabine for 3 cycles and then radiation to the chest wall. At this time, the patient had lung metastases and was treated with taxol and bevacizumab yielding a mixed response. Local invasive growth continued and a malignant pleural effusion developed four months later. Analyzing the genetic and molecular characteristics of this progression model in vitro, its tumorigenicity and metastasis in vivo, and interrogating lead findings in a growing collection of metaplastic tumors helps us to dissect the genetic heterogeneity in breast cancer, and potentially to identify the cell types that drive disease progression and treatment resistance. Our gene expression analyses and genomic evaluations identified epithelial to mesenchymal transition (EMT) as a key characteristic in the progression and treatment resistance of this cancer. Major changes in cytoskeletal genes, chemokines and their receptors, amplification of drug transporter proteins, metalloproteinases and matrix proteins seen with increasing motility and invasiveness along with recruitment of host inflammatory responses in the in vivo model, loss of chromosomal regions harboring known and putative tumor suppressors, and deletions of genes encoding proteins for metabolic inactivation of sex hormones in the breast tissue, along with specific loss of clusters of desmosomal genes are guiding our understanding of metaplastic breast cancer progression. The results provide insight into the development, the extremely invasive nature, and treatment resistance of these tumors. Our collaborative network of clinicians, pathologists, translational genomic researchers and bioinformatics specialists will enable us to identify and prioritize genetic events as disease drivers, prognostic biomarkers of disease progression, and determinants of treatment resistance. Our goal is to identify molecular and functional targets for effective therapy and evaluate them in the clinic. Lessons learned from metaplastic breast cancer will improve our understanding of breast cancer progression in general, and could translate into effective treatments for advanced breast cancer where current standard of care is failing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD03-07.

Molecular evolution of breast cancer

Molecular analysis of invasive breast cancer and its precursors has furthered our understanding of breast cancer progression. In the past few years, new multi-step pathways of breast cancer progression have been delineated through genotypic-phenotypic correlations. Nuclear grade, more than any other pathological feature, is strongly associated with the number and pattern of molecular genetic abnormalities in breast cancer cells. Thus, there are two distinct major pathways to the evolution of low- and high-grade invasive carcinomas: whilst the former consistently show oestrogen receptor (ER) and progesterone receptor (PgR) positivity and 16q loss, the latter are usually ER/PgR-negative and show Her-2 overexpression/amplification and complex karyotypes. The boundaries between the evolutionary pathways of well-differentiated/low-grade ductal and lobular carcinomas have been blurred, with changes in E-cadherin expression being one of the few distinguishing features between the two. In addition, lesions long thought to be precursors of breast carcinomas, such as hyperplasia of usual type, are currently considered mere risk indicators, whilst columnar cell lesions are now implicated as non-obligate precursors of atypical ductal hyperplasia (ADH) and well-differentiated ductal carcinoma in situ (DCIS). However, only through the combination of comprehensive morphological analysis and cutting-edge molecular tools can this knowledge be translated into clinical practice and patient management.