Breast Cancer Research update - june 9
Written by admin on Jun 14th, 2008 | Filed under: oncology UPDATE
Mammosphere culture of metastatic breast cancer cells enriches for tumourigenic breast cancer cells.
Background: The identification of potential breast cancer stem cells is of importance as the characteristics of stem cells suggest that they are resistant to conventional forms of therapy. Several techniques have been proposed to isolate or enrich for tumorigenic breast cancer stem cells including i) culture of cells in non-adherent, non-differentiating conditions to form mammospheres, and ii) sorting of the cells by their surface phenotype (expression of CD24 and CD44). Methods: We have cultured metastatic cells found in pleural effusions from breast cancer patients in non-adherent conditions without serum to form mammospheres. Dissociated cells from these mammospheres were used to determine the tumorigenicity of these cultures. Expression of CD24 and CD44 on uncultured cells and mammospheres derived from the pleural effusions was documented. Results: We found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged. After dissociation and plating with serum onto adherent dishes the cells can differentiate, as determined by the increased expression of cytokeratins and MUC1. Analysis of surface expression of CD24 and CD44 on uncultured cells from 21 of the samples showed that the cells from some samples separated into two populations, but some did not. The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres. Of 8 pleural effusion mammospheres tested in SCID mice, 4 were found to induce tumours when only 5000 or fewer cells were injected, while the same number of uncultured cells did not form tumours. The ability to induce tumours appeared to correlate with the ability to produce the larger mammospheres. Uncultured cells from a highly tumorigenic sample (PE14) were uniformly negative for surface expression of both CD24 and CD44. Conclusions: This paper shows, for the first time, that mammosphere culture of pleural effusions enriches for cells capable of inducing tumours in SCID mice. The data suggest that mammosphere culture of these metastatic cells could provide a highly appropriate model for studying the sensitivity of the tumorigenic “stem” cells to therapeutic agents, and for further characterisation of the tumour inducing subpopulation of breast cancer cells.
Jab1 is a target of EGFR signaling in ER-alpha negative breast cancer
Introductionc-Jun activation domain binding protein-1 (Jab1) is a multifunctional signaling protein that has previously been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in ER-alpha negative breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ER-alpha negative phenotype. Methods: MDA-MB-231 and MDA-MB-468 ER-alpha negative/EGFR positive cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry. Results: EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with co-localization of pERK and Jab1, as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ER-alpha negative subset (n=154, p=0.019). The same association was also confirmed for S100A7 and Jab1 (p=0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (p=0.004). Conclusions: Jab1 is a target of EGFR signaling in ER-alpha negative cell lines and breast tumors and therefore may be a common, central factor and potential therapeutic target for important cell signaling pathways in ER-alpha negative breast cancer.
Are the so-called low penetrance breast cancer genes, ATM, BRIP1, PALB2 and CHEK2, high risk for women with strong family histories?
A woman typically presents for genetic counselling because she has a strong family history and is interested in knowing the probability she will develop disease in the future; that is, her absolute risk. Relative risk for a given factor refers to risk compared with either population average risk (sense a), or risk when not having the factor, with all other factors held constant (sense b). Not understanding that these are three distinct concepts can result in failure to correctly appreciate the consequences of studies on clinical genetic testing. Several studies found that the frequencies of mutations in ATM, BRIP1, PALB2 and CHEK2 were many times greater for cases with a strong family history than for controls. To account for the selected case sampling (ascertainment), a statistical model that assumes that the effect of any measured variant multiplies the effect of unmeasured variants was applied. This multiplicative polygenic model in effect estimated the relative risk in the sense b, not sense a, and found it was in the range of 1.7 to 2.4. The authors concluded that the variants are “low penetrance”. They failed to note that their model fits predicted that, for some women, absolute risk may be as high as for BRCA2 mutation carriers. This is because the relative risk multiplies polygenic risk, and the latter is predicted by family history. Therefore, mutation testing of these genes for women with a strong family history, especially if it is of early onset, may be as clinically relevant as it is for BRCA1 and BRCA2.
Mammary carcinoma behavior is programmed in the precancer stem cell
IntroductionThe “MINO” mouse model of ductal carcinoma in situ (DCIS) consists of six lines with distinct morphologic phenotypes and behavior each meeting experimentally defined criteria for “pre-cancer”. Specifically, these lines grow orthotopically in cleared mammary fat pads and consistently progress to an invasive phenotype capable of ectopic growth. Transition to carcinoma has a consistent latency for each line and three of the lines also show pulmonary metastatic potential. Methods: Gland cleared orthotopic transplanted precancer MINO tissues were analyzed by BAC and oligo array CGH, microsatellite PCR, and telomerase repeat amplification assay. MINO cells were dissociated and cultured in three dimensional (3D) culture and transplanted in syngeneic gland cleared mammary fat pads. Results: Comparative genomic hybridization shows that the pre-cancer and invasive tumors are genetically stable with low level changes including whole chromosome gains in some lines. No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally. Microsatellite analysis shows a low frequency of alterations which are predominantly permanent within a MINO line. Telomerase activity is increased in both the MINO and the derived tumors when compared to normal mouse mammary gland. Dissociation of the precancer lesion cells and three dimensional “spheroid” culture of single cells reveals a bipotential for myoepithelial and luminal differentiation and the formation of unique 3D “MINOspheres.” These “MINOspheres” show features that are intermediate between spheroids that are derived from normal and carcinoma cells. Transplantation of a single cell derived MINOsphere recapitulates the outgrowth of the precancer morphology and progression to carcinoma. Conclusion: These data establish a pre-cancer stem cell capable of self renewal and multilineage differentiation as the origin of invasive cancer. In the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic hits for transformation.
EGFR and phosphorylated EGFR (pEGFR) expression in invasive breast carcinomas; pEGFR relation to invasiveness and angiogenesis and the impact of EGFR/pEGFR phenotype on overall survival
IntroductionEpidermal growth factor receptor (EGFR) is involved in regulating cell growth in breast carcinomas. Its activated form (pEGFR) is correlated with poor prognosis in lung cancer, whereas it has not yet been fully investigated in breast cancer. The aim of this study was to investigate the expression of EGFR and pEGFR and their correlation with overall and disease free, survival, clinicopathological parameters and biological markers of invasion and angiogenesis (pAkt, uPAR, MMP-14, VEGFR-1/Flt-1). Methods: A three-step immunohistochemical method (ABC/HPR) was applied on paraffin-embedded sections from 154 patients with invasive breast carcinoma to detect the expression of the proteins EGFR, pEGFR, ER, PR, c-erbB-2, pAkt, VEGFR-1/Flt-1, MMP-14 and uPAR. The results were statistically processed using chi-square test. Overall and disease-free survival distribution curves were assessed by Kaplan-Meier test and log-rank statistics followed by Cox’s proportional hazards regression model. Results: EGFR and pEGFR proteins were immunodetected in the membrane of the malignant cells (11.9% and 35.7% respectively). EGFR expression was positively correlated with nuclear grade (p=0.004) and negatively correlated with hormonal receptors ER (p=0.005). pEGFR was positively related to the Akt pathway (p=0.008) and appeared to participate in invasion and metastasis (uPAR: p=0.049, MMP-14: p=0.025 and VEGFR-1/Flt-1: p=0.016). Univariate analysis showed that EGFR/pEGFR phenotype was associated with poor overall survival (p=0.019), a finding further supported by multivariate analysis (p=0.013). Conclusions: These data provide the evidence that pEGFR expression is related to angiogenesis (via VEGFR-1/Flt-1, MMP-14 and pAkt pathways) and invasiveness (via uPAR, MMP-14 and pAkt pathways) and EGFR/pEGFR phenotype is associated with poor patient survival in invasive breast cancer.
Alternative splicing and the progesterone receptor in breast cancer
Progesterone receptor status is a marker for hormone responsiveness and disease prognosis in breast cancer. Progesterone receptor negative tumours have generally been shown to have a poorer prognosis than progesterone receptor positive tumours. The observed loss of progesterone receptor could be through a range of mechanisms, including the generation of alternatively spliced progesterone receptor variants that are not detectable by current screening methods. Many progesterone receptor mRNA variants have been described with deletions of various whole, multiple or partial exons that encode differing protein functional domains. These variants may alter the progestin responsiveness of a tissue and contribute to the abnormal growth associated with breast cancer. Absence of specific functional domains from these spliced variants may also make them undetectable or indistinguishable from full length progesterone receptor by conventional antibodies. A comprehensive investigation into the expression profile and activity of progesterone receptor spliced variants in breast cancer is required to advance our understanding of tumour hormone receptor status. This, in turn, may aid the development of new biomarkers of disease prognosis and improve adjuvant treatment decisions.
Surface-enhanced laser desorption/ionization time-of-flight proteomic profiling of breast carcinomas identifies clinicopathologically relevant groups of patients similar to previously defined clusters from cDNA expression
IntroductionMicroarray-based gene expression profiling represents a major breakthrough for understanding the molecular complexity of breast cancer. cDNA expression profiles cannot detect changes in activities that arise from post-translational modifications, however, and therefore do not provide a complete picture of all biologically important changes that occur in tumors. Additional opportunities to identify and/or validate molecular signatures of breast carcinomas are provided by proteomic approaches. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) offers high-throughput protein profiling, leading to extraction of protein array data, calling for effective and appropriate use of bioinformatics and statistical tools. Methods: Whole tissue lysates of 105 breast carcinomas were analyzed on IMAC 30 ProteinChip Arrays (Bio-Rad, Hercules, CA, USA) using the ProteinChip Reader Model PBS IIc (Bio-Rad) and Ciphergen ProteinChip software (Bio-Rad, Hercules, CA, USA). Cluster analysis of protein spectra was performed to identify protein patterns potentially related to established clinicopathological variables and/or tumor markers. Results: Unsupervised hierarchical clustering of 130 peaks detected in spectra from breast cancer tissue lysates provided six clusters of peaks and five groups of patients differing significantly in tumor type, nuclear grade, presence of hormonal receptors, mucin 1 and cytokeratin 5/6 or cytokeratin 14. These tumor groups resembled closely luminal types A and B, basal and HER2-like carcinomas. Conclusion: Our results show similar clustering of tumors to those provided by cDNA expression profiles of breast carcinomas. This fact testifies the validity of the SELDI-TOF MS proteomic approach in such a type of study. As SELDI-TOF MS provides different information from cDNA expression profiles, the results suggest the technique’s potential to supplement and expand our knowledge of breast cancer, to identify novel biomarkers and to produce clinically useful classifications of breast carcinomas.
The future of mammary stem cell biology: the power of in vivo transplants – authors’ response
The letter from Drs. Lindeman, Visvader, Smalley and Eaves conveys their concern regarding the future of the prospective isolation and characterization of individual cells that may be characterized as mammary stem cells upon in vivo transplantation. As they have pointed out, the difficulties encountered in identifying specific mammary epithelial subtypes by different levels of fluorescence (particularly for membrane components that decorate most if not all mammary epithelial cells) leads to differential reporting and “resultant confusion” and “underscores the need for improved standardization”.
Effects of common germline genetic variation in cell cycle control genes on breast cancer survival: results from a population-based cohort
IntroductionSomatic alterations have been shown to correlate with breast cancer prognosis and survival, but less is known about the effects of common inherited genetic variation. Of particular interest are genes involved in cell cycle pathways, which regulate cell division. Methods: We examined associations between common germline genetic variation in 13 genes involved in cell cycle control (CCND1, CCND2, CCND3, CCNE1, CDK2 [p33], CDK4, CDK6, CDKN1A [p21, Cip1], CDKN1B [p27, Kip1], CDKN2A [p16], CDKN2B [p15], CDKN2C [p18], and CDKN2D [p19]) and survival among women diagnosed with invasive breast cancer participating in the SEARCH (Studies of Epidemiology and Risk factors in Cancer Heredity) breast cancer study. DNA from up to 4,470 women was genotyped for 85 polymorphisms that tag the known common polymorphisms (minor allele frequency > 0.05) in the genes. The genotypes of each polymorphism were tested for association with survival using Cox regression analysis. Results: The rare allele of the tagging single nucleotide polymorphism (SNP) rs2479717 is associated with an increased risk of death (hazard ratio = 1.26 per rare allele carried, 95% confidence interval: 1.12 to 1.42; P = 0.0001), which was not attenuated after adjusting for tumour stage, grade, and treatment. This SNP is part of a large linkage disequilibrium block, which contains CCND3, BYSL, TRFP, USP49, C6ofr49, FRS3, and PGC. We evaluated the association of survival and somatic expression of these genes in breast tumours using expression microarray data from seven published datasets. Elevated expression of the C6orf49 transcript was associated with breast cancer survival, adding biological interest to the finding. Conclusion: It is possible that CCND3 rs2479717, or another variant it tags, is associated with prognosis after a diagnosis of breast cancer. Further study is required to validate this finding.
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