The Diagnosis of Breast Pre-Cancer By the Chronobra- II. The Breast Pre-Cancer Test

Contemporary screening programmes for early breast cancer based on X-rays (mammography) are very expensive and have two serious limitations in their potential to control the disease. First, soft-tissue radiology imaging is poor at resolving a tumour in the youngest third of cancer patients because of breast density; second, by the time size of a tumour enables a diagnosis to be made, there are already hundreds of thousands of malignant cells; depending on the particular biology of the tumour they may have disseminated.

For effective control of the disease there must be some understanding of breast cancer biology so that appropriate pre-cancer therapeutic strategies can be implemented. There is persuasive evidence from the growth kinetics of excised tumours; the prognosis of the breast cancer process; the protection afforded by an early first pregnancy; the age-related sensitivity to diagnostic X-rays; and, the age-correlated presence of oestrogen receptors in tumours, that the genesis of cancer is usually in the pre-menopause (the second or third decade). Breast cancer prevention programmes have to take this early genesis into their reckoning. Also, to see any progenitor or pre-cancerous lesions the histopathologist must look in the cancer mastectomy specimens of the younger patient. When this is done, such mastectomies reveal multiple benign focal hyperplasias and cysts very much more numerous than in age-matched consecutive autopsy breasts. These abnormalities may represent a failure of acinisation and an abnormal maturation pathway with a large number of uncommitted epithelial cells vulnerable to mutation since they are not fully differentiated.     

Tumour Biological Aspects of CD24, A Mucin-Like Adhesion Molecule

CD24 is a molecule that recently has raised considerable attention in tumour biology. It is involved in cell adhesion and metastatic tumour spread. It has also been described as a new diagnostic marker of tumours, of neuroendocrine differentiation and, possibly most intriguing of all, of patient prognosis. High rates of CD24 expression detected by immunohistochemistry have been found in epithelial ovarian cancer (83%), breast cancer (85%), non-small cell lung cancer (45%), prostate cancer (48%) and pancreatic cancer (72%). With the exception of pancreatic cancer, high rates of CD24 are significantly associated with a more aggressive course of the disease, a finding that remains significant in a multivariate analysis. The aim of this review is to summarize relevant work covering these aspects of CD24.     

Microcalcifications in breast cancer: From pathophysiology to diagnosis and prognosis

The implementation of mammographic screening programmes in many countries has been linked to a marked increase in early detection and improved prognosis for breast cancer patients. Breast tumours can be detected by assessing several features in mammographic images but one of the most common are the presence of small deposits of calcium known as microcalcifications, which in many cases may be the only detectable sign of a breast tumour. In addition to their efficacy in the detection of breast cancer, the presence of microcalcifications within a breast tumour may also convey useful prognostic information. Breast tumours with associated calcifications display an increased rate of HER2 overexpression as well as decreased survival, increased risk of recurrence, high tumour grade and increased likelihood of spread to the lymph nodes. Clearly, the presence of microcalcifications in a tumour is a clinically significant finding, suggesting that a detailed understanding of their formation may improve our knowledge of the early stages of breast tumourigenesis, yet there are no reports which attempt to bring together recent basic science research findings and current knowledge of the clinical significance of microcalcifications. This review will summarise the most current understanding of the formation of calcifications within breast tissue and explore their associated clinical features and prognostic value.     

Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal

Circulating tumour cells (CTCs) are independent predictor of prognosis in metastatic breast cancer. Nevertheless, in one third of patients, circulating tumour cells are undetected by conventional methods. Aim of the study was to assess the prognostic value of circulating tumour cells expressing mesenchymal markers in metastatic breast cancer patients. We isolated CTC from blood of 55 metastatic breast cancer patients. CTC were characterized for cytokeratins and markers of epithelial mesenchymal transition. The gain of mesenchymal markers in CTC was correlated to prognosis of patients in a follow-up of 24 months. The presence of mesenchymal markers on CTC more accurately predicted worse prognosis than the expression of cytokeratins alone. Because of the frequent loss of epithelial antigens by CTC, assays targeting epithelial antigens may miss the most invasive cell population. Thus, there is an urgent need to improve detection methods to identify CTC which undergone epithelial mesenchymal transition program.     

M-CSF (monocyte colony stimulating factor) and M-CSF receptor expression by breast tumour cells: M-CSF mediated recruitment of tumour infiltrating monocytes?

Infiltrating immune cells in 30 primary human epithelial breast tumours were studied using specific anti-CD3 (T cells), anti-CD68 (macrophages), anti-CD57 (NK cells), and an anti-pan-B cell antibody (L26). The majority of tumour infiltrating inflammatory cells are T cells (40-50%) and monocytes/macrophages (15-35%). The macrophage specific chemo-attractant and growth factor CSF-1 is detected by immunohistochemical techniques (IHC) at the level of invasive breast cancer cells in 46/50 tumours but not at the level of in-situ (pre-invasive) cancer. A mosaic staining pattern was usually observed, with a very high expression in areas of obvious stromal invasion (90% cells positive) and absent or trace staining in intraductal carcinoma. Macrophages and plasma cells are equally intensely positive. In-situ hybridisation experiments confirm the production of CSF-1 (mRNA) by tumour cells and show the same pattern of expression. Expression of the CSF-1 receptor protein (fms) was also observed by IHC in 41/48 invasive tumours, albeit at weaker intensities than in tumour infiltrating monocytes/macrophages. A concomitant expression of both CSF-1 and fms in in-situ carcinoma was never seen (n = 14). It is therefore proposed that the associated expression of CSF-1 and its receptor may be linked to the invasive potential of breast cancer, the monocytic infiltrate being an indication of the quantitative importance of CSF-1 production by the tumour.     

Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β‐catenin signalling and apoptosis regulation in breast cancer cells

Dickkopf‐related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour‐specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial‐mesenchymal transition (EMT) and down‐regulating stem cell markers. DKK3 inhibited canonical Wnt/β‐catenin signalling through mediating β‐catenin translocation from nucleus to cytoplasm and membrane, along with reduced active‐β‐catenin, further activating non‐canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.     

Characterization and phenotypic variation with passage number of cultured human endometrial adenocarcinoma cells

Despite numerous endometrial cancer cell lines, little is know about the progression and transition of primary cultured endometrial tumours. Herein, a stage I grade III endometrial adenocarcinoma was maintained in primary culture and the phenotypic and protein expression changes were observed in relation to passage number. At early passage numbers, cultured human endometrial cancer (CHEC) cells displayed classic epithelial cell morphology, growing in groups in a glandular structure and staining positive for cytokeratin. However, with increasing passage number, CHEC cells changed in morphology to display a stromal phenotype which was accompanied by a significant reduction in cytokeratin and increases in alpha-actin and vimentin expression. Simultaneous culture of stromal cells isolated from the original tumour failed to show the same morphological characteristics or protein expression patterns. We further characterised CHEC cells through a screening of cancer related proteins, among others, caveolin-1 and Tissue factor in comparison with established cancer cell lines and corresponding non-cancerous cells. This report demonstrates that endometrial adenocarcinoma cells in culture can undergo phenotypic and protein expression changes reminiscent of epithelial-mesenchymal transition. This work suggests that primary tumours and cell lines displaying stromal morphologies may have undergone epithelial-mesenchymal transition from an adenocarcinoma origin.     

Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2

The prognosis of breast cancer in young women is influenced by reproductive history. Women diagnosed within 5 years postpartum have worse prognosis than nulliparous women or women diagnosed during pregnancy. Here we describe a mouse model of postpartum breast cancer that identifies mammary gland involution as a driving force of tumor progression. In this model, human breast cancer cells exposed to the involuting mammary microenvironment form large tumors that are characterized by abundant fibrillar collagen, high cyclooxygenase-2 (COX-2) expression and an invasive phenotype. In culture, tumor cells are invasive in a fibrillar collagen and COX-2-dependent manner. In the involuting mammary gland, inhibition of COX-2 reduces the collagen fibrillogenesis associated with involution, as well as tumor growth and tumor cell infiltration to the lung. These data support further research to determine whether women at high risk for postpartum breast cancer would benefit from treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) during postpartum involution.     

The anti-apoptotic protein lifeguard is expressed in breast cancer cells and tissues

Lifeguard (LFG) is an anti-apoptotic protein that inhibits Fas-mediated death in tumour cells. However, the molecular function of human LFG in the carcinogenesis of human breast cells is uncertain. We studied the expression and function of endogenous LFG in four breast cancer cell lines (MCF-7, MDA-MB-231, T-47D and HS 578T), a human breast epithelial cell line (HS 578Bst), and in healthy and cancerous breast tissues. Molecular (Western blot and RT-PCR) and immunohistochemical techniques were used to investigate the LFG expression. To investigate the breast cancer cell proliferation in the presence of Fas, we performed fluorescent cell viability assays. The possible association of Fas with LFG was analyzed by immunofluorescence microscopy. In this paper, we provide convincing evidence that LFG is overexpressed in several human breast cancer cell lines. More importantly, we found that the LFG expression correlates with high tumour grades in primary breast tumours. Finally, we demonstrated that Fas sensitivity is reduced in breast cancer cell lines expressing LFG. Our results indicated that LFG is strongly expressed in breast cancer epithelial cells. Moreover, the overexpression of LFG correlated with tumour grade and reduced Fas sensitivity. Our findings support the idea that LFG may have a role in the downregulation of apoptosis in breast cancer cells.     

Endothelial cell differentiation of human breast tumour stem/progenitor cells

Breast tumour stem cells have been reported to differentiate in the epithelial lineage but a cross-lineage potential has not been investigated. We aimed to evaluate whether breast tumour stem cells were able to differentiate also into the endothelial lineage. We isolated and cloned a population of breast tumour stem cells, cultured as mammospheres that expressed the stem markers nestin and Oct-4 and not epithelial and endothelial differentiation markers, and formed serially transplantable tumours in SCID mice. When cultured in the presence of serum, mammosphere-derived clones differentiated in the epithelial lineage. When cultured in the presence of VEGF, the same clones were also able to differentiate in the endothelial lineage acquiring endothelial markers and properties, such as the ability to organize in Matrigel into capillary-like structures. In the transplanted tumours, originated from mammospheres, we demonstrate that some of the intratumour vessels were of human origin, suggesting an in vivo endothelial differentiation of mammosphere-derived cells. Finally, endothelial cell clones originated from mammospheres were able, when implanted in Matrigel in SCID mice, to form after 7 days a human vessel network and, after 3–4 weeks, an epithelial tumour suggesting that in the endothelial-differentiated cells a tumourigenic stem cell population is maintained. In conclusion, the results of the present study demonstrate that stem cells of breast cancer have the ability to differentiate not only in epithelial but also in endothelial lineage, further supporting the hypothesis that the tumour-initiating population possesses stem cell characteristics relevant for tumour growth and vascularization.     

Proteomic Comparison of MCF-7 Tumoursphere and Monolayer Cultures

Breast cancer is a heterogenous disease, composed of tumour cells with differing gene expressions and phenotypes. Very few antigens have been identified and a better understanding of tumour initiating-cells as targets for therapy is critically needed. Recently, a rare subpopulation of cells within tumours has been described with the ability to: (i) initiate and sustain tumour growth; (ii) resist traditional therapies and allow for secondary tumour dissemination; and (iii) display some of the characteristics of stem cells such as self-renewal. These cells are termed tumour-initiating cells or cancer stem cells, or alternatively, in the case of breast cancer, breast cancer stem cells. Previous studies have demonstrated that breast cancer stem cells can be enriched for in “tumoursphere” culture. Proteomics represents a novel way to investigate protein expression between cells. We hypothesise that characterisation of the proteome of the breast cancer line MCF-7 tumourspheres compared to adherent/differentiated cells identifies proteins of novel interest for further isolating or targeting breast cancer stem cells. We present evidence that: (i) the proteome of adherent cells is different to the proteome of cells grown in sphere medium from either early passage (passage 2) or late passage (passage 5) spheres; (ii) that spheres are enriched in expression of a variety of tumour-relevant proteins (including MUC1 and Galectin-3); and (iii) that targeting of one of these identified proteins (galectin-3) using an inhibitor (N-acetyllactosamine) decreases sphere formation/self-renewal of MCF-7 cancer stem cells in vitro and tumourigenicity in vivo. Hence, proteomic analysis of tumourspheres may find use in identifying novel targets for future therapy. The therapeutic targeting of breast cancer stem cells, a highly clinically relevant sub-population of tumour cells, has the potential to eliminate residual disease and may become an important component of a multi-modality treatment of cancer.     

A rat cell line derived from DMBA-induced mammary carcinoma

A new cell line, designated UHKBR-01, was successfully established from a 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumour. DMBA was administered orally at a dose of 4 mg/ml per rat on the first day of the experiment and thereafter at weekly intervals of same dosage, until the rats have reached a weight of around 150-200 g. The tumours grew rapidly after the injection, and were transplanted into nude mice one the harvest size (2.5 x 2 x 1 mm(3)) was reached, it was transplanted onto nude mice. We have developed a cell line from a portion of the DMBA-induced carcinoma of the nude mice. The UHKBR-01 cell exhibited a slow increase in growth rate during the time of culture and was highly tumourigenic in nude mice. The cells have been grown in culture for over 40 passages. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, tumour antigen expression and xenograft implantation into nude mice. These cells exhibit ultrastructural and immunohistochemical features of epithelial cells of mammary origin. The above analyses also demonstrated that UHKBR-01 cells were oestrogen- and progesterone-receptor positive, in likeness to other established breast cancer cell lines such as MDA-MB-231 and MCF-7. The cell line grows as monolayers of oval-shaped cells with large folded nuclei accompanied by a rich supply of mitochondria. This report describes the first in vitro cell line from transplantable DMBA-induced mammary carcinoma of nude mice, which presents unique characteristics that may prove to be a good experimental model for investigating breast cancer biology.     

Serum exosomal lncRNA XIST is a potential non-invasive biomarker to diagnose recurrence of triple-negative breast cancer

Exosomal lncRNAs secreted by cancer cells can serve as potential biomarkers in the diagnosis and prognosis of various tumours. Here, we are committed to explore the diagnostic and prognostic value of serum exosomal XIST secreted by tumour cells to predict recurrence in patients with triple-negative breast cancer (TNBC). Significant increments in XIST and exo-XIST from tumour tissues and blood serum were found in reoccurring TNBC patients by comparison with non-recurrences. Levels of serum exo-XIST were only significantly increased in TNBC recurrence and no association with other clinicopathological parameters. Additionally, serum exo-XIST levels could be served as an assessment of change in the load of triple-negative breast cancer. Expressions of exo-XIST were markedly decreased after resection of the primary breast tumours and obviously elevated at the time of recurrence. Finally, an obvious association was identified between serum exo-XIST levels and a poorer overall survival (OS) in TNBC patients. Levels of serum exo-XIST may serve as a diagnostic and prognostic biomarker to predict the recurrent TNBC-loading status.     

Control of aromatase activity in breast tumours: the role of the immune system

Cytokines such as interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF), have been identified as important regulators of aromatase activity in fibroblasts derived from normal and malignant breast tissues, and may play an important role in controlling aromatase activity in breast tumours. The major source of such cytokines within breast tumours remains to be established but macrophages and lymphocytes, which can infiltrate tumours, have been identified as a potential source of aromatase stimulatory cytokines. To obtain further insight into the possible role played by the immune system in cancer development, and in particular its ability to regulate aromatase activity via cytokine production, we have obtained peripheral blood monocytes and lymphocytes from an immunosuppressed kidney transplant recipient, receiving cyclosporin A therapy, and a woman with breast cancer. Monocytes and lymphocytes were stimulated with lipopolysaccharide (LPS), and the conditioned medium (CM) collected from these cells was tested for its ability to stimulate aromatase activity in fibroblasts derived from normal breast tissue from a woman undergoing lumpectomy for the removal of a breast tumour. The white blood cell count was lower for the immunosuppressed patient, mainly because of the reduction in the number of monocytes and lymphocytes. The ability of CM from the monocytes and lymphocytes of the immunosuppressed patient to stimulate aromatase activity was significantly reduced (68% and 82% for monocytes and lymphocytes, respectively) compared with that of CM from the cells of the woman with breast cancer. It is possible, therefore, that immunosuppression, which has been found to be associated with a reduction in the incidence of de novo breast cancer in kidney transplant recipients, may exert its effect by inhibiting cytokine production by the cells of the immune system and thus oestrogen synthesis. In contrast to the stimulatory effects that TNF has on aromatase activity in breast fibroblasts, in MCF-7 breast cancer cells, which possess low aromatase activity, it reduced activity. However, the extent of inhibition of aromatase activity in these epithelial cells was much lower than the marked stimulation which it can induce in breast fibroblasts.     

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study

Summary Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumours cells surrounded by connective tissue. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions are reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.     

SYK expression in human breast cancer

BACKGROUND: Syk (Splenic Tyrosine Kinase) is an intracellular receptor protein kinase involved in cell proliferation, differentiation and phagocytosis. It has been studied in T and B lymphocytes, NK cells and platelets. The strong expression of Syk in mammary gland prompted research into its potential role in mammary carcinogenesis. There have been very few studies about its role in breast cancer with conflicting results. This study aims to investigate the hypothesis that Syk expression is down-regulated in breast cancer compared with ANCT and the association between its expression and clinicopathological parameters. MATERIALS AND METHODS: mRNA was extracted from 48 breast cancer specimens. Relative Syk to ribosomal RNA expression was determined by RT-PCR and Taqman methodology. Mann-Whitney U test was used to examine the association between Syk expression in cancer and ANCT. Spearman's rank correlation test was used to examine the association between Syk expression in tumours and patients' age, tumour size, tumour grade, estrogen and progesterone receptor status, lymph node metastasis, vascular invasion and clinical outcome. RESULTS: The median for the relative value of Syk expression was 0.17 and 0.18 (range: 0.12 - 0.56 and 0.0 - 1.77) for tumours and ANCT respectively. There was no significant association between Syk expression in cancers and ANCT (p= 0.598) nor between Syk expression in tumours and patients' age, tumour size, tumour grade, estrogen and progesterone receptor status, lymph node metastasis, vascular invasion or prognosis. CONCLUSION: This study shows that Syk mRNA expression does not seem to vary between breast tumours and ANCT. Furthermore, we observed no significant association between Syk expression and clinicopathological parameters.     

miR‐516a‐3p inhibits breast cancer cell growth and EMT by blocking the Pygo2/Wnt signalling pathway

miR‐516a‐3p has been reported to play a suppressive role in several types of human tumours. However, the expression level, biological function and fundamental mechanisms of miR‐516a‐3p in breast cancer remain unclear. In the present study, we found that miR‐516a‐3p expression was down‐regulated and Pygopus2 (Pygo2) expression was up‐regulated in human breast cancer tissues and cells. Through analysing the clinicopathological characteristics, we demonstrated that low miR‐516a‐3p expression or positive Pygo2 expression was a predictor of poor prognosis for patients with breast cancer. The results of a dual luciferase reporter assay and Western blot analysis indicated that Pygo2 was a target gene of miR‐516a‐3p. Moreover, overexpression of miR‐516a‐3p inhibited cell growth, migration and invasion as well as epithelial‐mesenchymal transition (EMT) of breast cancer cells, whereas reduced miR‐516a‐3p expression promoted breast cancer cell growth, migration, invasion and EMT. Furthermore, we showed that miR‐516a‐3p suppressed cell proliferation, metastasis and EMT of breast cancer cells by inhibiting Pygo2 expression. We confirmed that miR‐516a‐3p exerted an anti‐tumour effect by inhibiting the activation of the Wnt/β‐catenin pathway. Finally, xenograft tumour models were used to show that miR‐516a‐3p inhibited breast cancer cell growth and EMT via suppressing the Pygo2/Wnt signalling pathway. Taken together, these results show that miR‐516a‐3p inhibits breast cancer cell growth, metastasis and EMT by blocking the Pygo2/ Wnt/β‐catenin pathway.     

Lymphangiogenesis and its role in cancer

In many tumour types, lymphatic vasculature serves as a major route for tumour metastasis. The dissemination of malignant cells to the regional lymph nodes is an early step in the progression of many solid tumours and is an important determinant of prognosis. Lymphangiogenesis (formation of new lymphatic vessels) is thought to be crucial for cancer cells to metastasise to the regional lymph nodes. However research in this important process has been neglected largely due to the lack of molecular markers specific to the lymphatic endothelium. Recently, several specific markers have been identified including LYVE-1, podoplanin and prox-1. Although the biology of lymphangiogeneis, particularly its regulation, is still far from clear, it is now well established that tumours are lymphangiogenic i.e. they could induce the generation of their own lymphatics and metastasise to the regional lymph nodes. It is thought that the interruption of the main signalling pathways involved in this process could help to prevent lymphatic spread of many tumours. Furthermore, understanding the molecular mechanisms in lymphangiogenesis might help to develop new therapeutic strategies against cancer lymphatic spread. Here, we reviewed the literature in regards to the biology of lymphangiogenesis, its molecular regulation, lymphatic markers and the significance in human solid tumours.