The tumor suppressor Zinc finger protein 471 suppresses breast cancer growth and metastasis through inhibiting AKT and Wnt/ß-catenin signaling.

BACKGROUND: Zinc-finger protein 471 (ZNF471) is a member of the Krüppel-associated box domain zinc finger protein (KRAB-ZFP) family. ZNF471 is methylated in squamous cell carcinomas of tongue, stomach and esophageal. However, its role in breast carcinogenesis remains elusive. Here, we studied its expression, functions, and molecular mechanisms in breast cancer. METHODS: We examined ZNF471 expression by RT-PCR and qPCR. Methylation-specific PCR determined its promoter methylation. Its biological functions and related molecular mechanisms were assessed by CCK-8, clonogenicity, wound healing, Transwell, nude mice tumorigenicity, flow cytometry, BrdU-ELISA, immunohistochemistry and Western blot assays. RESULTS: ZNF471 was significantly downregulated in breast cell lines and tissues due to its promoter CpG methylation, compared with normal mammary epithelial cells and paired surgical-margin tissues. Ectopic expression of ZNF471 substantially inhibited breast tumor cell growth in vitro and in vivo, arrested cell cycle at S phase, and promoted cell apoptosis, as well as suppressed metastasis. Further knockdown of ZNF471 verified its tumor-suppressive effects. We also found that ZNF471 exerted its tumor-suppressive functions through suppressing epithelial-mesenchymal transition, tumor cell stemness and AKT and Wnt/ß-catenin signaling. CONCLUSIONS: ZNF471 functions as a tumor suppressor that was epigenetically inactivated in breast cancer. Its inhibition of AKT and Wnt/ß-catenin signaling pathways is one of the mechanisms underlying its anti-cancer effects.

ZMYND10, an epigenetically regulated tumor suppressor, exerts tumor-suppressive functions via miR145-5p/NEDD9 axis in breast cancer.

BACKGROUND: Recent studies suggested that ZMYND10 is a potential tumor suppressor gene in multiple tumor types. However, the mechanism by which ZMYND10 inhibits breast cancer remains unclear. Here, we investigated the role and mechanism of ZMYND10 in breast cancer inhibition. RESULTS: ZMYND10 was dramatically reduced in multiple breast cancer cell lines and tissues, which was associated with promoter hypermethylation. Ectopic expression of ZMYND10 in silenced breast cancer cells induced cell apoptosis while suppressed cell growth, cell migration and invasion in vitro, and xenograft tumor growth in vivo. Furthermore, molecular mechanism studies indicated that ZMYND10 enhances expression of miR145-5p, which suppresses the expression of NEDD9 protein through directly targeting the 3'-untranslated region of NEDD9 mRNA. CONCLUSIONS: Results from this study show that ZMYND10 suppresses breast cancer tumorigenicity by inhibiting the miR145-5p/NEDD9 signaling pathway. This novel discovered signaling pathway may be a valid target for small molecules that might help to develop new therapies to better inhibit the breast cancer metastasis.

Recent insights into the molecular pathogenesis of mammary phyllodes tumours.

Phyllodes tumours (PTs) of the breast are true biphasic neoplasms within which interactions between the epithelium and stroma are critical for tumour development and progression. Despite numerous studies reporting the results of ancillary marker investigations in PTs, the current histological grading systems remain unreliable at predicting clinical outcome even when supplemented by these markers. As a consequence, there has been much interest in the prospect of using molecular/genetic techniques to develop a more robust "grading" system. This review focuses on recent cytogenetic and molecular studies investigating the pathogenesis of PTs and those correlating molecular findings with clinicopathological features of the tumours. Recent data highlight that intratumoural genetic heterogeneity is common in PTs and may account for the reported lack of correlation between histological grading and clinical behaviour. The entire spectrum of molecular aberrations in PTs are yet to be fully defined, however recent array-based studies using comparative genomic hybridisation have reported that copy number changes increase with the progression from benign PT to malignancy. Tumour recurrence and progression is likely to reflect the presence of under-recognised subclones. p(16INK4a) (CDKN2A) inactivation also appears to be important in PT pathogenesis. Further additional studies will be required to identify and validate new prognostic markers and therapeutic targets in order to improve the diagnosis, classification, prediction of outcome and management of patients with this rare neoplasm. Data generated from modern sequencing technologies are likely to provide new insights into the disease and assist in this endeavour.

Magnetic resonance in the detection of breast cancers of different histological types.

Breast cancer incidence is increasing worldwide. Early detection is critical for long-term patient survival, as is monitoring responses to chemotherapy for management of the disease. Magnetic resonance imaging and spectroscopy (MRI/MRS) has gained in importance in the last decade for the diagnosis and monitoring of breast cancer therapy. The sensitivity of MRI/MRS for anatomical delineation is very high and the consensus is that MRI is more sensitive in detection than x-ray mammography. Advantages of MRS include delivery of biochemical information about tumor metabolism, which can potentially assist in the staging of cancers and monitoring responses to treatment. The roles of MRS and MRI in screening and monitoring responses to treatment of breast cancer are reviewed here. We rationalize how it is that different histological types of breast cancer are differentially detected and characterized by MR methods.

E-cadherin expression in the epithelial components of mammary phyllodes tumors.

Phyllodes tumors are rare but clinically important fibroepithelial tumors of the breast. Both epithelial and stromal components actively interact with each other to participate in phyllodes tumor development. Accumulated evidence suggests that the Wnt signaling pathway is important in this stromal-epithelial interaction. Given that Wnt signaling also affects E-cadherin-dependent cellular adhesion and alteration of E-cadherin is common in epithelial cancers, it is possible that alteration of E-cadherin occurs also in the epithelial components of phyllodes tumor. We assessed epithelial E-cadherin expression in 155 phyllodes tumor cases, including 92 benign (59%), 42 borderline (27%), and 21 malignant phyllodes tumor (14%), by immunohistochemistry. Its expression was correlated with clinicopathologic features and phyllodes tumor recurrence. Significant correlations of both membranous and cytoplasmic E-cadherin expression were found with stromal cellularity (P = .009 and .013, respectively), overgrowth (P = .005 and .009, respectively), and mitotic counts (P = .023 and .029, respectively) but not tumor grade, margin, and nuclear atypia. Interestingly, a significantly higher level of cytoplasmic epithelial E-cadherin expression was found in those tumors with recurrence (score, 278.79±40.91 versus 250.00±63.46) and shorter specific disease-free survival (172.24±12.63 versus 207.24±19.71 months). Further multivariate analysis showed epithelial E-cadherin expression as an independent prognostic factor for phyllodes tumor-specific survival (P<.001 for cytoplasmic staining and .001 for membranous staining). In conclusion, we have demonstrated an association of epithelial E-cadherin expression with stromal histologic features and disease recurrence in phyllodes tumor. These findings provide further evidence of the importance of stromal-epithelial interactions in phyllodes tumors and highlight the potential value of epithelial components in prognostication.

Involvement of α- and ß-catenins and E-cadherin in the development of mammary phyllodes tumours.

AIMS: Phyllodes tumours (PT) are rare but clinically important fibroepithelial tumours of the breast. ß-Catenin, a key component in Wnt signalling, has been shown to be important in the development of PT. It also functions as a component of the cadherin complex, which may therefore be implicated in PT pathogenesis. By assessing stromal α-catenin, ß-catenin and E-cadherin expression in 158 PT cases using immunohistochemistry and examining associations with clinicopathological features, we aimed to determine the role of these proteins in PT pathogenesis. METHODS AND RESULTS: Cytoplasmic ß-catenin correlated with α-catenin expression. A significantly higher expression of both markers was observed in borderline than in benign PT (P = 0.003 and <0.001, respectively), but a lower level was found in malignant PT. Cytoplasmic E-cadherin expression was significantly higher in borderline and malignant than in benign PT (P = 0.001 and 0.012, respectively), but was not correlated with other markers. Both E-cadherin and α-catenin showed stronger correlations with histological parameters than ß-catenin. α-Catenin showed a significant correlation with recurrence (P = 0.005 and 0.016, respectively). CONCLUSION: α- and ß-catenins may be important in the early stages of PT development, while E-cadherin may be required for malignant development. The correlation of α-catenin expression with tumour recurrence may be relevant in predicting PT behaviour.

The ubiquitin peptidase UCHL1 induces G0/G1 cell cycle arrest and apoptosis through stabilizing p53 and is frequently silenced in breast cancer.

BACKGROUND: Breast cancer (BrCa) is a complex disease driven by aberrant gene alterations and environmental factors. Recent studies reveal that abnormal epigenetic gene regulation also plays an important role in its pathogenesis. Ubiquitin carboxyl- terminal esterase L1 (UCHL1) is a tumor suppressor silenced by promoter methylation in multiple cancers, but its role and alterations in breast tumorigenesis remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: We found that UCHL1 was frequently downregulated or silenced in breast cancer cell lines and tumor tissues, but readily expressed in normal breast tissues and mammary epithelial cells. Promoter methylation of UCHL1 was detected in 9 of 10 breast cancer cell lines (90%) and 53 of 66 (80%) primary tumors, but rarely in normal breast tissues, which was statistically correlated with advanced clinical stage and progesterone receptor status. Pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. Ectopic expression of UCHL1 significantly suppressed the colony formation and proliferation of breast tumor cells, through inducing G0/G1 cell cycle arrest and apoptosis. Subcellular localization study showed that UCHL1 increased cytoplasmic abundance of p53. We further found that UCHL1 induced p53 accumulation and reduced MDM2 protein level, and subsequently upregulated the expression of p21, as well as cleavage of caspase3 and PARP, but not in catalytic mutant UCHL1 C90S-expressed cells. CONCLUSIONS/SIGNIFICANCE: UCHL1 exerts its tumor suppressive functions by inducing G0/G1cell cycle arrest and apoptosis in breast tumorigenesis, requiring its deubiquitinase activity. Its frequent silencing by promoter CpG methylation may serve as a potential tumor marker for breast cancer.

ERp29 induces breast cancer cell growth arrest and survival through modulation of activation of p38 and upregulation of ER stress protein p58IPK.

Endoplasmic reticulum protein 29 (ERp29) is an ER luminal protein that has a role in protein unfolding and secretion, but its role in cancer is unclear. Recently, we reported that overexpression of ERp29 significantly inhibited cell proliferation and prevented tumorigenesis in highly proliferative MDA-MB-231 breast cancer cells. Here, we show that ERp29-induced cancer cell growth arrest is modulated by the interplay between the concomitant phosphorylation of p38 and upregulation of the inhibitor of the interferon-induced, double-stranded RNA-activated protein kinase, p58(IPK). In this cell model, ERp29 overexpression significantly downregulates modulators of cell proliferation, namely urokinase plasminogen activator receptor, ß(1)-integrin and epidermal growth factor receptor. Furthermore, ERp29 significantly (P<0.001) increases phosphorylation of p38 (p-p38) and reduces matrix metalloproteinase-9 secretion. The role of ERp29 in upregulating cyclin-dependent kinase inhibitors (p15 and p21) and in downregulating cyclin D(2) is demonstrated in slowly proliferating ERp29-overexpressing MDA-MB-231 cells, whereas the opposite response was observed in ERp29-knockdown MCF-7 cells. Pharmacological inhibition of p-p38 downregulates p15 and p21 and inhibits eIF2α phosphorylation, indicating a role for p-p38 in this process. Furthermore, p58(IPK) expression was increased in ERp29-overexpressing MDA-MB-231 cells and highly decreased in ERp29-knockdown MCF-7 cells. This upregulation of p58(IPK) by ERp29 suppresses the activation of p-p38/p-PERK/p-eIF2α by repressing eIF2α phosphorylation. In fact, reduction of p58(IPK) expression by RNA interference stimulated eIF2α phosphorylation. The repression of eIF2α phosphorylation by p58(IPK) prevents ERp29-transfected cells from undergoing ER-dependent apoptosis driven by the activation of ATF4/CHOP/caspase-3. Hence, the interplay between p38 phosphorylation and p58(IPK) upregulation has key roles in modulating ERp29-induced cell-growth arrest and survival.

Aurora-A expression, hormone receptor status and clinical outcome in hormone related cancers.

AIMS: We investigated the correlation between protein expression of Aurora-A with hormone receptor expression and clinicopathological parameters in ovarian, breast and prostate cancer. METHODS: Subcellular expression of Aurora-A, and androgen receptor (AR), oestrogen receptor (ER) and progesterone receptor (PR) expression, were examined by immunohistochemistry in human tissue microarrays of the three cancer types and by Western blot in cancer cell lines and selected patient tissues. RESULTS: Subgroups of all three cancer types exhibited both nuclear and cytoplasmic expression of Aurora-A. Nuclear presence of Aurora-A was observed in ER positive and negative breast cancer cell lines and tissues. Eighteen of the 126 (14%) tumour tissues that showed nuclear expression of Aurora-A were strongly associated with ER and PR positive breast tumours (p = 0.001). Cytoplasmic expression of AR and Aurora-A was strongly associated in prostate cancer tissues (45% versus 0, p = 0.015). Ovarian tumours (n = 45) with Aurora-A nuclear expression had decreased patient survival (mean survival, 29.5 versus 106.7 months; p < 0.0005) and showed a significant association with recurrence-free survival (mean survival 19.7 versus 95.9 months; p = 0.002). CONCLUSION: Association between nuclear Aurora-A with hormone receptors in breast cancer and with poor clinical outcome in ovarian cancer suggests the significance of active Aurora-A in disease initiation and progression.

Over-expression of ERp29 attenuates doxorubicin-induced cell apoptosis through up-regulation of Hsp27 in breast cancer cells.

The endoplasmic reticulum protein 29 (ERp29) has a critical role in regulating protein folding, maturation and secretion. However, its role in carcinogenesis remains elusive. Recently, we reported that ERp29 is a novel tumor suppressor and regulates mesenchymal-epithelial transition in MDA-MB-231 breast cancer cells. Here, we investigated whether ERp29 plays a role in the response of breast cancer cells to chemotherapeutic agents. We found that expression of ERp29 increased the resistance to doxorubicin, but not cisplatin and paclitaxel, and decreased the doxorubicin-induced cell apoptosis in MDA-MB-231 cells, whereas knockdown of ERp29 in MCF-7 cells increased the doxorubicin cytotoxicity. A proteomics study identified up-regulation of Hsp27 and down-regulation of stathmin-1, galectin and prohibitin in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells. Further, we demonstrated that ERp29 up-regulated expression of Hsp27 by down-regulating eukaryotic translational initiation factor 2α (eIF2α). When Hsp27 was knocked down by siRNA in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells and parental MCF-7 cells, cell viability was significantly decreased and doxorubicin-induced cell apoptosis was enhanced. These results indicate that Hsp27 is involved in the ERp29-mediated resistance to doxorubicin. Therefore, targeting of Hsp27, with a combination of other chemotherapeutic agents, is a rational strategy in treating doxorubicin-resistant cancer cells.

PLCD1 is a functional tumor suppressor inducing G(2)/M arrest and frequently methylated in breast cancer.

Chromosome 3p harbors multiple tumor-suppressor genes. PLCD1, located at 3p22, encodes an enzyme that mediates regulatory signaling of energy metabolism, calcium homeostasis and intracellular movement. We investigated the epigenetic alterations of PLCD1 and its tumor suppressor function in breast cancer. Frequent downregulation/silencing of PLCD1 was shown in most breast cancer cell lines by using semi-quantitative PCR. Promoter methylation of PLCD1 was detected in 78% (7/9) of cell lines and 52% (13/25) of primary tumors by Methylation-specific PCR (MSP), but not in any tumor adjacent breast tissues and normal breast tissues, which was further confirmed by bisulfite genomic sequencing (BGS). The silencing of PLCD1 could be reversed by pharmacological demethylation, indicating a methylation-mediated mechanism. Ectopic expression of PLCD1 in silenced breast cancer cells significantly inhibited their colony formation. In addition, PLCD1 inhibited tumor cell migration and induced cell cycle G(2)/M arrest. Thus, this study for the first time demonstrates the frequent inactivation of PLCD1 by promoter methylation and its tumor inhibitory function in breast cancer. Tumor-specific methylation of PLCD1 might serve as a biomarker for possible early detection and prognosis prediction of breast cancer.

Molecular-assisted immunohistochemical optimization.

Immunohistochemistry (IHC) is an essential tool in diagnostic surgical pathology, allowing analysis of protein subcellular localization. The use of IHC by different laboratories has lead to inconsistencies in published literature for several antibodies, due to either interpretative (inter-observer variation) or technical reasons. These disparities have major implications in both clinical and research settings. In this study, we report our experience conducting an IHC optimization of antibodies against five proteins previously identified by proteomic analysis to be breast cancer biomarkers, namely 6PGL (PGLS), CAZ2 (CAPZA2), PA2G4 (EBP1) PSD2 and TKT. Large variations in the immunolocalizations and intensities were observed when manipulating the antigen retrieval method and primary antibody incubation concentration. However, the use of an independent molecular analysis method provided a clear indication in choosing the appropriate biologically and functionally relevant "staining pattern". Without this latter step, each of these contradictory results would have been a priori "technically acceptable" and would have led to different biological and functional interpretations of these proteins and potentially different applications in a routine pathology setting. Thus, we conclude that full validation of immunohistochemical protocols for scientific and clinical use will require the incorporation of biological knowledge of the biomarker and the disease in question.

A cognitive virtual microscopic framework for knowledge-based exploration of large microscopic images in breast cancer histopathology.

Histopathological examination is a powerful method for prognosis of major diseases such as breast cancer. Analysis of medical images largely remains the work of human experts. Current virtual microscope systems are mainly an emulation of real microscopes with annotation and some image analysis capabilities. However, the lack of effective knowledge management prevents such systems from being computer-aided prognosis platforms. The cognitive virtual microscopic framework, through an extended modeling and use of medical knowledge, has the capacity to analyse histopathological images and to perform grading of breast cancer, providing pathologists with a robust and traceable second opinion.

Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies.

Transplantation of human embryonic stem cells (hESC) into immune-deficient mice leads to the formation of differentiated tumors comprising all three germ layers, resembling spontaneous human teratomas. Teratoma assays are considered the gold standard for demonstrating differentiation potential of pluripotent hESC and hold promise as a standard for assessing safety among hESC-derived cell populations intended for therapeutic applications. We tested the potency of teratoma formation in seven anatomical transplantation locations (kidney capsule, muscle, subcutaneous space, peritoneal cavity, testis, liver, epididymal fat pad) in SCID mice with and without addition of Matrigel, and found that intramuscular teratoma formation was the most experimentally convenient, reproducible, and quantifiable. In the same experimental setting, we compared undifferentiated hESC and differentiated populations enriched for either beating cardiomyocytes or definitive endoderm derivatives (insulin-secreting beta cells), and showed that all cell preparations rapidly formed teratomas with varying percentages of mesoderm, ectoderm, and endoderm. In limiting dilution experiments, we found that as little as two hESC colonies spiked into feeder fibroblasts produced a teratoma, while a more rigorous single-cell titration achieved a detection limit of 1/4000. In summary, we established core parameters essential for facilitating safety profiling of hESC-derived products for future therapeutic applications.

Annexin-1 regulates growth arrest induced by high levels of estrogen in MCF-7 breast cancer cells.

Estrogen, a naturally occurring female steroid growth hormone, has been implicated as a major risk factor for the development of breast cancer. Recent research into this disease has also correlated Annexin-1 (ANXA1), a glucocorticoid-inducible protein, with the development of breast tumorigenesis. ANXA1 is lost in many cancers, including breast cancer, and this may result in a functional promotion of tumor growth. In this study, we investigated the expression of ANXA1 in MCF-7 cells treated with estrogen and the regulation of estrogen functions by ANXA1. Exposure of MCF-7 breast cancer cells to high physiologic levels (up to 100 nmol/L) of estrogen leads to an up-regulation of ANXA1 expression partially through the activation of cyclic AMP-responsive element binding protein and dependency on activation of the estrogen receptor. In addition, treatment of MCF-7 cells with physiologic levels of estrogen (1 nmol/L) induced proliferation, whereas high pregnancy levels of estrogen (100 nmol/L) induced a growth arrest of MCF-7 cells, associated with constitutive activation of extracellular signal-regulated kinase 1/2 and up-regulation of cell cycle arrest proteins such as p21(waf/cip). Silencing of ANXA1 with specific small interfering RNA reverses the estrogen-dependent proliferation as well as growth arrest and concomitantly modulates extracellular signal-regulated kinase 1/2 phosphorylation. We confirm that ANXA1 is lost in clinical breast cancer, indicating that the antiproliferative protective function of ANXA1 against high levels of estrogen may be lost. Finally, we show that ANXA1-deficient mice exhibit faster carcinogen-induced tumor growth. Our data suggest that ANXA1 may act as a tumor suppressor gene and modulate the proliferative functions of estrogens.

Pathogenic mechanisms in the initiation and progression of mammary phyllodes tumours.

Mammary phyllodes tumours (PTs) are rare fibroepithelial neoplasms of the breast. They have a propensity to recur locally and the ability to metastasise. There is some correlation between the histological parameters of PTs and their biological behaviour; however, no single feature or grading scheme can accurately predict the behaviour of a PT. The PTs that will recur and/or metastasise are not being precisely delineated with the currently available diagnostic tools. A number of pathways/markers have been implicated in the pathogenesis of PTs including hormone receptors, members of the Wnt pathway, cell cycle proteins, factors involved in angiogenesis, tyrosine kinase receptors and matrix metalloproteases. The currently available evidence supports a model where initiation of PTs involves interactions between the epithelium and stroma and these interactions are lost with the progression to malignancy such that growth of the stroma becomes autonomous of the epithelium. Loss of the stromal-epithelial interdependancy, increased stromal proliferation, angiogenesis and matrix alterations appear to be involved in the progression to malignancy.

Increased epidermal growth factor receptor (EGFR) expression in malignant mammary phyllodes tumors.

Mammary phyllodes tumors are uncommon stromal-epithelial neoplasms, and are divided into benign, borderline malignant and frankly malignant groups on the basis of their histological features. Accumulating evidence shows that epidermal growth factor receptor (EGFR) is involved in the pathogenesis and progression of many malignancies. This study investigated 453 phyllodes tumors (296 benign, 98 borderline, 59 malignant) for EGFR expression using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for gene amplification. The staining was correlated to tumor margin status, degree of malignancy, stromal cellularity, mitotic activity, nuclear pleomorphism and stromal overgrowth. Cases with strong positive IHC staining were selected for FISH. The overall positive rate for EGFR was 16.2% (48/296), 30.6% (30/98) and 56% (33/59) for benign, borderline malignant and frankly malignant phyllodes tumors, respectively. FISH demonstrated egfr gene amplification in 8% of immunohistochemically positive cases. The results of this study provide strong evidence that EGFR overexpression is involved in the pathogenesis of phyllodes tumors, although gene amplification may not be the major underlying mechanism for overexpression.