Annexins are associated with metastasis and infiltration of cancer cells. Proteomic analysis and immunohistochemical staining were used to understand whether several annexins play important roles in cancer alone and/or synergistically. Seven fresh breast cancer samples with 23 paraffin specimens, three fresh pancreatic samples and five fresh laryngeal carcinoma samples with 25 paraffin specimens were obtained from humans, as well as ten golden hamster pancreatic cancer tissue samples, and they were used to observe differential expression of annexins compared with normal tissues using proteomics and immunohistochemical staining. Annexin A2, A4 and A5 were overexpressed in human breast cancer and laryngeal carcinoma tissues and in golden hamster pancreatic cancer tissue samples, respectively, as shown by proteomics and immunohistochemical staining. In addition, annexin A4 and A5 were expressed in breast cancer tissues, while annexin A1 was not expressed. Annexin A1, A2 and A4 were expressed in human laryngeal carcinoma tissues as shown by immunohistochemical staining. Annexin A1, A2, A4 and A5 played important roles in breast cancer, pancreatic cancer and laryngeal carcinoma, alone and/or synergistically, and they may be targets of therapy for malignant tumors. The choice of which annexins to target should depend on their respective biological behaviors.
breast cancer; pancreatic cancer; laryngeal carcinoma; annexins; proteomics; immunohistochemical staining; overexpression
Annexins are calcium and phospholipid binding proteins that form an evolutionary conserved multigene family. Considerable evidence indicates that annexin A10 (ANXA10) is involved in tumoral progression, although little is known about its role in human oral carcinogenesis. In this study, we investigated the involvement of ANXA10 in oral squamous cell carcinoma (OSCC).
ANXA10 mRNA and protein expressions were assessed by quantitative reverse transcriptase polymerase chain reaction and immunoblotting, and we conducted a proliferation assay and cell-cycle analysis in ANXA10 knockdown cells in vitro. We evaluated the correlation between the ANXA10 expression status in 100 primary OSCCs and the clinicopathological features by immunohistochemistry. ANXA10 mRNA and protein expression levels were up-regulated in all cellular lines examined (n = 7, p<0.05). ANXA10 knockdown cells showed that cellular proliferation decreased by inactivation of extracellular regulated kinase (ERK) (p<0.05), and cell-cycle arrest at the G1 phase resulted from up-regulation of cyclin-dependent kinase inhibitors. ANXA10 protein expression in primary OSCCs was also significantly greater than in normal counterparts (p<0.05), and higher expression was correlated with tumoral size (p = 0.027).
Our results proposed for the first time that ANXA10 is an indicator of cellular proliferation in OSCCs. Our results suggested that ANXA10 expression might indicate cellular proliferation and ANXA10 might be a potential therapeutic target for the development of new treatments for OSCCs.
Our recent research identified the protein annexin A2 to be regulated by ovarian cancer-peritoneal cell interactions. This study investigated the role of annexin A2 in ovarian cancer metastasis and its potential utility as a novel therapeutic target, using in vitro and in vivo ovarian cancer models. Annexin A2 expression was examined by qRT-PCR and western blotting in ovarian cancer cell lines and immunohistochemistry in serous ovarian carcinoma tissues. Annexin A2 siRNAs were used to evaluate the effects of annexin A2 suppression on ovarian cancer cell adhesion, motility, and invasion. Furthermore, annexin A2 neutralizing antibodies were used to examine the role of annexin A2 in tumor invasion and metastasis in vivo using a chick chorioallantoic membrane assay and an intraperitoneal xenograft mouse model. Strong annexin A2 immunostaining was observed in 90% (38/42) of the serous ovarian cancer cells and was significantly increased in the cancer-associated stroma compared to non-malignant ovarian tissues. Annexin A2 siRNA significantly inhibited the motility and invasion of serous ovarian cancer cells and adhesion to the peritoneal cells. Annexin A2 neutralizing antibodies significantly inhibited OV-90 cell motility and invasion in vitro and in vivo using the chick chorioallantoic membrane assay. The growth of SKOV-3 cells and their peritoneal dissemination in nude mice was significantly inhibited by annexin A2 neutralizing antibodies. Annexin A2 plays a critical role in ovarian cancer metastasis and is therefore a potential novel therapeutic target against ovarian cancer.
A candidate oncogene GIG47, previously known as a neudesin with a neurotrophic activity, was identified by applying the differential expression analysis method.
As a first step to understand the molecular role of GIG47, we analyzed the expression profile of GIG47 in multiple human cancers including the breast cancer and characterized its function related to human carcinogenesis. Based on this oncogenic role of GIG47, we then embarked on determining the high-resolution structure of GIG47. We have applied multidimensional heteronuclear NMR methods to GIG47.
GIG47 was over-expressed in primary breast tumors as well as other human tumors including carcinomas of the uterine cervix, malignant lymphoma, colon, lung, skin, and leukemia. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 cells. The ectopic expression of GIG47 in MCF7 cells promoted the invasiveness in the presence of 50% serum. In addition, it also resulted in the increased tumorigenicity in in vivo tumor formation assay. The tumorigenesis mechanism involving GIG47 might be mediated by the activation of MAPK and PI3K pathways. These results indicate that GIG47 plays a role in the breast tumorigenesis, thus representing a novel target for the treatment of breast cancer. To facilitate the development of GIG47-targeted therapeutics, we determined the structural configuration of GIG47. The high-resolution structure of GIG47 was obtained by combination of NMR and homology modeling. The overall structure of GIG47 has four α-helices and 6 β-strands, arranged in a β1-α1-β2-β3-α2-β4-α3-α4-β5-β6 topology. There is a potential heme/steroid binding pocket formed between two helices α2 and α3.
The determined three-dimensional structure of GIG47 may facilitate the development of potential anti-cancer agents.
Breast cancer; Oncogene; GIG47; Three-dimensional structure; Anti-cancer agents
Annexin VI is one of a family of calcium-dependent phospholipid-binding proteins. Although the function of this protein is not known, various physiological roles have been proposed, including a role in the budding of clathrin-coated pits (Lin et al., 1992. Cell. 70:283-291.). In this study we have investigated a possible endocytotic role for annexin VI in intact cells, using the human squamous carcinoma cell line A431, and report that these cells do not express endogenous annexin VI, as judged by Western and Northern blotting and PCR/Southern blotting. To examine whether endocytosis might in some way be either facilitated or inhibited by the presence of annexin VI, a series of A431 clones were isolated in which annexin VI expression was achieved by stable transfection. These cells expressed annexin VI at similar levels to other human cell types. Using assays for endocytosis and recycling of the transferrin receptor, we report that each of these cellular processes occurs with identical kinetics in both transfected and wild- type A431 cells. In addition, purified annexin VI failed to support the scission of coated pits in permeabilized A431 cells. We conclude that annexin VI is not an essential component of the endocytic pathway, and that in A431 cells, annexin VI fails to exert any influence on internalization and recycling of the transferrin receptor.
Hepatitis C virus (HCV) infection is a global health problem that affects more than 170 million people worldwide. It is a major cause of cirrhosis and hepatocellular carcinoma, making the virus the most common cause of liver failure and transplantation. The standard-of-care treatment for chronic hepatitis C (CHC) has been changed during the last decade and direct acting antiviral drugs have already been used. Besides, understanding of the pathogenesis of CHC has evolved rapidly during the last years and now several host factors are known to affect the natural history and response to treatment. Recent genome-wide association studies have shown the important role of interleukin-28B and inosine triphosphatase in HCV infection. The present review article attempts to summarize the current knowledge on the role of host factors towards individualization of HCV treatment.
Chronic hepatitis C; Hepatitis C virus host factors; Interleukin 28B; Inosine triphosphatase; Single nucleotide polymorphism
Annexin A5 is thought to have a role in the pathophysiology of the antiphospholipid syndrome (APS)—a syndrome characterised by recurrent thrombosis and pregnancy morbidity.
To investigate whether anti‐annexin A5 immunoglobulin (Ig)M or IgG antibodies, or the −1C→T polymorphism of annexin A5, is a risk factor for thrombosis or miscarriage, and whether the −1C→T polymorphism is correlated with APS.
A cohort study was carried out with a population of 198 patients with primary APS, systemic lupus erythematosus or lupus‐like disease. For the detection of anti‐annexin A5 antibodies and the measurement of annexin A5 plasma levels, ELISA‐type methods were used. The annexin A5 −1C→T mutation was detected by restriction fragment length polymorphism.
71 patients were positive for annexin A5 IgM or IgG antibodies, of whom 53 patients were positive for anti‐annexin A5 IgG antibodies and 27 of 198 patients were positive for anti‐annexin A5 IgM antibodies. The prevalence of IgM or IgG anti‐annexin A5 antibodies was not significantly associated with thrombosis or miscarriage on multivariate analysis. The prevalence of the −1C→T mutation in the annexin A5 gene (46/198 patients) was significantly associated with miscarriage (odds ratio 2.7, 95% confidence interval 1.1 to 6.7, independent risk factor).
The detection of anti‐annexin A5 antibodies does not seem relevant for estimating the risk for thrombosis or miscarriage in APS. The −1C→T mutation was an independent risk factor for miscarriage, which is independent of APS.
The steroid hormones, estrogens and progesterone are key drivers of postnatal breast development and are linked to breast carcinogenesis. Experiments in the mouse mammary gland have revealed that they rely on paracrine factors to relegate their signal locally and to amplify it. In particular, RANKL is a key mediator of progesterone action. Systemic inhibition of RANKL blocked proliferation in the mammary epithelium with potential clinical implications: a RANKL-inhibiting antibody, Denosumab (Amgen), has been approved by the US Food and Drug Administration for osteoporosis treatment. Two publications now provide evidence that progestin-driven mouse mammary tumorigenesis can be blocked by ablating RANK signaling. Can the osteoporosis drug help breast cancer patients? The burning question now is whether the role of this pathway is conserved in the human breast and whether RANKL signaling has a role in the pathogenesis of one or more subtypes of breast cancer.
The expression of Annexin A1 (ANXA1) is known to be reduced in human breast cancer; however, the role of ANXA1 expression in the development of breast cancer remains unclear. In this study, we determined the relationship between the expression features of ANXA1 and the prognostic factors of breast cancer.
Human breast tissues were obtained from patients specimens who had undergone breast surgery or core needle biopsies. The patterns of ANXA1 expression were analyzed by immunohistochemical staining in relation to histopathological diagnosis, clinical characteristics and outcomes.
One hundred eighty-two cases were included and the mean age of the patients was 46.34 ± 11.5 years. A significant loss of ANXA1 expression was noted in both ductal carcinoma in situ (DCIS) and invasive carcinomas compared to normal breast tissues (p<0.001) and benign breast diseases (p<0.001). There was a significant alteration in ANXA1 expression according to hormone receptor status (p<0.001), cancer intrinsic type (p<0.001), and nuclear grade (p=0.004) in invasive cancer. In a univariate analysis, ANXA1 positivity tended to be related with poor breast cancer-related survival (p=0.062); however, the same results was not realized in multivariate results (p=0.406). HER2 overexpression and TNM staging were significantly associated with relapse-free survivals (RFS) in the multivariate analysis (p=0.037, p=0.048, respectively). In particular, in node-positive patients (p=0.048), HER2 overexpressed patients (p=0.013), and non-triple negative breast cancer patients (p=0.002), ANXA1 overexpression was correlated with poor RFS.
Although significant loss of ANXA1 expression was noted in breast cancer including DCIS and invasive carcinoma, in cases of invasive cancer, overexpression of ANXA1 was related to unfavorable prognostic factors. And these results imply that ANXA1 plays dualistic roles and is involved in variable mechanisms related to cancer development and progression.
Annexin A1; Breast neoplasms
Nuclear factor-kappa B (NF-κB) signaling constitutes a key event in the multistep process of carcinogenesis, progression and treatment in many cancer types. However, the significance of NF-κB pathway for complex and tissue-specific aspects of head and neck cancer progression, such as invasion and metastasis, is less understood.
The expression of NF-κB p65 in squamous cell carcinoma of the head and neck (SCCHN) clinical specimens by immunohistochemistry. The role of NF-κB activity in head and neck squamous cell carcinoma was determined by western blot, reporter assay and EMSA analysis in vitro and metastasis assays in vivo in different metastatic potential tumor cells. Furthermore, the apoptosis rate and expression of metastasis-related protein such as MMP9 and VEGF were examined by Annexin V/PI staining and Western blot, respectively.
A higher level of active nuclear-localized NF-κB was observed in the metastatic SCCHN specimens group (p < 0.01). The NF-κB activities of SCCHN cell lines with different metastatic potentials were then determined and in excellent agreement with results found in SCCHN specimens, highly metastatic SCCHN cell lines expressed high level of NF-κB activity. The treatment of highly metastatic SCCHN cells with NF-κB inhibitors reduced the in vitro cell invasion capacity of the cells without affecting the apoptotic rate. Additionally, the NF-κB inhibitors significantly inhibited the experimental lung metastasis of Tb cells and lymph node metastasis of TL cells in nude mice. Furthermore, the expression of metastasis-related proteins, such as matrix metalloproteinase 9 and vascular endothelial growth factor, was inhibited by pyrrolidine dithiocarbonate.
This study suggests that NF-κB activity significantly contributes to tumor hematologic and lymphatic metastases and may aid in the development of early detection methods or therapies targeting non-conventional molecular targets.
Multiple genetic alterations play a role in the pathogenesis of ovarian cancer. Although many key proteins and pathways involved in ovarian carcinogenesis and metastasis have been discovered, knowledge of the early steps leading to malignancy remains poorly understood. This poor understanding stems from lack of data from early-stage cancers and absence of a well-established premalignant state universal to all ovarian cancer subtypes. Existing evidence suggests that ovarian cancers develop either through a stepwise mutation process (low-grade pathway), through genetic instability resulting in hastened metastasis (high-grade pathway), or more recently through what has been described as the “‘fimbrial-ovarian’ serous neoplasia theory.” In this latter model, ovarian serous cancers evolve from premalignant lesions in the distal fallopian tube called tubal intraepithelial carcinoma. In this manuscript, we review key genetic and molecular changes that occur in cancer cell progression and suggest a model of ovarian cancer pathogenesis involving both tumor cell mutations and microenvironmental factors.
Carcinogenesis; Ovarian cancer
Annexin 2 is a calcium-dependent phospholipid-binding protein that has been implicated in a number of membranerelated events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.
Myocardial infarction is the combined result of environmental factors and personal predispositions. Prothrombotic factors might play an important role in this phenomenon. Annexin V (ANV) is a calcium-dependent glycoprotein widely present in various tissues exerting a potent anticoagulant effect in vitro by reducing plaque adhesion and aggregation. Anti-annexin V antibodies (aANVAs) are detected in various diseases like rheumatoid arthritis, systemic lupus erythematosus and anti-phospholipid antibody syndrome. The study of ANV in Acute Myocardial Infarction (AMI) might shed light on hypercoagulability mechanisms in the pathogenesis of acute coronary syndromes. This study was conducted to investigate the association of plasma ANV, aANVAs and anti-cardiolipin antibodies (aCLAs) with AMI.
This study recruited 45 patients with the diagnosis of AMI according to WHO criteria in their first 24 hours of admission. 36 matched individuals were studied as the control group with normal coronary artery angiography. Plasma levels of ANV, aANVAs and aCLAs were determined by enzyme-linked immunosorbent assay and the results were compared.
Plasma ANV levels in the patients with AMI on admission were significantly lower than those in the control group (p = 0.002). Positive test for aANVAs were found to be present in a significant number of our patients (p = 0.004). The studied groups were similar in their rate of patients with positive aCLAs tests. ANV, aANVAs and aCLAs were not correlated with hypertension, diabetes mellitus, hyperlipidemia, sex, age and smoking.
Our findings suggest that low plasma ANV levels along with positive aANVAs tests in patients with AMI are indicative of hypercoagulable state that is not related to the traditional cardiovascular risk factors.
In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE), we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells. This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells. Here, we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations. In brief, granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C. After 30–90 min at 37°C more than 80–90% of the cells become early apoptotic (Annexin V positive/propidium iodide negative). After 24 h of rewarming at 37°C 98% of the cells were late apoptotic (secondary necrotic; Annexin V positive/propidium iodide positive). Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C. Inter-nucleosomal chromatin cleavage and caspase activation were other characteristics of this cold-shock-induced process of apoptosis. Consequently, apoptosis could be inhibited by a caspase inhibitor. Finally, SLE-derived anti-chromatin autoantibodies showed a high affinity for apoptotic blebs generated by cold-shock. Overall, cold-shock induced apoptosis is achieved without the addition of toxic compounds or antibodies, and quickly leads to synchronized homogeneous apoptotic cell populations, which can be applied for various research questions addressing apoptosis.
Apoptosis; Cold-shock; Autoantibodies; Apoptotic blebs; Lupus
Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The development of a HCV cell culture system enabled us to investigate its whole HCV life cycle and develop a better understanding of the pathogenesis of this virus. Post-translational modification plays a crucial role in HCV replication and in the maturation of viral particles. There is growing evidence also suggesting that the ubiquitin-proteasome pathway and the ubiquitin-independent proteasome pathway are involved in the stability control of HCV proteins. Many viruses are known to manipulate the proteasome pathways to modulate the cell cycle, inhibit apoptosis, evade the immune system, and activate cell signaling, thereby contributing to persistent infection and viral carcinogenesis. The identification of functional interactions between HCV and the proteasome pathways will therefore shed new light on the life cycle and pathogenesis of HCV. This review summarizes the current knowledge on the involvement of the ubiquitin-dependent and -independent proteasome pathways in HCV infection and discusses the roles of these two distinct mechanisms in HCV pathogenesis.
Hepatitis C virus; Ubiquitin; Proteasome; Degradation; Hepatitis
Annexins are Ca2+-binding, membrane-interacting proteins, widespread among eukaryotes, consisting usually of four structurally similar repeated domains. It is accepted that vertebrate annexins derive from a double genome duplication event. It has been postulated that a single domain annexin, if found, might represent a molecule related to the hypothetical ancestral annexin. The recent discovery of a single-domain annexin in a bacterium, Cytophaga hutchinsonii, apparently confirmed this hypothesis. Here, we present a more complex picture. Using remote sequence similarity detection tools, a survey of bacterial genomes was performed in search of annexin-like proteins. In total, we identified about thirty annexin homologues, including single-domain and multi-domain annexins, in seventeen bacterial species. The thorough search yielded, besides the known annexin homologue from C. hutchinsonii, homologues from the Bacteroidetes/Chlorobi phylum, from Gemmatimonadetes, from beta- and delta-Proteobacteria, and from Actinobacteria. The sequences of bacterial annexins exhibited remote but statistically significant similarity to sequence profiles built of the eukaryotic ones. Some bacterial annexins are equipped with additional, different domains, for example those characteristic for toxins. The variation in bacterial annexin sequences, much wider than that observed in eukaryotes, and different domain architectures suggest that annexins found in bacteria may actually descend from an ancestral bacterial annexin, from which eukaryotic annexins also originate. The hypothesis of an ancient origin of bacterial annexins has to be reconciled with the fact that remarkably few bacterial strains possess annexin genes compared to the thousands of known bacterial genomes and with the patchy, anomalous phylogenetic distribution of bacterial annexins. Thus, a massive annexin gene loss in several bacterial lineages or very divergent evolution would appear a likely explanation. Alternative evolutionary scenarios, involving horizontal gene transfer between bacteria and protozoan eukaryotes, in either direction, appear much less likely. Altogether, current evidence does not allow unequivocal judgement as to the origin of bacterial annexins.
Metadherin (MTDH) has been reported to be associated with cancer progression in various types of human cancers including breast cancer. Whether MTDH contributes to carcinogenesis of breast cancer is still unknown. In the present study, we investigated the expression of MTDH in normal, UDH (usual ductal hyperplasia), ADH (atypical ductal hyperplasia), DCIS (ductal carcinoma in situ) and invasive cancer to explore the possible role of MTDH for breast cancer carcinogenesis.
Immunohistochemistry was employed on paraffin sections of surgical removed breast samples.
The immunohistochemical results showed almost no staining in normal tissue, moderate staining in ADH and UDH, intense MTDH stains in DCIS and cancer. Statistical analysis demonstrated significant different MTDH expression between proliferative and cancerous breast lesions (p < 0.001). MTDH was positively correlated with the histological differentiation of DCIS (p = 0.028). In breast cancer, statistical analysis revealed a significant correlation between MTDH expression with patients' age (p = 0.042), ER status (p = 0.018) and p53 status (p = 0.001). We also examined the effect of MTDH on cell proliferation in DCIS and cancer, and we found that MTDH overexpression was significantly correlated with high Ki67 index (p = 0.008 and p = 0.036, respectively).
MTDH overexpression could be identified in proliferative breast lesions and may contribute to breast cancer progression.
Inflammation has been linked to the etiology of many organ-specific cancers. Indirect evidence suggests a possible role for inflammation in breast cancer. We investigated whether the systemic inflammation induced by Freund's adjuvant (FA) promotes mammary carcinogenesis in a rat model in which cancer is induced by the neu oncogene.
The effects of FA on hyperplastic mammary lesions and mammary carcinomas were determined in a neu-induced rat model. The inflammatory response to FA treatment was gauged by measuring acute phase serum haptoglobin. In addition, changes in cell proliferation and apoptosis following FA treatment were assessed.
Rats receiving FA developed twice the number of mammary carcinomas as controls. Systemic inflammation following FA treatment is chronic, as shown by a doubling of the levels of the serum biomarker, haptoglobin, 15 days following initial treatment. We also show that this systemic inflammation is associated with the increased growth of hyperplastic mammary lesions. This increased growth results from a higher rate of cellular proliferation in the absence of changes in apoptosis.
Our data suggests that systemic inflammation induced by Freund's adjuvant (FA) promotes mammary carcinogenesis. It will be important to determine whether adjuvants currently used in human vaccines also promote breast cancer.
Hepatocellular carcinoma (HCC), the most common primary malignant liver tumor, is the third leading cause of cancer deaths. The pathogenesis of HCC is closely associated with chronic liver inflammation fired by a variety of stimulates such as virus infection and metabolic stress. Recent work indicates that autophagy, a homeostatic self-degradation process, which decides cell survival or death upon stress, acts as an effector machinery of immune systems in defending microbial invasion and carcinogenesis. SQSTM1 is a selective target and receptor of autophagy, and the protein content of SQSTM1 reflects the level of autophagic flux in cells. Through degrading SQSTM1, decreasing SQSTM1 aggregates, and therefore interrupting the positive feedback between SQSTM1 aggregates and ROS production, autophagy plays a protective role against hepatocellular carcinoma. Indeed, our studies indicate that toll-like receptor 2 (TLR2)-mediated immune activities in the genotoxic carcinogen diethylnitrosamine (DEN)-injured liver tissue provide essential nutrient stimulates to induce intracellular senescence, which can ensure the activation and maturation of autophagy in liver cells. Loss of TLR2-mediated immune activity and senescence leads to the attenuation of autophagic flux, which cannot eliminate SQSTM1 aggregates, ROS accumulation, and DNA damage, and facilitates the development and progression of HCC.
DEN; SQSTM1; autophagic flux; hepatocellular carcinoma; immune network; senescence; toll-like receptor 2
Aberrant methylation of promoter CpG islands is a hallmark of human cancers and is an early event in carcinogenesis. We examined whether promoter hypermethylation contributes to the pathogenesis of benign breast lesions along a progression continuum to invasive breast cancer. The exploratory study cohort comprised 17 breast cancer patients with multiple benign and/or in situ lesions concurrently present with invasive carcinoma within a tumor biopsy. DNA from tumor tissue, normal breast epithelium when present, benign lesions (fibroadenoma, hyperplasia, papilloma, sclerosing adenosis, apocrine metaplasia, atypical lobular hyperplasia or atypical ductal hyperplasia), and in situ lesions of lobular carcinoma and ductal carcinoma were interrogated for promoter methylation status in 22 tumor suppressor genes using the multiplex ligation-dependent probe amplification assay (MS-MLPA). Methylation specific PCR was performed to confirm hypermethylation detected by MS-MLPA. Promoter methylation was detected in 11/22 tumor suppressor genes in 16/17 cases. Hypermethylation of RASSF1 was most frequent, present in 14/17 cases, followed by APC in 12/17, and GSTP1 in 9/17 cases with establishment of an epigenetic monocloncal progression continuum to invasive breast cancer. Hypermethylated promoter regions in normal breast epithelium, benign, and premalignant lesions within the same tumor biopsy implicate RASSF1, APC, GSTP1, TIMP3, CDKN2B, CDKN2A, ESR1, CDH13, RARB, CASP8, and TP73 as early events. DNA hypermethylation underlies thepathogenesis of step-wise transformation along a monoclonal continuum from normal to preneoplasia to invasive breast cancer.
benign; premalignant; transformation; DNA methylation; progression; continuum
Aberrant methylation of promoter CpG islands is a hallmark of human cancers and is an early event in carcinogenesis. We examined whether promoter hypermethylation contributes to the pathogenesis of benign breast lesions along a progression continuum to invasive breast cancer. The exploratory study cohort comprised 17 breast cancer patients with multiple benign and/or in situ lesions concurrently present with invasive carcinoma within a tumor biopsy. DNA from tumor tissue, normal breast epithelium when present, benign lesions (fibroadenoma, hyperplasia, papilloma, sclerosing adenosis, apocrine metaplasia, atypical lobular hyperplasia or atypical ductal hyperplasia), and in situ lesions of lobular carcinoma and ductal carcinoma were interrogated for promoter methylation status in 22 tumor suppressor genes using the multiplex ligation-dependent probe amplification assay (MS-MLPA). Methylation specific PCR was performed to confirm hypermethylation detected by MS-MLPA. Promoter methylation was detected in 11/22 tumor suppressor genes in 16/17 cases. Hypermethylation of RASSF1 was most frequent, present in 14/17 cases, followed by APC in 12/17, and GSTP1 in 9/17 cases with establishment of an epigenetic monocloncal progression continuum to invasive breast cancer. Hypermethylated promoter regions in normal breast epithelium, benign, and premalignant lesions within the same tumor biopsy implicate RASSF1, APC, GSTP1, TIMP3, CDKN2B, CDKN2A, ESR1, CDH13, RARB, CASP8, and TP73 as early events. DNA hypermethylation underlies the pathogenesis of step-wise transformation along a monoclonal continuum from normal to preneoplasia to invasive breast cancer.
benign; premalignant; transformation; DNA methylation; progression; continuum
Heat shock proteins (Hsps) are overexpressed in a wide range of human cancers and are implicated in tumor cell proliferation, differentiation, invasion, metastasis, death, and recognition by the immune system. We review the current status of the role of Hsp expression in cancer with special emphasis on the clinical setting. Although Hsp levels are not informative at the diagnostic level, they are useful biomarkers for carcinogenesis in some tissues and signal the degree of differentiation and the aggressiveness of some cancers. In addition, the circulating levels of Hsp and anti-Hsp antibodies in cancer patients may be useful in tumor diagnosis. Furthermore, several Hsp are implicated with the prognosis of specific cancers, most notably Hsp27, whose expression is associated with poor prognosis in gastric, liver, and prostate carcinoma, and osteosarcomas, and Hsp70, which is correlated with poor prognosis in breast, endometrial, uterine cervical, and bladder carcinomas. Increased Hsp expression may also predict the response to some anticancer treatments. For example, Hsp27 and Hsp70 are implicated in resistance to chemotherapy in breast cancer, Hsp27 predicts a poor response to chemotherapy in leukemia patients, whereas Hsp70 expression predicts a better response to chemotherapy in osteosarcomas. Implication of Hsp in tumor progression and response to therapy has led to its successful targeting in therapy by 2 main strategies, including: (1) pharmacological modification of Hsp expression or molecular chaperone activity and (2) use of Hsps in anticancer vaccines, exploiting their ability to act as immunological adjuvants. In conclusion, the present times are of importance for the field of Hsps in cancer, with great contributions to both basic and clinical cancer research.
The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell-cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell-cell and/or cell-ECM contacts and anchorage-independent cell proliferation.
Numerous hypermethylated genes have been reported in breast cancer, and the silencing of these genes plays an important role in carcinogenesis, tumor progression and diagnosis. These hypermethylated promoters are very rarely found in normal breast. It has been suggested that aberrant hypermethylation may be useful as a biomarker, with implications for breast cancer etiology, diagnosis, and management. The relationship between primary neoplasm and metastasis remains largely unknown. There has been no comprehensive comparative study on the clinical usefulness of tumor-associated methylated DNA biomarkers in primary breast carcinoma and metastatic breast carcinoma. The objective of the present study was to investigate the association between clinical extension of breast cancer and methylation status of Estrogen Receptor1 (ESR1) and Stratifin (14-3-3-σ) gene promoters in disease-free and metastatic breast cancer patients.
We studied two cohorts of patients: 77 patients treated for breast cancer with no signs of disease, and 34 patients with metastatic breast cancer. DNA was obtained from serum samples, and promoter methylation status was determined by using DNA bisulfite modification and quantitative methylation-specific PCR.
Serum levels of methylated gene promoter 14-3-3-σ significantly differed between Control and Metastatic Breast Cancer groups (P < 0.001), and between Disease-Free and Metastatic Breast Cancer groups (P < 0.001). The ratio of the 14-3-3-σ level before the first chemotherapy cycle to the level just before administration of the second chemotherapy cycle was defined as the Biomarker Response Ratio [BRR]. We calculated BRR values for the "continuous decline" and "rise-and-fall" groups. Subsequent ROC analysis showed a sensitivity of 75% (95% CI: 47.6 - 86.7) and a specificity of 66.7% (95% CI: 41.0 - 86.7) to discriminate between the groups for a cut-off level of BRR = 2.39. The area under the ROC curve (Z = 0.804 ± 0.074) indicates that this test is a good approach to post-treatment prognosis.
The relationship of 14-3-3-σ with breast cancer metastasis and progression found in this study suggests a possible application of 14-3-3-σ as a biomarker to screen for metastasis and to follow up patients treated for metastatic breast cancer, monitoring their disease status and treatment response.
17β-hydroxysteroid dehydrogenase isoform 12 (HSD17B12) overexpression is associated with poor clinical outcome in invasive ductal carcinoma of the breast. Here, we evaluated HSD17B12 overexpression and its activity in ovarian carcinoma (OvCa) to determine its role in growth and progression of this tumor.
Immunohistochemical analysis of HSD17B12 expression was performed in 100 tissue samples of untreated OvCa and was correlated with clinicopathologic characteristics and patient outcome. In A2780 OvCa cell line expressing HSD17B12, siRNA knockdown of the enzyme was performed, and its effects on tumor cell growth and Annexin V binding were determined.
HSD17B12 expression was detected in all tumor samples, but the staining intensity was variable. Normal ovarian epithelium was negative. Patients with tumor showing weak/moderate expression of HSD17B12 had a better overall survival than those with strongly positive tumors (p<0.001). The time to first recurrence was longer for patients with tumors with heterogenous staining relative to patients with tumors that were uniformly positive (p<0.001). Upon silencing of HSD17B12 in tumor cells, their growth was inhibited (p<0.005) and apoptosis was increased (p<0.05). Arachidonic acid but not estradiol reversed the growth inhibition mediated by HSD17B12 knockdown.
HSD17B12 overexpression is shown to be a marker of poor survival in patients with OvCa. Expression in the tumor and function of this enzyme facilitates OvCa progression.
HSD17B12; ovarian carcinoma; prognosis; HSD17B12 silencing