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.
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.
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 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.
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.
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
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.
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
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.
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.
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
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.
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.
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.
The role of MRI in the management of breast carcinoma is rapidly evolving from its initial use for specific indications only to a more widespread use on all women with newly diagnosed early stage breast cancer. However, there are many concerns that such widespread use is premature since detailed correlation of MRI findings with the underlying histopathology of the breast lesions is still evolving and clear evidence for improvements in management and overall prognosis of breast cancer patients evaluated by breast MRI after their initial cancer diagnosis is lacking. In this paper, we would like to bring attention to a benign lesion that is frequently present on MRI-guided breast biopsies performed on suspicious MRI findings in the affected breast of patients with a new diagnosis of breast carcinoma.
Two clinically relevant high-risk HPV (HR-HPV) types 16 and 18 are etiologically associated with the development of cervical carcinoma and are also reported to be present in many other carcinomas in extra-genital organ sites. Presence of HPV has been reported in breast carcinoma which is the second most common cancer in India and is showing a fast rising trend in urban population. The two early genes E6 and E7 of HPV type 16 have been shown to immortalize breast epithelial cells in vitro, but the role of HPV infection in breast carcinogenesis is highly controversial. Present study has therefore been undertaken to analyze the prevalence of HPV infection in both breast cancer tissues and blood samples from a large number of Indian women with breast cancer from different geographic regions.
The presence of all mucosal HPVs and the most common high-risk HPV types 16 and 18 DNA was detected by two different PCR methods - (i) conventional PCR assays using consensus primers (MY09/11, or GP5+/GP6+) or HPV16 E6/E7 primers and (ii) highly sensitive Real-Time PCR. A total of 228 biopsies and corresponding 142 blood samples collected prospectively from 252 patients from four different regions of India with significant socio-cultural, ethnic and demographic variations were tested.
All biopsies and blood samples of breast cancer patients tested by PCR methods did not show positivity for HPV DNA sequences in conventional PCRs either by MY09/11 or by GP5+/GP6+/HPV16 E6/E7 primers. Further testing of these samples by real time PCR also failed to detect HPV DNA sequences.
Lack of detection of HPV DNA either in the tumor or in the blood DNA of breast cancer patients by both conventional and real time PCR does not support a role of genital HPV in the pathogenesis of breast cancer in Indian women.
The role of the gastrin peptide hormones (G17, G34) and their precursors (progastrins, PG; gly-extended gastrin, G-gly), in gastrointestinal (GI) cancers has been extensively reviewed in recent years (1–3). A possible important role of progastrin peptides in colon carcinogenesis has become evident from experiments with transgenic mouse models (1;3). It is now known that growth stimulatory and co-carcinogenic effects of gastrin/PG peptides are mediated by both proliferative and anti-apoptotic effects of the peptides on target cells (4;5). Several receptor subtypes have been described that mediate growth effects of gastrin peptides (1;2). Recently, we identified Annexin II as a high affinity binding protein for gastrin/PG peptides (6). Importantly, the expression of Annexin II was required for mediating growth stimulatory effects of gastrin and PG peptides on intestinal epithelial and colon cancer cells (6), suggesting that Annexin II may represent the elusive novel receptor for gastrin/PG peptides. The importance of this finding in relation to the structure and function of Annexin II, especially in GI cancers, is described below. Since this surprising finding represents a new front in our understanding of the mechanisms involved in mediating growth effects of gastrin/PG peptides in GI cancers, our current understanding of the role of Annexin II in proliferation and metastasis of cancer cells is additionally reviewed.
Annexin-II; Progastrin; Tenascin-C; Colon Cancer; GI cancers; signalling
Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, 3H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.
LIM-only 4 (LMO4), a member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors, was initially reported to have an oncogenic role in breast cancer. We hypothesized that LMO4 may be related to pancreatic carcinogenesis as it is in breast carcinogenesis. If so, this could result in a better understanding of tumorigenesis in pancreatic cancer.
We measured LMO4 mRNA levels in cultured cells, pancreatic bulk tissues and microdissected target cells (normal ductal cells; pancreatic intraepithelial neoplasia-1B [PanIN-1B] cells; PanIN-2 cells; invasive ductal carcinoma [IDC] cells; intraductal papillary-mucinous adenoma [IPMA] cells; IPM borderline [IPMB] cells; and invasive and non-invasive IPM carcinoma [IPMC]) by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR).
9 of 14 pancreatic cancer cell lines expressed higher levels of LMO4 mRNA than did the human pancreatic ductal epithelial cell line (HPDE). In bulk tissue samples, expression of LMO4 was higher in pancreatic carcinoma than in intraductal papillary-mucinous neoplasm (IPMN) or non-neoplastic pancreas (p < 0.0001 for both). We carried out microdissection-based analyses. IDC cells expressed significantly higher levels of LMO4 than did normal ductal epithelia or PanIN-1B cells (p < 0.001 for both) or PanIN-2 cells (p = 0.014). IPMC cells expressed significantly higher levels of LMO4 than did normal ductal epithelia (p < 0.001), IPMA (p < 0.001) and IPMB cells (p = 0.003).
Pancreatic carcinomas (both IDC and IPMC) expressed significantly higher levels of LMO4 mRNA than did normal ductal epithelia, PanIN-1B, PanIN-2, IPMA and IPMB. These results suggested that LMO4 is overexpressed at late stages in carcinogenesis of pancreatic cancer.