MiRNA-615-5p Functions as a Tumor Suppressor in Pancreatic Ductal Adenocarcinoma by Targeting AKT2

Background

Aberrant microRNA (miRNA) expression is associated with tumor development. This study aimed to elucidate the role of miR-615-5p in the development of pancreatic ductal adenocarcinoma (PDAC).

Methods

Locked nucleic acid in situ hybridization (LNA-ISH) was performed to compare miR-615-5p expression in patients between PDAC and matched adjacent normal tissues. Effects of miR-615-5p overexpression on cell proliferation, apoptosis, colony formation, migration, and invasion were determined in the pancreatic cancer cell lines PANC-1 and MIA PaCa-2. Effects of miR-615-5p on AKT2 were examined by dual-luciferase reporter assay. Lentivirus expressing miR-615 was used to create stable overexpression cell lines, which were subsequently used in mouse xenograft and metastasis models to assess tumor growth, apoptosis and metastasis.

Results

miR-615-5p expression was significantly lower in PDAC than in adjacent normal tissues. Low levels of miR-615-5p were independently associated with poor prognosis (HR: 2.243, 95% CI: 1.190-4.227, P=0.013). AKT2 protein expression was inversely correlated with miR-615-5p expression (r=-0.3, P=0.003). miR-615-5p directly targeted the 3’-untranslated region of AKT2 mRNA and repressed its expression. miR-615-5p overexpression inhibited pancreatic cancer cell proliferation, migration, and invasion in vitro, and tumor growth and metastasis in vivo. Furthermore, miR-615-5p overexpression also induced pancreatic cancer cell apoptosis both in vitro and in vivo.

Conclusions

These results show that miR-615-5p inhibits pancreatic cancer cell proliferation, migration, and invasion by targeting AKT2. The data implicate miR-615-5p in the prognosis and treatment of PDAC.     

Activation of c-Met and Upregulation of CD44 Expression Are Associated with the Metastatic Phenotype in the Colorectal Cancer Liver Metastasis Model

Background

Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized.

Methods

HT29 LM1, HT29 LM2, HT29 LM3 cell lines were derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection in immunodeficient mice.

Results

CD44 expression, a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesions, and cancer cells adhesion to endothelial cells was increased in all in vivo selected cell lines, with maximum CD44 expression and cancer cells adhesion to endothelial cells in the highly metastatic HT29 LM3 cell line. Activation of c-Met upon hepatocyte growth factor (HGF) stimulation in the in vivo selected cell lines is CD44 independent. In vitro separation of CD44 high and low expression cells from HT29 LM3 cell line with FACS sorting confirmed that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation. Furthermore, in vivo evaluation of CD44 low and high expressing HT29 LM3 cells demonstrated no difference in liver metastasis penetrance.

Conclusions

Taken together, our findings indicate that the aggressive metastatic phenotype of in vivo selected cell lines is associated with overexpression of CD44 and activation of c-MET. We demonstrate that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation and confirm that CD44 expression in HT29 LM3 cell line is not responsible for the increase in metastatic penetrance in HT29 LM3 cell line.     

MiRNA-21 Expression Decreases from Primary Tumors to Liver Metastases in Colorectal Carcinoma

Objective

Metastasis is the major cause of death in colorectal cancer patients. Expression of certain miRNAs in the primary tumors has been shown to be associated with progression of colorectal cancer and the initiation of metastasis. In this study, we compared miRNA expression in primary colorectal cancer and corresponding liver metastases in order to get an idea of the oncogenic importance of the miRNAs in established metastases.

Methods

We analyzed the expression of miRNA-21, miRNA-31 and miRNA-373 in corresponding formalin-fixed paraffin-embedded (FFPE) tissue samples of primary colorectal cancer, liver metastasis and healthy tissues of 29 patients by quantitative real-time PCR.

Results

All three miRNAs were significantly up-regulated in the primary tumor tissues as compared to healthy colon mucosa of the respective patients (p < 0.01). MiRNA-21 and miRNA-31 were also higher expressed in liver metastases as compared to healthy liver tissues (p < 0.01). No significant difference of expression of miRNA-31 and miRNA-373 was observed between primary tumors and metastases. Of note, miRNA-21 expression was significantly reduced in liver metastases as compared to the primary colorectal tumors (p < 0.01).

Conclusion

In the context of previous studies demonstrating increased miRNA-21 expression in metastatic primary tumors, our findings raise the question whether miRNA-21 might be involved in the initiation but not in the perpetuation and growth of metastases.     

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer

Background

Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer.

Methodology

In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens.

Results

Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002).

Conclusion

Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer.     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.     

MicroRNA Signature Characterizes Primary Tumors That Metastasize in an Esophageal Adenocarcinoma Rat Model

Objective

To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC).

Background

The incidence of esophageal adenocarcinoma (EAC) has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies.

Methods

The modified Levrat’s surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA) along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5) and metastasis negative (n=28) primary tumors.

Results

The epithelial origin of distant metastasis was established by IF using villin (VIL1) and mucin 5AC (MUC5AC) antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001), miR-141-3p (p=0.0022), miR-451-1a (p=0.0181) and miR133a-3p (p=0.0304). Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2.

Conclusion

In vivo metastasis was confirmed in the modified Levrat’s model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.     

Adult Stromal Cells Derived from Human Adipose Tissue Provoke Pancreatic Cancer Cell Death both In Vitro and In Vivo

Background

Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC) spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC) on pancreatic tumor cell proliferation.

Principal Findings

Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate). ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth.

Conclusion

These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.     

Combined Treatment with Exendin-4 and Metformin Attenuates Prostate Cancer Growth

Introduction

Recently, the pleiotropic benefits of incretin-based therapy have been reported. We have previously reported that Exendin–4, a glucagon-like peptide–1 (GLP–1) receptor agonist, attenuates prostate cancer growth. Metformin is known for its anti-cancer effect. Here, we examined the anti-cancer effect of Exendin–4 and metformin using a prostate cancer model.

Methods

Prostate cancer cells were treated with Exendin–4 and/or metformin. Cell proliferation was quantified by growth curves and 5-bromo–2′-deoxyuridine (BrdU) assay. TUNEL assay and AMP-activated protein kinase (AMPK) phosphorylation were examined in LNCaP cells. For in vivo experiments, LNCaP cells were transplanted subcutaneously into the flank region of athymic mice, which were then treated with Exendin–4 and/or metformin. TUNEL assay and immunohistochemistry were performed on tumors.

Results

Exendin–4 and metformin additively decreased the growth curve, but not the migration, of prostate cancer cells. The BrdU assay revealed that both Exendin–4 and metformin significantly decreased prostate cancer cell proliferation. Furthermore, metformin, but not Exendin–4, activated AMPK and induced apoptosis in LNCaP cells. The anti-proliferative effect of metformin was abolished by inhibition or knock down of AMPK. In vivo, Exendin–4 and metformin significantly decreased tumor size, and further significant tumor size reduction was observed after combined treatment. Immunohistochemistry on tumors revealed that the P504S and Ki67 expression decreased by Exendin–4 and/or metformin, and that metformin increased phospho-AMPK expression and the apoptotic cell number.

Conclusion

These data suggest that Exendin–4 and metformin attenuated prostate cancer growth by inhibiting proliferation, and that metformin inhibited proliferation by inducing apoptosis. Combined treatment with Exendin–4 and metformin attenuated prostate cancer growth more than separate treatments.     

Lgr5 Methylation in Cancer Stem Cell Differentiation and Prognosis-Prediction in Colorectal Cancer

Objective

Leucine-rich-repeat-containing G-protein-coupled receptor 5 (lgr5) is a candidate marker for colorectal cancer stem cells (CSC). In the current study, we investigated the methylation status within thelgr5 promoter and evaluated its relationship with CSC differentiation, prognosis for colorectal cancer, and its clinicopathological features.

Methods

The methylation status within Lgr5 promoter was detected with a methylation-specific PCR in six colorectal cancer cell lines as well as 169 primary colorectal tumor tissues. Differentiation of CSC was examined with immunofluorescence and immunocytochemistry. Down-regulation of lgr5 was achieved with gene-specific siRNA. The associations between lgr5 methylation and the clinicopathological features as well as survival of patients were analyzed with statistical methods.

Results

The lgr5 promoter was methylated to different degrees for the six colorectal cell lines examined, with complete methylation observed in HCT116 cells in which the lgr5 expression was partially recovered following DAC treatment. The stem-cell sphere formation from HCT116 cells was accompanied by increasing methylation within the lgr5 promoter and decreasing expression of lgr5. Knocking down lgr5 by siRNA also led to stem-cell spheres formation. Among primary colorectal tumors, 40% (67/169) were positive for lgr5 methylation, while none of the normal colon tissues were positive for lgr5 methylation. Furthermore, lgr5 methylation significantly associated with higher tumor grade, and negative distant metastasis (p < 0.05), as well as better prognosis (p = 0.001) in patients with colorectal cancer.

Conclusions

Our data suggests that lgr5 methylation, through the regulation of lgr5 expression and colorectal CSC differentiation, may constitute a novel prognostic marker for colorectal cancer patients.     

Does Radiotherapy for the Primary Tumor Benefit Prostate Cancer Patients with Distant Metastasis at Initial Diagnosis?

Purpose/Objectives

Treatment of the primary tumor reportedly improves survival in several types of metastatic cancer. We herein evaluated the efficacy and toxicity of radiotherapy for the primary tumor in prostate cancer with metastasis.

Materials/Methods

The study cohort included 140 men with metastatic prostate cancer at initial diagnosis. Metastatic sites were divided into 4 groups as follows: solitary bone, 2–4 bones, ≥5 bones, and visceral organs. Patient, tumor, and treatment characteristics, and clinical outcomes were compared between patients treated with (prostate radiotherapy [PRT] group) or without radiotherapy to the primary tumor.

Results

Patients in PRT group presented with a statistically significantly younger age (p = .02), whereas other characteristics showed no significant difference. Overall survival (OS) and biochemical failure-free survival (BCFFS) were improved in PRT patients (3-year OS: 69% vs. 43%, p = 0.004; 3-year BCFFS: 52% vs. 16%, p = 0.002). Multivariate analysis identified PRT as a significant predictor of both OS (hazard ratio [HR] = 0.43, p = 0.015). None of the 38 PRT patients experienced severe (grade ≥3) genitourinary or gastrointestinal toxicity.

Conclusions

Our data suggest that radiotherapy to the primary tumor was associated with improved OS and BCFFS in metastatic prostate cancer. The results of this study warrant prospective controlled clinical trials of this approach in stage IV prostate cancer patients with limited extent of bone metastasis and good performance status.     

Breastfeeding and Immunohistochemical Expression of ki-67, p53 and BCL2 in Infiltrating Lobular Breast Carcinoma

Background/Aim

Invasive lobular breast carcinoma is the second most common type of breast cancer after invasive ductal carcinoma. According to the American Cancer Society, more than 180,000 women in the United States find out they have invasive breast cancer each year. Personal history of breast cancer and certain changes in the breast are correlated with an increased breast cancer risk. The aim of this work was to analyze breastfeeding in patients with infiltrating lobular breast carcinoma, in relation with: 1) clinicopathological parameters, 2) hormonal receptors and 3) tissue-based tumor markers

Materials and Methods

The study included 80 women with ILC, 46 of which had breastfeed their children. Analyzed parameters were: age, tumor size, axillary lymph node (N), distant metastasis (M), histological grade (HG), estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), Ki-67, p53 and BCL2

Results

ILC of non-lactating women showed a larger (p = 0.009), lymph node involvement (p = 0.051) and distant metastasis (p = 0.060). They were also more proliferative tumors measured by Ki-67 (p = 0.053). Breastfeeding history did not influence the subsequent behavior of the tumor regardless of histological subtype

Conclusion

Lactation seems to influence the biological characteristics of ILC defining a subgroup with more tumor size, axillary lymph node involvement, distant metastasis and higher proliferation measured by ki-67 expression.     

Fibroblast Activation Protein Overexpression and Clinical Implications in Solid Tumors: A Meta-Analysis

Objective

Fibroblast activation protein (FAP) plays a vital role in tumor invasion and metastasis. Previous studies have reported its prognostic value in different tumors. However, the results of these reports remain controversial. In this study, a meta-analysis was performed to clarify this issue.

Methods

A search of the PubMed, Embase and CNKI databases was conducted to analyze relevant articles. The outcomes included the relations between FAP expression and histological differentiation, tumor invasion, lymph node metastasis, distant metastasis and overall survival (OS). Sensitivity analysis by FAP expression in different cells and tumor types were further subjected to sensitivity analyses as subgroups. Pooled odds ratios (ORs) and hazard ratios (HRs) were evaluated using the random-effects model.

Results

The global analysis included 15 studies concerning various solid tumors. For global analysis, FAP overexpression in tumor tissue displayed significant associations with poor OS and tumor progression (OS: HR = 2.18, P = 0.004; tumor invasion: OR = 4.48, P = 0.007; and lymph node metastasis: OR = 3.80, P = 0.004). The subgroup analyses yielded two notable results. First, the relation between FAP overexpression and poor OS and tumor lymph node metastasis was closer in the patients with FAP expression in tumor cells. Second, the pooled analyses of colorectal cancers or pancreatic cancers all indicated that FAP overexpression was associated with a detrimental OS (HR: 1.72, P = 0.009; HR: 3.18, P = 0.005, respectively). The magnitude of this effect was not statistically significant compared with that in patients with non-colorectal cancers or non-pancreatic cancers. These analyses did not display a statistically significant correlation between FAP expression and histological differentiation and distant metastasis in all of the groups.

Conclusions

FAP expression is associated with worse prognosis in solid tumors, and this association is particularly pronounced if FAP overexpression is found in the tumor cells rather than the stroma.     

Re-expression of AKAP12 inhibits progression and metastasis potential of colorectal carcinoma in vivo and in vitro

Background

AKAP12/Gravin (A kinase anchor protein 12) is one of the A-kinase scaffold proteins and a potential tumor suppressor gene in human primary cancers. Our recent study demonstrated the highly recurrent loss of AKAP12 in colorectal cancer and AKAP12 reexpression inhibited proliferation and anchorage-independent growth in colorectal cancer cells, implicating AKAP12 in colorectal cancer pathogenesis.

Methods

To evaluate the effect of this gene on the progression and metastasis of colorectal cancer, we examined the impact of overexpressing AKAP12 in the AKAP12-negative human colorectal cancer cell line LoVo, the single clone (LoVo-AKAP12) compared to mock-transfected cells (LoVo-CON).

Results

pCMV6-AKAP12-mediated AKAP12 re-expression induced apoptosis (3% to 12.7%, p<0.01), migration (89.6±7.5 cells to 31.0±4.1 cells, p<0.01) and invasion (82.7±5.2 cells to 24.7±3.3 cells, p<0.01) of LoVo cells in vitro compared to control cells. Nude mice injected with LoVo-AKAP12 cells had both significantly reduced tumor volume (p<0.01) and increased apoptosis compared to mice given AKAP12-CON. The quantitative human-specific Alu PCR analysis showed overexpression of AKAP12 suppressed the number of intravasated cells in vivo (p<0.01).

Conclusion

These results demonstrate that AKAP12 may play an important role in tumor growth suppression and the survival of human colorectal cancer.     

Expression of Lipid Metabolism-Related Proteins in Metastatic Breast Cancer

Purpose

The tumor biology of metastatic breast cancers differ according to the metastatic sites, and the features of cancer metabolism may also be different. The aim of this study is to investigate the expression of lipid metabolism-related proteins in metastatic breast cancer according to metastatic site and discuss the clinical significance thereof.

Methods

Immunohistochemical staining for lipid metabolism-related proteins [fatty acid synthase (FASN), hormone-sensitive lipase (HSL), carnitine palmitoyltransferase IA (CPT-1A), acyl-CoA oxidase 1 (ACOX1), fatty acid binding protein 4 (FABP4,) and perilipin 1 (PLIN1)] was performed using a tissue microarray of 149 cases of metastatic breast cancer (bone metastasis = 39, brain metastasis = 37, liver metastasis = 21, and lung metastasis = 52).

Results

The expression levels of ACOX1 (p = 0.009) and FASN (p = 0.007) varied significantly according to metastatic site, with the highest expression in brain metastasis and the lowest expression in liver metastasis. ACOX1 positivity (p = 0.005) and FASN positivity (p = 0.003) correlated with HER-2 positivity. The expression of FASN was significantly higher in HER-2 type breast cancer, and lower in luminal A and TNBC type breast cancer (p<0.001). Among lipid metabolism-related proteins, PLIN1 positivity was found to be an independent poor prognostic factor on multivariate analysis (Hazard ratio: 4.979, 95% CI: 1.054–22.59, p = 0.043).

Conclusion

Different expression levels of lipid metabolism-related proteins were observed according to metastatic site. The expression of ACOX1 and FASN was highest in brain metastasis. These results suggest that the metastatic site should be considered when using lipid metabolism inhibitors for targeted therapy.     

The p70S6K Specific Inhibitor PF-4708671 Impedes Non-Small Cell Lung Cancer Growth

Background

As a serine/threonine protein kinase, p70S6K plays an important role in tumor cells. Evidence has revealed overexpression of p70S6K and phosphorylated p70S6K (p-p70S6K) in various tumor tissues, with these proteins identified as independent prognostic markers in non-small cell lung cancer (NSCLC). In this study, we explored the role of the p70S6K specific inhibitor PF-4708671 in NSCLC.

Methods

Three NSCLC cell lines (A549, SK-MES-1, and NCI-H460) were treated with PF-4708671 at five different concentrations, including 0.1μM, 0.3μM, 1μM, 3μM and 10μM, and protein levels were determined by Western-blot. Then, PF-4708671’s effects were assessed both in vitro (cell proliferation, apoptosis, cell cycle distribution, and invasion) and in vivo.

Results

The expression levels of p-p70S6K and the downstream effector S6 were significantly reduced by PF-4708671. Diametrically opposite, the downstream protein levels of BAD, Caspase3 and ERK had increased after treatment with PF-4708671. In addition, PF-4708671 drastically inhibited cell proliferation and invasion ability in A549, SK-MES-1 and NCI-H460 cells in vitro, causing cell cycle arrest in G0-G1 phase. Limited effects of PF-4708671 were observed on apoptosis in the three NSCLC cell lines assessed. Importantly, PF-4708671 could inhibit tumorigenesis in nude mice in vivo.

Conclusion

These findings demonstrated that the p70S6K specific inhibitor PF-4708671 has inhibitory effects on NSCLC tumorigenesis in vitro and in vivo. Therefore, P70S6K should be considered a new potential therapeutic target, and PF-470867 may be used as targeted drug for cancer treatment.     

RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

Objective

To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis.

Methods

RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression.

Results

The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01).

Conclusions

RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts.     

Bone Marrow Stromal Antigen 2 (BST-2) DNA Is Demethylated in Breast Tumors and Breast Cancer Cells

Background

Bone marrow stromal antigen 2 (BST-2) is a known anti-viral gene that has been recently identified to be overexpressed in many cancers, including breast cancer. BST-2 is critical for the invasiveness of breast cancer cells and the formation of metastasis in vivo. Although the regulation of BST-2 in immune cells is unraveling, it is unknown how BST-2 expression is regulated in breast cancer. We hypothesized that meta-analyses of BST-2 gene expression and BST-2 DNA methylation profiles would illuminate mechanisms regulating elevated BST-2 expression in breast tumor tissues and cells.

Materials and Methods

We performed comprehensive meta-analyses of BST-2 gene expression and BST-2 DNA methylation in The Cancer Genome Atlas (TCGA) and various Gene Expression Omnibus (GEO) datasets. BST-2 expression levels and BST-2 DNA methylation status at specific CpG sites on the BST-2 gene were compared for various breast tumor molecular subtypes and breast cancer cell lines.

Results

We show that BST-2 gene expression is inversely associated with the methylation status at specific CpG sites in primary breast cancer specimens and breast cancer cell lines. BST-2 demethylation is significantly more prevalent in primary tumors and cancer cells than in normal breast tissues or normal mammary epithelial cells. Demethylation of the BST-2 gene significantly correlates with its mRNA expression. These studies provide the initial evidence that significant differences exist in BST-2 DNA methylation patterns between breast tumors and normal breast tissues, and that BST-2 expression patterns in tumors and cancer cells correlate with hypomethylated BST-2 DNA.

Conclusion

Our study suggests that the DNA methylation pattern and expression of BST-2 may play a role in disease pathogenesis and could serve as a biomarker for the diagnosis of breast cancer.     

Semaphorin 3A suppresses tumor growth and metastasis in mice melanoma model

Background

Recent understanding on cancer therapy indicated that targeting metastatic signature or angiogenic switch could be a promising and rational approach to combat cancer. Advancement in cancer research has demonstrated the potential role of various tumor suppressor proteins in inhibition of cancer progression. Current studies have shown that axonal sprouting inhibitor, semaphorin 3A (Sema 3A) acts as a potent suppressor of tumor angiogenesis in various cancer models. However, the function of Sema 3A in regulation of melanoma progression is not well studied, and yet to be the subject of intense investigation.

Methodology/Principal Findings

In this study, using multiple in vitro and in vivo approaches we have demonstrated that Sema 3A acts as a potent tumor suppressor in vitro and in vivo mice (C57BL/6) models. Mouse melanoma (B16F10) cells overexpressed with Sema 3A resulted in significant inhibition of cell motility, invasiveness and proliferation as well as suppression of in vivo tumor growth, angiogenesis and metastasis in mice models. Moreover, we have observed that Sema 3A overexpressed melanoma clone showed increased sensitivity towards curcumin and Dacarbazine, anti-cancer agents.

Conclusions

Our results demonstrate, at least in part, the functional approach underlying Sema 3A mediated inhibition of tumorigenesis and angiogenesis and a clear understanding of such a process may facilitate the development of novel therapeutic strategy for the treatment of cancer.     

Aberrations Involving Chromosome 1 as a Possible Predictor of Odds Ratio for Colon Cancer - Results from the Krakow Case-Control Study

Background

There is still an open question how to predict colorectal cancer risk before any morphological changes appear in the colon.

Objective

The purpose was to investigate aberrations in chromosomes 1, 2 and 4 in peripheral blood lymphocytes analyzed by fluorescence in situ hybridization technique as a tool to assess the likelihood of colorectal cancer.

Methods

A hospital-based case-control study included 20 colon cancer patients and 18 hospital-based controls. Information about potential covariates was collected by interview. The frequency of stable and unstable chromosome aberrations in chromosome 1, 2 and 4 was assessed by fluorescence in situ hybridization technique.

Results

Colorectal cancer patients, as compared to controls, had a relatively higher frequency of chromosome 1 translocations (median: 3.5 versus 1.0 /1000 cells, p = 0.006), stable aberrations (3.8 versus 1.0 /1000 cells, p = 0.007) and total aberrations (p = 0.009). There were no differences observed for chromosomes 2 and 4. Our results showed an increase in the odds of having colon cancer by about 50–80% associated with an increase by 1/1000 cells in the number of chromosome 1 aberrations.

Conclusions

The results revealed that the frequency of chromosomal aberrations, especially translocations in chromosome 1, seems to be a promising method to show a colon cancer risk. Additionally, our study suggests the reasonableness of use of biomarkers such as chromosome 1 aberrations in peripheral blood lymphocytes in screening prevention programs for individuals at higher colon cancer risk to identify those who are at increased risk and require more frequent investigations, e.g. by sigmoidoscopy.