High-risk human papillomavirus in non-melanoma skin lesions from renal allograft recipients and immunocompetent patients

Background:

High-risk human papillomaviruses (HR-HPVs) can be detected in a proportion of non-melanoma skin cancers. Data on prevalence are inconclusive, but are essential to estimate the relevance of HR-HPV, particularly with regard to prophylactic HPV vaccines for skin cancer prevention.

Methods:

High-risk human papillomavirus DNA was investigated in 140 non-melanoma skin lesions from 54 immunocompetent patients and 33 immunosuppressed renal allograft recipients. Expression of p16^INK4a, a marker for HR-HPV oncogene expression in the uterine cervix, and of p53 and pRB was evaluated immunohistochemically.

Results:

The highest prevalence of HR-HPV was found in squamous cell cancer (SCC) (46.2% (6 out of 13) in immunosuppressed and 23.5% (4 out of 17) in immunocompetent patients). High-risk human papillomavirus positivity was accompanied by diffuse p16^INK4a expression in most SCC (P<0.001) and basal cell cancers (P=0.02), while almost all SCC in situ were p16^INK4a positive irrespective of HR-HPV presence (P=0.66). Diffuse p16^INK4a expression was associated with lack of pRB expression (P=0.001). p53 was strongly expressed in 40.0% (56 out of 140) of the lesions irrespective of HR-HPV presence.

Conclusion:

High-risk human papillomavirus can be detected in lesions of keratinised squamous epithelia. The association of HR-HPV with diffuse p16^INK4a expression might indicate HR-HPV oncogene expression in a proportion of lesions. Overexpression of p53 suggests p53 pathway alterations in HR-HPV-positive and -negative lesions.     

Loss of has-miR-337-3p expression is associated with lymph node metastasis of human gastric cancer

Background

Metastasis is the major cause of cancer-related death in patients with gastric cancer, and aberrant expression of various microRNAs (miRNAs) is associated with cancer metastasis.

Methods

Profiling of differentially expressed miRNAs was performed in three cases of primary gastric cancer and the corresponding metastatic lymph node tissues. Then, the five most altered miRNAs were further verified in 16 paired samples. Two of these five miRNAs were further assessed for their effects on the regulation of gastric cancer cell growth and invasion.

Results

The miRNA profile data showed 151 upregulated miRNAs (≥ 1.5-fold) and 285 downregulated miRNAs (≤ 0.67-fold) in the metastatic tissues compared to the primary gastric cancer tissues. Among these five miRNAs (i.e., hsa-miR-508-5p, hsa-miR-30c, hsa-miR-337-3p, hsa-miR-483-5p, and hsa-miR-134), expression of hsa-miR-337-3p and hsa-miR-134 was significantly downregulated in these 16 lymph node metastatic tissues compared to their primary tumor tissues (P<0.05) and in nine gastric cancer cell lines compared to the nonmalignant GES cell line. Furthermore, induction of hsa-miR-134 or hsa-miR-337-3p expression did not dramatically affect gastric cancer cell proliferation, but transfection of the hsa-miR-337-3p mimic did reduce gastric cancer cell invasion capacity.

Conclusions

These findings indicate that hsa-miR-337-3p plays a role in the reduction of gastric cancer cell invasion capacity, and further studies on the mechanism of hsa-miR-337-3p in gastric cancer metastasis are warranted.     

Human papillomavirus,p16INK4A,and Ki-67 in relation to clinicopathological variables and survival in primary carcinoma of the vagina

Background:

This study aimed to determine human papillomavirus (HPV) status and to investigate p16^INK4A and Ki-67 expression and their correlation with clinical parameters and survival in women with primary carcinoma of the vagina (PCV).

Methods:

The presence of HPV DNA was evaluated by PCR. Genotyping was performed by Luminex in 68 short-term (⩽2 years) and long-term (⩾8 years) PCV survivors. p16^INK4A and Ki-67 expression was evaluated by immunohistochemistry.

Results:

Human papillomavirus DNA was detected in 43% of patients, the majority (63%) of whom were HPV16 positive. High p16^INK4A expression was significantly correlated with low histopathological grade (P=0.004), HPV positivity (P=0.032), and long-term survival (P=0.045). High Ki-67 expression was negatively correlated with histopathological grade (P<0.001) and tumour size (P=0.047). There was an association between HPV positivity and low histopathological grade, but not between HPV positivity and survival.

Conclusion:

High p16^INK4A expression was associated with long-term survival, but the only independent predictors for survival were tumour size and histopathological grade. Our results indicate that p16^INK4A and Ki-67 expression might be useful in tumour grading, and that it might be possible to use p16^INK4A expression as a marker for HPV positivity, but this has to be further elucidated.     

Human Papillomaviruses,p16INK4a and Akt expression in basal cell carcinoma

Background

The pathogenic role of beta-HPVs in non melanoma skin cancer (NMSC), is not still completely understood, and literature data indicate that they might be at least cofactors in the development of certain cutaneous squamous cell carcinomas. However, only few reports contain data on basal cell carcinoma (BCC). The HPVs interact with many cellular proteins altering their function or the expression levels, like the p16^INK4a and Akt. Our study aimed to determine the presence of different beta -HPV types and the expression of p16^INK4a and Akt in BCC, the commonest NMSC, in the normal appearing perilesional skin and in forehead swab of 37 immunocompetent patients.

Methods

The expression of p16^INK4a and Akt, by immunohistochemistry, and the HPV DNA, by nested PCR, were investigated in each sample.

Results

No correspondence of HPV types between BCC and swab samples was found, whereas a correspondence between perilesional skin and BCC was ascertained in the 16,7% of the patients. In BCC, 16 different types of beta HPV were found and the most frequent types were HPV107 (15,4%), HPV100 (11,5%) and HPV15 (11,5%) all belonging to the beta HPV species 2. Immunohistochemistry detected significant p16^INK4a expression in almost all tumor samples (94,3%) with the highest percentages (> 30%) of positive cells detected in 8 cases. A statistically significant (p = 0,012) increase of beta HPV presence was detected in p16^INK4a strongly positive samples, in particular of species 2. pAkt expression was detected in all tumor samples with only 2 cases showing rare positive cells, whereas Akt2 expression was found in 14 out of 35 BCC (40%); in particular in HPV positive samples over-expressing p16^INK4a.

Conclusions

Our data show that p16^INK4a and pAkt are over-expressed in BCC and that the high expression of p16^INK4a and of Akt2 isoform is often associated with the presence of beta-HPV species 2 (i.e. HPV 15). The association of these viruses with the up-regulation of p16^INK4a and Akt/PI3K pathway suggests that in a subtype of BCC these viruses may exert a role in the carcinogenesis or in other, still undefined, biological property of these tumors. If this particular type of BCC reflects a different biology it will remain undisclosed until further studies on a larger number of samples will be performed.     

FAT4 functions as a tumour suppressor in gastric cancer by modulating Wnt/β-catenin signalling

Background:

FAT4, a cadherin-related protein, was shown to function as a tumour suppressor; however, its role in human gastric cancer remains largely unknown. Here, we investigated the role of FAT4 in gastric cancer and examined the underlying molecular mechanisms.

Methods:

The expression of FAT4 was evaluated by immunohistochemistry, western blotting, and qRT–PCR in relation to the clinicopathological characteristics of gastric cancer patients. The effects of FAT4 silencing on cell proliferation, migration, and invasion were assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay, and migration and invasion assays in gastric cancer cell lines in vitro and in a mouse xenograft model in vivo.

Results:

Downregulation of FAT4 expression in gastric cancer tissues compared with adjacent normal tissues was correlated with lymph-node metastasis and poor survival. Knockdown of FAT4 promoted the growth and invasion of gastric cancer cells via the activation of Wnt/β-catenin signalling, and induced epithelial-to-mesenchymal transition (EMT) in gastric cancer cells, as demonstrated by the upregulation and downregulation of mesenchymal and epithelial markers. Silencing of FAT4 promoted tumour growth and metastasis in a gastric cancer xenograft model in vivo.

Conclusions:

FAT4 has a tumour suppressor role mediated by the modulation of Wnt/β-catenin signalling, providing potential novel targets for the treatment of gastric cancer.     

Death-associated protein-3, DAP-3, correlates with preoperative chemotherapy effectiveness and prognosis of gastric cancer patients following perioperative chemotherapy and radical gastrectomy

Background:

DAP3 is a member of the death-associated protein (DAP) family and is characterised by proapoptotic function. It is involved in both exogenous and endogenous apoptotic pathways. In our previous studies, apoptotic level was found to be correlated with the effectiveness of preoperative chemotherapy. The effectiveness of preoperative chemotherapy was also associated with the overall effectiveness of the combined therapy and prognosis. The present study aimed to investigate the role of DAP3 in the evaluation of preoperative chemotherapy effectiveness and its ability to predict prognosis in gastric cancer.

Methods:

Quantitative PCR and immunohistochemistry staining were performed in 87 patients who received combined therapy. Knockdown of DAP3 was conducted in gastric cancer cell lines to investigate its impact on cell growth, migration, adhesion and invasion. Tolerance to chemotherapy agents was determined by assessing apoptosis and caspase-3.

Results:

Higher DAP3 expression in gastric tumours was correlated with better prognosis. Knockdown of DAP3 expression promoted cell migration and enhanced resistance to chemotherapy by inhibiting apoptosis.

Conclusion:

DAP3 is a potential molecular marker for response to preoperative chemotherapy and for predicting prognosis in gastric cancer patients treated with neoadjuvant chemotherapy and gastrectomy.     

Serpin peptidase inhibitor clade A member 1 is a biomarker of poor prognosis in gastric cancer

Background:

In a previous study, we reported that serpin peptidase inhibitor clade A member 1 (serpinA1) is upregulated in Snail-overexpressing gastric cancer. Although serpinA1 has been studied in several types of cancer, little is known about its roles and mechanisms of action. In this study, we examined the role of serpinA1 in the migration and invasion of gastric cancers and determined its underlying mechanism.

Methods:

Expression levels were assessed by western blot analyses and real-time PCR. Snail binding to serpinA1 promoter was analysed by chromatin immunoprecipitation (ChIP) assays. The roles of serpinA1 were studied using cell invasion and migration assays. In addition, the clinicopathologic and prognostic significance of serpinA1 expression were validated in 400 gastric cancer patients using immunohistochemical analysis.

Results:

Overexpression of Snail resulted in upregulation of serpinA1 in gastric cancer cell lines, AGS and MKN45, whereas knockdown of Snail inhibited serpinA1 expression. Chromatin immunoprecipitation analysis showed that overexpression of Snail increased Snail recruitment to the serpinA1 promoter. Overexpression of serpinA1 increased the migration and invasion of gastric cancer cells, whereas knockdown of serpinA1 decreased invasion and migration. Moreover, serpinA1 increased mRNA levels and release of metalloproteinase-8 in gastric cancer cells. Serpin peptidase inhibitor clade A member 1 was observed in the cytoplasm of tumour cells and the stroma by immunohistochemistry. Enhanced serpinA1 expression was significantly associated with increased tumour size, advanced T stage, perineural invasion, lymphovascular invasion, lymph node metastases, and shorter overall survival.

Conclusions:

Serpin peptidase inhibitor clade A member 1 induces the invasion and migration of gastric cancer cells and its expression is associated with the progression of gastric cancer. These results may provide a potential target to prevent invasion and metastasis in gastric cancer.     

p16INK4a immunohistochemistry is a promising biomarker to predict the outcome of low grade cervical intraepithelial neoplasia: comparison study with HPV genotyping

Objective

In cervical intraepithelial neoplasia (CIN), p16^INK4a immunohistochemistry has been reported to be a useful diagnostic biomarker. However, limited information is available about the association between the p16^INK4a immunohistochemistry and the outcomes of CIN. Here, we report p16^INK4a immunohistochemistry as an effective biomarker to predict the outcomes of CIN.

Methods

p16^INK4a immunohistochemistry was performed in patients with CIN from January 2000 to August 2009. Among these patients, we have performed a retrospective analysis of the medical records to evaluate the outcome of CIN 1-2 and performed statistical analysis to determine the correlation between p16^INK4a expression and the outcomes. We also performed HPV genotyping and analyzed the relation between the infecting human papillomavirus (HPV) genotype and the outcomes.

Results

A total of 244 patients, including 82 with CIN 1, 60 with CIN 2, and 102 with CIN 3, were examined. The rate of p16^INK4a overexpression increased with increasing CIN grade, 20.7% for CIN 1, 80.0% for CIN 2, and 89.2% for CIN 3, with significant differences between CIN 1 and CIN 2-3 group. In the 131 CIN 1-2 patients, the progression rate was significantly higher for the patients showing p16^INK4a overexpression than for those not showing p16^INK4a overexpression (p=0.005); the regression rate was also found to be significantly lower for the patients showing p16^INK4a overexpression (p=0.003). High-risk HPV genotypes were detected in 73 patients (73.7%). Both progression and regression rates were not significantly different between the high-risk HPV-positive and HPV-negative groups (p=0.401 and p=0.381, respectively).

Conclusion

p16^INK4a overexpression was correlated with the outcome of CIN 1-2, and p16^INK4a is considered to be a superior biomarker for predicting the outcome of CIN 1-2 compared with HPV genotyping.     

The candidate tumor suppressor gene ECRG4 inhibits cancer cells migration and invasion in esophageal carcinoma

Background

The esophageal cancer related gene 4 (ECRG4) was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"AF325503","term_id":"11991655","term_text":"AF325503"}}AF325503). ECRG4 was a new tumor suppressor gene in esophageal squamous cell carcinoma (ESCC) associated with prognosis. In this study, we investigated the novel tumor-suppressing function of ECRG4 in cancer cell migration, invasion, adhesion and cell cycle regulation in ESCC.

Methods

Transwell and Boyden chamber experiments were utilized to examined the effects of ECRG4 expression on ESCC cells migration, invasion and adhesion. And flow cytometric analysis was used to observe the impact of ECRG4 expression on cell cycle regulation. Finally, the expression levels of cell cycle regulating proteins p53 and p21 in human ESCC cells transfected with ECRG4 gene were evaluated by Western blotting.

Results

The restoration of ECRG4 expression in ESCC cells inhibited cancer cells migration and invasion (P < 0.05), which did not affect cell adhesion capacity (P > 0.05). Furthermore, ECRG4 could cause cell cycle G1 phase arrest in ESCC (P < 0.05), through inducing the increased expression of p53 and p21 proteins.

Conclusion

ECRG4 is a candidate tumor suppressor gene which suppressed tumor cells migration and invasion without affecting cell adhesion ability in ESCC. Furthermore, ECRG4 might cause cell cycle G1 phase block possibly through inducing the increased expression of p53 and p21 proteins in ESCC.     

Aquaporin-3 positively regulates matrix metalloproteinases via PI3K/AKT signal pathway in human gastric carcinoma SGC7901 cells

Background

matrix metalloproteinases (MMPs) are produced by tumor cells, so they may be associated with tumor progression including invasion, migration, angiogenesis and metastasis. Aquaporin-3 (AQP3) also plays a critical role in gastric cancer cell migration and proliferation.

Methods

In this study, AQP3 was silenced or over-expressed in SGC7901 cells.

Results

We found a significant decrease in MT1-MMP, MMP-2, and MMP-9 expression after AQP3 knockdown, and a significant increase in MT1-MMP, MMP-2, and MMP-9 expression after AQP3 over-expression in SGC7901 cells. We also found that AQP3 silence led to a significant decrease of phosphorylation of ser⁴⁷³ in AKT in SGC7901 cells.

Conclusion

Our findings showed that AQP3 might positively regulate MMPs proteins expression through PI3K/AKT signal pathway in human gastric carcinoma SGC7901 cells.     

The function and mechanism of COX-2 in angiogenesis of gastric cancer cells

Background

Here we aimed to investigate the effect of COX-2 siRNA on proliferation and angiogenesis of gastric cancer cells.

Methods

The gastric cancer cell line SGC7901 was transfected with COX-2 siRNA, then the growth and angiogenesis of cells were detected by in vitro and in vivo assay. Human microarray, RT-PCR and western blot were used to identify differentially expressed angiogenesis-related molecules in cells with decreased expression of COX-2.

Results

Down-regulation of COX-2 could significantly inhibit the in vitro and in vivo growth of gastric cancer cells, and suppress the migration and tube formation of human umbilical vein endothelial cells. Totally 23 angiogenesis-related molecules were found involved in COX-2-induced angiogenesis suppression. The results of RT-PCR and western blot showed that down-regulation of COX-2 might inhibit VEGF, Flt-1, Flk-1/KDR, angiopoietin-1, tie-2, MMP2 and OPN.

Conclusions

COX-2 might mediate tumor angiogenesis and growth, and could be considered as a target for gastric cancer therapy.     

G-CSF and G-CSFR are highly expressed in human gastric and colon cancers and promote carcinoma cell proliferation and migration

Background:

Granulocyte colony-stimulating factor (G-CSF) is a pro-inflammatory cytokine that stimulates myeloid stem cell maturation, proliferation, and migration into circulation. Despite being a known growth factor, the impact of G-CSF on solid tumours has not been well examined. G-CSF receptor (G-CSFR) is expressed by some tumours, and thus the aim of this study was to examine the expression and impact of G-CSF and G-CSFR on gastrointestinal tumours.

Methods:

In this study, G-CSF expression was examined in human gastric and colon tumours and by tumour-derived stromal myofibroblasts and carcinoma cells. G-CSFR expression was examined on carcinoma cells isolated from human tissues. The effects of G-CSF on gastric and colon carcinoma cell proliferation, migration, and signalling were examined.

Results:

G-CSFR was highly expressed in 90% of human gastric and colon carcinomas. G-CSF was also found to be highly produced by stromal myofibroblasts and carcinoma cells. Exposure of carcinoma cells to G-CSF led to increased proliferation and migration, and expansion of a sub-population of carcinoma cells expressing stem-like markers. These processes were dependent on ERK1/2 and RSK1 phosphorylation.

Conclusions:

These data suggest that the G-CSF/R axis promotes gastric and colorectal cancer development and suggest they are potential tumour targets.     

Expression of BMP-7 in human gastric cancer and its clinical significance

Background:

Bone morphogenetic protein-7 (BMP-7) is a signalling molecule belonging to the transforming growth factor--superfamily. Recent studies have demonstrated the clinical impact of BMP-7 expression in various human cancers. However, there have been few reports detailing this in gastric cancer.

Methods:

We immunohistochemically investigated the expression of BMP-7 in 233 gastric cancer patients to disclose the clinicopathological features of BMP-7-positive gastric cancer.

Results:

Immunohistochemically, in human gastric cancer, BMP-7 expression was identified in cellular membranes but also in the cytoplasm of cancer cells. Bone morphogenetic protein-7-positive expression was found in 129 of 233 patients (55%). Bone morphogenetic protein-7 expression was correlated with tumour size, nodal involvement, lymphatic invasion, venous invasion and histology (P<0.05). Bone morphogenetic protein-7 expression was significantly correlated with patient postoperative outcome, especially in the undifferentiated group. Multivariate analysis revealed BMP-7 expression as one of the independent prognostic factors next to the depth of invasion and nodal involvement (P<0.01).

Conclusions:

From the data collected, it would be appropriate to conclude on the possible regulation of gastric cancer progression by autocrine or paracrine BMP-7 loops. We can use BMP-7 expression as one of the strong predictors of risk of tumour recurrence in gastric cancer.     

Erythropoietin-driven signalling and cell migration mediated by polyADP-ribosylation

Background:

Recombinant human erythropoietin (EPO) is the leading biotechnology engineered hormone for treatment of anaemia associated with chronic conditions including kidney failure and cancer. The finding of EPO receptors on cancer cells has raised the concern that in addition to its action in erythropoiesis, EPO may promote tumour cell growth. We questioned whether EPO-induced signalling and consequent malignant cell manifestation is mediated by polyADP-ribosylation.

Methods:

Erythropoietin-mediated PARP (polyADP-ribose polymerase-1) activation, gene expression and core histone H4 acetylation were examined in UT7 cells, using western blot analysis, RT–PCR and immunofluorescence. Erythropoietin-driven migration of the human breast epithelial cell line MDA-MB-435 was determined by the scratch assay and in migration chambers.

Results:

We have found that EPO treatment induced PARP activation. Moreover, EPO-driven c-fos and Egr-1 gene expression as well as histone H4 acetylation were mediated via polyADP-ribosylation. Erythropoietin-induced cell migration was blocked by the PARP inhibitor, ABT-888, indicating an essential role for polyADP-ribosylation in this process.

Conclusions:

We have identified a novel pathway by which EPO-induced gene expression and breast cancer cell migration are regulated by polyADP-ribosylation. This study introduces new possibilities regarding EPO treatment for cancer-associated anaemia where combining systemic EPO treatment with targeted administration of PARP inhibitors to the tumour may allow safe treatment with EPO, minimising its possible undesirable proliferative effects on the tumour.     

Clinical implications of DLL4 expression in gastric cancer

Background

Delta-like ligand 4 (DLL4)-Notch signaling plays a key role in tumor neovascular development and angiogenesis during tumor growth. The clinical significance of DLL4 expression in gastric cancer has not been clarified.

Methods

Gastric cancer cell lines and 180 gastric cancer patients were enrolled. DLL4 expression in gastric cancer cells and stroma was identified and evaluated immunohistochemically. The association between DLL4 and clinicopathological factors was also assessed.

Results

DLL4 expression was identified in the cellular membrane and cytoplasm of gastric cancer cells by immunoblotting and immunohistochemical staining. DLL4 positivity in cancer cells and stroma was found in 88 (48%) and 41 (22%) of the 180 gastric cancer patients respectively. Both cancer and stromal DLL4 expression significantly correlated with more advanced tumor depth, nodal involvement, and lymphatic and venous invasion. A strongly positive association between cancerous and stromal DLL4 expression was identified (p < 0.01). Both cancerous and stromal DLL4 expression were prognostic markers in gastric cancer as determined by univariate analysis.

Conclusions

Cancerous and stromal DLL4 expression was found in 48% and 22% in gastric cancer, and significantly affected postoperative clinical outcomes. Cancerous and stromal DLL4 expression may be an effective target of anti-DLL4 treatment in gastric cancer.     

Identification of macrophage migration inhibitory factor and human neutrophil peptides 1�C3 as potential biomarkers for gastric cancer

Background:

Proteomic methods have the potential to meet the urgent need for better cancer biomarkers. We have used a range of proteomic analyses of serum and tissue from gastric cancer patients and relevant controls to discover biomarkers for gastric cancer.

Methods:

Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI) and antibody arrays were used to compare protein expression in 21 pairs of gastric cancer tissue and adjacent normal mucosa and serum from 51 gastric cancer patients and 29 patients with benign gastric diseases. Expression differences were confirmed by enzyme-linked immunosorbent assay.

Results:

Tissue analysis shows human neutrophil peptides 1–3 (HNPs 1–3) elevated 10-fold (P=0.001) in gastric cancer relative to adjacent normal mucosa. Macrophage migration inhibitory factor (MIF) was increased five-fold (P=1.84 × 10⁻⁷) in the serum of gastric cancer patients relative to individuals with benign gastric disease. The large increase in MIF concentration in serum gives an area under the receiver operating characteristic curve of 0.85.

Conclusions:

Proteomic analyses of serum and tissue indicate that HNPs 1–3 and MIF have potential as biomarkers for gastric cancer. In particular MIF may be useful, either alone or in combination with other markers, for diagnosing and monitoring gastric cancer.