RUNX3 is a prognostic marker and potential therapeutic target in human breast cancer

Purpose

To evaluate the role of RUNX3 in breast cancer pathogenesis, we examined the RUNX3 expression in breast cancer tissues and analyzed the correlation between RUNX3 expression and clinicopathologic variables and patients survival.

Methods

We evaluated the RUNX3 expression by immunohistochemistry using a tissue microarray containing 256 specimens of breast cancer patients. We also studied the role of RUNX3 in cell migration and invasion by performing cell migration and invasion assay. Differential expression of metastasis-related genes after RUNX3 restoration was analyzed using the Human Tumor Metastasis PCR Array.

Results

The RUNX3 expression was significantly correlated with breast cancer histology grade (P = 0.000), and low RUNX3 expression strongly correlated with worse 5-year overall and disease-specific survival rates (P = 0.000 and P = 0.001, respectively). Furthermore, we found that RUNX3 restoration suppressed breast cancer metastasis by controlling cell migration and invasion capacity. Finally, gene expression profiles of RUNX3-549 and Ctrl-549 cells showed matrix metalloproteinase-2 (MMP-2) was the most significant gene among the 84 metastasis-related genes influenced by RUNX3 reintroduction.

Conclusions

Reduced RUNX3 expression is significantly correlated with breast cancer progression and predicts worse survival. RUNX3 regulates breast cancer cell migration and invasion through the MMP-2 pathway.     

HOXA10 Promotes Cell Invasion and MMP-3 Expression Via TGFβ2-Mediated Activation of the p38 MAPK Pathway in Pancreatic Cancer Cells

Background

HOXA10 is closely related to tumor progression in many human cancers. However, the role of HOXA10 in pancreatic cancer remains unclear. The aim of this study was to determine the involvement of HOXA10 in pancreatic cancer cell invasion and migration.

Methods

The effect of HOXA10 on the invasion and migration of pancreatic cancer cells was assessed by invasion and migration assays. The protein of transforming growth factor beta-2 (TGFβ2) was neutralized by TGFβ2 blocking antibody. The activation of p38 was inhibited by SB239063.

Results

HOXA10 could promote the invasion and migration of pancreatic cancer cells. Knockdown of HOXA10 decreased the expressions of TGFβ2 and matrix metallopeptidase-3 (MMP-3) and suppressed the activation of p38. Conversely, overexpression of HOXA10 increased the levels of TGFβ2 and MMP-3. Further experiments identified that TGFβ2 contributed to the HOXA10-promoted invasion and migration and regulated MMP-3 expression and p38 activation. Additionally, inhibition of p38 suppressed cell invasion and MMP-3 expression in pancreatic cancer cells.

Conclusions

HOXA10 promotes cell invasion and MMP-3 expression of pancreatic cancer cells via TGFβ2-p38 MAPK pathway. Thus, HOXA10 could be a useful target for the treatment of pancreatic cancer.     

Receptor for activated C kinase 1 (RACK1): a regulator for migration and invasion in oral squamous cell carcinoma cells

Purpose

Receptor of activated protein kinase C 1 (RACK1) has been identified as an anchoring or adaptor protein in multiple intracellular signal transduction pathways. Our previous study has showed that the expression of RACK1 was paralleled with proliferation and correlated with metastasis and clinical outcome. However, the underlined mechanism has not been uncovered.

Materials and methods

We first selected a most effective siRNA among three siRNAs (siRNA-1, siRNA-2 and siRNA-3) targeting different regions in the RACK1 mRNA and re-evaluated the anticancer effect of RACK1 silencing on HSC-3 and Cal-27 cell lines by cell growth inhibition. And then, we investigated whether knockdown of RACK1 could inhibit cell adhesion, migration and invasion in these two cell lines. To further understand the molecular mechanism of RACK1 in these processes, the expressions of EGFR, pEGFR, HER2, MMP-2 and MMP-9 were detected by western blot.

Results

We verified that the silence of RACK1 gene in two OSCC cell lines could not only inhibit cell proliferation but also decrease the invasion, migration and adhesion capability of the tumor cells. Further, western blot analysis deduced that it might be related to the decrease in protein expression of EGFR, pEGFR, HER2, MMP-2 and MMP-9.

Conclusion

Our results clearly showed the significance of RACK1-induced OSCC cell migration, invasion and adhesion, which could explain the underlined mechanism of the effect of the gene on metastasis and clinical outcome. Also, our results confirmed its role to be a prognostic indicator and a promising drug target for OSCC cell metastasis.     

BMP7 Expression in Esophageal Squamous Cell Carcinoma and Its Potential Role in Modulating Metastasis

Background

Our previous study showed that BMP7 revealed significantly higher levels in esophageal squamous cell carcinoma (ESCC) tissues with lymph node metastasis compared with non-lymph node metastasis, using gene expression profiling assays. The roles of BMP7 in ESCC is not fully understood.

Aim

The aim of this study was to investigate the effect of BMP7 on lymph node metastasis of ESCC and to explore its potential mechanism.

Methods

Expression of BMP7 in ESCC tissues was evaluated by immunohistochemistry. BMP7 were down-regulated by RNA interference. The protein and mRNA levels of BMP7 were detected by western blot and RT-PCR, respectively. High content screening and transwell assay were used to identify the metastatic ability of tumor cells.

Results

Positivity of BMP7 staining was 57.5 % in the tissues of primary carcinoma with lymph node metastasis compared to tissues without lymph node metastasis, and expression of BMP7 was significantly higher in the cell lines with highly metastatic capacity than that in the cell lines without metastatic ability. Suppression of endogenous BMP7 expression by siRNA in the highly metastatic cell lines resulted in significant reduction in ability of cell migration and invasion in both in vitro and in vivo studies. In addition, inhibition of BMP7 by siRNA also leads to up-regulation of E-cadherin and down-regulation of MMP-9 in the highly metastatic cell lines.

Conclusions

These findings indicate that BMP7 modulates the expression of E-cadherin and MMP-9, and by which mechanism it may regulate cell migration and metastasis of ESCCs.     

miR-145 functions as tumor suppressor and targets two oncogenes,ANGPT2 and NEDD9, in renal cell carcinoma

Purpose

Abnormal expression of miRNAs is closely related to a variety of human cancers. The purpose of this study is to identify new tumor suppressor miRNA and elucidate its physiological function and mechanism in renal cell carcinoma (RCC).

Methods

The expression of miR-145 in 45 RCC and adjacent normal tissues was performed by quantitative RT-PCR. Cell proliferation, migration, invasion, apoptosis and cycle assays were carried out for functional analysis after miR-145 transfection. Two target genes of miR-145 were identified by luciferase reporter assay. The altered expression of 84 epithelial to mesenchymal transition (EMT)-related genes after miR-145 transfection was detected by RT² Profiler EMT PCR array.

Results

The expression of miR-145 was downregulated in RCC compared to their normal adjacent tissues. Restoring miR-145 expression in RCC cell lines dramatically suppressed cell proliferation, migration and invasion, and induced cell apoptosis and G2-phase arrest. We further validated those miR-145 targets two oncogenes, ANGPT2 and NEDD9 in RCC. In addition, miR-145 was found to regulate numerous genes involved in the EMT.

Conclusions

These findings demonstrate that miR-145 functions as tumor suppressor in RCC, suggesting that miR-145 may be a potential therapeutic target for RCC.     

Matrix Metalloproteinase-14 Is a Negative Prognostic Marker for Patients with Gastric Cancer

Background

Matrix metalloproteinase-14 (MMP-14) has been considered to play an important role in invasion and metastasis of human solid tumor.

Aim

The present study aimed to investigate the association of MMP-14 with overall survival in human gastric cancer.

Methods

Gastric cancer and adjacent normal specimens were collected from 205 patients who had not received neoadjuvant chemotherapy. MMP-14 expression was investigated by immunohistochemistry assay and staining evaluation results were analyzed statistically in relation to overall survival of patients.

Results

MMP-14 expression proved to be increased in gastric cancer compared with that in normal tissues. It was also proved that MMP-14 expression was associated with tumor invasion, metastasis, and TNM stage while no correlations were detected between MMP-14 expression and age, sex, differentiation status, or Lauren’s classification. Moreover, patients with gastric cancer of MMP-14-positive expression tend to have worse overall survival compared with those with MMP-14 negative expression.

Conclusions

The present study confirmed the over-expression of MMP-14 in human gastric cancer and its association with tumor progression. It also provided the first evidence that MMP-14 expression in gastric cancer was an independent negative prognostic factor of patients.     

MiR-181d acts as a tumor suppressor in glioma by targeting K-ras and Bcl-2

Purpose

Recently, several microRNAs (miRNAs) were reported to be involved in the modulation of glioma development. The aim of our study was to determine the effect of miR-181d on the growth of glioma and to investigate whether this growth is modulated by targeting K-ras and Bcl-2.

Methods

Real-time PCR was used to analyze the expression of miR-181d in human glioma samples and glioma cell lines. Apoptosis, cell cycle, and proliferation (MTT) assays were performed to assess the phenotypic changes in glioma cells. Immunohistochemistry was used to determine the expression of K-ras and Bcl-2 in glioma tissues, and a luciferase reporter assay was carried out to confirm whether K-ras and Bcl-2 are direct targets of miR-181d. Western blotting was used to identify the potential signaling pathways affected glioma cell growth by miR-181d. In vivo, xenograft tumors were examined for an anti-glioma effect of miR-181d.

Results

MiR-181d was down-regulated in human glioma samples and up-regulated in transfected glioma cells. Ectopic expression of miR-181d suppressed proliferation and triggered cell cycle arrest and apoptosis in glioma cell lines. K-ras and Bcl-2 were identified as direct targets of miR-181d and were up-regulated in glioma samples. The results showed evidence linking the tumor suppressor activity of miR-181d in glioma cells with the K-ras-related PI3K/AKT and MAPK/ERK pathways. Furthermore, xenograft tumors from miR-181d-treated U251 cells were suppressed in vivo.

Conclusion

MiR-181d may act as a glioma suppressor by targeting K-ras and Bcl-2.     

The opposite prognostic significance of nuclear and cytoplasmic p21 expression in resectable gastric cancer patients

Background

Protein p21^Cip1/Waf1 is a cyclin-dependent kinase inhibitor, which plays important roles in cell cycle arrest, senescence, and apoptosis. Interestingly, the nuclear and cytoplasmic p21 executes various functions in the cell. In this study, we investigated the prognostic impact of subcellular p21 expression in gastric cancer (GC).

Methods

Expressions of subcellular p21 was assessed by immunohistochemistry using a tissue microarray in a training cohort and it went into a second testing cohort and finally to a validating cohort. Prognostic and predictive role of subcellular p21 expression status was evaluated. We also studied the roles of subcellular p21 in GC cell migration and invasion.

Results

Nuclear and cytoplasmic p21 protein levels were significantly reduced and increased in GC lesions compared with adjacent non-cancerous tissues, respectively. Low nuclear p21 or high cytoplasmic p21 expression significantly correlated with shorter overall survival (OS), as well as with clinicopathologic characteristics in patients. Multivariate regression analysis showed that low nuclear and high cytoplasmic p21 expression, separately and together, were independent negative markers of OS. Finally, we found that nuclear p21 inhibits but cytoplasmic p21 promotes cell migration and invasion abilities.

Conclusions

These findings suggest that nuclear and cytoplasmic p21 protein expression in tumor are novel candidate prognostic markers in resectable human gastric carcinoma, and they exert distinct roles in cell migration and invasion.     

SULF1 Inhibits Proliferation and Invasion of Esophageal Squamous Cell Carcinoma Cells by Decreasing Heparin-Binding Growth Factor Signaling

Background

Heparin-binding growth factor signaling is involved in the pathogenesis and development of human cancers. It can be regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPG). SULF1 is a heparin-degrading endosulfatase which can modulate the sulfation of HSPGs.

Aim

The purpose of this study was to elucidate the role of SULF1 in modulating proliferation and invasion of esophageal squamous cell carcinoma (ESCC) by decreasing heparin-binding growth factor signaling.

Methods

We restored SULF1 expression in the ESCC cell line KYSE150, and examined the effects of SULF1 expression on the proliferation and invasion of KYSE150 cells. In addition, we investigated the expression of SULF1 in human ESCC tissues and analyzed the correlation of SULF1 expression with clinicopathologic characteristics of ESCC.

Results

Our study shows that re-expression of SULF1 in ESCC cell line results in the downregulation of hepatocyte growth factor-mediated activation of MAPK pathways with a resultant decrease in cell invasiveness. Cell proliferation was also inhibited in SULF1-transfected KYSE150 cells. Immunohistochemical assays reveal that SULF1 is expressed in nearly half of the human ESCC tissues but not in normal esophageal epithelial cells. SULF1 expression in human ESCC tissues is negatively correlated with tumor size and tumor invasion.

Conclusion

This study identified that SULF1 inhibits proliferation and invasion of ESCC by decreasing heparin-binding growth factor signaling and suggested that SULF1 plays an inhibiting role in the pathogenesis of ESCC.     

LASP-1 promotes tumor proliferation and metastasis and is an independent unfavorable prognostic factor in gastric cancer

Purpose

The LIM and SH3 protein 1 (LASP-1) is a focal adhesion protein, and its expression has been reported to be increased in many malignant tumors. However, the role of LASP-1 in gastric cancer is still unknown. The aim of this study was to determine the relationship of LASP-1 expression with the progression and prognosis of gastric cancer.

Methods

Expression of LASP-1 was evaluated in gastric cancer tissues and cell lines by immunohistochemistry and Western blot analysis. The relationship between LASP-1 expression and clinicopathological characteristics was analyzed. Using RNA interference, the effects of LASP-1 on cell proliferation, migration and invasion were investigated in gastric cancer cell lines both in vitro and in vivo.

Results

The LASP-1 was overexpressed in gastric cancer tissues and cell lines. LASP-1 expression was significantly associated with tumor size, invasive depth, TNM stage, lymph node metastasis and p53 expression (all P < 0.05). Multivariate survival analysis showed that LASP-1 expression was recognized as an independent prognostic factor of patient’s survival. Knockdown of LASP-1 inhibited cell proliferation, migration and invasion in vitro as well as tumorigenesis and metastasis in vivo.

Conclusions

Our study showed that LASP-1, overexpressed in gastric cancer and associated with poor prognosis, plays an important role in the growth and metastasis of gastric cancer.     

N-myc downstream-regulated gene 1/Cap43 may function as tumor suppressor in endometrial cancer

Purpose

N-myc downstream-regulated gene 1 (NDRG1) reportedly regulates tumor progression in various cancers. Our previous studies showed that NDRG1 was aberrantly overexpressed in human endometrial cancer tissues. The purpose of the present study was to investigate the role of NDRG1 in endometrial carcinogenesis.

Methods

A short hairpin RNA (shRNA)-mediated gene silencing strategy was employed to stably suppress the expression of NDRG1 in endometrial cancer Ishikawa cells. The influence of NDRG1 silencing on cancer cell biological behaviors was examined through observing in vitro tumor cell proliferation, colony formation, cell migration and invasion. Moreover, the mammalian NDRG1 expression vector pcDNA3.1(+)/NDRG1 was constructed to determine the effects of NDRG1 overexpression on cell proliferation and migration. Additionally, gene expression microarray analysis was conducted to identify NDRG1 downstream target genes after NDRG1 knockdown.

Results

It was demonstrated that NDRG1 knockdown significantly enhanced Ishikawa cell proliferation and dramatically promoted cell migration and invasion. Furthermore, overexpression of NDRG1 in Ishikawa cells greatly inhibited cell proliferation and migration. Through microarray analysis and data mining, a large cohort of NDRG1-repressed target genes were identified. Additionally, through comparing the current microarray results with those obtained previously in studies of cervical and ovarian cancer cells conducted by us, 19 more specific common downstream target genes were identified.

Conclusions

It was demonstrated that NDRG1 might carry out a tumor suppressor function during endometrial carcinogenesis. The identification of downstream target genes should afford meaningful hints for prospective investigations. The tumor suppressor function of NDRG1 may open a new window for the target therapy of endometrial cancer.     

miR-449a Regulates Proliferation and Chemosensitivity to Cisplatin by Targeting Cyclin D1 and BCL2 in SGC7901 Cells

Background

Recently, several miRNAs have been determined as tumor suppressors in various cancers, such as microRNA-449a. However, the exact molecular mechanisms underlying miR-449a regulated cell proliferation and chemosensitivity in gastric cancer cells have not been well documented.

Aim

The present study was designed to test whether miR-449a mediates cell proliferation and chemosensitivity in gastric cancer cells via regulating cyclin D1 and BCL2.

Methods

In vitro, the ability of cell proliferation and cell viability were measured by MTT assay; cell cycle and cell apoptosis was detected by FCM. qRT-PCR was used to measure the expression of miR-449a. Western blot and real-time PCR assays were used to detect the expression of cyclin D1 and BCL2 in gastric cancer cell line SGC7901.

Results

miR-449a expression was downregulated in gastric cancer cell line SGC7901 and human gastric cancer tissues, compared to the gastric epithelial cell line GES-1 and matched non-tumor associated tissues. Upregulation of miR-449a reduced the proliferation of SGC7901 cells. Ectopic expression of miR-449a decreased the percentage of S phase cells, increased the percentage of G1/G0 phase cells and increased the apoptosis induced by cisplatin. Moreover, miR-449a inhibited SGC7901 cells proliferation and enhanced cisplatin chemosensitivity by downregulating expression of BCL2 and cyclin D1, respectively, via directly targeting the 3′-untranslated regions of BCL2 and cyclin D1 mRNA.

Conclusions

This is the first report to provide evidence that miR-449a could modulate cell cycle and apoptosis through regulating cyclin D1 and BCL2 expression in SGC7901 cells.     

Neural cell adhesion molecule isoform 140 declines with rise of WHO grade in human gliomas and serves as indicator for the invasion zone of multiform glioblastomas and brain metastases

Purpose

Gliomas are highly invasive neuroepithelial tumors with a propensity of malignant transformation and very restricted treatment options. The neural cell adhesion molecule (NCAM) modulates cellular migration, proliferation, and synaptic plasticity by homophilic and heterophilic interactions. Hereby, we investigated its relevance as a glioma tissue marker for the biological aggressiveness of these tumors and compared these features with the carcinoma brain metastasis invasion zone.

Materials and methods

We analyzed 194 human brain samples. Human tumor-free brain specimens served as control for the white and gray matter. In addition to that, we used human glioblastomas from nude rats. All tissues were investigated immunohistochemically for the expression of the NCAM isoform 140. Additionally, the multiplanar MRI-CT fusion neuronavigation-guided serial stereotactic biopsy was performed and completed by histopathological workup.

Results

Human gliomas loose NCAM-140 with the rise of their WHO grade. Meningiomas are NCAM-140 negative. As the most striking feature, human brain metastases and the majority of human glioblastomas of our patients and of nude rats were totally NCAM-140 negative. This NCAM negativity led us to the conclusion of three different main glioblastoma invasion patterns. Surprisingly, the majority of brain metastasis samples that contained surrounding brain parenchyma demonstrated invasive tumor cell nests beyond the sharply demarcated metastasis border. We also found invasive metastatic cell nests outside the contrast enhancing tumor zone by means of the MRI-CT fusion neuronavigation-guided serial stereotactic biopsy.

Conclusion

The expression of NCAM-140 inversely correlates with the WHO grade of human gliomas. The lost expression of NCAM-140 in human glioblastomas and in brain metastases enables the investigation of the brain?Ctumor interface and the definition of glioblastoma invasion patterns and shows that brain metastases are more invasive than ever thought.     

Overexpression of macrophage migration inhibitory factor in adenoid cystic carcinoma: correlation with enhanced metastatic potential

Objectives

Adenoid cystic carcinoma (ACC) is a malignant tumor frequently arising in salivary glands with poor long-term prognosis due to high rates of local recurrences and distant metastases. Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine and has recently emerged as a pro-tumorigenic factor in various cancers. This study is designed to investigate the expression status and functional significance of MIF in ACC.

Methods

Immunohistochemical staining was performed to evaluate the expression levels of MIF, HIF-1α, MMP-9, p53, and p-JNK in ACC tissues. In vitro, ACC-2 cells were exposed to recombinant human MIF (rMIF) or ISO-1 (an inhibitor of MIF) at different concentrations and times, followed by the detection of cell growth, viability, migration, and invasion, as well as the expression levels of several cellular signals.

Results

The immunohistochemical results demonstrated the overexpression of MIF in ACC tissues as well as its association with the distant metastasis. Further analyses showed a significant correlation between the staining of MIF and p-JNK. Moreover, the in vitro studies revealed that the treatment for ACC cells with ISO-1 significantly attenuated cell migratory and invasive capacity, as opposed to the limited promotive effects of rMIF. More importantly, MIF inhibition could cause the activation of JNK, correlating with the immunohistochemical findings on ACC tissues.

Conclusions

The results suggest that MIF is likely to be an important player in the pathogenesis of ACC and may promote cancer metastasis, which possibly involves JNK inactivation. Further investigation of MIF-mediated molecular events may provide novel insights into the treatment for ACC.     

miR-1303 Targets Claudin-18 Gene to Modulate Proliferation and Invasion of Gastric Cancer Cells

Background

MicroRNAs have emerged as important gene regulators and are recognized as important molecules in carcinogenesis. However, the effects of microRNA-1303 (miR-1303) on gastric cancer (GC) cells and the upstream regulation of GC-associated claudin-18 gene (CLDN18) remain unclear. miR-1303 may be involved in the tumorigenesis of GC by targeting CLDN18.

Aims

The purpose of this study was to explore the effect of miR-1303 targeting of CLDN18 on the proliferation, migration and invasion of human GC cells.

Methods

The expression of miR-1303 and claudin-18 in GC tissues and gastric cancer cell lines were detected by qRT-PCR and western blotting, respectively. CCK8 and colony formation assays were performed to study the influence of miR-1303 on the proliferation of the GC cell lines. Transwell and wound-healing assays were carried out to investigate the effect of miR-1303 on the invasion and migration of GC cell lines. Luciferase reporter assays, restore assays and western blotting were used to demonstrate whether CLDN18 is a direct target of miR-1303.

Results

miR-1303 was significantly overexpressed whereas claudin-18 was downregulated in GC tissues and cell lines, which was significantly associated with tumor size, location invasion, histologic type and tumor-node-metastasis stage. Cell proliferation rates were reduced, and cell invasion and migratory ability was significantly restricted in miR-1303 inhibitor-transfected groups. miR-1303 could bind to the putative binding sites in CLDN18 mRNA 3′-UTR and visibly lower the expression of claudin-18. The introduction of claudin-18 without 3′-UTR restored the miR-1303 promoting migration function.

Conclusions

Downregulation of miR-1303 can inhibit proliferation, migration and invasion of GC cells by targeting CLDN18.