CD26 Induces Colorectal Cancer Angiogenesis and Metastasis through CAV1/MMP1 Signaling.

CD26 has been reported as a marker for colorectal cancer stem cells endowed with tumor-initiating properties and capable of colorectal cancer (CRC) metastasis. In this study, we investigated the functional effect of CD26 on CRC angiogenesis and metastasis, and the potential underlying mechanism. The functional effects of CD26 overexpression or repression were determined by a wound healing experiment, and cell migration and invasion assays in vitro and in mouse models. Differentially expressed genes regulated by CD26 were identified by genome-wide mRNA expression array and validated by quantitative PCR. CD26 functionally regulated CRC cell migration and invasion in vitro and angiogenesis and metastasis in vivo. Genome-wide mRNA expression array and qPCR showed that MMP1 was up-regulated in CD26+ subpopulation, and a subsequent experiment demonstrated the regulatory effect of CD26 on MMP1 in CRC cell lines with CD26 repression or overexpression. Furthermore, overexpression of CAV1 abrogated the CD26-regulated MMP1 induction in CRC cell lines. This study demonstrated the functional roles of CD26 in inducing CRC migration, invasion, angiogenesis and metastasis and identified the potential involvement of MMP1 and CAV1 in such process. CD26 is an attractive therapeutic target for combating tumor progression to improve the prognosis of CRC patients.

Emergence of CD26+ Cancer Stem Cells with Metastatic Properties in Colorectal Carcinogenesis.

Colorectal cancer results from genetic aberrations which accumulate over a long period of time, with malignant and metastatic properties acquired at a relatively late stage. A subpopulation of CD26+ colorectal cancer stem cells are known to be implicated in metastasis. We quantified CD26+ cancer cells in 11 primary tumor samples by flow cytometry, and showed that tumors having confirmed or suspected metastases harbored a relatively high CD26+ level in these samples. We hypothesized that this subpopulation of cancer stem cells arises in the late stage of carcinogenesis from the bulk of tumor daughter cells which are CD26-. The manipulation of PIK3CA and TP53, two genes commonly deregulated in the late stage, had an effect on the maintenance of the CD26+ cell population. When CD26- tumor daughter cells were sorted and cultured, the emergence of tumor spheres containing CD26+ cells occurred. These findings shed light to the origin of colorectal cancer stem cells with metastatic properties, which has an implication on conventional treatments by surgery or adjuvant chemotherapy for tumor debulking.

Nine-year experience of doxorubicin-eluting beads chemoembolization for hepatocellular carcinoma.

BACKGROUND: Chemoembolization with doxorubucin-eluting beads (DEB) has been used to treat hepatocellular carcinoma (HCC) since 2007. This study compared the efficacy and survival between transarterial chemoembolization (TACE) with DEB and conventional approach (cTACE) in HCC treatment. METHODS: This retrospective case-control study compared the overall survival and tumor response of HCC patients to cTACE (n=190) and DEB (n=143) by the reassessment of computed tomography and serum alpha-fetoprotein (AFP). Multivariate analysis was used to determine the factors affecting tumor response. RESULTS: The median post-treatment to pre-treatment AFP level was 0.8 for a DEB session (n=258) and 1.0 for a cTACE session (n=452), showing a significantly greater decrease in AFP after DEB (P<0.05). More patients in the DEB group achieved objective response (complete and partial) compared with those in the cTACE group (P<0.05). Objective tumor response after DEB vs cTACE was 34.8% vs 15.4% in 0-3 months (P=0.001), 37.1% vs 20.0% in 3-6 months (P<0.05), and 50.0% vs 30.0% in 6-12 months (P=0.093). DEB predicted a 3.604 times odds of achieving at least one objective tumor response in a patient when compared to cTACE (P<0.0001). The median survival from first transcatheter therapy of patients having undergone at least once DEB was 12.53 months, while those having received cTACE only was 10.53 months (P=0.086). A tendency of improved survival appeared to maintain until >80 months after the first TACE session in the DEB group. CONCLUSION: DEB is a safe alternative to cTACE in HCC patients with better therapeutic efficacy.

Age-dependent accumulation of oligomeric SNCA/α-synuclein from impaired degradation in mutant LRRK2 knockin mouse model of Parkinson disease: role for therapeutic activation of chaperone-mediated autophagy (CMA).

Parkinson disease (PD) is an age-related neurodegenerative disorder associated with misfolded SNCA/α-synuclein accumulation in brain. Impaired catabolism of SNCA potentiates formation of its toxic oligomers. LRRK2 (leucine-rich repeat kinase-2) mutations predispose to familial and sporadic PD. Mutant LRRK2 perturbs chaperone-mediated-autophagy (CMA) to degrade SNCA. We showed greater age-dependent accumulation of oligomeric SNCA in striatum and cortex of aged LRRK2R1441G knockin (KI) mice, compared to age-matched wildtype (WT) by 53% and 31%, respectively. Lysosomal clustering and accumulation of CMA-specific LAMP2A and HSPA8/HSC70 proteins were observed in aged mutant striatum along with increased GAPDH (CMA substrate) by immunohistochemistry of dorsal striatum and flow cytometry of ventral midbrain cells. Using our new reporter protein clearance assay, mutant mouse embryonic fibroblasts (MEFs) expressing either SNCA or CMA recognition 'KFERQ'-like motif conjugated with photoactivated-PAmCherry showed slower cellular clearance compared to WT by 28% and 34%, respectively. However, such difference was not observed after the 'KFERQ'-motif was mutated. LRRK2 mutant MEFs exhibited lower lysosomal degradation than WT indicating lysosomal dysfunction. LAMP2A-knockdown reduced total lysosomal activity and clearance of 'KFERQ'-substrate in WT but not in mutant MEFs, indicating impaired CMA in the latter. A CMA-specific activator, AR7, induced neuronal LAMP2A transcription and lysosomal activity in MEFs. AR7 also attenuated the progressive accumulation of both intracellular and extracellular SNCA oligomers in prolonged cultures of mutant cortical neurons (DIV21), indicating that oligomer accumulation can be suppressed by CMA activation. Activation of autophagic pathways to reduce aged-related accumulation of pathogenic SNCA oligomers is a viable disease-modifying therapeutic strategy for PD.Abbreviations: 3-MA: 3-methyladenine; AR7: 7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; CMA: chaperone-mediated autophagy; CQ: chloroquine; CSF: cerebrospinal fluid; DDM: n-dodecyl ß-D-maltoside; DIV: days in vitro; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GWAS: genome-wide association studies; HSPA8/HSC70: heat shock protein 8; KFERQ: CMA recognition pentapeptide; KI: knockin; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LDH: lactate dehydrogenase; LRRK2: leucine-rich repeat kinase 2; MEF: mouse embryonic fibroblast; NDUFS4: NADH:ubiquinone oxidoreductase core subunit S4; NE: novel epitope; PD: Parkinson disease; RARA/RARα: retinoic acid receptor, alpha; SNCA: synuclein, alpha; TUBB3/TUJ1: tubulin, beta 3 class III; WT: wild-type.

Combined LRRK2 mutation, aging and chronic low dose oral rotenone as a model of Parkinson's disease.

Aging, genetics and environmental toxicity are important etiological factors in Parkinson's disease (PD). However, its pathogenesis remains unclear. A major obstacle is the lack of an appropriate experimental model which incorporates genetic susceptibility, aging and prolonged environmental toxicity. Here, we explored the interplay amongst these factors using mutant LRRK2R1441G (leucine-rich-repeat-kinase-2) knockin mice. We found that mutant primary cortical and mesencephalic dopaminergic neurons were more susceptible to rotenone-induced ATP deficiency and cell death. Compared with wild-type controls, striatal synaptosomes isolated from young mutant mice exhibited significantly lower dopamine uptake after rotenone toxicity, due to reduced striatal synaptosomal mitochondria and synaptic vesicular proton pump protein (V-ATPase H) levels. Mutant mice developed greater locomotor deficits in open-field tests than wild-type mice following low oral rotenone doses given twice weekly over 50 weeks (half their lifespan). The increased locomotor deficit was associated with specific reduction in striatal mitochondrial Complex-I (NDUFS4) in rotenone-treated mutant but not in similarly treated wild-type mice. Our unique experimental model which incorporates genetic effect, natural aging and prolonged oral environmental toxicity administered to mutant knockin LRRK2 mice over half their life span, with observable and measurable phenotype, is invaluable in further studies of the pathogenic process and therapeutics of PD.

Identification of microRNA 885-5p as a novel regulator of tumor metastasis by targeting CPEB2 in colorectal cancer.

Colorectal cancer is the third most common cancer in the world and liver is the most frequent site of distant metastasis with poor prognosis. The aim of this study is to investigate microRNAs leading to liver metastasis. We applied microarray analysis and quantitative PCR to identify and validate dysregulated miRNAs in liver metastases when compared to primary CRCs. Functional significance and the underlying molecular mechanism of selected miRNA was demonstrated by a series of in vitro and in vivo assays. Our microarray analysis and subsequent quantitative PCR validation revealed that miR-885-5p was strongly up-regulated in liver metastases and in CRC cell-lines derived from distant metastases. Overexpression of miR-885-5p significantly induced cell migration, cell invasion, formation of stress fibre in vitro and development of liver and lung metastases in vivo. MiR-885-5p induced metastatic potential of CRC by repressing cytoplasmic polyadenylation element binding protein 2 transcription through directly binding to two binding sites on its 3' untranslated region, and consequently led to up-regulation of TWIST1 and hence epithelial-mesenchymal transition. Our findings demonstrated the overexpression of miR-885-5p in liver metastasis and its roles in inducing CRC metastasis, potentiating development of miR-885-5p inhibitor to treat advanced CRC in the future.

Post-operative plasma osteopontin predicts distant metastasis in human colorectal cancer.

BACKGROUND: The overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients. METHODS: This randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay. RESULTS: Our findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration. CONCLUSIONS: The results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.

Prognostic significance of CD26 in patients with colorectal cancer.

BACKGROUND: CD26, dipeptidyl peptidase IV, was discovered firstly as a membrane-associated peptidase on the surface of leukocyte. We previously demonstrated that a subpopulation of CD26+ cells were associated with the development of distant metastasis, enhanced invasiveness and chemoresistance in colorectal cancer (CRC). In order to understand the clinical impact of CD26, the expression was investigated in CRC patient's specimens. This study investigated the prognostic significance of tumour CD26 expression in patients with CRC. Examination of CD26+ cells has significant clinical impact for the prediction of distant metastasis development in colorectal cancer, and could be used as a selection criterion for further therapy. METHODS: Tumour CD26 expression levels were studied by immunohistochemistry using Formalin-fixed paraffin embedded (FFPE) tissues in 143 patients with CRC. Tumour CD26 expression levels were correlated with clinicopathological features of the CRC patients. The prognostic significance of tumour tissue CD26 expression levels was assessed by univariate and multivariate analyses. RESULT: CD26 expression levels in CRC patients with distant metastasis were significantly higher than those in non-metastatic. High expression levels of CD26 were significantly associated with advanced tumour staging. Patients with a high CD26 expression level had significantly worse overall survival than those with a lower level (p<0.001). CONCLUSIONS: The expression of CD26 was positively associated with clinicopathological correlation such as TNM staging, degree of differentiation and development of metastasis. A high CD26 expression level is a predictor of poor outcome after resection of CRC. CD26 may be a useful prognostic marker in patients with CRC.

The clinicopathological significance of miR-133a in colorectal cancer.

This study determined the expression of microRNA-133a (MiR-133a) in colorectal cancer (CRC) and adjacent normal mucosa samples and evaluated its clinicopathological role in CRC. The expression of miR-133a in 125 pairs of tissue samples was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and correlated with patient's clinicopathological data by statistical analysis. Endogenous expression levels of several potential target genes were determined by qRT-PCR and correlated using Pearson's method. MiR-133a was downregulated in 83.2% of tumors compared to normal mucosal tissue. Higher miR-133a expression in tumor tissues was associated with development of distant metastasis, advanced Dukes and TNM staging, and poor survival. The unfavorable prognosis of higher miR-133a expression was accompanied by dysregulation of potential miR-133a target genes, LIM and SH3 domain protein 1 (LASP1), Caveolin-1 (CAV1), and Fascin-1 (FSCN1). LASP1 was found to possess a negative correlation (γ = -0.23), whereas CAV1 exhibited a significant positive correlation (γ = 0.27), and a stronger correlation was found in patients who developed distant metastases (γ = 0.42). In addition, a negative correlation of FSCN1 was only found in nonmetastatic patients. In conclusion, miR-133a was downregulated in CRC tissues, but its higher expression correlated with adverse clinical characteristics and poor prognosis.

The Enhanced metastatic potential of hepatocellular carcinoma (HCC) cells with sorafenib resistance.

Acquired resistance towards sorafenib treatment was found in HCC patients, which results in poor prognosis. To investigate the enhanced metastatic potential of sorafenib resistance cells, sorafenib-resistant (SorR) cell lines were established by long-term exposure of the HCC cells to the maximum tolerated dose of sorafenib. Cell proliferation assay and qPCR of ABC transporter genes (ABCC1-3) were first performed to confirm the resistance of cells. Migration and invasion assays, and immunoblotting analysis on the expression of epithelial to mesenchymal transition (EMT) regulatory proteins were performed to study the metastatic potential of SorR cells. The expression of CD44 and CD133 were studied by flow cytometry and the gene expressions of pluripotency factors were studied by qPCR to demonstrate the enrichment of cancer stem cells (CSCs) in SorR cells. Control (CTL) and SorR cells were also injected orthotopically to the livers of NOD-SCID mice to investigate the development of lung metastasis. Increased expressions of ABCC1-3 were found in SorR cells. Enhanced migratory and invasive abilities of SorR cells were observed. The changes in expression of EMT regulatory proteins demonstrated an activation of the EMT process in SorR cells. Enriched proportion of CD44(+) and CD44(+)CD133(+) cells were also observed in SorR cells. All (8/8) mice injected with SorR cells demonstrated lung metastasis whereas only 1/8 mouse injected with CTL cells showed lung metastasis. HCC cells with sorafenib resistance demonstrated a higher metastatic potential, which may be due to the activated EMT process. Enriched CSCs were also demonstrated in the sorafenib resistant cells. This study suggests that advanced HCC patients with acquired sorafenib resistance may have enhanced tumor growth or distant metastasis, which raises the concern of long-term sorafenib treatment in advanced HCC patients who have developed resistance of sorafenib.

Over-expression of miR-106b promotes cell migration and metastasis in hepatocellular carcinoma by activating epithelial-mesenchymal transition process.

Hepatocellular carcinoma (HCC) is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated. In vitro functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs) microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis in vivo. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005), and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018). Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. In vivo functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT) process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration in vitro and metastasis in vivo.