Baocun Sun

CD133 + cells with cancer stem cell characteristics associates with vasculogenic mimicry in triple-negative breast cancer

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. In the study we demonstrated that CD133 expression was the highest in triple-negative (TN) breast cancer specimens. Importantly, VM showed statistical correlation with CD133+ expression. The presence of the close relationship between VM and CD133+ expression might be central for TN tumor relapse and progression. The TN breast cancer cell line, MDA-MB-231 cells developed a range of colony morphologies paralleling the holoclone, meroclone and paraclone morphologies produced by normal keratinocytes and other epithelial cancer cell lines when plated at clonal densities. Holoclone cells were capable of forming more colonies on soft agar than meroclone cells and paraclone cells, suggesting that holoclone cells had higher self-renew potential and might harbors cancer stem cells (CSCs) subpopulation. Strikingly, it was holoclone that displayed CD133+ phenotype and formed VM. In addition, holoclone acquired endothelial cell marker vascular endothelial-cadherin expression and upregulated VM mediators matrix metalloproteinase (MMP)-2 and MMP-9 expression. The subpopulation with holoclone morphology, CD133+ phenotype and CSCs characteristics might have the capacity of transdifferentiation and contributed to VM in TN breast cancer. The related molecular pathways may be used as novel therapeutic targets for the inhibition of angiogenesis and metastasis in TN breast carcinoma.

Generation of cancer stem-like cells through the formation of polyploid giant cancer cells

Polyploid giant cancer cells (PGCCs) have been observed by pathologists for over a century. PGCCs contribute to solid tumor heterogeneity, but their functions are largely undefined. Little attention has been given to these cells, largely because PGCCs have been generally thought to originate from repeated failure of mitosis/cytokinesis and have no capacity for long-term survival or proliferation. Here we report our successful purification and culture of PGCCs from human ovarian cancer cell lines and primary ovarian cancer. These cells are highly resistant to oxygen deprivation and could form through endoreduplication or cell fusion, generating regular-sized cancer cells quickly through budding or bursting similar to simple organisms like fungi. They express normal and cancer stem cell markers, they divide asymmetrically and they cycle slowly. They can differentiate into adipose, cartilage and bone. A single PGCC formed cancer spheroids in vitro and generated tumors in immunodeficient mice. These PGCC-derived tumors gained a mesenchymal phenotype with increased expression of cancer stem cell markers CD44 and CD133 and become more resistant to treatment with cisplatin. Taken together, our results reveal that PGCCs represent a resistant form of human cancer using an ancient, evolutionarily conserved mechanism in response to hypoxia stress; they can contribute to the generation of cancer stem-like cells, and also play a fundamental role in regulating tumor heterogeneity, tumor growth and chemoresistance in human cancer.

Hypoxia promotes vasculogenic mimicry formation by inducing epithelial-mesenchymal transition in ovarian carcinoma.

OBJECTIVES The functions of hypoxia and subsequent hypoxia-inducible factor-1α (HIF-1α) activation in vasculogenic mimicry (VM) are currently unclear. This study aimed to investigate the effects of hypoxia on VM formation in ovarian cancer, and explore the possible mechanism involved. METHODS The expression levels of HIF-1α, E-cadherin, vimentin, Twist1, Slug, and VE-cadherin proteins were analyzed by immunohistochemistry in 71 specimens of epithelial ovarian cancer. The results were correlated with VM and survival analysis. We used a well-established in vitro model of a three-dimensional culture to compare VM formation under hypoxia and normoxia in ovarian cancer cell lines SKOV3 and OVCAR3. To explore the potential mechanism, we examined the effects of hypoxia on the mRNA and protein expression levels of both E-cadherin and vimentin. RESULTS HIF-1α expression was correlated with loss of E-cadherin expression and up-regulated vimentin expression in 11 of the 18 VM-positive patients. Ovarian cancer with evidence of VM was significantly more likely to have high Twist1, Slug, and VE-cadherin expression levels. VM was observed in vitro under hypoxia. The ovarian cancer cells presented morphological epithelial-mesenchymal transition (EMT)-like changes (more fibroblastoid morphology and loss of cellular cohesiveness) under hypoxic conditions. The mRNA and protein levels demonstrated the induction of EMT after hypoxia. Clinicopathological analysis revealed that both VM and HIF-1α expression levels presented shorter survival durations. CONCLUSIONS Hypoxia contributed to VM formation by inducing EMT. These results may offer new insights for consideration in ovarian cancer treatment strategies.

Genetic amplification of the vascular endothelial growth factor (VEGF) pathway genes, including VEGFA, in human osteosarcoma.

Osteosarcoma is the most common primary tumor of bone. It is a highly vascular and extremely destructive malignancy that mainly affects children and young adults. The authors conducted microarray‐based comparative genomic hybridization (aCGH) and pathway analyses to gain a systemic view of pathway alterations in the genetically altered genes.

MiR-506 inhibits multiple targets in the epithelial-to-mesenchymal transition network and is associated with good prognosis in epithelial ovarian cancer.

Extensive investigations have shown that miRNAs are important regulators of epithelial‐to‐mesenchymal transition (EMT), mainly targeting the transcriptional repressors of E‐cadherin (E‐cad). Less is known about the post‐transcriptional regulation of vimentin or N‐cadherin (N‐cad) in EMT. Our previous study identified miR‐506 as a key EMT inhibitor through directly targeting the E‐cad transcriptional repressor SNAI2. In this study, we provide evidence that miR‐506 simultaneously suppresses vimentin and N‐cad. The knockdown of vimentin using siRNA reversed EMT, suppressed cell migration and invasion, and increased E‐cad expression on the cell membrane in epithelial ovarian cancer (EOC) cells. In a set of tissue microarrays that included 204 EOCs of all major subtypes (eg serous, endometrioid, clear cell, and mucinous), miR‐506 was positively correlated with E‐cad and negatively correlated with vimentin and N‐cad in all subtypes of EOC. A high level of miR‐506 was positively associated with early FIGO stage and longer survival in EOC. Introduction of miR‐506, mediated by nanoparticle delivery, in EOC orthotopic mouse models resulted in decreased vimentin, N‐cad, and SNAI2 expression and increased E‐cad expression; it also suppressed the dissemination of EOC cells. Thus, miR‐506 represents a new class of miRNA that regulates both E‐cad and vimentin/N‐cad in the suppression of EMT and metastasis. Copyright

Mutational landscape of gastric adenocarcinoma in Chinese: implications for prognosis and therapy.

Significance We have identified a lethal subtype of gastric cancer (GC) that is characterized by high levels of clonal heterogeneity and TP53 (tumor protein P53) mutation. We have also uncovered key novel mutations in the targetable NRG1 (neuregulin-1) and ERBB4 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 4) ligand-receptor pair and identified BRCA2 (breast cancer 2, early onset) mutations as new genetic markers to predict better survival for GC. Our study represents a novel approach for GC personalized medicine and identified novel clinical actionable therapies for GC therapy. Gastric cancer (GC) is a highly heterogeneous disease. To identify potential clinically actionable therapeutic targets that may inform individualized treatment strategies, we performed whole-exome sequencing on 78 GCs of differing histologies and anatomic locations, as well as whole-genome sequencing on two GC cases, each with three primary tumors and two matching lymph node metastases. The data showed two distinct GC subtypes with either high-clonality (HiC) or low-clonality (LoC). The HiC subtype of intratumoral heterogeneity was associated with older age, TP53 (tumor protein P53) mutation, enriched C > G transition, and significantly shorter survival, whereas the LoC subtype was associated with younger age, ARID1A (AT rich interactive domain 1A) mutation, and significantly longer survival. Phylogenetic tree analysis of whole-genome sequencing data from multiple samples of two patients supported the clonal evolution of GC metastasis and revealed the accumulation of genetic defects that necessitate combination therapeutics. The most recurrently mutated genes, which were validated in a separate cohort of 216 cases by targeted sequencing, were members of the homologous recombination DNA repair, Wnt, and PI3K-ERBB pathways. Notably, the drugable NRG1 (neuregulin-1) and ERBB4 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 4) ligand-receptor pair were mutated in 10% of GC cases. Mutations of the BRCA2 (breast cancer 2, early onset) gene, found in 8% of our cohort and validated in The Cancer Genome Atlas GC cohort, were associated with significantly longer survivals. These data define distinct clinicogenetic forms of GC in the Chinese population that are characterized by specific mutation sets that can be investigated for efficacy of single and combination therapies.

Dickkopf‐1 inhibits epithelial‐mesenchymal transition of colon cancer cells and contributes to colon cancer suppression

This study aimed to determine the expression pattern of dickkopf‐1 (Dkk1), a potent inhibitor of Wnt signaling, in colon cancer and to assess the function and mechanism of Dkk1 in tumor progression in vitro and in vivo. We detected the protein expression of Dkk1 and some epithelial‐mesenchymal transition (EMT)‐associated markers (E‐cadherin, vimentin and β‐catenin) in 217 tissue samples of human colon cancer, upregulated Dkk1 expression in HCT116 colon cancer cells, and established a nude mouse xenograft model. Dkk1 protein overexpression was inversely related to tumor grade and the presence of metastasis and recurrence of colon cancer. Notably, the expression of Dkk1 was concomitant with reduced immunohistochemical features of EMT (e.g. increased expression of epithelial marker E‐cadherin, decreased expression of mesenchymal marker vimentin, and cytoplasmic distribution of β‐catenin). Furthermore, Dkk1 overexpression resulted in restoration of the epithelial phenotype, decreased expression of EMT transcription factors Snail and Twist, and decreased expression of markers suggestive of intestinal stem cells (e.g. cluster of differentiation 133 [CD133] and leucine‐rich‐repeat‐containing G‐protein‐coupled receptor 5 [Lgr5]). Functional analysis showed overexpression of Dkk1 reduced proliferation, migration, and invasion of colon cancer cells. Moreover, upregulation of Dkk1 led to decreased tumor‐initiating ability and suppressed colon tumor growth in nude mice. Our findings indicate that Dkk1 can suppress the progression of colon cancer, possibly through EMT inhibition, and could therefore serve as a target for tumor therapy. (Cancer Sci 2012; 103: 828–835)

NGAL Expression Is Elevated in Both Colorectal Adenoma–Carcinoma Sequence and Cancer Progression and Enhances Tumorigenesis in Xenograft Mouse Models

Purpose: There is growing evidence implicating that neutrophil gelatinase–associated lipocalin (NGAL) plays a role in the development and progression of cancers. However, the effect of NGAL in colorectal carcinoma (CRC) has not been clearly elucidated. In this study, we investigated the role of NGAL in the tumorigenesis and progression of CRC and evaluated the clinical value of NGAL expression. Experimental Design: We examined NGAL expression in 526 colorectal tissue samples, including 53 sets of matched specimens (histologically normal mucosa, adenomas, and carcinomas) using immunohistochemical analysis. In CRCs, correlations between NGAL expression and clinicopathologic parameters were analyzed, and survival analysis was conducted. The role of NGAL was further tested using mouse xenograft models. Results: NGAL expression was elevated during the colorectal adenoma–carcinoma sequence both among the 526 cases (rs = 0.66, P < 0.001) and in the 53 sets of matched specimens (rs = 0.60, P < 0.001). In CRCs, NGAL expression was associated with cancer stage (P = 0.041) and tumor recurrence in stage II patients (P = 0.037). Survival analysis revealed that NGAL expression was an independent prognostic factor for overall survival (HR = 1.84, P = 0.004) and for disease-free survival of stage II patients (HR = 5.88, P = 0.021). In mouse models, the xenografts in cecum and spleen were heavier and more numerous in the group injected with NGAL-overexpressing CRC cells (P < 0.05). Conclusions: NGAL overexpression may promote the tumorigenesis and progression of CRC. Detecting NGAL expression in tumor tissues may be useful for evaluating prognosis of patients with CRC. Clin Cancer Res; 17(13); 4331–40. ©2011 AACR.

Zinc finger E-box binding homeobox 1 promotes vasculogenic mimicry in colorectal cancer through induction of epithelial-to-mesenchymal transition

Our previous studies have shown that epithelial–mesenchymal transition (EMT) may be involved in the vasculogenic mimicry (VM) formation in hepatocellular carcinoma. Here, we hypothesize that zinc finger E‐box binding homeobox 1 (ZEB1) promotes VM formation in colorectal carcinoma (CRC) by inducing EMT. We identified VM in 39 (19.2%) out of 203 CRC patients. The presence of VM was associated with aggressive biological behavior and was an unfavorable prognostic indicator. By immunohistochemical analysis, we found that the VM‐positive CRC samples showed increased ZEB1 expression compared with the VM‐negative samples and the ZEB1 expression occurred concomitantly with features of EMT. In vitro, knockdown of ZEB1 in poorly differentiated HCT116 CRC cells destroyed the vessel‐like structures in the 3‐D culture, a property associated with VM formation. Knockdown of ZEB1 resulted in restoration of epithelial phenotypes and significantly inhibited the ability to migrate and invade. In addition, ZEB1 underexpression decreased the expression of vascular endothelial (VE)‐cadherin and Flk‐1, which are characteristics of endothelial cells. Taken together, our results suggest that ZEB1 can promote VM formation by inducing EMT in CRC and might represent an important target in CRC. (Cancer Sci 2012; 103: 813–820)

The diagnostic value of SYT-SSX detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) for synovial sarcoma: A review and prospective study of 255 cases

This study aimed to evaluate the diagnostic value of SYT‐SSX detected by reverse transcriptase–polymerase chain reaction (RT‐PCR) and fluorescence in situ hybridization (FISH) for synovial sarcoma (SS) in known and potential cases. SYT‐SSX was analyzed in formalin‐fixed, paraffin‐embedded tissues of 62 known SS, 60 non‐SS and 133 potential SS by RT‐PCR and FISH. FISH was mainly performed on a tissue microarray with some modifications. SYT‐SSX was detected in 94.7% (54/57) of known SS and 70.5% (86/122) of potential SS by RT‐PCR and in 96.7% (58/60) of known SS and 78.1% (100/128) of potential SS by FISH. Moreover, SYT‐SSX was negative in 100% (58/58) of non‐SS by RT‐PCR and in 100% (59/59) of non‐SS by FISH. Accordingly, SYT‐SSX was detected in 106 potential SS by RT‐PCR or FISH, including 80 cases manifested by both methods, 20 specimens verified only by FISH and 6 samples confirmed only by RT‐PCR. Clinical findings and immunohistochemistry data were analyzed in potential SS with final molecular diagnosis. The positive ratio of cytokeratin (CK) and epithelial membrane antigen (EMA) in finally diagnosed SS was 51.9% (55/106) and 61.3% (65/106), respectively. Except EMA, clinical parameters (age, sex, tumor size, tumor sites) and other immunohistochemistry indexes (CK, S‐100, neurone specific enolase (NSE), CD99, myoglobin, smooth muscle actin (SMA), cluster of differentiation (CD) 68 and mesothelial cell) had no significant difference between finally diagnosed SS and non‐SS. It is indicated that the efficiency of FISH is comparable to or even higher than that of RT‐PCR for SYT‐SSX detection. The detection of SYT‐SSX by RT‐PCR or FISH is very useful for the final diagnosis of potential synovial sarcomas. (Cancer Sci 2008; 99: 1355–1361)