Kwok Wah Chan

miR-130b Promotes CD133+ Liver Tumor-Initiating Cell Growth and Self-Renewal via Tumor Protein 53-Induced Nuclear Protein 1

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133⁻ counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133⁻ cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133⁻ cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133⁻ cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.

CD133 + liver tumor-initiating cells promote tumor angiogenesis, growth, and self-renewal through neurotensin/interleukin-8/CXCL1 signaling

A novel theory in the field of tumor biology postulates that cancer growth is driven by a population of stem‐like cells, called tumor‐initiating cells (TICs). We previously identified a TIC population derived from hepatocellular carcinoma (HCC) that is characterized by membrane expression of CD133. Here, we describe a novel mechanism by which these cells mediate tumor growth and angiogenesis by systematic comparison of the gene expression profiles between sorted CD133 liver subpopulations through genome‐wide microarray analysis. A significantly dysregulated interleukin‐8 (IL‐8) signaling network was identified in CD133+ liver TICs obtained from HCC clinical samples and cell lines. IL‐8 was found to be overexpressed at both the genomic and proteomic levels in CD133+ cells isolated from HCC cell lines or clinical samples. Functional studies found enhanced IL‐8 secretion in CD133+ liver TICs to exhibit a greater ability to self‐renew, induce tumor angiogenesis, and initiate tumors. In further support of these observations, IL‐8 repression in CD133+ liver TICs by knockdown or neutralizing antibody abolished these effects. Subsequent studies of the IL‐8 functional network identified neurotensin (NTS) and CXCL1 to be preferentially expressed in CD133+ liver TICs. Addition of exogenous NTS resulted in concomitant up‐regulation of IL‐8 and CXCL1 with simultaneous activation of p‐ERK1/2 and RAF‐1, both key components of the mitogen‐activated protein kinase (MAPK) pathway. Enhanced IL‐8 secretion by CD133+ liver TICs can in turn activate an IL‐8‐dependent feedback loop that signals through the MAPK pathway. Further, in its role as a liver TIC marker CD133 also plays a functional part in regulating tumorigenesis of liver TICs by way of regulating NTS, IL‐8, CXCL1, and MAPK signaling. Conclusion: CD133+ liver TICs promote angiogenesis, tumorigenesis, and self‐renewal through NTS‐induced activation of the IL‐8 signaling cascade. (Hepatology 2012)

Upregulation of Twist in oesophageal squamous cell carcinoma is associated with neoplastic transformation and distant metastasis

Background: The antiapoptotic and epithelial–mesenchymal transition activities of Twist have been implicated in the neoplastic transformation and the development of metastasis, respectively. Upregulation of Twist, described in several types of human cancer, also acts as a prognostic marker of poor outcome. Aim: To investigate Twist expression in oesophageal squamous cell carcinoma (SCC) and its prognostic value in a Chinese cohort of patients with oesophageal SCC. Methods: Twist expression in primary oesophageal SCC of 87 Chinese patients was investigated by immunohistochemical staining. Twist protein level in one immortalised normal oesophageal epithelial cell line and six oesophageal SCC cell lines was measured by western blot analysis. Twist mRNA level in 30 pairs of frozen specimens of primary oesophageal SCC and non-neoplastic oesophageal epithelium from the upper resection margin of corresponding oesophagectomy specimen was also determined by semiquantitative reverse transcription-PCR. Results: It was found that Twist was upregulated in oesophageal SCC cell lines, and its mRNA and protein levels were both increased in oesophageal SCC and the non-neoplastic oesophageal epithelium (p<0.001). In addition, a high level of Twist expression in oesophageal SCC was significantly associated with a greater risk for the patient of developing distant metastasis within 1 year of oesophagectomy (OR 3.462, 95% CI 1.201 to 9.978; p = 0.022). Conclusions: Our results suggest that upregulation of Twist plays a role in the neoplastic transformation to oesophageal SCC and subsequent development of distant metastasis. Twist may serve as a useful prognostic marker for predicting the development of distant metastasis in oesophageal SCC.

Identification of PTK6, via RNA Sequencing Analysis, as a Suppressor of Esophageal Squamous Cell Carcinoma

BACKGROUND & AIMS Esophageal squamous cell carcinoma (ESCC) is the most commonly observed histologic subtype of esophageal cancer. ESCC is believed to develop via accumulation of numerous genetic alterations, including inactivation of tumor suppressor genes and activation of oncogenes. We searched for transcripts that were altered in human ESCC samples compared with nontumor tissues. METHODS We performed integrative transcriptome sequencing (RNA-Seq) analysis using ESCC samples from 3 patients and adjacent nontumor tissues to identify transcripts that were altered in ESCC tissue. We performed molecular and functional studies of the transcripts identified and investigated the mechanisms of alteration. RESULTS We identified protein tyrosine kinase 6 (PTK6) as a transcript that was significantly down-regulated in ESCC tissues and cell lines compared with nontumor tissues or immortalized normal esophageal cell lines. The promoter of the PTK6 gene was inactivated in ESCC tissues at least in part via hypermethylation and histone deacetylation. Knockdown of PTK6 in KYSE30 ESCC cells using small hairpin RNAs increased their ability to form foci, migrate, and invade extracellular matrix in culture and form tumors in nude mice. Overexpression of PTK6 in these cells reduced their proliferation in culture and tumor formation in mice. PTK6 reduced phosphorylation of Akt and glycogen synthase kinase (GSK)3β, leading to activation of β-catenin. CONCLUSIONS PTK6 was identified as a transcript that is down-regulated in human ESCC tissues via epigenetic modification at the PTK6 locus. Its product appears to regulate cell proliferation by reducing phosphorylation of Akt and GSK3β, leading to activation of β-catenin. Reduced levels of PTK6 promote growth of xenograft tumors in mice; it might be developed as a marker of ESCC.

Octamer 4/microRNA-1246 signaling axis drives Wnt/β-catenin activation in liver cancer stem cells

Wnt/β‐catenin signaling is activated in CD133 liver cancer stem cells (CSCs), a subset of cells known to be a root of tumor recurrence and therapy resistance in hepatocellular carcinoma (HCC). However, the regulatory mechanism of this pathway in CSCs remains unclear. Here, we show that human microRNA (miRNA), miR‐1246, promotes cancer stemness, including self‐renewal, drug resistance, tumorigencity, and metastasis, by activation of the Wnt/β‐catenin pathway through suppressing the expression of AXIN2 and glycogen synthase kinase 3β (GSK3β), two key members of the β‐catenin destruction complex. Clinically, high endogenous and circulating miR‐1246 was identified in HCC clinical samples and correlated with a worse prognosis. Further functional analysis identified octamer 4 (Oct4) to be the direct upstream regulator of miR‐1246, which cooperatively drive β‐catenin activation in liver CSCs. Conclusion: These findings uncover the noncanonical regulation of Wnt/β‐catenin in liver CSCs by the Oct4/miR‐1246 signaling axis, and also provide a novel diagnostic marker as well as therapeutic intervention for HCC. (Hepatology 2016;64:2062‐2076).

ANXA3/JNK signaling promotes self-renewal and tumor growth, and its blockade provides a therapeutic target for hepatocellular carcinoma

Summary Frequent tumor relapse in hepatocellular carcinoma (HCC) has been commonly attributed to the presence of residual cancer stem cells (CSCs) after conventional treatments. We have previously identified and characterized CD133 to mark a specific CSC subset in HCC. In the present study, we found endogenous and secretory annexin A3 (ANXA3) to play pivotal roles in promoting cancer and stem cell-like features in CD133+ liver CSCs through a dysregulated JNK pathway. Blockade of ANXA3 with an anti-ANXA3 monoclonal antibody in vitro as well as in human HCC xenograft models resulted in a significant reduction in tumor growth and self-renewal. Clinically, ANXA3 expression in HCC patient sera closely associated with aggressive clinical features. Our results suggest that ANXA3 can serve as a novel diagnostic biomarker and that the inhibition of ANXA3 may be a viable therapeutic option for the treatment of CD133+ liver-CSC-driven HCC.

Trehalose, an mTOR Independent Autophagy Inducer, Alleviates Human Podocyte Injury after Puromycin Aminonucleoside Treatment

Glomerular diseases are commonly characterized by podocyte injury including apoptosis, actin cytoskeleton rearrangement and detachment. However, the strategies for preventing podocyte damage remain insufficient. Recently autophagy has been regarded as a vital cytoprotective mechanism for keeping podocyte homeostasis. Thus, it is reasonable to utilize this mechanism to attenuate podocyte injury. Trehalose, a natural disaccharide, is an mTOR independent autophagy inducer. It is unclear whether trehalose alleviates podocyte injury. Therefore, we investigated the efficacy of trehalose in puromycin aminonucleoside (PAN)-treated podocytes which mimic cell damage in minimal change nephrotic syndrome in vitro. Human conditional immortalized podocytes were treated with trehalose with or without PAN. Autophagy was investigated by immunofluorescence staining for LC3 puncta and Western blotting for LC3, Atg5, p-AMPK, p-mTOR and its substrates. Podocyte apoptosis and necrosis were evaluated by flow cytometry and by measuring lactate dehydrogenase activity respectively. We also performed migration assay to examine podocyte recovery. It was shown that trehalose induced podocyte autophagy in an mTOR independent manner and without reactive oxygen species involvement. Podocyte apoptosis significantly decreased after trehalose treatment, while the inhibition of trehalose-induced autophagy abolished its protective effect. Additionally, the disrupted actin cytoskeleton of podocytes was partially reversed by trehalose, accompanying with less lamellipodias and diminished motility. These results suggested that trehalose induced autophagy in human podocytes and showed cytoprotective effects in PAN-treated podocytes.

Overexpression of microRNA-1288 in oesophageal squamous cell carcinoma

PURPOSE This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. METHODS In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS was examined using immunofluorescence and western blot. RESULTS Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. CONCLUSIONS Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC.

Neuropilin-2 promotes tumourigenicity and metastasis in oesophageal squamous cell carcinoma through ERK-MAPK-ETV4-MMP-E-cadherin deregulation.

Oesophageal squamous cell carcinoma (ESCC) is the most common histological subtype of oesophageal cancer. The disease is particularly prevalent in southern China. The incidence of the disease is on the rise and its overall survival rate remains dismal. Identification and characterization of better molecular markers for early detection and therapeutic targeting are urgently needed. Here, we report levels of transmembrane and soluble neuropilin‐2 (NRP2) to be significantly up‐regulated in ESCC, and to correlate positively with advanced tumour stage, lymph node metastasis, less favourable R category and worse overall patient survival. NRP2 up‐regulation in ESCC was in part a result of gene amplification at chromosome 2q. NRP2 overexpression promoted clonogenicity, angiogenesis and metastasis in ESCC in vitro, while NRP2 silencing by lentiviral knockdown or neutralizing antibody resulted in a contrary effect. This observation was extended in vivo in animal models of subcutaneous tumourigenicity and tail vein metastasis. Mechanistically, overexpression of NRP2 induced expression of ERK MAP kinase and the transcription factor ETV4, leading to enhanced MMP‐2 and MMP‐9 activity and, as a consequence, suppression of E‐cadherin. In summary, NRP2 promotes tumourigenesis and metastasis in ESCC through deregulation of ERK–MAPK–ETV4–MMP–E‐cadherin signalling. NRP2 represents a potential diagnostic or prognostic biomarker and therapeutic target for ESCC. Copyright

MiR-498 in esophageal squamous cell carcinoma: clinicopathological impacts and functional interactions

MicroRNA-498 plays a crucial role in progression of many carcinomas. The signaling pathways by which miR-498 modulates carcinogenesis are still unknown. Also, miR-498-associated molecular pathogenesis has never been studied in esophageal squamous cell carcinoma (ESCC). Herein, we aimed to examine the expression and functional roles of miR-498 in ESCC as well as its influences on the clinicopathological features in patients with ESCC. Expression of miR-498 was investigated in 93 ESCC tissues and 5 ESCC cell lines using quantitative real-time polymerase chain reaction. In vitro effects of miR-498 on cellular process were studied followed by overexpression of miR-498. Western blot and immunofluorescence techniques were used to identify the interacting targets for miR-498 in ESCC. miR-498 expression was significantly reduced in ESCC when compared with the nonneoplastic esophageal tissues (P<.05). Patients with low miR-498 expression showed different histological grading of cancer and survival rates when compared with the patients with high miR-498 expression. Overexpression of miR-498 in ESCC cell lines induced remarkable reductions of cell proliferation, barrier penetration, and colony formation when compared with control and wild-type counterparts. Also, miR-498 activated the FOXO1/KLF6 transcriptional axis in ESCC. In addition, miR-498 overexpression increased p21 protein expression and led to reduced cancer cell growth. To conclude, reduced expression of miR-498 in ESCC and in vitro analysis have confirmed the tumor suppressor properties of miR-498 by modulating the FOXO1/KLF6 signaling pathway. The changes in miR-498 expression may have impacts on the clinical pathological parameters of ESCC as well as in the management of the patients with ESCC.