Pak Shing Kwan

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)

A CD90+ Tumor-Initiating Cell Population with an Aggressive Signature and Metastatic Capacity in Esophageal Cancer

Tumor-initiating cells (TIC), also known as cancer stem cells, are regarded widely as a specific subpopulation of cells needed for cancer initiation and progression. TICs have yet to be identified in esophageal tumors that have an increasing incidence in developed countries. Here, we report a CD90(+) cell population found in esophageal squamous cell carcinoma (ESCC), which is endowed with stem cell-like properties and high tumorigenic and metastatic potential. mRNA profiling of these cells suggested pathways through which they drive tumor growth and metastasis, with deregulation of an Ets-1/MMP signaling pathway and epithelial-mesenchymal transition figuring prominently. These cells possessed higher self-renewal activity and were sufficient for tumor growth, differentiation, metastasis, and chemotherapeutic resistance. CD90(+) TICs were isolated and characterized from ESCC clinical specimens as well as ESCC cell lines. In freshly resected clinical specimens, they represented a rare cell population, the levels of which correlated with strong family histories and lymph node metastasis. Our results prompt further study of this CD90(+) population of esophageal TICs as potential therapeutic targets.

Rab25 Is a Tumor Suppressor Gene with Antiangiogenic and Anti-Invasive Activities in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is a devastating disease characterized by distinctly high incidences and mortality rates. However, there remains limited understanding of molecular events leading to development and progression of the disease, which are of paramount importance to defining biomarkers for diagnosis, prognosis, and personalized treatment. By high-throughout transcriptome sequence profiling of nontumor and ESCC clinical samples, we identified a subset of significantly differentially expressed genes involved in integrin signaling. The Rab25 gene implicated in endocytic recycling of integrins was the only gene in this group significantly downregulated, and its downregulation was confirmed as a frequent event in a second larger cohort of ESCC tumor specimens by quantitative real-time PCR and immunohistochemical analyses. Reduced expression of Rab25 correlated with decreased overall survival and was also documented in ESCC cell lines compared with pooled normal tissues. Demethylation treatment and bisulfite genomic sequencing analyses revealed that downregulation of Rab25 expression in both ESCC cell lines and clinical samples was associated with promoter hypermethylation. Functional studies using lentiviral-based overexpression and suppression systems lent direct support of Rab25 to function as an important tumor suppressor with both anti-invasive and -angiogenic abilities, through a deregulated FAK-Raf-MEK1/2-ERK signaling pathway. Further characterization of Rab25 may provide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target in ESCC treatment.

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.

Daxx regulates mitotic progression and prostate cancer predisposition

Mitotic progression of mammalian cells is tightly regulated by the E3 ubiquitin ligase anaphase promoting complex (APC)/C. Deregulation of APC/C is frequently observed in cancer cells and is suggested to contribute to chromosome instability and cancer predisposition. In this study, we identified Daxx as a novel APC/C inhibitor frequently overexpressed in prostate cancer. Daxx interacts with the APC/C coactivators Cdc20 and Cdh1 in vivo, with the binding of Cdc20 dependent on the consensus destruction boxes near the N-terminal of the Daxx protein. Ectopic expression of Daxx, but not the D-box deleted mutant (DaxxΔD-box), inhibited the degradation of APC/Cdc20 and APC/Cdh1 substrates, leading to a transient delay in mitotic progression. Daxx is frequently upregulated in prostate cancer tissues; the expression level positively correlated with the Gleason score and disease metastasis (P = 0.027 and 0.032, respectively). Furthermore, ectopic expression of Daxx in a non-malignant prostate epithelial cell line induced polyploidy under mitotic stress. Our data suggest that Daxx may function as a novel APC/C inhibitor, which promotes chromosome instability during prostate cancer development.

Abstract LB-53: Regulatory role of miR-142-3p on the functional hepatic cancer stem cell marker CD133

Tumor relapse after therapy typifies hepatocellular carcinoma (HCC) and is believed to be attributable to residual cancer stem cells (CSCs) that survive treatment. We have previously identified a CSC population derived from HCC that is characterized by CD133. Despite our growing knowledge of the importance of this subset of cells in driving HCC, the regulatory mechanism of CD133 is not known. Epigenetic changes are believed to be essential in the control of cancer and stem cells. Here, we report the epigenetic regulation of CD133 by miR-142-3p. The interaction between CD133 and miR-142-3p was identified by in silico prediction and substantiated by luciferase reporter analysis. Expression of CD133 was found to be inversely correlated with miR-142-3p in HCC clinical samples as well as in cell lines. Importantly, lower miR-142-3p expression in HCC was significantly associated with worst survival. Functional studies with miR-142-3p stably transduced in HCC cells demonstrated a diminished ability to self-renew, initiate tumor growth, invade, migrate, induce angiogenesis and resist chemotherapy. Rescue experiments whereby CD133 and miR-142-3p is simultaneously overexpressed compensated the deregulated ability of the cells to confer these features. Thus, miR-142-3p directly targets CD133 to regulate its ability to confer cancer and stem cell-like features in HCC.

Abstract 3023: RNA-Seq identifies neuropilin-2 as a novel oncogene in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is one of the most common and deadliest cancers in the world, with survival rates of less than 15%. Like many other solid tumors, the pathogenesis of ESCC is believed to be a multistep process with accumulation of numerous genetic alterations involving de-regulation of both tumor-suppressor genes and oncogenes. An understanding of the molecular events that result in the development and progression of the disease may lead to treatments that will increase survival rates of ESCC patients. Recent advances in sequencing technologies offer the opportunity to characterize the cancer genome at unprecedented depth and sensitivity. RNA-Seq has proved an effective means of precisely quantifying the changing expression levels of each transcript. In this study, we performed integrative RNA-Seq analysis on 3 paired patient-derived non-tumor and ESCC clinical specimens and discovered a number of commonly differentially expressed genes, of which included a significantly de-regulated neuropilin (NRP) family including neuropilin-1 (NRP1) and neuropilin-2 (NRP2). NRP is a single-spanned membrane glycoprotein. Members of the family have previously been shown to play an important role in various cancer types and to be a co-receptor of vascular endothelial growth factors. Yet to date, its role in ESCC has not been explored. Up-regulation of both NRP1 and NRP2 in ESCC was subsequently validated in the original 3 pairs of clinical samples used for RNA-Seq as well as in a larger cohort of samples (n = 40) by qPCR. Given that NRP2 de-regulation in ESCC is more significant, we chose to first focus our study on this member of the family. Immunohistochemical studies likewise found NRP2 to be up-regulated in ESCC as compared with its non-tumor counterparts in a tissue microarray. Consistently, expression studies in a panel of esophageal cell lines found NRP2 to be expressed at a significantly higher level in 7 of the 8 ESCC cell lines examined compared with immortalized normal esophageal cell lines, NE1 and NE3. Functional studies in ESCC cells, KYSE180 and KYSE140, with NRP2 stably repressed by lentiviral-based shRNA approach impaired proliferation and metastatic abilities in vitro, as demonstrated by foci formation and chemotaxis migration assays, respectively. Moreover, conditioned medium collected from NRP2 repressed cells displayed a reduced ability to induce capillary tube formation in HUVEC cells. These results suggest that NRP2 plays an important role in regulating cell proliferation, migration and angiogenesis in ESCC. Additional work on the mechanism by which NRP2 drives ESCC and the effectiveness of the use of a NRP2 neutralizing antibody against ESCC is currently being studied. We believe with further study, NRP2 may provide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target for ESCC treatment. Citation Format: Tsun Ming Fung, Man Tong, Pak Shing Kwan, Yuen Piu Chan, Xin-Yuan Guan, Stephanie Ma. RNA-Seq identifies neuropilin-2 as a novel oncogene in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2013-3023

Abstract 2105: RNA-seq identifies protein tyrosine kinase 6 (PTK6) as a candidate tumor-suppressor gene in esophageal squamous cell carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is one of the most common and deadliest cancers in the world, with survival rates of less than 15%. Like many other solid tumors, the pathogenesis of ESCC is also believed to be a multistep process with accumulation of numerous genetic alterations involving inactivation of tumor-suppressor genes and activation of oncogenes. An understanding of the molecular events that result in the development and progression of the disease may lead to treatments that will increase survival rates of ESCC patients. Recent advances in sequencing technologies offer the opportunity to characterize the cancer genome at unprecedented depth and sensitivity. Large-scale transcriptome sequencing (RNA-Seq) has proved an effective means of precisely quantifying the changing expression levels of each transcript. In this study, we performed integrative RNA-Seq analysis on 14 patient-derived ESCC clinical specimens (4 paired ESCC and their adjacent non-tumorous (NT) counterparts, 1 unpaired NT and 5 unpaired ESCC) and discovered a number of commonly differentially expressed genes in ESCC compared with NT tissues. A total of 526 genes were found to be significantly aberrantly expressed, including 452 genes that were up-regulated and 74 genes that were down-regulated. One candidate tumor suppressor gene, protein tyrosine kinase 6 (PTK6), was chosen for further characterization. By both quantitative real-time PCR and immunohistochemistry analysis, PTK6 was found to be significantly down-regulated in a larger cohort of ESCC tumors compared with NT counterparts. Consistently, expression studies in a panel of esophageal cell lines found PTK6 to be either absent or expressed in low amounts in all eight ESCC cell lines examined compared with two immortalized normal esophageal cell lines, NE1 and NE3. Down-regulation of PTK6 in both ESCC cell lines and clinical samples was found to be significantly associated with promoter hypermethylation, as evident by results obtained from studies involving demethylation treatment with 5-aza-dC, methylation specific PCR as well as bisulfite genomic sequencing. Functional studies in ESCC cell lines, EC109 and KYSE30, with PTK6 stably repressed by lentiviral-based approach stimulated both in vitro and in vivo tumorigenicity ability of the cells including foci formation, colony formation in soft agar as well as tumor formation in nude mice. Taken together, our findings define a function for PTK6 as an important tumor suppressor gene in ESCC development. Additional work on the mechanism by which PTK6 drives ESCC is currently being studied in our laboratory. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2105. doi:10.1158/1538-7445.AM2011-2105

Abstract 2451: CD133+ liver tumor-initiating cells promote tumor growth, angiogenesis and self-renewal through activation of the neurotensin / interleukin-8 signaling cascade

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs). We have previously identified a TIC population from hepatocellular carcinoma (HCC) that is marked by a CD133 surface phenotype and bears features that include the abilities to self-renew, differentiate, initiate tumors in vivo and resist standard chemotherapy through AKT pathway dysregulation (Ma et al., Gastroenterology 2007; Oncogene 2008 and Cell Stem Cell in press). Our present study aims to investigate the underlying molecular mechanism by which CD133 + liver TICs mediate tumor growth and maintenance. cDNA microarray approach was employed to compare gene expression profiles between sorted CD133 subpopulations in HCC cell lines Huh7 and PLC8024. A dysregulated interleukin-8 (IL-8) signaling functional network was preferentially identified in CD133 + liver TICs. IL-8 was found to be overexpressed at both genomic and proteomic (endogenous and secretory) levels in CD133 + cells isolated from HCC cell lines and clinical samples; while its expression also correlated positively with CD133 expression in a panel of liver cell lines. Functional studies on sorted CD133 HCC cells found enhanced IL-8 secretion in CD133 + liver TICs to possess a greater ability to self-renew and induce tumor angiogenesis and growth in vitro and in vivo. These observations were further validated when silencing of IL-8 in CD133 + liver TICs by knockdown or neutralizing antibody approach led to abolishment of these effects. Subsequent studies in the IL-8 functional network also found neurotensin (NTS) and chemokine ligand 1 (CXCL1) to be preferentially expressed in CD133 + liver TICs. Their expression also correlated with that of CD133 and IL-8 across a panel of liver cell lines. Upon addition of exogenous NTS, both expression of IL-8 and CXCL1 was dramatically up-regulated, with concomitant activation of phospho-ERK1/2, a key player of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, enhanced IL-8 secretion in CD133 + liver TICs can in turn activate an IL-8 positive feedback loop through MAPK signaling. Lastly, we also provide evidence to show that the transmembrane protein, CD133, apart from serving as a liver TIC marker, also plays a critical functional role in conferring TIC properties. Stable repression of CD133 in CD133 + liver TICs by lentiviral based approach resulted in inactivation of the NTS / IL-8 / CXCL1 / MAPK signaling cascade and attenuation of TIC self-renewal and tumorigenic properties. Taken together, these results suggest that CD133 + liver TICs promote tumor growth, angiogenesis and self-renewal through activation of NTS / IL-8 signaling cascade. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2451. doi:10.1158/1538-7445.AM2011-2451