Kwan Ho Tang

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)

CHD1L promotes hepatocellular carcinoma progression and metastasis in mice and is associated with these processes in human patients.

Chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) is a recently identified oncogene localized at 1q21, a frequently amplified region in hepatocellular carcinoma (HCC). To explore its oncogenic mechanisms, we set out to identify CHD1L-regulated genes using a chromatin immunoprecipitation-based (ChIP-based) cloning strategy in a human HCC cell line. We then further characterized 1 identified gene, ARHGEF9, which encodes a specific guanine nucleotide exchange factor (GEF) for the Rho small GTPase Cdc42. Overexpression of ARHGEF9 was detected in approximately half the human HCC samples analyzed and positively correlated with CHD1L overexpression. In vitro and in vivo functional studies in mice showed that CHD1L contributed to tumor cell migration, invasion, and metastasis by increasing cell motility and inducing filopodia formation and epithelial-mesenchymal transition (EMT) via ARHGEF9-mediated Cdc42 activation. Silencing ARHGEF9 expression by RNAi effectively abolished the invasive and metastatic abilities of CHD1L in mice. Furthermore, investigation of clinical HCC specimens showed that CHD1L and ARHGEF9 were markedly overexpressed in metastatic HCC tissue compared with healthy tissue. Increased expression of CHD1L was often observed at the invasive front of HCC tumors and correlated with venous infiltration, microsatellite tumor nodule formation, and poor disease-free survival. These findings suggest that CHD1L-ARHGEF9-Cdc42-EMT might be a novel pathway involved in HCC progression and metastasis.

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.

Lupeol targets liver tumor-initiating cells through phosphatase and tensin homolog modulation†

Liver tumor‐initiating cells (T‐ICs) are capable of self‐renewal and tumor initiation and are more chemoresistant to chemotherapeutic drugs. The current therapeutic strategies for targeting stem cell self‐renewal pathways therefore represent rational approaches for cancer prevention and treatment. In the present study, we found that Lup‐20(29)‐en‐3β‐ol (lupeol), a triterpene found in fruits and vegetables, inhibited the self‐renewal ability of liver T‐ICs present in both hepatocellular carcinoma (HCC) cell lines and clinical HCC samples, as reflected by hepatosphere formation. Furthermore, lupeol inhibited in vivo tumorigenicity in nude mice and down‐regulated CD133 expression, which was previously shown to be a T‐IC marker for HCC. In addition, lupeol sensitized HCC cells to chemotherapeutic agents through the phosphatase and tensin homolog (PTEN)–Akt–ABCG2 pathway. PTEN plays a crucial role in the self‐renewal and chemoresistance of liver T‐ICs; down‐regulation of PTEN by a lentiviral‐based approach reversed the effect of lupeol on liver T‐ICs. Using an in vivo chemoresistant HCC tumor model, lupeol dramatically decreased the tumor volumes of MHCC‐LM3 HCC cell line‐derived xenografts, and the effect was equivalent to that of combined cisplatin and doxorubicin treatment. Lupeol exerted a synergistic effect without any adverse effects on body weight when combined with chemotherapeutic drugs. Conclusion: Our results suggest that lupeol may be an effective dietary phytochemical that targets liver T‐ICs. (HEPATOLOGY 2011.)

Liver Tumor-Initiating Cells/Cancer Stem Cells: Past Studies, Current Status, and Future Perspectives

In the past decade, the hypothesis of tumor-initiating cell (TIC) or cancer stem cell (CSC)-driven tumorigenesis has been widely acknowledged in both hematological malignancies as well as solid epithelial tumors. In this chapter, we will first review in detail the current scientific knowledge in CSC research in hepatocellular carcinoma (HCC) and the molecular machinery underlying CSC-driven hepatocarcinogenesis. We will then discuss the relevance of liver cancer stem cells to the diagnosis and treatment of the disease and finally, consider the outstanding challenges and potential opportunities that remain ahead of us.

Abstract 2445: CD24+ liver tumor-initiating cells drives self-renewal and tumor initiation through Stat3-mediated Nanog regulation

Tumor-initiating cells (TICs) are a subpopulation of chemoresistant tumor cells that have been shown clinically to cause tumor recurrence upon chemotherapy. Identification of T-ICs and their related pathways are therefore priorities for the development of novel therapeutic paradigms. We established chemoresistant hepatocellular carcinoma (HCC) xenograft tumors in immunocompromised mice in which an enriched T-IC population was capable of tumor initiation and self-renewal. Using this model, we found CD24 to be up-regulated in residual chemoresistant tumor tissue when compared with bulk tumor upon cisplatin treatment. The determine whether CD24 is a candidate TIC marker for HCC, cell sorting approach was employed to separate CD24-/CD24+ populations derived from HCC cell lines and clinical samples. CD24 + HCC cells were found to be critical for the maintenance of tumor growth, self-renewal, differentiation, chemoresistance and metastasis of tumors. Using quantitative PCR, CD24 was over-expressed in HCCs when compared with their non-tumor counterparts, and CD24 expression was significantly correlated with poor patients’ survival. Using a lentiviral-based knockdown approach, CD24 was found to be a functional liver T-IC marker that drives tumor initiation and self-renewal. Notably, whether by cell sorting or gene-knockdown approach, it was shown by quantitative PCR that the CD24-enriched population consistently overexpressed Nanog. Upon transfection of Nanog cDNA into CD24-knockdown cells, self-renewal and tumor formation were functionally recovered, suggesting Nanog as the downstream effecter of CD24. By Ingenuity Pathway analysis, ‘acute phase response signaling’ was found to be the most significantly altered upon CD24 knockdown in which phosphorylation of Stat3 at the Tyrosine705 residue is critical. Using Stat3 inhibitor (S3I-201), we found that Nanog promoter and its protein were down-regulated in dose-dependent manner in CD24+ HCC cells. CD24 was found to be a functional liver T-IC marker that drives T-IC genesis through Stat3-mediated Nanog regulation. In conclusion, we identified CD24 + HCC cells within bulky tumor, and they functioned to initiate tumor growth and self-renewal through Stat3-mediated Nanog up-regulation. The identification of novel CD24 signaling pathways provides an attractive therapeutic strategy against this deadly disease. 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 2445. doi:10.1158/1538-7445.AM2011-2445

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 LB-129: Loss of Rab25 expression promotes tumor formation and angiogenesis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide and is particularly prevalent in some areas of Asian countries. With the advent of next-generation sequencing technologies, it is now possible for us to integrate and analyze large amounts of data at genome-wide scale, with the goal for a better understanding of cellular function and disease development. In order to elucidate the molecular mechanisms that drive tumorigenesis in ESCC, we employed a high-throughput sequencing technology named RNA-Seq (RNA sequencing) to identify dysregulated gene expression in 14 patient-derived ESCC or adjacent non-tumor (NT) clinical specimens of Chinese origin. The expression of Rab25, a family member of Ras-associated binding GTPases, was found to be downregulated in tumour tissues significantly. By both qPCR and immunohistochemistry analysis on a tissue microarray, expression of Rab25 was found to be significantly decreased in a larger cohort of ESCC tumors compared with NT counterparts. Patients with higher levels of Rab25 expression were also significantly associated with a better overall survival. Consistently, absent or downregulated Rab25 expression was also detected in all ESCC cell lines examined as compared to a pooled normal tissue control. Rab25 expression in ESCC cell lines could be restored by demethylation treatment with 5-aza-dC, suggesting Rab25 is silenced via epigenetic alterations. Rab25 functions as an important mediator of vesicle trafficking, regulating cell polarity and distribution of membrane proteins, which are important for the maintenance of normal epithelial lining of the esophagus. We hypothesized that loss of Rab25 expression could lead to dysregulation in the maintenance of this normal epithelium and thus result in tumor development. The tumor suppressive function of Rab25 and the tumorigenic effect of its loss were evaluated by lentiviral-based overexpression and shRNA-knockdown of Rab25 in ESCC cell lines. In vivo studies found Rab25 to suppress tumor formation and angiogenesis in nude mice. Rab25-knockdwon cells gave rise to significantly more and larger tumors as compared to negative control cells. Immunohistochemical staining of resected xenografts revealed an increase in microvessel density and tumor proliferation as shown by higher expressions of endothelial marker CD34 and proliferation marker PCNA, respectively, in Rab25-knockdown cells as compared to negative controls. Similarly, conditioned medium from Rab25-knockdown cells enhanced tube-forming capability of HUVECs, suggesting potential regulation of angiogenic factors by Rab25-mediated pathway. Work on the identification of potential angiogenic factors regulated by Rab25 is now currently underway using a human angiogenesis antibody array. In summary, Rab25 functions as an important tumor suppressor gene in ESCC development by regulating angiogenesis. 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 LB-129. doi:10.1158/1538-7445.AM2011-LB-129