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 identifies liver TICs/CSCs and is a prognostic marker for HC ► miR-130b promotes CD133
+ liver TICs growth and self renewal ► miR-130b regulates CD133
+ liver TICs via silencing TP53INP1
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.)
Hepatocellular carcinoma (HCC) is an aggressive tumour with a poor prognosis. Current therapeutic strategies against this disease target mostly rapidly growing differentiated tumour cells. However, ...the result is often dismal due to the chemoresistant nature of this tumour type. Recent research efforts on stem cells and cancer biology have shed light on new directions for the eradication of cancer stem cells (CSCs) in HCC. The liver is a distinctive organ with the ability of tissue renewal in response to injury. Based on the hypothesis that cancer development is derived from the hierarchy of the stem cell system, we will briefly discuss the origin of liver stem cells and its relation to HCC development. We will also summarize the current CSC markers in HCC and discuss their relevance to the treatment of this deadly disease.
Tumor-initiating cells (T-ICs) are a subpopulation of chemoresistant tumor cells that have been shown to cause tumor recurrence upon chemotherapy. Identification of T-ICs and their related pathways ...are therefore priorities for the development of new 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. With this model, we found CD24 to be upregulated in residual chemoresistant tumors when compared with bulk tumor upon cisplatin treatment. CD24
+ HCC cells were found to be critical for the maintenance, self-renewal, differentiation, and metastasis of tumors and to significantly impact patients' clinical outcome. With a lentiviral-based knockdown approach, CD24 was found to be a functional liver T-IC marker that drives T-IC genesis through STAT3-mediated NANOG regulation. Our findings point to a CD24 cascade in liver T-ICs that may provide an attractive therapeutic target for HCC patients.
► Enrichment of CD24
+ population in chemoresistant HCC xenograft model ► CD24
+ cells are able to initiate tumor, self-renew, differentiate, and metastasize ► Targeting CD24 attenuates characteristics of stem/progenitor in HCC cells ► CD24 drives T-IC genesis through Stat3-mediated Nanog regulation
Tumor-initiating cells (T-ICs) are a subpopulation of chemoresistant tumor cells that have been shown to cause tumor recurrence upon chemotherapy. Identification of T-ICs and their related pathways ...are therefore priorities for the development of new 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. With this model, we found CD24 to be upregulated in residual chemoresistant tumors when compared with bulk tumor upon cisplatin treatment. CD24 super(+ HCC cells were found to be critical for the maintenance, self-renewal, differentiation, and metastasis of tumors and to significantly impact patients' clinical outcome. With a lentiviral-based knockdown approach, CD24 was found to be a functional liver T-IC marker that drives T-IC genesis through STAT3-mediated NANOG regulation. Our findings point to a CD24 cascade in liver T-ICs that may provide an attractive therapeutic target for HCC patients.)
Abstract
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
Hepatocellular carcinoma (HCC) is a disease with poor prognosis attributed to the high recurrence rate post-resection. Chemotherapy is often ineffective due to chemoresistance, conferred by ...the presence of tumor-initiating cells (TICs). Hence, to improve outcome, it is imperative that markers and their pivotal pathways involved in tumor initiation.
Using HCC chemoresistant nude mice model, CD24 was found highly upregulated when compared to untreated tumor by cDNA microarray analysis. CD24 expression was first examined in HCC cell lines and human clinical HCC. CD24 expression across a HCC cell line panel ranged from 6.0% to 99.8%. Notably, no expression was detected in the non-tumorigenic hepatic cell line MIHA. Expression of CD24 was also found to represent only a minority (<1%) of the tumor cell population in 40% human HCC by immunohistochemistry. Using antibody-based magnetic and FACS-sorting respectively, CD24- and CD24+ tumor cells were isolated from three HCC patients and two HCC cell lines. The CD24+ HCC cells displayed the phenotype of self-renewing CSCs, including enhanced sphere-forming ability, higher anchorage-independent growth ability, capability of differentiation, and preferential expression of “stem-ness’ genes. Functional roles of CD24 in self-renewal and tumor initiation were further demonstrated by stable knockdown of CD24 expression in both HCC cell lines and clinical samples using lentiviral-based shRNA. When shCD24-Huh7 cells were injected subcutaneously into SCID mice, only 7/31 (22%) mice formed tumors, as compared to 20/31 (65%) tumors of significantly greater size, for controls. In addition, CD24-knockdown cells exhibited more rapid proliferation and chemo-sensitivity towards chemotherapeutic drugs. By quantitative PCR, CD24 mRNA was implicated in poor prognosis of 36 clinical HCC samples including tumor stages (p=0.024) and tumor recurrence(p=0.047).
Notably, whether by cell sorting or gene-knockdown approach, it was shown by quantitative PCR and western blotting that CD24 and Nanog, a gene important for the self-renewal of embryonic stem cells, were co-expressed in HCC cells. We therefore hypothesize that the functional role of CD24 in liver tumor initiation is mediated through regulation of Nanog expression. Upon transfection of Nanog cDNA into CD24-knockdown cells, self-renewal and tumor formation were functionally recovered, suggesting Nanog as the downstream effector of CD24. Moreover, CD24 acts through nanog via phosphorylation of Stat3, which is the main upstream regulator of Nanog in embryonic stem (ES) cells
Findings from this study indicate the role of CD24 as a marker of chemoresistant TICs in HCC, and introduce a novel mechanism through which tumor initiation may be effected by this marker. This opens the window towards further studies on HCC TICs, which may have important ramifications in future therapeutics against this deadly disease.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4314.
Abstract
Metastasis is the major cause of death for cancer patients. Understanding the molecular mechanism of tumor metastasis is urgently awaited. Using hepatocellular carcinoma as our cancer model, ...the mechanism of tumor metastasis was studied by establishment of a pair of primary and its corresponding metastatic lung counterparts (PLC-PT and PLC-LM) by orthotopic injection of parental PLC into the liver of SCID mice. These two cell lines share the same genetic background but differ in invasive ability evidenced by invasion and wound healing assay. Since protein kinases and their phosphorylated substrates play key role in signalling process leading to cancer metastasis, we employed Celluspot™ Serine/Threonine peptide array to evaluate the phosphorylation profiling between these two matched HCC cell lines. Phosphorylation profiling revealed significant higher phosphorylated level of nucleophosmin (NPM) at Threonine 234 in PLC-LM than PLC-PT, which was phosphorylated by cyclin-dependent kinase (CDK1). Western blot analysis confirmed elevated phosphorylated level of NPM at Threonine 234 (Thr234) in PLC-LM using phospho-NPM (T234) antibody. Consistent NPM (Thr234) over-expression was also found in higher metastatic HCC cell line (MHCC-97H) when compared with its corresponding primary one (MHCC-97L), further suggesting the role of NPM (Thr234) in HCC metastasis. The role of NPM (Thr234) was further evaluated in 30 matched primary and metastatic HCC clinical samples by immunohistochemistry. Over-expression (24/30) of NPM (Thr234) was significantly correlated with HCC metastasis (p<0.001). Using a tissue microarray consisting of 31 cases of primary and their metastatic colorectal cancer, the role of NPM (Thr234) in metastasis was further evaluated in colorectal cancer. Tissue microarray revealed significant correlation between NPM (Thr234) over-expression (20/31) with tumor metastasis (p<0.001). Using immunohistochemical approach, prognostic significance of NPM (Thr234) was examined in 61 primary tissues derived from patients with prostate cancer. Interestingly, NPM (Thr234) over-expression not only correlated with tumor metastasis (p<0.001) but also with poor patients’ survival (5.6 months Vs 54.9 months for low and high NPM (Thr234) expression). Functional role of NPM (Thr234) in tumor invasion was examined by creation of non-phosphorylated mutant of NPM. Lentiviral-based transfection of NPM (Thr234) to Ala mutant in PLC-LM inhibited cell migration and invasion when compared with wild-type NPM and empty vector control. Wild-type NPM was found to physically interact with a novel metastatic gene, Rho-kinase II, thereby altering its transcriptional activity. The physical interaction between NPM (Thr234) and Rho-kinase II is being investigated. In conclusion, NPM (Thr234) is a novel inducer of metastasis and may be a novel phsophorylated substrate for prognosis and therapeutic intervention for cancer patients.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3396.
This study reports on investigation of the magnetic properties of layer-by-layer (LbL) assembled nanofilms comprising polyvinyl alcohol (PVA) and citrate-coated magnetite (cit-MAG) nanoparticles ...deposited onto silicon (SF sample) and glass (GF sample) substrates. DC magnetization measurements were performed over the temperature range of 4 K to 300 K, in the applied magnetic field range of ±60 kOe. The magnetic data of the as-synthesized cit-MAG nanoparticles (F sample) are also collected for comparison. The three as-fabricated samples reveal perfect superparamagnetic (SPM) behavior only around room temperature; at temperatures lower than 200 K the SPM scaling is not observed and all samples behave as interacting superparamagnetic (ISPM) materials. The evolution from the ISPM to the SPM regime is marked by a steady decrease in the hysteretic properties of all samples, with the temperature-dependence of the coercivity decreasing slower than the T
behavior predicted for non-interacting superparamagnetic particles. The modified Bloch's law used to assess information on nanoparticles' surface spins gives the Bloch's exponent close to 2 (for the F and SF samples) and close to 1 (for the GF sample). Interestingly, the surface spin freezing temperature (T
) is 8 ± 1 K for all samples. The magnetic behavior of all three samples can be described within the model picture of a core-shell structure for the cit-MAG nanoparticles; the core comprising magnetically-ordered spins whereas the shell behaving as a spin-glass-like system. However, the contribution of the shell magnetism to the effective magnetic properties is much more evident in the GF sample in which magnetic dipole-dipole interaction is three-times weaker than in the SF sample and two times weaker than in the F sample. In contrast, the strong magnetic dipole-dipole interaction in the SF sample affects the surface spins, hindering the onset of magnetically-ordered regions in the nanoparticle's shell, making the surface magnetism contribution negligible. The LbL-fabricated nanofilms herein reported and the presented analysis of their magnetic properties we envisage can support the engineering of magnetic nanofilms for multiple applications.
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