Jianxin Jiang

CD44+CD133+ population exhibits cancer stem cell-like characteristics in human gallbladder carcinoma

Cancer stem cells (CSCs)/tumor-initiating cells have been defined as a subset of tumor cells responsible for initiating and sustaining tumor development. Emerging evidence strongly supports the existence of CSCs in various solid tumors, but they have not yet been identified in human gallbladder carcinomas (GBC). In this study, we identified CSCs in primary GBC and in the cell line GBC-SD using the cell surface markers CD44 and CD133. The percentages of CD44+CD133+ cells were 1.76-3.05% in primary tumors and 40.29% in GBC-SD cells. These cells showed stem cell properties, including self-renewal, differentiation potential, and high tumorigenicity. In vitro culture experiments revealed that CD44+CD133+ GBC cells possessed a higher spheroid-colony forming ability in serum-free media than other subpopulations. When injected into nonobese diabetic/severe combined immunodeficient mice, these cells formed new tumors and generated CD44+CD133+, CD44-, and CD133- progeny. CD44+CD133+ cells also showed a high degree of chemoresistance, possibly due to upregulation of the breast cancer resistance protein (ABCG2) and the transcription factor Gli1 in these highly tumorigenic cells. These results suggest that the CD44+CD133+ population exhibited CSC-like characteristics and may thus provide a novel approach to the diagnosis and treatment of GBC.

Direct targeting of SUZ12/ROCK2 by miR-200b/c inhibits cholangiocarcinoma tumourigenesis and metastasis.

Background:The multidrug resistance and distant metastasis of cholangiocarcinoma result in high postoperative recurrence and low long-term survival rates. It has been demonstrated that the ectopic expression of miR-200 suppresses the multidrug resistance and metastasis of cancer. However, the expression and function of miR-200 in cholangiocarcinoma has not yet been described.Methods:In this study, we identified dysregulated microRNAs (miRNAs, miR) in cholangiocarcinoma tissue by microarray analysis, and subsequent real-time PCR and northern blot analyses validated the expression of candidate miR. We performed functional analyses and investigated the relationship between miR-200b/c expression and the properties of cholangiocarcinoma cells. A dual luciferase assay was applied to examine the effect of miRNAs on the 3′-UTR of target genes, and we demonstrated the function of the target gene by siRNA transfection identifying the downstream pathway via western blotting.Results:We found significantly downregulated expression of four miR-200 family members (miR-200a/b/c/429) and then confirmed that ectopic miR-200b/200c inhibits the migration and invasion of cholangiocarcinoma cells both in vitro and in vivo. We found that miR-200b/c influenced the tumourigenesis of cholangiocarcinoma cells including their tumour-initiating capacity, sphere formation, and drug resistance. We further found that miR-200b/c regulated migration and invasion capacities by directly targeting rho-kinase 2 and regulated tumorigenic properties by directly targeting SUZ12 (a subunit of a polycomb repressor complex).Conclusion:Our study shows that miR-200b/c has a critical role in the regulation of the tumorigenic and metastatic capacity of cholangiocarcinoma and reveals the probable underlying mechanisms.

Synergistic antitumor activity of withaferin A combined with oxaliplatin triggers reactive oxygen species-mediated inactivation of the PI3K/AKT pathway in human pancreatic cancer cells

Application of oxaliplatin for the treatment of pancreatic cancer (PC) is restricted owing to its toxic side effects and drug resistance. We investigated how withaferin A (WA), a bioactive component isolated from the medicinal plant Withania somnifera, acts synergistically with oxaliplatin on human PC in vitro and in vivo. We found that WA enhanced oxaliplatin-induced growth suppression and apoptosis in PC cells dramatically through a mechanism involving mitochondrial dysfunction and inactivation of the PI3K/AKT pathway. Combination treatment resulted in significant accumulation of intracellular reactive oxygen species (ROS). Pretreatment of cells with the ROS scavenger N-acetylcysteine completely blocked the apoptosis induced by combination treatment, and recovered expression of AKT inactivation, which revealed the important role of ROS in apoptosis and AKT regulation. In vivo, combination therapy showed the strongest anti-tumor effects compared with single agents, without obvious additional toxicity. These results support the notion that combination treatment with oxaliplatin and WA could facilitate development of an effective strategy for PC treatment.

MicroRNA-138-5p regulates pancreatic cancer cell growth through targeting FOXC1

PurposeThe prognosis of pancreatic cancer ranks among the worst of all cancer types, which is primarily due to the fact that during the past decades little progress has been made in its diagnosis and treatment. Here, we set out to investigate the role of microRNA 138 (miR-138-5p) in the regulation of pancreatic cancer cell growth and to assess its role as putative therapeutic target.MethodsqRT-PCR was used to examine the expression of miR-138-5p in 8 pancreatic cancer cell lines and 18 primary human pancreatic cancer samples. A lentivirual vector containing miR-138-5p mimics (lv-miR-138-5p) was used to exogenously over-express miR-138-5p in the pancreatic cancer cells lines Capan-2 and PANC-1. The effect of this over-expression on cell proliferation was examined using an in vitro propidium iodide fluorescence assay. Capan-2 cells exogenously over-expressing miR-138-5p were transplanted into nude mice to examine its in vivo effect on tumor growth. A predicted target of miR-138-5p (FOXC1) was first validated using a luciferase assay and, subsequently, down-regulated by siRNA to assess its effect on pancreatic cancer cell growth.ResultsWe found that miR-138-5p was markedly down-regulated in both pancreatic cancer cell lines and primary human pancreatic cancer samples, compared to a human pancreas ductal epithelial (HPDE) cell line and normal pancreatic tissues, respectively (P < 0.05). In addition, we found that in the pancreatic cancer cells lines Capan-2 and PANC-1 lentiviral transfection of miR-138-5p mimicked up-regulation of the endogenous expression of miR-138-5p and, concomitantly, inhibited cancer cell proliferation (P < 0.05). The exogenous over-expression of miR-138-5p also led to a significant inhibition of tumor formation in vivo. Using a luciferase assay, we found that miR-138-5p directly targets FOXC1. In conformity with this notion, we found that FOXC1 was down-regulated upon miR-138-5p over-expression in pancreatic cancer cells. Finally, we found that silencing of FOXC1 by siRNA had an inhibitory effect on pancreatic cancer cell growth.ConclusionsOur data indicate that miR-138-5p may play an important role in regulating pancreatic cancer cell growth, possibly through targeting FOXC1. Over-expression of miR-138-5p may serve as a novel approach for the treatment of patients with pancreatic cancer.

MiR-1181 inhibits stem cell-like phenotypes and suppresses SOX2 and STAT3 in human pancreatic cancer.

Recent studies have shown that cancer stem cells (CSCs) play an important role in the development of pancreatic cancer. Multiple oncogenes and signaling pathways have been confirmed to participate in the stemness maintenance and tumorigenicity of CSCs, including sex-determining region Y-box 2 (SOX2) and signal transduction and activation of transcription 3 (STAT3), which may provide novel therapeutic targets on pancreatic cancer. Here, we reported in pancreatic cancer tissues and cells that miR-1181 expression was markedly downregulated, and the low miR-1181 expression was associated with poorer overall survival and disease-free survival in pancreatic cancer patients. Furthermore, overexpression of miR-1181 inhibited, whereas downregulation of miR-1181 promoted, CSCs-like phenotypes in vitro and tumorigenicity in vivo in pancreatic cancer cells. Moreover, we demonstrated that miR-1181 directly suppressed SOX2 and STAT3 expression, resulting in downregulation of SOX2 and inhibition of the STAT3 pathway. Hence, our results suggest that miR-1181 plays a vital role in inhibiting the CSCs-like phenotypes in pancreatic cancer and might represent a potential target for anti-pancreatic cancer.

Upregulation of miR-181c contributes to chemoresistance in pancreatic cancer by inactivating the Hippo signaling pathway

The Hippo signaling pathway plays a crucial role in regulating tissue homeostasis, organ size, tumorigenesis and cancer chemoresistance when deregulated. Physiologically, the Hippo core kinase cassette that consists of mamma-lian STE20-like protein kinase 1/2 (MST1/2), and large tumour suppressor 1/2 (LATS1/2), together with the adaptor proteins Salvador homologue 1 (SAV1) and MOB kinase activator 1 (MOB1), tightly restricts the activities of homologous oncoproteins Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) to low levels. However, how the Hippo kinase cassette core components are simultaneously inhibited, to exhibit constitutively inactivated Hippo signaling and activated YAP/TAZ in cancer remains puzzling. Herein, we reported that miR-181c directly repressed MST1, LATS2, MOB1 and SAV1 expression in human pancreatic cancer cells. Overexpression of miR-181c induced hyperactivation of the YAP/TAZ and enhanced expression of the Hippo signaling downstream genes CTGF, BIRC5 and BLC2L1, leading to pancreatic cancer cell survival and chemoresistance in vitro and in vivo. Importantly, high miR-181c levels were significantly correlated with Hippo signaling inactivation in pancreatic cancer samples, and predicted a poor patient overall survival. These findings provide a novel mechanism for Hippo signaling inactivation in cancer, indicating not only a potentially pivotal role for miR-181c in the progression of pancreatic cancer, but also may represent a new therapeutic target and prognostic marker.

Balanced Tiam1-rac1 and RhoA drives proliferation and invasion of pancreatic cancer cells.

Tiam1 is a rac1-specific guanine nucleotide exchange factor, and Tiam1-rac1 is involved in a number of cellular processes. Rac1 and RhoA act as molecular switches that cycle between GTP- and GDP-bound states to balance the activities of rac1 and RhoA. The downregulation of rac1 activity leads to upregulation of RhoA activity, which promotes invasion and migration of pancreatic cancers cells. At present, however, the role of Tiam1-rac1 and RhoA in pancreatic cancers is not fully understood. We found that Tiam1 was upregulated in pancreatic cancers and was significantly expressed in tumors without lymph node involvement or distant metastasis compared with cancers where there was involvement. Although Tiam1-rac1 signaling promoted pancreatic cancer cell proliferation and tumor growth via the Wnt signaling pathway in vitro and in vivo, inhibiting Tiam1-rac1 signaling did not prolong the overall survival time in vivo. This provided evidence that there was a balance between rac1 and RhoA activities in pancreatic cancers. Furthermore, only the combined inhibition of Tiam1-rac1 and RhoA had a beneficial effect on the growth of pancreatic cancers in vivo. Taken together, these results suggest that the progression of pancreatic tumors is partially controlled by the balance between Tiam1-rac1 and RhoA. Mol Cancer Res; 11(3); 230–9. ©2012 AACR.

The promotion of salinomycin delivery to hepatocellular carcinoma cells through EGFR and CD133 aptamers conjugation by PLGA nanoparticles

AIMS To develop salinomycin-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with both CD133 aptamers A15 and EGFR aptamers CL4 (CESN), to target hepatocellular carcinoma (HCC) cells simultaneously expressing EGFR and CD133. MATERIALS & METHODS The antitumor activity and mechanism of CESN were investigated. RESULTS & CONCLUSION The cytotoxicity of CESN in HCC cells and CD133(+) HCC cells was superior to that of A15 or CL4-conjugted or nontargeted salinomycin-loaded nanoparticles. The antitumor assay in mice bearing HCC xenograft tumors confirmed the superior antitumor activity of CESN over other controls. We speculated that the improved therapeutic effect of CESN may be attributed to both targeting a higher percentage of HCC cells and increased delivery of salinomycin to HCC cells.

MicroRNA-100 regulates pancreatic cancer cells growth and sensitivity to chemotherapy through targeting FGFR3

We intended to investigate the role of microRNA 100 (miR-100) in regulating pancreatic cancer cells’ growth in vitro and tumor development in vivo. QTR-PCR was used to examine the expression of miR-100 in pancreatic cancer cell lines and tumor cells from human patients. Lentivirual vector containing miR-100 mimics (lv-miR-100) was used to overexpress miR-100 in MIA PaCa-2 and FCPAC-1 cells. The effects of overexpressing miR-100 on pancreatic cancer cell proliferation and chemosensitivity to cisplatin were examined by cell proliferation essay in vitro. MIA PaCa-2 cells with endogenously overexpressed miR-100 were transplanted into null mice to examine tumor growth in vivo. The predicted target of miR-100, fibroblast growth factor receptor 3 (FGFR3), was downregulated by siRNA to examine its effect on pancreatic cancer cells. We found miR-100 was markedly underexpressed in both pancreatic cancer cell lines and tumor cells from patients. In cancer cells, transfection of lv-miR-100 was able to upregulate endogenous expression of miR-100, inhibited cancer cell proliferation, and increased sensitivities to cisplatin. Overexpressing miR-100 led to significant inhibition on tumor formation in vivo. Luciferase essay showed FGFR3 was direct target of miR-100. FGFR3 was significantly downregulated by overexpressing miR-100 in pancreatic cancer cells and knocking down FGFR3 by siRNA exerted similar effect as miR-100. Our study demonstrated that miR-100 played an important role in pancreatic cancer development, possibly through targeting FGFR3. It may become a new therapeutic target for gene therapy in patients suffered from pancreatic cancer.

Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells

ABSTRACT In contrast to normal tissue, cancer cells display profound alterations in protein synthesis and degradation. Therefore, proteins that regulate endoplasmic reticulum (ER) homeostasis are being increasingly recognized as potential therapeutic targets. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. However, interactions between autophagy, the proteasome, and ER stress pathways in cancer remain largely undefined. This study demonstrated that withaferin-A (WA), the biologically active withanolide extracted from Withania somnifera, significantly increased autophagosomes, but blocked the degradation of autophagic cargo by inhibiting SNARE-mediated fusion of autophagosomes and lysosomes in human pancreatic cancer (PC) cells. WA specifically induced proteasome inhibition and promoted the accumulation of ubiquitinated proteins, which resulted in ER stress-mediated apoptosis. Meanwhile, the impaired autophagy at early stage induced by WA was likely activated in response to ER stress. Importantly, combining WA with a series of ER stress aggravators enhanced apoptosis synergistically. WA was well tolerated in mice, and displayed synergism with ER stress aggravators to inhibit tumor growth in PC xenografts. Taken together, these findings indicate that simultaneous suppression of 2 key intracellular protein degradation systems rendered PC cells vulnerable to ER stress, which may represent an avenue for new therapeutic combinations for this disease.