Xiaoqian Jing

Pseudomonas aeruginosa -mannose-sensitive hemagglutinin inhibits pancreatic cancer cell proliferation and induces apoptosis via the EGFR pathway and caspase signaling

Pseudomonas aeruginosa-mannose-sensitive hemagglutinin (PA-MSHA) has demonstrated efficacy against several solid tumors. In this study, we found that PA-MSHA inhibited the proliferation of PANC-1 and SW1990 pancreatic cancer cells, but had no obvious effects on HPDE6-C7 normal human pancreatic duct epithelial cells. Electron microscopy revealed the presence of apoptotic bodies and intracellular vacuole formation in PA-MSHA-treated pancreatic cancer cells. Flow cytometric analysis indicated the rate of apoptosis correlated with the PA-MSHA concentration. We observed a decrease in cell fractions in G0/G1 and G2/M phases, and an increase in the fraction in S phase (p < 0.01). PA-MSHA thus caused cell cycle arrest. Increasing concentrations of PA-MSHA did not alter total levels of EGFR, AKT or ERK, but levels of the corresponding phosphoproteins decreased. PA-MSHA also reduced tumor volume in a xenograft mouse model of pancreatic cancer (p < 0.01). Furthermore, caspase-3 levels decreased while the levels of cleaved caspase-3 increased (p < 0.01). These data suggest that by blocking cell cycle progression, PA-MSHA induces apoptosis and inhibits tumor growth. PA-MSHA-mediated inhibition of EGFR signaling and activation of the caspase pathway may play an important role in the induction of apoptosis in pancreatic cancer cells.

FGFR3 promotes angiogenesis-dependent metastasis of hepatocellular carcinoma via facilitating MCP-1-mediated vascular formation

The biological role of fibroblast growth factor receptor 3 (FGFR3) in tumor angiogenesis of hepatocellular carcinoma (HCC) has not been discussed before. Our previous work had indicated FGFR3 was overexpressed in HCC, and silencing FGFR3 in Hu7 cells could regulate tumorigenesis via down-regulating the phosphorylation level of key members of classic signaling pathways including ERK and AKT. In the present work, we explored the role of FGFR3 in angiogenesis-dependent metastasis by using SMMC-7721 and QGY-7703 stable cell lines. Our results indicated FGFR3 could regulate in vitro cell migration ability and in vivo lung metastasis ability of HCC, which was in accordance with increased angiogenesis ability in vitro and in vivo. Using the supernatant from SMMC-7721/FGFR3 cells, we conducted a human angiogenesis protein microarray including 43 angiogenesis factors and found that FGFR3 modulated angiogenesis and metastasis of HCC mainly by promoting the protein level of monocyte chemotactic protein 1 (MCP-1). Silencing FGFR3 by short hairpin RNA (shRNA) could reduce MCP-1 level in lysates and supernatant of QGY-7703 cells and SMMC-7721 cells. Silencing MCP-1 in QGY-7703 or SMMC-7721 cells could induce similar phenotypes compared with silencing FGFR3. Our results suggested FGFR3 promoted metastasis potential of HCC, at least partially if not all, via facilitating MCP-1-mediated angiogenesis, in addition to previously found cell growth and metastasis. MCP-1, a key medium between HCC cells and HUVECs, might be a novel anti-vascular target in HCC.

Phenotypic and Signaling Consequences of a Novel Aberrantly Spliced Transcript FGF Receptor-3 in Hepatocellular Carcinoma.

Fibroblast growth factor receptor 3 (FGFR3) plays important roles in cell proliferation, differentiation, and angiogenesis. FGFR3 is abnormally upregulated in hepatocellular carcinoma (HCC), where it correlates positively with clinicopathologic index, HCC differentiation, and advanced nuclear grade. In this study, we describe an aberrantly spliced transcript of FGFR3, termed FGFR3Δ7-9, was identified as a high frequency even in HCC. FGFR3Δ7-9 lacks exons encoding the immunoglobulin-like III domain and promoted the proliferation, migration, and metastasis of HCC cells both in vitro and in vivo Coimmunoprecipation and surface plasmon resonance assays demonstrated that the binding affinity of the aberrant FGFR3Δ7-9 receptor to FGFs was significantly higher than wild-type FGFR3IIIc Furthermore, FGFR3Δ7-9 could be self-activated by homodimerization and autophosphorylation even in the absence of ligand. Finally, FGFR3Δ7-9 more potently induced phosphorylation of the ERK and AKT kinases, leading to abnormal downstream signaling through the ERK and PI3K/AKT/mTOR pathways. FGFR3Δ7-9 also upregulated the metastasis-associated molecules Snail, MMP-9, and downregulated E-cadherin, which associated directly with FGFR3Δ7-9 Thus, as a ligand-dependent or -independent receptor, FGFR3Δ7-9 exerted multiple potent oncogenic functions in HCC cells, including proliferation, migration, and lung metastatic capacity. Overall, FGFR3 mRNA missplicing in HCC contributes significantly to its malignant character, with implications for therapeutic targeting. Cancer Res; 76(14); 4205-15. ©2016 AACR.

Up-regulation of chemokine receptor CCR4 is associated with Human Hepatocellular Carcinoma malignant behavior

Studies indicate that the chemokine receptor is responsible for poor prognosis of hepatocellular carcinoma (HCC) patients. In this study, we initially demonstrated that CCR4 is overexpressed in HCC specimens, and its elevation in HCC tissues positively correlates with tumor capsule breakthrough and vascular invasion. Although overexpression of CCR4 failed to influent proliferation of HCC cells in vitro apparently, the prominent acceleration on HCC tumor growth in vivo was remarkable. The underlying mechanism may be involved in neovascularization. Interestingly, different from effect on proliferation, CCR4 overexpression could trigger HCC metastasis both in vitro and in vivo also induced HCC cell epithelial-mesenchymal transition (EMT) as well. Then we identified matrix metalloproteinase 2 (MMP2) as a direct target of CCR4 which plays an important role in CCR4-mediated HCC cell invasion, which was up-regulated by ERK/AKT signaling. Positive correlation between CCR4 and MMP2 expression was also observed in HCC tissues. In conclusion, our study suggested that chemokine receptor CCR4 promotes HCC malignancy and facilitated HCC cell metastases via ERK/AKT/MMP2 pathway. These findings suggest that CCR4 may be a potential new diagnostic and prognostic marker in HCC, and targeting CCR4 may be a potential therapeutic option for blocking HCC metastasis.

Asporin enhances colorectal cancer metastasis through activating the EGFR/Src/cortactin signaling pathway

Asporin has been implicated as an oncogene in various types of human cancers; however, the roles of asporin in the development and progression of colorectal cancer (CRC) have not yet been determined. With clinical samples, we found that asporin was highly expressed in CRC tissues compared to adjacent normal tissues and the asporin expression levels were significantly associated with lymph node metastasis status and TNM stage of the patients. Through knockdown of asporin in CRC cell lines RKO and SW620 or overexpression of asporin in cell lines HT-29 and LoVo, we found that asporin could enhance wound healing, migration and invasion abilities of the CRC cells. Further more, with the human umbilical vein endothelial cells (HUVECs) tube formation assays and the xenograft model, we found that asporin promoted the tumor growth through stimulating the VEGF signaling pathway. The portal vein injection models suggested that asporin overexpression stimulated the liver metastasis of HT29 cell line, while asporin knockdown inhibited the liver metastasis of RKO cell line. In addition, asporin was found to augment the phosphorylation of EGFR/Src/cortactin signaling pathway, which might be contributed to the biological functions of asporin in CRC metastasis. These results suggested that asporin promoted the tumor growth and metastasis of CRC, and it could be a potential therapeutic target for CRC patients in future.

Cortactin promotes colorectal cancer cell proliferation by activating the EGFR-MAPK pathway

Cortactin (CTTN) is overexpressed in various tumors, including head and neck squamous cell carcinoma and colorectal cancer (CRC), and can serve as a biomarker of cancer metastasis. We observed that CTTN promotes cancer cell proliferation in vitro and increases CRC tumor xenograft growth in vivo. CTTN expression increases EGFR protein levels and enhances the activation of the MAPK signaling pathway. CTTN expression also inhibits the ubiquitin-mediated degradation of EGFR by suppressing the coupling of c-Cbl with EGFR. CoIP experiments indicate CTTN can interact with c-Cbl in CRC cells. These results demonstrate that CTTN promotes the proliferation of CRC cells and suppresses the degradation of EGFR.

Abstract 3958: The phenotypic and signaling consequences of a novel aberrantly spliced transcript of fibroblast growth factor receptor 3 in hepatocellular carcinoma

Aims: To identify the phenotypic and signaling consequences of a novel aberrantly spliced transcript of fibroblast growth factor receptor 3 (FGFR3) in hepatocellular carcinoma (HCC). Methods: The nested RT-PCR was employed to analyze the splicing of FGFR3 from 35 HCC cases and 11 cell lines. The bridging method was utilized to construct the sequence of FGFR3 Δ7-9 (deleting exon7, 8, and 9 from the full length of FGFR3 ORF). Based on the establishment of the FGFR3Δ7-9 lentiviral overexpress and knockdown models, the influence on bio-characters of HCC cell lines was inspected in vitro. The changes of tumorigenicity and metastases were examined quantitatively in vivo on nude mice using pathological study and Micro-PET/CT. Co-immunoprecipitation and surface plasmon resonance (SPR) binding analysis were employed to understand the ligand affinities between FGFR3Δ7-9 and FGFs. Interacting proteins and signal transduction pathway were detected by mass spectrometry and dual luciferase reporter assay. Results: FGFR3Δ7-9, lacking exons encoding the immunoglobulin-like domain III, was identified with high frequency in HCC. FGFR3Δ7-9 can apparently promote the proliferation, migration, and metastases of HCC cells both in vitro and in vivo. In contrast, knockdown of FGFR3Δ7-9 can restrict the abnormal malignancies of HCC cells. Co-immumoprecipation and SPR assay showed the binding affinity of FGFR3Δ7-9 to FGFs was significantly higher than that of wild type FGFR3 (P Discussion/Conclusion: Dysregulation of mRNA splicing may generate an abnormal aberrant FGFR3 transcript, FGFR3Δ7-9. The joining of exon 6 to exon 10 in FGFR3Δ7-9 is in-frame, leading to the expression of a novel transmembrane form of FGFR3 containing an intact intracellular TK domain. As a ligand-independence or lowly ligand-dependent receptor, FGFR3Δ7-9 may function importantly in HCC tumorigenesis, proliferation, invasion and distant lung metastases. Altogether, our research strongly supports the idea that the dysregulation of FGFR3 expression by aberrant splicing of mRNA in a significant subset of HCC is an alternative pathway to neoplastic transformation. Note: This abstract was not presented at the meeting. Citation Format: Weihua Qiu, Weiping Yang, Xiaoqian Jing, Bingrui Wang, Xinyu Liu, Ding Ma, Helen Lin. The phenotypic and signaling consequences of a novel aberrantly spliced transcript of fibroblast growth factor receptor 3 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3958. doi:10.1158/1538-7445.AM2015-3958

MLLT10 promotes tumor migration, invasion, and metastasis in human colorectal cancer

Abstract Objectives: Colorectal cancer (CRC), one of the most aggressive gastrointestinal malignancies, is a frequently diagnosed life-threatening cancer worldwide. Most CRC patients have poor prognosis mainly because of frequent metastasis and recurrence. Thus, it is crucial to find out some new biomarkers and to show deeper insights into the mechanisms of CRC. MLLT10, Myeloid/lymphoid or mixed-lineage leukemia translocated to 10, also known as AF10, a recurrent MLL partner. In this study, we found that MLLT10 promotes CRC tumor invasion and metastasis both in vitro and in vivo. Methods: Here, the expression of MLLT10 was evaluated by immunohistochemistry. Then, the plasmid and lentivirus particles for MLLT10 overexpression or knockdown were designed and constructed into SW620 and HT29 cells. Finally, cell proliferation assay, cell adhesion assay, transwell migration, and invasion assay were used to detect the migration and invasion ability of MLLT10 in CRC cells. A tail vein injection assay was employed to evaluate the role of MLLT10 in tumor metastases. Results: MLLT10 expression was significantly higher in CRC tissues than in noncancerous tissues and was associated with some clinicopathological factors. In vitro, the overexpression of MLLT10 promoted CRC cell migration and invasion, while after MLLT10 was knocked down, the opposite results were observed. Furthermore, we used animal metastasis models to detect the function of MLLT10 in vivo, the results are same with the outcomes in vitro. In lung metastasis sites, the knockdown of MLLT10 in SW620 cells significantly inhibited Vimentin expression, whereas the E-Cadherin was increased. Conclusions: These results indicate that MLLT10 regulates the metastasis of CRC cells via EMT.

Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer

Apatinib, a novel tyrosine kinase inhibitor (TKI), has been confirmed for its efficacy and safety in the treatment of advanced gastric carcinoma and some other solid tumors. However, the direct functional mechanisms of tumor lethality mediated by apatinib have not yet been fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, in this study, we demonstrated that apatinib could induce both apoptosis and autophagy in human colorectal cancer (CRC) via a mechanism that involved endoplasmic reticulum (ER) stress. Moreover, activation of the IRE1α pathway from apatinib-induced ER stress is responsible for the induction of autophagy; however, blocking autophagy could enhance the apoptosis in apatinib-treated human CRC cell lines. Furthermore, the combination of apatinib with autophagy inhibitor chloroquine (CQ) tends to have the most significant anti-tumor effect of CRC both in vitro and in vivo. Overall, our data show that because apatinib treatment could induce ER stress-related apoptosis and protective autophagy in human CRC cell lines, targeting autophagy is a promising therapeutic strategy to relieve apatinib drug resistance in CRC.

LIFR promotes tumor angiogenesis by up-regulating IL-8 levels in colorectal cancer

Leukemia inhibitory factor receptor (LIFR) has been documented as a cancer promoter and to be present at high levels in various types of tumor tissues. In our search for molecules prognostic of colorectal cancer (CRC), we found high levels of LIFR in CRC tissue samples. Further analyses revealed that LIFR was indeed prognostic of CRC patient survival, and was associated with tumor size, lymphatic metastasis and stages. LIFR was found to promote tumor growth, metastasis and angiogenesis both in vitro and in vivo. High levels of LIFR in CRC facilitated proliferation and migration of endothelial cells, resulting in an increase in angiogenic activity. Moreover, interleukin 8 (IL-8) was found to play a role in the LIFR induced angiogenesis. IL-8 levels were correlated with LIFR levels in CRC tissues, whereas depletion of IL-8 led to a reduced angiogenic activity of LIFR in CRC cells. In addition, LIFR increased phosphorylation level of Erk, which regulates il-8 transcription. We conclude that LIFR is possibly a valuable prognostic marker for CRC. Our results also implicate a mechanism by which LIFR regulates tumor angiogenesis through Erk/IL-8 pathway, and that LIFR could be a potential therapeutic target for CRC.