Li Sheng Zheng

Cancer stem-like cell characteristics induced by EB virus-encoded LMP1 contribute to radioresistance in nasopharyngeal carcinoma by suppressing the p53-mediated apoptosis pathway

Emerging evidence confirms that cancer stem cells (CSCs) are responsible for the chemoradioresistance of malignancies. EBV-encoded latent membrane protein 1 (LMP1) is associated with tumor relapse and poor prognosis of nasopharyngeal carcinoma (NPC). However, whether LMP1 induces the development of CSCs and the mechanism by which this rare cell subpopulation leads to radioresistance in NPC remain unclear. In the present study, LMP1-transformed NPC cells showed significant radioresistance compared to the empty vector control. We found that LMP1 up-regulated the expression of several stemness-related genes, increased the cell number of side population (SP) by flow cytometry analysis, enhanced the self-renewal properties of the cells in a spherical culture and enhanced the in vivo tumor initiation ability. We also found that LMP1 positively regulated the expression of the CSC marker CD44. The CD44(+/High) subpopulation of the LMP1-transformed NPC cells displayed more significant CSC characteristics than the CD44(-/Low) subpopulation of the LMP1-transformed NPC cells; these characteristics included the upregulation of stemness-related genes, in vitro self-renewal and in vivo tumor initiation ability. Importantly, the CD44(+/High) subpopulation displayed more radioresistance than the CD44(-/Low) subpopulation. Our results also demonstrated that phosphorylation of the DNA damage response (DDR) proteins, ATM, Chk1, Chk2 and p53, was inactivated in the LMP1-induced CD44(+/High) cells in response to DNA damage, and this was accompanied by a downregulation of the p53-targeted proapoptotic genes, which suggested that the inactivation of the p53-mediated apoptosis pathway was responsible for the radioresistance in the CD44(+/High) cells. Taken together, we found that LMP1 induced an increase in CSC-like CD44(+/High) cells, and we determined the molecular mechanism underlying the radioresistance of the LMP1-activated CSCs, highlighting the need of CSC-targeted radiotherapy in EBV-positive NPC.

RASSF6 promotes p21Cip1/Waf1-dependent cell cycle arrest and apoptosis through activation of the JNK/SAPK pathway in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a highly aggressive and common pathological subtype of renal cancer. This cancer is characterized by biallelic inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene, which leads to the accumulation of hypoxia-inducible factors (HIFs). Although therapies targeted at HIFs can significantly improve survival, nearly all patients with advanced ccRCC eventually succumb to the disease. Thus, additional oncogenic events are thought to be involved in the development of ccRCC tumors. In this study, we investigated the role of RASSF6 in ccRCC. Downregulation of RASSF6 was commonly observed in primary tumors relative to matched adjacent normal tissues. Moreover, functional studies established that ectopic re-expression of RASSF6 in ccRCC cells inhibited cell proliferation, clonogenicity, and tumor growth in mice, whereas silencing of RASSF6 dramatically enhanced cell proliferation in vitro and in vivo. Mechanistic investigation suggested that RASSF6 triggers p21Cip1/Waf1 accumulation to induce G1 cell cycle arrest and promote apoptosis upon exposure to pro-apoptotic agents, and both of these mechanisms appear to be mediated by activated JNK signaling. Together, these findings suggest that RASSF6 may play a tumor suppressor role in the progression of ccRCC.

Urokinase-type plasminogen activator receptor signaling is critical in nasopharyngeal carcinoma cell growth and metastasis.

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in southern China and Southeast Asia, with the highest metastasis rate among head and neck cancers. The mechanisms underlying NPC progression remain poorly understood. Genome-wide expression profiling on 18 NPC vs. 18 noncancerous nasopharyngeal tissues together with GeneGo pathway analysis and expression verification in NPC cells and tissues revealed a potential role of urokinase-type plasminogen activator receptor (uPAR) in NPC progression, which has not been investigated in NPC. We then observed that uPAR expression is increased in poorly differentiated, highly metastatic NPC cells compared with lowly metastatic cells or differentiated NPC cells. In vitro studies demonstrated that uPAR regulates NPC cell growth, colony formation, migration, and invasion and promotes the epithelial–mesenchymal transition (EMT). Additional tumor xenograft and spontaneous metastasis experiments revealed that uPAR promotes NPC cell growth and metastasis in vivo. The JAK–STAT pathway is involved in uPAR-regulated signaling in NPC cells as determined by immunoblotting. Moreover, uPAR-mediated growth and motility is partially abolished upon treatment with the Jak1/Jak2 inhibitor INCB018424. We suppressed uPA expression in uPAR-overexpressing NPC cells and found that uPAR-mediated cellular growth and motility is not exclusively dependent on uPA. In summary, uPAR is a significant regulator of NPC progression and could serve as a promising therapeutic target.

SPINK6 promotes metastasis of nasopharyngeal carcinoma via binding and activation of epithelial growth factor receptor.

Nasopharyngeal carcinoma has the highest rate of metastasis among head and neck cancers, and distant metastasis is the major reason for treatment failure. The underlying molecular mechanisms of nasopharyngeal carcinoma metastasis are not fully understood. Here, we report the identification of serine protease inhibitor Kazal-type 6 (SPINK6) as a functional regulator of nasopharyngeal carcinoma metastasis via EGFR signaling. SPINK6 mRNA was upregulated in tumor and highly metastatic nasopharyngeal carcinoma cells. Immunohistochemical staining of 534 nasopharyngeal carcinomas revealed elevated SPINK6 expression as an independent unfavorable prognostic factor for overall, disease-free, and distant metastasis-free survival. Ectopic SPINK6 expression promoted in vitro migration and invasion as well as in vivo lymph node metastasis and liver metastasis of nasopharyngeal carcinoma cells, whereas silencing SPINK6 exhibited opposing effects. SPINK6 enhanced epithelial-mesenchymal transition by activating EGFR and the downstream AKT pathway. Inhibition of EGFR with a neutralizing antibody or erlotinib reversed SPINK6-induced nasopharyngeal carcinoma cell migration and invasion. Erlotinib also inhibited SPINK6-induced metastasis in vivo Notably, SPINK6 bound to the EGFR extracellular domain independent of serine protease-inhibitory activity. Overall, our results identified a novel EGFR-activating mechanism in which SPINK6 has a critical role in promoting nasopharyngeal carcinoma metastasis, with possible implications as a prognostic indicator in nasopharyngeal carcinoma patients. Cancer Res; 77(2); 579-89. ©2016 AACR.

PDZ binding kinase (PBK) is a theranostic target for nasopharyngeal carcinoma: driving tumor growth via ROS signaling and correlating with patient survival

Nasopharyngeal carcinoma (NPC) is well known as one of the most common malignancies in southern China and Southeast Asia. However, the mechanisms underlying NPC progression remain poorly understood. Herein, through overlapping the differentially expressed genes from 3 microarray data sets with the human kinome, we identified PBK, a serine-threonine kinase, is highly upregulated and has not been intensively investigated in NPC. PBK was required for malignant phenotypes of NPC, as PBK depletion by RNAi and inhibition by specific inhibitor HI-TOPK-032 obviously reduced cell proliferation and xenograft tumor growth in mice. Moreover, we determined that targeting PBK could accelerate apoptosis by inducing ROS that activates JNK/p38 signaling pathway. In NPC patients, elevated PBK expression in primary tumor positively correlated to clinical severity such as advanced T stage, high death risk and disease progression, and it could serve as an unfavorable independent indicator of overall survival and disease-free survival. Altogether, our results indicate that PBK is a novel significant regulator of NPC progression and a potential therapeutic target for NPC patients.

Promoting tumorigenesis in nasopharyngeal carcinoma, NEDD8 serves as a potential theranostic target

Nasopharyngeal carcinoma (NPC), is one of the most common human malignancies in south China, it has the highest recurrence rate and treatment resistance. The underlying molecular mechanisms of NPC relapse and treatment tolerance are not fully understood. In this study, the effects of NEDD8 and NEDD8-activating enzyme inhibitor (MLN4924) on NPC were studied both in vitro and in vivo. Immunohistochemical staining of 197 NPC tissues revealed an elevated NEDD8 expression as an unfavorable independent factor in overall survival and disease-free survival rates. NEDD8 expression was positively correlated with a high risk of death and positivity of lymph node metastasis. Depleted NEDD8 expression by shRNA and inhibited by specific inhibitor MLN4924 dramatically suppressed cell proliferation, cell apoptosis, cell cycle arrest, while ectopic NEDD8 exhibited opposing effects. NEDD8 affected cancer stem cell phenotypes of NPC as assessed in vitro using the cell number of side population (SP) by flow cytometry analysis, colony formation assay, sphere formation assay, and tumor initiation ability in vivo. Downregulation of NEDD8 enhanced the susceptibility of NPC cells to cisplatin and radiation. Moreover, we found that MLN4924 suppressed c-Jun degradation in human NPC cells. Taken together, this report revealed that NEDD8 may act as a novel prognostic marker and MLN4924 may serve as a promising therapeutic target for patients with NPC.

Downregulation of Ras association domain family member 6 (RASSF6) underlies the treatment resistance of highly metastatic nasopharyngeal carcinoma cells.

Radiation and cisplatin-based chemotherapy are major treatments for nasopharyngeal carcinoma (NPC). However, a major impediment for further improving the cure rate is the development of treatment resistance with an undetermined molecular mechanism in metastatic NPC cells. Our established, highly metastatic NPC cells have been reported to be more resistant to cisplatin chemotherapy. In the present study, we found that Ras association domain family member 6 (RASSF6) was downregulated in highly metastatic cells but upregulated in low metastatic cells in comparison to their parental cell line. Ectopic-expression of RASSF6 enhanced the sensitivity of highly metastatic NPC cells to cisplatin or radiation by enhancing apoptosis. RASSF6 depletion conversely reduced treatment sensitivity by decreasing the apoptosis rate. Over-expression of RASSF6 in highly metastatic NPC cells could enhance the phosphorylation of JNK when exposed to cisplatin or radiation treatment, while knocking down RASSF6 in low metastatic NPC cells could reduce the level of phospho-JNK when exposed to the same treatments. The activation of JNK signaling by RASSF6 and its subsequent sensitivity to apoptosis in NPC cells could be inhibited by applying the JNK inhibitor SP600125. In conclusion, the downregulation of RASSF6 in highly metastatic NPC cells contributed to their treatment resistance, and over-expression of RASSF6 conferred treatment sensitivity to highly metastatic NPC cells by activating JNK signaling. RASSF6 could be a valuable molecular marker for identifying sensitive metastatic NPC tumors during cisplatin treatment or radiotherapy.

CDC42-interacting protein 4 promotes metastasis of nasopharyngeal carcinoma by mediating invadopodia formation and activating EGFR signaling

BackgroundNasopharyngeal carcinoma (NPC) is a common malignancy in Southern China and Southeast Asia. In this study, we investigated the functional and molecular mechanisms by which CDC42-interacting protein 4 (CIP4) influences NPC.MethodsThe expression levels of CIP4 were examined by Western blot, qRT-PCR or IHC. MTT assay was used to detect the proliferative rate of NPC cells. The invasive abilities were examined by matrigel and transwell assay. The metastatic abilities of NPC cells were revealed in BALB/c nude mice.ResultsWe report that CIP4 is required for NPC cell motility and invasion. CIP4 promotes the activation of N-WASP that controls invadopodia formation and activates EGFR signaling, which induces downstream MMP2 (matrix metalloproteinase 2) upregulation. In addition, CIP4 could promote NPC metastasis by activating the EGFR pathway. In nude mouse models, distant metastasis was significantly inhibited in CIP4-silenced groups. High CIP4 expression is an independent adverse prognostic factor of overall survival (OS) and distant metastasis-free survival (DMFS).ConclusionWe identify the critical role of CIP4 in metastasis of NPC which suggest that CIP4 may be a potential therapeutic target of NPC patients.

RNA-binding protein QKI-5 inhibits the proliferation of clear cell renal cell carcinoma via post-transcriptional stabilization of RASA1 mRNA

ABSTRACT Clear cell renal cell carcinoma (ccRCC) is a common pathological subtype of renal cancer. Although the recent application of molecular-targeted agents has modestly improved the prognosis of ccRCC patients, their outcome is still poor. It is therefore important to characterize the molecular and biological mechanisms responsible for the development of ccRCC. Approximately 25% ccRCC patients involves the loss of RNA-binding protein QKI at 6q26, but the role of QKI in ccRCC is unknown. Here, we found that QKI-5 was frequently downregulated in ccRCC patients and its down-regulation was significantly associated with clinical features including T status, M status, and differentiation grade, and poorer patient prognosis. Moreover, QKI-5 inhibited the proliferation of kidney cancer cells both in vitro and in vivo. The subsequent functional studies showed that QKI-5 stabilized RASA1 mRNA via directly binding to the QKI response element region of RASA1, which in turn prevented the activation of the Ras-MAPK signaling pathway, suppressed cellular proliferation and induced cell cycle arrest. Overall, our data demonstrate a suppressive role of QKI in ccRCC tumourigenesis that involves the QKI-mediated post-transcriptional regulation of the Ras-MAPK signaling pathway.

Along with its favorable prognostic role, CLCA2 inhibits growth and metastasis of nasopharyngeal carcinoma cells via inhibition of FAK/ERK signaling

BackgroundCLCA2 was reported as a tumor suppressor and disregulated in breast cancer. However, its function in tumor growth and metastasis in NPC has rarely been reported. In this study, we investigated the functional and molecular mechanisms by which CLCA2 influences NPC.MethodsCLCA2 expression in human NPC cell lines and tissues was examined via real-time PCR (RT-PCR), Western blot and IHC. The biological roles of CLCA2 in proliferative, migration and invasion of NPC cell lines was evaluated in 5-8F, S18, S26 and SUNE-1 cells. Cell viability, migration and invasion were assessed in vitro by MTS, colony formation and transwell assay, respectively. CLCA2 in growth and metastasis of NPC were evaluated in vivo through NPC xenograft tumor growth, lung metastatic mice model and popliteal lymph node (LN) metastasis model.ResultsOverexpression of CLCA2 significantly decreased proliferation, migration and invasion of NPC cells. In contrast, knockdown of CLCA2 elicited the opposite effects. CLCA2 overexpression suppressed xenograft tumor growth and lung, popliteal lymph node (LN) metastasis in vivo. CLCA2 inhibited tumor metastasis through suppressing epithelial-Mesenchymal transition (EMT) and in-activating FAK/ERK1/2 signaling pathway in NPC cells. Immunohistochemical staining of 143 NPC samples revealed that CLCA2 expression was an independent, favorable prognostic factor for overall survival and distant metastasis-free survival of patients. In addition, inhibition of FAK and ERK1/2 reversed CLCA2 silencing-induced tumor cell migration. Furthermore, inhibitors against chloride channels suppressed NPC cellular migration which could have been enhanced by the presence of CLCA2.ConclusionCLCA2 suppress NPC proliferation, migration, invasion and epithelial-mesenchymal transition through inhibiting FAK/ERK signaling.