The Role And Mechanism Of Capn4in The Malignant Behavior Of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a malignancy arising from the epithelial cells ofthe nasopharynx. It has a distinct geographic distribution with a remarkably highdisease incidence in southern China and Southeast Asia, where the annual incidence ismore than20–30cases per100,000people. According to global cancer statistics fromthe International Agency for Research on Cancer, there were over84,000new NPCcases in2008, with80%of the cases located in Asia and5%in Europe. and thispattern persists in those who have emigrated. The etiology of NPC seems to follow amultistep process, in which EBV infection, ethnic background, consumption of foodand alcohol, and environmental carcinogens all seem to play a role. Nowadays,Technological advances in the fields of diagnostic imaging, radiation therapy, andconcurrent chemoradiotherapy have achieved better locoregional control. However,the majority of NPC cases present with locally advanced stages, and the treatmentoutcomes for locoregionally advanced NPC remain unsatisfactory. The overallsurvival (OS) rates at5years were53%–80%and28%–61%in NPC stages III and IV,respectively. Thus it is important to search for new therapeutic targets and efforts thatpromise to lead to a better understanding of the molecular mechanisms involved incarcinogenesis and, possibly, to clinical applications aimed at secondary prevention ortreatment.Calpain represents a family of calcium-dependent cytosolic cysteine proteases. So far, there are14calpain isoforms identified in human, most of which are found at focaladhesions and involved in cell spreading and migration, proliferation, cell cyclecontrol, and apoptosis. Capn4(also known as CapnS1) is a small regulatory subunit ofthe calpain proteolytic system and plays a critical role in regulation of calpain stabilityand activity. Several studies have demonstrated that deficiency of Capn4leads todysfunction of calpain-1and calpain-2, which is embryonic lethal. Knockdown ofCapn4results in decreased migration and focal adhesion of fibroblasts cells. Recently,overexpression of Capn4was found in hepatocellular carcinoma (HCC) andintrahepatic cholangiocarcinoma (ICC). Moreover, whereas Capn4overexpression isclosely correlated with prognosis of patients with HCC or ICC, siRNA-mediatedsilencing of the Capn4gene results in marked inhibition of invasion and metastasis inHCC and ICC cells. Therefore, Capn4may play a critical role in migration andadhesion of metastatic cancer cells, and it remains unknown whether Capn4plays afunctional role in pathogenesis of NPC.【Objectives】1、To investigate the expression and clinical significance of Capn4in humannasopharyngeal carcinoma.2、 To explore the possible role of Capn4innasopharyngeal carcinoma and the molecular mechanism underlying it.【Methods】The protein and mRNA level of Capn4in fresh NPC tissues and NPC cell lines wereexamined by Western-blot and RT-PCR, respectively.2、The expression of Capn4inhuman NPC tissues and the normal nasopharyngeal tissues was examined byimmunohistochemistry and the relationship between the expression of Capn4and theclinical characteristics was statistically analyzed by using SPSS version17.0software.3、The small interference RNA of Capn4were constructed and further indentified bysequence test.4、The effects of Capn4on the adhesive, invasive and in vivo metastaticabilities of NPC cells5-8F and CNE2were respectively investigated by transwellassay and tail vein metastatic assay.5、The expression levels of genes associated withtumor metastasis in different transfectant cells were determined by western blot.6、The mRNA expression and enzyme activity of MMP2were determined by RT-PCR and gelatin zymography assay, respectively.7、The effects of MMP2on Capn4mediate on the migration of NPC cells5-8F and CNE2were investigated by transwellassay.8、The phosphrylated NF-κB p65subunit in different transfectants weredetermined by Western blot.9、Studying influence of Capn4on tumor formationability of NPC cell by in vitro and in vivo cell proliferation assay, plating cloneformation assay, nude mouse tumor-bearing assay.10、Detecting the effect of Capn4on cell cycle and cell DNA replication by flow cytometry analysis.11、The expressionlevels of genes associated with cell cycles in different transfectant cells weredetermined by western blot.【Results】1、The expression level of Capn4in NPC tissues is significant higher than innormal nasopharyngeal tissues.Capn4mRNA and protein expression level was significantly up-regulated in NPCtissues (n=7) compared with normal nasopharyngeal tissues (n=2). In parallel,up-regulation of Capn4was also observed in three NPC cell lines,5-8F, CNE2, and6-10B, when compared to immortalized normal human nasopharyngeal epithelial celllines NP69. We next detected expression of Capn4protein in archivedparaffin-embedded NPC tumor (n=153) and non-tumoral nasopharyngeal specimens(n=30) by immunohistochemical staining. The expression levels of Capn4in NPCsamples were significantly higher than that in non-tumoral tissues (P﹤0.001). Proteinlevels of Capn4in tumors were significantly higher in patients with positive EB virusinfection, advanced tumors (T3/T4,), lymph node metastasis (N2/N3), distantmetastasis (M1), or TNM stage III/IV, compared to patients with negative EB virusinfection (P<0.001), early tumors (T1/T2, P<0.001), no or a few lymph nodemetastasis (N0/N1, P<0.001), no distant metastasis (M0, P<0.001), or TNM stage I/II(P=0.007). Moreover, Kaplan-Meier survival analyses demonstrated that patients withhigher expression of Capn4had a significantly shorter overall survival (OS),compared to those with lower levels of Capn4(P=0.002). Last, high levels of Capn4in NPC tumor were significantly correlated with reduced progression-free survival(PFS, P=0.003). 2、Knockdown of Capn4reduces migration and invasion of NPC cells in vitroand in vivoTo determine the functional role of Capn4in metastasis of NPC, we first generatedtwo clones of5-8F cells and CNE2cells stably transfected with Capn4siRNA(Capn4/siRNA-1and Capn4/siRNA-2). RT-PCR and Western blot analysesdemonstrated that both protein and mRNA levels of Capn4were substantiallydownregulated in Capn4/siRNA-1and Capn4/siRNA-2cells, compared to siRNAcontrol cells. Then, the transwell assays were performed to assess the effects of Capn4knockdown on capabilities of invasion and migration in NPC cells. Notably,Capn4/siRNA cells displayed significantly lower rate of invasion and migration thanthe parental cells and siRNA control cells. To validate the role of Capn4in NPCmetastasis in vivo,5-8F cells stably expressing Capn4or control siRNA wereimplanted into nude mice via lateral tail vein injection. Results show that lung andliver metastasis was observed in7of10mice injected with siRNA control cells, butonly in1or2of10mice with Capn4siRNA1cells. Collectively, these in vitro and invivo findings argue that Capn4might play a functional role in regulation of NPCmetastasis.3、Capn4promotes invasion of NPC cells via up-regulation of MMP2To gain further insight into the mechanism by which Capn4regulates migration andinvasion of NPC cells, Western blot analysis was performed to examine expression ofpotential Capn4-targeting proteins that are involved in cell migration and invasion.The results showed down-regulation of Capn4by siRNA led to decreased expressionof MMP2, Snail, and Vimentin, but increased expression of E-cadherin in NPC cells.In contrast, there was no change observed in expression of MMP9, N-cadherin, orβ-catenin. We then tested whether MMP2contributes to Capn4-mediated regulation ofNPC cell invasion. First, RT-PCR analysis confirmed a reduction in MMP2mRNAafter Capn4was knocked down in NPC cells. Second, MMP2enzyme activity wasdetermined using the gelatin zymography in media obtained from5-8F cells andCNE2cells transfected with Capn4siRNA1and siRNA2. The results showed lower enzyme activity in these cells than siRNA control cells, manifested by a band at72kDa. Last, knockdown of MMP2by siRNA significantly suppressed invasioncapability of5-8F cells and CNE2cells in transwell assays. Conversely,overexpression of MMP2in cells, in which Capn4was silenced by siRNA, rescuedthe functional effect of Capn4on cell invasion and subsequently restored the invasionactivity of NPC cells. Thus, these results suggest that Capn4may promote metastasisof NPC via regulation of MMP2expression.4、Capn4regulates MMP2expression via NF-kB activationWe further examined whether Capn4upregulates MMP2via activation of NF-kB inNPC cells. Western blotting analysis revealed that Capn4knockdown by siRNAdramatically decreased levels of phosphorylated p65, a critical component of NF-κB,compared to control siRNA. Conversely, ectopic expression of Capn4increased p65phosphorylation. Moreover, the NF-kB inhibitor helenalin blocked MMP2upregulation induced by ectopic expression of Capn4. Together, these findings arguethat Capn4induces MMP2expression at least in part via activation of NF-kB in NPCcells.5、Capn4promotes proliferation of NPC cells in vitro and in vivo.Capn4knockdown by siRNA dramatically decreased the expression of cellproliferation marker PCNA by western blot in5-8F/siRNA and CNE2/siRNA cellscompared to the parental cells and siRNA control cells. Subsequently, we examinedthe effect of decreased Capn4expression on NPC cell growth in vitro. Capn4knockdown decreased proliferation of Capn4/siRNA1and Capn4/siRNA2cellscompared with the parental cell line and control pSilencer vector cells (p＜0.05). Ourdata also showed that depletion of Capn4dramatically decreased the ability of colonyformation in these cells in plate and in soft agar (P <0.05). We next examined the invivo effect of Capn4in tumorigenicty in nude mice. The tumor sizes of mice injectedwith5-8F cells expressing siRNA1were smaller and slower than those injected with5-8F cells expressing control pSilencer vector (P <0.05). Immunoblotting analysis oftumor sections derived from mice receiving different treatments also confirmed that Capn4was down-regulated in siRNA1expressing groups compared to control group.6、Inhibition of Capn4expression induces the Cell Cycle Arrest of NPC CellsTo further probe the mechanism by which Capn4promotes NPC cell growth, westudied the effects of down-regulation of Capn4expression on the cell cycle byFlowcytometry. The results indicated that at24hours after the release of synchronizedcultures,68.5±1.7%of5-8F cells expressing siRNA1and60.7±1.3%of5-8F cellsexpressing siRNA2were in G0/G1-phase, respectively, whereas51.2±1.1%of5-8Fcells expressing pSilencer were in G0/G1-phase (P <.05). To further investigate themechanism by which Capn4induced cell cycle progress in NPC cells, we nextanalyze cell cycle effectors expressions by Western blot analysis. Our data showedthat down-regulation of CacyBP/SIP protein was associated with a reducing in cyclinD1, cyclin D2and cyclin E proteins, but with an increase in p27proteins, and it hasno effect on the level of CDK2、CDK4、P57、P21proteins.【Conclusion】1、The expression of Capn4protein in NPC tissues was significant higher than innormal nasopharyngea tissues. There are correlated with EB virus infection, advancedtumors, worse TNM classification，lymph node metastasis, distance metastasis, andadvanced stages of NPC patients.2、Down-regulation of Capn4by small interferenceRNA severely suppresses the migration, invasion, proliferation and tumorigenesis inNPC.3、They also unveil a potential mechanism involving upregulation of MMP2viaan NF-κB-dependent mechanism, underlying Capn4-mediated promotion of NPCprogression.4、G1/S transition arrest induced by inhibition of Capn4is at least partlymediated by down-regulation of Cyclin D1、Cyclin D2and Cyclin E as well asup-regulation of p27.