The Study On The Mechanisms Underlying The Effects Of STK33 And NGFR In Head And Neck Squamous Carcinoma

PART I:STUDY ON THE POSSIBLE RELATION OF STK33-POTENTIATED MALIGNANCY OF HYPOPHARYNGEAL SQUAMOUS CARCINOMA TO CALCIUMBackground:Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common human malignancies worldwide. Despite rapid development in therapy, the survival rate has not been dramatically improved during the last two decades. It is widely recognized that most of cancers result from a multifactorial process that includes the activation of oncogenes as well as the inactivation of tumor suppressor genes. Although the initiation and progression of HSCC involves numerous genetic and epigenetic factors, the molecules that participate in tumorigenesis of this kind of carcinoma are still little known. Protein kinases, a protein super-family, have been extensively explored in biomedical field. As a member of this super-family, the function of serine/threonine kinase is to phosphorylate serine and threonine residues on target proteins. This plays an important role in many physiological processes, such as DNA replication, signal transducing pathway, cell proliferation, differentiation, death, and tumorigenesis. Serine/threonine kinase33 (STK33) regulates the signal transduction pathway in K-ras-dependent cancer cells and plays a special role in tumor proliferation process. STK33 is a member of the calcium/calmodulin dependent kinase (CAMK) family. Data show that by specifically phosphorylating intermediate filament protein vimentin, STK33 participates in the dynamic changes in depolymerization of cytoskeleton and affects the cell structure and functions. Recently, compelling evidence has indicated the involvement of STK33 in tumorigenesis. Nevertheless, the precise function and mechanism of STK33 in HSCC remain unclear as yet and need more study. Therefore, the present study was designed to investigate the role of STK33 by an in vivo experiment and microarray analysis in STK33-RNAi Fadu cells, with special attention given to the possible relationship between STK33 and calcium.Methods:HSCC cells (Fadu cells) were infected by Lentiviral Vector with specific shRNA for STK33. PCR was employed to assess the STK33 expression level after lentivirus infection. Microarray analysis was performed to investigate the impact of STK33 knockdown (STK33-RNAi) on the gene profile alterations of Fadu cells. Subcutaneous injected same number of Fadu cells, Mock Fadu cells and STK33-RNAi Fadu cells into 5 weeks old nu/nu male nude mice. Tumor volume (in mm3) was determined by caliper measurements performed every other day and calculated. The tumors that arose in those mice were harvested when they all reached 1.5 cm in diameter in control group. The tumors and organs were taken from the mice. After H&E staining, changes of basic morphology of the lungs and tumors were observed under the light-microscopy. IHC was used to assess the expression of STK33 in tumor in vivo. MTT and Acridine orange staining were taken to observe the effects of Ionomycin on the cell viability and morphological changes. [Ca2+]i was quantified under the laser scanning confocal microscope by using Fluo-3/AM. qRT-PCR and western blot were used to evaluate the mRNA and protein expression of relative genes.Results:The STK33-RNAi Fadu and Mock Fadu cells were subcutaneously injected into the right flank of male nude mice. The Fadu cells developed into a tumor mass on 14th day after injection and grew for 39 days. Mock group exhibited the similar growth rate as the control group, and the volume reached about 570 mm3 on 39th day. However, the tumor of STK33-RNAi group grew slowly in the bodies of the nude mice, and on the 39th day, only small tumors formed, the volume was about 90 mm3. There was a significant difference in tumor size between control cells and STK33-RNAi cells. These data indicate that STK33 promotes tumor i genes is of HSCC in vivo. The IHC result showed that STK33-RNAi group tumors express less STK33 than the mock group. Dissemination of the lung of mice in the control group was found, but the mice with STK33-RNAi cells showed no dissemination.By microarray analyses, noticeable expression changes were identified in 140 symbolized genes in STK33-RNAi cells. These genes were significantly (p<0.05) up-(n=86) and down-regulated (n=54). By pathway analyses, we found that STK33 affects the expression of many genes, and regulates a lot of signal pathways. Interestingly, calpainl (CAPN1) was found, and the mRNA expression of CAPN1 was confirmed by PCR. The expression of CAPN1 was significantly decreased in STK33-RNAi Fadu cells (p<0.05). Ionomycin induced rapid increase of [Ca2+]i. After treatment with 1.5 μM Ionomycin for 6 hours, the effects of Ionomycin on morphological changes of Fadu cells were examined by acridine orange staining. Untreated cells showed polygonal shape. However, typical characteristics of apoptosis, including irregular cell shape, cell shrinkage, chromatin condensation, apoptotic bodies, were presented in the cells treated with Ionomycin in Fadu cells, indicating that Ionomycin can induce apoptosis of Fadu cells. Cell viability of Fadu cells was shown a trend of significantly decreasing with expending treatment-times, indicating that Ionomycin inhibited cell proliferation in a time-dependent manner. The cell viability significantly decreased with the treatment of STK33-RNAi. Furthermore, the exposure of Fadu cells to STK33-RNAi and Ionomycin resulted in a less decrease of cell viability compared with Ionomycin only (p<0.05). Compared with untreated Fadu cells, the protein expression of STK33 significantly increased after exposure to 1.5 μM Ionomycin for 1,2,4 and 6 hours(p<0.05). This suggests that Ionomycin can increase the protein expression of STK33. The expression of CAPN1 was significantly increased in Fadu cells after treated with 1.5 μM Ionomycin for 1,2,4,6 and 24 hours (p<0.05). But for the STK33-RNAi group, there’s no significant difference before and after the treatment.Discussion:shRNA-mediated silencing of STK33 gene decreases the repopulation capacity of the Fadu cells in vivo. This proves that STK33 itself contributes to the promotion of tumor growth, implies that STK33 plays an important role in tumorigenesis. Furthermore, by using IHC, STK33 expression in xenograft tumors was markedly decreased in the STK33-RNAi group. This suggests that STK33-RNAi still possesses its innovative role of STK33 depletion, whereby rendering the less proliferative ability to Fadu cells which underpins the antitumorigenic effect of STK33-RNAi in vivo. Taken together, in vivo study further validates our previous results that STK33 is a potential oncogene from the study in vitro and on resected specimen. STK33 gene has effects on Fadu cells via multiple signaling networks, in which numerous genes were involved and had critical specific functions for tumor progression. In response to STK33-RNAi, CAPN1 mRNA expression was markedly down-regulated as further evidenced by PCR in this work. The findings from this study indicate that STK33-RNAi can alter CAPN1, implying that CAPN1 is involved in the STK33-initiated tumorigenesis in Fadu cells. This finding might be an interesting clue by which STK33 possibly links to Ca21 in Fadu cells. Because STK33 is the member of the CAMK family, promoting us to focus on the investigation of the correlation between STK33 activity and the intracellular calcium alteration in Fadu cells after treated with Ionomycin. Ionomycin can inhibit the growth of Fadu cells in vitro, implying that Ionomycin exerts its cytotoxicity on Fadu cells by the elevation of [Ca2+]i level. Of particular interest is that STK33-RNAi could counterbalance the Ionomycin-induced injury on the cells in certain degree, this implies that STK33 knockdown may protect cell from high concentration of [Ca2+]i damage triggered by Ionomycin. This offers evidence that by STK33 activity, the intracellular free calcium may impact the expression of CAPN1, thereby this implicaties several fundamental signal transduction pathways, which are closely related to the critical role of STK33 in tumorigenesis.Findings from this work further validate that STK33 is a potential oncogene and plays an important role in tumorigenesis of HSCC via regulation of numerous genes. In addition, there exists the reciprocal influence between STK33 and [Ca2+]i in Fadu cells.PART Ⅱ:STUDY ON THE ROLE OF NGFR IN ESM1-MEDIATED TUMORIGENICITY OF ORAL SQUAMOUS CANCER CELLSBackground:Oral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms. Despite rapid development in therapy, the survival rates have not been dramatically improved for many years. Such a situation is mainly due to the development of distant metastases and the emergence of therapy-resistant recurrences. A biologic explanation for why certain cells are more resistant to chemotherapy and radiation therapy comes from work supporting the idea that a subpopulation of cells within a malignant tumor is endowed with "stem cell"-like properties. Our laboratory has recently identified a novel marker of CICs in OSCC that may address the obstacles just mentioned. This marker is the low-affinity nerve growth factor receptor (NGFR). NGFR is a functional and targetable marker for tumor-initiating cells (TICs) subpopulation in tumor progression, metastasis and survival of head and neck squamous cell carcinoma. Activation of NGFR results in activation of the NF-κB (nuclear factor-κB), Jun kinase as well as other signaling pathways. However, the precise function and mechanism of NGFR in OSCC have been sparingly studied and remained unclear as yet. Therefore, the present study, on the basis of our previous study, was designed to further investigate the role of NGFR via a microarray analysis and in vivo experiment in mice oral squamous cell carcinoma (MOC) cells.Methods:The relative amount of gene mRNA and protein was analyzed by quantitative real-time reverse Transcription-Polymerase Chain Reaction (qRT-PCR), Flow cytometry analysis and Enzyme-linked immunosorbent assay. To study NGFR signaling mechanism, we examined the altered gene expression profiles between MOC2 and NGFR overexpression MOC2 (MOC2T) by exome-sequencing microarray. By using lentivirus infection, ESM1 knockdown MOC2 cell line (ESM1-SH) and ESM1 overexpression MOC2 cell line (ESM1-OVER) were stably established. The effect of ESM1 and NGFR on cell proliferation, metastasis and invasion abilities was assessed by MTT assay, invasion and metastasis assay. Subcutaneously injected MOC2, MOC2-7, MOC2-10, MOC2-ESM1-SH, MOC2T and MOC2T-ESM1-SH cells to 6-11 weeks old B10; B6-Rag2-/-II2rg-/- mice. Tumor volume (in mm3) was determined by caliper measurements performed every two to three days and calculated by using the following formula:volume=length×width2×0.5. After H&E staining, changes of basic morphology of the lungs were observed under the light-microscopy. To test whether ESM1 may participates in the angiogenesis or not, vascular endothelial growth factor (VEGF) expression was measured in tumor sections.Results:By using qRT-PCR, we found that, NGFR is expressed in mouse oral squamous carcinoma cell lines (MOC2, MOC2-7 and MOC2-10). By lentivirus infection, the stable NGFR overexpression MOC2 cell line was made. By exome-sequencing microarray, we examined the altered gene expression profiles between MOC2 and NGFR overexpression MOC2 (MOC2T) cells. By microarray analysis, Endothelial cell specific molecule 1(ESM1) was found. ESM1 is a novel endothelium derived soluble dermatan sulfate proteoglycan, has the property of binding to a wide range of bioactive molecules associated with cellular signaling and adhesion, thus regulating proliferation, differentiation, migration, and adhesion of different cell types in health and disease. NGFR-dependent ESM1 expression was validated on mRNA and protein level in NGFR overexpressed-and NGF treated-MOC2 cells. ESM1 expression has positive correlation with tumor progression and invasion in MOC cells. ESM1 overexpression induced cell viability, migration and invasion of MOC2 cells, whereas ESM1 knockdown reduced proliferation, migration and invasive abilities. Subcutaneous transplanted immunodeficiency mice showed that ESM1 knockdown reduced tumor size and lung metastasis. Decreased expression of ESM1 in NGFR overexpressed-MOC2 cells (MOC2T) also reduced tumor size and invasion activity of MOC2T cells.Taken together, this is the first report identifying ESM1 as a target of NGFR, thereby providing the new interconnection between NGFR and ESM1. We demonstrate that ESM1 knockdown reduce cell proliferation and metastasis of MOC2 cells in vivo and in vitro, thereby underlining that NGFR regulates genes able to induce proliferation and metastasis by themselves. Furthermore, ESM1-alone or in combination with NGFR-might serve as novel prognostic biomarker for oral squamous carcinoma, and may represent an innovative therapeutic approach toward oral squamous carcinoma.