Novel Tumor Biomarkers CAPS And ASCL2 Predict The Prognosis Of Patients And The Underlying Mechanisms

Part 1. Calcium binding protein calcyphosine (CAPS) regulates self-renewal of glioma stem cellsObjective:Glioma is the most common brain malignancy, accounting for 27.5% of all primary brain tumors and 80% of primary malignant brain tumors. Despite the therapies including surgical resection, radiotherapy, chemotherapy and anti-angiogenesis therapy have been notably improved, the overall patient survival remains poor. The glioblastoma multiform(GBM), which accounts for 54.9% of glioma, suffers a 5-year overall survival rate less than 5%. The unfavorable outcome is mainly due to the less understanding of cytological bases and molecular mechanisms for glioma initiation and progression. Glioma stem cells (GSCs),which involved in glioma initiation, therapeutic resistance, tumor recurrence and poor outcome, are considered as novel target for the treatment. Our previous work revealed that calcyphosine (CAPS), a member of calcium binding protein (CBP) with four EF-hand motifs,was aberrantly expressed in GSCs than in monolayer glioma cells. However, the clinical relevance and biological functions of CAPS in glioma remain unclear.Methods and results:1. The data from The Cancer Genomic Atlas (TCGA) database, The Repository for Molecular BRAin Neoplasia DaTa (REMBRANDT) database and the Chinese Glioma Genomic Atlas (CGGA) database was obtained to evaluate the expression pattern and clinical significance of CAPS mRNA expression. In TCGA cohort,671 glioma patients and 5 normal controls were analyzed for the association between CAPS mRNA expression and clinicopathological features. The median intensity of CAPS mRNA in glioma vs. normal controls was -0.27 ±1.31 vs. -1.46 ± 0.74 (p=0.020). The expression level of CAPS was elevated as the WHO grade increase (p<0.001). The mean intensity of CAPS in patients with grade ?,grade ? and grade ? was -0.64 ± 1.04, -0.32 ± 1.32 and 0.19 ± 1.42, respectively.Kaplan-Meier analysis showed that patients with high CAPS expression (CAPShigh) suffered poorer overall survival rate than those with low CAPS expression (CAPSlow). Moreover, the CAPShigh group possessed higher recurremt rate than the CAPSlow group (p=0.019).Accordingly, the median disease-free survival time of CAPShigh group (35.0 (95% CI 25.9-44.0) months) was obviously shorter than that in the CAPSlow group (55.1 (95% CI 47.5-62.8)months).In REMBRANDT cohort, 588 glioma specimens (including 138 astrocytoma, 59 oligodendroglioma, 11 mixed glioma and 380 glioblastoma multiform) and 28 normal controls were interrogated to determine the CAPS expression pattern and its prognostic value.CAPS expression was significantly elevated in glioma compared with normal brain tissues(4.30 ± 6.03 vs. 1.36 ± 1.13, p<0.001). The intensity of CAPS mRNA was found low in patients with grade ?/? (3.34 ± 6.20),middle in patients with grade ? (3.50 ± 4.81),and high in patients with grade IV (4.43 ± 5.02). Similarly, the overall survival was poorer in CAPS group than in CAPSlow group (p<0.001).In CGGA cohort, 220 glioma sepcimens and 5 normal controls were interrogated to evaluate the CAPS mRNA expression and its prognostic significance in glioma. Consistent with the results in TCGA and REMBRANDNT cohorts, the data in CGGA cohort also revealed an elevated level of CAPS expression in glioma compared to normal controls (0.20± 1.19 vs. -0.68 ± 0.66, p<0.001),and the intensity of CAPS mRNA was elevated grade by grade (p<0.001). Kaplan-Meier analysis and Cox-regression analysis indicated that CAPS was an independent poor prognostic factor for the patients with glioma (HR=2.057, p=0.003).2. We detected the expression of endogenous CAPS in primary GBM cells (GBM-1/2),glioma cell lines (U251 and U87) and normal human glial cell line (HEB). High levels of CAPS expression were observed in glioma cells than in the normal glial cells both in mRNA and protein levels. Moreover, GSCs isolated from GBM-1,U87 and U251 possessed higher levels of CAPS than that in the monolayer cells.3. U251 and GBM-1 cells with CAPS-knockdown were established by lentivirus targeting CAPS gene. Compared to control cells, the CAPS-knockdown cells of U251 and GBM-1 showed significantly decreased clone formation efficiency and neurosphere formation efficiency in vitro. The tumorigenicity assay also revealed that knockdown of CAPS expression hampered the tumorigenesity capability of GSCs in vivo.4. Human glioma cell line U87 with CAPS-overexpression was well established and vertified by qRCR and western blotting assay. Overexpression of CAPS significantly enhanced the clone formation efficiency and neurosphere formation efficiency in vitro and tumorigenesis capability in vivo.5. To clarify the molecular mechanisms how CAPS regulates the self-renewal of GSCs,we explored the alteration of stemness-related genes and Notchl signaling. In the CAPS-knockdown cells of U251 and GBM-1, the expression of sternness-related molaculars Oct4,Sox2 and Nanog were markedly reduced, meanwhile, Notchl signaling was downregulated with the deficiency of Hes1, Hes5 and CBF1 expression. In the U87 cells with CAPS-overexpressed, expression of Oct4, Sox2 and Nanog were enhanced both in mRNA and protein levels, and Notchl signaling was upregulated with elevated levels of Hesl, Hes5 and CBF1.CAPS was considered as a CBP and the cross-talk of cAMP cascade and PIP3 cascade.Thus, we applied immunoprecipitation assay to determine whether it interacted with other CBPs. The mass spectrum and co-immunoprecipitation assay revealed that CAPS could form complex with ANXA2. The CAPS-ANXA2 complex might be involved in the regulation process of GSCs.Conclusion:The expression levels of CAPS are significantly increased in glioma when compared with the normal brain tissues in TCGA, REMBRANDT and CGGA cohorts. Elevated expression of CAPS is correlated with tumor grade positively. Kaplan-Meier analysis reveals that patients with CAPShigh suffers poorer overall survival than those with CAPSlow. Cellularly,CAPS is significantly overexpressed in GSCs compared with the monolayer cells. Reducing CAPS expression hampers the charcteristics of GSCs, while overexpression of CAPS promotes self-renewal ability of GSCs in vitro and in vivo. Molecularly, CAPS upregulates the expression of sternness-related moleculars Oct4, Sox2 and Nanog, as well as the Notchl signaling by increasing the expression of Notch1. In addition, interaction between CAPS and ANXA2 may be involved in the regulation process of GSCs.Part II. The expression pattern and prognostic value of ASCL2 innon-small cell lung cancerObjective:Lung cancer is the second most common (accounting for 13.3%) cancer type and the first leading cause (accounting for 26.8%) of cancer-related deaths worldwide. The lung squamous cell carcinoma (SCC) and lung adenocarcinoma (AC) are two main subtypes of non small-cell lung cancer (NSCLC), which accounts for more than 80% of lung cancer.Despite various therapies are applied, the 5-year overall survival rate of the NSCLC patients remains under 20%. Recently, Achaetescute-like 2 (ASCL2), a member of the basic helix-loop-helix (bHLH) family of transcription factors, was reported to be aberrantly overexpressed and involved in tumor progression in gastrointestinal carcinomas. However,the expression pattern and clinical significance of ASCL2 in NSCLC remain unclear. This study aimed to evaluate the protein expression levels of ASCL2 in SCC and AC, and determine the prognostic value for the patients.Methods and results:1. Immunochemical (IHC) staining was performed to detect the expression levels of ASCL2 in 10 samples of normal lung tissue,79 cases of SCC and 67 cases of AC specimens.The mean IHC scores of ASCL2 in SCC vs. normal bronchia and AC vs. normal alveoli were 5.89 ± 2.79 vs. 2.40 ± 1.02 (p<0.001) and 4.78 ± 2.92 vs. 2.40 ± 0.92 (p=0.014), respectively.The SCC exhibited higher expression levels than that in AC (5.89 ± 2.79 vs. 4.78 ? 2.92,p=0.002).2. The relationship between ASCL2 expression and various clinicopathological characters was evaluated. High expression of ASCL2 was correlated with advanced TNM stages (? and ?) (p=0.010) and worse differentiation status (p=0.010) in SCC, but not with age, gender, smoking, T status and N status (all p>0.05). Within AC, high expression of ASCL2 correlated to advanced TNM stages (? and ?) (p=0.018) and T status (p=0.004),but not to age, gender, smoking, differentiation status and N status (all p>0.05).3. Kaplan-Meier analysis showed that the patients with high levels of ASCL2 had significant worse overall survival than those with low levels of ASCL2 (p=0.004) in SCC.Accordingly, median overall survival time in ASCL2low group (74.8 (95% CI 65.9-83.7)months) was obviously longer than that in the ASCL2high group (53.3 (95% CI 43.4-63.2)months). However, we did not find significant association between ASCL2 expression and overall survival time in AC (p=0.172). Multivariate Cox-regression analysis also revealed that elevated expression of ASCL2 could be an independent prognostic factor (HR=2.692,p=0.041) in SCC,but not in AC (HR= 1.528,p=0.256).4. By querying the TCGA database through the Open-Access and Controlled-Access Data Tiers Portal, 351 SCC and 439 AC paired with 50 normal bronchia and 58 normal alveoli, were interrogated to evaluate the mRNA expression level and prognostic significance of ASCL2 mRNA in SCC and AC. The mean intensity of ASCL2 in SCC vs. normal bronchia and AC vs. normal alveoli were 0.97 ± 1.44 vs. -0.65 ± 0.68 (p<0.001) and -0.04 ± 1.47 vs.-0.4 ± 0.81 (p<0.001). Consistent with the results in IHC corhot, the data in TCGA cohort also revealed that ASCL2high patients suffered significant worse (p=0.016) overall survival rate than ASCL2low patients in SCC, while no significant association was observed between ASCL2 mRNA expression and patients outcome in AC.5. The IHC staining revealed that ASCL2 mainly located in the cytoplasm, but rarely in nucleus both in cancer and normal tissue. Our observation was differnent from previous reports, which revealed that ASCL2 was a transcription factor and localized in the nucleus.Then, we applied immunofluorescent staining in lung caner cells and frozen tissues to explore the subcellular localization of ASCL2. The IF staining showed that ASCL2 was mainly located in the cytoplasm in cells including normal bronchial epithelium cell line HBE, AC cell line A549 and lung lare cell carcinoma cell line NCI-H460. The positive staining signals in frozen tissues were also mainly located in cytoplasm.Conclusion:The expression levels of ASCL2 are significantly increased both in SCC and AC when compared with the normal lung tissue. Elevated expression of ASCL2 is correlated with advanced TNM stages (? and ?) and worse differentiation status in SCC, whereas, only a positive correlation between ASCL2 expression levels and advanced TNM stages is observed in AC. Kaplan-Meier analysis and Cox-regression analysis indicates that ASCL2 is an independent prognostic indicator in SCC exclusively. Subcelluarly, ASCL2 is mainly localized in the cytoplasm in NSCLC tissues.