Cripto-1 Research Articles

Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions.

Background/Objectives: Cripto-1 (CR1) is a plurifunctional embryonic protein required for implantation and re-expressed in the adult during wound repair, inflammation, and tumorigenesis. CR1 and its predicted CR1 pseudogene product Cripto-3/CR3 are highly homologous proteins, and given this physical attribute, commercially available antibodies cannot discriminate between CR1 and CR3. Methods: A series of mouse monoclonal antibodies [MoAbs] were developed with a high-affinity binding that can differentiate human CR1/CR3 proteins and showed no measurable cross-reactivity. Results: Using these reagents, we confirm that CR3 is a bona fide translated protein found in human tumor tissue, cancer cell lysates, and in normal/cancer patient donor sera. We also reveal that CR1 and CR3 compete for binding to signal transduction protein Nodal, glucose-regulated protein 78Da (GRP78), and activin receptor-like kinase 4 (Alk4). Our discriminatory MoAbs provide new reagents to help clarify current CR1/CR3 protein expression vagaries in the Cripto field of study, challenging established CR1 conventions. In addition, our data validate CR3 involvement in human carcinogenesis and cell signaling pathways, with potential clinical relevance in determining cancer patient prognosis and disease severity.

Expression and prognostic value of Cripto-1 in early non-small cell lung cancer.

We aim to explore the expression of Cripto-1 (CR-1) protein in patients with early stage non-small cell lung cancer (NSCLC). We investigated CR-1 expression status in specimens obtained from 240 patients with resected NSCLC and 30 cases of para-carcinous normal lung tissues. Compared with normal lung tissue, the positive expression of CR-1 protein in NSCLC was significantly increased (p < 0.005). Cox multivariate regression analysis showed that the expression of CR-1 protein was an independent prognostic factor for early stage NSCLC (p = 0.002). Detecting CR-1 protein can predict the prognosis and recurrence in patients with NSCLC.

Elevated serum Cripto-1 and VEGF levels in patients with non-small cell lung cancer.

Cripto‐1 (CR‐1) facilitates vascular endothelial growth factor (VEGF) expression, and these markers are associated with various tumor cell proliferation, angiogenesis, and metastasis. The main aim of our study was to investigate the clinical value of CR‐1 and VEGF for non‐small cell lung cancer (NSCLC) patients. Serum samples were collected from 312 patients with NSCLC and 120 healthy controls. The levels of CR‐1 and VEGF were measured by enzyme‐linked immunosorbent assay (ELISA). The serum levels of CR‐1 and VEGF in NSCLC patients were significantly higher than those of healthy controls (p < 0.05). Elevated CR‐1 levels were associated with progression of NSCLC stage and higher CR‐1 was detected more in patients with distant metastasis (p < 0.05). Patients exhibiting low levels of serum CR‐1 had better overall survival than those with high levels (p < 0.05). The CR‐1 levels of NSCLC patients with postoperative recurrent were higher than those of nonrecurrent NSCLC patients. Our study suggests that serum CR‐1 and VEGF are useful biomarker for NSCLC patients.

Cripto-1/Glucose-Regulated Protein 78 Affects Proliferation, Migration and Apoptosis of Ovarian Carcinoma Cells

Background: The Cripto-1 (CR-1)/glucose-regulated protein 78 (GRP78) complex was involved in enhancing survival in different types of cells. CR-1 presented increased levels in ovarian carcinoma tissue. However, the potential mechanism of CR-1/GRP78 was unclear in ovarian cancer. Thus, the study aimed to analyze the role of CR-1/GRP78 in ovarian carcinoma cells. Methods and materials: The CR-1 and GRP78 expression in different ovarian cancer cell lines were detected by RT-qPCR and Western blot (WB). Immunoprecipitation assay was performed to analyze whether Cripto-1 interacted with GRP78. The CR-1 interfering plasmids or GRP-78 overexpressing plasmids transfected into cells were used to decrease endogenous CR-1 levels and increase GRP-78 levels. Cell clonogenicity and proliferation capabilities were separately evaluated by clone growth assay, along with the detection of cell migration and invasion abilities by transwell and wound healing assay. In addition, Matrix Metalloproteinases (MMPs) levels were detected by WB. The cell apoptosis was analyzed by Flow Cytometer and the detection of apoptosis-related proteins. Results: The results showed that CR-1 and GRP78 levels were higher in SKOV3 than other cell lines. Furthermore, CR-1 interacted with GRP78 in cells, which formed protein complex. CR-1 silence significantly decreased GRP-78 levels. Moreover, GRP78 overexpression blocked the anti-survival effects caused by CR-1 knockdown. Conclusion: CR-1 silence inhibited cell proliferation and promoted apoptosis via GRP78. It replied that GRP-78 overexpression might enhance the biological functions of CR-1/GRP78 complex ameliorated by CR-1 silence. Thus, CR-1/GRP78 could be a potential target for treating ovarian carcinoma.

Liver-specific over-expression of Cripto-1 in transgenic mice promotes hepatocyte proliferation and deregulated expression of hepatocarcinogenesis-related genes and signaling pathways.

In this study, we investigated the role of embryonic gene Cripto-1 (CR-1) in hepatocellular carcinoma (HCC) using hepatocyte-specific CR-1-overexpressing transgenic mice. The expression of truncated 1.7-kb CR-1 transcript (SF-CR-1) was significantly higher than the full-length 2.0-kb CR-1 transcript (FL-CR-1) in a majority of HCC tissues and cell lines. Moreover, CR-1 mRNA and protein levels were significantly higher in HCC tissues than adjacent normal liver tissues. Hepatocyte-specific over-expression of CR-1 in transgenic mice enhanced hepatocyte proliferation after 2/3 partial hepatectomy (2/3 PHx). CR-1 over-expression significantly increased in vivo xenograft tumor growth of HCC cells in nude mice and in vitro HCC cell proliferation, migration, and invasion. CR-1 over-expression in the transgenic mouse livers deregulated HCC-related signaling pathways such as AKT, Wnt/β-catenin, Stat3, MAPK/ERK, JNK, TGF-β and Notch, as well as expression of HCC-related genes such as CD5L, S100A8, S100A9, Timd4, Orm2, Orm3, PDK4, DMBT1, G0S2, Plk2, Plk3, Gsta1 and Gsta2. However, histological signs of precancerous lesions, hepatocyte dysplasia or HCC formation were not observed in the livers of 3-, 6- or 8-month-old hepatocyte-specific CR-1-overexpressing transgenic mice. These findings demonstrate that liver-specific CR-1 overexpression in transgenic mice deregulates signaling pathways and genes associated with HCC.

New Insights into Cancer Targeted Therapy: Nodal and Cripto-1 as Attractive Candidates

The transforming growth factor beta (TGF-β) signaling is fundamental for correct embryonic development. However, alterations of this pathway have been correlated with oncogenesis, tumor progression and sustaining of cancer stem cells (CSCs). Cripto-1 (CR-1) and Nodal are two embryonic proteins involved in TGF-β signaling. Their expression is almost undetectable in terminally differentiated cells, but they are often re-expressed in tumor cells, especially in CSCs. Moreover, cancer cells that show high levels of CR-1 and/or Nodal display more aggressive phenotypes in vitro, while in vivo their expression correlates with a worse prognosis in several human cancers. The ability to target CSCs still represents an unmet medical need for the complete eradication of certain types of tumors. Given the prognostic role and the selective expression of CR-1 and Nodal on cancer cells, they represent archetypes for targeted therapy. The aim of this review is to clarify the role of CR-1 and Nodal in cancer stem populations and to summarize the current therapeutic strategy to target CSCs using monoclonal antibodies (mAbs) or other molecular tools to interfere with these two proteins.

The role of Nodal and Cripto-1 in human oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common epithelial malignancy of the oral cavity. Nodal and Cripto-1 (CR-1) are important developmental morphogens expressed in several adult cancers and are associated with disease progression. Whether Nodal and CR-1 are simultaneously expressed in the same tumor and how this affects cancer biology are unclear. We investigate the expression and potential role of both Nodal and CR-1 in human OSCC. Immunohistochemistry results show that Nodal and CR-1 are both expressed in the same human OSCC sample and that intensity of Nodal staining is correlated with advanced-stage disease. However, this was not observed with CR-1 staining. Western blot analysis of lysates from two human OSCC line experiments shows expression of CR-1 and Nodal, and their respective signaling molecules, Src and ERK1/2. Treatment of SCC25 and SCC15 cells with both Nodal and CR-1 inhibitors simultaneously resulted in reduced cell viability and reduced levels of P-Src and P-ERK1/2. Further investigation showed that the combination treatment with both Nodal and CR-1 inhibitors was capable of reducing invasiveness of SCC25 cells. Our results show a possible role for Nodal/CR-1 function during progression of human OSCC and that targeting both proteins simultaneously may have therapeutic potential.

Expression of Cripto-1 predicts poor prognosis in stage I non-small cell lung cancer.

Cripto‐1 (CR‐1) is related to the biological behaviour and prognosis of carcinomas. The purpose of this study was to investigate the significance of CR‐1 expression in surgically resected stage I non‐small cell lung cancer (NSCLC). One hundred and forty‐eight patients with completely resected stage I NSCLC and available clinical follow‐up data were assessed. The protein expression of CR‐1 in the tumours was detected by immunohistochemistry. CR‐1 was highly expressed in 64 of 148 tumours. Among patients with high CR‐1 expression, progression‐free survival and overall survival rate were significantly lower than those of patients with low CR‐1 levels (P = .013 and P = .019, respectively). The incidence of distant metastasis in patients with high CR‐1 expression was significantly higher than that of in patients with low CR‐1 expression (57.13% vs 21.43%, P = .001). The results of the multivariate analysis confirmed that a high CR‐1 was a significant factor for poor prognosis. In conclusion, CR‐1 could be a useful prognostic factor in patients with stage I NSCLC, likely as an indicator of the metastatic propensity of the tumour.

Role of Oncofetal Protein CR-1 as aPotential Tumor Marker for Oral Squamous Cell Carcinoma.

Cripto-1 (CR-1) is an oncofetal protein with its role as a key factor in early process of carcinoma has been evaluated in cases of various cancers. However, very few studies have reported its role in oral cancer, which is the sixth most common cancer around the world, particularly with high prevalence in developing countries. Oral squamous cell carcinoma (OSCC) is the most predominant (90%) of all the histological types of oral cancer. Late detection, associated with increased morbidity and mortality, is mainly attributed to non-availability of a suitable biomarker for the disease. In the present pilot study, we have evaluated the role of soluble CR-1, in serum as a potential tumor marker for OSCC. CR-1 was estimated using sandwich ELISA in serum samples of 50 biopsy proven OSCC patients (pre and post treatment) along with age and gender matched healthy controls. Immunohistochemistry was also done in corresponding tumor tissue sections to check the expression of CR-1. Pre-treatment CR-1 was found to be 2.25-fold higher in serum of OSCC patients as compared to control (p < 0.0001***), which was reduced to 1.6 folds post treatment (p = 0.0006***). CR-1 levels were comparatively higher in early stage of disease. Upon IHC 80% of the cases were found to be positive for CR-1. This study provides evidence that serum levels of CR-1 are elevated in patients of Oral Squamous Cell Carcinoma, which decrease post treatment. Also, the association of expression of protein with tumor progression predicts CR-1 as a molecule that can be further evaluated as a potential tumor maker in OSCC.

KRAS mutation and abnormal expression of Cripto-1 as two potential candidate biomarkers for detection of colorectal cancer development.

Colorectal cancer (CRC), regardless of standard procedures of treatment and screening, is still considered one of the deadliest cancers in the Western world, and in economically developed Asian countries, especially Iran. The current study was undertaken to investigate whether changes in the level of Cripto-1 (CR-1) expression and KRAS mutations have a cumulative effect on the onset and progression of CRC. Fifty colorectal tissue samples, including 35 colorectal carcinomas with matching adjacent mucosa, and 15 colorectal adenomas, were chosen for analysis. Twenty-five CRC biopsies and 15 adenoma were analyzed for KRAS mutations by DNA sequencing (Sanger sequencing), and all 50 patients (35 CRCs and 15 adenomas) were evaluated by immunohistochemistry for the CR-1 protein expression. The inducible somatic KRAS mutation (G12D) was observed in nine (36%) of CRC patients, and in two (13.3%) of adenoma patients. The CR-1 expression level in both adenomas (P < .05) and carcinomas (P < .001), were significantly different, compared with the matching adjacent mucosa. The intensity of CR-1 staining in adenomas was less than the intensity of staining, detected in the CRCs (P < .001). The G12D KRAS mutation and CR-1 abnormalities are significantly associated as two signature biomarkers with potential clinical characteristics for the detection of CRC development.

Overexpression levels of cripto-1 predict poor prognosis in patients with prostate cancer following radical prostatectomy.

Overexpression of cripto-1 (CR-1), an epidermal growth factor-cripto-1/FRL-1/Cryptic family protein, has been reported in multiple types of malignancy. However, the clinical functions of CR-1 in prostate cancer (PCa) remain largely unclear. The objective of the present study was to investigate the association between CR-1 expression and the clinicopathological features and prognosis of PCa. CR-1 expression was evaluated in 138 PCa tissues and 67 benign prostate hyperplasia (BPH) tissues using immunohistochemistry. The association between the clinicopathological features of patients with PCa and CR-1 expression was analyzed using a χ2 test. Receiver operating characteristic (ROC) curve and Cox regression model were used to analyze the association between CR-1 expression and biochemical recurrence (BCR)-free survival. It was revealed that the protein expression of CR-1 was markedly higher in PCa tissues than in BPH tissues. The mRNA expression of CR-1 in PCa tissue and cells was also significantly higher than in BPH tissue and the normal RWPE-1 prostate cell line (P<0.05). In addition, high CR-1 expression was significantly associated with prostate-specific antigen level (P=0.008), Gleason score (P=0.011) and lymph node metastasis (P=0.025) in patients with PCa. ROC curve indicated that patients with elevated expression of CR-1 exhibited shorter BCR-free survival (P<0.001). Furthermore, multivariate statistical analysis demonstrated that overexpression of CR-1 may be a novel predictor for prognosis of patients with PCa. Accordingly, the present study considered CR-1 to be a valuable predictor of poor prognosis and progression in PCa, and a potential therapeutic target for patients with PCa.

Abstract LB-248: A novel multiplex assay for detection and quantitation of fatty acid synthase (FASN) in serum or plasma samples

Abstract Many cancers overexpress FASN which catalyzes the final synthetic step of de novo fatty acid synthesis pathway. FASN has been considered as a potential drug target for several decades now. Many publications provide evidence of the elevated FASN with aggressive tumors, but still, there are very few inhibitors of FASN that have successfully been utilized as therapeutics in the clinical arena. An accurate measurement of FASN in association with tumor-specific markers in solid tumors and serum may help to propel forward the therapeutic significance of FASN inhibitors. With this objective in mind, we are currently working on the development of a multiplex assay for detection and quantitation of FASN and Cripto-1 in serum samples. We have optimized a magnetic fluorescent bead based multiplex sandwich immunoassay for detection and quantitation of FASN and Cripto-1 (CR-1) in human serum and plasma samples. We selected Cripto-1 to pair with FASN because it is an EGF-like domain carrying growth factor associated with a number of different types of human carcinomas. We use two different monoclonal antibodies for FASN and one for CR-1 as capture antibodies covalently coupled to magnetic fluorescent beads and a mixture of two biotin-labeled detection antibodies in this assay. The streptavidin-phycoerythrin (SAPE) conjugate is used to report the positive capture of a target in this assay. We have also included various internal controls in this assay to monitor the assay performance and assure quality of the reagents used in each well. The dynamic range of this assay was determined to be 100pg/mL to 100 ng/mL using recombinant FASN and Cripto-1 from commercial sources. The R2 values were 0.98 and above for each of the analytes and recovery of each standard were between 90 % and 125%. We spiked recombinant FASN and Cripto-1 in normal human serum and plasma at different concentrations and spike recovery was between 70 % and 130 %. We further evaluated this assay with some commercially obtained clinical samples. This presentation includes detailed results of this study. Citation Format: Neeraja Venkateswaran, William M. Nelson. A novel multiplex assay for detection and quantitation of fatty acid synthase (FASN) in serum or plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-248.

Diagnostic and prognostic value of serum Cripto‐1 in patients with non‐small cell lung cancer

Cripto-1 (CR-1) is a member of the epidermal growth factor (EGF)-CFC protein family, which is involved in tumor pathogenesis. This study aimed to explore the diagnostic and prognostic value of serum CR-1 level in patients with non-small cell lung cancer (NSCLC). Serum specimens from 312 NSCLC patients and 120 healthy controls were collected. Serum CR-1 level was measured using enzyme-linked immunosorbent assay. The serum CR-1 level was significantly elevated in NSCLC patients compared with healthy controls (P<.001). Higher serum CR-1 level was associated with advanced TNM stage, lymph node metastasis and distant metastasis. With a cutoff value of 1.67ng/mL, CR-1 showed a good diagnostic performance for NSCLC. Kaplan-Meier log rank analysis revealed that the low serum CR-1 patients had a better overall survival (OS) and progression-free survival (PFS) compared with high CR-1 patients (P=.004 and .001, respectively). Further univariate and multivariate Cox regression analyses showed that serum CR-1 level was an independent risk factor of prognosis of NSCLC patients. Our study suggests that serum CR-1 level is a useful diagnostic and prognostic marker for NSCLC patients.

High level of serum Cripto-1 in hepatocellular carcinoma, especially with hepatitis B virus infection.

Background:Human Cripto-1 (CR-1), a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic protein family (EGF-CFC), is highly expressed in a variety of human cancers. We aimed to detect serum CR-1 level in liver diseases especially in hepatocellular carcinoma (HCC) patients.Methods:Serum CR-1 level was Sandwich-type enzyme-linked immuno sorbent assay (ELISA) detected in 330 patients with liver diseases including HCC, cirrhosis, and chronic hepatitis and 50 volunteers without hepatitis B virus (HBV) or hepatitis C virus (HCV) infection as control.Results:The serum CR-1 level was significantly higher in HCC patients than volunteer controls and it was also significantly higher in HBV-related HCC than HCV-related HCC. In addition, serum CR-1 level was correlated with serum alpha-feto-protein (AFP) in HBV-related HCC patients. The serum CR-1 was also higher in cirrhosis and chronic hepatitis than volunteer controls. The serum CR-1 in HBV-related cirrhosis was higher than chronic hepatitis B, but there was no significant difference between HCV-related cirrhosis and chronic hepatitis C.Conclusions:Serum CR-1 was higher in HCC patients and might serve as a complementary biomarker to clinical diagnosis of HBV-related HCC. The high level of serum CR-1 in HBV-related liver disease might be partly attributed to HBV infection.

Abstract 823: Anti-melanoma effects of novel Cripto-1 CFC small peptide mimetics

Abstract The diagnosis of melanoma is increasing and current therapies for advanced disease remain unsuccessful. Thus, research aimed at developing novel targeting approaches is needed. Cripto-1 (CR-1), an epidermal growth factor (EGF)-like developmental morphogen, functions as a co-receptor for the tumor growth factor (TGF)-beta related molecule, Nodal. CR-1/Nodal signaling pathways can induce cell survival, proliferation and migration of cancer cells. Studies have shown that Nodal is expressed at high levels in advanced melanoma. CR-1 has also been shown to be expressed in melanoma but limited to a small subpopulation of melanoma cells with more stem cell-like characteristics. Recent work has demonstrated that targeting Nodal in melanoma is associated with reduction of a significant population of melanoma cells. However, a residual population continues to persist. We hypothesize that interference with CR-1/Nodal receptor binding dynamics will negatively affect downstream Nodal-dependent signaling events important for cancer growth and further reduce the number of surviving melanoma cells. To test this hypothesis, we have developed small peptides that mimic CR-1 CFC binding domain and prevent CR-1 from binding to the ALK4 receptor necessary for proper CR-1 co-receptor function for Nodal. We show that in vitro treatment of human melanoma cells with our prototype CFC mimetic B3 reduced melanoma cell viability and negatively affected melanoma cell cycle progression. We also show that treatment with B3 resulted in reduction of active Smad2/3 and ERK1/2, known to be involved in downstream Nodal signaling, as a result of B3 interference with CR-1 co-receptor function. These preliminary data provide the scientific rationale for the development of anti-CR-1 and Nodal therapeutics that can synergize with or complement current treatment approaches in melanoma. Citation Format: Emanuela Iaccarino, Gustavo Untiveros, Annamaria Sandomenico, Andrea Caporale, Luisa Calvanese, Lucia Falcigno, Gabriella D'Auria, Menotti Ruvo, Luigi Strizzi. Anti-melanoma effects of novel Cripto-1 CFC small peptide mimetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 823.

Knockdown of Cripto-1 inhibits the proliferation, migration, invasion, and angiogenesis in prostate carcinoma cells.

Cripto-1 (CR-1) is a member of the epidermal growth factor-Cripto-1/FRL1/Cryptic gene family that plays a key role in the various malignant cancers. However, the role of CR-1 in prostate carcinoma (PCa) remains limited. The expression of CR-1 was down-regulated by small interfering RNA (siRNA). Western blot measured the expression levels of CR-1 and some related proteins. We performed Cell Counting Kit-8, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and flow cytometry to detect the cellular proliferation and cycle. The transwell assay was used to observe cellular migration and invasion. The ability of angiogenesis was evaluated by tube formation assay. Our results showed that CR-1 knockdown markedly inhibited cell proliferation and induced cycle arrest in G1 phase, as p21 and p27 were up-regulated, whereas cyclin D1 and cyclin E1 were diminished. Moreover, silencing of CR-1 dramatically inhibited cell migration and invasion, repressed matrix metalloproteinases, and disturbed epithelial-mesenchymal transition. CR-1 siRNA suppressed the secreted level of vascular endothelial growth factor, and reduced protein level of Vascular endothelial growth factor receptor 2. We further found that decreased CR-1 expression inhibited FAK/Src/PI3K and Wnt/b-catenin signalling in PCa cells. These results suggested CR-1 might be served as an effective therapeutic target in PCa.

Cripto-1 acts as a functional marker of cancer stem-like cells and predicts prognosis of the patients in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is highly malignant with highly invasive and metastatic capabilities and poor prognosis. It is believed that the ESCC cancer stem-like cells (ECSLCs) are critical for tumorigenicity, invasion and metastasis of ESCC. However, the properties of ECSLCs vary with different markers used in isolation, so that new and more effective markers of ECSLCs need to be identified. This study aimed to estimate the potentiality of Cripto-1 (CR-1) as an ECSLC surface marker and investigate the clinical significance of CR-1 expression in ESCC.MethodsESCC cells with CR-1 high or CR-1low were obtained by flow cytometry then their self-renewal capability and tumorigenicity were compared by colony and limiting dilution sphere formation analysis in vitro and xenograft in nude mice in vivo, respectively. Knockdown of CR-1 expression in ESCC cells was conducted with short hairpin RNA. Cell migration and invasion were examined by scratch test and matrigel transwell assay, respectively. Metastatic capability of ESCC cells was assayed by a mouse tail vein metastasis model. The levels of CR-1 expression in cancerous and paired adjacent normal tissues were assessed by IHC and qRT-RCR.ResultsCR-1high subpopulation of ESCC cells isolated by FACS expressed high level of genes related to stemness and epithelial-mesenchymal transition (EMT), and possessed high capacities of self-renewal, tumorigenesis, invasion and metastasis. Suppression of CR-1 expression significantly reduced the expression of stemness- and EMT-related genes and the capabilities of self-renewal in vitro, tumorigenicity and metastasis in vivo in ESCC cells. In the clinical ESCC specimens, the expression levels of CR-1 in cancerous tissues were positively correlated to TNM stage, invasive depth, and lymph node metastasis. Cox regression analysis indicated that CR-1 was an independent indicator of prognosis. The expression of CR-1 was found overlapping with aldehyde dehydrogenase 1A1 (ALDH1A1), an intracellular marker for ESCLCs, in ESCC cell lines and specimens.ConclusionsCR-1 is a functional and cell surface ECSLC marker, and an independent prognostic indicator as well as a potential therapeutic target for ESCC.

Cripto-1 promotes epithelial-mesenchymal transition in prostate cancer via Wnt/β-catenin signaling.

The Cripto-1 (CR-1) derived EGF-CFC family was overexpressed in tumor development enhancing proliferation, epithelial-mesenchymal transition (EMT) and migration of tumor cells. However, correlation between CR-1 and prostate cancer (PCa) remains still unclear. In the present study, we proved that CR-1 was expressed in PCa and its function was in the progression of PCa. Compared with benign prostatic hyperplasia (BPH) tissues, we confirmed that PCa tissues had high expression of CR-1 by immunohistochemistry and statistical data showed that CR-1 promoted properties of EMT in PCa tissues, including the downregulation of the cell adhesion molecules β-catenin (membrane) and E-cadherin while upregulating transcription factors β-catenin. Overexpression of CR-1 had close relationship with PSA, Gleason, clinical staging and lymph node metastasis in PCa patients. Then, we found that PC-3 cells transfected with CR-1-shRNA inhibited EMT using RT-PCR, RT-qPCR, western blotting and immunofluorescence. Also, we evaluated cell invasive ability invitro by transwell and wound-healing assay. Our data showed that transfected CR-1-shRNA altered EMT including β-catenin, E-cadherin, c-myc, GSK-3, p-GSK and Wnt/β-catenin pathway in PC-3. It also suppressed PC-3 cell migration. Additionally, our results displayed that Licl had antitumor activity against PC-3 through the inhibition of Wnt/β-catenin pathway. Inhibition of cell viability was dose-time dependent. The present study proved that CR-1 regulates EMT of PCa by Wnt/β-catenin pathway. Hence, CR-1 may provide a new biological marker, and possibly contributes to clinical treatment against PCa.

Cripto-1 and RUNX2 Expressions in Non-small Cell Lung Cancer, their Roles in its Progression and Patients Outcome

Background: Non-small cell carcinoma of lung (NSCLC) is the commonest and most lethal lung cancer type; it includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma subtypes. The five-year survival rate in NSCLC patients is still very low although improvements in treatment modalities are still emerging. Hence, new prognostic markers and therapies need to be brought to light aiming to improve patients' outcome. Cripto-1 (CR-1) is one of the family members of epidermal-growth-factor; cripto FRL1 cryptic-(EGF-CFC) is needed for embryogenesis. Runt-related-transcription-factor [RUNX] family members make core-binding factor complex (CBFC) that attach to DNA to stimulate or inhibit many genes transcription, regulate the survival, differentiation and maturation of many tissues. The aim of this work is to detect the clinical significance and prognostic role of CR-1 and RUNX2 expressions in NSCLC using immunohistochemistry. Method: CR-1 and RUNX2 expressions were evaluated in 59 paraffin blocks sections of NSCLC. The relationship between their level of expressions and patient's prognosis was analyzed. Results: CR-1 and RUNX2 were highly expressed in NSCLC patients, 59.3% and 67.8%, respectively. There was a significant positive association between their expressions in NSCLC patients (p=0.015). Both markers were significantly correlated with size, grade, stage, site of the tumor within the lung, malignant (pleural and/or pericardial) effusion, presence of distant metastases, ECOG performance status of the patients (p<0.001) and existence of hepatic metastases (p=0.004). Both markers expressions were significantly correlated with poor response to treatment (p<0.001). After a median follow up of 30 months, mean PFS of NSCLC patients having elevated CR-1 and RUNX2 expressions was shorter (p<0.001). Patients with high RUNX2 expressing have significantly shorter mean OS (p=0.025). High CR-1 expression negatively affected OS but that was not statistically significant (p=0.2). Conclusion: NSCLC patients with elevated CR-1 and RUNX2 expression values had unfavorable prognosis.

Serum cripto-1 as a clinical marker for lung cancer.

Cripto-1 (CR-1) plays an important role in angiogenesis related to tumor growth, in concert with vascular endothelial growth factor (VEGF), and enhanced expression of CR-1 has been reported in lung cancer tissue. Patients with lung cancer (n = 156) and healthy volunteers (n = 60) were enrolled in the study. Serum CR-1 and VEGF concentrations were measured using enzyme-linked immunosorbent assay. Patients with lung cancer had higher serum CR-1 (4.03 ± 1.49 ng/mL vs. 1.13 ± 0.43 ng/mL, p&lt;0.05) and VEGF (503.62 ± 112.74 pg/mL vs. 398.62 ± 117.84 pg/mL, p&lt;0.05) levels than the control group. With stage progression in non-small cell lung cancer (NSCLC), serum CR-1 levels increased, and patients with distant metastasis had higher levels than those without metastasis (p&lt;0.05). In NSCLC, the low CR-1 group (&lt;1.54 ng/mL) had a better overall survival rate compared with the high CR-1 group (&gt;1.54 ng/mL, p&lt;0.05). Our study suggests that serum CR-1 is a useful diagnostic and prognostic marker for NSCLC patients.