Cripto-1 Expression Research Articles

Abstract 2140: Mesenchymal stromal cells expressing a PEAK1/Cripto axis sustain pro-survival NF-κB signaling in adjacent tumor cells to promote disease progression and therapy resistance

Abstract The cellular and molecular heterogeneity of solid tumors, like breast cancer, is a significant hurdle in the effort to develop effective therapies. The complex breast cancer microenvironment (BCME) includes mesenchymal stromal cells (MSCs) that engage in paracrine/juxtacrine signaling with adjacent tumor cells to support disease progression. Since PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is a cytoskeleton-associated kinase that regulates growth factor-integrin signaling crosstalk in mesenchymal cell types and recent evidence demonstrates that stromal expression of PEAK1 correlates with breast cancer recurrence, we hypothesized that PEAK1 expression in the mesenchymal compartment of the BCME promotes tumorigenesis. We report that PEAK1 is expressed in patient-derived cancer-associated fibroblasts (CAFs) from all breast cancer subtypes. Additionally, PEAK1hi CAF conditioned media promoted breast cancer cell (BCC) proliferation, migration and therapy resistance in vitro. We established in vitro co-culture and in vivo co-xenografting models for evaluating the effects of defined MSC populations on BCC proliferation/survival, metastasis and therapy response. Co-xenografting PEAK1hi CAFs or MSCs with BCCs increased the mass of HER2-positive and ER-positive primary tumors. In agreement with these data, co-culturing MSCs with BCCs promoted cancer cell proliferation and resistance to lapatinib or tamoxifen treatment, while MSC conditioned media supported BCC expansion under serum-free conditions in vitro. Notably, PEAK1 knockdown in MSCs abrogated their ability to promote tumor growth in vivo and therapy/stress resistance in vitro. Using the highly multiplexed cyclic immunofluorescence (CycIF) platform, we analyzed tumor cell states in these MSC-BCC co-cultures at different time points following lapatinib treatment. We demonstrate that MSCs sustain NF-κB (p65) signaling and anti-apoptotic gene expression within BCCs in the presence of lapatinib. Finally, we discovered that PEAK1 is required for MSC expression of Cripto, a GPI-anchored glycoprotein previously reported to activate NF-κB signaling in trans. Taken together, this work identifies new PEAK1-dependent stroma-tumor signaling vulnerabilities that may be exploited for improving patient responses to current therapeutic interventions. Citation Format: Sarkis Hamalian, Robert Güth, Ioannis Zervantonakis, Erika Duell, Jia-Ren Lin, Preston Shisgal, Justin Molnar, Cameron Geller, Megan Agajanian, Julia Tchou, Peter K. Sorger, Joan S. Brugge, Jonathan A. Kelber. Mesenchymal stromal cells expressing a PEAK1/Cripto axis sustain pro-survival NF-κB signaling in adjacent tumor cells to promote disease progression and therapy resistance [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 2140.

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cripto-1 is overexpressed in carcinoma ex pleomorphic adenoma of salivary gland.

Cripto-1 is a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic family. Besides being critical for early embryonic development, Cripto-1 is also associated with the development and behavior of several cancers. We analyzed the immunoexpression of Cripto-1 in normal salivary glands (NSGs), pleomorphic adenomas (PAs), and carcinoma ex pleomorphic adenomas (CaExPAs) of salivary glands. A total of 12 NSGs, 16 PAs and 12 CaExPAs underwent immunohistochemical study by the polymeric biotin-free technique. Immunopositive cells were evaluated semiquantitatively (scores 0-3). For statistical analysis, Mann-Whitney and Kruskal-Wallis tests were performed and a significance level of p ≤ 0.05 was established. Most CaExPAs (n = 10) were strong positive (score 3) for Cripto-1, and only three cases of PAs and two specimens of NSGs exhibited some expression (score 1), being statistically significant these findings (p < 0.001). No difference between the expression of this protein in tumors of major and minor salivary glands was observed. Overexpression was found mainly in cases of CaExPAs with invasive growth (n = 8) when compared to those without capsular invasion (intracapsular pattern) (p = 0.036). Patients with or without lymph node metastasis showed no difference (p = 0.294). The results revealed a significantly higher expression of Cripto-1 in CaExPA compared to PA and NSG, suggesting this protein is possibly deregulated in PA malignant transformation. Furthermore, the increased expression of this protein is associated with a more aggressive behavior (invasive growth) in salivary gland tumors.

Identification of Trophectoderm-Derived Cripto as an Essential Mediator of Embryo Implantation.

Cripto-1 (TDGF1) is a multifunctional signaling factor that stimulates cellular effects, including proliferation, migration, survival, epithelial-to-mesenchymal transition, and angiogenesis, to regulate embryogenesis, tissue homeostasis, and tumorigenesis. Those cell behaviors are also associated with implantation of the embryo into the uterine wall, and this led us to investigate the role of embryo-derived Cripto in embryo attachment and implantation. In this study, we show that Cripto and its signaling mediator GRP78 are uniquely localized to embryo implantation sites. We knocked down Cripto expression specifically in trophoblast cells and found that this resulted in a corresponding decrease in the levels of its downstream signaling mediators, phosphorylated (phospho-)SMAD2, phospho-SRC, phospho-extracellular signal-regulated kinase, and phospho-AKT, which are also known mediators of embryo implantation. We then transplanted Cripto knockdown and control embryos into uteri of pseudopregnant female mice and found that embryos with Cripto-depleted trophoblast cells had dramatically impaired capacity to attach to the uterine wall when compared with controls. This loss of appropriate embryo attachment following Cripto knockdown in trophoblast cells was associated with abnormally enlarged implantation sites that were almost completely devoid of microvessels. A role for Cripto in embryo implantation was further supported by our demonstration that attachment of trophoblast-derived spheroids to endometrial cells in vitro was stimulated by Cripto treatment and diminished by treatment with either of two mechanistically distinct Cripto blocking agents. Collectively, our findings identify Cripto as a novel and critical embryo attachment factor and suggest that modulation of Cripto signaling may have significant therapeutic potential for the treatment of infertility and other related disorders.

The expression of the embryonic gene Cripto-1 is regulated by OCT4 in human embryonal carcinoma NCCIT cells.

Cripto-1 and OCT4, expressed in stem cells and cancers, play important roles in tumorigenesis. Here, we demonstrate that Cripto-1 expression is regulated by OCT4 in human embryonic carcinoma NCCIT cells. The endogenous expression of Cripto-1 and OCT4 is significantly reduced during differentiation. Cripto-1 expression is increased by OCT4 overexpression, but decreased by shRNA-mediated OCT4 knockdown. OCT4 overexpression significantly activates Cripto-1 transcriptional activity. A 5'-upstream minimal promoter sequence in the gene-encoding Cripto-1 is significantly activated by OCT4 overexpression. Mutation of the putative OCT4-binding site abolishes OCT4-mediated activation of the Cripto-1 promoter. The OCT4 transactivation domains mediate transcriptional activity of the Cripto-1 minimal promoter through direct interaction. Taken together, OCT4 plays an important role as a transcriptional activator of Cripto-1 expression in NCCIT cells.

High cripto-1 and low miR-205 expression levels as prognostic markers in early stage non-small cell lung cancer

ObjectivesCripto-1 (CR-1) plays a critical role in the activation of SMAD, SRC, and epithelial-to-mesenchymal transition (EMT) pathways and has been shown to be prognostic in several cancer types. In addition, we showed that CR-1 renders EGFR-mutated NSCLC cells resistant to EGFR-TKI through the activation of SRC and EMT via miR-205 downregulation. This study aimed to investigate the correlation between expression of CR-1 and miR-205 and prognosis of NSCLC patients with or without EGFR mutations. Materials and methodsA total of 265 patients with stage I (AJCC 6th edition) radically resected NSCLC were tested for CR-1 expression and EGFR mutations by immunohistochemistry and miR-205 expression via qPCR assay. ResultsCR-1 expression was evaluated with immunohistochemistry using a tissue microarray on 265 T1-2N0 surgical NSCLC samples. Of the 265 tumors, 250 (94%) expressed various levels of CR-1. A significant inverse correlation was identified between expression of miR-205 and CR-1. NSCLC patients (T1N0, n = 106) with high CR-1 expression had worse prognosis (shorter recurrence-free survival, p = .045) than those with low CR-1 expression. A similar trend was observed in NSCLC patients with normal preoperative carcinoembryonic antigen (CEA) levels (serum CEA levels <5 ng/ml; n = 179; p = .085); however, no significant correlation was found between CR-1 expression and survival rate in the T2N0 or high CEA groups. In addition, NSCLC patients with low miR-205 expression (n = 126) had poorer prognosis in terms of recurrence than those with high miR-205 expression (n = 127; p = .001). ConclusionHigh CR-1 expression is correlated with poor prognosis in NSCLC with low tumor burden and may be used to select high-risk patients for adjuvant chemotherapy in early NSCLC. Moreover, low miR-205 expression likely related to high CR-1 expression could be a prognostic marker for patients with NSCLC.

54P - Cripto-1 expression and its correlation with outcomes in oropharyngeal carcinoma treated by chemoradiation

54P - Cripto-1 expression and its correlation with outcomes in oropharyngeal carcinoma treated by chemoradiation

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 and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer

CRIPTO (CR-1, TDGF1) is a cell surface/secreted oncoprotein actively involved in development and cancer. Here, we report that high expression of CRIPTO correlates with poor survival in stratified risk groups of prostate cancer (PCa) patients. CRIPTO and its signaling partner glucose-regulated protein 78 (GRP78) are highly expressed in PCa metastases and display higher levels in the metastatic ALDHhigh sub-population of PC-3M-Pro4Luc2 PCa cells compared with non-metastatic ALDHlow. Coculture of the osteotropic PC-3M-Pro4Luc2 PCa cells with differentiated primary human osteoblasts induced CRIPTO and GRP78 expression in cancer cells and increases the size of the ALDHhigh sub-population. Additionally, CRIPTO or GRP78 knockdown decreases proliferation, migration, clonogenicity and the size of the metastasis-initiating ALDHhigh sub-population. CRIPTO knockdown reduces the invasion of PC-3M-Pro4Luc2 cells in zebrafish and inhibits bone metastasis in a preclinical mouse model. These results highlight a functional role for CRIPTO and GRP78 in PCa metastasis and suggest that targeting CRIPTO/GRP78 signaling may have significant therapeutic potential.

Biological and clinicopathological significance of Cripto-1 expression in the progression of human ESCC

Human Cripto-1, a member of the EGF-CFC family, is involved in embryonic development, embryonic stem cell maintenance, and tumor progression. It also participates in multiple cell signaling pathways including Wnt, Notch, and TGF-β. Remarkably, it is expressed in cancer stem cell (CSC) compartments, boosting tumor cell migration, invasion, and angiogenesis. Although Cripto-1 is overexpressed in a variety of human malignant tumors, its expression in esophageal squamous cell carcinoma (ESCC) remains unclear. Our aim in this study was to evaluate the possible oncogenic role of Cripto-1 in ESCC progression and elucidate its association with clinicopathological parameters in patients. In this study, Cripto-1 expression in 50 ESCC tissue samples was analyzed and compared to corresponding margin-normal esophageal tissues using quantitative real-time PCR. Cripto-1 was overexpressed in nearly 40% of ESCC samples compared with normal tissue samples. Significant correlations were observed between Cripto-1 expression and tumor differentiation grade, progression stage, and location (p < 0.05). Our results indicate that overexpression of Cripto-1 is involved in the development of ESCC. Further assessment will be necessary to determine the role of Cripto-1 cross talk in ESCC tumorigenesis.

Clinical significance of cripto-1 expression in lung adenocarcinoma.

Cripto-1 can promote tumourigenesis and may be a potential prognostic biomarker in several malignancies, yet little is known about this protein in lung adenocarcinoma (LAC). The aim of this study was to evaluate the prognostic value of cripto-1 expression in a cohort of patients with LAC. Tumours from 290 patients with pathologically confirmed LAC were used for an immunohistochemical analysis of cripto-1 expression. The correlation between cripto-1 expression and the clinicopathological parameters of patients, EGFR-TKI sensitivity was analysed. Significant associations between cripto-1 expression and pT status, pN status, pTNM status, E-cadherin expression and EGFR-TKI sensitivity were identified. Compared with patients with low cripto-1 expression, patients with high cripto-1 expression exhibited significantly poorer progression-free survival (PFS) and overall survival (OS). Moreover, multivariate analyses showed that high cripto-1 expression was an independent predictor of worse survival of patients with LAC. The combination of cripto-1 expression and serum CEA level was correlated with both PFS and OS. In conclusion, cripto-1 may be a potential prognostic biomarker of survival in patients with LAC.

Clinical significance of expression of Cripto-1 and evaluation of its role as biomaker in oropharyngeal squamous cell carcinoma

Clinical significance of expression of Cripto-1 and evaluation of its role as biomaker in oropharyngeal squamous cell carcinoma

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.

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells

Objective: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. Methods: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. Results: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. Conclusion: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

34P The expression and functional role of Cripto-1 in human colorectal cancer

34P The expression and functional role of Cripto-1 in human colorectal cancer

34P The expression and functional role of Cripto-1 in human colorectal cancer

34P The expression and functional role of Cripto-1 in human colorectal cancer

35P Clinical significance of expression of cripto-1 in patients of squamous cell carcinoma of oropharynx

35P Clinical significance of expression of cripto-1 in patients of squamous cell carcinoma of oropharynx

35P Clinical significance of expression of cripto-1 in patients of squamous cell carcinoma of oropharynx

35P Clinical significance of expression of cripto-1 in patients of squamous cell carcinoma of oropharynx