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Effects of anti-Nodal antibody treatment on SK-MEL-28 cells.

DTIC and anti-Nodal antibody combination treatment promotes cell death in SK-MEL-28 cells.

Nodal in SK-MEL-28 cells surviving treatment with the B-RAF inhibitor, RG7204.

Nodal expression in DTIC-treated melanoma cell lines.

Effects on DTIC-treated melanoma cell lines.

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Figure legends for supplementary figures 1-5

Supplementary Tables 1-4 from Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

Supplementary Figure 5 from Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

Metastatic melanoma is an aggressive skin cancer associated with poor prognosis. The reactivation of the embryonic morphogen Nodal in metastatic melanoma has previously been shown to regulate the aggressive behavior of these tumor cells. During the establishment of left-right asymmetry in early vertebrate development, Nodal expression is specifically regulated by a Notch signaling pathway. We hypothesize that a similar relationship between Notch and Nodal may be reestablished in melanoma. In this study, we investigate whether cross talk between the Notch and Nodal pathways can explain the reactivation of Nodal in aggressive metastatic melanoma cells. We show a molecular link between Notch and Nodal signaling in the aggressive melanoma cell line MV3 via the activity of an RBPJ-dependent Nodal enhancer element. We show a precise correlation between Notch4 and Nodal expression in multiple aggressive cell lines but not poorly aggressive cell lines. Surprisingly, Notch4 is specifically required for expression of Nodal in aggressive cells and plays a vital role both in the balance of cell growth and in the regulation of the aggressive phenotype. In addition, Notch4 function in vasculogenic mimicry and anchorage-independent growth in vitro is due in part to Notch4 regulation of Nodal. This study identifies an important role for cross talk between Notch4 and Nodal in metastatic melanoma, placing Notch4 upstream of Nodal, and offers a potential molecular target for melanoma therapy. Cancer Res; 70(24); 10340–50. ©2010 AACR.

Supplementary Figure Legends 1-5 from Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

Supplementary Figure 3 from Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

Supplementary Figure 1 from Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

Supplementary Figure 2 from Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

Supplementary Methods from Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

Supplementary Figure 4 from Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

Nodal is a TGF-β–related embryonic morphogen that is expressed in multiple human cancers. Detection of Nodal expression in these tissues can be challenging if issues related to Nodal transcription and protein processing are not considered. Here, we discuss certain characteristics related to Nodal expression and function and how these can facilitate acquisition and interpretation of expression data, contributing to our understanding of the potential role of Nodal in human cancer. We also discuss how Nodal could be exploited clinically as a novel biomarker for cancer progression and therapeutic target. Cancer Res; 72(8); 1915–20. ©2012 AACR.

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.

Aggressive tumor cells express a plastic, multipotent phenotype similar to embryonic stem cells. However, the absence of major regulatory checkpoints in these tumor cells allows aberrant activation of embryonic signaling pathways, which seems to contribute to their plastic phenotype. Emerging evidence showing the molecular cross-talk between two major stem cell signaling pathways Nodal and Notch suggests a promising therapeutic strategy that could target aggressive tumor cells on the basis of their unique plasticity, and provide new insights into the mechanisms underlying the re-emergence of developmental signaling pathways during tumor progression. [Cancer Res 2009;69(18):7131–4]

Pluripotent cells within embryonal carcinoma (EC) can differentiate in vivo or in vitro on treatment with specific agents. Differentiating EC cells express lower levels of stem cell–related genes, such as Cripto-1. We show that migration of human EC cells (NTERA/2 and NCCIT) can be reduced following treatment with the guidance molecule Netrin-1. Moreover, Netrin-1 treatment increased the levels of β-III tubulin, glial filament acidic protein, Nestin, and γ-aminobutyric acid and reduced the expressions of Cripto-1, Nanog, and Oct4 in EC cells. These Netrin-1–induced effects in the EC cells were mediated via binding of Netrin-1 to the Neogenin receptor and activation of SHP-2, resulting in increased levels of inactive phosphorylated c-src(Y527). These results suggest that Netrin-1 can induce neuroectodermal-like differentiation of human EC cells by affecting c-src signaling via SHP-2 activation and regulation of Nanog, Oct4, and Cripto-1 expressions. [Cancer Res 2009;69(5):1717–21]

Purpose: Human Cripto-1 (CR-1), a cell membrane glycosylphosphatidylinositol-anchored glycoprotein that can also be cleaved from the membrane, is expressed at high levels in several different types of human tumors. We evaluated whether CR-1 is present in the plasma of patients with breast and colon cancer, and if it can represent a new biomarker for these malignancies. Experimental Design: We determined CR-1 plasma levels using a sandwich-type ELISA in 21 healthy volunteers, 54 patients with breast cancer, 33 patients with colon carcinoma, and 21 patients with benign breast lesions. Immunohistochemical analysis was also used to assess CR-1 expression in cancerous tissues. Results: Very low levels of CR-1 (mean ± SD) were detected in the plasma of healthy volunteers (0.32 ± 0.19 ng/mL). A statistically significant increase in the levels of plasma CR-1 was found in patients with colon carcinoma (4.68 ± 3.5 ng/mL) and in patients with breast carcinoma (2.97 ± 1.48 ng/mL; P < 0.001). Although moderate levels of plasma CR-1 were found in women with benign lesions of the breast (1.7 ± 0.99 ng/mL), these levels were significantly lower than in patients with breast cancer (P < 0.001). Finally, immunohistochemical analysis and real-time reverse transcription-PCR confirmed strong positivity for CR-1 in colon and/or breast tumor tissues. Conclusion: This study suggests that plasma CR-1 might represent a novel biomarker for the detection of breast and colon carcinomas.

Triple negative breast cancer represents an aggressive malignancy for which few targeted therapies exist. The embryonic morphogen Nodal is re-expressed in highly aggressive breast cancers and reduction of Nodal signaling decreases tumor cell metastatic characteristics. This represents a potential avenue for the development of therapeutics aimed at inhibiting the Nodal pathway. To explore this further, we evaluated Nodal expression in breast cancer cells following chemotherapy. In vitro treatment of MDA-MB-231 and MDA-MB-468 human breast cancer cell lines with Doxorubicin, a chemotherapy commonly used for the treatment of malignant breast cancer, resulted in the generation of surviving subpopulations that retained Nodal expression (>80%) and remained viable. To validate these findings in vivo, we analyzed Doxorubicin treated metastatic patient derived breast cancer xenografts (PDX) which displayed a residual population of Nodal positive cells when analyzed by immunohistochemistry. Based on these findings, we hypothesize that targeting Nodal in a combinatorial approach with chemotherapy will lead to a reduction in remaining viable populations of cancer cells. When we utilized an anti-Nodal neutralizing antibody to inhibit Nodal signaling, antibody treatment reduced cell proliferation, attachment and increased rates of apoptosis in Doxorubicin treated breast cancer cell lines. Decreases in phosphorylation of Histone 3, a marker of cellular proliferation, and increases in the cleavage of PARP, correlating to induction of apoptosis, were noted in response to antibody treatment. These data suggest that Nodal signaling plays a critical role in the growth of metastatic breast cancer cells and targeting Nodal may improve therapeutic outcome in malignancies such as triple negative breast cancer. The initial results highlight translational potential for the development of humanized antibodies capable of inhibiting Nodal function. Citation Format: Thomas M. Bodenstine, Grace S. Chandler, Naira V. Margaryan, Luigi Strizzi, Alina Gilgur, Elisabeth A. Seftor, Richard E B Seftor, Zhila Khalkhali-Ellis, Andrey Ugolkov, Andrew P. Mazar, Mary J C Hendrix. Targeting the embryonic morphogen Nodal reduces viability of doxorubicin-treated breast cancer cells in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2015-4378

Neuroblastoma is a devastating pediatric cancer and most patients older than 18 months present with multi-organ metastatic disease. High grade or recurrent disease is refractory to treatment with chemotherapy and almost uniformly fatal. Because GSK-3beta has been shown to be a positive regulator of NF-kappaB-mediated survival and chemoresistance in cancer cells, we hypothesize that the inhibition of GSK-3 may have potential therapeutic effects in neuroblastoma. To test this premise we used small molecule inhibitors, Western blotting, apoptotic and MTS assays to study the effect(s) of GSK-3 inactivation in neuroblastoma cell lines SK-N-DZ and SK-N-BE(2). Using chemically distinct GSK-3 inhibitors (AR-A014418, TDZD8 and 9-ING-41), we found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of neuroblastoma cells. We observed that inhibition of GSK-3 results in decreased expression of the anti-apoptotic molecules Bcl-XL and XIAP (NF-kappaB target genes) and a subsequent increase in neuroblastoma cell apoptosis. Moreover, we show that our novel GSK-3 inhibitor 9-ING-41 enhances the antitumor effects of irinotecan in neuroblastoma cells. Our results demonstrate that GSK-3 positively regulates neuroblastoma cell survival and proliferation and suggest that the inhibition of GSK-3 is a promising new approach for the treatment of neuroblastoma. Citation Format: Oleksii Dubrovskyi, Andrey Ugolkov, Irina Gaisina, Gennadiy Bondarenko, Luigi Strizzi, Naira Margaryan, Thomas O'Halloran, Alan Kozikowski, Mary Hendrix, Andrew Mazar. Targeting GSK-3: a new approach for the treatment of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3287. doi:10.1158/1538-7445.AM2015-3287

It is widely accepted that the microenvironment/niche exert genetic and/or epigenetic effects on stem cells. Current studies have demonstrated that an aberrant microenvironment may contribute to the genesis of cancer stem cells (CSCs). In the case of cancer, growing evidence indicate a cancerous niche can play an active role in the regulation of tumor cell maintenance and progression through exosomes/microvesicles-based intercellular communication. It has not been reported, however, whether this vesicle-mediated communication induces the malignant transformation of normal stem cells/differentiating progenitors, result in CSC generation. Recentry, we have reported that the conditioned medium derived from the mouse Lewis Lung Carcinoma (LLC) cell line can convert mouse induced pluripotent stem cells (miPSCs) into CSCs. Here, we investigated the contribution of tumor-derived exosomes/microvesicles (TEMVs) that are secreted from LLC cells to induce the transformation of miPSCs into CSCs. The differentiating miPSCs were exposed to TEMVs derived from LLC cells for 4 weeks. We used Nanog-GFP miPS, in which GFP expression is under control of Nanog promoter, indicative of undifferentiation state. During this period, we observed the reemergence of the GFP-positive colonies, whereas most of cells were differentiated and GFP-negative without TEMV-treatment. The resultant TEMVs treated cells (miPS-LLCemv) expressed Nanog and Oct3/4 proteins comparable to miPSCs assessed by Western blotting. The sphere formation of the miPS-LLCemv cells in suspension culture indicated the selfrenewal capacity of the miPS-LLCemv cells. When the miPSLLCemv cells were subcutaneously allografted into immunodeficient mice, malignant tumors with extensive angiogenesis developed. Phathological features such as vementin, Ki67, PPAR gamma expression, and accumulation of lipid droplets in tumor revealed these tumors were liposarcomas. Tumor cells established from liposarcomas showed the capacity of spheroid formation in suspension culture and the capacity of differntiation into adipocytes and endothelial cells. These results indicate that miPS-LLCemv cells and established tumor cells possess CSC properties. Interestingly, these cells expressed Sox2 and Klf4 genes significantly higher than parental miPS cells, might be related to selfrenewal and addipogenesis. Thus, we concluded that TEMVs should have the potential to induce CSC properties in normal stem cells. Citation Format: Yan Ting, Junko Masuda, Akifumi Mizutani, Ling Chen, Tsukasa Shigehiro, Shuichi Matsuda, Tomonari Kasai, Takayuki Kudoh, Hiroshi Murakami, Mary J.C. Hendrix, Luigi Strizzi, David S. Salomon, Li Fu, Masaharu Seno. Characterization of cancer stem-like cells derived from mouse induced pluripotent stem cells transformed by tumor-derived exosomes/microvesicles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3016. doi:10.1158/1538-7445.AM2014-3016

Aggressive tumor cells can show characteristics similar to normal embryogenic progenitor cells, and de-regulated embryonic developmental signaling networks have been linked to cancer initiation and progression. Thus, cell fate regulation in embryonic development and cell transformation during oncogenesis may share common signaling pathways. Several tumor types have been suggested to be initiated from stem-like cells. Exploring in tumors, the re-emergence of embryonic signaling pathways involved in stem cell self-renewal can produce new insight into the initiation and progression of cancer and may lead to development of new therapeutic strategies. The Nodal pathway is required for maintenance of undifferentiated embryonic stem cells. Nodal is a morphogen that belongs to the TGF-beta family. Its signaling can be regulated by co-factors such as the Epidermal Growth Factor - like member, Cripto-1 and another TGF-beta member, Lefty. Nodal and Cripto-1 are critical in embryonic development and is generally poorly detected in differentiated tissues. However, an increased expression has been detected in different human tumor types. The dismal prognosis of glioblastomas calls for increased biological knowledge and novel therapeutic targets. Gene expression studies in a glioblastoma xenograft model, where multicellular spheroids from glioblastoma patient biopsies were generated and implanted intracranially in nude rats and where highly infiltrative non-angiogenic tumors can switch to angiogenic tumors upon in vivo intracranial passaging, has revealed importance of gene ontology categories connected to development and negative regulators of differentiation. In this model we detected several significant overrepresented morphogenesis categories, and we hypothesized that increased activity in Nodal signaling also could be found in human glioblastomas. We assessed the gene expression of Nodal, Cripto-1 and Lefty in microarrays of infiltrative and angiogenic xenograft samples developed from four glioblastoma patients. The genes were expressed both in the infiltrative and angiogenic xenografts. Further we examined Nodal, Cripto-1 and Lefty expression in 199 primary glioblastomas by immunohistochemistry, and assessed their potential clinical relevance by analyzing the association between their protein expression and patient survival. A weak to moderate protein expression of Nodal and Lefty and a stronger expression of Cripto-1 was found in the patient samples. Interestingly a significant association between patient age, Cripto-1 expression and survival was demonstrated, where higher Cripto-1 scores were associated with poorer survival in younger patients. These findings suggest that Cripto-1 may represent a novel negative prognostic marker in younger glioblastoma patients and also a potential therapeutic target in this patient group. Citation Format: Berit B. Tysnes, Hege AA Sætran, Sverre J. Mørk, Naira V. Margaryan, Geir E. Eide, Rolf Bjerkvig, Luigi Strizzi, Mary J.C. Hendrix. Age-dependent association between protein expression of the embryonic stem cell marker Cripto-1 and survival of glioblastoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2013-2650

Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. The current lack of effective therapies for treating metastatic melanoma exemplifies the desperate need for the identification of new targets and the development of new therapies. Nodal is an embryonic signaling molecule from the TGFβ superfamily that is not typically expressed in adult tissues but is reactivated in a number of aggressive cancers including melanoma, and breast and prostate carcinomas. In a study recently published by our group, we determined a connection between the Notch and Nodal pathways in aggressive melanoma. We identified a specific correlation between the expression of Notch4 and Nodal in aggressive melanoma cell lines and in advanced stage human melanomas. By inhibiting Notch4 expression or function, we established a link between Notch4 and Nodal signaling in promoting the aggressive phenotype of metastatic melanoma cells in vitro. Notch4 regulation of Nodal expression was identified as important in cellular plasticity, since inhibition of Notch4 function impaired the ability of aggressive cells to engage in vasculogenic mimicry (de novo formation of vascular-like networks by non-endothelial tumor cells). Notably, vascular-like network formation could be partially rescued by recombinant human Nodal, suggesting both pathways are important for this phenomenon. To extend our published observations, we utilized neutralizing antibodies to Notch4 and to Nodal, and compared gene expression profiles. From this, we identified a mechanism of feedback between signaling pathways that controls the expression of components of both pathways, including Nodal, Notch4, and the Notch ligand, Jagged2. Since Notch4 is enriched in the subpopulation of cells that form vascular-like networks in melanoma, we specifically analyzed the expression of angiogenesis signature genes previously associated with vascular-like network formation, and determined that inhibition of Notch4 or Nodal function downregulates a large group of these genes including members of the VEGF, angiopoietin, MMP, and cadherin families. We are currently investigating the hypothesis that the Notch4-Nodal signaling axis may be a master regulator of the vascular-like phenotype in aggressive melanoma cells. We suggest that inhibition of Notch4 and/or Nodal function may offer a potential molecular targeting strategy for metastatic melanoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5204. doi:1538-7445.AM2012-5204

The embryonic morphogen Nodal is a potent inducer of EMT and critical mediator of stem cell pluripotency in the developing embryo, but is not expressed in normal adult tissues. Intriguingly, our lab has recently shown that Nodal, a TGF-beta family member, is in fact secreted by melanoma and breast cancer cells and that Nodal expression positively correlates with tumor grade in human patient samples. Our current studies demonstrate a positive role for Nodal in regulating breast cancer cell renewal and aggressiveness. Specifically, short hairpin (shRNA) knockdown of Nodal expression in human breast cancer cell lines diminishes proliferation, invasion and clonogenicity of these cell lines in vitro and dramatically curtails tumor formation in a mouse xenograft model. Additionally, our data suggest that these effects may be caused, in part, by alterations in the expression of adhesion receptors known to function in tumor engraftment and metastasis. Taken together, our data suggest that Nodal signaling may facilitate the acquisition of self-renewal and migratory capabilities in breast cancer cells and represent a novel therapeutic target for disrupting these processes in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3353. doi:1538-7445.AM2012-3353

The diagnosis of melanoma is becoming more frequent. Although surgical excision of early lesions is associated with relatively significant high cure rates, treatment modalities are largely unsuccessful for advanced disease. Characteristics such as cellular heterogeneity, phenotypic plasticity and the expression of embryonic related molecules and signaling pathways that are important for self renewal and pluripotency, present a challenge in targeting the most aggressive tumor cells. However, the absence of major regulatory checkpoints in these tumor cells allows aberrant activation of embryonic signaling pathways. In particular, the embryonic morphogen Nodal, belonging to the TGF-β superfamily, is an important regulator of embryonic stem cell fate. We have recently demonstrated that Nodal is also expressed in aggressive melanoma, although unlike embryonic stem cells, melanoma does not express Lefty, a natural inhibitor of Nodal, thus allowing Nodal to be expressed in an unregulated manner. Further analysis revealed that Lefty is silenced in these tumor cells by DNA methylation. Down-regulation of Nodal expression in melanoma cells results in a significant reduction in clonogenicity and suppression of tumorigenesis. Additional translational studies indicate that antibodies against Nodal are capable of targeting and inducing apoptosis in human melanoma tumors lodged in the lungs of nude mice. Furthermore, our data show another stem cell signaling pathway, Notch, is expressed in these aggressive melanoma cells, and there is direct evidence demonstrating the molecular cross-talk between Nodal and Notch. Thus, these two prominent stem cell pathways contributing to melanoma cell plasticity may serve as promising dual therapeutic targets for clinical intervention.