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Melanoma is a lethal form of skin cancer triggered by genetic and environmental factors. Excision of early-stage, poorly aggressive melanoma often leads to a successful outcome, however, left undiagnosed these lesions can progress to metastatic disease. This research investigates whether the exposure of poorly aggressive melanoma to certain normal skin cells can explain how non-metastatic melanoma becomes more aggressive while still confined to the skin. To this end, we used a serial co-culture approach to sequentially expose cells from two different, poorly aggressive human melanoma cell lines against normal cells of the skin beginning with normal melanocytes, then epidermal keratinocytes, and finally dermal fibroblasts. Protein extraction of melanoma cells occurred at each step of the co-culture sequence for western blot (WB) analysis. In addition, morphological and functional changes were assessed to detect differences between the serially co-cultured melanoma cells and non-co-cultured cells. Results show that the co-cultured melanoma cells assumed a more mesenchymal morphology and displayed a significant increase in proliferation and invasiveness compared to control or reference cells. WB analysis of protein from the co-cultured melanoma cells showed increased expression of Snail and decreased levels of E-cadherin suggesting that epithelial-to-mesenchymal transition (EMT) is occurring in these co-cultured cells. Additional WB analysis showed increased levels of Nodal protein and signaling and signs of increased Wnt activity in the co-cultured melanoma cells compared to reference cells. These data suggest that interaction between poorly aggressive melanoma cells with normal cells of the skin may regulate the transition from localized, poorly aggressive melanoma to invasive, metastatic disease via Nodal and/or Wnt induced EMT.

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.

Breast cancer is the most common invasive cancer in women worldwide. Surgical removal of the breast tumor and subsequent reconstructive surgery can result in complications such as infection or necr...

The center of tumors, stem cell niches and mucosal surfaces all represent areas of the body that are reported to be anoxic. However, long-term study of anoxic cell physiology is hindered by the lack of a sustainable method permitting cell cultivation in the complete absence of oxygen. A novel methodology was developed that enabled anoxic cell cultivation (17d maximum time tested) and cell passage. In the absence of oxygen, cell morphology is significantly altered. All cells tested exhibited morphologic changes, i.e., a combination of tethered (monolayer-like) and runagate (suspension-like) morphologies. Both morphologies replicated (Vero and HeLa cells tested) and could be passaged anaerobically. In the absence of exogenous oxygen, anoxic cells produced reactive oxygen species (ROS). Anaerobic runagate HeLa and Vero cells increased ROS production from day 3 to day 10 by 2- and 3-fold, respectively. In contrast, anoxic tethered HeLa and Vero cells either showed no significant change in ROS production between days 3 and 10 or exhibited a 3-fold decrease in ROS, respectively. Detection of ROS was inversely related to detection of hypoxia-inducible factor-1α (HIF1) mRNA and HIF-1 protein expression which cycled over a 10-day period. This methodology has broad applications for the study of tumor and stem cell physiology as well as gastrointestinal cell-microbiome interactions. In addition, sustainable anaerobic cell culture may lead to the identification of novel pathways and targets for chemotherapeutic drug development.

The TGF-β associated growth factor Nodal is highly expressed in aggressive metastatic melanoma. Determining the risk for melanomagenesis from Nodal expression in nevi prior to the development of melanoma may be useful for both the screening and prevention of melanoma. Tissue sections of human adult nevi with or without a history of melanoma were stained by immunohistochemistry (IHC) for Nodal, the Nodal co-receptor Cripto-1, and Notch4, which have previously been shown to be associated with Nodal expression in melanoma. The degree of Nodal, Cripto-1 and Notch4 staining was scored and correlated with available clinical data. Median IHC scores for Nodal, Cripto-1 and Notch4 expression were significantly higher in nevi removed from patients who eventually developed melanoma compared with nevi from patients with no history of melanoma. In addition, the degree of Nodal expression in nevi from patients who eventually developed melanoma correlated significantly with the Breslow depth of the melanoma. Expression of Nodal and components of its signaling pathway in nevi may represent a biomarker for selecting a unique subset of patients requiring increased surveillance for screening and prevention of melanoma.

Triple-negative breast cancer (TNBC) represents an aggressive cancer subtype characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The independence of TNBC from these growth promoting factors eliminates the efficacy of therapies which specifically target them, and limits TNBC patients to traditional systemic neo/adjuvant chemotherapy. To better understand the growth advantage of TNBC - in the absence of ER, PR and HER2, we focused on the embryonic morphogen Nodal (associated with the cancer stem cell phenotype), which is re-expressed in aggressive breast cancers. Most notably, our previous data demonstrated that inhibition of Nodal signaling in breast cancer cells reduces their tumorigenic capacity. Furthermore, inhibiting Nodal in other cancers has resulted in improved effects of chemotherapy, although the mechanisms for this remain unknown. Thus, we hypothesized that targeting Nodal in TNBC cells in combination with conventional chemotherapy may improve efficacy and represent a potential new strategy. Our preliminary data demonstrate that Nodal is highly expressed in TNBC when compared to invasive hormone receptor positive samples. Treatment of Nodal expressing TNBC cell lines with a neutralizing anti-Nodal antibody reduces the viability of cells that had previously survived treatment with the anthracycline doxorubicin. We show that inhibiting Nodal may alter response mechanisms employed by cancer cells undergoing DNA damage. These data suggest that development of therapies which target Nodal in TNBC may lead to additional treatment options in conjunction with chemotherapy regimens - by altering signaling pathways critical to cellular survival.

Human Cripto-1 (Cripto-1), the founding member of the EGF-CFC superfamily, is a key regulator of many processes during embryonic development and oncogenesis. Cripto-1 is barely present or even absent in normal adult tissues while it is aberrantly re-expressed in various tumors. Blockade of the CFC domain-mediated Cripto-1 functions is acknowledged as a promising therapeutic intervention point to inhibit the tumorigenic activity of the protein. In this work, we report the generation and characterization of murine monoclonal antibodies raised against the synthetic folded CFC [112−150] domain of the human protein. Through subtractive ELISA assays clones were screened for the ability to specifically recognize “hot spot” residues on the CFC domain, which are crucial for the interaction with Activin Type I receptor (ALK4) and GRP78. On selected antibodies, SPR and epitope mapping studies have confirmed their specificity and have revealed that recognition occurs only on a conformational epitope. Furthermore, FACS analyses have confirmed the ability of 1B4 antibody to recognize the membrane-anchored and soluble native Cripto-1 protein in a panel of human cancer cells. Finally, we have evaluated its functional effects through in vitro cellular signaling assays and cell cycle analysis. These findings suggest that the selected anti-CFC mAbs have the potential to neutralize the protein oncogenic activity and may be used as theranostic molecules suitable as tumor homing agents for Cripto-1-overexpressing cancer cells and tissues and to overcome drug-resistance in routine cancer therapies.

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.

Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.

Nodal is a potent embryonic morphogen belonging to the TGF-beta superfamily. Typically, it also binds to the ALK4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells, is absent in normal cells but re-emerges in several human cancers, including melanoma, breast, and colon cancer. Our aim was to obtain mAbs able to recognize Nodal on a major CBR (Cripto-Binding-Region) site and to block the Cripto-1-mediated signalling. To achieve this, antibodies were raised against hNodal(44-67) and mAbs generated by the hybridoma technology. We have selected one mAb, named 3D1, which strongly associates with full-length rhNodal (KD 1.4 nM) and recognizes the endogenous protein in a panel of human melanoma cell lines by western blot and FACS analyses. 3D1 inhibits the Nodal-Cripto-1 binding and blocks Smad2/3 phosphorylation. Data suggest that inhibition of the Nodal-Cripto-1 axis is a valid therapeutic approach against melanoma and 3D1 is a promising and interesting agent for blocking Nodal-Cripto mediated tumor development. These findings increase the interest for Nodal as both a diagnostic and prognostic marker and as a potential new target for therapeutic intervention.

Human melanoma is the most rapidly increasing malignant skin disease in Caucasians (Siegel et al. 2015). Once considered a rare disease, the lifetime risk for developing melanoma in the US has increased from approximately 1 in 1500 during the 1930s to its present risk of approximately 1 in 60 (Giblin and Thomas 2007). The American Cancer Society’s recent cancer report estimates that 76,380 new cases of melanoma will be diagnosed and 10,130 deaths will result from melanoma during 2016 in the United States (American Cancer Society 2016). Important risk factors for developing melanoma include increased number of melanocytic nevi, a family history of melanoma, or a history of previous melanoma (Seykora and Elder 1996; Psaty et al. 2010). Prolonged sun exposure associated with increased outdoor activity has been suggested to play an important role in the epidemiologic increase in the incidence of melanoma (Leiter and Garbe 2008; Moan et al. 2008). Acute exposure of the skin to ultraviolet radiation (UVR) can induce varying degrees of erythema, pigmentation, and impairment of immune function (Matsumura and Ananthaswamy 2004). Increased numbers of melanocytic nevi associated with sunburn and intermittent or “holiday” sun exposure has been suggested as a major risk factor for developing melanoma in different studies (Elwood and Jopson 1997; Newton-Bishop et al. 2010). In fact, the Clark model for melanoma suggests a stepwise progression from hyperplastic and dysplastic nevi to melanoma (Clark et al. 1984).

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However, mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study, we investigated the effects of two nuclear receptors, liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter, while GCNF strongly suppressed CR-1 expression in these cells. In addition, the CR-1 promoter was unmethylated in NTERA-2 cells, while T47D, ZR75-1, and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells, promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors, suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively, these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer.

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.

Cripto-1 is a membrane-bound protein that is highly expressed in embryonic stem cells and in human tumors. in the present study, we investigated the effect of low levels of oxygen, which occurs naturally in rapidly growing tissues, on Cripto-1 expression in mouse embryonic stem (mES) cells and in human embryonal carcinoma cells. During hypoxia, Cripto-1 expression levels were significantly elevated in mES cells and in Ntera-2 or NCCIT human embryonal carcinoma cells, as compared with cells growing with normal oxygen levels. The transcription factor hypoxia-inducible factor-1 alpha directly regulated Cripto-1 expression by binding to hypoxia-responsive elements within the promoter of mouse and human Cripto-1 genes in mES and NCCIT cells, respectively. Furthermore, hypoxia modulated differentiation of mES cells by enhancing formation of beating cardiomyocytes as compared with mES cells that were differentiated under normoxia. However, hypoxia failed to induce differentiation of mES cells into cardiomyocytes in the absence of Cripto-1 expression, demonstrating that Cripto-1 is required for hypoxia to fully differentiate mES cells into cardiomyocytes. Finally, cardiac tissue samples derived from patients who had suffered ischemic heart disease showed a dramatic increase in Cripto-1 expression as compared with nonischemic heart tissue samples, suggesting that hypoxia may also regulate Cripto-1 in vivo.

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]

Epithelial-mesenchymal transition (EMT) is a process occurring during both embryogenesis and early stages of invasive cancer. Epithelial cells that undergo EMT become more migratory and invasive with a mesenchymal morphology. Herein we assess EMT induction in a mouse mammary epithelial cell line driven by Msx2, a homeobox-containing transcription factor important during mammary gland development. NMuMG cells, a normal mouse mammary epithelial cell line, stably transfected with a Msx2 cDNA showed downregulation of an epithelial marker E-cadherin and upregulation of the mesenchymal markers vimentin and N-cadherin. Furthermore, overexpression of Cripto-1, a member of the epidermal growth factor-CFC protein family already known to be involved in EMT, was detected in Msx2-transfected cells. The expression of Cripto-1 was accompanied by activation of the tyrosine kinase c-Src pathway and an increase in the invasive ability of the cells. Functional assays also demonstrated inhibition of the invasive behavior of the Msx2-transfected cells by a c-Src specific inhibitor. Moreover, immunohistochemistry of human infiltrating breast carcinomas showed positive staining for Msx2 only in the infiltrating tumor cells while the non-infiltrating tumor cells were negative. These results suggest that Msx2 may play a significant role in promoting EMT in epithelial cells that acquire properties involved in tumor invasion. J. Cell. Physiol. 219: 659-666, 2009. Published 2009 Wiley-Liss, Inc.

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]

The greatest health threat from malignant melanoma is death due to metastatic disease. Consequently, the identification of markers predictive of metastatic disease is essential for identifying new therapeutic targets. EphA2, a protein tyrosine kinase receptor commonly expressed in epithelial cells, has been found to be overexpressed and constitutively active in melanoma tumor cells having a metastatic phenotype as characterized by increased invasion, proliferation and vasculogenic mimicry (VM). Based on this observation, we hypothesized that increased expression of EphA2 by melanoma tumor cells could promote these characteristics of a metastatic phenotype in addition to promoting tumorigenicity as a whole. We analyzed a panel of human melanoma tumor cell lines derived from patient tissues classified as primary (either radial growth phase or vertical growth phase) and/or metastatic for the expression of EphA2 and found a correlation between increased EphA2 expression and metastatic potential. Experiments using the most metastatic of the human melanoma cell lines demonstrated that downregulation of EphA2 results in a significant decrease in invasion, proliferation, clonogenicity and VM in vitro, in addition to suppressed tumorigenicity in an orthotopic mouse model. Lastly, utilization of a human phospho-kinase array revealed increased phosphorylation of several different protein kinases involved in mediating various aspects of cellular proliferation. To the best of our knowledge these results provide the first direct in vivo evidence demonstrating a role for EphA2 in promoting melanoma tumorigenicity and suggest EphA2 as a significant molecular target for the therapeutic intervention of malignant melanoma.

Transgenic mice expressing the Notch 4 intracellular domain (ICD) (Int3) in the mammary gland have two phenotypes: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. We have conditionally ablated the Rbpj gene in the mammary glands of mice expressing whey acidic protein (Wap)-Int3. Interestingly, Rbpj knockout mice (Wap-Cre(+)/Rbpj(-/-)/Wap-Int3) have normal mammary gland development, suggesting that the effect of endogenous Notch signaling on mammary gland development is complete by day 15 of pregnancy. RBP-J heterozygous (Wap-Cre(+)/Rbpj(-/+)/Wap-Int3) and Rbpj control (Rbpj(flox/flox)/Wap-Int3) mice are phenotypically the same as Wap-Int3 mice with respect to mammary gland development and tumorigenesis. In addition, the Wap-Cre(+)/Rbpj(-/-)/Wap-Int3-knockout mice also developed mammary tumors at a frequency similar to Rbpj heterozygous and Wap-Int3 control mice but with a slightly longer latency. Thus, the effect on mammary gland development is dependent on the interaction of the Notch ICD with the transcription repressor/activator Rbpj, and Notch-induced mammary tumor development is independent of this interaction.

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.

Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. Despite a complete response in fewer than 5% of patients, the chemotherapeutic agent dacarbazine (DTIC) remains the reference drug after almost 40 years. More recently, FDA-approved drugs have shown promise but patient outcome remains modest, predominantly due to drug resistance. As such, combinatorial targeting has received increased attention, and will advance with the identification of new molecular targets. One attractive target for improving melanoma therapy is the growth factor Nodal, whose normal expression is largely restricted to embryonic development, but is reactivated in metastatic melanoma. In this study, we sought to determine how Nodal-positive human melanoma cells respond to DTIC treatment and to ascertain whether targeting Nodal in combination with DTIC would be more effective than monotherapy. A single treatment with DTIC inhibited cell growth but did not induce apoptosis. Rather than reducing Nodal expression, DTIC increased the size of the Nodal-positive subpopulation, an observation coincident with increased cellular invasion. Importantly, clinical tissue specimens from patients with melanomas refractory to DTIC therapy stained positive for Nodal expression, both in pre- and post-DTIC tumors, underscoring the value of targeting Nodal. In vitro, anti-Nodal antibodies alone had some adverse effects on proliferation and apoptosis, but combining DTIC treatment with anti-Nodal antibodies decreased cell growth and increased apoptosis synergistically, at concentrations incapable of producing meaningful effects as monotherapy. Implications: Targeting Nodal in combination with DTIC therapy holds promise for the treatment of metastatic melanoma. Mol Cancer Res; 13(4); 670–80. ©2015 AACR.

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.

Overexpression of Cripto-1 (CR-1) in FVB/N mice using the MMTV-LTR promoter results in increased mammary tumourigenesis in these female transgenic mice (MMTV-CR-1). Here, we characterize uterine tumours that developed in 15/76 (19.7%) of MMTV-CR-1 female nulliparous or multiparous mice during 24 months of observation compared with 0/33 (0%) of FVB/N normal control mice observed during the same time period (p < 0.01). The uterine tumours collected from the MMTV-CR-1 mice were classified as leiomyosarcomas and found to express the CR-1 transgene by polymerase chain reaction analysis and immunohistochemistry. Detection by western blot analysis showed higher levels of phosphorylated (P) forms of c-src, Akt, GSK-3β, and dephosphorylated (DP)-β-catenin in lysates from MMTV-CR-1 uterine leiomyosarcomas in comparison with lysates from normal control FVB/N uteri. Immunostaining showed increased nuclear localization of β-catenin in the MMTV-CR-1 uterine leiomyosarcomas. Increased immunostaining for CR-1 was detected in 9/13 (69.2%) cases of human leiomyosarcoma compared with staining in 3/15 (20%) human leiomyoma sections. Stronger immunostaining for P-src, P-Akt, P-GSK-3β and increased nuclear localization of β-catenin was also seen in human leiomyosarcomas in comparison with leiomyomas. Normal human uterine smooth muscle (UtSM) cells treated with exogenous soluble rhCR-1 showed increased levels of P-src, P-Akt, P-GSK-3β and dephosphorylated (DP)-β-catenin and increased proliferation (p < 0.05) and migration (p < 0.01) in comparison with untreated control UtSM cells. Inhibitors against c-src, Akt or β-catenin, individually or in combination, significantly reduced CR-1-induced migration. These results suggest a role for CR-1 during uterine tumourigenesis either directly by activating c-src and Akt and/or via cross-talk with the canonical Wnt signalling pathway, as suggested by the increased expression of P-GSK-3β, DP-β-catenin, and increased nuclear localization of β-catenin in human and MMTV-CR-1 mice leiomyosarcomas. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Overexpression of Cripto-1 has been reported in several types of human cancers including breast cancer. To investigate the role of human Cripto-1 (CR-1) in mammary gland development and tumorigenesis, we developed transgenic mice that express the human CR-1 transgene under the regulation of the whey acidic protein (WAP) promoter in the FVB/N mouse background. The CR-1 transgene was detected in the mammary gland of 15-week-old virgin WAP-CR-1 female mice that eventually developed hyperplastic lesions. From mid-pregnancy to early lactation, mammary lobulo-alveolar structures in WAP-CR-1 mice were less differentiated and delayed in their development due to decreased cell proliferation as compared to FVB/N mice. Early involution, due to increased apoptosis, was observed in the mammary glands of WAP-CR-1 mice. Higher levels of phosphorylated AKT and MAPK were detected in mammary glands of multiparous WAP-CR-1 mice as compared to multiparous FVB/N mice suggesting increased cell proliferation and survival of the transgenic mammary gland. In addition, more than half (15 of 29) of the WAP-CR-1 multiparous female mice developed multifocal mammary tumors of mixed histological subtypes. These results demonstrate that overexpression of CR-1 during pregnancy and lactation can lead to alterations in mammary gland development and to production of mammary tumors in multiparous mice.

The Ras-ERK pathway is deregulated in approximately a third of human cancers, particularly those of epithelial origin. In aggressive, triple-negative, basal-like breast cancers, most tumors display increased MEK and ERK phosphorylation and exhibit a gene expression profile characteristic of Kras or EGFR mutant tumors; however, Ras family genetic mutations are uncommon in triple-negative breast cancer and EGFR mutations account for only a subset of these tumors. Therefore, the upstream events that activate MAPK signaling and promote tumor aggression in triple-negative breast cancers remain poorly defined. We have previously shown that a secreted TGF-β family signaling ligand, Nodal, is expressed in breast cancer in correlation with disease progression. Here we highlight key findings demonstrating that Nodal is required in aggressive human breast cancer cells to activate ERK signaling and downstream tumorigenic phenotypes both in vitro and in vivo. Experimental knockdown of Nodal signaling downregulates ERK activity, resulting in loss of c-myc, upregulation of p27, G1 cell cycle arrest, increased apoptosis and decreased tumorigenicity. The data suggest that ERK activation by Nodal signaling regulates c-myc and p27 proteins post-translationally and that this cascade is essential for aggressive breast tumor behavior in vivo. As the MAPK pathway is an important target for treating triple-negative breast cancers, upstream Nodal signaling may represent a promising target for breast cancer diagnosis and combined therapies aimed at blocking ERK pathway activation.

Methionine aminopeptidase-2 (MetAP2) is the molecular target of the angiogenesis inhibitors, fumagillin and ovalacin. Fumagillin can also inhibit cancer cell proliferation, implying that MetAP2 may play a quite complex role in tumor progression. Here, we examined the expression and function of MetAP2 in an in vitro model of human mesothelioma. We found that mesothelioma cells expressed higher MetAP2 mRNA levels than primary normal mesothelial cells. Consistently, fumagillin induced apoptosis, owing to early mitochondrial damage, in malignant, but not in normal mesothelial cells. Transfection of mesothelioma cells with a MetAP2 anti-sense oligonucleotide determined a time-dependent inhibition of cell survival and induced nucleosome formation. Interestingly, mRNA and protein levels of the anti-apoptotic gene bcl-2 as well as telomerase activity were selectively reduced after MetAP2 inhibition in mesothelioma cells, whereas bcl-2 overexpression counteracted the effect of MetAP2 inhibition on telomerase activity and apoptosis. MetAP2 inhibition also increased caspase activity and the caspase inhibitor, zVAD-fmk, prevented fumagillin-induced apoptosis, but it did not alter telomerase activity. These results indicate that MetAP2 is a main regulator of proliferative and apoptotic pathways in mesothelioma cells and suggest that MetAP2 inhibition may represent a potential target for therapeutic intervention in human mesothelioma.

The prognosis of patients with malignant pleural mesothelioma (MM) is dependent more on tumor extension and differentiation than on therapeutic effects. Reduplication of the basal lamina (RBL) is an ultrastructural feature of some benign and malignant tumors that has been inversely correlated with aggressiveness and was recently described in MM. To investigate whether RBL is important for predicting the survival of patients with MM, transmission electron microscopy was used to identify the presence of basal lamina or RBL in biopsy specimens obtained by thoracoscopy from 35 patients. Cox's regression analysis was used to study the relation of these ultrastructural features to survival. Better outcomes were found for patients whose tumors expressed either basal lamina (HR 0.48; 95% CI, 0.09-2.47) or RBL (HR 0.38; 95% CI 0.12-1.22) compared with the reference category, where basal lamina or RBL was not found. The expression of basal lamina and RBL is an important novel prognostic factors in MM. HUM PATHOL 31:1341-1345.

Previous report indicated that Interleukin-2 (IL-2) is able to inhibit the growth of IL-2-receptor-positive cancer cell lines without any involvement of the immune system, through IL-2-induced alterations of the cell cycle kinetics. In this study we provide evidence that IL-2 exerts anti-proliferative effect on three human malignant mesothelioma (MMe) cells in vitro, while no effects were observed on normal human mesothelial cell (HMC) primary cultures. The growth inhibitory effect of IL-2 on neoplastic cells appeared to depend on the baseline proliferative status of these cells. Indeed, in highly proliferating MMe cells, we observed a reduction of malignant cells in the S-phase of the cell cycle, with an accumulation in G0/G1, followed by apotosis for longer incubations or exposure to higher doses. On the contrary, in MMe cells proliferating at lower rate, IL-2 induces only a late cytotoxic effect, leading to apoptosis, without significantly affecting the cell cycle. IL-2Rbeta mRNA was detectable by RT-PCR in all MMe cells, IL-2Ralpha mRNA in one only out the three assayed and IL-2Rgamma mRNA in none. In addition, mRNA specific for the IL-2Rbeta-associated Jak-1 tyrosine kinase was expressed in all MMe cell lines, further suggesting that IL-2Rbeta may play a role in the observed effects. Very low, albeit detectable, levels of IL-2Rbeta chain appeared to be expressed at the cell surface of MMe cells by indirect immunofluorescence and FACS analyses. Finally, Ca(++) fluxes were rapidly induced when MMe cells were exposed to exogenous IL-2.

Cripto-1 (CR-1) protein function differs according to cellular or extracellular expression. In this study, we explore the significance of cell surface CR-1 expression in human melanoma cells. Cell surface CR-1-expressing human melanoma cells (CR1-CS+) were selected by fluorescence-activated cell sorting (FACS) and grown in vitro and in vivo in nude mice to study their growth characteristics. The CR1-CS+ melanoma cells were found to express increased levels of Oct4, MDR-1 and activated c-Src compared with cells lacking this subpopulation (CR1-CS−) or unsorted cells, used as control. CR1-CS+ show reduced proliferation rates and diminished spherical colony formation compared with control cells when cultured in vitro. Orthotopic injections of CR1-CS+ in nude mice formed slow growing tumors with histologic variability across different areas of the CR1-CS+ xenografts. CR-1-expressing cells from first generation CR1-CS+ tumors showed significantly increased tumor-forming rate and aggressiveness following subsequent transplants in nude mice. These data demonstrate that within a heterogeneous melanoma cell population there resides a slow proliferating, cell surface CR-1-expressing subpopulation capable of giving rise to a fast growing, aggressive progeny that may contribute to disease recurrence and progression.

Expression of Nodal, a Transforming Growth Factor-beta (TGF-β) related growth factor, is associated with aggressive melanoma. Nodal expression in adult dysplastic nevi may predict the development of aggressive melanoma in some patients. A subset of pediatric patients diagnosed with giant or large congenital melanocytic nevi (LCMN) has shown increased risk for development of melanoma. Here, we investigate whether Nodal expression can help identify the rare cases of LCMN that develop melanoma and shed light on why the majority of these patients do not. Immunohistochemistry (IHC) staining results show varying degree of Nodal expression in pediatric dysplastic nevi and LCMN. Moreover, median scores from Nodal IHC expression analysis were not significantly different between these two groups. Additionally, none of the LCMN patients in this study developed melanoma, regardless of Nodal IHC levels. Co-culture experiments revealed reduced tumor growth and lower levels of Nodal and its signaling molecules P-SMAD2 and P-ERK1/2 when melanoma cells were grown in vivo or in vitro with normal melanocytes. The same was observed in melanoma cells cultured with melanocyte conditioned media containing pigmented melanocyte derived melanosomes (MDM). Since MDM contain molecules capable of inactivating radical oxygen species, to investigate potential anti-oxidant effect of MDM on Nodal expression and signaling in melanoma, melanoma cells were treated with either N-acetyl-l-cysteine (NAC), a component of the anti-oxidant glutathione or synthetic melanin, which in addition to providing pigmentation can also exert free radical scavenging activity. Melanoma cells treated with NAC or synthetic melanin showed reduced levels of Nodal, P-SMAD2 and P-ERK1/2 compared to untreated melanoma cells. Thus, the potential role for Nodal in melanoma development in LCMN is less evident than in adult dysplastic nevi possibly due to melanocyte cross-talk in LCMN capable of offsetting or delaying the pro-melanoma effects of Nodal via anti-oxidant effects of MDM.

The re-emergence of the tumour growth factor-beta (TGF-beta)-related embryonic morphogen Nodal has recently been reported in several different human cancers. In this study, we examined the expression of Nodal in a series of benign and malignant human breast tissues to determine the clinical significance of this expression and whether Nodal could represent a potential therapeutic target in breast cancer. Tissue sections from 431 therapeutically naive patients diagnosed with benign or malignant breast disease were stained for Nodal by immunohistochemistry and analysed in a blinded manner. The degree of Nodal staining was subsequently correlated with available clinical data, such as diagnoses and disease stage. These tissue findings were further explored in breast cancer cell lines MDA-MB-231 and MDA-MB-468 treated with a Nodal blocking antibody to determine biological effects for target validation. A variable degree of Nodal staining was detected in all samples. The intensity of Nodal staining was significantly greater in undifferentiated, advanced stage, invasive breast cancer compared with benign breast disease or early stage breast cancer. Treatment of human breast cancer cells in vitro with Nodal blocking antibody significantly reduced proliferation and colony-forming ability in soft agar, concomitant with increased apoptosis. These data suggest a potential role for Nodal as a biomarker for disease progression and a promising target for anti-Nodal therapy in breast cancer.

Background: CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma. Methods: Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers. Results: Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth. Conclusion: These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.

As the frequency of melanoma diagnosis increases, current treatment strategies are still struggling to significantly impact patient survival. Some promise has been shown in treating certain melanomas by targeting activated signaling pathways resulting from specific mutations in proteins, such as in BRAF and NRAS. Recently, the identification of embryonic signaling pathways in melanomas has helped us better understand certain biological characteristics, such as cellular heterogeneity and phenotypic plasticity, and has provided novel insight pertinent to diagnosis and therapy. For instance, our studies have shown that the TGF-beta family member, Nodal, is expressed in melanoma and is responsible, at least in part, for tumor cell plasticity and aggressiveness. Since the majority of normal adult tissues do not express Nodal, we reason that this embryonic morphogen could be used to identify and target aggressive melanoma cells. We have also identified that molecular cross-talk between the Notch and Nodal pathways may represent a mechanism responsible for the overexpression of Nodal in melanoma. Further exploitation of the relationship between embryonic signaling pathways and cancer pathogenesis could lead to novel approaches for diagnosis and therapy in cancers, such as melanoma.

The plastic phenotype of aggressive melanoma has presented a significant challenge in the detection and targeting of tumor cells exhibiting stem cell-like characteristics. As the molecular signaling pathways underlying tumor cell plasticity become more transparent, our understanding of how to suppress this elusive phenotype will be enhanced. Indeed, we are making progress in identifying critical embryonic pathways, such as the Nodal signaling pathway, that reemerge in aggressive tumor cells – in the absence of regulatory check points. Because Nodal is not expressed by the majority of normal adult tissues, and is over-expressed by aggressive tumor cells, it represents a valuable new therapeutic target. Collectively, we have learned a great deal from studies that focus our attention on the convergence of embryonic and tumorigenic signaling pathways. At this interaction of normal development and tumor formation reside the clues to suppressing cancer progression.

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.

Activation of the ErbB family of receptor tyrosine kinases via cognate Epidermal Growth Factor (EGF)-like peptide ligands constitutes a major group of related signaling pathways that control proliferation, survival, angiogenesis and metastasis of breast cancer. In this respect, clinical trials with various ErbB receptor blocking antibodies and specific tyrosine kinase inhibitors have proven to be partially efficacious in the treatment of this heterogeneous disease. Induction of an embryonic program of epithelial-to-mesenchymal transition (EMT) in breast cancer, whereupon epithelial tumor cells convert to a more mesenchymal-like phenotype, facilitates the migration, intravasation, and extravasation of tumor cells during metastasis. Breast cancers which exhibit properties of EMT are highly aggressive and resistant to therapy. Activation of ErbB signaling can regulate EMT-associated invasion and migration in normal and malignant mammary epithelial cells, as well as modulating discrete stages of mammary gland development. The purpose of this review is to summarize current information regarding the role of ErbB signaling in aspects of EMT that influence epithelial cell plasticity during mammary gland development and tumorigenesis. How this information may contribute to the improvement of therapeutic approaches in breast cancer will also be addressed.

Nodal, an embryonic morphogen belonging to the TGF-β superfamily, is an important regulator of embryonic stem cell fate. We have recently demonstrated that Nodal is expressed significantly in aggressive melanoma. Surprisingly, expression of the Nodal coreceptor, Cripto-1, was detected in only a small fraction of the melanoma tumor cell population, indicating a primary role for Cripto-1-independent signaling of Nodal in melanoma. In this review, we discuss how regulatory factors present in an embryonic environment, such as Lefty, can downregulate Nodal expression and inhibit tumorigenicity and plasticity of melanoma cells. Our translational studies show that antibodies against Nodal are capable of repressing melanoma vasculogenic mimicry and of inducing apoptosis in melanoma tumors in an in vivo lung-colonization assay. Our previous work and ongoing studies suggest that Nodal may represent a novel diagnostic marker and therapeutic target in melanoma.

Members of the Epidermal Growth Factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) family, such as human Cripto-1, are important mediators of crucial events that take place during embryonic pattern formation. New evidences from gene expression and transgenic mouse studies have also shown that perturbation of Cripto-1 signaling may lead to cell transformation and tumor formation in vivo. In addition, Cripto-1 is expressed at high levels in a wide variety of human carcinomas including early and late breast cancers. Despite the clear correlation between Cripto-1 overexpression and human and mouse tumors, the exact molecular mechanism of Cripto-1 contribution to the cell transformation process is not clear. Cripto-1 has been shown to activate multiple signaling pathways to promote either differentiation during embryogenesis or cancer growth. In this review we will discuss the multifunction properties of the EGF-CFC family of proteins and the complex network of signaling molecules activated by Cripto-1 focusing in particular on the mammary gland. A better understanding of the intracellular signaling pathways that mediate Cripto-1 activity in human tumors might identify novel points of intervention to target Cripto-1 in human malignancies.

Aggressive cancer cells manifest stem-cell-like qualities that allow them to self-renew and to derive a heterogeneous tumor. Ultimately, this multipotent phenotype facilitates metastasis and resistance to therapy. Cancer cells likely acquire and maintain multipotent phenotypes by aberrantly expressing embryonic factors, such as Nodal and Notch, which maintain pluripotency in normal embryonic stem cell types. Recent studies have shown that Nodal, an embryonic morphogen belonging to the transforming growth factor-beta (TGF-β) superfamily, is aberrantly expressed in melanoma and breast carcinoma cells. Moreover, Nodal facilitates breast cancer and melanoma tumorigenesis. During development, Nodal is regulated by the spatial and temporal expression of inhibitors such as Lefty. In aggressive cancer cells, this balance of regulatory mediators is disrupted, leading to unchecked Nodal expression. By exposing aggressive cancer cells to embryonic microenvironments, inclusive of Lefty, Nodal expression is decreased and tumorigenesis is suppressed. Embryonic stem cell-derived factors, such as Lefty, may provide therapeutic modalities that may be used to specifically differentiate and eradicate aggressive cancers.

The diagnosis of melanoma is becoming ever 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 and plasticity, expression of certain molecules such as the multidrug resistance protein-1 (MDR1) or the aberrant expression of embryonic signaling molecules and morphogens like Nodal, important for self renewal and pluripotency, suggest that a stem cell-like population may reside in aggressive melanomas. This perspective focuses on preliminary findings obtained in our laboratory which indicate that the expression of the Nodal co-receptor, Cripto-1, in a subset of malignant melanoma cells may be exploited to identify possible melanoma stem cells (MSC). In fact, the use of anti-Cripto-1 antibodies to cell sort Cripto-1-positive cells in the metastatic melanoma cell line C8161 has identified a slow growing, sphere forming subpopulation that expresses increased levels of Oct4, Nanog and MDR1. If current in vivo studies confirm the self renewal and tumorigenic characteristics of these cells, the expression of Cripto-1 may represent a useful marker to identify cancer stem cells in melanoma, and possibly other aggressive tumors as well.

Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland.

Breast carcinoma cells and embryonic progenitors similarly implement stem cell-associated signaling pathways to sustain continued growth and plasticity. Indeed, recent studies have implicated signaling pathways, including those associated with the Notch, and Transforming Growth Factor-Beta (TGF-beta) superfamilies, as instrumental to both embryological development and breast cancer progression. In particular, Nodal, an embryonic morphogen belonging to the TGF-beta superfamily, and its co-receptor, Cripto-1, are requisite to both embryogenesis and mammary gland maturation. Moreover, these developmental proteins have been shown to promote breast cancer progression. Here, we review the role of Nodal and its co-receptor Cripto-1 during development and we describe how this signaling pathway may be involved in breast cancer tumorigenesis. Moreover, we emphasize the potential utility of this signaling pathway as a novel target for the treatment and diagnosis of breast cancer.

The epidermal growth factor receptor (EGFR) is expressed in the majority of human carcinomas, where it regulates proliferation and survival of cancer cells. Clinical studies have failed, however, to demonstrate a direct correlation between expression of this receptor and prognosis of cancer patients. In this regard, pre-clinical studies suggest that the transforming ability of the EGFR depends on the levels of expression of EGFR ligands and that it is greatly enhanced when different ErbB receptors are co-expressed. Indeed, co-expression of different ErbB receptors and EGF-like growth factors has been demonstrated to occur in several human carcinomas. This observation leads to hypothesize that the growth of human tumors might be regulated by a network of receptors and ligands of the EGFR family. Therefore, the global levels of expression of these proteins should be determined for prognostic and therapeutic applications. Finally, recent findings suggest that the EGFR system might regulate the functions of the tumor microenvironment that are important for tumor progression such as neo-angiogenesis and osteoclast activation.

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

Malignant mesothelioma (MM) is a primary tumor of the pleura and peritoneum. Malignant mesotheliomas that are limited to other organs are extremely rare, though several cases of pericardial MM have been reported (1). A unique feature of MM is its strong relationship with asbestos exposure (2,3), which has recently led to great public concern in view of the ubiquitous presence of that mineral. Insulation, construction, shipyard industries, and automobile brakes are among the many sources of occupational exposure (4). Exposure can be not only occupational but also environmental, or even familial by household contamination (5). The mechanisms of MM pathogenesis have not been fully elucidated. Asbestos fibers could work their way through the lung tissues to damage pleura and produce adhesions and plaques. Changes observed in target tissues exposed to asbestos include hyperplasia, metaplasia, DNA synthesis, and increased production of oxygen free radicals. Activation of diacylglycerol, protein kinase C, and ornithine decarboxylase also has been reported in a pathway similar to classic tumor promoters, such as phorbol esters (6–8). Moreover, crocidolite fibers, which are the major tumorigenic asbestos fibers, induce angiogenesis in the peritoneal lining of MM animal models (9). Thus, ingrowth of new blood vessels around clusters of asbestos fibers may also facilitate the later emergence of MM at these sites. Angiogenesis plays an important role in the growth, progression, and metastasis of solid tumors (10). Further, quantitative histologic studies have suggested that angiogenesis, as assessed by intratumoral microvascular density (IMD) and total microvascular area (MVA), correlates with poor prognosis in several human neoplasms (11,12). Malignant mesothelioma demonstrated a higher IMD than colon and breast tumors (13–15). This value was significantly and independently related to survival adjusted for other known prognostic variables in MM, such as histologic type, stage, and age (16). This might call for an IMD profile to be provided as part of the pathologic evaluation of tumor specimens from patients with MM. Currently, many angiogenic factors have been identified to be released from tumor-associated inflammatory cells, extracellular matrix

The neuronal guidance molecule, Netrin-1, has been suggested to play a role in the adhesion and migration of the mammary gland epithelium. Human and mouse Cripto-1 induce proliferation, migration, invasion and colony formation by epithelial cells in 3D matrices. Here we investigate whether Netrin-1 affects these Cripto-1-dependent activities in mouse mammary epithelial cells. Overexpression of Cripto-1 in EpH4 and HC-11 cells (EpH4/Cripto-1 or HC-11/Cripto-1) was associated with low expression of Netrin-1 and increased expression of its receptor Neogenin compared to that of wild-type cells. No change was observed in the expression of the other Netrin-1 receptor, UNC5H1. Treating EpH4/Cripto-1 or HC-11/Cripto-1 mammary cells with exogenous soluble Netrin-1 resulted in increased expression of E-cadherin and UNC5H1, decreased expression of vimentin and decreased activation of Akt as determined by western blotting. Colony formation by Eph4/Cripto-1 cells in 3D gels was significantly reduced in proximity to a Netrin-1 source, and mammary glands of transgenic mice overexpressing human Cripto-1 showed altered ductal growth in proximity to implanted Netrin-1-releasing pellets. Terminal end buds in the treated transgenic mice mammary glands also showed increased expression of E-cadherin and UNC5H1 and decreased expression of active Akt determined by immunohistochemistry. Together, these results suggest that regulation of Netrin-1 expression is important in regulating Cripto-1-dependent invasion and migration of mammary epithelial cells.