Netrin-1 Stimulates Migration of Neogenin Expressing Aggressive Melanoma Cells.

Netrin-1 is a neural guidance factor that regulates migration and positioning of neural crest-derived cells during embryonic development. Depending on the type of Netrin-1 receptor expression, cells are either attracted or repulsed by Netrin-1. Postnatal expression of Netrin-1 is detected in brain, colon, liver, and kidney, which are common sites of cancer metastasis, including melanoma. Thus, understanding the dynamics between Netrin-1 and its receptors could explain the attraction of melanoma towards these Netrin-1-expressing tissues. Here, we investigate whether the Netrin-1-attractive receptor Neogenin can affect migration of melanoma cells towards a Netrin-1 source. Results from Western blot (WB) analysis show higher expression of Neogenin in aggressive compared to non-aggressive melanoma cells. Cell migration experiments show increased migration of Neogenin-expressing aggressive melanoma cells towards exogenous, soluble recombinant human Netrin-1 and towards a Netrin-1-expressing cell line. Furthermore, WB reveals ERK1/2 activation and increased N-cadherin expression in Neogenin-expressing aggressive melanoma cells treated with rhNetrin-1. Moreover, treatment with anti-Neogenin blocking antibody caused decreased migration towards Netrin-1-expressing cells and reduced ERK1/2 activity in Neogenin-expressing aggressive melanoma cells. These results suggest Neogenin may play a role during migration of melanoma cells towards Netrin-1 via ERK1/2 signaling.

Role of Presenilin-1 in Aggressive Human Melanoma.

Presenilin-1 (PS-1), a component of the gamma (γ)-secretase catalytic complex, has been implicated in Alzheimer's disease (AD) and in tumorigenesis. Interestingly, AD risk is inversely related to melanoma, suggesting that AD-related factors, such as PS-1, may affect melanomagenesis. PS-1 has been shown to reduce Wnt activity by promoting degradation of beta-catenin (ß-catenin), an important Wnt signaling partner. Since Wnt is known to enhance progression of different cancers, including melanoma, we hypothesized that PS-1 could affect Wnt-associated melanoma aggressiveness. Western blot results showed that aggressive melanoma cells expressed significantly lower levels of both PS-1 and phosphorylated-ß-catenin (P-ß-catenin) than nonaggressive melanoma cells. Immunohistochemistry of human melanoma samples showed significantly reduced staining for PS-1 in advanced stage melanoma compared with early stage melanoma. Furthermore, γ-secretase inhibitor (GSI) treatment of aggressive melanoma cells was followed by significant increases in PS-1 and P-ß-catenin levels, suggesting impaired Wnt signaling activity as PS-1 expression increased. Finally, a significant reduction in cell migration was associated with the higher levels of PS-1 and P-ß-catenin in the GSI-treated aggressive melanoma cells. We demonstrate for the first time that PS-1 levels can be used to assess melanoma aggressiveness and suggest that by enhancing PS-1 expression, Wnt-dependent melanoma progression may be reduced.

Normal Skin Cells Increase Aggressiveness of Cutaneous Melanoma by Promoting Epithelial-to-Mesenchymal Transition via Nodal and Wnt Activity.

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.

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

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

Structure-based design of small bicyclic peptide inhibitors of Cripto-1 activity.

Bicyclic peptides assembled around small organic scaffolds are gaining an increasing interest as new potent, stable and highly selective therapeutics because of their uncommon ability to specifically recognize protein targets, of their small size that favor tissue penetration and of the versatility and easiness of the synthesis. We have here rationally designed bicyclic peptides assembled around a common tri-bromo-methylbenzene moiety in order to mimic the structure of the CFC domain of the oncogene Cripto-1 and, more specifically, to orient in the most fruitful way the hot spot residues H120 and W123. Through the CFC domain, Cripto-1 binds the ALK4 receptor and other protein partners supporting uncontrolled cell growth and proliferation. Soluble variants of CFC have the potential to inhibit these interactions suppressing the protein activity. A CFC analog named B3 binds ALK4 in vitro with an affinity in the nanomolar range. Structural analyses in solution via NMR and CD show that B3 has rather flexible conformations, like the parent CFC domain. The functional effects of B3 on the Cripto-1-positive NTERA cancer cell line have been evaluated showing that both CFC and B3 are cytotoxic for the cells and block the Cripto-1 intracellular signaling. Altogether, the data suggest that the administration of the soluble CFC and of the structurally related analog has the potential to inhibit tumor growth.

Development of conformational antibodies targeting Cripto-1 with neutralizing effects in vitro.

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.

Micro-RNA-195 and -451 regulate the LKB1/AMPK signaling axis by targeting MO25.

BACKGROUND: Recently, MicroRNAs (miR) and AMP-kinase (AMPK) have emerged as prominent players in the development of cardiac hypertrophy and heart failure. We hypothesized that components of the adenosine monophosphate-activated kinase (AMPK) pathway are targeted by miRs and alter AMPK signaling during pathological cardiac stress. METHODOLOGY/PRINCIPAL FINDINGS: Using a mouse model of hypertrophic cardiomyopathy (HCM), we demonstrated early elevation of miR-195 and miR-451 in HCM hearts, which targets MO25, a central component of the MO25/STRAD/LKB1 complex that acts as an upstream kinase for AMPK. We show functional targeting of MO25 by miR-195 and -451. Further in vitro interrogation of MO25 as a functional target validated this hypothesis where over-expression of miR-195 in C2C12 cells knocked down MO25 expression levels and downstream AMPK signaling (phosphorylation of Acetyl CoA carboxylase [ACC] and AMPK activity assay), similar to MO25 knockdown in C2C12 cells by siRNA. Parallel changes were measured in 60 day R403Q HCM male hearts that were rescued by short-term administration of AICAR, an AMPK agonist. CONCLUSIONS/SIGNIFICANCE: Elevated miR-195 targets the LKB1/AMPK signaling axis in HCM progression and implicates a functional role in HCM disease progression. MiR-195 may serve as potential therapeutics or therapeutic targets for heart disease.