NRN1 interacts with Notch to increase oncogenic STAT3 signaling in melanoma.

BACKGROUND: Melanoma is a highly heterogeneous cancer, in which frequent changes in activation of signaling pathways lead to a high adaptability to ever changing tumor microenvironments. The elucidation of cancer specific signaling pathways is of great importance, as demonstrated by the inhibitor of the common BrafV600E mutation PLX4032 in melanoma treatment. We therefore investigated signaling pathways that were influenced by neurotrophin NRN1, which has been shown to be upregulated in melanoma. METHODS: Using a cell culture model system with an NRN1 overexpression, we investigated the influence of NRN1 on melanoma cells' functionality and signaling. We employed real time cell analysis and spheroid formation assays, while for investigation of molecular mechanisms we used a kinase phosphorylation kit as well as promotor activity analysis followed by mRNA and protein analysis. RESULTS: We revealed that NRN1 interacts directly with the cleaved intracellular domain (NICD) of Notch1 and Notch3, causing a potential retention of NICD in the cytoplasm and thereby reducing the expression of its direct downstream target Hes1. This leads to decreased sequestration of JAK and STAT3 in a Hes1-driven phosphorylation complex. Consequently, our data shows less phosphorylation of STAT3 while presenting an accumulation of total protein levels of STAT3 in association with NRN1 overexpression. The potential of the STAT3 signaling pathway to act in both a tumor suppressive and oncogenic manner led us to investigate specific downstream targets - namely Vegf A, Mdr1, cMet - which were found to be upregulated under oncogenic levels of NRN1. CONCLUSIONS: In summary, we were able to show that NRN1 links oncogenic signaling events between Notch and STAT3 in melanoma. We also suggest that in future research more attention should be payed to cellular regulation of signaling molecules outside of the classically known phosphorylation events.

The Role of T-Cadherin (CDH13) in Treatment Options with Garcinol in Melanoma.

Targeted therapies with chemotherapeutic agents and immunotherapy with checkpoint inhibitors are among the systemic therapies recommended in the guidelines for clinicians to treat melanoma. Although there have been constant improvements in the treatment of melanoma, resistance to the established therapies continues to occur. Therefore, the purpose of this study was to explore the function of garcinol with regards to specific cancer properties such as proliferation and apoptosis. Garcinol, a natural compound isolated from the plant also known as mangosteen (Garcinia mangostana), is a newly discovered option for cancer treatment. Numerous pharmaceutical substances are derived from plants. For example, the derivates of camptothecin, extracted from the bark of the Chinese tree of happiness (Camptotheca acuminate), or paclitaxel, extracted from the bark of the Western yew tree (Taxus brevifolia), are used as anti-cancer drugs. Here, we show that garcinol reduced proliferation and induced apoptosis in melanoma cell lines. In addition, we found that those cells that are positive for the expression of the cell-cell adhesion molecule T-cadherin (CDH13) respond more sensitively to treatment with garcinol. After knock-down experiments with an siRNA pool against T-cadherin, the sensitivity to garcinol decreased and proliferation and anti-apoptotic behavior of the cells was restored. We conclude that patients who are T-cadherin-positive could especially benefit from a therapy with garcinol.

Nuclear AREG affects a low-proliferative phenotype and contributes to drug resistance of melanoma.

AMPHIREGULIN (AREG) is a multifaceted molecule, which acts not only as an extracellular ligand for EGF receptor (EGFR), but also as an intracellular signaling molecule. It remains elusive, however, whether AREG has a tumor suppressive or oncogenic role in melanoma. Here, we found that several melanoma cell lines express AREG, but the expression does not correlate with that of EGFR. Recombinant AREG and the neutralizing antibody experiments showed that intracellular AREG plays an important role in melanoma, implying a divergent function of AREG in addition to the role as a ligand for EGFR. Further investigation of this mechanism revealed that particularly nuclear-localized AREG regulates IGF-1R, P21 (Cip1/Waf1), TP53 and JARID1B protein accumulation in the nucleus. Furthermore, manipulation of nuclear AREG levels has influence on heterochromatin condensation (HP1beta, SETDB1) and trimethylation of histones H3K9 and H3K4. As these molecules correspond to previously identified markers for slow-cycling drug resistant cells, we speculate that nuclear AREG predisposes cells to resistance to therapy. According to the hypothesis, we detected the accumulation of AREG in the nucleus of SK-Mel-28-VR, which was cultured under Vemurafenib (VR) selection pressure, and this correlates with JARID1B expression. Here, knockdown of AREG makes the previously resistant cells more sensitive to VR treatment, resulting in inhibited proliferation. Taken together, we suggest that nuclear AREG affects a slow-cycling phenotype and increases resistance to VR, raising a possibility that AREG might be a potential therapeutic target for resistance in melanoma.

miR-488-5p and its role in melanoma.

Due to their ability to regulate dozens to hundreds of target genes simultaneously and, therefore, influence several oncogenic pathways at the same time, microRNAs are a fascinating research object in melanoma. MicroRNAs have been identified as regulators of tumor proliferation, invasion and metastasis in melanoma. More precisely, it has been published that dysregulation of miR-488 contibutes to the progression of several cancer entities. However, the biological functions of miR-488, in special miR-488-5p in melanoma, remain unclear. This study showed the involvement of miR-488-5p in Wnt/ß-catenin pathway and the function as a tumor suppressor. Transfection of miR-488-5p mimic led to inhibition of proliferation, migration, anchorage independent growth and led to induction of apoptosis. These data indicated that miR-488-5p acts as a tumor suppressor and is lost during melanoma development. The loss of miR-488-5p was confirmed in vivo by in situ hybridization on melanoma tissue.

Inhibition of peptidyl-prolyl isomerase (PIN1) and BRAF signaling to target melanoma.

PIN1 is a phosphorylation-dependent peptidyl-prolyl cis/trans isomerase, overexpressed in many cancers, including melanoma. Our immunohistochemistry data of melanoma patient tissue underline the up-regulation of PIN1 in metastases. Here, we demonstrate important functions of PIN1 and its selective and cell permeable inhibitor 37 for the treatment of melanoma. To analyze its possible role in oncogenesis and as a therapeutic target, we first suppressed PIN1 expression by a siRNA pool. PIN1 knockdown potently inhibited melanoma cell proliferation and vascular mimicry by influencing several cancer-relevant pathways. Furthermore, inhibitor 37 inhibited cell growth in melanoma and induced apoptosis. Normal healthy melanocytes, keratinocytes and fibroblasts are not affected by the PIN1 inhibitor 37. Combinatorial treatment of melanoma cells is with Vemurafenib as a common therapeutic option for BRAF-mutated melanoma and inhibitor 37 resulted in a strong, synergistic effect on apoptosis of melanoma cell lines. In summary, targeting PIN1 offers a promising therapeutic approach to simultaneously downregulate multiple cancer-driving pathways in cancer.

UVB radiation represses CYLD expression in melanocytes.

CYLD lysine 63 deubiquitinase (CYLD) was originally identified as a tumor suppressor that is mutated in familial cylindromatosis. Unlike in cylindromatosis, downregulation of the deubiquitinase CYLD in melanoma, a highly aggressive tumor, is not caused by mutations in the CYLD gene, but rather by a constitutive and high expression of the snail family transcriptional repressor 1 (SNAIL1). A reduced CYLD level leads to B-cell lymphoma-3/p50/p52-dependent nuclear factor-κB activation, which in turn triggers the expression of genes such as cyclin D1 and N-cadherin. Elevated levels of cyclin D1 and N-cadherin promote melanoma proliferation and invasion. By analyzing the regulation of CYLD expression in melanocytes, the present study identified a signaling pathway that is regulated in response to ultraviolet B (UVB) radiation in melanocytes. UVB light leads to an extracellular signal-regulated kinase-mediated induction of SNAIL1 and subsequent downregulation of CYLD expression in normal human epithelial melanocytes. The UVB-mediated suppression of CYLD in melanocytes may have a key role in the reaction to UV stimuli, and may also potentially be involved in the early malignant transformation processes.

The neurotrophin Neuritin1 (cpg15) is involved in melanoma migration, attachment independent growth, and vascular mimicry.

The neurotrophin Neuritin1 (NRN1; cpg15) belongs to the candidate plasticity gene (CPG) family and is expressed in postmitotic-differentiating neurons of the developmental nervous system and neuronal structures associated with plasticity in the brain of human adult.Our newest findings document that NRN1 deregulation could contribute also to disease development and have impact on malignant melanoma. Our analyses displayed the over-expression of NRN1 in melanoma in vitro and in vivo, shown by immunohistochemistry and qRT-PCR on microdissected melanoma tissue; furthermore, soluble NRN1 was detectable in tissue culture supernatant and serum of melanoma patients.To investigate the role of NRN1 in melanoma we performed knockdown, over-expression and recombinant-NRN1-treatment experiments affiliated by functional assays. Our results show that migration, attachment independent growth and vasculogenesis were affected after manipulation of NRN1 on endogenous and extrinsic level. Interestingly, high NRN1 serum levels correlate with low MIA serum levels (< 10ng/ml). Therefore, we speculate that NRN1 could be a marker for early melanoma stages, in particular.In summary, we detected an overexpression of NRN1 in melanoma patient. In functional cell culture experiments we found a correlation between NRN1 expression and the cancerous behavior of melanoma cells.