Mayuko Y. Kumasaka

RAS/RAF/MEK/ERK and PI3K/PTEN/AKT Signaling in Malignant Melanoma Progression and Therapy

Cutaneous malignant melanoma is one of the most serious skin cancers and is highly invasive and markedly resistant to conventional therapy. Melanomagenesis is initially triggered by environmental agents including ultraviolet (UV), which induces genetic/epigenetic alterations in the chromosomes of melanocytes. In human melanomas, the RAS/RAF/MEK/ERK (MAPK) and the PI3K/PTEN/AKT (AKT) signaling pathways are two major signaling pathways and are constitutively activated through genetic alterations. Mutations of RAF, RAS, and PTEN contribute to antiapoptosis, abnormal proliferation, angiogenesis, and invasion for melanoma development and progression. To find better approaches to therapies for patients, understanding these MAPK and AKT signaling mechanisms of melanoma development and progression is important. Here, we review MAPK and AKT signaling networks associated with melanoma development and progression.

Molecular Network Associated with MITF in Skin Melanoma Development and Progression

Various environmental and genetic factors affect the development and progression of skin cancers including melanoma. Melanoma development is initially triggered by environmental factors including ultraviolet (UV) light, and then genetic/epigenetic alterations occur in skin melanocytes. These first triggers alter the conditions of numerous genes and proteins, and they induce and/or reduce gene expression and activate and/or repress protein stability and activity, resulting in melanoma progression. Microphthalmia-associated transcription factor (MITF) is a master regulator gene of melanocyte development and differentiation and is also associated with melanoma development and progression. To find better approaches to molecular-based therapies for patients, understanding MITF function in skin melanoma development and progression is important. Here, we review the molecular networks associated with MITF in skin melanoma development and progression.

c-RET Molecule in Malignant Melanoma from Oncogenic RET-Carrying Transgenic Mice and Human Cell Lines

Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf) and Gdnf receptor alpha 1 (Gfra1) transcripts in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in human malignant melanoma cell lines (HM3KO and MNT-1) were higher than those in primary cultured normal human epithelial melanocytes (NHEM), while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT) sited potentially downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of malignant melanoma.

CD109 expression levels in malignant melanoma.

Yoshiki Tokura Department of Dermatology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan *Corresponding author at: Department of Dermatology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawara, Sakyo, Kyoto 606-8507, Japan. Tel.: +81 75 7513310/93 6917445; fax: +81 75 7613002/93 6910907 E-mail address: kaba@kuhp.kyoto-u.ac.jp (Kenji Kabashima).

A Novel Mouse Model for De novo Melanoma

Nevus-associated melanomas arise from pre-existing benign lesions, but de novo melanomas can also develop in the absence of such lesions. Few studies have addressed the latter phenomenon because no animal models have been described in which melanomas clearly develop in a de novo manner. In this study, we have address this need in defining RFP-RET-transgenic mice (RET mice) as a mouse model for multi-step melanomagenesis that proceeds via tumor-free, benign, premalignant, and malignant stages. Melanomas from RET mice exhibited decreased expression levels of endothelin receptor B (Ednrb) compared with benign tumors. In RET mice that were heterozygous for Ednrb (Ednrb+/-;RET mice), >80% of the arising primary tumors were malignant. Life span after tumor development in the mice was significantly shorter than in RET mice. Lung metastasis after tumor development was significantly higher than in RET mice. The observed process of melanomagenesis in Ednrb+/-;RET mice, which proceeded without a pre-existing benign lesion, along with the emergent characteristics in the model after tumor development corresponded well with the formation of de novo melanoma in humans. Our findings define a novel transgenic mouse model for de novo melanoma and suggest that reduced expression of Ednrb might facilitate the development of de novo melanoma in humans.

Mitf contributes to melanosome distribution and melanophore dendricity.

Mitf is a transcription factor of the basic/helix‐loop‐helix/leucine‐zipper family which is indispensable for development of melanocytes and the retinal pigment epithelium. Our previous work using Xenopus laevis as a model system suggested that Mitf regulates melanosome dispersal in vivo though whether this was via melanosome transport or melanophore dendricity was not obvious. To better understand the role of Mitf, we have now characterized neural tube cultures from wild‐type Mitf‐injected or a dominant‐negative Mitf‐injected embryos and compared them with controls. In vitro, lower levels of Mitf activity induced less dendritic melanophores with aggregated melanosomes, whereas melanophores overexpressing Mitf had an extensive dendritic morphology with dispersed melanosomes. Moreover, immunorfluoresence assays reveal that expression of a dominant‐negative Mitf leads to decreased Rab27a expression. These results suggest that Mitf is involved in the regulation of melanosome transport and the level of dendricity in melanophores.

Barium Promotes Anchorage-Independent Growth and Invasion of Human HaCaT Keratinocytes via Activation of c-SRC Kinase

Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl2) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5–50 µM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 µM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5–5 µM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro.

Zinc finger protein 28 as a novel melanoma-related molecule

Since melanoma is an aggressive cancer with highly metastatic ability, the increase in its incidence is a threat to public health. Therefore, it is important to find new biomarkers and effective therapies. The c-RET proto-oncogene encodes a receptor-tyrosine kinase, and glial cell line-derived neurotrophic factor (GDNF)related ligands, including GDNF, neurturin, artemin and persephin, have been reported to be ligands of RET [1]. RFP-RET is a hybrid oncogene between c-RET and RFP, and its kinase activity is highly up-regulated compared with the activity of c-RET tyrosine kinase [2]. Previously, we established a metallothionein-I/RFP-RETtransgenic mouse of line 304/B6 (RET-mice) [3], in which systemic skin melanosis, benign melanocytic tumor(s) and malignant melanoma stepwise develop [4]. The transgenic mouse line is a powerful tool for analyzing the effects of molecules on melanomagenesis. In fact, we performed DNA microarray analysis for

L-cysteine as a regulator for arsenic-mediated cancer-promoting and anti-cancer effects.

Previous studies have shown that activities of tyrosine kinases and secretion of the active form of matrix metalloproteinase-2 (MMP-2) are correlated with promotion of tumor growth, while apoptotic cell death in cancer cells is correlated with anti-cancer effects. Although arsenic has been reported to have both cancer-promoting and anti-cancer effects, the mechanisms of the arsenic-mediated bidirectional effects remain unknown. We examined the effects of arsenic on both proto-oncogene c-RET-transfected NIH3T3 cells with benign characters and oncogenic RET-MEN2A-transfected NIH3T3 cells with malignant characters. Arsenic promoted not only c-RET tyrosine kinase activity but also genetically activated RET-MEN2A kinase activity with promotion of dimer formation of RET proteins. Arsenic also increased secretion of the active form of MMP-2 in both RET-MEN2A-transfectants and c-RET-transfectants. On the other hand, arsenic promoted poly-(ADP-ribose) polymerase (PARP) degradation and cell death in both malignant and non-malignant cells. Interestingly, l-cysteine inhibited the arsenic-mediated tumor-promoting effects (activation of kinases and MMP-2 secretion) but not arsenic-mediated anti-cancer effects (PARP degradation and cell death). Our results suggest redox-linked regulation of arsenic-mediated activities of kinases and MMP-2 secretion but not arsenic-mediated cell death. Our results also suggest that l-cysteine is an ideal supplement that inhibits arsenic-mediated tumor-promoting effects without affecting arsenic-mediated anti-cancer effects.

A redox-linked novel pathway for arsenic-mediated RET tyrosine kinase activation

We examined the biochemical effects of arsenic on the activities of RET proto‐oncogene (c‐RET protein tyrosine kinases) and RET oncogene (RET‐MEN2A and RET‐PTC1 protein tyrosine kinases) products. Arsenic activated c‐RET kinase with promotion of disulfide bond‐mediated dimerization of c‐RET protein. Arsenic further activated RET‐MEN2A kinase, which was already 3‐ to 10‐fold augmented by genetic mutation compared with c‐RET kinase activity, with promotion of disulfide bond‐mediated dimerization of RET‐MEN2A protein (superactivation). Arsenic also increased extracellular domain‐deleted RET‐PTC1 kinase activity with promotion of disulfide bond‐mediated dimerization of RET‐PTC1 protein. Arsenic increased RET‐PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic‐mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET‐PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol‐dependent dimer formation. As arsenic (14.2 µM) was detected in the cells cultured with arsenic (100 µM), direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox‐linked mechanism of activation of arsenic‐mediated RET proto‐oncogene and oncogene products. J. Cell. Biochem. 110: 399–407, 2010.