Machiko Iida

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.

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.

Sunlight exposure-mediated DNA damage in young adults.

Background: Previous experimental studies showed that single ultraviolet B (UVB) light irradiation increased levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a well-established biomarker of carcinogenesis and oxidative DNA damage, in epithelial cells in animals and humans. We conducted for the first time an epidemiologic study to investigate the correlations among levels of oxidative DNA damage, skin pigmentation, and sunlight exposure in human daily life. Methods: Digitalized skin pigmentation levels and creatinine-adjusted urinary 8-OHdG levels were examined in 127 healthy young adults aged 20 to 24 years and in hairless mice with normal pigmented skin (HL-mice; n = 20) and hyperpigmented skin (HL-HPS-mice; n = 20). Data obtained by a questionnaire were also analyzed for the 127 subjects. Results: Binary logistic regression analysis showed that increased sunlight intensity, but not sunlight-exposed time or sunlight-exposed skin area, was correlated with elevation in creatinine-adjusted urinary 8-OHdG levels. In contrast, increased skin pigmentation level, but not the use of sunscreen, was correlated with reduction in urinary 8-OHdG level in humans. UVB irradiation corresponding to several minutes of sunlight exposure significantly increased urinary 8-OHdG levels in HL-mice but not in HL-HPS-mice. Conclusions: We showed that increase in intensity of sunlight in human daily life increased levels of DNA damage. We also showed a protective effect of skin pigmentation on sunlight exposure–mediated DNA damage. Impact: We have provided more reliable evidence of routine sunlight exposure–mediated DNA damage in humans through the combination of epidemiologic and experimental studies. Cancer Epidemiol Biomarkers Prev; 20(8); 1622–8. ©2011 AACR.

Chronic Exposure to Low Frequency Noise at Moderate Levels Causes Impaired Balance in Mice

We are routinely exposed to low frequency noise (LFN; below 0.5 kHz) at moderate levels of 60–70 dB sound pressure level (SPL) generated from various sources in occupational and daily environments. LFN has been reported to affect balance in humans. However, there is limited information about the influence of chronic exposure to LFN at moderate levels for balance. In this study, we investigated whether chronic exposure to LFN at a moderate level of 70 dB SPL affects the vestibule, which is one of the organs responsible for balance in mice. Wild-type ICR mice were exposed for 1 month to LFN (0.1 kHz) and high frequency noise (HFN; 16 kHz) at 70 dB SPL at a distance of approximately 10–20 cm. Behavior analyses including rotarod, beam-crossing and footprint analyses showed impairments of balance in LFN-exposed mice but not in non-exposed mice or HFN-exposed mice. Immunohistochemical analysis showed a decreased number of vestibular hair cells and increased levels of oxidative stress in LFN-exposed mice compared to those in non-exposed mice. Our results suggest that chronic exposure to LFN at moderate levels causes impaired balance involving morphological impairments of the vestibule with enhanced levels of oxidative stress. Thus, the results of this study indicate the importance of considering the risk of chronic exposure to LFN at a moderate level for imbalance.

Non-equilibrium atmospheric pressure plasmas modulate cell cycle-related gene expressions in melanocytic tumors of RET-transgenic mice

The incidence of cutaneous malignant melanoma is increasing at a greater rate than that of any other cancer in the world. However, an effective therapy for malignant melanoma has not been established. Recently, some studies have shown an antitumor effect of non‐equilibrium atmospheric pressure plasmas (NEAPPs) in vitro. Here, we examined the in vivo effect of NEAPP on cell cycle regulators, key elements for malignant transformation, in spontaneously developed benign melanocytic tumors in a hairless animal model. NEAPP irradiation decreased expression levels of cell cycle promoters, Cyclin D1, E1 and E2, and increased expression level of a cell cycle repressor, p27KIP1. Cyclin D1, E1 and E2 and p27KIP expression levels were associated with malignant transformation of the benign tumor in the animal model. Our results suggest that NEAPP irradiation suppresses malignant transformation of a benign melanocytic tumor via control of the expression levels of cell cycle regulators.

Epidemiological analysis of the association between hearing and barium in humans

Our previous study experimentally showed barium (Ba)-mediated hearing loss in mice. To our knowledge, however, it remains unknown whether Ba affects hearing in humans. This epidemiological study aimed at investigating ototoxicity of Ba in humans. Associations of Ba levels in hair, toenails and urine with hearing levels (1, 4, 8 and 12 kHz) were analyzed in 145 Bangladeshi subjects. Binary logistic regression analysis with adjustment for age, sex, body mass index (BMI) and smoking showed that Ba levels in hair had significant associations with hearing loss at 8 kHz (OR=4.75; 95% CI: 1.44, 17.68) and 12 kHz (OR=15.48; 95% CI: 4.04, 79.45). Ba levels in toenails were also associated with hearing loss at 8 kHz (OR=3.20; 95% CI: 1.35, 7.85) and 12 kHz (OR=3.63; 95% CI: 1.58, 8.55), whereas there was no correlation between Ba level in urinary samples and hearing. There was a significant correlation between hearing loss and Ba levels in hair and toenails in the model adjusted with arsenic levels as the confounder. In conclusion, this study suggested that Ba levels could be a new risk factor for hearing loss, especially at high frequencies of 8 and 12 kHz, in humans.

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.

Non-thermal atmospheric pressure plasmas as a novel candidate for preventive therapy of melanoma

Due to the increased ultraviolet radiation, the incidence of melanoma is increasing worldwide more than that of any other cancer. In this study, the effects of irradiation of non-thermal atmospheric pressure plasmas (NEAPPs) on benign melanocytic tumors from our original hairless model mice (HL-RET-mice), in which benign melanocytic tumors and melanomas spontaneously develop in the skin stepwise, were examined. Expression levels of melanoma cell adhesion molecule (MCAM) and matrix metalloproteinase-2 (MMP-2) mRNA in melanomas were higher than those in benign melanocytic tumors in the mice. Repeated irradiation of non-thermal atmospheric pressure plasmas (NEAPPs) for the benign tumors decreased the expression levels of MCAM and MMP-2 mRNA in the tumors from the mice. Previous studies showed that MCAM sites are upstream of MMP-2, that MCAM regulates transcription of MMP-2 in melanoma cells and that MMP-2 is associated with the conversion of a benign tumor to a malignant tumor. Therefore, our results suggest that the NEAPP irradiation-mediated decrease in the expression level of MMP-2 in benign melanocytic tumors is associated with decreased expression levels of MCAM. Moreover, NEAPP irradiation might be a potential candidate for therapy to prevent melanoma development through suppression of malignant conversion in benign melanocytic tumors.