Operative therapy of acral melanomas

Acral lentiginous melanomas (ALM) represent 4?C10% of cutaneous melanomas in white populations. Patients with ALM seem to have a poor prognosis, often due to late diagnosis. Micrographic surgery (3D-histology) is not seen as surgical procedure but more as histopathological technique. With micrographic surgery, continuously spreading ALM can be excised with smaller excision margins and good functional and cosmetic outcomes. In a recent study, 244 patients with ALM were compared using conventional histology versus 3D-histology. Clinical and surgical risk factors influence the prognosis of ALM. Tumor thickness and ulceration are the most important prognostic factors. 3D-histology with paraffin technique (optionally combined with immunohistological methods) can reduce excision margins and avoid local recurrences. Subungual melanomas represent only 2?C3% of cutaneous melanomas in Caucasian and 20% in African or Asian skin type and are often clinically misdiagnosed. They are often localized on the thumb or great toe, which are most important for the function of the affected limb. The excision of subungual melanoma with 3D-histology and tumor-free excision margins including the nail matrix can be seen as a safe surgical strategy, which does not hazard the prognosis of the patient. Function and cosmesis of the finger or toe are preserved. Amputation in subungual melanoma is not recommended and should be reserved only for infiltrating melanomas with affection of the bone or joint.     

Dual role of protein kinase C on mitogen-activated protein kinase activation and human keratinocyte proliferation

Abstract: Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K#5 and K#14. In keratinocyte autocrine culture, the exogenously added epidermal growth factor (EGF) has no effect on cell proliferation and mitogen‐activated protein kinase (MAPK) activity. PD98059 inhibits MAPK pathway and autocrine keratinocyte proliferation. Staurosporine and Gö6976 strongly inhibit autonomous keratinocyte proliferation. In contrast, Gö6983 (which does not inhibit PKCµ) inhibits only 20% of autocrine keratinocyte proliferation. Staurosporine inhibits MAPK activity, whereas Gö6976 and Gö6983 strongly increase it. We have concluded that MAPK, PKCµ and probably PKCα take part in autocrine keratinocyte proliferation. The effect of Gö6976 and Gö6983 on MAPK activity could be explained by the inhibition of PKC‐dependent MAPK‐phosphatase expression. The effect of staurosporine could be explained by its paradoxical action (activation) on protein kinase C (PKC) in keratinocytes.     

Mutational profiling of acral melanomas in Korean populations

The proportion of acral melanoma (AM) is much higher in Asian populations than in Caucasian populations. Although mutational profiles associated with AM have been discovered in Caucasian populations, knowledge of its genetic alterations in Asian populations is limited. To describe the molecular nature of AM in Korean patients, we performed mutational profiling of AM and matched normal tissues in patients. Fifty‐one formalin‐fixed paraffin‐embedded AM samples and 32 matched pairs from patients’ saliva DNA were analysed by next‐generation sequencing. Only mutations confirmed via digital droplet PCR or in BRAF, KIT and NRAS, the most frequently altered cancer genes in cutaneous melanoma, were considered as positive. The relationship between mutational status and clinicopathological features were examined. Of the 47 AM patients screened, alteration of BRAF, NRAS and KIT genes was observed in 6.4%, 4.3% and 8.5%, respectively. We also tested matched normal tissues of patients to identify tumor‐specific mutations. Examination of the mutational profile in a cohort of 28 primary melanomas and matched normal controls found BRAF mutations in two cases (7.1%), KIT mutations in three cases (10.7%) and CTNNB1 mutations in one case (3.6%). The BRAF, NRAS and KIT mutation status did not correlate with clinicopathological characteristics. Our results show that KIT, NRAS and BRAF hotspot mutations occur at a low frequency in Korean populations. We also observed a case with the CTNNB1 mutation, which raises the possibility that other pathways are associated with AM development.     

Expression of soluble adenylyl cyclase in acral melanomas

Soluble adenylyl cyclase (sAC) regulates melanocytic cells, and is a diagnostic marker for pigmented skin lesions. Because only a few studies on sAC expression in acral melanomas have been performed, we investigated the histopathological significance of sAC expression in 33 cases of acral melanoma, and assessed its diagnostic value in distinguishing melanoma in situ (MIS, n = 17) from acral invasive melanomas (n = 16) and melanocytic naevi (n = 11). Acral melanomas exhibited more marked nuclear immunopositivity compared with acral melanocytic naevi. sAC expression significantly correlated with the nuclear morphology of melanocytes and melanoma cells, namely, hyperchromatic nuclei and prominent nucleoli within vesicular nuclei. sAC expression was predominantly observed in the hyperchromatic nuclei of MIS and the prominent nucleoli invasive melanomas, respectively. In vitro culture models of melanocytes and melanoma cell lines exhibited sAC staining patterns similar to those of acral melanomas. Differentiation induction showed that nuclear and nucleolar expression varied depending on cell morphology. sAC immunostaining may be useful for the differential diagnosis of acral melanocytic lesions, and sAC expressed in the nucleus and nucleolus might be related to cytological and nuclear changes associated with invasion and progression of acral melanomas.     

Stratifin-induced matrix metalloproteinase-1 in fibroblast is mediated by c-fos and p38 mitogen-activated protein kinase activation

Previously, we have demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, stimulates matrix metalloproteinase (MMP)-1 expression in dermal fibroblasts. In this study, we showed that stratifin induced fibroblast MMP-1 messenger ribonucleic acid (mRNA) and protein levels through p38 mitogen-activated protein kinase (MAPK). Our data indicated that treatment of dermal fibroblasts with stratifin resulted in rapid and transient upregulation of c-jun and c-fos mRNA levels. We also demonstrated that SB203580 (SB), a specific inhibitor of p38 MAPK activity, inhibited the activation of fibroblast MMP-1 mRNA expression by stratifin. Subsequently, western blot analysis revealed phosphorylation of p38 at 90 min after stratifin stimulation and this was decreased to approximately 50% of the maximum value by 120 min. Stratifin was demonstrated to increase MMP-1 protein levels starting at 4 h and reaching its peak at 12-24 h. Furthermore, SB significantly blocked the stratifin induction of MMP-1 protein levels (***p<0.005, n=3). Microarray analysis of stratifin-treated fibroblasts shows an increase in Elk4/Sap1 mRNA expression and this finding was confirmed by northern blot analysis. Our results indicate that stratifin markedly increase Elk4/Sap1 mRNA expression in a time-dependent fashion. In conclusion, stratifin stimulates fibroblast MMP-1 levels through the activation of c-fos and MAPK pathway.     

皮肤病中c-Jun氨基末端蛋白激酶通路的研究现况

c-Jun氨基末端蛋白激酶是丝裂原活化蛋白激酶家族的成员,调节细胞的生长、发育、增殖和分化.c-Jun氨基末端蛋白激酶的异常表达与人皮肤疾病的发生、发展密切相关.研究证实,c-Jun氨基末端蛋白激酶在多种皮肤疾病患者的真/表皮中表达过度增高及异常活化,进而导致细胞的生长、发育、增殖、分化异常以及炎症反应和细胞凋亡的发生.选择阻断c-Jun氨基末端蛋白激酶通路的异常活化可以使病情得到改善.针对c-Jun氨基末端蛋白激酶信号通路的靶向治疗已经成为目前多种皮肤病的研究热点.     

Harnessing autophagy to overcome mitogen-activated protein kinase kinase inhibitor-induced resistance in metastatic melanoma

Malignant melanoma tumour cells often carry mutations in the BRAF protein, which activates a second protein called MEK to fuel tumour growth. Using drugs that block the mutant form of BRAF and MEK, we can specifically target and kill most of these tumour cells, but a subset of tumour cells often become resistant to BRAF and MEK inhibiting drugs, and cause the tumour to regrow. The aim of this study was to determine how changes in a protein called CD271, which has previously been shown to increase tumour growth and aggressiveness, contributes to the resistance of BRAF mutant melanoma cells to the MEK inhibiting drug, trametinib. Furthermore, we have previously shown that a cell survival process within tumour cells called autophagy is increased in BRAF mutant melanoma cells, so we also aimed to determine the contribution of autophagy to the resistance of BRAF mutant melanoma cells to trametinib. We show that CD271 and autophagy are increased in advanced malignant melanoma tumours compared to normal moles, and that treatment of melanoma cells in the laboratory with trametinib results in the emergence of a subset of melanoma cells with increased CD271 and autophagy. However, specific inhibition of CD271 reduced the number of resistant melanoma cells following trametinib treatment, while either blocking or activating autophagy resulted in death of trametinib-resistant melanoma cells. We also show that combined treatment with drugs that specifically block both MEK and autophagy reduced the invasion of trametinib-resistant melanoma cells in embryonic zebrafish up to 5 days old. Our results show that embryonic zebrafish at an early stage of development may replace mice as a more ethical alternative animal model for the study of cancer metastasis (spreading) and drug response, and highlight an important new way to prevent drug-resistance by blocking autophagy in patients with BRAF mutant melanoma cells.     

Hematopoietic progenitor kinase 1, mitogen-activated protein/extracellular signal-related protein kinase kinase kinase 1, and phosphomitogen-activated protein kinase kinase 4 are overexpressed in extramammary Paget disease

The c-Jun amino-terminal kinase (JNK) pathway seems to play important roles in the pathogenesis of several tumors, but its significance in extramammary Paget disease (EMPD) has not been investigated yet. The purpose of the study was to investigate the potential contribution of the JNK-associated molecules, such as hematopoietic progenitor kinase 1 (HPK1), mitogen-activated protein/extracellular signal-related protein kinase kinase kinase1 (MEKK1), transforming growth factor-β activated kinase 1 (TAK1), and phosphomitogen-activated protein kinase kinase 4 (p-MKK4) to the development of EMPD. Thirty-five paraffin-embedded EMPD specimens were subjected to immunohistochemical staining for HPK1, MEKK1, TAK1, and p-MKK4. All the 35 EMPD, including 13 dermal invasive EMPD and 2 lymph node metastasis, showed cytoplasmic overexpression of HPK1, MEKK1, and p-MKK4. The expression (%positive cells) of HPK1, MEKK1, and p-MKK4 in EMPD (92.3% ± 8.6%, 92.9% ± 8.6%, and 92.7% ± 7.4%, respectively) were significantly higher than in normal eccrine sweat gland cells (51.6% ± 10.4%, 44.7% ± 11.7%, 0% ± 0%). In addition, the expression of HPK1-, MEKK1-, and p-MKK4 in invasive EMPD was significantly higher than in noninvasive EMPD. Meanwhile, the expression of TAK1 was basically low and no significantly different between EMPD and normal controls. In conclusion, these results indicate that JNK pathway may play a role in the pathogenesis of EMPD.     

Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) inhibitor KN-62 suppresses the activity of mitogen-activated protein kinase (MAPK), c-myc activation and human keratinocyte proliferation

Autocrine growth of human epidermal keratinocytes can be maintained in subconfluent cell cultures in the absence of exogenous growth factors. We used this culture model to investigate the interactions between the mitogen-activated protein kinase (MAPK) pathway and Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) in autocrine keratinocyte proliferation. We have previously demonstrated that MAPK and protein kinase C (PKC) are both involved in keratinocyte proliferation in a complex set of interactions. Treatment of keratinocytes with PD98059, a potent inhibitor of MAPK kinase, inhibited the MAPK pathway, c-myc activation and autocrine keratinocyte proliferation. Application of the CaM-kinase inhibitor KN-62 also led to a strong inhibition of MAPK/c-myc activation and autocrine keratinocyte proliferation. Other inhibitors, such as wortmannin (selective and potent inhibitor of phosphatidylinositol 3-kinase) and AG 490 (JAK2 inhibitor) had weak effects on autocrine keratinocyte proliferation, MAPK and c-myc activation. Our results clearly demonstrate a crosstalk between CaM-kinase/MAPK pathways in transducing keratinocyte proliferation stimuli.     

蛋白激酶B和丝裂原活化蛋白激酶在烟酸保护的HaCaT细胞抵抗中波紫外线损伤中的作用

目的 探讨烟酸保护由UVB诱导的角质形成细胞损伤的胞内信号传导分子机制。 方法 UVB照射和烟酸处理HaCaT细胞,TUNEL法检测细胞凋亡,Western印迹检测蛋白激酶B（Akt）/丝裂原活化蛋白激酶（MAPK）通路相关蛋白Akt、P38、JNK、ERK1/2的磷酸化水平变化。ELISA检测细胞分泌内皮素1（ET-1）及碱性成纤维细胞生长因子（bFGF）的水平。结果 Western印迹结果表明,UVB照射和烟酸处理HaCaT细胞后,p-Akt、p-P38、p-JNK、p-ERK1/2蛋白在60 min内都显著激活（P < 0.01）。烟酸预处理后的HaCaT细胞再经UVB照射,可以发现p-Akt、p-P38、p-ERK1/2信号分子在2 h内激活更显著（P < 0.01）。３个抑制剂加UVB照射组较3个单独抑制剂组ET-1、bFGF表达降低,差异均有统计学意义,其中LY294002组、SB203580组ET-1、bFGF水平最低;烟酸预保护的抑制剂处理组HaCaT细胞在UVB照射后,LY294002和U0126组没有出现ET-1、bFGF水平回升,SB203580组bFGF水平出现回升。结论　Akt信号分子在烟酸保护的HaCaT细胞抵抗UVB损伤中起一定的调控作用。