Sensing of the melanoma biomarker TROY using silicon nanowire field-effect transistors

Antibody-functionalized silicon nanowire field-effect transistors have been shown to exhibit excellent analyte detection sensitivity enabling sensing of analyte concentrations at levels not readily accessible by other methods. One example where accurate measurement of small concentrations is necessary is detection of serum biomarkers, such as the recently discovered tumor necrosis factor receptor superfamily member TROY (TNFRSF19), which may serve as a biomarker for melanoma. TROY is normally only present in brain but it is aberrantly expressed in primary and metastatic melanoma cells and shed into the surrounding environment. In this study, we show the detection of different concentrations of TROY in buffer solution using top-down fabricated silicon nanowires. We demonstrate the selectivity of our sensors by comparing the signal with that obtained from bovine serum albumin in buffer solution. Both the signal size and the reaction kinetics serve to distinguish the two signals. Using a fast-mixing two-compartment reaction model we are able to extract the association and dissociation rate constants for the reaction of TROY with the antibody immobilized on the sensor surface.The authors thank Biosite Diagnostics (San Diego, CA) for providing TROY antibodies. The authors acknowledge NIH, NSF, and Battelle Memorial Institute for support of this work. (NIH; NSF; Battelle Memorial Institute)https://pubs.acs.org/doi/pdf/10.1021/acssensors.6b00017Accepted manuscrip

Differences in telomere length between sporadic and familial cutaneous melanoma

BACKGROUND: Several pieces of evidence indicate that a complex relationship exists between constitutional telomere length (TL) and the risk of cutaneous melanoma. Although the general perception is that longer telomeres increase melanoma risk, some studies do not support this association. We hypothesise that discordant data are due to the characteristics of the studied populations. OBJECTIVES: To evaluate the association of telomere length with familial and sporadic melanoma. METHODS: TL was measured by multiplex quantitative PCR in leukocytes from 310 melanoma patients according to familial/sporadic and single/multiple cancers and 216 age-matched controls. RESULTS: Patients with sporadic melanoma were found to have shorter telomeres as compared to those with familial melanoma. In addition, shorter telomeres, while tending to reduce the risk of familial melanoma regardless of single or multiple tumors, nearly trebled the risk of single sporadic melanoma. CONCLUSIONS: This is the first time that TL has been correlated to opposite effects on melanoma risk according to the presence or absence of familial predisposition. Individual susceptibility to melanoma should be taken into account when assessing the role of TL as a risk factor. This article is protected by copyright. All rights reserved

m6A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti-PD-1 blockade

Melanoma is one of the most deadly and therapy-resistant cancers. Here we show that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy. FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice. FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway. Knockdown of FTO increases m6A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity. Our findings demonstrate a crucial role of FTO as an m6A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma. © 2019, The Author(s)

A Targeted Quantitative Proteomic Method Revealed a Substantial Reprogramming of Kinome during Melanoma Metastasis.

Kinases are involved in numerous critical cell signaling processes, and dysregulation in kinase signaling is implicated in many types of human cancers. In this study, we applied a parallel-reaction monitoring (PRM)-based targeted proteomic method to assess kinome reprogramming during melanoma metastasis in three pairs of matched primary/metastatic human melanoma cell lines. Around 300 kinases were detected in each pair of cell lines, and the results showed that Janus kinase 3 (JAK3) was with reduced expression in the metastatic lines of all three pairs of melanoma cells. Interrogation of The Cancer Genome Atlas (TCGA) data showed that reduced expression of JAK3 is correlated with poorer prognosis in melanoma patients. Additionally, metastatic human melanoma cells/tissues exhibited diminished levels of JAK3 mRNA relative to primary melanoma cells/tissues. Moreover, JAK3 suppresses the migration and invasion of cultured melanoma cells by modulating the activities of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). In summary, our targeted kinome profiling method provided by far the most comprehensive dataset for kinome reprogramming associated with melanoma progression, which builds a solid foundation for examining the functions of other kinases in melanoma metastasis. Moreover, our results reveal a role of JAK3 as a potential suppressor for melanoma metastasis

Pigmented melanoma cell migration study on murine syngeneic B16F10 melanoma cells or tissue transplantation models

Melanoma is a lethal form of skin cancer with poor prognosis, especially due to the early metastatic feature. Recent studies have shown that the melanin pigment influences the nanomechanical properties and, therefore, the metastatic behavior of the melanoma cells. We aimed to study the growth of subcutaneously transplanted syngeneic melanoma tissue in female C57BL/6 mice harvested from a mouse with a four-week B16F10 melanoma. Also, we studied the effect of the melanin pigment loading on the peritumoral migratory abilities of melanoma cells. Even when the syngeneic transplant was different (cultured cells vs. tumor tissue), the morphological features and the tumor growth were similar in both groups of mice. Heavily pigmented melanoma cells had low migration abilities. Angiogenesis, the depigmentation phenomenon, and the cell shape changes were related to pigmented melanoma cell migration along the matrix collagen fibers of peritumoral structures: the abluminal face of the vessels (angiotropism), the endomysium, and the nerves (neurotropism). The replacement of the histopathological growth pattern, the absence of angiogenesis, and rapidly tumor-bearing emboli were correlated with amelanotic and low pigmented melanoma cells. This study demonstrated that syngeneic melanoma tissue transplantation was a viable technique, and that the melanin pigment loading level can affect the melanoma cell migration profile

KMT2A promotes melanoma cell growth by targeting hTERT signaling pathway.

Melanoma is an aggressive cutaneous malignancy, illuminating the exact mechanisms and finding novel therapeutic targets are urgently needed. In this study, we identified KMT2A as a potential target, which promoted the growth of human melanoma cells. KMT2A knockdown significantly inhibited cell viability and cell migration and induced apoptosis, whereas KMT2A overexpression effectively promoted cell proliferation in various melanoma cell lines. Further study showed that KMT2A regulated melanoma cell growth by targeting the hTERT-dependent signal pathway. Knockdown of KMT2A markedly inhibited the promoter activity and expression of hTERT, and hTERT overexpression rescued the viability inhibition caused by KMT2A knockdown. Moreover, KMT2A knockdown suppressed tumorsphere formation and the expression of cancer stem cell markers, which was also reversed by hTERT overexpression. In addition, the results from a xenograft mouse model confirmed that KMT2A promoted melanoma growth via hTERT signaling. Finally, analyses of clinical samples demonstrated that the expression of KMT2A and hTERT were positively correlated in melanoma tumor tissues, and KMT2A high expression predicted poor prognosis in melanoma patients. Collectively, our results indicate that KMT2A promotes melanoma growth by activating the hTERT signaling, suggesting that the KMT2A/hTERT signaling pathway may be a potential therapeutic target for melanoma

Therapeutic applications of SAMMSON lncRNA inhibition in uveal melanoma

Uveal melanoma is the most common intraocular malignancy in adults. The lack of an effective treatment results in a median survival time less than one year for patients with metastatic disease. Recently, our lab identified the melanoma-specific long non-coding RNA (lncRNA) SAMMSON as a novel therapeutic target in skin melanoma. Analysis of a PAN cancer RNA-sequencing dataset revealed consistent expression of SAMMSON in uveal melanoma tumors. Although SAMMSON expression was lower in uveal compared to skin melanoma, over 90% of uveal melanoma tumors showed detectable SAMMSON expression. Further analysis also revealed SAMMSON expression in conjunctival melanoma, another form of ocular melanoma. To evaluate the therapeutic potential of SAMMSON inhibition in uveal and conjunctival melanoma, we treated 8 representative cell lines with SAMMSON-specific ASOs and observed a strong reduction in cell viability, accompanied by induction of apoptosis. In line with the role of SAMMSON in modulating mitochondrial metabolism, SAMMSON knock down resulted in decreased mitochondrial oxygen consumption. Uveal melanomas are characterized by activated MEK-signaling through mutations in GNA11/GNAQ. Combining SAMMSON-specific ASOs with the MEK-inhibitor Trametinib, induced strong synergistic effects, resulting in a nearly complete abrogation of tumor cells at nanomolar concentrations of Trametinib. The in vivo effects of SAMMSON inhibition, whether or not in combination with Trametinib, are currently being investigated in a uveal melanoma PDX model. Together, our results demonstrate the efficacy of SAMMSON inhibition as a novel treatment option for uveal melanoma and demonstrate its synergism with MEK inhibition making SAMMSON a promising anti-cancer target for uveal melanoma patients

WIPI1, BAG1 and PEX3 autophagy-related genes are relevant melanoma markers

ROS and oxidative stress may promote autophagy; on the other hand, autophagy may help reduce oxidative damages. According to the known interplay of ROS, autophagy, and melanoma onset, we hypothesized that autophagy-related genes (ARGs) may represent useful melanoma biomarkers. We therefore analyzed the gene and protein expression of 222 ARGs in human melanoma samples, from 5 independent expression databases (overall 572 patients). Gene expression was first evaluated in the GEO database. Forty-two genes showed extremely high ability to discriminate melanoma from nevi (63 samples) according to ROC (AUC ≥ 0.85) and Mann-Whitney (p < 0.0001) analyses. The 9 genes never related to melanoma before were then in silico validated in the IST online database. BAG1, CHMP2B, PEX3, and WIPI1 confirmed a strong differential gene expression, in 355 samples. A second-round validation performed on the Human Protein Atlas database showed strong differential protein expression for BAG1, PEX3, and WIPI1 in melanoma vs control samples, according to the image analysis of 80 human histological sections. WIPI1 gene expression also showed a significant prognostic value (p < 0.0001) according to 102 melanoma patients' survival data. We finally addressed in Oncomine database whether WIPI1 overexpression is melanoma-specific. Within more than 20 cancer types, the most relevant WIPI1 expression change (p = 0.00002; fold change = 3.1) was observed in melanoma. Molecular/functional relationships of the investigated molecules with melanoma and their molecular/functional network were analyzed via Chilibot software, STRING analysis, and gene ontology enrichment analysis. We conclude that WIPI1 (AUC = 0.99), BAG1 (AUC = 1), and PEX3 (AUC = 0.93) are relevant novel melanoma markers at both gene and protein levels

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement