Biogenesis of fibrils requires C-mannosylation of PMEL.

Premelanosome protein (PMEL), a melanocyte-specific glycoprotein, has an essential role in melanosome maturation, assembling amyloid fibrils for melanin deposition. PMEL undergoes several post-translational modifications, including N- and O-glycosylations, which are associated with proper melanosome development. C-mannosylation is a rare type of protein glycosylation at a tryptophan residue that might regulate the secretion and localization of proteins. PMEL has one putative C-mannosylation site in its core amyloid fragment (CAF); however, there is no report focusing on C-mannosylation of PMEL. To investigate this, we expressed recombinant PMEL in SK-MEL-28 human melanoma cells and purified the protein. Mass spectrometry analyses demonstrated that human PMEL is C-mannosylated at multiple tryptophan residues in its CAF and N-terminal fragment (NTF). In addition to the W153 or W156 residue (CAF), which lies in the consensus sequence for C-mannosylation, the W104 residue (NTF) was C-mannosylated without the consensus sequence. To determine the effects of the modifications, we deleted the PMEL gene by using CRISPR/Cas9 technology and re-expressed wild-type or C-mannosylation-defective mutants of PMEL, in which the C-mannosylated tryptophan was replaced with a phenylalanine residue (WF mutation), in SK-MEL-28 cells. Importantly, fibril-containing melanosomes were significantly decreased in W104F mutant PMEL-re-expressing cells compared with wild-type PMEL, observed using transmission electron microscopy. Furthermore, western blot and immunofluorescence analysis suggested that the W104F mutation may cause mild endoplasmic reticulumretention, possibly associated with early misfolding, and lysosomal misaggregation, thus reducing functional fibril formation. Our results demonstrate that C-mannosylation of PMEL is required for proper melanosome development by regulating PMEL-derived fibril formation.

Recombinant human Hsp110-gp100 chaperone complex vaccine is nontoxic and induces response in advanced stage melanoma patients.

Heat shock proteins (hsp) are intracellular chaperones that possess extracellular immunostimulatory properties when complexed with antigens. A recombinant Hsp110-gp100 chaperone complex vaccine showed an antitumor response and prolonged survival in murine melanoma. A phase Ib dose-escalation study of a recombinant human Hsp110-gp100 vaccine in advanced-stage melanoma patients was performed to evaluate toxicity, immunostimulatory potential and clinical response. Patients with pretreated, unresectable stage IIIB/C/IV melanoma received the chaperone complex vaccine in a dose-escalation protocol; three vaccinations over a 43-day-period. Tumor response, clinical toxicity and immune response were measured. Ten patients (eight female, median age 70 years) were enrolled and two patients had grade 1 adverse events; minor skin rash, hyperhidrosis and fever (no grade 2 or higher adverse events). Median progression-free survival was longer for lower vaccine doses as compared to the maximum dose of 180 mcg (4.5 vs. 2.9 months; P = 0.018). The lowest dose patients (30 and 60 mcg) had clinical tumor responses (one partial response, one stable disease). CD8+ T cell interferon-γ responses to gp100 were greater in the clinically responding patients. A pattern of B cell responses to vaccination was not observed. Regulatory T cell populations and co-stimulatory molecules including cytotoxic T-lymphocyte-associated protein 4 and PD-1 appeared to differ in responders versus nonresponders. A fully recombinant human Hsp110-gp100 chaperone complex vaccine had minimal toxicity, measurable tumor responses at lower doses and produced peripheral CD8+ T cell activation in patients with advanced, pretreated melanoma. Combination with currently available immunotherapies may augment clinical responses.

The impaired unfolded protein-premelanosome protein and transient receptor potential channels-autophagy axes in apoptotic melanocytes in vitiligo.

Vitiligo is an autoimmune skin disease, characterized by depigmentation and epidermal melanocytes loss. The specific mechanisms underlying vitiligo have not been fully understood. As a result, treating vitiligo is a dermatological challenge. Recently, much attention has been paid to the dysfunction and interaction of organelles under environmental stress. The impaired organelles could generate misfolded proteins, particularly accumulated toxic premelanosome protein (PMEL) amyloid oligomers, activating the autoimmune system and cause melanocyte damage. Unfolded protein response (UPR) dysfunction accelerates toxic PMEL accumulation. Herein, we presented a narrative review on UPR's role in vitiligo, the misfolded PMEL-induced attack of the autoimmune system under autophagy dysfunction caused by abnormal activation of transient receptor potential (TRP) channels and the background of UPR system defects in melanocytes. All of these mechanisms were integrated to form UPR/PMEL-TRP channels/autophagy axis, providing a new understanding of vitiligo pathogenesis.

Is immunohistochemistry always the panacea to morphologic mimics? Two, distinct soft tissue tumors exhibiting alveolar pattern and TFE3 immunoreactivity.

Alveolar soft part sarcoma (ASPS) and certain perivascular epithelioid cell neoplasms (PEComas) exhibit overlapping histopathological features, including immunohistochemical expression of TFE3, as well as TFE3 gene rearrangement. PEComas with an epithelioid morphology are known to exhibit variable immunoexpression of muscle markers. At the same time, aberrant immunoreactivity of HMB45 immunostain, which is invariably, used to substantiate a diagnosis of a PEComa, has been reported in various other tumors. Herein, we discuss two rare cases of soft tissue tumors with overlapping morphological and immunohistochemical features. Case1: A 34-year-old male underwent a biopsy for a recurrent, right-sided nasal polyp. Biopsy showed polygonal tumor cells, containing prominent nucleoli, arranged in a "nesting-type"/alveolar growth pattern. Immunohistochemically, tumor cells displayed TFE3 positivity and an aberrant positivity for HMB45. Special stain (PAS-diastase) highlighted intracytoplasmic granules and crystals. Diagnosis of ASPS was offered. Furthermore, the tumor cells displayed TFE3 gene rearrangement. Case 2: A 29-year-old female underwent an aural polypectomy. Microscopic examination revealed a tumor with a "nesting-type"/alveolar arrangement of tumor cells with vacuolated cytoplasm, arranged around thin-walled blood vessels. Immunohistochemically, tumor cells were diffusely positive for HMB45 and TFE3 and focally for SMA. A diagnosis of a PEComa was offered. This report constitutes the first documentation of aberrant HMB45 immunoreactivity in case of ASPS, and one of the first reported cases of a PEComa in the ear. It emphasizes the value of integrating clinicopathological features with immunohistochemical and molecular results in differentiating two rare, but distinct soft tissue tumors with overlapping features. An exact diagnosis of both these tumor entities has therapeutic implications.

Comparison of two routes of administration of a cationic liposome formulation for a prophylactic DC vaccination in a murine melanoma model.

Dendritic cell (DC) vaccination can be achieved via straight loading of vaccine into DCs ex vivo or administration to DCs in vivo. However, there is no certain consensus on which approach is preferable, and each strategy has its advantages and disadvantages, which affect the efficacy and safety of vaccines. It will also be more complicated when a vaccine delivery system is included. In this study, the efficacy of ex vivo pulsed DC-based vaccine compared with in vivo subcutaneous administration of a cationic liposomes (CLs) formulation containing gp100 antigen (gp100-CLs) was evaluated in a murine melanoma model. In combination with an anti-PD-1 antibody, the ex vivo approach of gp100-CLs yielded a significant (P < 0.01) increase in the number of antigen-specific tumors infiltrated lymphocytes (TILs) with a significant upregulation of IFN-γ (P < 0.0001) and PD-1 (P < 0.0001) expression level. They also dampened the function of immunosuppressive regulatory T cells (Tregs) via significant downregulation of IL-10 and TGF-ß (P < 0.0001) expression level compared to in vivo approach in the tumor microenvironment (TME). Furthermore, prophylactic immunization with gp100-CLs pulsed DCs ex vivo delayed tumor growth and induced the survival benefit over in vivo immunization. Collectively, the ex vivo DC-based vaccination pulsed with gp100 encapsulated in liposomes synergizes with anti-PD-1 antibody and represents a preferable approach against melanoma.

Functional Domains and Evolutionary History of the PMEL and GPNMB Family Proteins.

The ancient paralogs premelanosome protein (PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.

Atypical Fibroxanthoma Demonstrating HMB45+ Staining.

ABSTRACT: Immunohistochemistry is useful and often necessary for the diagnosis of many histopathological entities, including atypical fibroxanthoma (AFX), which is typically considered a diagnosis of exclusion after ruling out spindle cell melanoma, sarcomatoid carcinoma, and other spindle cell tumors. AFX is a superficial fibrohistiocytic tumor previously believed to be related to pleomorphic sarcoma (formerly known as malignant fibrous histiocytoma), but is now considered a distinct clinicopathological entity. AFXs commonly express CD68, smooth muscle actin, and lysozyme and are usually negative for melanocytic markers such as HMB45 and S100. However, immunohistochemistry can sometimes be misleading, especially when used without other relevant markers in making a histopathologic diagnosis. HMB45 is a glycoprotein marker of premelanosomes and is often helpful in identifying melanoma because it stains melanosomes in the epidermis, dermis, and nevi glycocomplexes. We report a case of AFX which was strongly positive for HMB45, but negative for all other melanocytic markers. This case emphasizes the potential pitfall of relying on a single immunohistochemical marker to make the diagnosis, especially of melanoma, and also is one of the only rare reported cases of AFXs which are HMB45+.

PMEL as a Prognostic Biomarker and Negatively Associated With Immune Infiltration in Skin Cutaneous Melanoma (SKCM).

Premelanosome protein (PMEL) is crucial for the formation of melanosomal fibrils through the transition from stage I to stage II melanosomes. It was used as a target antigen in some adoptive T-cell therapy of melanoma. The correlation of PMEL to prognosis and immune cell infiltration level are unknown in melanoma. The PMEL expression was evaluated via Tumor Immune Estimation Resource, Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA). We also evaluate the influence of PMEL on overall survival via GEPIA, PrognoScan, and immunohistochemistry in human tissue microarray. The correlation between PMEL expression level and immune cell or gene markers of immune infiltration level was explored on Tumor Immune Estimation Resource and GEPIA. PMEL expression was significantly higher in skin cutaneous melanoma (SKCM) and SKCM-metastasis in comparison with the other cancers. In SKCM, PMEL expression in high levels was associated with poor overall survival. In both SKCM and SKCM-metastasis patients, PMEL expression is negatively correlated with the infiltration cells of CD8+ T cells, macrophages, and neutrophils. Programmed cell-death protein 1 just showed response rates ranging from 20% to 40% in patients with melanoma, so it is critical to discover a new therapeutic target. PMEL is negatively associated with immune cell infiltration and can be as a negative prognosis marker or new immunotherapy target in SKCM and SKCM-metastasis.

Identification of critical amino acid residues in the regulatory N-terminal domain of PMEL.

The pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.

Dermal melanocytic tumor with CRTC1-TRIM11 fusion: Report of two additional cases with review of the literature of an emerging entity.

"Cutaneous melanocytic tumor with CRTC1-TRIM11 fusion" (CMTCT) is a newly described, potentially novel entity that typically presents as a dermal nodule on the head and neck, extremities, and trunk of adults. Histopathologically, it is reported as a nodular or multinodular tumor composed of epithelioid and spindle cells that are variably immunoreactive for S100-protein, SOX10, and MITF along with more specific melanocytic markers such as MelanA and HMB45. With only 11 cases reported in the English literature so far, the neoplasm appears to behave in a relatively indolent fashion. Nevertheless, in one case, local recurrence and synchronous distant metastasis were evident after 13 years. Additional cases with longer follow-up are essential to determine the neoplasm's biologic behavior with more accuracy. Herein, two cases of CMTCT, one arising on the lower back of a 65-year-old female and the other on the arm of a 33-year-old female in addition to a comprehensive literature review are reported.

In silico approach of modified melanoma peptides and their immunotherapeutic potential.

Melanoma is a type of skin cancer with increasing incidence worldwide and high lethality. Conventional forms of treatment are not effective in advanced cancer stages. Hence, immunotherapeutic approaches have been tested to modulate immune response against tumor cells. Some vaccine models using tumor-associated antigens (TAAs) such as glycoprotein 100 (gp100) have been studied, but their expected effectiveness has not been shown until now. Antigen immunogenicity is a crucial point to improve the immune response, and therefore mutations are inserted in peptide sequences. It is possible to understand the interactions which occur between peptides and immune system molecules through computer simulation, and this is essential in order to guide efficient vaccine models. In this work, we have calculated the interaction binding energies of crystallographic data based on modified gp100 peptides and HLA-A*0201 using density functional theory (DFT) and the molecular fractionation with conjugated caps (MFCC) approach. Our results show the most relevant residue-residue interactions, the impact of three mutations in their binding sites, and the main HLA-A*0201 amino acids for peptide-HLA binding.

Histological and Immunohistochemical Changes of Congenital Melanocytic Nevi With Age.

ABSTRACT: Clinical but not histological changes of congenital melanocytic nevi (CMN) with age are well characterized. Our objective was to analyze histological changes of CMN with age and discuss possible clinical implications of our findings. We investigated serial excisions of 21 patients with CMN and compared histological and immunohistochemical features over time. The median number of serial excisions was 6 [interquartile range (IQR) 5-7], the median age at the first excision was 12 months (IQR 5-98), and the median time between the first and last analyzed excision was 53 months (IQR 45-64). The projected adult size of the excised CMN was "large" or "giant" in 14 of the 21 CMN (67%) and "medium" in the remaining lesions (33%). Nineteen CMN (90%) involved the subcutaneous fat, and 16 of the 21 CMN (76%) reached the lower surgical margin. The histological pattern and depth did not change over time but the cellularity and HMB-45 expression of dermal melanocytes decreased in 16 of the 21 patients (76%) and in 15 of the 21 patients (71%), respectively (both P < 0.001). Patients with decreasing HMB-45 expression were significantly younger at the first excision (median 6 months, IQR 4-28) than patients with unchanged HMB-45 expression (median 176 months, IQR 12-186; P = 0.018). The expression of Ki-67 and p16 did not change significantly with age. Our study demonstrates that (1) the cellularity and pigment production of CMN decreases with age, (2) the histological pattern and extension in depth remain stable, and (3) clear resection margins can rarely be achieved in larger CMN.

The Silk Protein Sericin Promotes Viability of ARPE-19 and Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells in vitro.

PURPOSE: Maintaining mature and viable retinal pigment epithelial cells (RPE) in vitro has proven challenging. Investigating compounds that can promote RPE-viability and maturation is motivated by RPE transplantation research, the quest to understand RPE physiology, and a desire to modulate RPE in pathological states. We have previously reported that the silk protein sericin promotes viability, maturation, and pigmentation of human fetal RPE. In the present study, our aim was to uncover whether these effects can be seen in adult retinal pigment epithelial cell line-19 (ARPE-19) and induced pluripotent stem cell-derived RPE (iPSC-RPE). METHODS: ARPE-19 and iPSC-RPE were cultured with or without 10 mg/mL sericin. After 7 days, viability was assessed with calcein-acetoxymethyl ester (CAM) and ethidium homodimer-1 (EH-1) assays, flow cytometry, and morphometric analysis. Expression levels of RPE65, tyrosinase, and Pmel17 were quantified to compare maturation between the sericin-treated and control cultures. Light microscopy and staining of the tight junction protein zonula occludens protein 1 (ZO-1) were employed to study sericin's effects on RPE morphology. We also measured culture medium pH, glucose, lactate, and extracellular ion content. RESULTS: Sericin-supplemented RPE cultures demonstrated significantly better viability compared to control cultures. Sericin appeared to improve ARPE-19 maturation and morphology in vitro. No effects were seen on RPE pigmentation with the concentration of sericin and duration of cell culture herein reported. CONCLUSIONS: This is the first study to demonstrate that supplementing the culture media with sericin promotes the viability of iPSC-RPE and ARPE-19. Sericin's viability-promoting effects may have important implications for retinal therapeutics and regenerative medicine research.

Mitotically Active Nevus and Nevoid Melanoma: A Clinicopathological and Molecular Study.

ABSTRACT: The distinction between nevoid melanoma and a mitotically active nevus can be challenging at the microscopic level. In this study, we performed cytogenetic testing on a cohort of 25 mitotically active melanocytic proliferations resembling common melanocytic nevus from 25 patients. Based on cytogenetic findings, the lesions were classified as "nevoid melanoma" (n = 13) or "mitotically active nevus" (n = 12). Subsequently, we compared the clinicopathological features between these 2 groups. Nevoid melanomas occurred in older patients (P = 0.007); however, there were no significant differences in gender, size, or anatomical distribution between the 2 groups. Histologically, deep/marginal mitoses (P = 0.006), lack of maturation with depth (P = 0.036), and pseudo-maturation (P = 0.006) were significantly more common in nevoid melanomas. Immunohistochemically, complete loss of p16 was an important divisive feature (P = 0.0004), seen in 70% of nevoid melanomas, and highly correlated with loss of CDKN2A gene (chromosome 9p21). Our findings suggest that such reproducible immunomorphological differences can be of value in distinguishing nevoid melanoma from mitotically active nevus. Nevoid melanomas demonstrated a spectrum of chromosomal aberrations similar to those seen in common subtypes of melanoma, which can serve as a powerful adjunct diagnostic tool in morphologically challenging lesions.

Type I interferon signaling limits viral vector priming of CD8+ T cells during initiation of vitiligo and melanoma immunotherapy.

Vitiligo is an autoimmune skin disease in which epidermal melanocytes are targeted for destruction by CD8+ T cells specific for melanocyte/melanoma-shared antigens. IFNγ is the central cytokine driving disease, but the role of type I IFN in vitiligo remains unclear. We investigated the functional role of type I IFN during vitiligo progression using two different mouse models: one induced with a vaccinia virus (VV) vaccine and one induced with dendritic cells to prime autoimmune T cells. Induction of vitiligo by VV in IFNaR-deficient mice led to the development of severe vitiligo compared with wild-type (WT) mice and was characterized by a significantly enhanced effector CD8+ T-cell response. Severe vitiligo in this model was a result of VV persistence, because exacerbation of disease in IFNaR-deficient mice was not observed when antigen-pulsed dendritic cells were used to induce vitiligo instead of virus. Treatment of B16F10 melanoma-inoculated mice with VV vaccine therapy also induced a significantly enhanced anti-tumor response in IFNaR-deficient mice compared with WT. These results not only help define the pathways responsible for vitiligo progression but also suggest that blockade of type I IFNs following administration of a VV vaccine may provide increased immunogenicity and efficacy for melanoma immunotherapy.

Aberrant expression of HMB45 and negative PRAME expression in halo nevi.

BACKGROUND: Traditionally, most cutaneous nevi show a gradient of HMB45 (human melanoma black 45) and negative PRAME (preferentially expressed antigen in melanoma) immunostaining, while melanomas often show irregularly positive, diffusely positive or completely negative HMB45 expression, and PRAME immunopositivity. However, we have occasionally observed benign halo nevi with loss of HMB45 gradient, raising diagnostic consideration for melanoma. The purpose of this study was to elucidate the expression pattern of HMB45 and PRAME in nevi with the halo phenomenon (NHP). METHODS: PRAME and HMB45 staining patterns in 20 cases of NHP and 16 cases of conventional nevi were evaluated using light microscopy. An HMB45 gradient was defined as immunopositivity in only superficial melanocytes. HMB45 aberrant expression consisted of superficial and deep immunopositivity. RESULTS: Aberrant HMB45 expression was observed in 10 of 20 NHP (50%). A gradient of HMB45 staining was seen in most conventional nevi, with only one showing focal weak expression in the deep dermis (6.3%). All cases of NHP and conventional nevi showed essentially negative immunostaining by PRAME. CONCLUSION: Aberrant HMB45 expression in NHP is not uncommon and may be a diagnostic pitfall. Negative PRAME immunostaining may be a reassuring finding to help differentiate halo nevus from malignant melanoma.

Hofmeister Ions Modulate the Autocatalytic Amyloidogenesis of an Intrinsically Disordered Functional Amyloid Domain via Unusual Biphasic Kinetics.

Hofmeister ions are thought to play fundamentally important roles in protein solubility, folding, stability, and function. Salt ions profoundly influence the course of protein misfolding, aggregation, and amyloid formation associated with devastating human diseases. However, the molecular origin of the salt-effect in protein aggregation remains elusive. Here, we report an unusual biphasic amyloidogenesis of a pH-responsive, intrinsically disordered, oligopeptide repeat domain of a melanosomal protein, Pmel17, that regulates the amyloid-assisted melanin synthesis in mammals via functional amyloid formation. We demonstrate that a symphony of molecular events involving charge-peptide interactions and hydration, in conjunction with secondary phenomena, critically governs the course of this biphasic amyloid assembly. We show that at mildly acidic pH, typical of melanosomes, highly amyloidogenic oligomeric units assemble into metastable, dendritic, fractal networks following the forward Hofmeister series. However, the subsequent condensation of fractal networks via conformational maturation into amyloid fibrils follows an inverse Hofmeister series due to fragmentation events coupled with secondary nucleation processes. Our results indicate that ions exert a strong influence on the aggregation kinetics as well as on the nanoscale morphology and also modulate the autocatalytic amplification processes during amyloid assembly via an intriguing dual Hofmeister effect. This unique interplay of molecular drivers will be of prime importance in delineating the aggregation pathways of a multitude of intrinsically disordered proteins involved in physiology and disease.

Defining an amyloid link Between Parkinson's disease and melanoma.

An epidemiological connection exists between Parkinson's disease (PD) and melanoma. α-Synuclein (α-syn), the hallmark pathological amyloid observed in PD, is also elevated in melanoma, where its expression is inversely correlated with melanin content. We present a hypothesis that there is an amyloid link between α-syn and Pmel17 (premelanosomal protein), a functional amyloid that promotes melanogenesis. Using SK-MEL 28 human melanoma cells, we show that endogenous α-syn is present in melanosomes, the organelle where melanin polymerization occurs. Using in vitro cross-seeding experiments, we show that α-syn fibrils stimulate the aggregation of a Pmel17 fragment constituting the repeat domain (RPT), an amyloidogenic domain essential for fibril formation in melanosomes. The cross-seeded fibrils exhibited α-syn-like ultrastructural features that could be faithfully propagated over multiple generations. This cross-seeding was unidirectional, as RPT fibrils did not influence α-syn aggregation. These results support our hypothesis that α-syn, a pathogenic amyloid, modulates Pmel17 aggregation in the melanosome, defining a molecular link between PD and melanoma.

MCF7 Spheroid Development: New Insight about Spatio/Temporal Arrangements of TNTs, Amyloid Fibrils, Cell Connections, and Cellular Bridges.

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme.