Somatic genomic changes in single Alzheimer's disease neurons.

Dementia in Alzheimer's disease progresses alongside neurodegeneration1-4, but the specific events that cause neuronal dysfunction and death remain poorly understood. During normal ageing, neurons progressively accumulate somatic mutations5 at rates similar to those of dividing cells6,7 which suggests that genetic factors, environmental exposures or disease states might influence this accumulation5. Here we analysed single-cell whole-genome sequencing data from 319 neurons from the prefrontal cortex and hippocampus of individuals with Alzheimer's disease and neurotypical control individuals. We found that somatic DNA alterations increase in individuals with Alzheimer's disease, with distinct molecular patterns. Normal neurons accumulate mutations primarily in an age-related pattern (signature A), which closely resembles 'clock-like' mutational signatures that have been previously described in healthy and cancerous cells6-10. In neurons affected by Alzheimer's disease, additional DNA alterations are driven by distinct processes (signature C) that highlight C>A and other specific nucleotide changes. These changes potentially implicate nucleotide oxidation4,11, which we show is increased in Alzheimer's-disease-affected neurons in situ. Expressed genes exhibit signature-specific damage, and mutations show a transcriptional strand bias, which suggests that transcription-coupled nucleotide excision repair has a role in the generation of mutations. The alterations in Alzheimer's disease affect coding exons and are predicted to create dysfunctional genetic knockout cells and proteostatic stress. Our results suggest that known pathogenic mechanisms in Alzheimer's disease may lead to genomic damage to neurons that can progressively impair function. The aberrant accumulation of DNA alterations in neurodegeneration provides insight into the cascade of molecular and cellular events that occurs in the development of Alzheimer's disease.

Leveraging the replication-competent avian-like sarcoma virus/tumor virus receptor-A system for modeling human gliomas.

Gliomas are the most common primary intrinsic brain tumors occurring in adults. Of all malignant gliomas, glioblastoma (GBM) is considered the deadliest tumor type due to diffuse brain invasion, immune evasion, cellular, and molecular heterogeneity, and resistance to treatments resulting in high rates of recurrence. An extensive understanding of the genomic and microenvironmental landscape of gliomas gathered over the past decade has renewed interest in pursuing novel therapeutics, including immune checkpoint inhibitors, glioma-associated macrophage/microglia (GAMs) modulators, and others. In light of this, predictive animal models that closely recreate the conditions and findings found in human gliomas will serve an increasingly important role in identifying new, effective therapeutic strategies. Although numerous syngeneic, xenograft, and transgenic rodent models have been developed, few include the full complement of pathobiological features found in human tumors, and therefore few accurately predict bench-to-bedside success. This review provides an update on how genetically engineered rodent models based on the replication-competent avian-like sarcoma (RCAS) virus/tumor virus receptor-A (tv-a) system have been used to recapitulate key elements of human gliomas in an immunologically intact host microenvironment and highlights new approaches using this model system as a predictive tool for advancing translational glioma research.

Elevated fibroblast growth factor-inducible 14 expression transforms proneural-like gliomas into more aggressive and lethal brain cancer.

High-grade gliomas (HGGs) are aggressive, treatment-resistant, and often fatal human brain cancers. The TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) signaling axis is involved in tissue repair after injury and constitutive signaling has been implicated in the pathogenesis of numerous solid cancers. The Fn14 gene is expressed at low levels in the normal, uninjured brain but is highly expressed in primary isocitrate dehydrogenase wild-type and recurrent HGGs. Fn14 signaling is implicated in numerous aspects of glioma biology including brain invasion and chemotherapy resistance, but whether Fn14 overexpression can directly promote tumor malignancy has not been reported. Here, we used the replication-competent avian sarcoma-leukosis virus/tumor virus A system to examine the impact of Fn14 expression on glioma development and pathobiology. We found that the sole addition of Fn14 to an established oncogenic cocktail previously shown to generate proneural-like gliomas led to the development of highly invasive and lethal brain cancer with striking biological features including extensive pseudopalisading necrosis, constitutive canonical and noncanonical NF-κB pathway signaling, and high plasminogen activator inhibitor-1 (PAI-1) expression. Analyses of HGG patient datasets revealed that high human PAI-1 gene (SERPINE1) expression correlates with shorter patient survival, and that the SERPINE1 and Fn14 (TNFRSF12A) genes are frequently co-expressed in bulk tumor tissues, in tumor subregions, and in malignant cells residing in the tumor microenvironment. These findings provide new insights into the potential importance of Fn14 in human HGG pathobiology and designate both the NF-κB signaling node and PAI-1 as potential targets for therapeutic intervention. MAIN POINTS: This work demonstrates that elevated levels of the TWEAK receptor Fn14 in tumor-initiating, neural progenitor cells leads to the transformation of proneural-like gliomas into more aggressive and lethal tumors that exhibit constitutive NF-κB pathway activation and plasminogen activator inhibitor-1 overexpression.

Incidence and clinicopathologic features of H3 K27M mutations in adults with radiographically-determined midline gliomas.

PURPOSE: H3 K27 mutations, most commonly in H3F3A, are common in diffuse midline glioma. The exact frequency of these mutations in adults with gliomas in the midline location is unknown. This study was conducted to define the incidence of H3 K27M mutations in this location and to compare clinicopathological features with those of patients who do not harbor this mutation. METHODS: Consecutive glioma cases from 2007 to 2017 were screened for gliomas in the midline location. Immunohistochemistry was performed on all available tissue for mutations of H3 K27M, IDH1, and ARTX. RESULTS: Of 850 gliomas screened, 163 cases had midline glioma on MRI. Sufficient FFPE tissue was available for 123 cases (75%). H3 K27M mutation was identified in 18 of 123 cases (15%). All except one H3 K27M-mutant tumors were WHO grade III or IV on histology, while non-mutant tumors encompassed all four grades. The most common midline locations for H3 K27M-mutated tumors were midbrain (2/3; 67%), pons (4/11; 36%), and cerebellum (6/24; 25%). As compared to H3 K27M-wildtype tumors, there were no differences in age at diagnosis, sex, tumor grade, contrast enhancement on MRI, extent of resection, or treatment received. In this cohort, median survival was longer for patients with H3 K27M-mutated tumors (n = 18; 17.6 months) compared with high-grade wildtype tumors (n = 74; 7.7 months, p = 0.03). CONCLUSIONS: H3 K27M mutations are common in midline gliomas in adults and can present in all midline locations. Survival comparison between H3 K27M-mutant and wildtype midline gliomas suggests that survival may be similar or possibly improved if the mutation is present.

Leveraging Surface Plasmon Resonance to Dissect the Interfacial Properties of Nanoparticles: Implications for Tissue Binding and Tumor Penetration.

Therapeutic efficacy of nanoparticle-drug formulations for cancer applications is significantly impacted by the extent of intra-tumoral accumulation and tumor tissue penetration. We advanced the application of surface plasmon resonance to examine interfacial properties of various clinical and emerging nanoparticles related to tumor tissue penetration. We observed that amine-terminated or positively-charged dendrimers and liposomes bound strongly to tumor extracellular matrix (ECM) proteins, whereas hydroxyl/carboxyl-terminated dendrimers and PEGylated/neutrally-charged liposomes did not bind. In addition, poly(lactic-co-glycolic acid) (PLGA) nanoparticles formulated with cholic acid or F127 surfactants bound strongly to tumor ECM proteins, whereas nanoparticles formulated with poly(vinyl alcohol) did not bind. Unexpectedly, following blood serum incubation, this binding increased and particle transport in ex vivo tumor tissues reduced markedly. Finally, we characterized the protein corona on PLGA nanoparticles using quantitative proteomics. Through these studies, we identified valuable criteria for particle surface characteristics that are likely to mediate their tissue binding and tumor penetration.

Differential neuronal susceptibility and apoptosis in congenital Zika virus infection.

To characterize the mechanism of Zika virus (ZIKV)-associated microcephaly, we performed immunolabeling on brain tissue from a 20-week fetus with intrauterine ZIKV infection. Although ZIKV demonstrated a wide range of neuronal and non-neuronal tropism, the infection rate was highest in intermediate progenitor cells and immature neurons. Apoptosis was observed in both infected and uninfected bystander cortical neurons, suggesting a role for paracrine factors in induction of neuronal apoptosis. Our results highlight differential neuronal susceptibility and neuronal apoptosis as potential mechanisms in the development of ZIKV-associated microcephaly, and may provide insights into the design and best timing of future therapy. Ann Neurol 2017;82:121-127.

The effect of machine learning tools for evidence synthesis on resource use and time-to-completion: protocol for a retrospective pilot study.

BACKGROUND: Machine learning (ML) tools exist that can reduce or replace human activities in repetitive or complex tasks. Yet, ML is underutilized within evidence synthesis, despite the steadily growing rate of primary study publication and the need to periodically update reviews to reflect new evidence. Underutilization may be partially explained by a paucity of evidence on how ML tools can reduce resource use and time-to-completion of reviews. METHODS: This protocol describes how we will answer two research questions using a retrospective study design: Is there a difference in resources used to produce reviews using recommended ML versus not using ML, and is there a difference in time-to-completion? We will also compare recommended ML use to non-recommended ML use that merely adds ML use to existing procedures. We will retrospectively include all reviews conducted at our institute from 1 August 2020, corresponding to the commission of the first review in our institute that used ML. CONCLUSION: The results of this study will allow us to quantitatively estimate the effect of ML adoption on resource use and time-to-completion, providing our organization and others with better information to make high-level organizational decisions about ML.

DHODH inhibition impedes glioma stem cell proliferation, induces DNA damage, and prolongs survival in orthotopic glioblastoma xenografts.

Glioma stem cells (GSCs) promote tumor progression and therapeutic resistance and exhibit remarkable bioenergetic and metabolic plasticity, a phenomenon that has been linked to their ability to escape standard and targeted therapies. However, specific mechanisms that promote therapeutic resistance have been somewhat elusive. We hypothesized that because GSCs proliferate continuously, they may require the salvage and de novo nucleotide synthesis pathways to satisfy their bioenergetic needs. Here, we demonstrate that GSCs lacking EGFR (or EGFRvIII) amplification are exquisitely sensitive to de novo pyrimidine synthesis perturbations, while GSCs that amplify EGFR are utterly resistant. Furthermore, we show that EGFRvIII promotes BAY2402234 resistance in otherwise BAY2402234 responsive GSCs. Remarkably, a novel, orally bioavailable, blood-brain-barrier penetrating, dihydroorotate dehydrogenase (DHODH) inhibitor BAY2402234 was found to abrogate GSC proliferation, block cell-cycle progression, and induce DNA damage and apoptosis. When dosed daily by oral gavage, BAY2402234 significantly impaired the growth of two different intracranial human glioblastoma xenograft models in mice. Given this observed efficacy and the previously established safety profiles in preclinical animal models and human clinical trials, the clinical testing of BAY2402234 in patients with primary glioblastoma that lacks EGFR amplification is warranted.

Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection.

The present protocol describes a standardized paradigm for rodent brain tumor resection and tissue preservation. In clinical practice, maximal tumor resection is the standard-of-care treatment for most brain tumors. However, most currently available preclinical brain tumor models either do not include resection, or utilize surgical resection models that are time-consuming and lead to significant postoperative morbidity, mortality, or experimental variability. In addition, performing resection in rodents can be daunting for several reasons, including a lack of clinically comparable surgical tools or protocols and the absence of an established platform for standardized tissue collection. This protocol highlights the use of a multi-functional, non-ablative resection device and an integrated tissue preservation system adapted from the clinical version of the device. The device applied in the present study combines tunable suction and a cylindrical blade at the aperture to precisely probe, cut, and suction tissue. The minimally invasive resection device performs its functions via the same burr hole used for the initial tumor implantation. This approach minimizes alterations to regional anatomy during biopsy or resection surgeries and reduces the risk of significant blood loss. These factors significantly reduced the operative time (<2 min/animal), improved postoperative animal survival, lower variability in experimental groups, and result in high viability of resected tissues and cells for future analyses. This process is facilitated by a blade speed of ~1,400 cycles/min, which allows the harvesting of tissues into a sterile closed system that can be filled with a physiologic solution of choice. Given the emerging importance of studying and accurately modeling the impact of surgery, preservation and rigorous comparative analysis of regionalized tumor resection specimens, and intra-cavity-delivered therapeutics, this unique protocol will expand opportunities to explore unanswered questions about perioperative management and therapeutic discovery for brain tumor patients.

The Alternative Splicing Factor, MBNL1, Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-Induced Stemness.

Muscleblind-like proteins (MBNL) belong to a family of tissue-specific regulators of RNA metabolism that control premessenger RNA splicing. Inactivation of MBNL causes an adult-to-fetal alternative splicing transition, resulting in the development of myotonic dystrophy. We have previously shown that the aggressive brain cancer, glioblastoma (GBM), maintains stem-like features (glioma stem cell, GSC) through hypoxia-induced responses. Accordingly, we hypothesize here that hypoxia-induced responses in GBM might also include MBNL-based alternative splicing to promote tumor progression. When cultured in hypoxia condition, GSCs rapidly exported muscleblind-like-1 (MBNL1) out of the nucleus, resulting in significant inhibition of MBNL1 activity. Notably, hypoxia-regulated inhibition of MBNL1 also resulted in evidence of adult-to-fetal alternative splicing transitions. Forced expression of a constitutively active isoform of MBNL1 inhibited GSC self-renewal and tumor initiation in orthotopic transplantation models. Induced expression of MBNL1 in established orthotopic tumors dramatically inhibited tumor progression, resulting in significantly prolonged survival. This study reveals that MBNL1 plays an essential role in GBM stemness and tumor progression, where hypoxic responses within the tumor inhibit MBNL1 activity, promoting stem-like phenotypes and tumor growth. Reversing these effects on MBNL1 may therefore, yield potent tumor suppressor activities, uncovering new therapeutic opportunities to counter this disease. SIGNIFICANCE: This study describes an unexpected mechanism by which RNA-binding protein, MBNL1, activity is inhibited in hypoxia by a simple isoform switch to regulate glioma stem cell self-renewal, tumorigenicity, and progression.

MCT4 regulates de novo pyrimidine biosynthesis in GBM in a lactate-independent manner.

BACKGROUND: Necrotic foci with surrounding hypoxic cellular pseudopalisades and microvascular hyperplasia are histological features found in glioblastoma (GBM). We have previously shown that monocarboxylate transporter 4 (MCT4) is highly expressed in necrotic/hypoxic regions in GBM and that increased levels of MCT4 are associated with worse clinical outcomes. METHODS: A combined transcriptomics and metabolomics analysis was performed to study the effects of MCT4 depletion in hypoxic GBM neurospheres. Stable and inducible MCT4-depletion systems were used to evaluate the effects of and underlining mechanisms associated with MCT4 depletion in vitro and in vivo, alone and in combination with radiation. RESULTS: This study establishes that conditional depletion of MCT4 profoundly impairs self-renewal and reduces the frequency and tumorigenicity of aggressive, therapy-resistant, glioblastoma stem cells. Mechanistically, we observed that MCT4 depletion induces anaplerotic glutaminolysis and abrogates de novo pyrimidine biosynthesis. The latter results in a dramatic increase in DNA damage and apoptotic cell death, phenotypes that were readily rescued by pyrimidine nucleosides supplementation. Consequently, we found that MCT4 depletion promoted a significant prolongation of survival of animals bearing established orthotopic xenografts, an effect that was extended by adjuvant treatment with focused radiation. CONCLUSIONS: Our findings establish a novel role for MCT4 as a critical regulator of cellular deoxyribonucleotide levels and provide a new therapeutic direction related to MCT4 depletion in GBM.

Decreased nonspecific adhesivity, receptor-targeted therapeutic nanoparticles for primary and metastatic breast cancer.

Development of effective tumor cell-targeted nanodrug formulations has been quite challenging, as many nanocarriers and targeting moieties exhibit nonspecific binding to cellular, extracellular, and intravascular components. We have developed a therapeutic nanoparticle formulation approach that balances cell surface receptor-specific binding affinity while maintaining minimal interactions with blood and tumor tissue components (termed "DART" nanoparticles), thereby improving blood circulation time, biodistribution, and tumor cell-specific uptake. Here, we report that paclitaxel (PTX)-DART nanoparticles directed to the cell surface receptor fibroblast growth factor-inducible 14 (Fn14) outperformed both the corresponding PTX-loaded, nontargeted nanoparticles and Abraxane, an FDA-approved PTX nanoformulation, in both a primary triple-negative breast cancer (TNBC) model and an intracranial model reflecting TNBC growth following metastatic dissemination to the brain. These results provide new insights into methods for effective development of therapeutic nanoparticles as well as support the continued development of the DART platform for primary and metastatic tumors.

Radiation-induced astrocyte senescence is rescued by Δ133p53.

BACKGROUND: Cellular senescence and the senescence-associated secretory phenotype (SASP) may contribute to the development of radiation therapy-associated side effects in the lung and blood vessels by promoting chronic inflammation. In the brain, inflammation contributes to the development of neurologic disease, including Alzheimer's disease. In this study, we investigated the roles of cellular senescence and Δ133p53, an inhibitory isoform of p53, in radiation-induced brain injury. METHODS: Senescent cell types in irradiated human brain were identified with immunohistochemical labeling of senescence-associated proteins p16INK4A and heterochromatin protein Hp1γ in 13 patient cases, including 7 irradiated samples. To investigate the impact of radiation on astrocytes specifically, primary human astrocytes were irradiated and examined for expression of Δ133p53 and induction of SASP. Lentiviral expression of ∆133p53 was performed to investigate its role in regulating radiation-induced cellular senescence and astrocyte-mediated neuroinflammation. RESULTS: Astrocytes expressing p16INK4A and Hp1γ were identified in all irradiated tissues, were increased in number in irradiated compared with untreated cancer patient tissues, and had higher labeling intensity in irradiated tissues compared with age-matched controls. Human astrocytes irradiated in vitro also experience induction of cellular senescence, have diminished Δ133p53, and adopt a neurotoxic phenotype as demonstrated by increased senescence-associated beta-galactosidase activity, p16INK4A, and interleukin (IL)-6. In human astrocytes, Δ133p53 inhibits radiation-induced senescence, promotes DNA double-strand break repair, and prevents astrocyte-mediated neuroinflammation and neurotoxicity. CONCLUSIONS: Restoring expression of the endogenous p53 isoform, ∆133p53, protects astrocytes from radiation-induced senescence, promotes DNA repair, and inhibits astrocyte-mediated neuroinflammation.

Mechanisms of invasion and motility of high-grade gliomas in the brain.

High-grade gliomas are especially difficult tumors to treat due to their invasive behavior. This has led to extensive research focusing on arresting glioma cell migration. Cell migration involves the sensing of a migratory cue, followed by polarization in the direction of the cue, and reorganization of the actin cytoskeleton to allow for a protrusive leading edge and a contractile trailing edge. Transmission of these forces to produce motility also requires adhesive interactions of the cell with the extracellular microenvironment. In glioma cells, transmembrane receptors such as CD44 and integrins bind the cell to the surrounding extracellular matrix that provides a substrate on which the cell can exert the requisite forces for cell motility. These various essential parts of the migratory machinery are potential targets to halt glioma cell invasion. In this review, we discuss the mechanisms of glioma cell migration and how they may be targeted in anti-invasion therapies.

INSM1 Expression Is Frequent in Primary Central Nervous System Neoplasms but Not in the Adult Brain Parenchyma.

Tumors with a neuronal component comprise a small percentage of central nervous system (CNS) neoplasms overall, but the presence of neuronal differentiation has important diagnostic, prognostic, and therapeutic implications. Insulinoma-associated protein 1 (INSM1) is a transcription factor with strong nuclear immunostaining in neuroendocrine cells and neoplasms of neuroendocrine origin; however, its expression in the CNS in normal brain and in neoplastic cells has not been fully explored. Here, we present immunostaining results from a large number of archival CNS tissue specimens, including 416 tumors. Nuclear immunostaining for INSM1 was frequently seen in medulloblastomas (87%, n = 94). Diffuse nuclear INSM1 immunostaining was detected in all central neurocytomas and pituitary adenomas. Patchy to rare staining with INSM1 was also seen in other high-grade embryonal tumors and high-grade gliomas. In normal brain tissue, specific nuclear INSM1 immunohistochemical staining was only seen in early neuronal development. Notably, nuclear INSM1 staining was not seen in adult normal brain, including areas of gliosis. These findings indicate that nuclear INSM1 immunostaining may serve as a strong nuclear marker in the brain for neoplasms of neuroendocrine or immature neuronal differentiation, when used in concert with other immunostains.

Tetrameric hub structure of postsynaptic scaffolding protein homer.

Homer is a crucial postsynaptic scaffolding protein involved in both maintenance and activity-induced plasticity of the synapse. However, its quaternary structure has yet to be determined. We conducted a series of biophysical experiments that provide the first evidence that Homer forms a tetramer via its coiled-coil domain, in which all subunits are aligned in parallel orientation. To test the importance of the tetrameric structure for functionality, we engineered dimeric and tetrameric Homer by deleting a part of coiled-coil domain or replacing it with artificially engineered dimeric or tetrameric coiled-coil domain from a yeast protein. The structure-activity relationship was determined by assaying cocluster formation with its ligand in heterologous cells, distribution in dendritic spines, and turnover rate of protein exist in dendritic spines. Our results provide the first insight into the structure of native Homer protein as a tetramer and the functional significance conferred by that structure.

Zincergic innervation of medial prefrontal cortex by basolateral projection neurons.

The basolateral amygdaloid complex is a site of origin for zinc-containing pathways in the brain; it is also known for its massive innervation of the medial prefrontal cortex. The presence, and potential neuromodulatory role, of zinc within this fundamental corticolimbic circuit has not been described. For this study, basolateral neurons innervating the medial prefrontal cortex were retrogradely labeled with FluoroGold, and zinc-containing neurons were identified using autometallography to visualize zinc selenium precipitates. Upon quantification of single-labeled and double-labeled cells, 35% of basolateral neurons projecting to medial prefrontal cortex were found to also contain zinc. We conclude that zinc may act as a neuromodulator for a substantial proportion of basolateral-medial prefrontal cortical innervation, therefore implicating zinc in corticolimbic function as well as pathology.

Huntingtin-interacting protein 1 phosphorylation by receptor tyrosine kinases.

Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor ß receptor (PDGFßR) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGFßR (H/P), and TEL/PDGFßR (T/P). We identified a four-tyrosine "HIP1 phosphorylation motif" (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGFßR but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1's only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1's role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival.

Huntingtin-interacting protein 1: a Merkel cell carcinoma marker that interacts with c-Kit.

Merkel cell carcinoma (MCC) is a neoplasm thought to originate from the neuroendocrine Merkel cells of the skin. Although the prevalence of MCC has been increasing, treatments for this disease remain limited because of a paucity of information regarding MCC biology. We have found that the endocytic oncoprotein Huntingtin-interacting protein 1 (HIP1) is expressed at high levels in ∼90% of MCC tumors and serves as a more reliable histological cytoplasmic stain than the gold standard, cytokeratin 20. Furthermore, high anti-HIP1 antibody reactivity in the sera of a cohort of MCC patients predicts the presence of metastases. Another protein that is frequently expressed at high levels in MCC tumors is the stem cell factor (SCF) receptor tyrosine kinase, c-Kit. In working toward an understanding of how HIP1 might contribute to MCC tumorigenesis, we have discovered that HIP1 interacts with SCF-activated c-Kit. These data not only identify HIP1 as a molecular marker for management of MCC patients but also show that HIP1 interacts with and slows the degradation of c-Kit.