Autophagy-based unconventional secretion in melanoma

Macroautophagy, or autophagy, is a catabolic process that sequesters damaged organelles and protein aggregates for degradation. Autophagy occurs at basal levels in most cells to perform homeostatic functions and adaptively responds to environmental stresses to confer cell survival, serving a pro-tumorigenic role in established tumors. Autophagy has recently been recognized for its role in unconventional secretion, a novel paradigm beyond its previously known catabolic cellular function. However, its broader role in modifying the tumor secretome, a component of the tumor microenvironment that influences tumor progression and immune responses, has yet to be investigated. This is particularly relevant in melanoma, where autophagy levels are highly variable across tumors, considerably impacting tumor behavior and therapy response. In this thesis, we sought to determine the effects of autophagy at basal levels and in response to anti-melanoma therapy on the melanoma secretome to gain insight into the context-dependent effects of autophagy on secretion. The conditioned medium of isogenic melanoma cell lines with intrinsically disparate levels of autophagy were compared using quantitative proteomics, detecting 26 proteins at significantly higher levels in the secretomes of cells harboring high autophagic flux. From functional bioinformatics analysis, IL1-β, IL-8, LIF, DKK3, and FAM3C were the focus of further investigation. Measurements of these proteins in independent melanoma cell lines profiled for autophagy confirmed the early correlation with autophagy. Further, secretion of these proteins increased in response to targeted autophagy induction and decreased in autophagy-deficient melanoma cells. Finally, these proteins were detected at higher levels in sera of melanoma patients with high pre-treatment tumor autophagy levels, demonstrating the potential for improved clinical detection of autophagy. Follow up studies evaluated the effects of inhibiting BRAF, a serine-threonine kinase mutated in melanoma, on autophagy-based remodeling of the secretome. BRAF inhibition resulted in acute increases in autophagic flux in mutant BRAF cells coupled with increased secretion of a number of proteins, notably Histone H4 and ERK2 which were mechanistically linked to autophagy. Our work demonstrates that autophagy-based unconventional secretion is an important mechanism for altering the extracellular milieu in melanoma, the plasticity of which may impact tumor progression and therapy response in the clinic

Autophagy-based unconventional secretion in melanoma

Macroautophagy, or autophagy, is a catabolic process that sequesters damaged organelles and protein aggregates for degradation. Autophagy occurs at basal levels in most cells to perform homeostatic functions and adaptively responds to environmental stresses to confer cell survival, serving a pro-tumorigenic role in established tumors. Autophagy has recently been recognized for its role in unconventional secretion, a novel paradigm beyond its previously known catabolic cellular function. However, its broader role in modifying the tumor secretome, a component of the tumor microenvironment that influences tumor progression and immune responses, has yet to be investigated. This is particularly relevant in melanoma, where autophagy levels are highly variable across tumors, considerably impacting tumor behavior and therapy response. In this thesis, we sought to determine the effects of autophagy at basal levels and in response to anti-melanoma therapy on the melanoma secretome to gain insight into the context-dependent effects of autophagy on secretion. The conditioned medium of isogenic melanoma cell lines with intrinsically disparate levels of autophagy were compared using quantitative proteomics, detecting 26 proteins at significantly higher levels in the secretomes of cells harboring high autophagic flux. From functional bioinformatics analysis, IL1-β, IL-8, LIF, DKK3, and FAM3C were the focus of further investigation. Measurements of these proteins in independent melanoma cell lines profiled for autophagy confirmed the early correlation with autophagy. Further, secretion of these proteins increased in response to targeted autophagy induction and decreased in autophagy-deficient melanoma cells. Finally, these proteins were detected at higher levels in sera of melanoma patients with high pre-treatment tumor autophagy levels, demonstrating the potential for improved clinical detection of autophagy. Follow up studies evaluated the effects of inhibiting BRAF, a serine-threonine kinase mutated in melanoma, on autophagy-based remodeling of the secretome. BRAF inhibition resulted in acute increases in autophagic flux in mutant BRAF cells coupled with increased secretion of a number of proteins, notably Histone H4 and ERK2 which were mechanistically linked to autophagy. Our work demonstrates that autophagy-based unconventional secretion is an important mechanism for altering the extracellular milieu in melanoma, the plasticity of which may impact tumor progression and therapy response in the clinic

Identification of secreted proteins that reflect autophagy dynamics within tumor cells

<div><p>Macroautophagy, a catabolic process of cellular self-digestion, is an important tumor cell survival mechanism and a potential target in antineoplastic therapies. Recent discoveries have implicated autophagy in the cellular secretory process, but potential roles of autophagy-mediated secretion in modifying the tumor microenvironment are poorly understood. Furthermore, efforts to inhibit autophagy in clinical trials have been hampered by suboptimal methods to quantitatively measure tumor autophagy levels. Here, we leveraged the autophagy-based involvement in cellular secretion to identify shed proteins associated with autophagy levels in melanoma. The secretome of low-autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu in physiological 3-dimensional cell culture using quantitative proteomics. These comparisons identified candidate autophagy biomarkers IL1B (interleukin 1, β), CXCL8 (chemokine (C-X-C motif) ligand 8), LIF (leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (dickkopf WNT signaling pathway inhibitor 3) with known roles in inflammation and tumorigenesis, and these proteins were subsequently shown to be elevated in supernatants of an independent panel of high-autophagy melanoma cell lines. Secretion levels of these proteins increased when low-autophagy melanoma cells were treated with the autophagy-inducing tat-BECN1 (Beclin 1) peptide and decreased when <i>ATG7</i> (autophagy-related 7) was silenced in high-autophagy cells, thereby supporting a mechanistic link between these secreted proteins and autophagy. In addition, serum from metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from patients with low-autophagy tumors. These results suggest that autophagy-related secretion affects the tumor microenvironment and measurement of autophagy-associated secreted proteins in plasma and possibly in tumors can serve as surrogates for intracellular autophagy dynamics in tumor cells.</p></div

BRCA locus-specific loss of heterozygosity in germline BRCA1 and BRCA2 carriers

Complete loss of BRCA1 or BRCA2 function is associated with sensitivity to DNA damaging agents. However, not all BRCA1 and BRCA2 germline mutation-associated tumors respond. Herein we report analyses of 160 BRCA1 and BRCA2 germline mutation-associated breast and ovarian tumors. Retention of the normal BRCA1 or BRCA2 allele (absence of locus-specific loss of heterozygosity (LOH)) is observed in 7% of BRCA1 ovarian, 16% of BRCA2 ovarian, 10% of BRCA1 breast, and 46% of BRCA2 breast tumors. These tumors have equivalent homologous recombination deficiency scores to sporadic tumors, significantly lower than scores in tumors with locus-specific LOH (ovarian, P = 0.0004; breast P &lt; 0.0001, two-tailed Student's t-test). Absence of locus-specific LOH is associated with decreased overall survival in ovarian cancer patients treated with platinum chemotherapy (P = 0.01, log-rank test). Locus-specific LOH may be a clinically useful biomarker to predict primary resistance to DNA damaging agents in patients with germline BRCA1 and BRCA2 mutations.Most tumours associated with germline BRCA1/BRCA2 loss of function mutations respond to DNA damaging agents, however, some do not. Herein, the authors identify that a subset of breast/ovarian tumors retain a normal allele, which is associated with decreased overall survival after DNA damage-inducing platinum chemotherapy

Analysis of matched primary and recurrent BRCA1/2 mutation-associated tumors identifies recurrence-specific drivers

Recurrence is a major cause of death among BRCA1/2 mutation carriers with breast (BrCa) and ovarian cancers (OvCa). Herein we perform multi-omic sequencing on 67 paired primary and recurrent BrCa and OvCa from 27 BRCA1/2 mutation carriers to identify potential recurrence-specific drivers. PARP1 amplifications are identified in recurrences (False Discovery Rate q = 0.05), and PARP1 is significantly overexpressed across primary BrCa and recurrent BrCa and OvCa, independent of amplification status. RNA sequencing analysis finds two BRCA2 isoforms, BRCA2-201/Long and BRCA2-001/Short, respectively predicted to be sensitive and insensitive to nonsense-mediated decay. BRCA2-001/Short is expressed more frequently in recurrences and associated with reduced overall survival in breast cancer (87 vs. 121 months; Hazard Ratio = 2.5 [1.18-5.5]). Loss of heterozygosity (LOH) status is discordant in 25% of patient's primary and recurrent tumors, with switching between both LOH and lack of LOH found. Our study reveals multiple potential drivers of recurrent disease in BRCA1/2 mutation-associated cancer, improving our understanding of tumor evolution and suggesting potential biomarkers. Carriers of pathogenic BRCA1/2 variants have a higher risk of breast and ovarian cancers, which recur frequently. Here, the authors sequence primary and recurrent tumours of BRCA1/2 mutation carriers, finding PARP1 amplifications, differential BRCA2 isoform usage, and discordant loss of heterozygosity that are associated with recurrenc

OpenPBTA: The Open Pediatric Brain Tumor Atlas

Pediatric brain and spinal cancers are collectively the leading disease-related cause of death in children; thus, we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Children\u27s Brain Tumor Network (CBTN) and Pacific Pediatric Neuro-Oncology Consortium (PNOC) created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to establish OpenPBTA, an open collaborative project with over 40 scalable analysis modules that genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals universal dysregulation in mismatch repair-deficient hypermutant high-grade gliomas and loss as a significant marker for poor overall survival in ependymomas and H3 K28-mutant diffuse midline gliomas. Already being actively applied to other pediatric cancers and PNOC molecular tumor board decision-making, OpenPBTA is an invaluable resource to the pediatric oncology community