Although B cell response is frequently found in cancer, there is little evidence that it alters tumor development or progression. The process through which tumor-associated antigens trigger humoral response is not well delineated. We investigate the repertoire of antigens associated with humoral immune response in pancreatic ductal adenocarcinoma (PDAC) using in-depth proteomic profiling of immunoglobulin-bound proteins from PDAC patient plasmas and identify tumor antigens that induce antibody response together with exosome hallmark proteins. Additional profiling of PDAC cell-derived exosomes reveals significant overlap in their protein content with immunoglobulin-bound proteins in PDAC plasmas, and significant autoantibody reactivity is observed between PDAC cell-derived exosomes and patient plasmas compared to healthy controls. Importantly, PDAC-derived exosomes induce a dose-dependent inhibition of PDAC serum-mediated complement-dependent cytotoxicity towards cancer cells. In summary, we provide evidence that exosomes display a large repertoire of tumor antigens that induce autoantibodies and exert a decoy function against complement-mediated cytotoxicity.

MYC is an oncogenic driver of development and progression in triple-negative breast cancer (TNBC). Ornithine decarboxylase, the rate-limiting enzyme in polyamine metabolism, is a transcriptional target of MYC. We therefore hypothesized that a plasma polyamine signature may be predictive of TNBC development and progression. Using liquid chromatography mass spectrometry, polyamine levels were determined in plasma samples from newly diagnosed patients with TNBC (n = 87) and cancer-free controls (n = 115). Findings were validated in plasma samples from an independent prospective cohort of 54 TNBC, 55 estrogen receptor negative (ER−) and progesterone receptor negative (PR−) and HER2 positive (HER2+), and 73 ER+ case patients, and 30 cancer-free control subjects. Gene expression data and clinical data for 921 and 2359 breast cancer tumors were obtained from The Cancer Genome Atlas repository and the Oncomine database, respectively. Relationships between plasma diacetylspermine (DAS) and tumor spermine synthase (SMS) mRNA expression with metastasis-free survival and overall survival were determined using Cox proportional hazard models; Fisher exact tests were used to assess risk of distant metastasis in relation to tumor SMS mRNA expression. An increase in plasma DAS, a catabolic product of spermine mediated through SMS, was observed in the TNBC subtype of breast cancer. Plasma levels of DAS in TNBC associated with increased risk of metastasis (plasma DAS value ≥ 1.16, hazard ratio = 3.06, 95% confidence interval [CI] = 1.15 to 8.13, two-sided P = .03). SMS mRNA expression in TNBC tumor tissue was also found to be predictive of poor overall survival (top 25th percentile hazard ratio = 2.06, 95% CI = 1.04 to 4.08, one-sided P = .04) and increased risk of distant metastasis in TNBC (comparison of lowest SMS quartile [reference] to highest SMS quartile relative risk = 1.90, 95% CI = 0.97 to 4.06, one-sided Fisher exact test P=.03). Metabolomic profiling identified plasma DAS as a predictive marker for TNBC progression and metastasis.

Approximately 20% of lung adenocarcinoma (LUAD) is negative for the lineage-specific oncogene Thyroid transcription factor 1 (TTF-1) and exhibits worse clinical outcome with a low frequency of actionable genomic alterations. To identify molecular features associated with TTF-1–negative LUAD, we compared the transcriptomic and proteomic profiles of LUAD cell lines. SRGN , a chondroitin sulfate proteoglycan Serglycin, was identified as a markedly overexpressed gene in TTF-1–negative LUAD. We therefore investigated the roles and regulation of SRGN in TTF-1–negative LUAD. Proteomic and metabolomic analyses of 41 LUAD cell lines were done using mass spectrometry. The function of SRGN was investigated in 3 TTF-1–negative and 4 TTF-1–positive LUAD cell lines and in a syngeneic mouse model (n = 5 to 8 mice per group). Expression of SRGN was evaluated in 94 and 105 surgically resected LUAD tumor specimens using immunohistochemistry. All statistical tests were 2-sided. SRGN was markedly overexpressed at mRNA and protein levels in TTF-1–negative LUAD cell lines (P < .001 for both mRNA and protein levels). Expression of SRGN in LUAD tumor tissue was associated with poor outcome (hazard ratio = 4.22, 95% confidence interval = 1.12 to 15.86, likelihood ratio test, P = .03), and with higher expression of Programmed cell death 1 ligand 1 (PD-L1) in tumor cells and higher infiltration of Programmed cell death protein 1–positive lymphocytes. SRGN regulated expression of PD-L1 as well as proinflammatory cytokines, including Interleukin-6, Interleukin-8, and C-X-C motif chemokine 1 in LUAD cell lines; increased migratory and invasive properties of LUAD cells and fibroblasts; and enhanced angiogenesis. SRGN was induced by DNA demethylation resulting from Nicotinamide N-methyltransferase–mediated impairment of methionine metabolism. Our findings suggest that SRGN plays a pivotal role in tumor–stromal interaction and reprogramming into an aggressive and immunosuppressive tumor microenvironment in TTF-1–negative LUAD.

Background Citrulline post-translational modification of proteins is mediated by protein arginine deiminase (PADI) family members and has been associated with autoimmune diseases. The role of PADI-citrullinome in immune response in cancer has not been evaluated. We hypothesized that PADI-mediated citrullinome is a source of neoantigens in cancer that induces immune response. Methods Protein expression of PADI family members was evaluated in 196 cancer cell lines by means of indepth proteomic profiling. Gene expression was assessed using messenger RNA data sets from The Cancer Genome Atlas. Immunohistochemical analysis of PADI2 and peptidyl-citrulline was performed using breast cancer tissue sections. Citrullinated 12–34-mer peptides in the putative Major Histocompatibility Complex-II (MHC-II) binding range were profiled in breast cancer cell lines to investigate the relationship between protein citrullination and antigen presentation. We further evaluated immunoglobulin-bound citrullinome by mass spectrometry using 156 patients with breast cancer and 113 cancer-free controls. Results Proteomic and gene expression analyses revealed PADI2 to be highly expressed in several cancer types including breast cancer. Immunohistochemical analysis of 422 breast tumor tissues revealed increased expression of PADI2 in ER− tumors (p<0.0001); PADI2 protein expression was positively correlated (p<0.0001) with peptidyl-citrulline staining. PADI2 expression exhibited strong positive correlations with a B cell immune signature and with MHC-II-bound citrullinated peptides. Increased circulating citrullinated antigen–antibody complexes occurred among newly diagnosed breast cancer cases relative to controls (p=0.0012). Conclusions An immune response associated with citrullinome is a rich source of neoantigens in breast cancer with a potential for diagnostic and therapeutic applications.

Activation of the NRF2 pathway through gain-of-function mutations or loss-of-function of its suppressor KEAP1 is a frequent finding in lung cancer. NRF2 activation has been reported to alter the tumor microenvironment. Here, we demonstrated that NRF2 alters tryptophan metabolism through the kynurenine pathway that is associated with a tumor-promoting, immune suppressed microenvironment. Specifically, proteomic profiles of 47 lung adenocarcinoma (LUAD) cell lines (11 KEAP1 mutant and 36 KEAP1 wild-type) revealed the tryptophan-kynurenine enzyme kynureninase (KYNU) as a top overexpressed protein associated with activated NRF2. The siRNA-mediated knockdown of NFE2L2, the gene encoding for NRF2, or activation of the NRF2 pathway through siRNA-mediated knockdown of KEAP1 or via chemical induction with the NRF2-activator CDDO-Me confirmed that NRF2 is a regulator of KYNU expression in LUAD. Metabolomic analyses confirmed KYNU to be enzymatically functional. Analysis of multiple independent gene expression datasets of LUAD, as well as a LUAD tumor microarray demonstrated that elevated KYNU was associated with immunosuppression, including potent induction of T-regulatory cells, increased levels of PD1 and PD-L1, and resulted in poorer survival. Our findings indicate a novel mechanism of NRF2 tumoral immunosuppression through upregulation of KYNU.