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Function and relevance of sushi domain-containing protein 2 (SUSD2) in circulating tumor cells of metastatic estrogen receptor positive breast cancer

ABSTRACT: Albeit responding well to therapy, estrogen receptor positive breast cancers frequently become subsequently resistant. This leads to distant metastasis and release of circulating tumor cells (CTCs) into the blood stream and ends with the death of the patient. Here we analyzed the proteome of CTC-ITB-01 by mass spectrometry and quantified 7954 different proteins using MCF-7 as a reference proteome. The data were deposited in a database for public access. CTC-ITB-01 is a CTC cell line derived from a patient with metastatic and estrogen receptor alpha (ER-alpha) positive breast cancer. We found remarkable high levels of the protein sushi domain-containing protein 2 (SUSD2) in CTC-ITB-01. Presence of SUSD2 in CTC of patients with breast cancer was confirmed by CellSearch (9/23 CTC positive cases). Ectonucleotide pyrophosphatase /phosphodiesterase family member 1 (ENPP1) was detected in CTC-ITB-01 and confirmed in CTC of breast cancer patients (n=3). ENPP1 can mediate immune evasion of tumor cells. Overexpression of SUSD2 in MCF-7 revealed upregulation of ER-alpha, 78 kDa glucose-regulated protein (GRP78) downregulation of programmed cell death protein 4 (PDCD4). GRP78 mediates resistance to chemotherapy, whereas loss of PDCD4 is involved in drug resistance and malignant transformation. Database search revealed significantly reduced overall survival in luminal breast cancer for low PDCD4 levels (n=348; p=0.0323). In conclusion, high levels of SUSD2 may confer improved cytoprotection of metastatic breast cancer cells and their CTC travelers to distant organs in the blood.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Kai Bartkowiak

LAB HEAD: Kai Bartkowiak

PROVIDER: PXD047444 | Pride | 2025-02-04

REPOSITORIES: Pride

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Project description:Infection or reactivation with human cytomegalovirus (HCMV) is still a major cause of morbidity and mortality in patients undergoing solid organ transplantation or allogeneic stem cell transplantation (alloSCT). Recent studies have shown that protection against latent HCMV infection relies on a much broader antigen spectrum than previously expected. The identification of novel immunogenic HCMV-derived peptides that facilitate HCMV control is therefore essential to improve immune monitoring of HCMV-specific T cells and HCMV vaccine development. In particular, only a few HCMV-derived peptides have been described for less common HLA alleles, such as HLA-A*03:01 and HLA-B*15:01, limiting the implementation of these techniques for a substantial number of patients. Here, we identified novel HCMV-derived HLA-A*03:01- and HLA-B*15:01-restricted immunogenic peptides by an innovative combined in vitro and in silico approach. We utilized the computational tools Peptide-PRISM (1) and PRICE (2) to analyze mass spectrometric (MS) and ribosome sequencing (ribo-seq) datasets, respectively. To refine our in silico approach, we utilized machine learning to rate the identified peptide candidates based on their translational activity, binding affinity and positioning within the small open reading frames (sORFs), with the goal of assessing their likelihood to be presented on HLA-A*03:01 and HLA-B*15:01 molecules. After identifying the highest-scoring 49 canonical and 49 cryptic potentially immunogenic peptides for each HLA allele, we assessed their immunogenicity by screening HCMV-seropositive and -seronegative healthy donors, as well as alloSCT patients for their peptide-specific T-cell responses. We used a stimulation procedure in two consecutive steps: peripheral blood cells (PBMCs) were stimulated starting with peptide pools and subsequently with potentially reactive single peptides. In vitro T-cell stimulation resulted in the direct identification of three canonical and one cryptic HLA-A*03-restricted immunogenic peptides as well as five canonical and one cryptic HLA-B*15-restricted immunogenic peptide, with a specific IFNγ+/CD8+ T cell response of ≥ 0.02%. Highest T-cell responses were identified against two HLA-A*03-restricted and three HLA-B*15-restricted canonical peptides with up to 3.42% of IFNγ+/CD8+ T cells in patients 180 days post alloSCT. In conclusion, we identified specific T-cell responses against eight canonical and two cryptic HLA-A*03:01- and HLA-B*15:01-restricted peptides in healthy individuals and post-transplanted patients and which were classified as immunogenic. Of these, six peptides are novel HCMV-derived, immunogenic peptides according to the UniProt database, broadening the spectrum of immunogenic HCMV-derived peptides for personalized immune monitoring and vaccine development.

Project description:Background: The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is activated by xenobiotic chemicals that function as AHR ligands. The response to xenobiotic AHR ligands is toxicity and the induction of drug metabolizing enzymes. The impact of AHR knockdown on gene expression and pathways in human breast cancer cells in absence of xenobiotic AHR ligands has not been investigated on a genome-wide scale. Methods: MCF-7 cells were used as a model of human breast cancer. The AHR was silenced with short interfering RNA against AHR (siAHR). RNA-sequencing coupled with Ingenuity pathway analysis (IPA) was used to determine the impact of AHR knockdown on gene expression and pathways in the absence of xenobiotic AHR ligands. Western blot analysis and recombinant tumor necrosis factor (TNF) was used to investigate the impact of AHR knockdown on TNF induction of MNSOD expression. Results: We found that the AHR is transcriptionally active in MCF-7 breast cancer cells in the absence of xenobiotic AHR ligands. In total, the expression of 634 genes was significantly changed in AHR knockdown cells compared to controls at a false discovery rate of < 10%. The analysis confirmed that drug metabolizing enzymes were AHR targets; however, we found that AHR also promoted the expression of genes that were not directly related to the metabolism of xenobiotics, such as those involved in lipid and eicosanoid synthesis. Gene pathway analysis of the AHR regulated gene dataset predicted TNF activity to be reduced in AHR knockdown cells. Our finding that AHR knockdown inhibited TNF-stimulated increases in MNSOD expression confirmed this IPA prediction. Conclusions: This is the first gene expression profiling study of AHR knockdown breast cancer cells. Several known and novel AHR targets were identified. The results suggest that endogenous AHR regulation impacts eicosanoid synthesis by regulating gene expression. The IPA prediction of reduced TNF activity in AHR knockdown cells was confirmed by showing that TNF-induced increases in MNSOD expression was inhibited in AHR knockdown cells. The requirement of AHR for MNSOD activation is novel and provides a new link by which AHR may impact reactive oxidative species (ROS) signaling. Expression profiles by mRNA sequencing were generated for human MCF-7 cells transfected with cRNAi (6 replicates) or AHR-siRNA (5 replicates) for 36hr. Sequencing was performed on an Illumina HiSeq 1000 using a 2x100 base paired-end strategy.

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Function and relevance of sushi domain-containing protein 2 (SUSD2) in circulating tumor cells of metastatic estrogen receptor positive breast cancer

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