Desmocollin-1 is associated with pro-metastatic phenotype of luminal A breast cancer cells and is modulated by parthenolide.

BACKGROUND: Desmocollin-1 (DSC1) is a desmosomal transmembrane glycoprotein that maintains cell-to-cell adhesion. DSC1 was previously associated with lymph node metastasis of luminal A breast tumors and was found to increase migration and invasion of MCF7 cells in vitro. Therefore, we focused on DSC1 role in cellular and molecular mechanisms in luminal A breast cancer and its possible therapeutic modulation. METHODS: Western blotting was used to select potential inhibitor decreasing DSC1 protein level in MCF7 cell line. Using atomic force microscopy we evaluated effect of DSC1 overexpression and modulation on cell morphology. The LC-MS/MS analysis of total proteome on Orbitrap Lumos and RNA-Seq analysis of total transcriptome on Illumina NextSeq 500 were performed to study the molecular mechanisms associated with DSC1. Pull-down analysis with LC-MS/MS detection was carried out to uncover DSC1 protein interactome in MCF7 cells. RESULTS: Analysis of DSC1 protein levels in response to selected inhibitors displays significant DSC1 downregulation (p-value ≤ 0.01) in MCF7 cells treated with NF-κB inhibitor parthenolide. Analysis of mechanic cell properties in response to DSC1 overexpression and parthenolide treatment using atomic force microscopy reveals that DSC1 overexpression reduces height of MCF7 cells and conversely, parthenolide decreases cell stiffness of MCF7 cells overexpressing DSC1. The LC-MS/MS total proteome analysis in data-independent acquisition mode shows a strong connection between DSC1 overexpression and increased levels of proteins LACRT and IGFBP5, increased expression of IGFBP5 is confirmed by RNA-Seq. Pathway analysis of proteomics data uncovers enrichment of proliferative MCM_BIOCARTA pathway including CDK2 and MCM2-7 after DSC1 overexpression. Parthenolide decreases expression of LACRT, IGFBP5 and MCM_BIOCARTA pathway specifically in DSC1 overexpressing cells. Pull-down assay identifies DSC1 interactions with cadherin family proteins including DSG2, CDH1, CDH3 and tyrosine kinase receptors HER2 and HER3; parthenolide modulates DSC1-HER3 interaction. CONCLUSIONS: Our systems biology data indicate that DSC1 is connected to mechanisms of cell cycle regulation in luminal A breast cancer cells, and can be effectively modulated by parthenolide.

A large-scale assay library for targeted protein quantification in renal cell carcinoma tissues.

Renal cell carcinoma (RCC) represents 2.2% of all cancer incidences; however, prognostic or predictive RCC biomarkers at protein level are largely missing. To support proteomics research of localized and metastatic RCC, we introduce a new library of targeted mass spectrometry assays for accurate protein quantification in malignant and normal kidney tissue. Aliquots of 86 initially localized RCC, 75 metastatic RCC and 17 adjacent non-cancerous fresh frozen tissue lysates were trypsin digested, pooled, and fractionated using hydrophilic chromatography. The fractions were analyzed using LC-MS/MS on QExactive HF-X mass spectrometer in data-dependent acquisition (DDA) mode. A resulting spectral library contains 77,817 peptides representing 7960 protein groups (FDR = 1%). Further, we confirm applicability of this library on four RCC datasets measured in data-independent acquisition (DIA) mode, demonstrating a specific quantification of a substantially increased part of RCC proteome, depending on LC-MS/MS instrumentation. Impact of sample specificity of the library on the results of targeted DIA data extraction was demonstrated by parallel analyses of two datasets by two pan human libraries. The new RCC specific library has potential to contribute to better understanding the RCC development at molecular level, leading to new diagnostic and therapeutic targets.

SWATH-MS Analysis of FFPE Tissues Identifies Stathmin as a Potential Marker of Endometrial Cancer in Patients Exposed to Tamoxifen.

A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.

Proteomics Identification and Validation of Desmocollin-1 and Catechol-O-Methyltransferase as Proteins Associated with Breast Cancer Cell Migration and Metastasis.

Biological treatment of many cancers currently targets membrane bound receptors located on a cell surface. To identify novel membrane proteins associated with migration and metastasis of breast cancer cells, a more migrating subpopulation of MDA-MB-231 breast cancer cell line is selected and characterized. A high-resolution quantitative mass spectrometry with SILAC labeling is applied to analyze their surfaceome and it is compared with that of parental MDA-MB-231 cells. Among 824 identified proteins (FDR < 0.01), 128 differentially abundant cell surface proteins with at least one transmembrane domain are found. Of these, i) desmocollin-1 (DSC1) is validated as a protein connected with lymph node status of luminal A breast cancer, tumor grade, and Her-2 status by immunohistochemistry in the set of 96 primary breast tumors, and ii) catechol-O-methyltransferase is successfully verified as a protein associated with lymph node metastasis of triple negative breast cancer as well as with tumor grade by targeted data extraction from the SWATH-MS data of the same set of tissues. The findings indicate importance of both proteins for breast cancer development and metastasis and highlight the potential of biomarker validation strategy via targeted data extraction from SWATH-MS datasets.

A hybrid DDA/DIA-PASEF based assay library for a deep proteotyping of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and deeper proteome coverage is needed for its molecular characterization. We present comprehensive library of targeted mass spectrometry assays specific for TNBC and demonstrate its applicability. Proteins were extracted from 105 TNBC tissues and digested. Aliquots were pooled, fractionated using hydrophilic chromatography and analyzed by LC-MS/MS in data-dependent acquisition (DDA) parallel accumulation-serial fragmentation (PASEF) mode on timsTOF Pro LC-MS system. 16 individual lysates were analyzed in data-independent acquisition (DIA)-PASEF mode. Hybrid library was generated in Spectronaut software and covers 244,464 precursors, 168,006 peptides and 11,564 protein groups (FDR = 1%). Application of our library for pilot quantitative analysis of 16 tissues increased identification numbers in Spectronaut 18.5 and DIA-NN 1.8.1 software compared to library-free setting, with Spectronaut achieving the best results represented by 190,310 precursors, 140,566 peptides, and 10,463 protein groups. In conclusion, we introduce assay library that offers the deepest coverage of TNBC proteome to date. The TNBC library is available via PRIDE repository (PXD047793).

Catechol-O-methyl transferase suppresses cell invasion and interplays with MET signaling in estrogen dependent breast cancer.

Catechol-O-methyl transferase (COMT) is involved in detoxification of catechol estrogens, playing cancer-protective role in cells producing or utilizing estrogen. Moreover, COMT suppressed migration potential of breast cancer (BC) cells. To delineate COMT role in metastasis of estrogen receptor (ER) dependent BC, we investigated the effect of COMT overexpression on invasion, transcriptome, proteome and interactome of MCF7 cells, a luminal A BC model, stably transduced with lentiviral vector carrying COMT gene (MCF7-COMT). 2D and 3D assays revealed that COMT overexpression associates with decreased cell invasion (p < 0.0001 for Transwell assay, p < 0.05 for spheroid formation). RNA-Seq and LC-DIA-MS/MS proteomics identified genes associated with invasion (FTO, PIR, TACSTD2, ANXA3, KRT80, S100P, PREX1, CLEC3A, LCP1) being downregulated in MCF7-COMT cells, while genes associated with less aggressive phenotype (RBPMS, ROBO2, SELENBP, EPB41L2) were upregulated both at transcript (|log2FC|> 1, adj. p < 0.05) and protein (|log2FC|> 0.58, q < 0.05) levels. Importantly, proteins driving MET signaling were less abundant in COMT overexpressing cells, and pull-down confirmed interaction between COMT and Kunitz-type protease inhibitor 2 (SPINT2), a negative regulator of MET (log2FC = 5.10, q = 1.04-7). In conclusion, COMT may act as tumor suppressor in ER dependent BC not only by detoxification of catechol estrogens but also by suppressing cell invasion and interplay with MET pathway.

How Different Are the Molecular Mechanisms of Nodal and Distant Metastasis in Luminal A Breast Cancer?

Lymph node status is one of the best prognostic factors in breast cancer, however, its association with distant metastasis is not straightforward. Here we compare molecular mechanisms of nodal and distant metastasis in molecular subtypes of breast cancer, with major focus on luminal A patients. We analyze a new cohort of 706 patients (MMCI_706) as well as an independent cohort of 836 primary tumors with full gene expression information (SUPERTAM_HGU133A). We evaluate the risk of distant metastasis, analyze targetable molecular mechanisms in Gene Set Enrichment Analysis and identify relevant inhibitors. Lymph node positivity is generally associated with NF-κB and Src pathways and is related to high risk (OR: 5.062 and 2.401 in MMCI_706 and SUPERTAM_HGU133A, respectively, p < 0.05) of distant metastasis in luminal A patients. However, a part (≤15%) of lymph node negative tumors at the diagnosis develop the distant metastasis which is related to cell proliferation control and thrombolysis. Distant metastasis of lymph node positive patients is mostly associated with immune response. These pro-metastatic mechanisms further vary in other molecular subtypes. Our data indicate that the management of breast cancer and prevention of distant metastasis requires stratified approach based on targeted strategies.