Dataset Information

Characterization of CD44s glycoproteoforms in bladder tumours and bladder cancer cell culture models

ABSTRACT: Bladder cancer (BC) is a major health concern retaining high mortality rates when diagnosed at advanced stages. In this context, the clinical management of BC requires the introduction of new biomarkers and molecular targets capable to elicit precision medicine therapeutical approaches. A promising candidate is cluster of differentiation 44 (CD44), a multifunctional and heavily glycosylated transmembrane protein, involved in cell-cell and cell-matrix adhesion, and a transducer of several oncogenic signalling cascades. CD44 has been widely explored as a marker of BC aggressiveness and cancer stem cells (CSC), but this is a highly heterogenic protein and the lack of molecular tools for precise molecular characterization has led to conflicting results, delaying clinical application. CD44 molecular variability results from the number of proteoforms arising from alternative splicing. The human CD44 gene consists of 17 exons, and while the exons 1-5 are constitutively expressed, exons 6-14 are subjected to alternative splicing, generating a myriad of different variants whose functional implications are yet to be fully understood. Furthermore, this number of proteoforms is greatly amplified by O-GalNAc glycosylation, which is predicted to mostly occur in the protein variable regions. This microheterogeneity and its relevance for BC can only be addressed by obtaining a comprehensive characterization at the protein level. Herein, we devised a strategy, combining transcriptomics, glycomics and mass spectrometry based proteomics data, to interrogate CD44 proteoform expression in a series of BC cell culture models showing different aggressiveness (5637 and T24) and a sample pool from formalin fixed paraffin embedded (FFPE) tumours. Glycoengeneered T24 cells expressing the Tn antigen (T24 C1GALT1 knockout (KO)) were also analysed.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urinary Bladder Urothelium

DISEASE(S): Urinary Bladder Cancer

SUBMITTER: André Silva

LAB HEAD: José Alexandre Ferreira

PROVIDER: PXD028307 | Pride | 2022-05-19

REPOSITORIES: Pride

ACCESS DATA

Publications

Glycoproteogenomics characterizes the CD44 splicing code associated with bladder cancer invasion.

Gaiteiro Cristiana C Soares Janine J Relvas-Santos Marta M Peixoto Andreia A Ferreira Dylan D Paulo Paula P Brandão Andreia A Fernandes Elisabete E Azevedo Rita R Palmeira Carlos C Freitas Rui R Miranda Andreia A Osório Hugo H Prieto Jesús J Lima Luís L Silva André M N AMN Santos Lúcio Lara LL Ferreira José Alexandre JA

Theranostics 20220328 7

<b>Rationale:</b> Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying cl ...[more]

PMID: 35547758

Similar Datasets

Project description:Breast cancers contain a minority population of cancer cells characterized by CD44 expression but low or undetectable levels of CD24 (CD44+CD24-/low) that have higher tumorigenic capacity than other subtypes of cancer cells. METHODS: We compared the gene-expression profile of CD44+CD24-/low tumorigenic breast-cancer cells with that of normal breast epithelium. Differentially expressed genes were used to generate a 186-gene invasiveness gene signature (IGS), which was evaluated for its association with overall survival and metastasis-free survival in patients with breast cancer or other types of cancer. RESULTS: There was a significant association between the IGS and both overall and metastasis-free survival (P<0.001, for both) in patients with breast cancer, which was independent of established clinical and pathological variables. When combined with the prognostic criteria of the National Institutes of Health, the IGS was used to stratify patients with high-risk early breast cancer into prognostic categories (good or poor); among patients with a good prognosis, the 10-year rate of metastasis-free survival was 81%, and among those with a poor prognosis, it was 57%. The IGS was also associated with the prognosis in medulloblastoma (P=0.004), lung cancer (P=0.03), and prostate cancer (P=0.01). The prognostic power of the IGS was increased when combined with the wound-response (WR) signature. CONCLUSIONS: The IGS is strongly associated with metastasis-free survival and overall survival for four different types of tumors. This genetic signature of tumorigenic breast-cancer cells was even more strongly associated with clinical outcomes when combined with the WR signature in breast cancer. Expression profling was performed on 6 tumorigenic, 3 non tumorigenic samples of breast tumors and 3 normal breast samples on two different platforms GPL96 and GPL97. A gene signature was derived by comparing the gene expressions of 6 tumorigenic samples with 3 normal breast samples.

Project description:Recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) has a poor prognosis with less than 1-year median survival. Platinum-based chemotherapy (cisplatin or carboplatin) remains the first-line treatment for HNSCC. The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self renewing cell populations that constitute the bulk of the tumor. A small population of CSCs exists within HNSCC that are relatively resistant to chemotherapy and clinically predicted to mediate tumor recurrence. These CSCs are identified by high cell-surface expression of CD44 and high intracellular activity of aldehyde dehydrogenase (ALDH) and termed ALDHhighCD44high. We investigated the molecular pathways active in ALDHhighCD44high cells, which remain poorly studied. Additionally, we performed a molecular examination of cisplatin-resistant ALDHhighCD44high cells, which has not been reported. Two HNSCC cell lines, UM-SCC-1 and UM-SCC-22b, were utilized in this study. For microarray analysis, UM-SCC-22b cells were treated for 5 days in vitro with 2uM cisplatin and analyzed by flow cytometry, sorted and submitted for microarray analysis of ALDHhighCD44high and ALDHlowCD44low cells from untreated and cisplatin treated cells. Four separate flow cytometry experiments were performed using Affymetrix Human Gene ST 2.1 microarrays. Microarray data was analyzed using R/Bioconductor. Files were preprocessed by Robust Multiarray Average (RMA) with background substraction, quantile normalization, and median polish (oligo package). Data was fitted with robust probe level linear models to all the probesets (oligo package). Experiment and processing batch differences were accounted for using 'ComBat' within the SVA package. Differentially expressed genes were identified using univariate comparisons after fitting data to a linear model (limma package). Initial statistics were determined using an empirical Bayesian model. Multiple testing comparisons were adjusted using Benjamini and Hochberg (aka FDR). Probes with an adjusted p-value <0.05 were considered statistically significant. Unsupervised hierarchical clustering with complete linkage and Euclidean distance was performed on only statistically significant probes. In four separate experiments, the head and neck squamous cell carcinoma cell line UM-SCC-22b were cultured for 5 days with or without 2uM (micromolar) cisplatin in 6-well plates. Media was replaced every other day. Control and cisplatin treated cells were trypsinized, procesed, and stained for CD44 cell-surface expression and intracellular aldehyde dehydrogenase (ALDH) activity to identify cancer stem cells (ALDH+CD44+). CSCs and non-CSCs (ALDH-CD44-) were collected by flow cytometry from both groups. Total RNA was collected from each fraction (ALDH+CD44+, ALDH-CD44-), treatment (control, cisplatin), and experiment (#1-4). A total of 16 samples were analyzed. One set of 4 (experiment #4) were analyzed on a Human Gene ST 2.1 strip and the rest on a Human Gene ST 2.1 plate. Differential gene expression was determined with R/Bioconductor with Robust Multiarray Average (RMA) and fitting the data to linear models (limma). Experimental and processing batch effects were accounted for using ComBat. Four sets of univariate comparisons were made: 1) Cisplatin ALDH+CD44+ vs Control ALDH+CD44+; 2) Control ALDH+CD44+ vs Control ALDH-CD44-; 3) Cisplatin ALDH+CD44+ vs Cisplatin ALDH-CD44-; 4) Cisplatin ALDH-CD44- vs Control ALDH-CD44-. Multiple testing comparisons were adjusted using Benjamini and Hochberg (aka FDR). Probes with an adjusted p-value <0.05 were considered statistically significant.

Dataset Information

Characterization of CD44s glycoproteoforms in bladder tumours and bladder cancer cell culture models

Publications

Glycoproteogenomics characterizes the CD44 splicing code associated with bladder cancer invasion.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure