Dataset Information

Histone post-translational modification analysis from low-abundance clinical samples

ABSTRACT: We streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells. We then applied this protocol to breast cancer patient tissue sections in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI-imaging profiling. These results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible, opening the way for the investigation of epigenetic features in the context of tissue and tumor heterogeneity.

INSTRUMENT(S): Q Exactive Plus, Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Breast Epithelium, Acinar Cell, Breast, Pancreas, Breast Cancer Cell

DISEASE(S): Breast Cancer

SUBMITTER: Roberta Noberini

LAB HEAD: Tiziana Bonaldi

PROVIDER: PXD024799 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples.

Noberini Roberta R Savoia Evelyn Oliva EO Brandini Stefania S Greco Francesco F Marra Francesca F Bertalot Giovanni G Pruneri Giancarlo G McDonnell Liam A LA Bonaldi Tiziana T

Clinical epigenetics 20210728 1

<h4>Background</h4>Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for subs ...[more]

PMID: 34315505

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Project description:To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in 3-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends upon specific ECM-interacting network hubs. Blockade of one such hub, the b1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression. There are 3 experiments in this study: (1a) a time course of human epidermal tissue transformed by oncogenic ER:H-Ras and IkBaM comprising days 0, 5, 20, and 35 post Ras activation using 4OHT; (1b) a matched time course of adjacent mouse stromal tissue during tumor progression; (2) Arrays comparing 4OHT-induced, Raf-1:ER/IkBaM transformed epidermal tissue and -4OHT controls; and (3) Arrays comparing Ras:ER/IkBaM grafts co-treated with 4OHT and either IgG control antibody or an anti-b1 integrin blocking antibody, P5D2 for 30 days. All arrays were done in biologic duplicate. Epidermal tissue co-expressing ER:H-RasG12V (ER:Ras) and IkBaM was regenerated on female scid/scid mouse recipients. Grafts were allowed to heal for at least 3 weeks before Ras activation via daily i.p. injections of 730ug of 4OHT (in 110ul of a corn oil/ethanol mixture). Duplicate grafts were harvested after 0, 5, 20, and 35 days of 4OHT treatment. Laser capture microdissection was utilized to independently isolate epithelial cells of the basal most layers and adajacent stromal tissue for each time point. RNA was subjected to one round of T7-based linear amplification (Ambion Message Amp II enhanced kit) and hybridized to either HG-U133A 2.0 or MG-430A affymetrix oligonucleotide arrays. Arrays from this experiment are labeled Ras and Stroma, respectively. The second experment was performed on Raf-1:ER/IkBaM expressing grafts with and without 4OHT treament for 30 days. The third experiment was done comparing ER:Ras/IkBaM grafts concomitantly treated with 4OHT and 1.5mg/week of either IgG control antibody or a mouse monoclonal blocking antibody against b1 integrin for 30 days. For experiments 2 and 3, HG-U133A 2.0 GeneChips were used; 4OHT treatment and RNA isolation/amplification was performed as above.

Dataset Information

Histone post-translational modification analysis from low-abundance clinical samples

Publications

Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples.

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