ABSTRACT
Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry-based proteomic approach and analyzed a breast cancer cohort of 113 formalin-fixed paraffin-embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor-adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post-treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER-positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/therapy , Neoadjuvant Therapy , Proteomics , Breast Neoplasms/pathology , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Cell Proliferation , Citric Acid Cycle , Female , Gene Regulatory Networks , Humans , Neoplasm Invasiveness , Neoplasm Proteins/metabolism , Prognosis , Protein Interaction Maps , Pyrroline Carboxylate Reductases/metabolism , Recurrence , Survival Analysis , delta-1-Pyrroline-5-Carboxylate ReductaseABSTRACT
BACKGROUND: The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. RESULTS: In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. CONCLUSIONS: Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.
Subject(s)
Cryopreservation , Gene Expression Profiling , RNA, Messenger/genetics , Sequence Analysis, RNA , Tissue Fixation/methods , Breast Neoplasms/genetics , Breast Neoplasms/pathology , HumansABSTRACT
OBJECTIVE: To examine the effects of LSC101, a botanical compound, on adaptive and innate immunity. MATERIALS AND METHODS: LCS101 preparations were tested for batch-to-batch consistency using high-performance liquid chromatography. T-cell activation was quantified in murine spleen cells using 3H-thymidine incorporation, and cytokine production analyzed with enzyme-linked immunosorbent assay. Natural killer cell activity was tested on human blood cells using flow cytometry, and cytotoxicity measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis using a FACSCalibur. Effects on interferon-γ production in fluorouracil/doxorubicin-treated mice were tested with enzyme-linked immunosorbent assay. RESULTS: High-performance liquid chromatography analysis demonstrated batch-to-batch consistency. T-cell proliferation was increased, and a dose-dependent activation of natural killer cells and macrophage tumor necrosis factor-α secretion were observed with LCS101 treatment. Interferon-γ levels, reduced following fluorouracil treatment, were corrected in treated animals. No toxicity or compromised treatment outcomes were associated with LCS101 exposure. CONCLUSIONS: LCS101 demonstrated significant effects on a number of immune processes. Further research is needed in order to understand the molecular immunomodulatory pathways affected by this compound, as well as clinical implications for treatment.
