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High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids
Published on: June 14, 2021
Molecular characterization of acquired tolerance of tumor cells to picropodophyllin (PPP)
Jamileh Hashemi1, Claire Worrall, Daiana Vasilcanu
1Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, CMM L8:01, Stockholm, Sweden. Jamileh.Hashemi@ki.se
Background:
Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment.
Methodology/Principal Findings:
Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines.
Conclusions:
Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions.
Insights
Picropodophyllin (PPP) resistance in cancer cells involves genomic and expression changes. Long-term PPP selection alters genes related to cell death, impacting drug sensitivity.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Picropodophyllin (PPP) is a novel anti-neoplastic agent with demonstrated efficacy in killing tumor cells.
- Previous studies indicated moderate PPP tolerance in only a fraction of cell lines.
- This study investigates the genomic and expression alterations underlying acquired PPP tolerance.
Purpose of the Study:
- To identify and characterize the genomic and gene expression changes associated with long-term picropodophyllin (PPP) selection in cancer cells.
- To understand the molecular mechanisms conferring PPP resistance.
- To validate the role of specific altered genes in modulating PPP sensitivity.
Main Methods:
- Comparative genomic hybridization (array-CGH) to detect copy number alterations.
- Gene expression profiling to identify changes in gene activity.
- Nexus software analysis to integrate array-CGH and gene expression data.
- siRNA validation to confirm the functional role of selected genes in PPP sensitivity.
Main Results:
- Overlapping copy number alterations were observed between parental and tolerant cells, notably gain/amplification of MYC and PVT1 loci at 8q24.21.
- Specific genomic abnormalities, including gains and losses, were identified in tolerant cell lines (e.g., 5q11.2 amplification, 11q12.1-q14.3 gain, 13q33.3-qter gain).
- Altered expression of genes such as ALDH1A3, ANXA1, TLR4, RAB5A (downregulated) and COX6A1, NFIX, ME1, MAPK, TAP2 (upregulated) correlated with genomic changes and modulated PPP sensitivity.
Conclusions:
- Long-term picropodophyllin (PPP) selection induces significant genomic and gene expression alterations in tolerant cancer cells.
- Changes in genes involved in cell death pathways, such as PTEN and BCL2, were observed.
- Acquired genomic copy number variations frequently corresponded with altered mRNA expression levels of genes within those regions, contributing to PPP resistance.
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