Data from: A calpain-6/YAP axis in sarcoma stem cells that drives the outgrowth of tumors and metastases
Data files
Oct 14, 2025 version files 15.20 MB
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143B_C6__1_R1_fastqc.html
300.81 KB
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143B_C6__1.ReadsPerGene.Gencode.txt
964.50 KB
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143B_C6__2_R1_fastqc.html
300.86 KB
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143B_C6__2.ReadsPerGene.Gencode.txt
963.38 KB
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143B_C6__3_R1_fastqc.html
302.65 KB
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143B_C6__3.ReadsPerGene.Gencode.txt
964.20 KB
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143B_GFP__1_R1_fastqc.html
299.55 KB
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143B_GFP__1.ReadsPerGene.Gencode.txt
961.76 KB
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143B_GFP__2_R1_fastqc.html
302.42 KB
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143B_GFP__2.ReadsPerGene.Gencode.txt
963 KB
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143B_GFP__3_R1_fastqc.html
300.75 KB
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143B_GFP__3.ReadsPerGene.Gencode.txt
964.25 KB
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143B_GFP-_1_R1_fastqc.html
306.01 KB
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143B_GFP-_1.ReadsPerGene.Gencode.txt
965.10 KB
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143B_GFP-_2_R1_fastqc.html
302.79 KB
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143B_GFP-_2.ReadsPerGene.Gencode.txt
962.40 KB
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143B_GFP-_3_R1_fastqc.html
308.29 KB
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143B_GFP-_3.ReadsPerGene.Gencode.txt
964.01 KB
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143B_shC6_1_R1_fastqc.html
300.08 KB
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143B_shC6_1.ReadsPerGene.Gencode.txt
965.44 KB
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143B_shC6_2_R1_fastqc.html
301.87 KB
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143B_shC6_2.ReadsPerGene.Gencode.txt
961.12 KB
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143B_shC6_3_R1_fastqc.html
298.53 KB
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143B_shC6_3.ReadsPerGene.Gencode.txt
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README.md
7.79 KB
Abstract
Sarcomas include cancer stem cells, but how these cells contribute to local and metastatic relapse is largely unknown. We previously showed the pro-tumor functions of calpain-6 in sarcoma stem cells. Here, we use an osteosarcoma cell model, osteosarcoma tissues and transcriptomic data from human tumors to study gene patterns associated with calpain-6 expression or suppression. Calpain-6 modulates the expression of Hippo pathway genes and stabilizes the hippo effector YAP. It also modulates the vesicular trafficking of β-catenin degradation complexes. Calpain-6 expression is associated with genes of the G2M phase of the cell cycle, supports G2M-related YAP activities and up-regulated genes controlling mitosis in sarcoma stem cells and tissues. In mouse models of bone sarcoma, most tumor cells expressed calpain-6 during early steps of tumor out-growth. YAP inhibition prevented the neoformation of primary tumors and metastases but had no effect on already developed tumors. It could even accelerate lung metastasis associated with large bone tumors by affecting tumor-associated inflammation in the host tissues. Our results highlight a specific mechanism involving YAP transcriptional activity in cancer stem cells that is crucial during the early steps of tumor and metastasis outgrowth and that could be targeted to prevent sarcoma relapse.
Dataset DOI: 10.5061/dryad.z34tmpghd
Description of the data and file structure
The 143B human osteosarcoma cell line that was modified to express GFP under control of the CAPN6 regulatory sequence to sort calpain-6 expressing cells (GFP+) from the other tumor cells (GFP-). These cells were compared to a homogeneous population with calpain-6 expression stably inhibited by a specific shRNA (Calp6shRNA cells). A homogeneous population of 143B cells that overexpressed calpain-6 by using a lentiviral vector (Calp6 + cells) was also included in the analysis. Cells were confluent and cultured in 3% O2 in DMEM + 10% FCS
Total RNAs was extracted from 3 independent cultures by using the ISOLATE II RNA Mini Kit (Bioline). Reverse transcription was performed with random primers with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RNA purity/quality (RIN = 7) was assessed with the 2100 Agilent Bioanalyzer by using the Agilent small RNA kit. RNAseq involved using the Integragen platform. Libraries were prepared with the Ultra II Directional RNA Library Prep Kit for Illumina protocol according to the supplier recommendations. Briefly, the key stages of this protocol are successive for the purification of PolyA containing mRNA molecules using poly-T oligo attached magnetic beads from 1 µg total RNA (with the Magnetic mRNA Isolation Kit from NEB), fragmentation by using divalent cations under elevated temperature to obtain approximately 300 bp pieces, double-strand cDNA synthesis and finally Illumina adapters ligation and cDNA library amplification by PCR for sequencing. Sequencing was then carried out on Paired End 100b reads of Illumina NovaSeq. Image analysis and base calling involved using Illumina Real Time Analysis v3.4.4 with default parameters.
Samples:
- 143B_GFP-_1 143B-Human osteosarcoma cell line Bone express GFP under control of the CAPN6 regulatory sequence FACS sorted GFP- cells
- 143B_GFP-_2 143B-Human osteosarcoma cell line Bone express GFP under control of the CAPN6 regulatory sequence FACS sorted GFP- cells
- 143B_GFP-_3 143B-Human osteosarcoma cell line Bone express GFP under control of the CAPN6 regulatory sequence FACS sorted GFP- cells
- 143B_GFP+__1 143B-Human osteosarcoma cell line Bone express GFP under control of the CAPN6 regulatory sequence FACS sorted GFP+ cells
- 143B_GFP+__2 143B-Human osteosarcoma cell line Bone express GFP under control of the CAPN6 regulatory sequence FACS sorted GFP+ cells
- 143B_GFP+__3 143B-Human osteosarcoma cell line Bone express GFP under control of the CAPN6 regulatory sequence FACS sorted GFP+ cells
- 143B_shC6_1 143B-Human osteosarcoma cell line Bone express Calpain-6 shRNA Calpain-6 Knockdown
- 143B_shC6_2 143B-Human osteosarcoma cell line Bone express Calpain-6 shRNA Calpain-6 Knockdown
- 143B_shC6_3 143B-Human osteosarcoma cell line Bone express Calpain-6 shRNA Calpain-6 Knockdown
- 143B_C6+__1 143B-Human osteosarcoma cell line Bone express Calpain-6 under control of CMV promoter Calpain-6 overexpressing cells
- 143B_C6+__2 143B-Human osteosarcoma cell line Bone express Calpain-6 under control of CMV promoter Calpain-6 overexpressing cells
- 143B_C6+__3 143B-Human osteosarcoma cell line Bone express Calpain-6 under control of CMV promoter Calpain-6 overexpressing cells
Files:
.txt
143B_C6__1.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the sample N°1 of 143B cells overexpressing Calpain-6.
143B_C6__2.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the sample N°2 of 143B cells overexpressing Calpain-6.
143B_C6__3.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the sample N°3 of 143B cells overexpressing Calpain-6.
143B_GFP__1.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the sample N°1 of sorted GFP+ 143B cells.
143B_GFP__2.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the sample N°2 of sorted GFP+ 143B cells.
143B_GFP__3.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the sample N°3 of sorted GFP+ 143B cells.
143B_GFP-_1.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the sample N°1 of sorted GFP- 143B cells.
143B_GFP-_2.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the sample N°2 of sorted GFP- 143B cells.
143B_GFP-_3.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the sample N°3 of sorted GFP- 143B cells.
143B_shC6_1.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the samples N°1 of 143B cells expressing Calpain-6 shRNA.
143B_shC6_2.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the samples N°2 of 143B cells expressing Calpain-6 shRNA.
143B_shC6_3.ReadsPerGene.Gencode.txt
these files contain the results of successful read mapping and the indications for number unmapped, multimapping, noFeature and ambiguous reads for the samples N°3 of 143B cells expressing Calpain-6 shRNA.
.html
143B_C6__1_R1_fastqc.html
these files contain quality control reports for the sample N°1 of 143B cells overexpressing Calpain-6.
143B_C6__2_R1_fastqc.html
these files contain quality control reports for the sample N°2 of 143B cells overexpressing Calpain-6.
143B_C6__3_R1_fastqc.html
these files contain quality control reports for the sample N°3 of 143B cells overexpressing Calpain-6.
143B_GFP__1_R1_fastqc.html
these files contain quality control reports for the sample N°1 of sorted GFP+ 143B cells.
143B_GFP__2_R1_fastqc.html
these files contain quality control reports for the sample N°2 of sorted GFP+ 143B cells.
143B_GFP__3_R1_fastqc.html
these files contain quality control reports for the sample N°3 of sorted GFP+ 143B cells.
143B_GFP-_1_R1_fastqc.html
these files contain quality control reports for the sample N°1 of sorted GFP- 143B cells.
143B_GFP-_2_R1_fastqc.html
these files contain quality control reports for the sample N°2 of sorted GFP- 143B cells.
143B_GFP-_3_R1_fastqc.html
these files contain quality control reports for the sample N°3 of sorted GFP- 143B cells.
143B_shC6_1_R1_fastqc.html
these files contain quality control reports for the sample N°1 of 143B cells expressing Calpain-6 shRNA.
143B_shC6_2_R1_fastqc.html
these files contain quality control reports for the sample N°2 of 143B cells expressing Calpain-6 shRNA.
143B_shC6_3_R1_fastqc.html
these files contain quality control reports for the sample N°3 of 143B cells expressing Calpain-6 shRNA.
Total RNAs was extracted from osteosarcoma cells by using the ISOLATE II RNA Mini Kit (Bioline).
RNA purity/quality (RIN ≥ 7) was assessed with the 2100 Agilent Bioanalyzer by using the Agilent small RNA kit. RNAseq involved using the Integragen platform. Libraries were prepared with the Ultra II Directional RNA Library Prep Kit for Illumina protocol according to the supplier recommendations. Briefly, the key stages of this protocol are successive for the purification of PolyA-containing mRNA molecules using poly-T oligo attached magnetic beads from 1µg total RNA (with the Magnetic mRNA Isolation Kit from NEB), fragmentation by using divalent cations under elevated temperature to obtain approximately 300 bp pieces, double-strand cDNA synthesis and finally Illumina adapters ligation and cDNA library amplification by PCR for sequencing. Sequencing was then carried out on Paired End 100b reads of Illumina NovaSeq. Image analysis and base calling involved using Illumina Real Time Analysis v3.4.4 with default parameters.
- Tchicaya-Bouanga, Joëlle; Hung, Yu-Jen; Schwartz, Jean-Marc et al. (2022). A calpain-6/YAP axis in sarcoma stem cells that drives the outgrowth of tumors and metastases. Cell Death & Disease. https://doi.org/10.1038/s41419-022-05244-3