DOI: 10.5061/dryad.z34tmpghd
title: Data from: A calpain-6/YAP axis in sarcoma stem cells that drives the outgrowth of tumors and metastases

description: 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.
description: 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.
description: # Data for: A calpain-6/YAP axis in sarcoma stem cells that drives the
 outgrowth of tumors and metastases 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.