TRPC3 shapes the ER-mitochondria Ca2+ transfer characterizing tumour-promoting senescence

Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca2+ load via negative regulation of IP3 receptor-mediated Ca2+ release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.

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Software and code
Policy information about availability of computer code Data collection Flow cytometry data were collected with the Summit Software version 4.3 from Beckman Coulter. In vivo bioluminescence recording was performed with the Living Image software version 4.5 from PerkinElmer. Calcium imaging data were collected with the MetaFluor version 4.5 software. Confocal images and data were collected with the Zen version software from Zeiss Microscopy. Microarray datasets were collected with Oncomine or the R version 3.6.0 software. Primers were designed with the qPrimerDepot version 1.0 software. qRT-PCR data were collected with the CFX Manager version 3.1 software from BioRad.

Data analysis
Flow cytometry data were analysed with the FlowJo version 7.0 software from BD. Statistic analysis were performed with the GraphPad Prism 8 software. Western blot densitometry was performed with the ImageJ/Fiji version 1.53 software. qRT-PCR data analysis were performed with the CFX Manager version 3.1 software from BioRad. GSEA analysis was performed using the GSEA software version 4.1.0 from UC San Diego and Broad Institute.
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April 2020
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability GSE130727 and GSE35988 datasets used to analyse TRPC3 expression (Supplementary Fig.1 I-L) are available in Gene Expression Omnibus (GEO) (https:// www.ncbi.nlm.nih.gov/geo/) and in Oncomine Database (https://www.oncomine.org/) identified as "Grasso Prostate" and "Tamura Prostate". All remaining data will be available from the corresponding author upon reasonable request. The dataset used to calculate gene enrichment ( Supplementary Fig. 1M) is available on the Human Cellular Senescence Database (HCSGD) (http://www.bioinfo-xwwang-thu.cn/qdong/HCSGD/). Data relative to the remaining figures are available on the Source File provided with this manuscript.

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For experiments involving calcium imaging and confocal microscopy, sample size was not predetermined. Depending on the number of cells in a single field of view, several technical replicates were produced for each biological replicate in order to give a number of cells sufficient to give statistical significance, if any. For flow cytometry, the count was stopped to at least 10000 events according to the expected elevated frequency of positive events and hence a low coefficient of variation ( Data exclusions No data were excluded from the analysis.

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For ethic reasons, the in vivo study was performed once on a group of 10 mice for each condition. All other experiments were done at least in triplicate. All attempts of replication were successful and gave similar results.
Randomization For in vivo studies, mice were earmarked and randomly assigned, by an independent person, to two groups of 10 subjects receiving PC3-Luc cells along with either shTRPC3 or siCTL prostate fibroblasts.

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The investigators were not blinded to the groups allocation during the experiments. Blinding was not possible as experimental conditions were evident from data (different morphology of senescent cells or use of concomitant senescence markers).

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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.   -Cancer-associated fibroblasts were not authenticated but confirmed as published in Mol. Carcinog. 56, 1851-1867(2017 -Human Embrionic Kidney GP-293 and Lenti-X 293T cells were bought from TakaraBio without further authentication -Sf9 cells were bought from Merck-Millipore without further authentication Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Were necessary, fibroblasts (MRC5 or Human Prostate Fibroblast) were were incubated with 5 μl of Pacific Blue Annexin-V and 5 ul of 7-AAD for 15 min at +4 °C or with DDAOG for 3 h at 37 °C 5% CO2 before analysis. Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.