Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jul 27:14:1215645.
doi: 10.3389/fgene.2023.1215645. eCollection 2023.

Characterization of the TRPV6 calcium channel-specific phenotype by RNA-seq in castration-resistant human prostate cancer cells

Clément Cordier  1 , Aurélien Haustrate  1 , Natalia Prevarskaya  1 , V'yacheslav Lehen'kyi  1
Affiliations

Characterization of the TRPV6 calcium channel-specific phenotype by RNA-seq in castration-resistant human prostate cancer cells

Clément Cordier et al. Front Genet. .

Abstract

Background: Transient receptor potential vanilloid subfamily member 6 (TRPV6), a highly calcium-selective channel, has been shown to play a significant role in calcium homeostasis and to participate both in vitro and in vivo in growth, cell survival, and drug resistance of prostate cancer. Its role and the corresponding calcium-dependent pathways were mainly studied in hormone-dependent human prostate cancer cell lines, often used as a model of early-stage prostate cancers. The goal of the present study was to describe the TRPV6-specific phenotype and signaling pathways it is involved in, using castration-resistant prostate cancer cell lines. Methods: RNA sequencing (RNA-seq) was used to study the gene expression impacted by TRPV6 using PC3Mtrpv6-/- versus PC3Mtrpv6+/+ and its derivative PC3M-luc-C6trpv6+/+ cell line in its native and TRPV6 overexpressed form. In addition to the whole-cell RNA sequencing, immunoblotting, quantitative PCR, and calcium imaging were used to validate trpv6 gene status and functional consequences, in both trpv6 -/- and TRPV6 overexpression cell lines. Results: trpv6 -/- status was validated using both immunoblotting and quantitative PCR, and the functional consequences of either trpv6 gene deletion or TRPV6 overexpression were shown using calcium imaging. RNA-seq analysis demonstrated that the calcium channel TRPV6, being a crucial player of calcium signaling, significantly impacts the expression of genes involved in cancer progression, such as cell cycle regulation, chemotaxis, migration, invasion, apoptosis, ferroptosis as well as drug resistance, and extracellular matrix (ECM) re-organization. Conclusion: Our data suggest that the trpv6 gene is involved in and regulates multiple pathways related to tumor progression and drug resistance in castration-resistant prostate cancer cells.

Keywords: RNA-seq analysis; TRPV6 channel; castration-resistant prostate cancer; expression profile; signaling pathway.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Validation of PC-3M and PC-3M-Luc-C6 cell line models and the store-operated capacitive calcium entry (SOCE) therein. The trpv6 gene expression was analyzed by immunoblotting and real-time PCR in PC-3M (A) and PC-3M-Luc-C6 (B) cell lines. (C) SOCE in both PC3M trpv6+/+ and PC3M trpv6−/− cell lines. (D) SOCE in both PC-3M-Luc-C6 trpv6+/+ and PC-3M-Luc-C6 trpv6+/+ + pTRPV6WT cell lines.
FIGURE 2
FIGURE 2
Transcriptional changes revealed by RNA-seq profiling of trpv6 expression-modulated cell lines. (A) Principal component analysis showing the overall transcriptomic similarity between the (a) PC-3M and (b) PC-3M-Luc-C6 cell lines. (B) Volcano plots showing the differentially expressed genes (DEGs; Log2FC > 1 or < −1, false discovery rate adjusted p-value <0.05) in PCa cells. (C,D) Kyoto Encyclopedia of Genes and Genomes (KEGG) functional clustering of genes that were dysregulated for biological processes in PC-3M versus PC-3M-Luc-C6 cell lines, respectively. (E,F) Gene Ontology biological process (GO BP) functional enrichment analysis of DEGs in PC-3M versus PC-3M-Luc-C6 cell lines, respectively (terms of top 15 counts ranked by GeneRatio). The color and size of circles indicate the p-adjust and gene number, respectively.
FIGURE 3
FIGURE 3
Differentially expressed genes (DEGs) in trpv6 expression-modulated cell lines and their roles in cancer cell biology. (A,B) Top 10 protein-coding differentially expressed genes, upregulated (orange) and downregulated (blue), in PC-3M versus PC-3M-Luc-C6 cell lines, respectively. (C) Intersection of sets between the DEGs in PC-3M trpv6+/+ versus PC-3M-Luc-C6 trpv6+/+ +pTRPV6wt cell lines.
FIGURE 4
FIGURE 4
A heat map of the representative key canonical pathway genes in the aggressive phenotype in PC-3M and PC-3M-Luc-C6 trpv6+/+ . Main genes involved in the ECM–receptor interaction (A,E), focal adhesion (B,F), positive regulation of cell migration which is involved in sprouting angiogenesis (C,G), and positive regulation of chemotaxis signaling (D,H) are represented in the heat map if expressed in samples (RPKM >0), according to both the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology biological process (GO BP) databases. Colors ranged from blue to red, corresponding from low to high expression levels, respectively (see scale bar).
FIGURE 5
FIGURE 5
A heat map of the representative key canonical genes involved in calcium metabolism and also ion channels in PC-3M and PC-3M-Luc-C6 trpv6+/+ cell lines. (A,B) Main genes involved in the “calcium signaling pathway” database according to Kyoto Encyclopedia of Genes and Genomes (KEGG). (C,D) Main ion channel genes in prostate cancer, realized with Morpheus, https://software.broadinstitute.org/morpheus. The genes were represented in the heat map if expressed in samples (RPKM >0). Colors range from blue to red, corresponding to low to high expression levels (see scale bar).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Akashi T., Koizumi K., Tsuneyama K., Saiki I., Takano Y., Fuse H. (2008). Chemokine receptor CXCR4 expression and prognosis in patients with metastatic prostate cancer. Cancer Sci. 99 (3), 539–542. 10.1111/j.1349-7006.2007.00712.x - DOI - PMC - PubMed
    1. Arbabian A., Iftinca M., Altier C., Singh P. P., Isambert H., Coscoy S. (2020). Mutations in calmodulin-binding domains of TRPV4/6 channels confer invasive properties to colon adenocarcinoma cells. Channels 14 (1), 101–109. 10.1080/19336950.2020.1740506 - DOI - PMC - PubMed
    1. Bai S., Wei Y., Liu R., Chen Y., Ma W., Wang M., et al. (2023). The role of transient receptor potential channels in metastasis. Biomed. Pharmacother. 158, 114074. 10.1016/j.biopha.2022.114074 - DOI - PubMed
    1. Basu G. D., Azorsa D. O., Kiefer J. A., Rojas A. M., Tuzmen S., Barrett M. T., et al. (2008). Functional evidence implicating S100P in prostate cancer progression. Int. J. Cancer 123 (2), 330–339. 10.1002/ijc.23447 - DOI - PubMed
    1. Bilusic M., Madan R. A., Gulley J. L. (2017). Immunotherapy of prostate cancer: Facts and hopes. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 23 (22), 6764–6770. 10.1158/1078-0432.CCR-17-0019 - DOI - PMC - PubMed