Abstract 2001: Preclinical investigation of a 15 gene hypoxia profile in different prostate cancer cell lines
Authors: Wittenborn Thomas R. , Sorensen Brita S. , Busk Morten , Thomsen Mathilde B. , Nielsen Steffen , Horsman Michael R. 
Published: July 2017

Purpose: Hypoxia is known to reduce the response to radiotherapy and has been found to be a common feature in prostate cancer. Identifying patients with hypoxic tumors will allow us to select those patients that require specific hypoxic modifiers. Previously our department has developed a gene profile consisting of 15 genes, which demonstrated prognostic and predictive impact for hypoxic modification in head and neck squamous cell carcinomas (HNSCC). In the current study we investigated the 15 gene profile in vitro in different prostate cancer cell lines, and subsequently in vivo in the PC3 xenograft model.

Methods: For the in vitro experiments the prostate cancer cell lines investigated were PC-3, LNCaP and DU-145. Cell lines were cultured under normoxic (21% O2) or varying hypoxic conditions (0%, 0.5%, 1.0%, 2.0%, and 5.0% O2) for 24 hours, totRNA was extracted and gene expression levels measured by qPCR. Individual reference genes were selected (PSMC4, TBP, NDFIP1) and applied in the normalization of the relative expression levels, together with the reference genes previously used in the HNSCC study. For in vivo experiments, the PC3 cell line was inoculated on the flank of female NMRI nu/nu mice. Two hypoxia-sensitive tracers (18F-FAZA and Pimonidazole) were administered in order to determine hypoxic and non-hypoxic regions on excised tumor sections. These regions were isolated by laser-assisted microdissection, after which totRNA was extracted and gene expression levels measured by qPCR.

Results: In the in vitro experiments, all three prostate cancer cell lines had 14 of the 15 genes induced by hypoxia, when comparing the anoxic cells (0% O2) to the normoxic.cells (21% O2). The only discrepancy was ALDOA, which was not upregulated in the hypoxic cells. In the PC3 and DU-145 cell lines genes were investigated at additional hypoxic levels (0.5%, 1.0%, 2.0%, and 5.0% O2), giving a more intricate expression of the 15 genes. In vivo experiments are still ongoing but preliminary results show a hypoxia induced upregulation in 10 out of the 15 genes, of which 4 were significantly upregulated (ADM, ANKRD37, FAM162A, and LOX).

Conclusion: We investigated the 15 gene hypoxic profile in different prostate cancer cell lines, both in vitro and in vivo. A hypoxia dependent induction of genes was observed in both types of experiments, however with a differential gene expression with regard to hypoxia level. From the performed experiments, and looking only at oxygen dependency, it appears that the gene profile could be suitable for prostate cancers as well as HNSCC.

Funding sources: Danish Cancer Society ; The Danish Council for Independent Research: Medical Sciences ; EC FP7 project METOXIA (project no. 222741) ; CIRRO: The Lundbeck Foundation Center for Interventional Research in Radiation Oncology and The Danish Council for Strategic Research.

Citation Format: Thomas R. Wittenborn, Brita S. Sorensen, Morten Busk, Mathilde B. Thomsen, Steffen Nielsen, Michael R. Horsman. Preclinical investigation of a 15 gene hypoxia profile in different prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2001. doi:10.1158/1538-7445.AM2017-2001