Unbiased analysis of pancreatic cancer radiation resistance reveals cholesterol biosynthesis as a novel target for radiosensitisation

Abstract

Background: Despite its promise as a highly useful therapy for pancreatic cancer (PC), the addition of external beam radiation therapy to PC treatment has shown varying success in clinical trials. Understanding PC radioresistance and discovery of methods to sensitise PC to radiation will increase patient survival and improve quality of life. In this study, we identified PC radioresistance-associated pathways using global, unbiased techniques.

Methods: Radioresistant cells were generated by sequential irradiation and recovery, and global genome cDNA microarray analysis was performed to identify differentially expressed genes in radiosensitive and radioresistant cells. Ingenuity pathway analysis was performed to discover cellular pathways and functions associated with differential radioresponse and identify potential small-molecule inhibitors for radiosensitisation. The expression of FDPS, one of the most differentially expressed genes, was determined in human PC tissues by IHC and the impact of its pharmacological inhibition with zoledronic acid (ZOL, Zometa) on radiosensitivity was determined by colony-forming assays. The radiosensitising effect of Zol in vivo was determined using allograft transplantation mouse model.

Results: Microarray analysis indicated that 11 genes (FDPS, ACAT2, AG2, CLDN7, DHCR7, ELFN2, FASN, SC4MOL, SIX6, SLC12A2, and SQLE) were consistently associated with radioresistance in the cell lines, a majority of which are involved in cholesterol biosynthesis. We demonstrated that knockdown of farnesyl diphosphate synthase (FDPS), a branchpoint enzyme of the cholesterol synthesis pathway, radiosensitised PC cells. FDPS was significantly overexpressed in human PC tumour tissues compared with healthy pancreas samples. Also, pharmacologic inhibition of FDPS by ZOL radiosensitised PC cell lines, with a radiation enhancement ratio between 1.26 and 1.5. Further, ZOL treatment resulted in radiosensitisation of PC tumours in an allograft mouse model.

Conclusions: Unbiased pathway analysis of radioresistance allowed for the discovery of novel pathways associated with resistance to ionising radiation in PC. Specifically, our analysis indicates the importance of the cholesterol synthesis pathway in PC radioresistance. Further, a novel radiosensitiser, ZOL, showed promising results and warrants further study into the universality of these findings in PC, as well as the true potential of this drug as a clinical radiosensitiser.

Figure 1

Response of PC cell lines to radiation and generation of radiation-resistant sublines. (A) Colony formation assays of PC cell lines exposed to the indicated dose of radiation reveal a heterogeneous response. It was observed that the immortalised human pancreatic ductal epithelial cell line (HPDE) was highly sensitive to radiation, with no colonies being observed with 2 Gy treatment (not shown). Cell line names are given with the mean inactivation dose, ( QUOTE ), given in parentheses. Mean inactivation dose has units of Gy. (B) Schematic of the generation of radiation-resistant sublines. Cell lines were exposed to 10 fractions of 2 Gy irradiation over a 2-week period and then allowed to recover. Subsequently, colony formation assays were done following exposure to varying doses of radiation to compare the sensitivity between the parental and radioresistant sublines. (C) Colony formation assay of Panc-1RR vs Panc-1. The Panc-1 subline exposed to fractionated radiation required 1.6-fold higher IR dose to elicit equivalent cell death as the parental Panc-1, and was thus referred to as radioresistant, Panc-1RR. The mean inactivation dose, ( QUOTE ), is given in parentheses. (D) Colony formation assay of BxPC3-RR vs BxPC3. No difference was observed between the naturally radioresistant parental line BxPC3 and the BxPC3 subline exposed to fractionated radiation (BxPC3-RR).

Figure 2

Cholesterol (mevalonate) synthesis pathway genes are upregulated in radiation-resistant cells. (A) Simplified cholesterol biosynthesis pathway. Asterisks (*) mark upregulated genes in Panc-1 radiation-resistant (Panc-1RR) cells. FDPS is a branchpoint enzyme in the pathway, producing farnesyl pyrophosphate that is used for cholesterol synthesis, post-translational modification of small GTPases, including KRAS, termed farnesylation or geranylgeranylation (collectively called prenylation), which helps traffic proteins to membranes, and recently discovered to be an agonist for several nuclear receptors. ZOL (Zometa) inhibits FDPS, whereas the cholesterol-lowering statins inhibit HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol pathway. (B) Immunoblot of FDPS protein expression in various PC cell lines. (C) Immunoblot of Panc-1RR cells transfected with control scrambled (Scr) siRNA and FDPS siRNA indicating efficient knockdown at 48 h. (D) Colony formation assay comparing Panc-1RR cells transfected with scrambled control (Scr) or FDPS siRNA. Cells were irradiated 48 h after transfection, and then seeded for colony assay. siFDPS cells were sensitised to radiation, with ER being 1.34. Surviving fraction is expressed relative to non-irradiated cells in the respective treatment group. Graph shows the mean±s.e. of triplicate samples of a representative experiment. (E) Immunohistochemical analysis of normal pancreas (left) and PC tissue (right) for FDPS. Normal pancreas samples were from donors for pancreas transplant while PC specimens were from the Rapid Autopsy Program at the University of Nebraska Medical Center. Samples were mounted as tissue arrays and all were stained at the same time. Scoring was done by one pathologist. (F) Table summarising the incidence of FDPS overexpression in pancreatic tumours. (G) Scatter plot indicating the distribution of composite score for FDPS staining in pancreatic tumours. The median composite score (black bar) was 7.5.

Figure 3

Inhibition of FDPS radiosensitises PC cells. (Left) CSAs of radioselected Panc-1RR, BxPC3, fractionated BxPC3-RR, UN-KPC-961, and radioselected L3.6-RR cells following radiation treatment in the absence (control) and presence of indicated concentration of ZOL. ZOL treatment was 24 h before and 72 h after radiation treatment for Panc-1RR, BxPC3, fractionated BxPC3-RR, and 72 h after radiation treatment for UN-KPC-961 and radioselected L3.6-RR cells. Surviving fraction is expressed relative to non-irradiated cells in the respective treatment group. Graph shows the mean±s.e. of triplicate samples of a representative experiment. Asterisks indicate P-value <0.05. (Right) ER for each cell line following ZOL treatment. A value significantly >1 indicates radiosensitisation.

Figure 4

Effect of ZOL on radiation therapy of subcutaneous mouse allograft tumours. Immune-competent mice were injected s.c. with UN-KPC-961 cells in two areas – one to be irradiated and one as unirradiated control. Half of the mice were given ZOL (2 mg kg⁻¹) 1–2 h before the first radiation dose. Each mouse was aligned to its CT scan by ExacTrac and then stereotactically irradiated on 5 consecutive days with 7 Gy fractions. Waterfall plot showing percentage change in tumour volume at day 17 as observed by caliper measurement of tumours for all mouse tumours examined in the study. Similar results were observed with volume measured by CT imaging. Median of each treatment group is marked with ‘m'.