The SGLT2 inhibitor canagliflozin suppresses growth and enhances prostate cancer response to radiotherapy

Radiotherapy is a non-invasive standard treatment for prostate cancer (PC). However, PC develops radio-resistance, highlighting a need for agents to improve radiotherapy response. Canagliflozin, an inhibitor of sodium-glucose co-transporter-2, is approved for use in diabetes and heart failure, but is also shown to inhibit PC growth. However, whether canagliflozin can improve radiotherapy response in PC remains unknown. Here, we show that well-tolerated doses of canagliflozin suppress proliferation and survival of androgen-sensitive and insensitive human PC cells and tumors and sensitize them to radiotherapy. Canagliflozin blocks mitochondrial respiration, promotes AMPK activity, inhibits the MAPK and mTOR-p70S6k/4EBP1 pathways, activates cell cycle checkpoints, and inhibits proliferation in part through HIF-1α suppression. Canagliflozin mediates transcriptional reprogramming of several metabolic and survival pathways known to be regulated by ETS and E2F family transcription factors. Genes downregulated by canagliflozin are associated with poor PC prognosis. This study lays the groundwork for clinical investigation of canagliflozin in PC prevention and treatment in combination with radiotherapy.


Supplementary figures
Average animal weight id Supplementary Figure 3. (a-b) Effects of canagliflozin and radiation (RT) treatments on (a) oxygen consumption rate (OCR), and (b) extracellular acidification rate (ECAR), raw data.(c) Regulation of expression of glycolytic related genes of the "glycolysis" Gene Ontology (GO) term and membrane transporter genes.All glycolysis related genes are not significantly regulated by canagliflozin, FDR q-value > 0.05.All membrane transporter genes are significantly downregulated by canagliflozin, FDR q-value < 0.05.(d) Immunoblotting assay quantification for phosphorylated-ACC(Ser 79 )/GAPDH.(e) Normalized basal OCR values from the glycolytic rate assay (GRA) experiment in PC3 cells treated with canagliflozin, IACS-010759, or BAY-872243.Cell number normalization was performed using crystal violet for the cells.The significance of the time course experiment at each treatment dosage was determined using a two-way analysis of variance (ANOVA), asterisks represent of p value, * = p<0.05,and ** = p<0.01.The data shown is mean ± SEM; n=3.Ordinary one-way ANOVA with the post hoc Tukey's multiple comparisons test was performed for figure e, to determine if there were significant changes between the treatment and control groups.4. Regulation of expression of genes genes involved in "cell cycle, DNA replication, p53 pathway and cellular response to starvation" Gene Ontology (GO) term.(a) Heatmap diagrams illustrating effects of canagliflozin (10M) (CANA), radiation (5Gy) (RT) or combined treatment in 22RV1 cells were generated from RNAseq analysis data (False Discovery Rate (FDR) p-value < 0.05).Normalized log2 feature counts are used to represent gene expression (with robust z-score).The blue to red scale represents downregulated to upregulated from -1 to 1.
food intake) and adding two more mice in control, RT.
the impact of Canagliflozin (CANA) and Radiotherapy (RT) Combination in vivo.(a) Average animal weight, (b-e) Ex vivo tumor weight (mg) and ex vivo tumor volume (mm 3 ), (b-c) in PC3 xenograft nude mice, and (d-e) 22RV1 xenografts NRG mice, both were measured at the endpoint.(f-g) Representative images of IHC examination of the necrosis and its quantification of tumors from 22RV1 NRG mice.(f) Tumors stained with H&E from 22RV1 NRG mice in the control, canagliflozin, radiation, and combination treatment groups.In the control and treatment groups, arrows on the images point to the necrotic area.Necrosis ratio images were created using ImageJ software.The red colour represents the non-necrotic vital areas, while the white colour represents the necrotic areas.(g) Quantification of necrosis ratio (%) images.To quantify the necrosis % in the 22RV1 xenograft tumor, all slides were stained with H&E and the whole section was quantified using ImageJ software, following the ImageJ user guide for tissue quantification found on the NIH ImageJ website (https://imagej.nih.gov/ij/index.html).We calculated necrotic area as follows: Necrotic Area = Total Area -Viable Tissue Area, and Necrotic Tissue Percentage = Necrotic Area / Total Area.Ordinary one-way anova, Tukey's multiple comparison, n=6/group were used for statistical analysis, asterisks represent of p value, * = p<0.05,and ** = p<0.01.The data shown is Mean ± SEM.
The supplementary blots b contain the original blots for Figure 5a (mTOR Pathway), including AKT (total and phosphorylation at thr308 and Ser473), Raptor (total and phosphorylation at Ser792), mTOR (total and phosphorylation at Ser2448), 4-EPBP1 (total and phosphorylation at Ser65), P70S6K (total and phosphorylation at Thr389), S6 (total and phosphorylation at Ser240/244), and a representative β-actin control blot.The blots on the right side shows the visible molecular weight ladder.Figures may have a different aspect ratio than the image in the manuscript due to the size of the blot, as we are showing the fully uncropped and unedited images here.Supplementary blots e: The supplementary blots 5 contain the original blots for Figure 6a and Figure 6c (Hif-1aplha), and the representative b-actin control blot.The blots on the right side shows the visible molecular weight ladder.Figures may have a different aspect ratio than the image in the manuscript due to the size of the blot, as we are showing the full image.
blots f: The supplementary blots 6 contain the original blots for Figure 7a for PC3 cells cell cycle checkpoints (P21 and P27), and the representative B-actin control blot.The blots on the right side shows the visible molecular weight ladder.Figures may have a different aspect ratio than the image in the manuscript due to the size of the blot, as we are showing the full image.

Clonogenic (Av. mean to control) Clonogenic capacity CANA (µM)
SynergySupplementary Figure1.Effects of canagliflozin (CANA) or radiotherapy (RT) on the cell proliferation and Clonogenic survival.(a-e) The synergy score for cellular proliferation induced by the combined treatment of CANA and RT, as well as their individual effects in prostate cancer cell lines (PrCa cells).Highest Single Agent (HSA) synergy score of four PrCa cell lines with different mutation profiles (LnCaP, 22RV1, PC3, DU145) and one radio-resistant cell line (DU145-RR) analyzed on proliferation assay.HSA mean score of (10 or higher indicates synergism, a score between (+10 and -10) indicates additivity, and a score =< -10 indicates antagonism).(f) Clonogenic assay performed with prostate cancer (PrCa) cell lines treated with single