Pharmacological targeting of polyamine and hypusine biosynthesis reduces tumour activity of endometrial cancer

Endometrial cancer (EC) is a common and deadly cancer in women and novel therapeutic approaches are urgently needed. Polyamines (putrescine, spermidine, spermine) are critical for mammalian cell proliferation and MYC coordinately regulates polyamine metabolism through ornithine decarboxylase (ODC). ODC is a MYC target gene and rate-limiting enzyme of polyamine biosynthesis and the FDA-approved anti-protozoan drug α-difluoromethylornithine (DFMO) inhibits ODC activity and induces polyamine depletion that leads to tumour growth arrest. Spermidine is required for the hypusine-dependent activation of eukaryotic translation initiation factors 5A1 (eIF5A1) and 5A2 (eIF5A2) and connects the MYC/ODC-induced deregulation of spermidine to eIF5A1/2 protein translation, which is increased during cancer cell proliferation. We show that eIF5A1 is significantly upregulated in EC cells compared to control cells (p=.000038) and that combined pharmacological targeting of ODC and eIF5A hypusination with cytostatic drugs DFMO and N1-guanyl-1,7-diaminoheptane (GC7), respectively, reduces eIF5A1 activation and synergistically induces apoptosis in EC cells. In vivo, DFMO/GC7 suppressed xenografted EC tumour growth in mice more potently than each drug alone compared to control (p=.002) and decreased putrescine (p=.045) and spermidine levels in tumour tissues. Our data suggest DFMO and GC7 combination therapy may be useful in the treatment or prevention of EC.</p

Supplementary Table S2 from Prediction of Lymph Node Metastasis in Patients with Endometrioid Endometrial Cancer Using Expression Microarray

EASE data</p

Fig. S4 from Nuclear PTEN Localization Contributes to DNA Damage Response in Endometrial Adenocarcinoma and Could Have a Diagnostic Benefit for Therapeutic Management of the Disease

Supplementary Figure 4. Effects of Zeocin treatment on levels of �H2AX and accumulation of cells in G2/M phase in PTEN-null Ishikawa cells after the overexpression of four different PTEN plasmids. Cells were transfected with plasmids for 24 h, treated with Zeocin for 16 h, then assayed. A-P Levles of �H2AX as determined by co-immunocytostaining with anti-PTEN antibody followed by Alexa Fluor 488 conjugated secondary antibody (green) and anti-phospho H2AX (Ser 139) antibody followed by Alexa Fluor 546 conjugated secondary antibody (red). Nuclei were stained with DAPI (blue). A-D, pEGFP-EV. E-H, PTEN-WT. I-L, PTEN-NLS. M-P, PTEN-NES. Scale bar; 200 μm. Q, Comparison of % �H2AX+ cells as determined by flow cytometry analysis in PTEN transfected cells with or without Zeocin treatment. R, Comparison of the percentage of cells in G2/M as determined by flow cytometry analysis in PTEN transfected cells with or without Zeocin treatment. Statistical analysis performed was paired T-test for each plasmid P<0.05</p

Fig. S1 from Nuclear PTEN Localization Contributes to DNA Damage Response in Endometrial Adenocarcinoma and Could Have a Diagnostic Benefit for Therapeutic Management of the Disease

Supplementary Figure 1. A, Oncoprints of PTEN and TP53 in four molecular subtypes of EndoCA based on TCGA. B, Mutational landscape of PTEN in EndoCA. DSPc; dual specificity phosphatase domain, C2; cell membrane binding domain. Results were generated using C-BioPortal website algorithms (48, 49).</p

Fig. S3 from Nuclear PTEN Localization Contributes to DNA Damage Response in Endometrial Adenocarcinoma and Could Have a Diagnostic Benefit for Therapeutic Management of the Disease

Supplementary Figure 3. PTEN localization in cytoplasm and nuclei of epithelial cells in ANT and EndoCA by grade. PTEN immunostaining was performed on adjacent normal tissues (ANT) and matched primary endometrial tumors (EndoCA). Quantification of staining intensities was assessed by histo-scoring (H-score) in cytoplasm and nuclei of epithelial cells. Average intensities are expressed as median with 95% CI, and statistical significance are determined by Wilcoxon matched-pairs signed rank test. A, n=10 matched ANT and Grade1 tumors. B, n=9 matched ANT and Grade 2 tumors. C, n=10 matched ANT and Grade 3 tumors.</p

Fig. S2 from Nuclear PTEN Localization Contributes to DNA Damage Response in Endometrial Adenocarcinoma and Could Have a Diagnostic Benefit for Therapeutic Management of the Disease

Supplementary Figure 2. A and B, PTEN immunostaining in adjacent normal tissue (ANT) and EndoCA, respectively. C and D, Immunostaining of phospho-AKT (Ser473) in ANT and EndoCA, respectively. E, TCGA analysis of PTEN expression in human endometrial tumors by high throughput reverse phase protein lysate microarray (RPPA). F-H, PTEN immunostaining of human normal endometrial tissue with the anti-human PTEN antibody generated from three different clones. EG; endometrial gland. Str; stroma.</p

Supplementary Figures S1 & S2 from Prediction of Lymph Node Metastasis in Patients with Endometrioid Endometrial Cancer Using Expression Microarray

Supplementary Figures S1 & S2 from Prediction of Lymph Node Metastasis in Patients with Endometrioid Endometrial Cancer Using Expression Microarra

Supplementary Table S1 from Prediction of Lymph Node Metastasis in Patients with Endometrioid Endometrial Cancer Using Expression Microarray

Class comparison data</p

TP53/ARID1A/PIK3CA mutant mouse survival and marker staining.

a, Survival curves for LtfCre0/+; (Gt)R26Pik3ca*H1047R; Trp53fl/fl mice with and without an additional Arid1afl/fl allele (TP53/PIK3CA mutant and TP53/ARID1A/PIK3CA mutant mice). Statistic is Cox log-rank test. b, Representative IHC marker analysis in TP53/ARID1A/PIK3CA mutant mice. Left, phospho-S6; right, ARID1A staining. Arrowheads denote mutant endometrial epithelia. (TIF)</p

Enrichment of gene expression alterations at TP53 core transcriptional program genes in mouse models.

Proportion of genes significantly differentially expressed (DE) in TP53 core transcriptional program gene mouse orthologs compared to all expressed genes, for (top) ARID1A/PIK3CA mutant and (bottom) TP53/PIK3CA mutant endometrial epithelia compared to cells from control mice. Statistic is hypergeometric enrichment test. (TIF)</p