Journal: Cancer Research

Article Title: Blocking Platelet-Derived Growth Factor-D/Platelet-Derived Growth Factor Receptor β Signaling Inhibits Human Renal Cell Carcinoma Progression in an Orthotopic Mouse Model

doi: 10.1158/0008-5472.can-04-4313

Figure Lengend Snippet: Figure 1. PDGF-D is overexpressed in human renal cell carcinoma samples. Normal kidney tissue in the tissue microarray stains with anti–PDGF-D antibody in collecting tubes (A-D). In clear cell type (E and G), granular cell type (F), and granular and clear cell type (H) tumor tissues, PDGF-D stains strongly and homogenously. The arrow indicates positive staining. I, isotype control staining of the tumor tissues. Antigoat immunoglobulin stains negatively of the tumor tissues. Bar, 50 Am.

Article Snippet: Tissue microarray slides bearing 50 human renal cell carcinoma tumor sections and nine normal human kidney sections were purchased from Imgenex (San Diego, CA).

Techniques: Microarray, Staining, Control

Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: (A) Schematic representation of polyamine biosynthesis. Each enzyme contains a corresponding box plot with TCGA mRNA data in normal (N) and ccRCC (T) samples. Each y-axis represents the normalized RNA-seq reads of the gene; n = 69 normal kidney and 480 tumor samples. *** p< 0.001 (B) 786-O cells grown under hypoxia (0.5% O2) and supplemented with various concentrations of putrescine (Put), andHK-2 cells grown in 1 mM glucose, 1% FBS conditions, supplemented with Put. Cell growth was assessed by WST-1 assays, and error bars represent SEM from 10 replicate wells. Prostate cancer (VCaP) cells were supplemented with indicated concentrations of putrescine, and cell counts performed over the indicated times. Error bars represent SEM from 4 replicate wells. (C) Polyamine metabolite abundance in primary ccRCC combining two independent datasets, n = 158 (Hakimi et al., 2016; Li et al., 2014). Data are displayed as the fold change in metabolite abundance of each tumor, normalized to its control sample. (D) Quantification of immunohistochemistry staining of primary ccRCC tissue microarray,demonstrating reduced ODC protein in ccRCC tumors. *** p <0.001, Welch’s t-test. See image in Figure S6B. (E) Kaplan-Meier survival analysis of OAZ1 expression (from TCGA data) in ccRCC. ‘Low’ versus ‘High’ data are segregated based on the median expression of OAZ1. Mantel-Cox log-rank test was performed. Abbreviations: OAZ, ornithine decarboxylase antizyme; AZIN1, ornithine decarboxylase antizyme inhibitor; ODC1, ornithine decarboxylase1; AMD1, adenosylmethionine decarboxylase1; SRM, spermidine synthase, SMS, spermine synthase. See also Figures S6 and S7.

Article Snippet: Normal and ccRCC kidney tissue sections (from Cooperative Human Tissue Network) and tissue microarray slides (KD802, KD804, KD481, KD482, KD244, KD901, KD601, KD701 from US Biomax) were deparaffinized by baking slides at 50°C for 20 min.

Techniques: RNA Sequencing Assay, Immunohistochemistry, Staining, Microarray, Expressing

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: Fig. 5 ISCA2 inhibition induces ferroptosis. A Effect of co-treatment of ISCA2 siRNA and DMSO or liproxstatin (2.5 μM) on cell viability determined using resazurin in ACHN cells. siRNA transfection was performed for a total of 5 days with liproxstatin (or DMSO) added for the last 24 h. B, C Cell viability assays (resazurin) of 786-0 cells treated with (B) #1 or (C) #25, ± DFO (100 μM), liproxstatin (1 μM) or ZVAD-FMK (20 μM); or (H) #25 ± DFO (100 μM), NAC (1 mM), liproxstatin (1 μM) or ZVAD-FMK (20 μM). Treatments were performed for 24 h. Average IC50 values (µM) are shown in brackets. D Impact of #25 treatment (48 h) on BODIPY 581/591 fluorescence detected by flow cytometry in 786-0 cells. E Impact of 48 hours’ treatment with #25, PT2385 (PT) or 6 hours’ treatment with RSL3 on malondialdehyde (MDA), an indicator of lipid peroxidation in RCC10 cells. F Transmission electron microscopy of RCC10 cells treated with DMSO or #25 for 24 h (2 representative micrographs of each condition). DMSO-treated cells show normal mitochondria whereas #25-treated cells show damaged mitochondria including lost or irregular cristae (white arrows) and irregular matrix with voids (yellow arrows).

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Inhibition, Transfection, Cytometry, Transmission Assay, Electron Microscopy

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: Fig. 7 ISCA2 is decreased in ccRCC and low ISCA2 is associated with poor patient prognosis. A Immunohistochemistry showing representative expression of ISCA2 in normal kidney (top) and in ccRCC (bottom). B Quantitation of ISCA2 H-Score in paired uninvolved and ccRCC cores from 19 patients. ****p < 0.0001 determined via Mann Whitney test. C, D Kaplan–Meier curves showing associations of (C) ISCA2 protein, or (D) ISCA2 transcript levels above (high) or below (low) the median with overall survival using a tumor microarray (TMA; 94 cases) or data from TCGA-KIRC (522 cases) respectively.

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Immunohistochemistry, Expressing, Quantitation Assay, MANN-WHITNEY, Microarray

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: Fig. 8 ISCA2 inhibition inhibits xenograft growth in vivo. A, B Effect of treatment of 786-0 subcutaneous xenografts with vehicle or indicated doses of compound #25 (8 mice/group) administered orally once daily, on tumor volume (A), or mouse body weights (B). **p < 0.01; ***p < 0.001; ****p < 0.0001 from Students’ T-tests of vehicle versus 60 mg/kg treated group. p was not significant (NS) for 30 mg/kg treated group versus vehicle. C Effect of treatment of RENCA subcutaneous xenografts in syngeneic Balb/c mice with vehicle or 60 mg/kg #25 (8 mice/ group) once daily. D Western blots showing HIF-1α and GAPDH with densitometric values normalized to GAPDH below. E MDA content of RENCA tumors from mice treated with vehicle or 60 mg/kg #25. F Proposed model for effects of ISCA2 blockade. Inhibition of ISCA2 using either small molecules or siRNA results in perceived iron deprivation, which induces upregulation of IRP2, TFRC and downregulation of FTH1, that together promote iron/metals accumulation that triggers cell death via ferroptosis, and also drives the later downregulation of IRP1, IRP2 and TFRC. This perceived iron deprivation also drives a shift to the IRE-binding form of IRP1 which inhibits HIF-2α translation. ISCA2 inhibition may also cause other mitochondrial/extramitochondrial defects dependent or independent of perceived iron deprivation that inhibits the translation of HIF-1α through unknown mechanisms. Thus, ISCA2 inhibition depletes both HIF-1α and HIF-2α and promotes cell death via ferroptosis.

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Inhibition, In Vivo, Western Blot, Binding Assay