Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Heatmap generated with qPCR data of DTFs and various genes related to three biological alterations. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black, as generated by Cluster 3.0. b. IHC of proteins related to normal renal function (KNG1, AQP2, SCNN1B). c. Immune function (TLR2, CXCR4). d. Metabolic function (ENO2, CYP2J2,ALDOB). This pattern of expression is in accord with the microarray findings. Sum scores are shown with n as indicated. *p<0.01 when comparing ccRCC to normal match.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Generated, Expressing, Microarray

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. A microarray heatmap showing significant downregulation of four developmental transcriptional factors in ccRCC and a table showing fold changes of DTFs and their known renal developmental function. b. IHC validation of decreased expression of TFAP2B, DMRT2, and TFCP2L1. Sum scores are shown with n , as indicated. *p<0.01 when comparing ccRCC to normal match. c. Microarray heatmap showing downregulation of TFCP2L1 and its regulated genes in ccRCC. S–Stage, N–normal, T–tumor. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in yellow, as generated by Genetree.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Microarray, Biomarker Discovery, Expressing, Generated

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Heatmap showing adipogenic gene expression signature in ccRCC. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black. b. IHC showing lipid-laden clear cell morphology of ccRCC, increased expression of ADFP, and decreased expression of GATA2 in ccRCC. Sum scores are shown with n , as indicated. *p<0.01 when comparing ccRCC to normal match.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Cellular differentiation experiments showing that KIJ-308 and KIJ-265 ccRCC cells are capable of adipogenic differentiation and become lipid-laden in adipogenic media, as indicated by Oil Red ‘O’ staining. Normal patient-matched cells were unable to differentiate. b. A498 ccRCC cells are also capable of adipogenic differentiation, as indicated by Oil Red ‘O’ staining, unlike normal renal canine MDCK cells. c. Under adipogenic media conditions, ccRCC also produces glycogen, as shown by a PASH stain.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Cell Differentiation, Staining

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Cellular differentiation experiments showing that KIJ-308 and KIJ-265 ccRCC cells are capable of osteogenic differentiation in osteogenic media by developing calcium deposits, as shown by Alizarin Red stain. Normal patient-matched cells were unable to differentiate. b. A498 ccRCC cells are also capable of osteogenic differentiation, as shown by Alizarin Red stain, unlike normal renal canine MDCK cells.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Cell Differentiation, Staining

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. A heatmap showing the increased expression of some markers associated with EMT. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black. b. IHC validation of two known markers of EMT: vimentin and N-cadherin. Sum scores are shown with n , as indicated. *p<0.01 when comparing ccRCC to normal match.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Expressing, Biomarker Discovery

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: During normal renal development, mesenchymal stem cells undergo mesenchymal epithelial transition (MET) to develop into normal renal epithelial cells (NREs). In renal carcinogenesis, NREs undergo de-differentiation and epithelial mesenchymal transition (EMT), followed by adipogenic differentiation to develop into ccRCC.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques:

Journal: Cancer discovery

Article Title: Cholesterol Auxotrophy as a Targetable Vulnerability in Clear Cell Renal Cell Carcinoma

doi: 10.1158/2159-8290.CD-21-0211

Figure Lengend Snippet: A, Simplified schematic of the mevalonate pathway. Mammalian cells maintain cholesterol homeostasis through direct synthesis, which can be inhibited by statins, or import from the extracellular environment. B, Gene set enrichment analysis (GSEA) of RNAseq data provided by the TCGA KIRC project indicating that genes belonging to the “cholesterol” and “mevalonate” pathways have lower expression in ccRCC tumors compared to normal kidney tissue. Generated metabolic gene sets were ranked based on normalized enrichment score changes in ccRCC compared to normal tissue. C, Metabolic gene set analysis of RNAseq data provided by the TCGA KIRC project. 538 ccRCC tumor and 72 adjacent normal tissues were included. 2,752 genes encoding all human metabolic enzymes and transporters were classified according to KEGG. Generated metabolic gene sets were ranked based on their log2 median fold expression changes in ccRCC compared to normal tissue. D, TCGA dataset analysis shows that expression of genes involved in the “mevalonate pathway” is significantly downregulated in ccRCC tumors vs. normal tissue. HMGCR , 3-hydroxy-3-methylglutaryl-coenzyme A reductase; FDFT1 , squalene synthase; DHCR24 , delta 24-sterol reductase; SQLE , squalene monooxygenase; LSS , lanosterol synthase. E, Real-time qPCR analysis performed on 12 tumor tissues and their normal counterparts, indicating mevalonate pathway gene expression is decreased in tumors compared to normal tissues. F, Real-time qPCR analysis performed on immortalized proximal tubular renal epithelial cells (HK-2) and two ccRCC cell lines, A498 and 786-O, evaluating expression of HMGCR, LSS, SQLE, DHCR24 and FDFT1 . G, Alteration frequency of HMGCR gene in several kidney cancer genomic datasets using cBio Cancer genomic portal. IRC, Nat Genet 2012; DFCI, Science 2019; TCGA pub, firehose legacy; TCGA, Nature 2013; Utokyo, Nat Genet 2013, TCGA PanCancer Atlas; BGI, Nat Genet 2012. H, Metabolomics analysis of squalene in 114 normal kidney tissues and 68 ccRCC tumors. I, J and K, A498, 786-O and HK-2 cell proliferation assays showing insensitivity of ccRCC cell lines to 72h of atorvastatin (ATOR) treatment (5μM). (All experiments were performed in at least triplicates and statistical analysis was applied with *=P<0.05, **=P<0.01, ***=<0.001, n.s=non-significant).

Article Snippet: Human ccRCC cell lines (786-O, A498), Caki-2, hTERT immortalized RPTEC and 293T cells were obtained from the American Type Culture Collection (ATCC) and were cultured in DMEM (ThermoFisher Scientific, cat. 11965092) supplemented with 10% FBS (Gemini Bio-Products, cat. 900–108) or 10% delipidated FBS, referenced as DLPS, (Gemini Bio-Products, cat. 900–123) supplemented with either cholesterol (10μg/ml) or HDL (100μg/ml).

Techniques: RNA sequencing, Expressing, Generated, Gene Expression

Journal: Cancer discovery

Article Title: Cholesterol Auxotrophy as a Targetable Vulnerability in Clear Cell Renal Cell Carcinoma

doi: 10.1158/2159-8290.CD-21-0211

Figure Lengend Snippet: A, Proliferation assay performed on A498 cells grown in media supplemented with 10% FBS, 10% DLPS, or 10% DLPS and cholesterol (CHOL) (10μg/mL). B, Representative photographs of A498 cells grown in media supplemented with 10% FBS, 10% DLPS, or 10% DLPS and cholesterol (CHOL) (10μg/mL) for 96h. Magnification (x100). C, Annexin-V/PI staining and flow cytometry analysis performed on A498 cells after 96h of incubation in 10% FBS, 10% DLPS, or 10% DLPS and cholesterol (CHOL) (10μg/mL) media (left). Representative annexin-V/PI flow plots of A498 cells after 96h of incubation in 10% FBS, 10% DLPS, or 10% DLPS and cholesterol (CHOL) (10μg/mL) media (right). D, Liquid chromatography-tandem mass spectrometry (LC/MS) analysis assessing various cholesterol ester species in A498 cells grown in 10% FBS or 10% DLPS media. E, Cholesterol content of A498 cells grown in 10% FBS or 10% DLPS. F, Proliferation assay performed on HK2 cells grown in media supplemented with 10% FBS, 10% DLPS, or 10% DLPS and cholesterol (CHOL) (10μg/mL). G, Mendelian Randomization analysis using GWAS summary statistics was performed and the effect of circulating metabolites on RCC odds estimated, revealing a significant association between HDL particles and RCC risk. Estimates reflect the OR (95% CI) for RCC per SD increase in circulating metabolite concentration. (red = significant) H, Tumor growth curves from A498 cells subcutaneously implanted in nude mice fed a no cholesterol (0%) or a high cholesterol (2%) diet. Tumor volume was assessed at the indicated timepoints using caliper measurements (n=5 mice per group, 2 tumors per mouse). I, Tumor weight from A498 cells subcutaneously implanted in nude mice, fed a no cholesterol (0%) or a high cholesterol (2%) diet, 55 days after implantation. J, Representative photographs of A498 tumors grown in mice fed a no cholesterol (0%) or a high cholesterol (2%) diet at day 55 after implantation. K, Analysis of serum HDL from nude mice subcutaneously implanted with A498 cells and fed a no cholesterol (0%) or a high cholesterol (2%) diet for 70 days. (All experiments were performed in at least triplicates and statistical analysis was applied with *=P<0.05, **=P<0.01, ***=<0.001, n.s=non-significant).

Article Snippet: Human ccRCC cell lines (786-O, A498), Caki-2, hTERT immortalized RPTEC and 293T cells were obtained from the American Type Culture Collection (ATCC) and were cultured in DMEM (ThermoFisher Scientific, cat. 11965092) supplemented with 10% FBS (Gemini Bio-Products, cat. 900–108) or 10% delipidated FBS, referenced as DLPS, (Gemini Bio-Products, cat. 900–123) supplemented with either cholesterol (10μg/ml) or HDL (100μg/ml).

Techniques: Proliferation Assay, Staining, Flow Cytometry, Incubation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

Journal: Cancer discovery

Article Title: Cholesterol Auxotrophy as a Targetable Vulnerability in Clear Cell Renal Cell Carcinoma

doi: 10.1158/2159-8290.CD-21-0211

Figure Lengend Snippet: A, HDL uptake assay using shSCR and sh SCARB1 A498 cells treated with doxycycline (DOX) (96h, 1μg/mL) showing reduced HDL uptake when SCARB1 is inhibited. B, Proliferation assay performed on shSCR and sh SCARB1 A498 cells grown in media with 10%FBS and supplemented with doxycycline (DOX) to induce SCARB1 knockdown. C, Real-time qPCR analysis of S CARB1 mRNA level in A498 cells after shSCR or sh SCARB1 lentiviral infection, puromycin selection (48h, 2μg/mL) and doxycycline (DOX) treatment for 4 days (1μg/mL). D, SCARB1 protein expression assessed by immunoblots in shSCR and sh SCARB1 A498 cells treated or not with doxycycline (DOX) (4 days, 1μg/mL). HSP90 was used as the loading control. E, Annexin-V/PI staining and flow cytometry analysis performed on shSCR and shSCARB1 A498 cells after 96h of doxycycline (DOX) treatment (1μg/mL). F, Cell cycle analysis for shSCR and sh SCARB1 A498 cells after 96h of doxycycline (DOX) treatment (1μg/mL) showing increased cell cycle arrest in G1 when SCARB1 is inhibited. G, Tumor growth curves from doxycycline-inducible shSCR and sh SCARB1 A498 cells subcutaneously implanted in nude mice fed a diet containing doxycycline (DOX) (200mg/kg) when tumors reached a volume of ~100mm 3 . Tumor volume was assessed at the indicated timepoints using caliper measurements (n=10 mice per group). H, Tumor weight from shSCR and sh SCARB1 A498 cells subcutaneously implanted in nude mice, fed a diet containing doxycycline (DOX) (200mg/kg), 48 days after implantation. I, Representative photographs of shSCR and sh SCARB1 A498 tumors grown in nude mice fed a diet containing doxycycline (DOX) (200mg/kg), 48 days after implantation. J, Proliferation assay performed on shSCR and sh SCARB1 A498 cells grown in 10% DLPS medium with or without HDL (100μg/mL), and supplemented with doxycycline (DOX) to induce SCARB1 knockdown. K, Annexin-V/PI staining and flow cytometry analysis performed on shSCR and shSCARB1 A498 cells treated with doxycycline (DOX) and grown in 10% FBS or 10% DLPS media supplemented with or without HDL (100μg/mL) for 4 days. L, Cell cycle analysis for shSCR and sh SCARB1 A498 cells treated with doxycycline (DOX) (1μg/mL) and grown in 10% FBS or 10% DLPS media supplemented with or without HDL (100μg/mL). Percentage of cells in G1, S and G2-M phases is displayed. (All experiments were performed in at least triplicates and statistical analysis was applied with *=P<0.05, **=P<0.01, ***=<0.001, n.s=non-significant).

Article Snippet: Human ccRCC cell lines (786-O, A498), Caki-2, hTERT immortalized RPTEC and 293T cells were obtained from the American Type Culture Collection (ATCC) and were cultured in DMEM (ThermoFisher Scientific, cat. 11965092) supplemented with 10% FBS (Gemini Bio-Products, cat. 900–108) or 10% delipidated FBS, referenced as DLPS, (Gemini Bio-Products, cat. 900–123) supplemented with either cholesterol (10μg/ml) or HDL (100μg/ml).

Techniques: In Vitro, In Vivo, Proliferation Assay, Knockdown, Infection, Selection, Expressing, Western Blot, Control, Staining, Flow Cytometry, Cell Cycle Assay

Journal: Cancer discovery

Article Title: Cholesterol Auxotrophy as a Targetable Vulnerability in Clear Cell Renal Cell Carcinoma

doi: 10.1158/2159-8290.CD-21-0211

Figure Lengend Snippet: A, Proliferation assay performed on A498 cells grown in media with 10%FBS and treated with the SCARB1 inhibitor, BLT-1 (5μM), or vehicle control (DMSO). B, Annexin-V/PI staining and flow cytometry analysis performed on A498 cells after 96h of BLT-1 treatment (5μM). C, Cell cycle analysis for A498 cells after 96h of BLT-1 treatment (5μM) showing increased cell cycle arrest in G1 when SCARB1 is inhibited. D, HDL uptake assay using BLT-1-treated (96h, 5μM) A498 cells showing reduced HDL uptake when SCARB1 is inhibited. E, Proliferation assay performed on A498 cells grown in media with 10%FBS or 10% DLPS media supplemented with or without HDL (100μg/mL) and treated with BLT-1 (5μM) or vehicle control (DMSO). F, Annexin-V/PI staining and flow cytometry analysis performed on A498 cells after 96h of DMSO or BLT-1 treatment (5μM) and grown in 10% FBS or 10% DLPS media supplemented with or without HDL (100μg/mL). G, Representative photographs of A498 cells grown in media supplemented with 10% FBS, 10% DLPS, or 10% DLPS and HDL (100μg/mL) and treated with BLT-1 (5μM) or DMSO for 96h. Magnification (100X). H, Cell cycle analysis for A498 cells after 96h of BLT-1 (5μM) or DMSO treatment and grown in 10% FBS or 10% DLPS media supplemented with or without HDL (100μg/mL). Percentage of cells in G1, S and G2-M phases is displayed. I, Tumor growth curves from A498 cells subcutaneously implanted in nude mice treated or not with BLT-1 (50 mg/kg) by oral gavage daily for 30 days after tumor volume reached ~100mm 3 . Tumor volume was assessed at the indicated timepoints using caliper measurements (n=5 mice per group). J, Tumor weight from A498 cells subcutaneously implanted in nude mice treated or not with BLT-1 (50 mg/kg) by oral gavage daily for 30 days. K, Representative photographs of A498 tumors grown in nude mice treated or not with BLT-1 (50 mg/kg) by oral gavage daily for 30 days. L, Tumor volume fold change over the course of 30 day-vehicle or 30 day-BLT-1 (50 mg/kg) treatments. M, Analysis of serum HDL from nude mice subcutaneously implanted with A498 cells and treated or not with BLT-1 (50 mg/kg) by oral gavage daily for 30 days. N, Body weight average of mice treated by oral gavage daily for 30 days with either vehicle control or BLT-1 (50 mg/kg). (All experiments were performed in at least triplicates and statistical analysis was applied with *=P<0.05, **=P<0.01, ***=<0.001, n.s=non-significant)

Article Snippet: Human ccRCC cell lines (786-O, A498), Caki-2, hTERT immortalized RPTEC and 293T cells were obtained from the American Type Culture Collection (ATCC) and were cultured in DMEM (ThermoFisher Scientific, cat. 11965092) supplemented with 10% FBS (Gemini Bio-Products, cat. 900–108) or 10% delipidated FBS, referenced as DLPS, (Gemini Bio-Products, cat. 900–123) supplemented with either cholesterol (10μg/ml) or HDL (100μg/ml).

Techniques: In Vitro, In Vivo, Proliferation Assay, Control, Staining, Flow Cytometry, Cell Cycle Assay

Journal: Cancer discovery

Article Title: Cholesterol Auxotrophy as a Targetable Vulnerability in Clear Cell Renal Cell Carcinoma

doi: 10.1158/2159-8290.CD-21-0211

Figure Lengend Snippet: A, ROS levels assessed by flow cytometry measuring DCFDA fluorescence in A498 cells cultured in 10% FBS or 10% DLPS media for 72h. Representative plots (left) and mean fluorescence intensity quantifications are shown (right). B, ROS levels assessed by flow cytometry measuring DCFDA fluorescence in A498 cells cultured in 10% FBS or 10% DLPS media supplemented with or without HDL (100μg/mL) for 72h. Representative plots (left) and mean fluorescence intensity quantifications are shown (right). C, ROS levels assessed by flow cytometry measuring DCFDA fluorescence in A498 cells cultured in 10% FBS or 10% DLPS media supplemented with or without α-tocopherol (0.5mM) for 72h. Representative plots (left) and mean fluorescence intensity quantifications are shown (right). D and E, ROS levels assessed by flow cytometry measuring DCFDA fluorescence in shSCR and sh SCARB1 A498 cells treated with doxycycline (DOX) (1μg/mL) and grown in 10% FBS or 10% DLPS media for 72h. F, ROS levels assessed by flow cytometry measuring DCFDA fluorescence in shSCR and sh SCARB1 A498 cells treated with doxycycline (DOX) (1μg/mL) and grown in 10% FBS or 10% DLPS media supplemented with or without HDL (100μg/mL) for 72h. G, Schematic representing SCARB1, the HDL receptor, as a central receptor in ccRCC cells for cholesterol import compensated by diminished biosynthetic mevalonate pathway. High intracellular cholesterol levels allow ccRCC cells to maintain PI3K/AKT pathway activation, control ROS homeostasis and store cholesterol surplus in lipid droplets (CE: Cholesterol ester, TG: Triglycerides). (All experiments were performed in at least triplicates and statistical analysis was applied with *=P<0.05, **=P<0.01, ***=<0.001, n.s=non-significant).

Article Snippet: Human ccRCC cell lines (786-O, A498), Caki-2, hTERT immortalized RPTEC and 293T cells were obtained from the American Type Culture Collection (ATCC) and were cultured in DMEM (ThermoFisher Scientific, cat. 11965092) supplemented with 10% FBS (Gemini Bio-Products, cat. 900–108) or 10% delipidated FBS, referenced as DLPS, (Gemini Bio-Products, cat. 900–123) supplemented with either cholesterol (10μg/ml) or HDL (100μg/ml).

Techniques: Flow Cytometry, Fluorescence, Cell Culture, Activation Assay, Control