Figure 5 from ANGPTL4 Suppresses Clear Cell Renal Cell Carcinoma via Inhibition of Lysosomal Acid Lipase

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posted on 2024-08-27, 10:40 authored by Zeng Jin, Umasankar De, Tanzia Islam Tithi, Jeremy Kleberg, Akhila Nataraj, Elena Jolley, Madison E. Carelock, Brandon S. Davies, Weizhou Zhang, Ryan Kolb

nANGPTL4 inhibits LAL to suppress ccRCC colony formation. A–C, Total intracellular lipids in the indicated CAKi-1 or 786O cells were stained with BODIPY 493/503 and the median fluorescence intensity (MFI) was determined. A, Representative histogram is presented for BODIPY staining of CAKi WT (gray) and A4KO (red) cells. B and C, Graph depicts the average BODIPY 493/503 MFI of the indicated CAKi-1 (B) and 786O (C) cells ± SD. Welch’s t test was used to determine significance. D, CAKi-1 WT and A4KO cells were incubated with BODIPY FL C12 and analyzed by flow cytometry to measure lipid uptake. Left: representative histogram showing BODIPY FL C12 fluorescent intensity of WT (gray) and A4KO (red) cells. Right: graph depicts the average BODIPY FL C12 MFI ± SD in the indicated 786O cells. Welch’s t test was done to determine significance. E, LAL activity was determined by incubating the indicated cells with lysolive-green and measuring the fluorescence Intensity by flow cytometry. Left: representative histogram depicting intensity of lysolive-green fluorescence in CAKi-1 WT (gray) and A4KO (red) cells. Right: graph depicts the average lysolive-green MFI as a fold change compared to WT ± SD. Welch’s t test was done to determine significance. F, Graph depicts the average lysolive-green MFI as a fold change compared to WT ± SD. Welch’s t test was done to determine significance. G, CAKi-1 A4KO cells were mock transfected or transfected with full length (FL) or N-terminus (N-term) ANGPTl4 and incubated with lysolive-green. Left representative histrogram depicting lysolive-green fluorescence intensity in the indicated cell lines. The peak fluorescence intensity in mock transfected cells is indicated by the black line. Right: Average lysolive-green MFI as a fold changed compared to mock transfected ± SD. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance. H, Graph depicts the average number of colonies/well of the indicated CAKi-1 cells grown in the presence or absence of the LAL inhibitor lalistat 1. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance. I, Graph depicts the average number of colonies/well of the indicated 786O cells grown in the presence or absence of the LAL inhibitor lalistat 1. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance. J, CAKi-1 A4KO cells were transfected with siRNA targeting LIPA (gene that encodes LAL) or nontargeting control siRNA (−). Cells were plated in non-adherent conditions, cultured for 72 hours and the number of colonies was counted. Top: western blot for LAL at the indicated time point post transfection. GAPDH blot is used as a loading control. Bottom: graph depicts the average number of colonies per well ± SD. Welch’s t test was done to determine significance. K, CAKi-1 A4KO cells were transfected with a lentivirus plasmid containing CRISPR-Cas9 and one of the three guide RNAs targeting LIPA. After selection total lysates were collected and a Western blot for LAL and GAPDH, as a loading control, was done. Arrow indicates LAL band. L, The indicated CAKi-1 cells (A4KO LIPA KO cells are g2 cells from K) were cultured in nonadherent conditions for 72 hours and the number of colonies was counted. Graph depicts the number of colonies per well ± SD. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance. M, The indicated CAKi-1 cells were cultured for 72 hours, stained with annexin V-FITC and PI and analyzed by flow cytometry. Graph depicts the % of FITC-positive cells as a percentage of single cells ± SD. One-way ANOVA with Dunnett’s test to correct for multiple comparisons was done to determine significance.

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ARTICLE ABSTRACT

Renal cell carcinoma (RCC), the most common form of kidney cancer, is a heterogeneous disease with clear cell RCC (ccRCC) being the most prevalent and aggressive subtype. While most ccRCC tumors have elevated expression of angiopoietin-like4 (ANGPTL4), in our study we identified a significant subset of patients whose cancers show no increase in ANGPTL4 expression. These patients have a worse prognosis compared to the patients with high expression of ANGPTL4. These ANGPTL4-low cancers are characterized by the increased frequency of wild-type Von Hippel-Lindau(WT VHL), a gene that is commonly mutated in ccRCC, and an enrichment for genes associated with lipid metabolism. Using RCC tumor models with WT VHL, we demonstrate that ANGPTL4 behaves as a tumor suppressor. The loss of ANGPTL4 in ccRCC cell lines results in increased tumor growth and colony formation in a lysosomal acid lipase (LAL)-dependent manner, a phenotype rescued by the expression of N-terminus ANGPTL4. At the mechanistic level, the loss of ANGPTL4 increases LAL activity in ccRCC cells. These data suggest that ANGPTL4 enacts its tumor-suppressive effects in ccRCC by regulating LAL activity. Importantly, the identified patient cohort with low ANGPTL4 expression may exhibit increased reliance on lipid metabolism, which can be a point of target for future therapy. Our data indicate angiopoietin-like 4 (ANGPTL4) acts as a tumor suppressor in clear cell renal cell carcinoma via regulating lipid metabolism and identifies a cohort of patients with lower expression of ANGPTL4 that are correlated with shorter survival.