Activation of PPARγ and inhibition of cell proliferation reduces key proteins associated with the basal subtype of bladder cancer in As3+-transformed UROtsa cells

Abstract

Environmental exposure to arsenite (As 3+) has a strong association with the development of human urothelial cancer (UC) and is the 5 th most common cancer in men and the 12 th most common cancer in women. Muscle invasive urothelial cancer (MIUC) are grouped into basal or luminal molecular subtypes based on their gene expression profile. The basal subtype is more aggressive and can be associated with squamous differentiation, characterized by high expression of keratins (KRT1, 5, 6, 14, and 16) and epidermal growth factor receptor (EGFR) within the tumors. The luminal subtype is less aggressive and is predominately characterized by elevated gene expression of peroxisome proliferator-activated receptorgamma (PPARγ) and forkhead box protein A1 (FOXA1). We have previously shown that As 3+-transformed urothelial cells (As-T) exhibit a basal subtype of UC expressing genes associated with squamous differentiation. We hypothesized that the molecular subtype of the As-T cells could be altered by inducing the expression of PPARγ and/or inhibiting the proliferation of the cells. Non-transformed and As-T cells were treated with Troglitazone (TG, PPARG agonist, 10 μM), PD153035 (PD, an EGFR inhibitor, 1 μM) or a combination of TG and PD for 3 days. The results obtained demonstrate that treatment of the As-T cells with TG upregulated the expression of PPARγ and FOXA1 whereas treatment with PD decreased the expression of some of the basal keratins. However, a combined treatment of TG and PD resulted in a consistent decrease of several proteins associated with the basal subtype of bladder cancers (KRT1, KRT14, KRT16, P63, and TFAP2A). Our data suggests that activation of PPARγ while inhibiting cell proliferation facilitates the regulation of genes involved in maintaining the luminal subtype of UC. In vivo animal studies are needed to address the efficacy of using PPARγ agonists and/or proliferation inhibitors to reduce tumor grade/stage of MIUC.

Chronic Arsenic Exposure Upregulates the Expression of Basal Transcriptional Factors and Increases Invasiveness of the Non-Muscle Invasive Papillary Bladder Cancer Line RT4

“…Droplet digital PCR yields ~20,000 droplets/20 µL sample where each droplet is an independent PCR reaction of equal and defined volume. Therefore, ddPCR technology increases the signal-to-noise ratio while providing precise absolute quantitation without the need for a standard curve or reference [ 69 ]. The final data is reported as copies/µL.…”

Pevonedistat Inhibits SOX2 Expression and Sphere Formation but Also Drives the Induction of Terminal Differentiation Markers and Apoptosis within Arsenite-Transformed Urothelial Cells

“…Gene expression analysis of the treated cells showed an increase in expression of genes associated with squamous/terminal differentiation such as KRT6, KRT16, KRT14, IVL, DSC2, GRHL1 and 2, SPRR1A, 2A and 3. The role of KRTs in the differentiation of epithelial cells is well established and in our previous studies, we showed that transformation of the UROtsa cells with arsenite induces the expression of basal KRTs 5, 6, 14 and 16 and the expression is associated with areas of squamous differentiation in tumor hetero-transplants [24,25,28,42,43]. Other markers such as IVL and DSC2 are also expressed in UCs with squamous differentiation [26,30,44,45].…”