SOX2 Regulates Growth, Expression of Basal/Luminal Markers, and Chemotherapy Response in Urothelial Carcinoma

Abstract

Urothelial carcinoma (UC) is a common genitourinary malignancy. Smoking, exposure to arsenic in drinking water, and age can increase the risk of developing UC. Neoadjuvant cisplatin-based chemotherapy prior to radical cystectomy is the standard treatment for the muscle invasive form of UC (MIUC). Tumors of the basal/squamous (Ba/Sq) subtype of MIUC are aggressive, express basal keratins (KRT5, 6, and 14), are associated with squamous differentiation (SD), and frequently develop chemotherapy resistance. The SOX2 transcription factor is a marker of UC stem cells, and its expression is associated with poor overall and disease-free survival. We hypothesized that the attenuation of SOX2 would reduce the expression of basal keratins and increase the chemotherapy response in human UC cells. For this study, we performed lentiviral knockdown (KD) of SOX2 expression in two separate arsenite (As³⁺)-transformed UROtsa (As_I, As_II), 5637, and RT4 cells. Cellular growth and colony-forming ability was inhibited in all UC cell lines after SOX2 KD. We demonstrate that SOX2 KD in the UC cells of the Ba/Sq subtype (As_I, As_II, 5637) decreased the expression of stem-associated proteins, oncoproteins, and basal keratins. Additionally, there was an induction of several luminal markers and enhanced cisplatin sensitivity following the repression of SOX2. Lastly, proteomics revealed reductions in lipid-, cholesterol-, and interferon-signaling pathways after SOX2 KD. This study provides a better understanding of the regulation of key genes responsible for defining the Ba/Sq subtype of UC and demonstrates that the inhibition of SOX2 improves chemotherapy response in UC.

Keywords: SOX2; arsenite; basal; cisplatin; squamous differentiation; urothelial carcinoma.

Figure 1

Knockdown of SOX2 in UC cells. (A) SOX2 gene and protein expression after SOX2 knockdown (KD) in (A) UROtsa As_I cells, (B) UROtsa As_II cells, (C) 5637 cells, and (D) RT4 cells. All data are plotted as fold-change compared to the scramble control for each cell line. Gene expression was normalized to the 18S housekeeping gene, and protein expression was normalized to the Jess system control. Gene data represent n = 3 and protein data represent n = 2. The values reported are mean ± SEM. A t-test was performed, and asterisks indicate significant differences from the control (** p < 0.01, *** p < 0.001, **** p < 0.0001). KD: knockdown; SOX2: transcription factor SOX2.

Figure 2

Morphology and growth after SOX2 knockdown in UC cells. (A) Brightfield microscope images (10× magnification, scale bar = 500 µm) of UC cells after SOX2 knockdown. (B,D,F,H) Luminescence intensity plotted over 24, 48, 72, 96, 120, and 144 h for control and SOX2-knockdown As_I, As_II, 5637, and RT4 cells. A total of 2500 cells were initially seeded for growth and measured according to their luminescence on a 96-well plate. (C,E,G,I) Calculated doubling times obtained from the luminescence intensity curve. Luminescence growth represents n = 8 and the mean intensity ± SEM is plotted. A t-test was performed, and asterisks indicate significant differences from the control (**** p < 0.0001). KD: knockdown; SOX2: transcription factor SOX2.

Figure 3

Effect of SOX2 knockdown on colony formation in UC cells. (A) Quantification of colonies (expressed as percentage of control). (B) Image of crystal-violet-stained colonies in the UC cells. Initially, 200 cells/well were seeded in a 6-well plate and cultured for 14 days before staining the resulting colonies. The colony counts for control and SOX2-knockdown cells for each cell line were performed with n = 3. The values reported are mean ± SEM. A t-test was performed, and asterisks indicate significant differences from the control (* p < 0.05, ** p < 0.01, *** p < 0.001). KD: knockdown; SOX2: transcription factor SOX2.

Figure 4

SOX2 regulates the expression of stem-associated proteins and oncoproteins in UC cells. Quantification and Western blot images of proteins in (A) UROtsa As_I cells, (B) UROtsa As_II cells, (C) 5637 cells, and (D) RT4 cells after SOX2 knockdown. All data are plotted as fold-change compared to the scramble control for each cell line. Protein expression was normalized to the Jess system control. Protein data represent n = 2. The values reported are mean ± SEM. A t-test was performed, and asterisks indicate significant differences from the control (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). KD: knockdown; SOX2: transcription factor SOX2; TRIM29: tripartite motif-containing protein 29; P63: tumor protein 63; YAP1: transcriptional coactivator YAP1; ALDH3A1: aldehyde dehydrogenase, dimeric NADP-preferring; BLIMP1: PR domain zinc finger protein 1.

Figure 5

Immunohistochemical staining of YAP1, ALDH3A1, and BLIMP1 in tumors derived from RT4 and UROtsa As_I UC cells. YAP1 displayed mainly cytoplasmic staining and some nuclear expression in both RT4 and As_I tumors. ALDH3A1 displayed mainly cytosolic staining in RT4 tumors but mostly nuclear staining As_I tumors. BLIMP1 expression was negative in RT4 tumors and had nuclear expression in As_I tumors. The asterisks (*) indicate well-differentiated/squamous areas, and the caret (^) indicates basal areas within the As_I tumors. The brown color indicates the presence of the protein, whereas the blue/purple color indicates the nuclei that are stained with the counterstain hematoxylin. All images are at a magnification of 20×. Data represent n = 5/group. YAP1: transcriptional coactivator YAP1; ALDH3A1: aldehyde dehydrogenase, dimeric NADP-preferring; BLIMP1: PR domain zinc finger protein 1.

Figure 6

Expression of basal keratins after SOX2 knockdown in UC cells. Quantification and Western blot images of proteins in (A) UROtsa As_I cells, (B) UROtsa As_II cells, and (C) 5637 cells after SOX2 knockdown. All data are plotted as fold-change compared to the scramble control for each cell line. Protein expression was normalized to the Jess system control. Protein data represent n = 2. The values reported are mean ± SEM. A t-test was performed and asterisks indicate significant differences from the control (**** p < 0.0001). KD: knockdown; SOX2: transcription factor SOX2; KRT5: keratin, type II cytoskeletal 5; KRT6: keratin, type II cytoskeletal 6; KRT14: keratin, type I cytoskeletal 14; KRT16: keratin, type I cytoskeletal 16.

Figure 7

Expression of proteins associated with the luminal subtype of UC after SOX2 knockdown. Quantification and Western blot images of proteins in (A) UROtsa As_I cells, (B) UROtsa As_II cells, (C) 5637, and (D) RT4 cells after SOX2 knockdown. All data are plotted as fold-change compared to the scramble control for each cell line. Protein expression was normalized to the Jess system control. Protein data represent n = 2. The values reported are mean ± SEM. A t-test was performed and asterisks indicate significant differences from the control (* p < 0.05, ** p < 0.01, **** p < 0.0001). KD: knockdown; SOX2: transcription factor SOX2; PPARG: peroxisome proliferator-activated receptor gamma; FOXA1: hepatocyte nuclear factor 3-alpha; GATA3: trans-acting T-cell-specific transcription factor GATA-3; KRT7: keratin, type II cytoskeletal 7; KRT8: keratin, type II cytoskeletal 8.

Figure 8

SOX2 regulates chemotherapy response in UC cells. (A,B) Viability (expressed in percentage) after 0.00 (control), 0.13, 0.25, 0.50, and 1 µM cisplatin treatment for 72 h in the UROtsa As_I and As_II cells, respectively. (C,D) Viability after 0.00 (control), 12.5, 25.0, 50.0, 100.0, and 200.0 nM pevonedistat treatment for 72 h in the UROtsa As_I and As_II cells, respectively. The IC50 values are reported in the top panel (next to sample names). Blue bars indicate control viability while red bars indicate viability in the SOX2 KD cells. In total, 2500 cells were seeded/well in a 96-well plate, and there were eight replicates (n = 8) for each dose. Twenty-four hours after seeding, the cells were dosed with either cisplatin or pevonedistat for 72 h. The reported values are mean ± SEM. A t-test was performed, and asterisks indicate significant differences from the control (* p < 0.05). KD: knockdown; SOX2: transcription factor SOX2; %: percentage.

Figure 9

Proteomics identifies that lipid/cholesterol- and interferon-signaling pathways are downregulated after SOX2 knockdown¯. (A) The top two biological process pathways downregulated or (B) upregulated after SOX2 KD in UROtsa As_II cells. (C) Western blot images and (D) quantification of FASN protein levels after SOX2 KD in UC cells. (E) The GEPIA2 boxplot of FASN transcript expression in normal and BLCA tissues. Red bars represent tumor (T) levels and grey bars represent normal (N) tissue levels. (F) The GEPIA2 survival curve for FASN. The blue dashed lines indicate the 95% confidence interval (CI) for the low FASN expression group and the red dashed lines indicate the 95% CI for the high FASN expression group. The proteomic data were generated using n = 5/group. Western blot data are plotted as fold-change compared to the scramble control for each cell line. Protein expression (from Western blot) was normalized to the Jess system control. Protein data represent n = 2. The Western blot values reported are mean ± SEM. A t-test was performed, and asterisks indicate significant differences from the control (* p < 0.01, **** p < 0.0001). GEPIA, Gene Expression Profiling Interactive Analysis; BLCA, bladder urothelial carcinoma; KD: knockdown; SOX2: transcription factor SOX2; FASN: fatty acid synthase.