Mutant Kras as a Biomarker Plays a Favorable Role in FL118-Induced Apoptosis, Reactive Oxygen Species (ROS) Production and Modulation of Survivin, Mcl-1 and XIAP in Human Bladder Cancer

Abstract

Tumor heterogeneity in key gene mutations in bladder cancer (BC) is a major hurdle for the development of effective treatments. Using molecular, cellular, proteomics and animal models, we demonstrated that FL118, an innovative small molecule, is highly effective at killing T24 and UMUC3 high-grade BC cells, which have Hras and Kras mutations, respectively. In contrast, HT1376 BC cells with wild-type Ras are insensitive to FL118. This concept was further demonstrated in additional BC and colorectal cancer cells with mutant Kras versus those with wild-type Kras. FL118 strongly induced PARP cleavage (apoptosis hallmark) and inhibited survivin, XIAP and/or Mcl-1 in both T24 and UMUC3 cells, but not in the HT1376 cells. Silencing mutant Kras reduced both FL118-induced PARP cleavage and downregulation of survivin, XIAP and Mcl-1 in UMUC3 cells, suggesting mutant Kras is required for FL118 to exhibit higher anticancer efficacy. FL118 increased reactive oxygen species (ROS) production in T24 and UMUC3 cells, but not in HT1376 cells. Silencing mutant Kras in UMUC3 cells reduced FL118-mediated ROS generation. Proteomics analysis revealed that a profound and opposing Kras-relevant signaling protein is changed in UMUC3 cells and not in HT1376 cells. Consistently, in vivo studies indicated that UMUC3 tumors are highly sensitive to FL118 treatment, while HT1376 tumors are highly resistant to this agent. Silencing mutant Kras in UMUC3 cell-derived tumors decreases UMUC3 tumor sensitivity to FL118 treatment. Together, our studies revealed that mutant Kras is a favorable biomarker for FL118 targeted treatment.

Keywords: FL118; Mcl-1; XIAP; apoptosis; bladder cancer; human bladder tumor mouse models; mutant Kras; proteomics analysis; reactive oxygen species (ROS); survivin.

Figure 1

Differential inhibition of human bladder cancer cell growth by FL118. Subconfluent bladder cancer cells HT1376 (wild-type Ras), T24 (Hras G12V mutant) and UMUC-3 (Kras G12C mutant) in 96-well plates were treated with either DMSO (control) or a series of FL118 concentrations for 24 h–72 h followed by MTT assay to determine cell viability. (A) HT-1376 cells with wild-type Ras were insensitive to FL118 treatment compared to other cell lines (T24, UMUC-3). (B,C) Ras mutant T24 and UMUC-3 cells are highly sensitive to FL118 treatment. Each bar in the histogram shown in (A–C) is the mean ± SD from three independent assays.

Figure 2

Induction of differential poly (ADP-ribose) polymerase (PARP) cleavage in bladder cancer cells by FL118. Subconfluent bladder cancer cells in 60 mm plates were treated with DMSO (as a vehicle) or with 10, 50 and 100 nM FL118 for 24 h and/or 48 h, as shown, followed by Western blot analyses to determine PARP cleavage. (A) HT-1376 cells with wild-type Ras show no significant PARP cleavage after 24 or 48 h FL118 treatment. (B) T24 cells with Hras G12V mutation showed the cleavage of PARP with all concentrations of FL118 treatment. (C) mutant Kras (UMUC-3) cells with Kras G12C mutation showed the maximum PARP cleavage with the disappearance of total PARP bands after 24 h FL118 treatment. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) shown in A, B and C were used as internal controls of total protein-loading.

Figure 3

Inhibition of survivin, Mcl-1 and/or XIAP by FL118 in bladder cancer cell lines by FL118. Cells were treated with DMSO or 10–100 nM FL118 for 24 h and/or 48 h, as shown, followed by Western blot analyses to determine the expression of survivin, Mcl-1, XIAP and cIAP2. (A) In HT1376 cells, FL118 treatment did not inhibit any of these anti-apoptotic proteins. (B) T24 cells showed inhibition of XIAP and survivin after FL118 treatment. (C) FL118 treatment showed strong inhibition of Mcl-1, survivin and XIAP in UMUC-3 cells after FL118 treatment. GAPDH shown in A, B and C is the internal control of total protein-loading.

Figure 4

Effect of FL118 on AKT and ERK1/2 expression and phosphorylation/activation in bladder cancer cells. Subconfluent bladder cancer cells were treated with (10, 100 nM) and without (C, control/vehicle) FL118 for 8 h, 16 h, 24 h and 48 h, as shown, followed by Western blot analyses to determine the expression of AKT and ERK1/2 as well as the phospho-AKT (ser473) and phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204). (A) Treatment of HT1376 cells with FL118 does not significantly modulate the expression and phosphorylation of Erk1/2. At the same time, FL118 reduces the expression of pAKT (ser473). (B) Treatment of T24 cells with FL118 enhances the phosphorylation of Akt and Erk1/2. (C) Treatment of UMUC-3 cells with FL118 downregulated the expression of active AKT and ERK1/2. GAPDH presented in (A–C) is internal controls for total protein-loading.

Figure 5

Sensitivity of bladder cancer cells to FL118 is irrelevant to Top1 level after FL118 treatment. Subconfluent bladder cancer cells in 6-well plates were treated with (10, 50, 100 nM) and without (C, vehicle control) FL118 for 24 h and/or 48 h, as shown, followed by Western blot analyses to determine the expression of Top1. (A) Treatment of HT1376 cells with FL118 resulted in the least inhibition of Top 1 expression after FL118 treatment. (B) Treatment of T24 cells with FL118 resulted in the inhibition of Top 1 expression after FL118 treatment. (C) Treatment of UMUC3 cells with FL118 resulted in the most inhibition of Top 1 expression after FL118 treatment. GAPDH presented in (A–C) is the internal control for total protein-loading.

Figure 6

Role of mutant Kras in FL118-mediated apoptosis and inhibition of anti-apoptotic protein expression and cell viability. (A) Silencing of Kras G12C via Kras-specific shRNA in UMUC-3 cells increases cell resistance to FL118-induced apoptosis (less PARP cleavage). UMUC-3 cells were infected with Kras-specific lentiviral shRNA identified in our previous studies [21] or control shRNA followed by treatment with DMSO or FL118 (10 and 100 nM) for 24 h. At the end of treatment, Western blot analysis was performed to check PARP cleavage and the expression of anti-apoptotic proteins (XIAP, Mcl-1, survivin). GAPDH was used as an internal control. (B) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine cell viability with 10 nM and 100 nM FL118 treatment in UMUC-3 cells infected with control scramble shRNA and Kras-specific shRNA. Each bar in the histogram data is the mean ± SD from three independent assays. (C) Effect of forced expression of Kras G12V in HT1376 cells. HT1376 cells were transfected with Kras G12V or control vectors followed by treatment with DMSO or 100 nM FL118 for 48 h. At the end of treatment, Western blot analysis was performed to check the expression of apoptosis-related proteins. GAPDH was used as an internal control of total protein-loading. * p-value < 0.05.

Figure 7

Determination of reactive oxygen species (ROS) production in bladder cancer cells. (A) FL118-induced ROS production in bladder cancer cell lines. HT1376, T24 and UMUC-3 cells in 96-well plates were treated with DMSO or 100 nM FL118 for different time points as indicated. At the end of treatment, ROS production was measured using Abcam’s 2′,7′-dichlorofluorescin diacetate (DCFDA) cellular reactive oxygen species detection kit, as described in the Materials and Methods section. Values were shown as mean ± SD derived from three experiments. (B) Silencing Kras G12C via Kras-specific shRNA in UMUC-3 cells decreases ROS production after FL118 treatment. UMUC-3 cells were infected with Kras-specific lentiviral shRNA or control shRNA followed by treatment with DMSO or 100 nM FL118 for different time periods. At the end of treatment, ROS production was measured as described in the previous experiment. Values were shown as mean ± SD derived from three experiments. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001

Figure 8

Effects of FL118 on Kras pathway-associated ubiquitination (Ub), de-Ub and proteasome-related proteins. HT1376 and UMUC3 cells were treated with FL118 (20 nM) for 24 h and 48 h. Proteomics analyses were then performed as described in the Method section. The data shown here is the effect of FL118 on the Kras pathway-associated Ub, de-Ub and proteasome-related proteins for 24 h (A) and 48 h (B). All data in proteomics analyses are in triple replicates in parallel with triple vehicle controls (refer to Table S1).

Figure 9

Antitumor efficacy of FL118 in severe combined immunodeficiency (SCID) mice bearing human bladder cancer xenograft tumors. The human bladder cancer xenograft tumors were first generated through implanting bladder cancer cells at the flank area of SCID mice. Then, the tumors were isolated, and individual experimental mice were subcutaneously implanted with 30–50 mg nonnecrotic tumor masses at the flank area of individual mice. Seven to 14 days after tumor transplantation at which the implanted bladder xenograft tumors were grown to 100–150 mm³ (defined as day 0), mice were randomly divided into the required groups (5 mice per group) for treatment vial oral administration of FL118 at the doses as shown or vehicles with a schedule of weekly × 3 or 4 (arrowed) as described in the “Materials and Methods” section. (A) FL118 efficacy against UMUC-3 bladder cancer cell-established xenograft tumors in SCID mice. (B) FL118 efficacy against HT1376 bladder cancer cell-established xenograft tumors in SCID mice. The tumor growth curves shown in A and B are the mean ± SD derived from 5 mice. (C) SCID mouse body weight changes after treatment with vehicle or with FL118. The mouse body weight curves shown in C are the mean ± SD derived from 5 mice. (D) Western blots validate the expression of mutant Kras in UMUC3 cells in the condition of no infection, with control shRNA lentiviral particle transfection or with Kras shRNA lentiviral particle transfection (Of note, this is the stably infected multiple-infectant mixture, which usually had the issue with some endogenous mutant Kras, due to some unclear reasons). GAPDH is the internal control. (E) FL118 exhibited was less effective in inhibiting UMUC3 tumors with a partial silencing of mutant Kras using Kras shRNA. The tumor growth curves shown in E are the mean ± SD derived from 5 mice.