Characterization and treatment of gemcitabine‐ and cisplatin‐resistant bladder cancer cells with a pan‐RAS inhibitor

Combination chemotherapy with gemcitabine and cisplatin (GC) is recommended as the primary treatment for advanced bladder cancer (BC). However, the benefits of this approach are limited owing to the acquisition of drug resistance. Here, we found that gemcitabine‐resistant and cisplatin‐resistant BCs do not exhibit cross‐resistance, and that these BCs exhibit different mRNA patterns, as revealed using RNA sequence analysis. To overcome drug resistance, we used the newly developed pan‐RAS inhibitor Compound 3144. Compound 3144 inhibited cell viability through suppression of RAS‐dependent signaling in gemcitabine‐ and cisplatin‐resistant BCs. RNA sequencing revealed that several genes and pathways, particularly those related to the cell cycle, were significantly downregulated in Compound 3144‐treated BCs. These findings provide insights into potential therapeutic strategies for treating BC.

The prognosis of patients with advanced bladder cancer (BC) is poor because of frequent disease relapse and death, even if antitumor drug therapy and radiotherapy are temporarily successful in patients with recurrent or metastatic disease [1]. In particular, combination chemotherapy with gemcitabine and cisplatin (GC) is used as the first-line antitumor therapy; however, its efficacy is limited [2,3], and clinical trials of other antitumor therapies and various moleculartargeted agents have failed to demonstrate efficacy [4,5]. Recently, anti-programmed death receptor-1 (PD-1) and programmed death ligand 1 (PD-L1) antibodies have been clinically applied for the treatment of advanced or metastatic BC; however, the response rates to these drugs are insufficient, and therapeutic efficacy is limited [6,7]. Thus, compared with other urological cancers, such as prostate cancer and renal cancer, there are fewer therapeutic options for patients with BC. Accordingly, further studies are needed to elucidate the mechanisms of acquisition of drug resistance and to develop novel therapeutic strategies.
We have previously generated several gemcitabineresistant BC cell lines (GR-BOY, GR-T24) [8] and cisplatin-resistant (CR) BC cell lines (CR-BOY, CR-T24) [9]. Using gemcitabine-resistant BC cells, we found that microRNA-99a-5p induces cellular senescence by targeting SWI/SNF-related, matrixassociated, actin-dependent regulator of chromatin, subfamily D, member 1, a member of the SWI/SNF chromatin remodeling complex family [8]. Additionally, in cisplatin-resistant BC cells, we reported that enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase, which is involved in the degradation of long-chain fatty acids in peroxisomes and in the boxidation of fatty acids, contributes to cisplatin resistance through regulation of tumor-suppressive microRNA-486-5p [9]. However, it is unclear whether there is cross-resistance between GC and whether there are independent or common mechanisms of acquisition of drug resistance in BC cell lines.
HRAS, an isoform of RAS, was the first human oncogene found to be mutated in the BC cell line T24 in 1982 [10], and HRAS mutations are reported to be present in approximately 4% of BC cases [11]. Because KRAS and NRAS mutations also occur in many other cancers [12], there have been worldwide efforts to develop RAS inhibitors, including salirasib, which selectively disrupts the binding of the active RAS protein to the plasma membrane. Previously, we reported that salirasib requires high volumes to suppress BC cell lines in in vitro experiments and fails to suppress tumor growth in vivo [13]. The difficulty in developing RAS inhibitors is associated with the lack of binding sites on the RAS protein surface; however, new compounds that bind simultaneously to multiple sites on the activated and conformationally altered surface of the RAS protein have recently been developed [14]. The newly developed pan-RAS inhibitor Compound 3144 suppresses RAS protein activity in vitro and in vivo and exhibits antitumor efficacy in cancer cells. However, it is unclear whether this novel inhibitor exerts antitumor effects in BC cells and in cells that have acquired drug resistance.
Accordingly, in this study, we first investigated whether there was cross-resistance between gemcitabine-and cisplatin-resistant BC cell lines. We then performed RNA sequencing (RNAseq) analysis to identify alterations in the cells that may support the presence or absence of cross-resistance. Furthermore, as a novel therapeutic strategy against gemcitabineand cisplatin-resistant BC cells, we used the novel pan-RAS inhibitor Compound 3144 in in vitro experiments. Finally, we investigated genes and pathways affected by Compound 3144 in gemcitabine-and cisplatinresistant BC cells using RNAseq analysis.

BC cell lines and culture
We used 2 human BC cell lines (BOY and T24). BOY cells were established in our laboratory from a 66-year-old Asian male patient, who was diagnosed with stage IV BC with many lung metastases. T24 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Using these cell lines, we established gemcitabine-resistant BC cell lines (GR-BOY and GR-T24) and cisplatinresistant BC cell lines (CR-BOY and CR-T24), as previously reported [8,9]. These cell lines were cultured in minimum essential medium Eagle (MEME) supplemented with 10% fetal bovine serum at 37°C in a humidified, 5% CO 2 incubator.

Pan-RAS inhibitor
For in vitro experiments, the pan-RAS inhibitor Compound 3144 (CAS 1835283-94-7; ProbeChem, Inc., Shanghai, China), solubilized in dimethyl sulfoxide (DMSO), was used. The Compound 3144/DMSO solution was prepared in MEME at different concentrations, and the solutions were added to cell culture plates at final inhibitor concentrations of 1, 2, 3, 4, or 5 lM. The DMSO concentration was adjusted to 0.1%.

Cell proliferation, migration, and invasion assays
To evaluate cell proliferation, we used XTT assays. BOY and T24 cells were seeded in 96-well plates (1 9 10 3 cells/ well) with 100 lL medium per well. We determined the extent of cell proliferation 96 h after seeding using a Cell Proliferation Kit II (Roche Diagnostics GmbH, Mannheim, Germany). When using cisplatin, gemcitabine, or Compound 3144, we added 10 lL of the stock solution, adjusted to 10-times the target concentration. Inhibition data were used to calculate the half-maximal inhibitory concentration (IC 50 ) values using nonlinear, fourparameter, variable slope equation software (GRAPHPAD PRISM ver. 8.00 for Windows; GraphPad Software, San Diego, CA, USA). Wound healing assays were used to assess cell migration activity. Cells (2 9 10 5 cells/well) were plated in 6-well plates, and after 48 h of incubation, the cell monolayer was scraped using a P-1000 micropipette tip. The initial gap length (0 h) and the residual gap length 24 h after wounding were calculated from photomicrographs. For cell invasion assays, we used modified Boyden chambers consisting of Matrigel-coated Transwell membrane filter inserts with 8-lM pores in 24-well tissue culture plates (BD Biosciences, San Jose, CA, USA). The cells that passed through the pores and attached to the surface of the chamber were counted from photomicrographs.

Three-dimensional spheroid culture system
To evaluate spheroid formation, we introduced threedimensional culture system (Cell-able TM ; Toyo Gosei Co. Ltd. Tokyo, Japan). According to the manufacturer's protocol, BC cells were seeded in 96-well plates (5 9 10 3 cells/ well) with 100 lL medium per well with control (DMSO) or Compound 3144 (5 lM). 48 h after incubation, spheroid formations were evaluated, respectively.

RNA sequencing
An Isogen kit (Nippon Gene, Tokyo, Japan) was used for extraction of total RNA following the manufacturer's protocol. Total RNA from each cell was subjected to RNAseq, which was performed by Eurofins Japan. mRNA profiles were generated by single-read deep sequencing using an Illumina HiSeq 4000 instrument (Illumina, San Diego, CA, USA).

Statistical analysis
Cluster analysis was performed in R (version 4.1.2) using Euclidean distances with the vegan package and Ward.D2 linkage with the COMPLEX HEATMAP package. Principal coordinate analysis (PCoA) was performed using the distance calculated with the altGower method. Data are presented as means AE standard deviations of at least three independent experiments. The relationships between two groups were analyzed using Mann-Whitney U tests. The relationships among three or more variables and numerical values were analyzed using Bonferroni-adjusted Mann-Whitney U tests. All analyses were performed using Expert STATVIEW software, version 5.0 (SAS Institute, Inc., Cary, NC, USA). Results with P values less than 0.05 were considered statistically significant.

Analysis of a BC cohort with The Cancer Genome Atlas
In order to evaluate the genetic alteration of RAS genes such as HRAS, KRAS, and NRAS, a The Cancer Genome Atlas (TCGA) cohort database of 476 patients with BLCA was used through cBioPortal (http://www.cbioportal.org/ public-portal/).

Ethics and standards for conducting of human
Our study was approved by the Bioethics Committee of Kagoshima University; written prior informed consent and approval were given by the patient from which the human BC cell line BOY was established. The study methodologies, approved by the ethics committee of Kagoshima University as 21S009, conformed to the standards set by the Declaration of Helsinki.

IC 50 values of cisplatin and gemcitabine for parental and drug-resistant cells
To determine whether there was cross-resistance of gemcitabine-resistant cells to cisplatin and cisplatinresistant cells to gemcitabine, we calculated the IC 50 values of both drugs in these cells. GR-BOY and GR-T24 did not show cisplatin resistance, similar to the parent cell lines, whereas CR-BOY and CR-T24 showed more than 5-and 2-fold higher IC 50 values compared with the GR cell lines (BOY IC 50 : 3.0 lM, GR-BOY IC 50 : 2.2 lM, CR-BOY IC 50 : 11.7 lM,

RNAseq of parental and drug-resistant cells
Cluster analysis of mRNA expression levels did not show distinct profiles for drug-resistant cell lines ( Fig. S1). However, cluster analysis using a select 402 gene dataset (2-or 0.5-fold differences in expression), demonstrated distinct clusters associated with gemcitabine resistance and cisplatin resistance ( Fig. 2A). PCoA showed clear differences on the y-axis between gemcitabine resistance and cisplatin resistance (Fig. 2B).  (Fig. 3D, Fig. S4). By contrast, the phosphorylation of ERK was not decreased by Compound 3144 in GR-BOY cells.    Fig. 4C). Western blot analysis showed that Compound 3144 decreased the phosphorylation of Ser473 on AKT and the phosphorylation of ERK in CR-BOY and CR-T24 cells (Fig. 4D, Fig. S4).     (Fig. 5A), CDDP-R-BOY/T24 (Fig. 5B), and parental BOY/T24 cells (Fig. 5C).

Compound 3144 inhibited signaling
We next performed RNAseq of the parental, gemcitabine-resistant, and cisplatin-resistant BC cell lines after treatment with Compound 3144. We identified 106 genes that were commonly downregulated by Compound 3144, which was less than half that in the control for all 6 cell lines; the expression of 22 genes was completely suppressed by Compound 3144 (Table 1). We also performed Gene Ontology biological processes pathway analyses with 106 genes, and pathways associated with the cell cycle were particularly enriched ( Table 2).

RAS status based on the BLCA cohort of TCGA
The analysis with TCGA database in BC indicated that HRAS and KRAS mutations were observed in  Fig. S4. The grouping of blots cropped from different parts of the same gel or from different gels, fields, or exposures was divided with white space. nearly 5%, whereas only 1% was mutated in NRAS. On the other hand, mRNA high expression or DNA amplification was more frequently observed than mutations in each RAS (Fig. S3). In total, one fourth of BCs had RAS mutation, its mRNA high expression or its DNA amplification.

Discussion
The mechanisms of acquisition of cisplatin resistance can be classified into 4 categories [16]. The first category is pre-target resistance, which involves steps preceding the binding of cisplatin to DNA, perhaps owing to lowered cisplatin uptake into cells. The second category is on-target resistance related to inadequate DNA/cisplatin adducts. The third category is post-target resistance owing to the lethal signaling pathways elicited by cisplatin-mediated DNA damage. The last category is off-target resistance, in which a nontarget signaling pathway is triggered by cisplatin [16]. Several mechanisms have also been identified for the mechanisms of acquisition of gemcitabine resistance [17]. The first mechanism of gemcitabine resistance is the dysregulation of proteins associated with gemcitabine metabolism through human equilibrative nucleoside transporter 1, downregulation of the ratelimiting enzyme deoxycytidine kinase (dCK), upregulation of ribonucleotide reductase catalytic subunit M (RRM) 1/RRM2, and Hu antigen R, an RNA-binding protein that post-transcriptionally regulates dCK. The second mechanism is high expression of drug efflux pumps, including ABC transporter family proteins, to protect CSCs from chemotherapeutic agents. The epithelial-mesenchymal transition has also been reported to be associated with CSCs. Another mechanism involves multiple genetic and epigenetic abnormalities, such as mutations in nuclear factor-jB, AKT, mitogen-activated protein kinase, and hypoxiainducible factor-1a pathways, which lead to inactivation of apoptosis. However, few reports have described concurrent GC resistance. Pan et al. [18] showed that the long noncoding RNA UCA1 promotes cisplatin/ gemcitabine resistance through CREB-modulating miR-196a-5p in BC cells. In our study, RNAseq showed that there were common alterations in gene expression related to acquisition of drug resistance to GC, even in different cell lines; the results also suggested that there may be different mechanisms mediating GC resistance. Although there was no apparent cross-resistance between gemcitabine-and cisplatinresistant cells because the functional mechanisms of these drugs differ, to the best of our knowledge, our finding that there was no cross-resistance between these resistant cell lines was the first report of this result obtained using drug-resistant cell lines.
Recently, the KRAS G12C inhibitor sotorasib was approved as the world's first drug targeting KRAS, showing efficacy against unresectable advanced or recurrent non-small cell lung cancer with KRAS G12C mutation-positive disease that progressed after cancer chemotherapy [19]. However, KRAS and NRAS mutations are less frequently observed in BC, whereas HRAS mutations occur in 4% of BC cases [11]. Actually, TCGA database in BC indicated that HRAS and KRAS mutations were observed in nearly 5%, whereas only 1% was mutated in NRAS. On the other hand, mRNA high expression or DNA amplification was more frequently observed than mutations in each RAS (Fig. S3). In total, one fourth of BCs had RAS mutation, mRNA high expression or DNA amplification. Therefore, a pan-RAS inhibitor, such as Compound 3144, may be beneficial for the treatment of BC, as shown in this study. However, Compound 3144 showed toxicity and off-target activity, despite its excellent antitumor efficacy, which it exerts via binding with wild-type KRAS, NRAS, and HRAS [14]. In addition, pan-RAS inhibitors are unlikely to be tolerated because RAS function is critical in normal cells [20]. Recently, enfortumab vedotin, a nectin-4-directed antibody and microtubule inhibitor conjugate, was approved for the treatment of urothelial cancer in patients who had previously received a platinumcontaining chemotherapy and a PD-1 or PD-L1 inhibitor [21,22]. Enfortumab vedotin is an antibody-drug conjugate (ADC) that combines the targeting capabilities of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs [22]. Because ADCs can be designed to distinguish between healthy and diseased tissue [23], these compounds may be an effective method for delivery of pan-RAS inhibitors to tumors for applications in the clinical setting. The novel pan-RAS inhibitor Compound 3144, which was designed to target multiple sites on RAS proteins, showed sufficient affinity and selectivity for pharmacological inhibition of RAS signaling, e.g., the RAS/PI3K/ AKT and RAS/RAF/MEK/ERK cascades [14]. In our study, phosphorylation of AKT was sufficiently suppressed by Compound 3144 in parental and drugresistant BC cells, whereas phosphorylation of ERK was not suppressed, particularly in BOY cells. These findings may be related to the specific characteristics of the cell lines. For example, T24 cells harbor the HRASG12V mutation (a substitution of glycine by valine at codon 12 of HRAS), and BOY cells contain wild-type HRAS [13]. RNAseq analysis of cells treated with Compound 3144 also identified several genes that showed complete downregulation, and several pathways, particularly cell cyclerelated pathways, were associated with Compound 3144 treatment. Because RNAseq analysis identified different genes and pathways for known mechanisms associated with GC resistance, Compound 3144 could inhibit GR and CR BC cells. Notably, Compound 3144 inhibited not only cell proliferation but also cell migration and invasion in this study. Therefore, further studies are needed to elucidate the functional roles of Compound 3144 in vitro and in vivo.

Conclusions
In this study, we found that cross-resistance did not occur between gemcitabine-and cisplatin-resistant BC cells. Moreover, Compound 3144 showed antitumor effects in both gemcitabine-and cisplatin-resistant BC cells. Our findings provided important insights into the mechanisms of GC resistance in BC and may facilitate the development of novel therapeutic strategies to treat progressive BC.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.  and 100 lm (C). The error bars represent SD. (D) Parental BOY and T24 cells were treated with control (DMSO) or Compound 3144 (5 lM) for 1 h, and RAS-dependent signaling was evaluated by western blotting. These blots are cropped and uncropped membranes are shown in Fig. S5. The grouping of blots cropped from different parts of the same gel or from different gels, fields, or exposures was divided with white space. Fig. S3. RAS status based on the BLCA cohort of TCGA. Genetic alteration of RAS genes in BC samples. Fig. S4. The full-length blots/gels of Figs 3D and 4D. Fig. S5. The full-length blots/gels of Fig. S2D.