2.1 Cell cultures and transfection

The human PC cell lines PC-3 (RRID: CVCL_0035) and DU145 (RRID: CVCL_0105) were obtained from the Cell Resource Centre for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan). Cabazitaxel-resistant PC-3-TxR/CxR (RRID: CVCL_ZX06) and DU145-TxR/CxR (RRID: CVCL_ZX04) cells were generated previously.⁴ Cabazitaxel-resistant 22Rv1-CR cells were also generated previously.⁵ Cell culturing was basically performed as previously reported.¹⁴ PC-3-TxR/CxR and DU145-TxR/CxR cells were cultured in RPMI-1640 medium (Fujifilm Wako, Osaka, Japan) with 3 nM cabazitaxel (Fujifilm Wako) to maintain their drug resistance. All human cell lines have been authenticated using short tandem repeat (STR) profiling within the past 3 years. The CycleavePCR Mycoplasma Detection Kit (Takara Bio, Shiga, Japan) was used to confirm that the cultures were mycoplasma-free.

2.2 Reagents and antibodies

Lipofectamine RNAiMax Transfection Reagent, OPTI-MEM, the small interfering RNA (siRNA) SiLAT1 (Stealth siRNAs: HSS112004 and HSS112005), SiCDK1 (Silencer Select SiRNAs: s464 and s465), SiCDK2 (Silencer Select SiRNAs: s205 and s206), and Stealth RNAi siRNA Negative Control Med GC Duplex #3 were obtained from Thermo Fisher Scientific (Waltham, MA). The plasmid vectors containing CDK1 and CDK2 were provided by Kazusa Genome Technologies (Clone ID oc00221 and oc00080; Chiba, Japan). The LAT1-specific inhibitor JPH203 was provided by J-Pharma (Yokohama, Japan). The catalog numbers and dilutions of all used antibodies are summarized in Table S1.

2.3 Reverse transcription-polymerase chain reaction

Total RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) were performed as described previously.¹⁴ The GAPDH mRNA level was quantified as a control for normalization. The PCR primers used in this study are listed in Table S2.

2.4 Western blot analysis

Protein extraction and western blotting were performed as described previously.⁷ The protein assay bicinchoninic acid kit (Nacalai Tesque, Kyoto, Japan) was used to quantify the protein content, and 20-mg protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis using a gel prepared with the TGX Fastcast Acrylamide Kit (10%; Bio-Rad, Hercules, CA) according to the manufacturers’ protocols. Blocking One-P (Nacalai Tesque) was used as a blocking agent for the detection of phosphorylation, and Bullet Blocking One (Nacalai Tesque) was used as a blocking agent for all of the other experiments. Original full images of membranes are shown in Figures S1–S3.

2.5 Cell growth inhibition assay

Cells were seeded into 96-well plates (1500 cells/well), and at each time point, cells were incubated with Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan) for 1.5 h, then the absorbance was measured using Microplate Manager v6.0 (Bio-Rad). To evaluate the inhibitory effect of JPH203, 24 h after seeding, the cells were treated with the inhibitors at the indicated concentrations or the same amount of dimethyl sulfoxide (DMSO; 0.5%; vehicle control). The half-maximal inhibitory concentration (IC₅₀) definition and measurements were as previously reported.⁸

To examine the effects of JPH203 in combination with a cyclin-dependent kinase (CDK) inhibitor (dinaciclib, flavopiridol, or milciclib), combination assays were performed using a fixed concentration of JPH203 and three concentrations of each CDK inhibitor based on previous literature.¹⁵ Of the three concentrations tested, only the one that had the largest effect in combination with JPH203 is shown in the results.

2.6 Cell migration and invasion assay

The protocol and reagents used for the migration and invasion assay were as described previously.¹⁴ A wound healing assay was also performed as described previously.¹⁶ In each assay, cells were incubated with 5 μg/mL of mitomycin C (Nacalai Tesque) for 1 h at the start to inhibit cell proliferation.

2.7 Phosphoproteomics analysis

2.8 Cell cycle and apoptosis assay

Cabazitaxel-resistant PC cells were cultured in six-well dishes for 24 h, then treated with JPH203 or DMSO as a control for 24 h before collection. The reagents and analysis methods used were as previously reported.¹⁶

2.9 L-Leucine uptake assay

Cabazitaxel-resistant PC-3 cells (1.5 × 10⁵ cells/well) were cultured in 24-well dishes for 48 h and examined by transporter assay using L-Leucine (containing [14C] leucine at 0.1 mCi/mL) (PerkinElmer, Boston, USA). The precise protocols of the experiment was reported previously.²⁴

2.10 Immunohistochemistry

Paraffin-embedded sections (4 mm in thickness) were used. The detailed immunohistochemistry (IHC) methods were as previously reported.²⁵ Anti-Cdc6 (phospho S54, 1:200 dilution) and anti-phospho Rb (Ser807/811, 1:100 dilution) were used as the primary antibodies. To assess the expression of these phosphoproteins, we employed a scoring method that we have used previously.⁹ The intensity of tumor cell staining was evaluated in at least five fields of view at 400× magnification. Two independent evaluators (AF and JR), who were blinded to the sample information, scored the intensity of tumor cell staining in the specimens.

2.11 Analysis of a clinical prostate cancer patient dataset

A dataset of clinical PC data from Grasso et al. (GSE35988)²⁶ was analyzed in this study as previously reported.¹⁴ Gene expression correlations were examined from reports of expression analysis in patients with advanced prostate cancer²⁷ (https://github.com/cBioPortal/datahub/tree/master/public/prad_su2c_2019.).

2.12 Mouse xenografts

Six-week-old male SCID mice (C.B-17/Icr-scid/scidJcl) were obtained from CLEA (Tokyo, Japan). After an acclimatization period, 2 × 10⁶ PC-3-TxR/CxR cells were implanted with 50% Matrigel (354,230, Corning, Corning, NY) in the dorsal subcutaneous region of the mice. When the tumor volumes reached approximately 100 mm³, the mice were divided into two groups: a control group and a JPH203-treated group (n = 4 each). The tumor size was calculated using the following formula: tumor volume = (major axis) × (minor axis) 2 × 0.5. For the JPH203-treated group, the mice were given an intravenous injection of JPH203 (25 mg/kg) once daily, while the control group was given an intravenous injection of the placebo once daily. The tumor size was measured daily with a Vernier caliper. After 15 days, the mice were killed, and the tumor xenografts were resected.

2.13 Statistical analysis

JMP Pro version 15.0.0 (SAS Institute, Cary, NC) was used for all statistical analyses. The unpaired Student's t-test was used for assessing differences between two groups with Bonferroni correction. Statistical significance was set at p-values below 0.05. p-values are indicated by asterisks in the figures: *p < 0.05, **p < 0.01, and ***p < 0.001.

4.1 Effects of L-type amino acid transporter 1 inhibition on cell migration, invasion, and the expression of related genes

Next, we validated the effects of JPH203 treatment on cell migration, invasion, and the expression of genes involved in epithelial–mesenchymal transition (EMT) in the cabazitaxel-resistant strains. Administration of JPH203 significantly reduced migration in the chamber assay (Figure 2A). The same effect was observed in the wound healing assay (Figure 2B). We subsequently investigated the invasive capacity of the cells, and similar results were obtained (Figure 2C). JPH203 treatment decreased the expression of N-cadherin, Slug, and vimentin in both cell lines (Figure 2D). E-cadherin expression was not observed in PC-3-TxR/CxR. In DU145-TxR/CxR, E-cadherin expression was slightly increased by JPH203 treatment. In addition, a similar assay was performed on a normal strain of PC-3. JPH203 treatment decreased the expression of N-cadherin, Slug, and vimentin and increased the expression of E-cadherin. It also significantly decreased cell migration and invasion (Figure S6).

4.2 Identification of kinases that were affected by the inhibition of L-type amino acid transporter 1 and pathway analysis

To explore how the administration of JPH203 affects intracellular phosphorylation, we performed a comprehensive LC–MS analysis using PC-3-TxR/CxR cells. Proteins were extracted from cells in the JPH203-treated and control groups, and finally, 3080 substrate proteins and 10,749 phosphorylated peptides were identified to have been affected. The MA plot for this assay is shown in Figure S8. First, GO and biological process analyses of the variable gene groups were performed. Among the top 10 GO terms that fluctuated were many elements related to the cell cycle (Figure 3A). The same was true for the biological process analysis (Figure 3B). Subsequent analyses included phosphorylation sites using KSEA. The kinase activity of CDK1 and CDK2 was significantly decreased (Figure 3C). In the molecular network analysis, CDK1 and CDK2 were each involved in many molecular pathways (Figure 3D,E). An overview of the molecular network is shown in Figure S9.

From the kinase-substrate links, the substrates of CDK1, CDK2, and mTOR that were markedly reduced were extracted (Figure 3F). The decreases in phosphorylation were confirmed by western blotting using antibodies recognizing these specific phosphorylation sites. The phosphorylation of cdc6, Rb, and 4EBP1 was decreased in a concentration-dependent manner by treatment with JPH203 (Figure 3G). We also observed a decrease in phosphorylation over time during the 24 h after the administration of JPH203 (Figure 3H). In the analysis of the public database, CDK1 expression was significantly higher in HSPC than in the normal cells and was also significantly higher in CRPC than in HSPC (Figure S5). In contrast, no such trend was observed for CDK2 (Figure S5). When the expression of CDK1 and CDK2 was compared between the normal and cabazitaxel-resistant cell lines of PC-3 and DU145, the expression was significantly higher in the cabazitaxel-resistant line than in the normal line in both PC-3 and DU145 (Figure S10). We subsequently examined whether administration of JPH203 in normal strains decreases phosphorylation of the mTOR pathway. In PC-3 cells, treatment with JPH203 decreased phosphorylation of 4EBP1 (Figure S10). Finally, we investigated whether the downstream phosphorylation signals of LAT1 that we identified in this study are associated with the mTOR pathway. Phosphorylation of 4EBP1 was decreased by JPH203 or by rapamycin, a specific inhibitor of the mTOR pathway. In contrast, phosphorylation of Rb was decreased by JPH203 but not by rapamycin (Figure S10).

4.3 Effects of L-type amino acid transporter 1 inhibition on the cell cycle and apoptosis

Because treatment with JPH203 reduced the CDK1 and CDK2 kinase activity, we investigated the effects of JPH203 treatment on the cell cycle and apoptosis by flow cytometry in cabazitaxel-resistant lines. Administration of JPH203 caused a significant increase of cells in the G0/G1 phase and a significant decrease of cells in the S and G2/M phases (Figure 4A). Furthermore, the percentage of apoptotic cells was significantly higher in the JPH203-treated group than in the control group (Figure 4B). JPH203 treatment increased cleaved PARP expression, suggesting that LAT1 inhibition induces apoptosis in cabazitaxel-resistant PC cells (Figure 4C). We subsequently prepared CDK1 and CDK2 plasmids and SiRNAs to show whether the LAT1 and CDK1 and CDK2 pathways are important for cabazitaxel-resistant prostate cancer. Each plasmid resulted in overexpression, while the SiRNAs nearly abolished expression (Figure 4D).

We then performed a rescue assay using the CDK plasmid. Compared to JPH203 alone, administration of JPH203 and CDK plasmid significantly increased cell proliferation (Figure 4E). Similarly, treatment with plasmids of SiLAT1 and CDK significantly enhanced cell growth compared to treatment with SiLAT1 alone (Figure 4F). Furthermore, administration of JPH203 plus SiCDK1 and SiCDK2 had no significant effect on cell proliferation compared to JPH203 alone (Figure 4G).

We then selected three CDK inhibitors (dinaciclib, flavopiridol, and milciclib), referring to previous reports,¹⁵ to explore their growth-inhibitory effects. The combined use of JPH203 and dinaciclib or flavopiridol significantly inhibited cell growth in the two cabazitaxel-resistant lines when compared to JPH203 or the CDK inhibitors alone (Figure S11). In contrast, the combination of JPH203 with milciclib significantly inhibited cell proliferation in DU145-TxR/CxR when compared to milciclib alone, but this was not seen in PC-3-TxR/CxR. Furthermore, there was no significant difference between the combination of JPH203 with milciclib and JPH203 alone in any of the cells (Figure S11). Finally, we performed a database analysis of patients with advanced prostate cancer and found no correlation in expression between Rb (RB1 gene) and the MDR1 gene, which is known to be involved in cabazitaxel resistance (Figure S12). In contrast, there was a negative correlation in expression between RB1 and LAT1 (Figure S12).

4.4 JPH203 inhibits tumor cell growth in vivo

To assess the growth inhibitory effect of JPH203 in a cabazitaxel-resistant strain in vivo, we implanted PC-3-TxR/CxR cells into C.B-17/Icr-scid/scidJcl mice and treated them with JPH203. JPH203 administration for 2 weeks significantly reduced the volume of the PC-3-TxR/CxR tumors when compared to the control group without causing weight loss (Figure 5A,B). The tumors were then extracted and paraffin-embedded, and IHC assays were performed. Both cdc6 phosphorylation and Rb phosphorylation were decreased in the JPH203 group when compared to the control group (Figure 5C). The IHC scores were also significantly lower in the JPH203 group than in the control group for both cdc6 and Rb phosphorylation (Figure 5D).