L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mTOR complex 1 (mTORC1) signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer and are involved in increasing nutrients and stimulating cell growth. In a study reported in the Journal of the National Cancer Institute, Qian Wang, PhD, of the Origins of Cancer Laboratory, Centenary Institute, in Newton, Australia, and colleagues assessed the role of LAT3 function in prostate cancer.¹ They found that inhibition of LAT transporters, and thus leucine uptake, may constitute a new strategy in metastatic castration-resistant prostate cancer.

Study Details

In the study, LAT3 protein expression was assessed in human prostate cancer tissue microarrays, which contained samples from patients with prostatic intraepithelial neoplasia or Gleason-graded prostate cancer (n = 88) and from patients at different stages after neoadjuvant hormone therapy (n = 72). LAT function was inhibited using the leucine analog BCH in androgen-dependent and androgen-independent environments, and gene expression was analyzed by microarray. The effects of inhibition of LAT1 and LAT3 expression were examined in a PC-3 xenograft mouse model.

ATF4-Mediated Upregulation of Amino Acid Transporters

LAT3 expression was detected at all stages of prostate cancer, with no differences in expression by Gleason grades. Compared with untreated patients (n = 123 cores), those undergoing neoadjuvant hormone therapy showed a decrease in LAT3 expression that became significant after 4 to 7 months (25.1% decrease; 4–7 month mean score = 1.571, 95% confidence interval [CI] = 1.155–1.987, vs 0 month mean score = 2.098, 95% CI = 1.962–2.235, P = .0187).

Inhibition of LAT function with BCH led to activating transcription factor 4 (ATF4)-mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated by the synthetic androgen R1881. ASCT1, ASCT2, and 4F2hc protein levels showed increased expression after leucine deprivation and in the presence of dihydrotestosterone, indicating that these proteins are regulated by both ATF4 and the androgen receptor.

M-Phase Cell-Cycle Genes

Analysis to determine which gene sets and pathways are altered by BCH treatment showed substantial enrichment of gene sets involved in cell-cycle regulation including spindle formation, microtubule cytoskeleton, M phase, mitosis, cell-cycle process, and cell-cycle phase genes and a significant enrichment in binding sites for E2F transcription factor, the family members of which regulate the cell cycle and metabolism in cancers.

Examination of genes showing significant changes after BCH treatment yielded 122 downregulated genes and 12 upregulated genes; of the 122 downregulated genes, which included transcription factors E2F1 and E2F2, 72 (59%) were related to the cell cycle and proliferation. Further analysis generated a network of downregulated genes from the E2F transcription factors, showing that approximately half of the genes are part of a common cell-cycle regulatory pathway. This analysis indicated that E2F transcription factors have a central role in the regulation of cell-cycle gene expression in prostate cancer.

Three of the cell-cycle genes—CDK1 (CDC2), CDC20, and UBE2C (ubiquitin-conjugating enzyme E2C)—are upregulated and control cell-cycle progression in androgen-independent prostate cancer cells and have been shown to be critical M-phase regulators of the cell cycle in metastasis. Western blot analysis showed that E2F1, E2F2, CDK1, UBE2C, and CDC20 protein levels were slightly decreased in both PC-3 and LNCaP human prostate cancer cells after BCH treatment, with all proteins exhibiting a dramatic decrease after leucine deprivation.

BCH-Downregulated Genes

Analysis of metastatic samples showed that the set of enriched genes in metastasis was highly similar to that inhibited by BCH treatment; these included M phase, mitosis, cell-cycle process, and cell-cycle phase genes. Analysis of transcription factor motif gene sets showed enrichment for genes with E2F binding sites, with 17 of 30 of the significantly enriched transcription factor motif gene sets being common to the metastasis-upregulated and BCH-downregulated genes.

This analysis suggested that E2F-regulated cell-cycle genes are also crucial for metastatic castration-resistant prostate cancer. Overall, 91% of BCH-downregulated genes in metastasis were significantly upregulated in metastatic patient samples, including E2F1, E2F2, CDK1, CDC20, and UBE2C; 83% of the BCH-upregulated genes were significantly downregulated in metastatic patient samples.

Knockdown of LAT1 and LAT3

The mouse xenograft model used to examine the role of LAT1 and LAT3 in castration-resistant prostate cancer tumor formation employed lentiviral shRNA constructs against either LAT1 or LAT3, which resulted in decreased transporter expression and decreased expression of cell cycle proteins including UBE2C, CDK1, and CDC20. Knockdown of both LAT1 and LAT3 caused a reduction in tumor take rate—ie, 70% for shLAT1 and 61% for shLAT3 cells compared with 83% for shControl cells. Xenografts expressing either shLAT1 or shLAT3 showed significantly decreased tumor growth compared with controls.

Analysis of Ki-67 expression in the tumors showed a significant decrease in proliferation for both the shLAT1 and shLAT3 tumors compared with the shControl group. In addition, the xenografts showed decreased expression of CDK1 and UBE2C in shLAT1 and shLAT3 tumors, with CDC20 expression also decreasing in shLAT3 tumors.

Organ harvesting showed that 8 of 14 mice with shControl tumors had metastases to the liver, lymph nodes, or lungs, compared with 3 of 13 with shLAT1 tumors (P =.1201) and 1 of 13 with shLAT3 tumors (P = .012) tumors. These findings suggest that depletion of LAT1 or LAT3 expression in prostate cancer cells may also suppress metastatic potential.

The investigators concluded: “[W]e have shown that prostate cancer cells use [androgen receptor] and ATF4 transcriptional pathways to maintain amino acid transporter levels during primary and metastatic prostate cancer. Targeting LAT transporters, thereby inhibiting leucine uptake, may offer a new therapeutic opportunity for metastatic [castration-resistant prostate cancer], affecting tumor growth and metastasis through inhibition of M-phase cell cycle and mTORC1 signaling pathways.”

Jeff Holst, PhD, of Origins of Cancer Laboratory, Centenary Institute, is the corresponding author for the Journal of the National Cancer Institute article. ■

Disclosure: The study authors reported no potential conflicts of interest.

Reference

1. Wang Q, Tiffen J, Bailey CG, et al: Targeting amino acid transport in metastatic castration-resistant prostate cancer: Effects on cell cycle, cell growth, and tumor development. J Natl Cancer Inst 105:1463-1473, 2013.