LAT1 is highly expressed in solid tumors, including lung, colon, and liver, and is associated with tumor proliferation, angiogenesis, and poor survival 21. The 5-year survival rate of LAT1-positive patients is worse than that of LAT1-negative patients in pathological stage I of non-small cell lung cancer (NSCLC) 22. LAT1 expression is positively associated with non-response to chemotherapy with platinum-based drugs 23. In addition, LAT1 silencing in NSCLC cells induces the downregulation of programmed cell death 1 ligand 1, an immune checkpoint inhibitor 24. Therefore, LAT1 is suggested to be closely associated with malignancy of NSCLC. In the present study, we found that CLDN1 expression correlated with LAT1 and LAT3 expression in A549 spheroids (Fig. 4). It is unclear whether LAT3 is associated with malignancy in NSCLC, but it is reported to be a prognosis marker for gastric 25, liver 26, and prostate cancers 27.
Inner cancer cells in the tumor microenvironment are generally exposed to malnutrition, oxidative stress, and hypoxia stress, leading to malignant transformations. LATs transport branched-chain AAs, especially leucine, into cells, resulting in the promotion of cell proliferation mediated by activation of the mammalian target of rapamycin (mTOR) pathway. Information about transcriptional regulation of LATs is limited 28. The induction of hypoxia-induced transcriptional regulators, HIF-1 and HIF-2, upregulates LAT1 expression in human glioblastoma cells 29. Glucose deprivation induces LAT1 expression in cultured rat retinal capillary endothelial cells 30. We found that both LAT1 and LAT3 expression levels in 2D cultured A549 cells were increased by extracellular AA deficiency, but not by hypoxia and glucose deficiency (Fig. 2). Thus, the regulatory mechanism of LAT1 expression in A549 cells may be different from the known regulatory mechanisms. We found that AA deficiency increases ROS generation and expression of the oxidative stress response element Nrf2 (Fig. 7B). The transcription of LAT1 has been reported to be increased by activation of the Nrf2 pathway in the striatum 31. Expression of LAT1 in A549 cells may be upregulated by the ROS-dependent Nrf2 pathway.
CLDN1 forms a paracellular barrier to aqueous small molecules 32. The regulatory mechanisms of AA flux by CLDNs are not fully understood. We recently reported that CLDN4 and CLDN8 silencing especially enhances basic AAs and middle molecular size AAs in mouse colonic epithelial cells, respectively 33,34. On the other hand, the current data indicate that CLDN1 silencing enhances paracellular AA flux except for Ser, Thr, and Tyr in A549 cells (Fig. 3C). The common property of these AAs is that they are neutral AAs and have a hydroxy residue. The second extracellular loop domain of CLDN1 contains more hydrophobic AAs than those in CLDN4 and CLDN8 8. Interaction of the second extracellular loop domain of CLDN1 and free AAs with a hydroxy residue may be weaker than that of CLDN4 and CLDN8.
CLDN1 silencing enhances sensitivity to DXR in A549 spheroids, but the mechanisms have not been clarified in detail. Our previous data indicated that CLDN1 blocks the influx of DXR into A549 spheroids 12. Here, we found that CLDN1 may function as a paracellular barrier to AAs (Fig. 3B). mTORC1, which can be activated by extracellular AAs and growth factors, is a negative regulator of autophagy 28. In response to the AA depletion, various cancer cells activate autophagy to overcome nutrient stress. There is a possibility that CLDN1 enhances chemoresistance mediated by the induction of autophagy because CLDN1 may block the influx of AAs into spheroids. However, this hypothesis was not supported because the AA contents in control spheroids were higher than that in CLDN1-silenced spheroids (Fig. 4E). CLDN1 silencing attenuates the expression of Nrf2 and downstream antioxidant genes (Fig. 6). DXR-induced cytotoxicity was enhanced by CLDN1 silencing in A549 spheroids, which was blocked by the Nrf2 activator sulforaphane (Fig. 8). Furthermore, DXR-induced cytotoxicity was enhanced by AA deficiency and sulforaphane treatment of 2D cultured cells. Cancer cells undergo reprogramming of glucose, fatty acid, and AA metabolism to survive and grow in stress conditions 35. The expression of Nrf2 has been reported to be correlated with mitochondrial redox status 36. Our data indicated that CLDN1 silencing downregulates mitochondrial ROS generation (Fig. 5B). We suggest that Nrf2 is involved in CLDN1-induced chemoresistance in A549 spheroids.
Saito et al. 37 reported that the cytotoxicity of gefitinib, a small-molecule epidermal growth factor receptor tyrosine kinase inhibitor, is enhanced by AA starvation in A549 cells, which is different from our results with DXR (Fig. 8B). This discrepancy may be caused by a difference in AA concentration; they used completely AA-deficient medium, whereas we used 50% AA-deficient medium. The AA content in spheroids was not completely zero regardless of whether CLDN1 was present or not (Fig. 4E). We also identified that AA starvation (0% AA) enhanced DXR-induced toxicity in A549 cells (data not shown). Our data suggest that mild deficiency of AAs induces chemoresistance in lung adenocarcinoma cells.
In conclusion, we found that both LAT1 and LAT3 expression levels were upregulated by AA deficiency in A549 cells. CLDN1 can function as a paracellular barrier to AAs except for Ser, Thr, and Tyr. The expression of LAT1 and LAT3, mitochondrial activity, ROS production, Nrf2 expression, and antioxidant enzyme expression were downregulated by CLDN1 silencing in A549 spheroids. The cytotoxicity of DXR was suppressed by extracellular AA deficiency and treatment with an Nrf2 activator. We suggest that CLDN1 enhances chemoresistance of lung adenocarcinoma cells mediated by a restriction in AA supply.