Fig 2: GCN2 regulates amino acid transporters ensuring sufficient amino acids for tumor growth in mouse prostate cancer (PCa) xenograft models.(A) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) (N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p = 0.05; ****p = 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t-test. Error bars indicate standard deviation (SD); **p = 0.01. (B) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t-test. Error bars indicate SD (N = 4); *p = 0.05; ***p = 0.001. (C) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM (N = 5); ***p = 0.001; ****p = 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t-test. Error bars indicate SD (N = 5); *p = 0.01. (D) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t-test. Error bars indicate SD (N = 4); **p = 0.01. (E) 22Rv1 WT (N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO (N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. (F) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD (N = 4); #p = 0.1, *p = 0.05. (G) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for (A), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT (N = 4) and 22Rv1 KO (N = 5) are the same tumor growth curves shown in (E). 22Rv1 WT + EAA (N = 5), 22Rv1 GCN2 KO + EAA (N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p = 0.0001. (H) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD (N = 4); #p = 0.1; *p = 0.05; **p = 0.01. (I) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM (N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p = 0.0001.

Fig 3: SLC3A2 promotes cell proliferation in ER+ breast cancer cells.a Kaplan–Meier plots of ER+/PR+ and ER−/PR− BC patients’ survival. b Overexpression of SLC3A2 in MCF-7 cells. c Cell viability assay of SLC3A2-OE MCF-7 cells. d Knockdown of SLC3A2 in MCF-7 cells. e Cell viability assay of SLC3A2-KD MCF-7 cells. f 2D growth of SLC3A2-KD MCF-7 cells. g Tumor growth of SLC3A2-KD MCF-7 cells in NSG mice. h Total protein levels of SLC7A5 in SLC3A2-KD MCF-7 cells. i Total protein levels of SLC3A2 in SLC7A5-KD MCF-7 cells. j Heatmap of metabolites in SLC3A2-KD MCF-7 cells. k Venn diagram representation of the number of intracellular metabolites downregulated by SLC3A2-KD in MCF-7 and T47D cells (left) and downregulated in both SLC3A2-KD and SCRIB-KD cells (right). l List of metabolites downregulated by SLC3A2-KD and SCRIB-KD. Data c, e, f, and g are shown as mean ± s.e.m.; c, and e; n = 9 from three biological replicates performed in triplicate, f; n = 3, g; n = 5 for control cells and n = 4 for SLC3A2-KD cells. Statistical analysis was conducted by t-test (c, e), and two-way ANOVA followed by Tukey’s posttest (f) or Sidak’s posttest (g).

Fig 4: Expression of CD98hc in normal and TNBC tumoral tissue. A Immunohistochemical stainings of CD98hc showing tumor tissue scored as high (+++), intermediate (++) and low (+), and normal tissue with negative (-) staining. Magnification: 40X. B Immunohistochemical staining of CD98hc in a sample of a patient, showing staining of CD98hc only in tumor tissue and not in normal tissue. Magnification: 20 X. C Box plot showing SLC3A2 gene expression levels in normal and tumor tissue of breast cancer patients. Data were obtained from the TNMplot database. D Box plot showing SLC3A2 gene expression levels in different breast cancer subtypes from patients with breast cancer. The data were obtained using the Xena online tool

Fig 5: SCRIB regulates intracellular localization of SLC3A2 in ER+ breast cancer cells.a Immunohistological images of SLC3A2 in ER+ and ER− breast cancer. The numbers of specimens were indicated in each image. b Total protein levels of SLC3A2 and SLC7A5 in breast cancer cell lines. Immunoblot images (left) and the relative ratio of signal intensities are shown (right). c Cellular surface protein levels of SLC3A2 and SLC7A5 in breast cancer cell lines. d Normalized intensity of surface protein levels of SLC7A5 and SLC3A2 in ER+ or ER− breast cancer cells. e Immunoprecipitation of HA-SCRIB using anti-HA antibody in HEK293T cells. f Total protein levels of SLC7A11 in breast cancer cell lines. Immunoblot image (left) and the relative ratio of signal intensities are shown (right).