Fig 1: KISS1R expression promotes primary breast tumor growth and glutamine metabolism in an orthotopic xenograft model.Six-week-old immunocompromised mice were injected with SKBR3pFLAG controls (2 × 106 cells/mouse) or SKBR3FLAG-KISS1R cells (106 cells/mouse) into the mammary fat pad. a Primary tumor volume after 6 weeks for animals injected with SKBR3FLAG-KISS1R cells and 8 weeks for animals injected with SKBR3pFLAG controls. b Representative images of orthotopic primary tumors subjected to hematoxylin and eosin, antihuman cytochrome C oxidase, or antihuman glutaminase (GLS1); magnified section in white boxed area shown in image below. Metabolites in primary breast tumors (c, d) and serum (e, f), respectively, from xenografts: c, e glutamate and d, f glutamine levels measured by LC–MS. Student’s unpaired t test, *p < 0.05. (n = 4 mice for SKBR3FLAG-KISS1R xenografts; n = 3 for pFLAG control xenografts).
Fig 2: KISS1R expression promotes purine synthesis.a Diagram of the purine synthesis pathway, including the amide nitrogen glutamine tracer. Diamonds indicate nitrogen, labeled for amide (red) and unlabeled (white). Diamond with subscript a denotes the alpha nitrogen of glutamine. Fractional enrichment of glutamine’s amide nitrogen contributing to metabolites in purine synthesis in SKBR3FLAG-KISS1R and pFLAG control cells (n = 3): b IMP, c ATP, and d 5-methylthioadenosine. Metabolite levels in orthotopic primary tumors of e 5-methylthioadenosine and f xanthosine-5'-phosphate and g serum guanosine levels in SKBR3FLAG-KISS1R and SKBR3pFLAG control xenografts (n = 4 mice for FLAG-KISS1R xenografts; n = 3 for pFLAG controls). Mean ± SEM shown. *p < 0.05, Student’s unpaired t test.
Fig 3: KISS1R expression regulates primary breast tumor growth and metastasis in xenograft models.a–e Depletion of endogenous KISS1R in human metastatic triple-negative MDA-MB-231 breast cancer cells inhibits primary tumor growth and lung metastases in a spontaneous metastasis xenograft model. a Representative western blot showing KISS1R expression in lysates from MDA-MB-231 cells stably expressing KISS1R shRNA and scrambled control used in xenograft experiments; ß-actin, loading control. Densitometric analysis of blots shown in Supplementary Fig. 1b. b Primary orthotopic tumor volumes: mouse mammary fat pad, 106 cells/mouse, n = 5 mice/group. Points represent each mouse mean tumor volume and bars represent mean volume ± SEM. c Representative images of lung metastasis (outlined in green, left panels) subjected to either hematoxylin and eosin, antihuman Ki67 (middle panels), or human anti-cytochrome C oxidase. Scale bar, 100 µm. The extent of lung metastasis measured by (d) number of lung metastases (normalized to 100 mm2) lung and (e) tumor burden in the lung (percentage of lung occupied by tumor); four sections/tissue/mouse, n = 5 mice per group). Bars represent lung area and number of metastases ± SEM, respectively. One-way ANOVA followed by Dunnett’s multiple comparison test: *p < 0.05. f KISS1R overexpression promotes lung colonization in a experimental metastasis xenograft model. Lung metastases (n = 5 mice/group, outlined in green) formed by human ERa-negative SKBR3 breast cancer cells stably expressing FLAG-KISS1R or pFLAG vector control. Bars represent mean ± SEM. Metastases quantified blindly in hematoxylin and eosin and human anti-cytochrome C oxidase stained slides. Student’s unpaired t test: *p < 0.05. Scale bars, 200 µm.
Fig 4: KISS1R induces c-Myc expression to regulate glutamate production via MAPK.a c-MYC mRNA and protein expression in SKBR3FLAG-KISS1R cells expressing c-MYC siRNA or control siRNA (Con) constructs, 72 h after transfection. (n = 5). b Representative images of the scratch assay to assess cell motility of SKBR3FLAG-KISS1R cells transfected with c-MYC siRNA or control siRNA at 0 and 18 h (n = 3). Scale bars, 300 µm. Scrambled cells migrated 548.2 ± 14.72 µM. Bars show mean ± SEM. Student’s unpaired t test, *p < 0.05. c-Myc-suppressed SKBR3FLAG-KISS1R cells are resistant to glutamine starvation. Cells expressing c-MYC siRNA were plated in the presence of glutamine and then cultured in the absence of glutamine and counted each day to assess cell proliferation. Differences in cell count are expressed as fold change, calculated by dividing the number of cells from each corresponding day, by that of day 0 *p < 0.05; two-way ANOVA with multiple comparisons followed by Bonferroni post hoc test. Mean and SEM shown (n = 3). a, c control vs. siMYC1; b, d control vs. siMYC2. d SLC1A5 mRNA expression in SKBR3FLAG-KISS1R cells expressing c-MYC siRNA or control siRNA. (n = 5), e c-MYC mRNA and protein expression, and f SLC1A5 mRNA in MDA-MB-231 cells expressing c-MYC siRNA or control siRNA constructs, 72 h after transfection. (n = 5). g Representative western blots showing endogenous c-Myc expression in SKBR3FLAG-KISS1R cells treated with vehicle, 10 µM U0126 (ERK1 and ERK2 inhibitor), and 10 µM U0124 (inactive U0126 analog). Densitometric analysis of blots shown for c-Myc normalized to housekeeping (GAPDH) and p-ERK normalized to total ERK expression (n = 3). h Glutamate production rate measured in the unlabeled conditioned media of SKBR3FLAG-KISS1R cells grown in the presence of U0126, U0124 or vehicle (n = 3). i Schematic of the KISS1R signaling pathway in cancer metabolism. Pathway shown in red is a potential mechanism by which KISS1R promotes glutamine uptake by the tumors to thereby regulate tumorigenesis.
Fig 5: KISS1R expression promotes glutaminolysis, increases glutamine flux to tricarboxylic acid (TCA) cycle metabolites, and stimulates glutamine-dependent nucleotide synthesis.a Production rate of glutamate measured in the conditioned media after 4 or 16 h of culturing SKBR3pFLAG controls or SKBR3FLAG-KISS1R cells grown in serum-free media (unlabeled). b Schematic showing the transfer of carbon atoms of the 13C5-labeled glutamine tracer used to detect glutamine flux into TCA cycle intermediates; labeled carbon atoms (red) and unlabeled carbon atoms (white). c Glutamate pool size from 13C5 glutamine tracer in cells. d–h Relative fractions of 13C5 glutamine tracer in TCA cycle metabolites d citrate, e aconitate, f fumarate, and g malate. Normalization of pool size was done to pack cell volume per dish. Mean ± SEM shown (n = 3). *p < 0.05, Student’s unpaired t test. h Schematic of the pyrimidine synthesis pathway showing the contribution of the amide nitrogen using a 15N-glutamine tracer. Diamonds indicate nitrogen, labeled for amide (red) and unlabeled (white). Diamond with subscript a denotes the alpha nitrogen of glutamine. i Representative western blot of the CAD enzyme complex in lysates from SKBR3FLAG-KISS1R and SKBR3pFLAG vector control cells. See Supplementary Fig. 2f for quantification of blots. Fraction of glutamine’s amide nitrogen contributing to pyrimidine synthesis metabolites: j UMP and k uracil in cultured SKBR3FLAG-KISS1R and SKBR3pFLAG control cells (n = 3). Levels of pyrimidine synthesis metabolites measured by LC–MS in orthotopic breast tumors and serum from xenografts: l serum uridine; primary tumor (m), UTP (n) ribose phosphate. Student’s unpaired t test, *p < 0.05. (n = 4 mice for SKBR3FLAG-KISS1R xenografts; n = 3 for SKBR3pFLAG control xenografts).
Supplier Page from Abcam for Anti-GPR54 antibody