Nortriptyline Inhibits Lysosomal Exocytosis-Mediated SASP During Gastric Cancer Progression via Targeting HOXA1-PITX2 Phase Separation

Abstract

Lysosomal exocytosis, a calcium-dependent secretory process, facilitates extracellular release of cargos that promote cancer progression, though its regulatory pathways and therapeutic strategies are poorly understood. Herein, using combined transcriptomic and proteomic approaches, we identify homeobox A1 (HOXA1) as a functional partner of paired like homeodomain 2 (PITX2) within biomolecular condensates forming via liquid-liquid phase separation. Mechanistically, HOXA1-PITX2 complex facilitates the expression of mucolipin 1 (MCOLN1) and RAS-related protein Rab-3A (RAB3A), which drive lysosomal exocytosis of galectin-1 (LGALS1) and insulin like growth factor binding protein 7 (IGFBP7) from senescent gastric cancer cells. This process potentiates AKT activation and epithelial-mesenchymal transition, accelerating tumorigenesis and aggressiveness of gastric cancer. Molecular docking and affinity purification assays reveal nortriptyline (Nor) as a potent phase separation disruptor of HOXA1-PITX2 complex. Preclinical studies demonstrate that Nor administration attenuates lysosomal exocytosis-mediated senescence-associated secretory phenotype (SASP) and reduces aggressive phenotypes in gastric cancer models, underscoring the HOXA1/PITX2 axis as a critical regulator of gastric cancer progression. Clinically, elevated expression of HOXA1, PITX2, MCOLN1, RAB3A, LGALS1, and IGFBP7 constitutes a prognostic signature correlating with poor outcomes of gastric cancer patients. Collectively, these results indicate that Nor impedes gastric cancer progression by suppressing HOXA1-PITX2 phase separation and subsequent lysosomal exocytosis-mediated SASP.

Keywords: cancer progression; homeobox A1; lysosomal exocytosis; paired like homeodomain 2; senescence‐ associated secretory phenotype.

Figure 1

PITX2 facilitates gene expression essential for lysosomal exocytosis in gastric cancer. A) Volcano plot of RNA‐seq assay indicating the differentially expressed genes (fold change >2, p <0.05) in gastric cancer AGS cells treated with complete medium (CM) or serum deprivation (SD) for 10 days (n = 3). B) GSEA revealing the involvement of differentially expressed genes in regulation of lysosomal lumen or exocytosis. C) Venn diagram (left panel) and Sankey diagram (right panel) showing the identification of differentially expressed lysosomal exocytosis genes and their potential transcription factors (TF) analyzed by ChIP‐X and JASPAR (http://jaspar.genereg.net/) programs. D) GSEA indicating PITX2 targets in differentially expressed genes of gastric cancer AGS cells treated with CM or SD for 10 days. E) Western blot assay showing the PITX2 levels in cultured HEK293T cells and cancer cell lines. F) Heatmap of real‐time qRT‐PCR assay indicating the levels (normalized to β‐actin, n = 5) of DOC2A, MCOLLN1, RAB3A, SNAPIN, SYNGR1, VAMP7, and VI1B in AGS and HGC‐27 cells stably transfected with empty vector (mock), PITX2, scramble shRNA (sh‐Scb), sh‐PITX2 #1, or sh‐PITX2 #2 under CM or SD condition. G‐I) ChIP‐qPCR assay (G, normalized to input, n = 3), dual‐luciferase (H, n = 3), and western blot (I) assays showing the PITX2 enrichment on MCOLLN1 or RAB3A promoter region, PITX2 activity, and protein levels of MCOLLN1 and RAB3A in AGS and HGC‐27 cells stably transfected with mock, PITX2, sh‐Scb, sh‐PITX2 #1, or sh‐PITX2 #2 under CM or SD condition. Fisher's exact test for overlapping analysis in C. Student's t‐test or one‐way ANOVA compared the difference in G and H. Data are shown as mean ± s.e.m. (error bars); ^*, p <0.05; ^**, p <0.01.

Figure 2

PITX2 promotes lysosomal exocytosis of senescent gastric cancer cells via up‐regulating MCOLN1 and RAB3A. A) Representative images (left panel) and quantification (right panel) of Fluo 8‐AM staining (arrowheads) in AGS and HGC‐27 cells stably transfected with empty vector (mock), PITX2, scramble shRNA (sh‐Scb), sh‐PITX2 #1, or sh‐PITX2 #2 under SD condition, and those treated with ML‐SI3 (10 µmol·L⁻¹) or ML‐SA1 (20 µmol·L⁻¹), with nuclei staining by DAPI. Scale bars: 10 µm. B) Quantification of Lyso‐Tracker Red staining (left panel) showing the number and anterograde transport distance of lysosomes in AGS and HGC‐27 cells stably transfected with mock, PITX2, sh‐Scb, sh‐PITX2 #1, or sh‐PITX2 #2 under SD condition (n = 15), and those treated with ML‐SI3 (10 µmol·L⁻¹) or ML‐SA1 (20 µmol·L⁻¹). Schematic diagram (right panel) for calculating the distance of lysosome to plasma membrane. C) Confocal images and quantification of Lyso‐Tracker Red staining (left panel, n = 15) and western blot (right panel) assays indicating the lysosomal anterograde transport and expression levels of PITX2 or RAB3A in living AGS cells stably transfected with mock or PITX2, and those co‐transfected with scramble siRNA (si‐Scb) or si‐RAB3A. Scale bars: 10 µm. D) Representative images of LAMP1 and Dil staining in AGS cells stably transfected with mock or PITX2 under SD condition, and those treated with vacuolin‐1 (Vac, 1.0 µmol·L⁻¹). Scale bars: 10 µm. E) Transmission electron microscopic observation (upper panel) and quantification (lower panel, n = 15) of lysosomal vesicles (red arrowheads) in AGS cells stably transfected with mock or PITX2 under SD condition, and those treated with Vac (1.0 µmol·L⁻¹). Scale bars: 2 µm. F) Western blot assay indicating the plasma membrane or cytoplasmic expression of LAMP1 in AGS cells stably transfected with mock or PITX2 under SD condition, and treated with Vac (1.0 µmol·L⁻¹). One‐way ANOVA compared the difference in A‐C and E. Data are shown as mean ± s.e.m. (error bars); ^*, p <0.05; ^**, p <0.01.

Figure 3

PITX2 drives tumorigenesis and aggressiveness of gastric cancer via lysosomal exocytosis‐mediated SASP. A) Venn diagram showing the over‐lapping analysis of altered proteins in mass spectrometry (MS) analysis of culture medium from AGS cells with stable PITX2 over‐expression or vacuoloin‐1 (1.0 µmol·L⁻¹) treatment under SD condition, SASP proteins in SASP Atlas database (http://www.SASPAtlas.com), and reported secretome. B) Western blot assay indicating the levels of p‐AKT³⁰⁸, p‐AKT⁴⁷³, E‐cadherin, N‐cadherin, or Vimentin in AGS cells with stably transfected with empty vector (mock) or PITX2 under SD condition, and those treated with neutralizing antibody against sLGALS1 or IGFBP7. C and D) Representative images (left panel) and quantitative (right panel) of soft agar (C) and matrigel invasion (D) assays showing the growth and invasion of AGS cells treated with culture medium from those stably transfected with mock or PITX2 under SD condition, and treated with vacuoloin‐1 (Vac, 1.0 µmol·L⁻¹) and neutralizing antibody against sLGALS1 or IGFBP7 (n = 5). E) Representative images (left upper panel), growth curve (right upper panel), weight at the end points (lower panel), LGALS1 and IGFBP7 secretion (lower panel), and Ki‐67 or CD31 expression (lower panel) of xenograft tumors formed by subcutaneous injection of AGS or HGC‐27 cells in nude mice (n = 5 for each group) that subsequently treated with intravenous injection of culture medium collected from senescent cells stably transfected with mock, PITX2, scramble shRNA (sh‐Scb), or sh‐PITX2 #1, with or without Vac (1.0 µmol·L⁻¹) treatment. F‐H) Representative images (F), hematoxylin‐eosin (HE) staining (G, arrowheads), quantification of lung metastatic colonization (H), and Kaplan‐Meier curves (H) of nude mice (n = 5 for each group) receiving vein tail injection of AGS or HGC‐27 cells that subsequently treated with intravenous injection of culture medium collected from senescent cells stably transfected with mock, PITX2, sh‐Scb, sh‐PITX2 #1, with or without Vac (1.0 µmol·L⁻¹) treatment. One‐way ANOVA or Student's t‐test compared the difference in C‐E and H. Log‐rank test for survival comparison in H. Data are shown as mean ± s.e.m. (error bars); ^*, p <0.05; ^**, p <0.01.

Figure 4

HOXA1 interacts with PITX2 protein to facilitate lysosomal exocytosis‐related gene expression in gastric cancer cells. A) Venn diagram showing the identification of consistent PIXT2 partners via comprehensive analysis of public databases HuRI (http://www.interactome‐ atlas.org/), InBioMap (https://inbio‐discover.com/), BioGRID (https://thebiogrid.org/), and IID (http://iid.ophid.utoronto.ca/). B) Co‐IP and Western blot analysis revealed endogenous interactions between PITX2 and HOXA1 proteins in HGC‐27 cells. IgG binding protein was used as a negative control. C) Representative images (upper panel) and quantification (lower panel) of immunofluorescence showing co‐localization of PITX2 and HOXA1 in AGS cells, as well as in those stably transfected with empty vectors (mock), HOXA1, or PITX2. Scale bars: 10 µm. D) Co‐IP and Western blot assays indicating interaction between GST‐tagged HOXA1 and MBP‐tagged PITX2 truncation proteins as indicated. E) Representative images of BiFC assay showing physical interaction (arrowheads) of PITX2 and HOXA1 in AGS cells co‐transfected with VN173‐HOXA1 and VC155‐PITX2. Scale bars: 10 µm. F) Western blot assay showing the levels of target genes MCOLN1 and RAB3A in HGC‐27 and AGS cells stably transfected with mock, HOXA1, scramble shRNA (sh‐Scb), or sh‐HOXA1 #1, and those co‐transfected with sh‐PITX2 #1 or PITX2. Data are shown as representative of three independent experiments in B‐F.

Figure 5

PITX2 and HOXA1 form liquid condensates in gastric cancer cells. A) IDRs within PITX2 and HOXA1 proteins analyzed by PONDR (http://www.pondr.com/) program. B) Fluorescence imaging assay indicating the condensate formation (circles) of EGFP‐PITX2 and HOXA1 in AGS cells stably transfected with wild‐type or IDR deficient (∆IDR) PITX2 construct, and those treated with SD. Scale bars: 10 µm. C) Fluorescence imaging assay indicating the condensate formation (circles) of EGFP‐PITX2 and HOXA1 in AGS cells treated with DMSO or 1.5% 1,6‐Hex. D) Representative images of droplet formation of wild‐type or IDR deficient (∆IDR) EGFP‐PITX2 and HOXA1‐mCherry in droplet formation buffer. Scale bars: 10 µm. E) Representative images of droplet formation of EGFP‐PITX2 and HOXA1‐mCherry in the presence or absence of 1.5% 1,6‐Hex. Scale bars: 10 µm. F) Representative images (left panel) and quantification (right panel) of FRAP assay showing the exchange kinetics (circles) of EGFP‐PITX2 and HOXA1 in AGS cells stably transfected with PITX2 construct. Scale bars: 10 µm. G) Representative images (left panel) and quantification (right panel) of FRAP assay showing the exchange kinetics (circles) of EGFP‐PITX2 and HOXA1‐mCherry proteins within condensates. Scale bars: 10 µm. Data are shown as representative of three independent experiments in B‐G.

Figure 6

HOAX1/PITX2 liquid condensates promote gastric cancer progression via lysosomal exocytosiss. A and B) Representative images (left panel) and quantification (right panel) of soft agar (A) and matrigel invasion (B) assays indicating anchorage‐independent growth and invasion capability of MKN‐45 cells treated by culture medium form senescent cells stably transfected with scramble shRNA (sh‐Scb) or sh‐HOXA1 #1, and those co‐transfected with empty vector (mock) or PITX2 (n = 5). C and D) Representative images (C, left panels), growth curve (C, middle panel), weight at the end points (C, middle panel), LGALS1 or IGFBP7 secretion (C, right panel), and immunohistochemical staining of Ki‐67 and CD31 (D, arrowheads) within tumor xenografts formed by subcutaneous injection of MKN‐45 cells in nude mice that subsequently treated with intravenous injection of culture medium collected from senescent cells stably transfected with sh‐Scb or sh‐HOXA1 #1, and those co‐transfected with mock or PITX2 (n = 5 for each group). Scale bars: 100 µm. E and F) Representative images (E), hematoxylin‐eosin (HE) staining (F, arrowheads), quantification of lung metastatic colonization (F), and Kaplan‐Meier curves (F) of nude mice (n = 4 for each group) receiving vein tail injection of MKN‐45 cells that subsequently treated with intravenous injection of culture medium collected from senescent cells stably transfected with sh‐Scb or sh‐HOXA1 #1, and those co‐transfected with mock or PITX2. One‐way ANOVA compared the difference in A‐D and F. Log‐rank test for survival comparison in F. Data are shown as mean ± s.e.m. (error bars); ^*, p <0.05; ^**, p <0.01.

Figure 7

Nortriptyline inhibits the interaction and phase separation of HOXA1 and PITX2 in gastric cancer cells. A) Schematic illustration (left panel), MTT colorimetric assay (middle panel), and dual‐luciferase assay (right panel) indicating the screening of PITX2 activity inhibitor from 1495 FDA‐approved drugs. B) BiFC assay using VN173‐HOXA1 and VC155‐PITX2 constructs (left panel) and molecular docking (right panel) via CB‐Dock2 program (http://183.56.231.194:8001/cb‐dock2) showing the screening of compounds repressing the HOXA1‐PITX2 interaction, as well as potential interaction of nortriptyline with PITX2 protein. C) Validating co‐IP and western blot assays indicating the binding of PITX2 to HOAX1 in HGC‐27 cells treated with DMSO or nortriptyline (Nor, 40 µmol·L⁻¹). D) Silver staining (left panel) and western blot (right panel) assays indicating affinity of proteins within HGC‐27 cell lysates or recombinant proteins with FG beads covalently conjugated with Nor (40 µmol·L⁻¹). E) DSF assay and temperature showing the fluroresence intensity of HEPES or recombinant HOXA1 or PITX2 protein treated with solvent (DMSO) or Nor (40 µmol·L⁻¹, n = 5). F) Representative images of droplet formation of EGFP‐PITX2 and HOXA1‐mCherry proteins incubated with vehicle or Nor (40 µmol·L⁻¹). Scale bar: 5 µm. G) Fluorescence imaging assay indicating the condensate formation (circles) of EGFP‐PITX2 and HOXA1 in AGS cells stably transfected with PITX2 construct, and those treated with vehicle or Nor (40 µmol·L⁻¹). Scale bars: 10 µm. H) ChIP‐qPCR (normalized to input, n = 3) and dual‐luciferase reporter (n = 3) assays showing the PITX2 enrichment on MCOLLN1 or RAB3A promoter regions as well as PITX2 activity in MKN‐45 cells stably transfected with empty vector (mock), PITX2, or HOXA1, and those treated with vehicle or Nor (40 µmol·L⁻¹). I and J) Heatmap of real‐time qRT‐PCR (I, normalized to β‐actin, n = 5) and western blot (J) assays indicating the transcript and protein levels of MCOLLN1 and RAB3A in MKN‐45 cells stably transfected with mock, PITX2, or HOXA1, and those treated with vehicle or Nor (40 µmol·L⁻¹). Student's t‐test or one‐way ANOVA compared the difference in E and H. Data are shown as mean ± s.e.m. (error bars); ^*, p <0.05; ^**, p <0.01; ns, non‐significant.

Figure 8

Nortriptyline suppresses lysosomal exocytosis and progression of gastric cancer. A) Representative images (left panel) and quantification (right panel) of LAMP1 and Dil staining in HGC‐27 cells treated with vehicle or Nor (40 µmol·L⁻¹). Scale bars: 10 µm. B) ELISA showing the levels of LGALS1 and IGFBP7 within culture medium of MKN‐45 cells stably transfected with empty vector (mock), PITX2, or HOXA1, and those treated with vehicle or Nor (40 µmol·L⁻¹, n = 3). C and D) Representative images (left panel) and quantitative (right panel) of soft agar (C) and matrigel invasion (D) assays showing the growth and invasion of MKN‐45 cells treated with culture medium from those stably transfected with mock, PITX2, or HOXA1, and those treated with vehicle or Nor (40 µmol·L⁻¹, n = 3). E and F) Representative images (E), growth curve (E), weight at the end points (E), LGALS1 or IGFBP7 secretion (E, right panel), and immunohistochemical staining of Ki‐67 and CD31 (F, arrowheads) within tumor xenografts formed by subcutaneous injection of HGC‐27 cells in nude mice that subsequently treated with Nor (50 mg·kg⁻¹, n = 5 for each group). Scale bars: 100 µm. G) Western blot assay showing the expression levels of PITX2, HOAX1, p‐AKT³⁰⁸, p‐AKT⁴⁷³, E‐cadherin, N‐cadherin, or vimention in xenograft tumors formed by subcutaneous injection of HGC‐27 cells in nude mice that subsequently treated with Nor (50 mg·kg⁻¹, n = 5 for each group). H) Representative images and hematoxylin‐eosin (HE) staining of lung metastatic colonization (arrowheads) of nude mice (n = 4 for each group) receiving vein tail injection of HGC‐27 cells and subsequent intravenous administration of Nor (50 mg·kg⁻¹). I) Schematic depicting the mechanisms underlying HOAX1/PITX2‐regulated cancer progression: as a transcription factor, PITX2 facilitates the expression of lysosomal exocytosis genes (MCOLN1 and RAB3A), while HOXA1 interacts with PITX2 to facilitate its activity, resulting in increase of lysosomal exocytosis‐mediated SASP (LGASL1 or IGFBP7) and subsequent activation of AKT signaling and EMT. Meanwhile, nortriptyline (Nor) is able to block HOXA1‐PITX2 interaction, resulting in inhibition of lysosomal exocytosis, tumorigenesis, and aggressiveness. Student's t test or one‐way ANOVA compared the difference in A‐F. Data are shown as mean ± s.e.m. (error bars); ^*, p <0.05; ^**, p <0.01.