p140Cap inhibits β-Catenin in the breast cancer stem cell compartment instructing a protective anti-tumor immune response

Abstract

The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of β-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of β-Catenin depends on its ability to localize in and stabilize the β-Catenin destruction complex, promoting enhanced β-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the β-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response.

Fig. 1. A positive p140Cap status directly correlates with the presence of TILs and inversely with inflammatory hallmarks in human BC.

a Representative images of p140Cap and H&E staining, showing TILs, in HER2 negative and HER2 positive tumor breast tissues from 390 female patients. Black arrows point to TILs; red arrows indicate to cancer. Mag, magnifications. Bars, 200, 100 µm. b Quantitative analysis of the presence of TILs (positive or negative) according to p140Cap status (p140Cap^LOW, IHC score <1; p140Cap^HIGH, IHC score ≥1), evaluated on H&E stained TMA cores in All patients, as well as in HER2-negative and HER2-positive patients. OR odds ratio, CI confidence interval; p-value by Pearson’s Chi-Squared Test. c GSEA^, plot showing the behavior of the “Hallmark_Inflammatory_Response” Gene Set from MSigDB~5.0 in the cohort of 1095 BC female patients from TCGA , stratified according to SRCIN1 transcript levels. The plot shows the distribution of genes in the set that are correlated or not with SRCIN1 expression; two-tailed unpaired t test.

Fig. 2. Reduced tumor growth and lung metastasis in p140Cap expressing cells and composition of the immune tumor microenvironment in mock and p140Cap tumors.

a Effect of p140Cap over-expression on tumor growth and metastasis in TuBo and 4T1 BC cell models. TuBo (10⁵) or 4T1 (10⁴) mock and p140Cap cells were inoculated into the fat pad of 8-weeks-old female BALB/c mice. Tumor growth was monitored and tumor size was measured every two days from tumor onset (TuBo, n = 11; 4T1, n = 5; data are represented for n = x mices, two-tailed unpaired t test). For metastasis analysis in TuBo mock and p140Cap tumor-bearing mice, tumors were surgically removed when they reached 10 mm diameter and mice were kept alive. After 5 weeks, mice were sacrified and lungs were explanted and analyzed. For metastasis analysis of 4T1 mock and p140Cap tumors-bearing mice, lungs were analyzed 22 and 30 days post-injection for mock and p140Cap cells, respectively. Representative dot plots show the number of lung metastasis as mean ± SEM (Standard Error of the Mean; TuBo, n = 5; 4T1, n = 10; 2way ANOVA). b, c Flow cytometry analysis for M1- and M2-macrophages, CD4⁺ and CD8⁺ T-Lymphocytes, Natural Killer cells in tumor-bearing mice. Representative dot plots show the percentage (%) of tumor infiltrated M1- and M2-macrophages, CD4⁺ and CD8⁺ T-Lymphocytes, Natural Killer (NK) cells, normalized on CD45⁺ cells in TuBo mock and p140Cap tumor-bearing mice in panel (b) (n = 8/M1, CD4⁺ and CD8⁺ and n = 9/NK, n = 5/M2) and 4T1 mock and p140Cap tumor-bearing mice in panel (c) (n = 5/group). Data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test). For TuBo mock and p140Cap tumors, the analysis was performed at day 26 or 32, respectively, while for 4T1 mock and p140Cap tumors at day 12. d Flow cytometry analysis for PMN-MDSCs (CD11b⁺Ly6G⁺Ly6C^low) and for M-MDSCs (CD11b⁺Ly6G⁻Ly6C⁺) normalized on CD45⁺ cells, in tumor-bearing mice. Representative dot plots show the percentage of tumor infiltrated PMN-MDSCs and M-MDSCs cells in TuBo and 4T1 mock and p140Cap tumor-bearing mice, as described in panels (b, c) (TuBo n = 8/group; 4T1 n = 7/PMN-MDSCs and n = 8/M-MDSCs). Data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test.

Fig. 3. G-CSF expression is strongly reduced in p140Cap positive BC model.

a RNASeq Data Analysis was performed on the transcriptomic profiles of mock- vs. p140Cap-expressing TuBo cells. Shown is the list of 18 differentially expressed genes (provided by BIOCARTA) involved in the inflammatory response upon p140Cap expression. b, c p140Cap modulates G-CSF expression and secretion. b G-CSF transcript was analyzed by quantitative real-time RT-PCR in mock and p140Cap TuBo (n = 4) and 4T1 cells (n = 3). G-CSF protein levels were measured in mock and p140Cap TuBo (n = 4) and 4T1 (n = 3) cell culture supernatants by ELISA. Data are represented for n = x experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. c G-CSF transcript levels were analyzed by RT-PCR in mock and p140Cap TuBo and 4T1 tumors. Data are represented for n = 4 mice/group as mean ± SEM; two-tailed unpaired t test.

Fig. 4. p140Cap expression affects systemic distribution of PMN-MDSCs.

a, b Flow cytometry analysis for PMN-MDSCs (CD11b⁺ Ly6G⁺ Ly6C^low), normalized on CD45⁺ cells, in lungs, blood, spleen and bone marrow of tumor-bearing mice. Representative flow cytometry bar plots show: a the percentage of blood, spleen and bone marrow PMN-MDSCs in TuBo (n = 8) and 4T1 (n = 7/blood and bone marrow; n = 9/spleen) mock and p140Cap tumor-bearing mice; (b) the percentage of lung PMN-MDSCs in 4T1 mock and p140Cap tumor-bearing mice (n = 5 mice/group; data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test). c G-CSF protein levels in sera of tumor-bearing mice. G-CSF protein levels were measured in sera collected from TuBo (n = 4) and 4T1 (n = 5) mock and p140Cap tumor-bearing mice by ELISA (Data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test).

Fig. 5. p140Cap impairs the breast TICs compartment.

a, b p140Cap effects on the Tumor-Initiating Cell (TIC) compartment. Mammosphere formation assay from panel (a) mock and p140Cap TuBo (n = 5), 4T1 (n = 5) and MDA-MB-231 (n = 7) cells and from panel (b) mock and p140Cap TuBo tumors (n = 5). Bar plots of mammosphere numbers are shown. Data are represented for n = x experimental repeats and shown as mean ± SEM; two-tailed unpaired t test). c In vivo Limiting Diluition Assay (LDA) of mock and p140Cap TuBo cells. Decreasing amounts (105, 104, 23 and 102) mock and p140Cap TuBo cells were injected into the mammary fat pad of 8-weeks-old female BALB/c mice. Tumor onset was monitored for 5 weeks post-transplantation. The Extreme Limiting Dilution Analysis (ELDA) software was used to calculate the LDA frequencies estimating the number of stem cells in the bulk population in mock and p140Cap TuBo tumors. d, e FACS analysis of stem cell/TIC markers. d FACS analysis of CD44+/CD24− cell populations in mock and p140Cap 4T1 (n = 4) and MDA-MB-231 (n = 3) cells. e FACS analysis of Sca1 expression in TuBo mock and p140Cap cells (n = 5). Bar plots of TIC marker quantification are shown. Data are represented for n = x experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. f GSEA^, plot of WONG_EMBRYONIC_STEM_CELL_CORE in TuBo RNAseq data, and in the CCLE and TCGA database. The plots show the distribution of genes correlated or not with SRCIN1 gene expression; two-tailed unpaired t test. g G-CSF protein levels in mammosphere supernatants. G-CSF protein levels were measured in mock and p140Cap TuBo (n = 4), 4T1 (n = 5) and MDA-MB-231 (n = 4) mammosphere culture supernatants by ELISA. Data are represented for n = x experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. h Bar plot represent the normalization of G-CSF (pg/ml), shown in panel (g), on mammosphere number generated from mock and p140Cap TuBo (n = 4), 4T1 (n = 5) and MDA-MB-231 (n = 4) cells shown in panel (a). Data are represented for n = x experimental repeats, two-tailed unpaired t test.

Fig. 6. p140Cap affects the TIC compartment through reduction of the β-Catenin active form.

a GSEA^, plot of FEVR_CTNNB1_TARGETS_DN in TuBo RNAseq, CCLE and TCGA database. Plots show the distribution of genes correlated or not with SRCIN1 gene expression; two-tailed unpaired t test. b–d Effect of p140Cap expression on β-Catenin activation. Immunoblot analysis in mammospheres from (b) mock and p140Cap TuBo cells, (c) tumors. Vinculin,Tubulin and GAPDH were used as loading controls. Act = active β-catenin (n = 7/cells, n = 3/tumors experiments), Tot = Total β-catenin (n = 5/cells, n = 3/tumors), GSK-3β S9 (n = 3/cells and tumors). d mock and p140Cap 4T1 cells. Act = active β-catenin (n = 6), Tot = Total β-catenin (n = 3); arb.units = arbitrary units. Data are representative of n = x experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. In (b) total β-catenin and GSK−3β S9 have been run on a separate blot. In (c) active and total β-catenin have been run on a separate blot. In (d) total β-catenin has been run on a separate blot. e Generation of p140Cap TuBo cells expressing a constitutive active (C.A.) form of β-Catenin or empty pcDNA vector, as control. C.A. expression evaluated by immunoblotting. GAPDH: loading controls. Total β-catenin has been run on a separate blot. Data are represented for n = 3 experimental repeats. f, g C.A. β-Catenin effect on G-CSF expression in p140Cap cells. f G-CSF mRNA by RT-PCR (n = 3) and (g) G-CSF protein levels by ELISA (n = 3). Data are represented for n = x experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. h Mammosphere formation assay from mock pcDNA, p140Cap pcDNA and p140Cap C.A β-Catenin in TuBo cells. Bar plots of mammosphere number are shown. Data are represented for n = 9 experimental repeats; data presented as mean ± SEM; two-tailed unpaired t test. i G-CSF protein levels were measured in mock pcDNA (n = 3), p140Cap pcDNA (n = 3) and p140Cap β-Catenin C.A (n = 6). TuBo mammosphere supernatants by ELISA. Data are represented for n = x experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. j Representative bar plots show the percentage of PMN-MDSC cells normalized on CD45 + cells, in tumor, blood and spleen of mock pcDNA, p140Cap pcDNA and p140Cap C.A β-Catenin. TuBo pcDNA tumor-bearing mice (n = 7/tumor, n = 10/blood, n = 6/spleen); TuBo p140Cap pcDNA tumor-bearing mice (n = 9/tumor, n = 8/blood, n = 6/spleen); TuBo p140Cap C.A β-Catenin tumor-bearing mice (n = 12/tumor, n = 18/blood, n = 8/spleen). Data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test. k C.A. β-Catenin expression on tumor growth. 10⁵ pcDNA (n = 8), p140Cap pcDNA (n = 8) and p140Cap C.A. β-Catenin (n = 13) cells were injected into 8-weeks-old female BALB/c mice. Tumor size was measured every two days. Data are represented for n = x mice as mean ± SEM; 2way ANOVA.

Fig. 7. p140Cap is a component of the destruction complex.

a–e Effect of p140Cap expression on the Destruction Complex stability. a, b Immunoprecipitation of p140Cap and immunoblot analysis with antibodies to β-Catenin, Axin1, GSK−3β in mock vs. p140Cap TuBo in (a) and mock vs. p140Cap 4T1 mammospheres in (b) (n = 3). Data are representative of n = x experimental repeats. c–e Immunoprecipitation of β-Catenin in (c), GSK−3β in (d), Axin1 in (e), or IgG (negative control) from mock and p140Cap TuBo mammospheres, and immunoblot analysis with antibodies to p140Cap, β-Catenin, GSK−3β, and Axin1. Bar plots are represented for n = 3 experimental repeats and shown as mean ± SEM; two-tailed unpaired t test). GAPDH was used as loading control; arb.units = Arbitrary Units. f Immunoprecipitation of β-Catenin from Mock and p140Cap TuBo mammospheres treated with MG132 followed by immunoblot analysis with antibodies to ubiquitin and β-Catenin. Vinculin was used as loading control. Data are represented for n = 3 experimental repeats. g BioID assay in HEK293 cells. Extracts from HEK293 cells transfected with p140Cap-BirA-HA and BirA-HA constructs in combination with Axin1-GFP were isolated with the BioID protocol and blotted with antibodies to Axin1, Total β-Catenin, Phospho β-Catenin, GSK-3β and HA. BirA-HA construct was used as negative control. Data are represented for n = 3 experimental repeats; bar plot represent biotinylated protein normalized on HA transfected amount as mean ± SEM; two-tailed unpaired t test. h Bar plot represents the percentage of biotinylated total or Phospho β-Catenin normalized on the total amount of total or Phospho β-Catenin, respectively. Data are represented for n = 3 experimental repeats and shown as mean ± SEM; two-tailed unpaired t test.

Fig. 8. p140Cap controls the stability of the destruction complex.

a Confocal images showing the number of Axin1-GFP punctae in Mock and p140Cap-TuBo cells. The nuclei were counterstained by DAPI. Scale bar 10 μm. Data are representative of n = 5 experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. b Confocal images showing the colocalization of Axin1-GFP and p140Cap-RFP in punctae in Mock-TuBo cells co-transfected with Axin1-GFP and p140Cap-RFP. Scale bar 10 μm. Data are representative of n = 5 experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. c Selected frames of FRAP experiments before and after bleaching, and throughout the recovery of Axin1-GFP in TuBo cells transfected with Axin1-GFP alone or in combination with p140Cap-RFP. Immobile fraction and half-life of GFP-Axin1. Scale bar 1 μm and 10 μm. Cell number > 31, data are representative of n = 3 experimental repeats and shown as mean ± SEM; two-tailed unpaired t test; 2way ANOVA for FRAP.

Fig. 9. IWR-1 Axin1 stabilizer mimics p140Cap ability to improve the destruction complex stability and to curb tumor progression.

a Effect of IWR-1 on the destruction complex stability. Immunoprecipitation of β-Catenin from Mock TuBo mammospheres treated for 5 consecutive days with 1 μM IWR-1 or DMSO (control vehicle). Immunoblot with indicated antibodies. GAPDH was used as loading control (n = 3). Data are representative of n = 3 experimental repeats. b Effect of IWR-1 treatment on mammosphere formation efficiency. Mammosphere formation assay from untreated Mock and p140Cap cells or Mock TuBo mammosphere treated with IWR-1 as in (a). Bar plots of mammosphere numbers are shown. Data are represented for n = 4 experimental repeats/group, as mean ± SEM; two-tailed unpaired t test. c G-CSF protein levels in mammosphere supernatants, treated as in (a). G-CSF protein levels were measured by ELISA in supernatants from untreated Mock and p140Cap or Mock TuBo mammospheres treated with IWR-1 as in (a). Data are represented for n = 3 experimental repeats/group, as mean ± SEM; two-tailed unpaired t test. d Immunoblot analysis of active β-Catenin in untreated Mock and p140Cap cells, or Mock TuBo mammospheres treated with IWR-1 as in (a). GAPDH was used as loading control. Data are representative of n = 5 experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. e 10⁵ cells were injected into the mammary fat pad of 6-weeks-old BALB/C mice, tumor growth was monitored and tumor size was measured. Mock TuBo tumor-bearing mice were treated with IWR-1 (n = 5) (5 mg/Kg) every two days, with six different injections, starting when the tumor size had reached approximately 80 mm³. Mock (n = 6) and p140Cap TuBo (n = 6) tumor-bearing mice were treated with DMSO as controls. Data are represented for n = x mice and shown as mean ± SEM; 2way ANOVA. f Representative flow cytometry bar plots show the percentage of PMN-MDSCs cells normalized on CD45⁺ cells, in tumor, blood and spleen of mice described in (e). TuBo vehicle (n = 7/tumor, n = 5/blood, n = 6/spleen); TuBo p140Cap vehicle (n = 6/tumor, n = 5/blood, n = 5/spleen); TuBo IWR-1 (n = 7/tumor, n = 6/blood, n = 6/spleen). Data are represented for n = x mice and shown as mean ± SEM; two-tailed unpaired t test. g G-CSF levels in sera of IWR-treated or untreated tumor-bearing mice as in (e), by ELISA. TuBo vehicle (n = 6); TuBo p140Cap vehicle (n = 4); TuBo IWR-1 (n = 6). Data are represented for n = x mice and shown as mean ± SEM; two-tailed unpaired t test.

Fig. 10. p140Cap exerts a cell-extrinsic influence on the immune infiltrate composition of the tumor microenvironment, thereby favoring an effective anti-tumor immune response at the expense of a tumor-promoting inflammatory microenvironment.

Overall, this study shows that p140Cap activity is able to prevent tumor microenvironment infiltration through immunosuppressive PMN-MDSCs by inhibiting their systemic mobilization and local infiltration. Our functional studies in vitro and in vivo revealed that the decrease in G-CSF content is a function of the p140Cap ability to reduce the TIC compartment, which in turn is responsible for the production of G-CSF. The effect on the TIC compartment depends on the ability of p140Cap to enter and stabilize the β-Catenin destruction machinery, thereby inhibiting β-Catenin activity. Created with Biorender.com.