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. 2015 Oct 1;75(19):4063-73.
doi: 10.1158/0008-5472.CAN-14-3394. Epub 2015 Aug 3.

Metastasis Suppressors Regulate the Tumor Microenvironment by Blocking Recruitment of Prometastatic Tumor-Associated Macrophages

Casey Frankenberger  1 , Daniel Rabe  1 , Russell Bainer  2 , Devipriya Sankarasharma  3 , Kiran Chada  3 , Thomas Krausz  4 , Yoav Gilad  2 , Lev Becker  5 , Marsha Rich Rosner  5
Affiliations

Metastasis Suppressors Regulate the Tumor Microenvironment by Blocking Recruitment of Prometastatic Tumor-Associated Macrophages

Casey Frankenberger et al. Cancer Res. .

Abstract

Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. TNBC is characterized by reduced expression of metastasis suppressors such as Raf kinase inhibitory protein (RKIP), which inhibits tumor invasiveness. Mechanisms by which metastasis suppressors alter tumor cells are well characterized; however, their ability to regulate the tumor microenvironment and the importance of such regulation to metastasis suppression are incompletely understood. Here, we use species-specific RNA sequencing to show that RKIP expression in tumors markedly reduces the number and metastatic potential of infiltrating tumor-associated macrophages (TAM). TAMs isolated from nonmetastatic RKIP(+) tumors, relative to metastatic RKIP(-) tumors, exhibit a reduced ability to drive tumor cell invasion and decreased secretion of prometastatic factors, including PRGN, and shed TNFR2. RKIP regulates TAM recruitment by blocking HMGA2, resulting in reduced expression of numerous macrophage chemotactic factors, including CCL5. CCL5 overexpression in RKIP(+) tumors restores recruitment of prometastatic TAMs and intravasation, whereas treatment with the CCL5 receptor antagonist Maraviroc reduces TAM infiltration. These results highlight the importance of RKIP as a regulator of TAM recruitment through chemokines such as CCL5. The clinical significance of these interactions is underscored by our demonstration that a signature comprised of RKIP signaling and prometastatic TAM factors strikingly separates TNBC patients based on survival outcome. Collectively, our findings identify TAMs as a previously unsuspected mechanism by which the metastasis-suppressor RKIP regulates tumor invasiveness, and further suggest that TNBC patients with decreased RKIP activity and increased TAM infiltration may respond to macrophage-based therapeutics.

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Conflict of interest statement

None of the authors have conflicts of interest to declare.

Figures

Figure 1
Figure 1. Non-metastatic RKIP+ tumors contain fewer macrophages
A) Quantile-quantile (qq)-plot showing ranked −log10 transformed p-values among macrophage-specific genes (y-axis) relative to a similar bootstrapped distribution in blue (x-axis). Distortion of the density above x = y (red line) indicates that the measured p-values are systematically lower than expected by chance. B) Representative images of relative macrophage presence in xenograft (BM1 and MDA-MB-436) and syngeneic (4T1.2) tumor models with and without RKIP expression were sectioned and immunostained for F4/80. C) Relative macrophage inflitration was quantified as the proportion of inner tumor mass positively stained with F4/80 via immunohistochemistry. D) Number of positively staining cells was determined by flow cytometry.
Figure 2
Figure 2. RKIP suppresses recruitment of a distinct TAM population that potentiates tumor cell invasion
A–C) BM1 or BM1+RKIP tumor cells were pretreated with conditioned media collected from various types of macrophages for 24h; A) THP1 human monocytic cell line (n=8 per group), B) TAMs isolated from BM1 tumors (n=5 per group), C) TAMs isolated from BM1 or BM1+RKIP tumors (n=6 per group). Relative invasion is against BM1 grown in a media control. P-values were calculated using an unpaired T-test with Welch’s correction. D–E) TAM conditioned media from four independent tumors was analyzed for protein levels using RayBiotech L308 Mouse Cytokine Arrays. Protein abundance for RKIP derived TAMs were normalized to control tumor TAMs. D) Proteins with 0.8 or lower relative abundance, E) Proteins with >1.2 relative abundance. F) Relative mRNA was measured from three independent TAM samples per group. Relative mRNA was calculated as compared to control TAMs, with Gapdh as the reference gene.
Figure 3
Figure 3. Overexpression of CCL5 restores TAMs and overcomes metastasis suppression in RKIP+ tumors
A) GSEA identifies enrichment of genes involved in cytokine-cytokine receptor interactions (black lines) among genes with anticorrelated expression levels in tumors and surrounding stroma. B) Tumor genes differentially expressed in RKIP tumors relative to control (p<0.05) are shown from the external stimulus (GO) category from our RNAseq data. C) qRT-PCR was performed on mRNA purified from xenograft tumors (BM1 & BM1+RKIP). Species-specific primers were used to detect relative mRNA of CCL5 (Hs) and Ccr5 (Mm) in the tumor and stroma, respectively. Relative mRNA was normalized to GAPDH (Hs) or Rpl4 (Mm). D) Relative mRNA was calculated relative to BM1 TAMs with Gapdh as the reference gene. Flow cytometry of BM1 (red) and BM1+RKIP (blue) isolated TAMs are also shown for CCR5. E) Representative images of macrophage presence in BM1 tumors with and without RKIP and CCL5 expression. F) Relative macrophages in BM1 and BM1+RKIP tumors with or without exogenous CCL5 expression in tumor cells. Infiltration was quantified as the proportion of total tumor area positively stained with F4/80 (n=3 per group). G) Effect of Maraviroc on BM1 tumor macrophage numbers was assessed by immunostaining for F4/80. Data are displayed as the %F4/80+ cells in the core of the tumor. H) BM1 cells were pretreated with TAM conditioned media (BM1, BM1+RKIP, BM1+RKIP+CCL5, or BM1+CCL5 TAMs) for 24 hours prior to invasion assays, using TAMs from four independent tumors each. P-values were obtained using an unpaired T-test with Welch’s correction, n=6. I) Relative intravasation of tumor cells into blood 4 weeks following injection was estimated by quantifying the ratio of human GAPDH (tumor) to mouse Gapdh by qRT-PCR (n=4 per group).
Figure 4
Figure 4. RKIP blocked TAM phenotype rescued by CCL5
A) TAM conditioned media from four independent tumors were analyzed for protein levels using RayBiotech L308 Mouse Cytokine Arrays. RKIP+CCL5 derived TAMs were normalized to RKIP derived TAMs with a cutoff set at greater than 3-fold expression. B) Relative protein is shown for each protein from the cytokine array; x-axis: RKIP TAMs relative to control TAMs, y-axis RKIP+CCL5 TAMs relative to RKIP TAMs. Both axes are shown on a log 2 scale. C) Relative mRNA was measured from three independent TAM samples per group. Relative mRNA was calculated as compared to control TAMs, with Gapdh as the reference gene. D) Relative invasion for BM1 cells pretreated with various concentrations of PGRN or sTNFR2 for 24 hours, results are plotted relative to an untreated control. Statistical significance was determined using a Student’s T-test, n=5 per group. E) Relative mRNA levels for bone marrow derived macrophages (BMMs) treated with or without 1 µg/ml of recombinant CCL5 for 24h. Gapdh is used as a reference gene. F) Protein levels (spectral counts) in the conditioned media collected from BMMs (M0), LPS -induced BMMs (M1), and IL-4-induced BMMs (M2) were quantified by mass spectrometry.
Figure 5
Figure 5. Suppression of metastasis and TAMs by RKIP is coordinated though HMGA2 signaling
A) BM1 cells were transduced with two separate shRNAs targeting HMGA2. Relative expression of HMGA2 and CCL5 was quantified by qRT-PCR and normalized to GAPDH. Results (n=3 per group) are relative to BM1 cells transduced with control shRNA. B) qRT-PCR analysis of gene expression in tumors isolated from wild type and Hmga2−/− mice (n=4). C) Representative images of macrophage infiltration in wild type and Hmga2−/− mice as determined by F4/80+ staining. Stromal regions are delimited by the dashed lines. Macrophage infiltration was quantified as the %F4/80+ area in the tumor and stromal regions. D) A schematic showing the regulation of CCL5 by RKIP through HMGA2.
Figure 6
Figure 6. An RKIP-HMGA2-CCL5-macrophage gene signature predicts metastasis-free survival
A) Gene expression estimates from a set of 871 breast cancer patients, stratified into either TNBC or non-TNBC patients. B) Individual scores for each gene in comparison to CCL5 expression are plotted for 12 separate genes. C) Heatmap identifying data sets (top) where breast cancer metastasis free survival is significantly stratified by classifier (right). D) Kaplan-Meier plots are shown for a set of all 871 breast cancer patients.
Figure 7
Figure 7. TNBC-TAM crosstalk
A schematic displaying the circular interplay between TNBC cells and TAMs.
See this image and copyright information in PMC

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