Activation of Robo1 signaling of breast cancer cells by Slit2 from stromal fibroblast restrains tumorigenesis via blocking PI3K/Akt/β-catenin pathway

Abstract

Tumor microenvironment plays a critical role in regulating tumor progression by secreting factors that mediate cancer cell growth. Stromal fibroblasts can promote tumor growth through paracrine factors; however, restraint of malignant carcinoma progression by the microenvironment also has been observed. The mechanisms that underlie this paradox remain unknown. Here, we report that the tumorigenic potential of breast cancer cells is determined by an interaction between the Robo1 receptor and its ligand Slit2, which is secreted by stromal fibroblasts. The presence of an active Slit2/Robo1 signal blocks the translocation of β-catenin into nucleus, leading to downregulation of c-myc and cyclin D1 via the phosphoinositide 3-kinase (PI3K)/Akt pathway. Clinically, high Robo1 expression in the breast cancer cells correlates with increased survival in patients with breast cancer, and low Slit2 expression in the stromal fibroblasts is associated with lymph node metastasis. Together, our findings explain how a specific tumor microenvironment can restrain a given type of cancer cell from progression and show that both stromal fibroblasts and tumor cell heterogeneity affect breast cancer outcomes.

Fig. 1. Stromal fibroblasts can either promote or suppress soft agar colony formation of breast cancer cells

A, B. Soft agar colony formation assays using MDA-MB-231 (A) and SKBR3 cells (B) co-cultured with primary carcinoma-associated fibroblasts (CAF) or normal breast-associated fibroblasts (NAF). C, D. Soft agar colony formation assays using breast cancer cell lines other than MDA-MB-231 and SKBR3 co-cultured with immortalized human CAF (199Ct). Colony formation in MDA-MB-361, Hs578T and MDA-MB-157 cells was inhibited (C), but increased in HCC1937, BT20 and MDA-MB-468 cells (D) when co-cultured with 199Ct. E, F. Soft agar colony formation assays using MDA-MB231 (E) and SKBR3 cells (F) co-cultured with two other human fibroblast cell lines, WI38 and Hs68. All data points were performed in triplicates and all experiments were performed at least three times with similar results. Data show means ± standard deviation. **, p < 0.01

Fig. 2. Robo1 is identified as a candidate receptor involved in fibroblast-mediated tumor suppression

A. Summary of cDNA microarray analyses. Total six hundred and ten genes expressed at least 2-fold higher in MDA-MB-231 (I), MDA-MD-361 (II) and Hs578T (III) than in SKBR3 were found by Affymetrix microarray analysis. B. Eight transmembrane receptors were identified among the 610 genes with their expression pattern matched with the profile that MDA-MB-231, MDA-MB-361 and Hs578T had a higher level than that of SKBR3. C. Reconfirming the expression profile of the eight transmembrane receptors in six different breast cancer cells. Their receptor mRNA expressions were measured directly by qRT-PCR with Gapdh as an internal control. D, E. mRNA and protein expressions of Robo1, 2 and 4 were measured by RT-PCR (D) and immunoblotting (E), respectively. Gapdh and α-tubulin served as loading controls for RNA and protein expression, respectively.

Fig. 3. Robo1 plays an essential role in fibroblast-mediated suppression of breast tumorigenesis

A. Immunoblotting analysis of Robo1 expression in MDA-MB-231 cells infected by two independent lentiviruses, #180 and #248, carrying shRobo1. Cells infected with lentiviral sh-luciferease (shCtrl) or uninfected (Mock) served as controls. α-tubulin was used as a loading control. B. Soft-agar colony formation assays using Mock, shCtrl and shRobo1 MDA-MB-231 cells co-cultured with 199Ct. C. Tumor growth assay in NOD/SCID mice. shCtrl or shRobo1 MDA-MB-231 cells were injected into mammary fat-pad with or without 199Ct and the tumor volumes were measured every 4–5 days. D. Immunoblotting analysis of Robo1 expression in MDA-MB-157 cells infected by shRobo1 #180. Cells infected with shCtrl served as the control. α-tubulin was used as a loading control. E. Soft-agar colony formation assays using shCtrl and shRobo1 MDA-MB-157 cells co-cultured with 199Ct. F. Immunoblotting analysis of ectopic expression of Robo1 in BT20 and HCC1937 cells. Cells were infected either with lentiviruses carrying Robo1 cDNA (Robo1) or empty vector (LentiV). β-actin was used as a loading control. G. Soft agar colony formation assay for LentiV and Robo1 overexpressing BT20 and HCC1937 cells co-cultured with or without 199Ct. H. Tumor growth assay in NOD/SCID mice. HCC1937 cells expressing Robo1 or control (LentiV) were injected into mammary fat-pad with or without 199Ct and the tumor volumes were measured every 4–5 days. All data points were performed in triplicates and all in vitro experiments were performed at least three times. Data show means ± standard deviation. For tumor growth assay in NOD/SCID mice, six mice per group were used. *, p < 0.05; **, p < 0.01.

Fig. 4. Slit2 secreted from stromal fibroblasts inhibits cancer cell tumorigenesis

A. Slit2 mRNA expression in breast cancer cell lines (MDA-MB-231, MDA-MB-361, SKBR3 and BT20), fibroblast cell lines (199Ct, WI38 and HS68) and primary fibroblasts (221C, 222N, 288N and 428N) were examined by qRT-PCR. B. IHC staining with antibody against Slit2 in breast cancer specimen. Low magnification (left panel) scale bar: 25 µm. High magnification (right panel) scale bar: 50 µm. Arrows indicate stromal fibroblasts. Arrow heads indicate cancer cells. C. Soft-agar colony formation assay for MDA-MB-231 cells treated with conditioned medium (199Ct cm) collected from 199Ct culture. D. Immunoblotting analysis of Slit2 proteins in harvested conditioned medium (cm-Slit2) from shCtrl or shSlit2 199Ct. β-actin was used as a loading control. E. Soft agar colony formation assay for MDA-MB-231 cells co-cultured with shCtrl or shSlit2 199Ct. F. Soft agar colony formation of shCtrl and shRobo1 MDA-MB-231 cells treated with PBS or 50 ng/ml of recombinant Slit2 (rSlit2) protein. All data points were performed in triplicates and all experiments were performed at least three times. Data show means ± standard deviation. *, p < 0.05; **, p < 0.01.

Fig. 5. Slit2/Robo1 signaling blocks β-catenin translocation through PI3K/Akt pathway

A. Robo1 overexpressing BT20 cells were treated with rSlit2 (200 ng/ml) for 10, 20 and 30 min. Co-immunoprecipitation (Co-IP) of Robo1 and p85 was detected using immunoblotting analysis in response to the rSlit2 treatment. Normal IgG (IgG) was used as a negative Co-IP control. β-actin served as a loading control. B. Immunoblotting analysis of phospho-Akt in shCtrl and shRobo1 MDA-MB-231 cells treated with rSlit2 (200 ng/ml). Total Akt was used as a quantification control. β-actin served as a loading control. Relative expression (RE) in phospho-Akt to total Akt protein levels is indicated. C. Immunoblotting assay of β-catenin in cytosolic and nuclear fractions of the shCtrl and shRobo1 MDA-MB-231 cells co-cultured with or without 199Ct. α-tubulin and histone deacetylase (HDAC) were used as cytosolic and nuclear markers, respectively. D. Immunofluorescence staining with antibody against β-catenin for shCtrl and shRobo1 MDA-MB-231 cells after treated with rSlit2 (200 ng/ml). Scale bar: 20µm. Arrows indicate nuclei. E. Immunoblotting analysis of shCtrl and shRobo1 MDA-MB-231 cells treated with rSlit2 using antibodies against cyclin D1 and c-myc. α-tubulin as a loading control. RE in cyclin D1 and c-myc to α-tubulin protein levels is indicated. F. Diagram summarized the pathway of how Slit2/Robo1 signal is transmitted from stromal fibroblasts to breast cancer cells.

Fig. 6. Clinical relevance of Robo1/Slit2 expression in breast cancer specimens

A. Kaplan-Meier survival analysis for 122 breast cancer patients divided into two groups based on their Robo1 mRNA expression levels. Blue line (n=93) represents Robo1 high and red line (n=29) represents Robo1 low patients. B. IHC staining of Robo1 in breast tumor specimens. Low magnification (left panel) scale bar: 100 µm. High magnification (right panel) scale bar: 50 µm. C. Kaplan-Meier survival analysis for 162 breast cancer specimens using IHC staining to detect Robo1 expression in cancer cell membrane. Blue line (n=63) represents Robo1 high and red line (n=99) represents Robo1 low patients. D. Fifty-one cancer-associated fibroblasts isolated from freshly obtained specimens were used to determine the correlation between Slit2 mRNA expression and metastasis status of the patients. Slit2 mRNA levels were measured by qRT-PCR and compared with the internal control gene, Gapdh. Patients were divided into two groups according to the metastasis status to correlate with the relative Slit2 mRNA expression. N-, no lymph node metastasis (n=26) and N+, with lymph node metastasis (n=25).