ROBO1 Expression in Metastasizing Breast and Ovarian Cancer: SLIT2-induced Chemotaxis Requires Heparan Sulfates (Heparin)

Abstract

Background: The members of the slit homolog (SLIT) and roundabout homolog (ROBO) families have emerged as important signaling molecules in tumor metastasis. This study analyzed their role in regulating breast cancer (BC) cell motility and chemotaxis and assessed expression of ROBO1 in brain metastases (BMs) of breast, lung, and colon cancer, and in peritoneal metastases (PMs) of ovarian cancer. Materials and Methods: The BC cell line MDA-MB231 was subjected to scratch, motility, and chemotaxis assays using heparin and a purified recombinant N-terminal SLIT2 fragment. Protein expression was assessed in primary tumors and metastases by immunohistochemistry. Results: Exposure to SLIT2 induced MDA-MB231 cell motility, but no significant chemotaxis without the presence of heparin. ROBO1 was expressed in 4/5 primary BC and in 18/21 BC-derived BM samples; 7/9 BM primary lung cancer samples also stained positive. In contrast, BMs from colorectal cancer were negative for ROBO1. Primary ovarian cancer and ovarian PM showed ROBO1 expression in 0/6 and in only 2/6 samples, respectively, whereas SLIT2 was observed in 1/6 primary cancer and in 6/6 PMs samples. Conclusion: SLIT2 can induce BC cell motility and chemotaxis, but the latter requires the presence of heparin. BM expression of ROBO1 is a common feature of some, but not all cancer types. SLIT2 expression appears to be a general feature of ovarian cancer-derived PMs.Metastasis is a process that involves multiple steps, starting with the mobilization and release of tumor cells from the primary tumor, which are then transported through the lymphatic system and blood vessels to distant sites, where they form new tumors after extravasation and tissue invasion. This process is not random, as it has been established that certain primary cancer types metastasize preferentially to distinct target organs (1). Within the sites of metastasis, distinct molecules, such as cytokines and chemokines play an important role in attracting tumor cells which express the corresponding cell surface receptors (2, 3). C-X-C motif chemokine 12 (CXCL12) with its receptor C-X-C chemokine receptor type 4 (CXCR4), originally identified as a major inducer and regulator of leukocyte chemotaxis (4), is an example of this process. Specifically, CXCR4 is expressed by malignant and pre-malignant breast tumors, such as those at the stage of atypical ductal hyperplasia (5). CXCR4-expressing tumor cells mediate targeted metastasis to bone and other tissues expressing the CXCR4 ligand CXCL12. A new candidate for a modulatory role in CXCR4-/CXCL12-mediated metastasis is the neuronal repellent slit homolog (SLIT) 2 protein. The SLIT proteins (SLIT1, -2, and -3) are a family of large, extracellular-matrix secreted glycoproteins that act as ligands to the Roundabout receptor family (ROBO1, -2, -3, and -4). They have been linked to neuronal development, myogenesis, leukocyte chemotaxis, and cancer (6, 7). SLIT2 (acting through its receptor ROBO1) was found to inhibit CXCL12-induced leukocyte chemotaxis (8). In pre...

Epigenetic loss of the transfer RNA-modifying enzyme TYW2 induces ribosome frameshifts in colon cancer

“…Among the identified TYW2 targets, we selected the roundabout guidance receptor 1 (ROBO1) for further validation and study. ROBO1 was selected because it is reportedly lost in some colorectal tumors ( 30 , 31 ) and because of its proposed role as an inhibitor of cell migration and epithelial-to-mesenchymal transition (EMT) ( 32 , 33 ). We first validated the RNA expression levels observed in the RNA-seq approach by quantitative real-time RT-PCR, and found that the TYW2 unmethylated/expressing HCT-116 and SW480 cells expressed the ROBO1 transcript, whereas CRISPR/Cas9-mediated inactivation of TYW2 induced the loss of ROBO1 transcript levels ( Fig.…”

Expression of the axon guidance factor Slit2 and its receptor Robo1 in patients with Hirschsprung disease

“…Numerous studies have shown that the Slit2-Robo1 signaling pathway participates in the growth, migration and distribution of axons of neurons in the central nervous system and malignant cancer cells. [15][16][17] However, details on the expression of Slit2 and Robo1 in the intestines of patients with HD have rarely been reported. Therefore, this study focused on the axon guidance factor Slit2 and assessed the expression and distribution of Slit2 and its receptor Robo1 in diseased segments (spastic and transitional segments) and normal segments of the intestinal tract from children with HD.…”

“…HD is a form of intestinal malformation caused by local ganglion cell loss due to inhibition of the differentiation of neurons migrating into the intestines into ganglion cells. Numerous studies have shown that the Slit2–Robo1 signaling pathway participates in the growth, migration and distribution of axons of neurons in the central nervous system and malignant cancer cells [15–17] . However, details on the expression of Slit2 and Robo1 in the intestines of patients with HD have rarely been reported.…”

Slit2-Mediated Metabolic Reprogramming in Bone Marrow-Derived Macrophages Enhances Antitumor Immunity

“…Moreover, Slit2/ROBO1 signaling is reported to exert antitumor activity (5,6). This antitumor effect is attributed to the regulation of b-catenin and chemotaxis (7). Recent studies also highlight the dual nature of Slit2 in cancer suppression or progression based on whether the tumor or surrounding cells produce and secrete this protein.…”