Semaphorin 4D Promotes Skeletal Metastasis in Breast Cancer.

Bone density is controlled by interactions between osteoclasts, which resorb bone, and osteoblasts, which deposit it. The semaphorins and their receptors, the plexins, originally shown to function in the immune system and to provide chemotactic cues for axon guidance, are now known to play a role in this process as well. Emerging data have identified Semaphorin 4D (Sema4D) as a product of osteoclasts acting through its receptor Plexin-B1 on osteoblasts to inhibit their function, tipping the balance of bone homeostasis in favor of resorption. Breast cancers and other epithelial malignancies overexpress Sema4D, so we theorized that tumor cells could be exploiting this pathway to establish lytic skeletal metastases. Here, we use measurements of osteoblast and osteoclast differentiation and function in vitro and a mouse model of skeletal metastasis to demonstrate that both soluble Sema4D and protein produced by the breast cancer cell line MDA-MB-231 inhibits differentiation of MC3T3 cells, an osteoblast cell line, and their ability to form mineralized tissues, while Sema4D-mediated induction of IL-8 and LIX/CXCL5, the murine homologue of IL-8, increases osteoclast numbers and activity. We also observe a decrease in the number of bone metastases in mice injected with MDA-MB-231 cells when Sema4D is silenced by RNA interference. These results are significant because treatments directed at suppression of skeletal metastases in bone-homing malignancies usually work by arresting bone remodeling, potentially leading to skeletal fragility, a significant problem in patient management. Targeting Sema4D in these cancers would not affect bone remodeling and therefore could elicit an improved therapeutic result without the debilitating side effects

Semaphorin 4D Promotes Skeletal Metastasis in Breast Cancer.

Bone density is controlled by interactions between osteoclasts, which resorb bone, and osteoblasts, which deposit it. The semaphorins and their receptors, the plexins, originally shown to function in the immune system and to provide chemotactic cues for axon guidance, are now known to play a role in this process as well. Emerging data have identified Semaphorin 4D (Sema4D) as a product of osteoclasts acting through its receptor Plexin-B1 on osteoblasts to inhibit their function, tipping the balance of bone homeostasis in favor of resorption. Breast cancers and other epithelial malignancies overexpress Sema4D, so we theorized that tumor cells could be exploiting this pathway to establish lytic skeletal metastases. Here, we use measurements of osteoblast and osteoclast differentiation and function in vitro and a mouse model of skeletal metastasis to demonstrate that both soluble Sema4D and protein produced by the breast cancer cell line MDA-MB-231 inhibits differentiation of MC3T3 cells, an osteoblast cell line, and their ability to form mineralized tissues, while Sema4D-mediated induction of IL-8 and LIX/CXCL5, the murine homologue of IL-8, increases osteoclast numbers and activity. We also observe a decrease in the number of bone metastases in mice injected with MDA-MB-231 cells when Sema4D is silenced by RNA interference. These results are significant because treatments directed at suppression of skeletal metastases in bone-homing malignancies usually work by arresting bone remodeling, potentially leading to skeletal fragility, a significant problem in patient management. Targeting Sema4D in these cancers would not affect bone remodeling and therefore could elicit an improved therapeutic result without the debilitating side effects

Xenografts with silenced Sema4D exhibit fewer and smaller lesions and longer survival compared to controls in a mouse model of skeletal metastasis.

<p><b>A.</b> MDA-MB-231 expressing luciferase, controls (C) and with silenced Sema4D (Sema4D shRNA), were introduced into the systemic circulation of immunocompromised mice and developing lesions tracked by BLI. Representative lesions developing after 7 days (top row) and 34 days (bottom row) are shown. Luminescence, related to the number of collected photons, is shown on the right. <b>B.</b> Radiographs of representative lesions are shown. <b>C.</b> Mice grafted with MDA-MB-231 with silenced Sema4D exhibited fewer lesions (tumor burden, 10⁶ photons/sec, Y-axis) compared to controls (**, p ≤ 0.01). <b>D.</b> Kaplan–Meier curve reveals that mice grafted with MDA-MB-231 with silenced Sema4D exhibited longer survival compared to controls.</p

Silencing Sema4D in breast cancer changes serum levels of mediators of bone remodeling.

<p><b>A.</b> ELISA performed for RANKL, an osteoblast-derived pro-osteoclastogenic factor, revealed higher levels in mice bearing tumors from MDA-MB-231 where Sema4D was silenced, suggestive of enhanced osteoblast activity, when compared to controls (RANKL levels in pg/ml, Y-axis). <b>B.</b> OPG levels are also higher in mice with tumors with silenced Sema4D (pg/ml, Y-axis). <b>C.</b> The ratio of the antagonists RANKL/OPG is not significantly different between the two populations, indicating the importance of other factors in the bone resorption observed in our model. <b>D.</b> In support of <i>in vitro</i> findings, mice bearing control tumors expressing Sema4D demonstrated significantly higher levels of serum LIX/CXCL5 (pg/ml, Y-axis). <b>E.</b> As expected, osteocalcin, a marker of osteoblast activity, is higher in the serum of Sema4D shRNA populations compared to controls (ng/ml, Y-axis; *, p ≤ 0.05; n.s., not significant).</p

LIX/CXCL5, the murine homologue of IL-8, is produced by MC3T3 in the presence of sSema4D and promotes osteoclastogenesis.

<p><b>A.</b> An ELISA performed on MC3T3 revealed increasing concentrations of LIX/CXCL5 (LIX, pg/ml, Y-axis) in increasing concentrations of sSema4D. <b>B.</b> Media conditioned by sSema4D treated MC3T3 (CM) induces osteoclastogenesis of RAW264.7 cells, compared to controls (C). TRAP assay, top; Quantification of results, bottom (average number of TRAP positive osteoclasts/ field, Y-axis; *, p ≤ 0.05; **, p ≤ 0.01). <b>C.</b> RAW264.7 cells are observed to fuse into multinucleated osteoclasts in the presence of media conditioned by sSema4D treated MC3T3.</p

Sema4D promotes larger metastatic lesions, with fewer osteoblasts and more osteoclasts.

<p><b>A.</b> Hematoxylin and eosin (H&E) stained sections of control (C) tumors or those with silenced Sema4D (Sema4D shRNA) metastatic to the femur (top row; T, tumor; BM, bone marrow; original magnification 10x). Control tumors on average were larger than those from cells where Sema4D was silenced. Sema4D silencing was confirmed by immunohistochemistry in tumor tissues (second row; original magnification 40x). Osteoclasts attached to bone in the tumor microenvironment were identified by TRAP immunohistochemistry (TRAP, bottom row, positive cells indicated by arrows; original magnification 40x). <b>B.</b> Histomorphometric analysis for total tumor area in formalin fixed paraffin embedded specimens confirmed that control tumors were larger (tumor area in mm², Y-axis). <b>C.</b> Tumors made up of cells with silenced Sema4D exhibited greater preservation of bone volume (bone volume expressed as a percentage of total tissue volume, Y-axis). <b>D.</b> The number of osteoclasts present in lesional bone, as determined by TRAP positivity, was greater in control tumors (number of osteoclasts per mm² of bone surface, Y-axis). (E) The number of osteoblasts in lesional bone was higher in tumors where Sema4D was silenced (number of osteoblasts per mm² of bone surface, Y-axis; *, p ≤ 0.05).</p

Sema4D produced by breast cancer cells inhibits mineralization and promotes RhoA-mediated production of IL-8 by osteoblasts.

<p><b>A.</b> Breast cancer cell line MDA-MB-231 expresses high levels of Sema4D in an immunoblot compared to the non-tumorigenic line MCF-12 and T47D, which rarely metastasize to bone (top panel). GAPDH was used as the loading control (lower panel). <b>B.</b> MDA-MB-231 cells infected with lentivirus coding for a scrambled shRNA (C) express Sema4D in whole cell lysates, levels of which are greatly reduced in cells infected with lentivirus expressing Sema4D shRNA (top left panel). MCF-12A cells, which express very little endogenous Sema4D (C), are induced to overexpress it through lentiviral-mediated transfer of the wild-type construct (Sema4D, top right panel). GAPDH was used as a loading control (middle panel). sSema4D is present in media conditioned by control MDA-MB-231 cells and MCF-12A overexpressing Sema4D, but not in MDA-MB-231 with silenced Sema4D or control MCF-12A infected with a lentivirus containing an empty vector (bottom panel). <b>C.</b> MC3T3-mediated matrix deposition and mineralization, shown by Alizarin Red stain, is inhibited by media conditioned by control MDA-MB-231 (C, first column, left panels), but not in those where Sema4D is silenced by shRNA (Sema4D shRNA, second column, left). When Plexin-B1 is silenced in MC3T3 (PB1 shRNA, second row), or anti-Sema4D blocking antibody is administered (bottom row), mineralization is restored. Media conditioned by control MCF-12A had no effect on mineralization (C, first column, right panels) unless these cells were over-expressing Sema4D, in which case there was inhibition (Sema4D, second column, right). Mineralization was restored when Plexin-B1 was silenced in osteoblasts (PB1 shRNA, second row) or blocking antibody was present (bottom row). Isotype matched IgG was used as a control for both experimental groups. <b>D.</b> Quantification of results by absorbance at 405 nm (Y-axis) of the results shown in (B) from three independent experiments. MDA-MB-231 cells, left bar graph; MCF-12A, right. <b>E.</b> Incubation of the human osteoblast line HOB with media conditioned by MDA-MB-231 induces production of IL-8 (concentration in ng/ml, Y-axis), unless Sema4D is silenced in MDA-MB-231 by shRNA or Plexin-B1 signaling to RhoA is inhibited by addition of the <i>Clostridium botulinum</i> toxin C3 (*, p ≤ 0.05; **, p ≤ 0.01).</p