Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Semaphorin 3A upregulates FOXO 3a-dependent MelCAM expression leading to attenuation of breast tumor growth and angiogenesis

Abstract

Semaphorin 3A (Sema 3A), a member of semaphorin family, serves as a guidance clue during embryonic development and is known as a candidate tumor suppressor that attenuates breast tumor progression by binding with its co-receptor, neuropilin-1 (NRP-1). However, the underlying mechanism by which Sema 3A suppresses breast tumor growth is still unexplored. In this study, we report that Sema 3A regulates phosphorylation and nuclear translocation of phosphatase and tensin homolog (PTEN) and FOXO 3a. Moreover, Sema 3A controls NRP-1-mediated PTEN-dependent FOXO 3a activation. Overexpression of PTEN and FOXO 3a enhances Sema 3A-induced attenuation of breast cancer cell migration. Chromatin immunoprecipitation and electrophoretic mobility shift assay data revealed that FOXO 3a regulates MelCAM at the transcriptional level. Furthermore, Sema 3A induces NRP-1-mediated MelCAM expression through PTEN and FOXO 3a. The data also showed that vascular endothelial growth factor-induced angiogenesis is inhibited by Sema 3A. Loss of or gain in function study revealed that Sema 3A modulates phosphorylation of PTEN and FOXO 3a and expression of MelCAM, leading to suppression of tumor growth and angiogenesis using in vivo mice model. Clinical specimen analysis revealed that reduced expression of Sema 3A and p-PTEN are correlated with enhanced breast cancer progression, further strengthening our in vitro and in vivo findings. Correlation of relapse-free survival of breast cancer patients (n=2878) with expression levels of Sema 3A, NRP-1, FOXO 3a and MelCAM were studied by Kaplan–Meier analysis. Statistical analysis revealed a close association between reduced expression of Sema 3A and MelCAM with that of poor patient’s survival. Our study demonstrated a novel mechanism of regulation of tumor suppression by Sema 3A in coordination with a chain of tumor-suppressor genes, which in turn inhibits breast cancer cell migration, tumor growth and angiogenesis.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D . Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90.

    Google Scholar 

  2. American Cancer Society Cancer Facts & Figures 2013, American Cancer Society: Atlanta, GA, USA, 2013.

  3. Kolodkin AL, Matthes DJ, Goodman CS . The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 1993; 75: 1389–1399.

    Article  CAS  Google Scholar 

  4. Yazdani U, Terman JR . The semaphorins. Genome Biol 2006; 7: 211.

    Article  Google Scholar 

  5. Serini G, Maione F, Giraudo E, Bussolino F . Semaphorins and tumor angiogenesis. Angiogenesis 2009; 12: 187–193.

    Article  CAS  Google Scholar 

  6. Staton CA, Shaw LA, Valluru M, Hoh L, Koay I, Cross SS et al. Expression of class 3 semaphorins and their receptors in human breast neoplasia. Histopathology 2011; 59: 274–282.

    Article  Google Scholar 

  7. Yacoub M, Coulon A, Celhay O, Irani J, Cussenot O, Fromont G . Differential expression of the semaphorin 3A pathway in prostatic cancer. Histopathology 2009; 55: 392–398.

    Article  Google Scholar 

  8. He Z, Tessier-Lavigne M . Neuropilin is a receptor for the axonal chemorepellent Semaphorin III. Cell 1997; 90: 739–751.

    Article  CAS  Google Scholar 

  9. Glauser DA, Schlegel W . The emerging role of FOXO transcription factors in pancreatic beta cells. J Endocrinol 2007; 193: 195–207.

    Article  CAS  Google Scholar 

  10. Fu Z, Tindall DJ . FOXOs, cancer and regulation of apoptosis. Oncogene 2008; 27: 2312–2319.

    Article  CAS  Google Scholar 

  11. Arden KC . FoxOs in tumor suppression and stem cell maintenance. Cell 2007; 128: 235–237.

    Article  CAS  Google Scholar 

  12. Ramaswamy S, Nakamra N, Sansal I, Bergeron L, Sellers WR . A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer Cell 2002; 2: 81–91.

    Article  CAS  Google Scholar 

  13. Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, Sellers WR . Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Mol Cell Bio 2000; 20: 8969–8982.

    Article  CAS  Google Scholar 

  14. Dijkers PF, Birkenkamp KU, Lam EW, Thomas NS, Lammers JW, Koenderman L et al. FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase-B enhanced cell survival through maintenance of mitochondrial integrity. J Cell Bio 2002; 156: 531–542.

    Article  CAS  Google Scholar 

  15. Lehmann JM, Riethmüller G, Johnson JP . MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily. Proc Natl Acad Sci USA 1989; 86: 9891–9895.

    Article  CAS  Google Scholar 

  16. Xie S, Luca M, Huang S, Gutman M, Reich R, Johnson JP et al. Expression of MCAM/MUC18 by human melanoma cells leads to increased tumor growth and metastasis. Cancer Res 1997; 57: 2295–2303.

    CAS  Google Scholar 

  17. Shih LM, Hsu MY, Palazzo JP, Herlyn M . The cell-cell adhesion receptor Mel-CAM acts as a tumor suppressor in breast carcinoma. Am J Pathol 1997; 151: 745–751.

    CAS  Google Scholar 

  18. Shih IM . The role of CD146 (Mel-CAM) in biology and pathology. J Pathol 1999; 189: 4–11.

    Article  CAS  Google Scholar 

  19. Chakraborty G, Rangaswami H, Jain S, Kundu GC . Hypoxia regulates cross-talk between Syk and Lck leading to breast cancer progression and angiogenesis. J Biol Chem 2006; 281: 11322–11331.

    Article  CAS  Google Scholar 

  20. Satyamoorthy K, Muyrers J, Meier F, Patel D, Herlyn M . Mel-CAM-specific genetic suppressor elements inhibit melanoma growth and invasion through loss of gap junctional communication. Oncogene 2001; 20: 4676–4684.

    Article  CAS  Google Scholar 

  21. Li G, Kalabis J, Xu X, Meier F, Oka M, Bogenrieder T et al. Reciprocal regulation of MelCAM and AKT in human melanoma. Oncogene 2003; 22: 6891–6899.

    Article  CAS  Google Scholar 

  22. Vazquez F, Ramaswamy S, Nakamura N, Sellers WR . Phosphorylation of the PTEN tail regulates protein stability and function. Mol Cell Bio 2000; 20: 5010–5018.

    Article  CAS  Google Scholar 

  23. Torres J, Pulido R . The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteosome-mediated degradation. J Biol Chem 2001; 276: 993–998.

    Article  CAS  Google Scholar 

  24. Chadborn NH, Ahmed AI, Holt MR, Prinjha R, Dunn GA, Jones GE et al. PTEN couples Sema3A signalling to growth cone collapse. J Cell Sci 2006; 119: 951–957.

    Article  CAS  Google Scholar 

  25. Bachelder RE, Lipscomb EA, Lin X, Wendt MA, Chadborn NH, Eickholt BJ et al. Competing autocrine pathways involving alternative neuropilin-1 ligands regulate chemotaxis of carcinoma cells. Cancer Res 2003; 63: 5230–5233.

    CAS  Google Scholar 

  26. Blanco-Aparicio C, Renner O, Leal JF, Carnero A . PTEN, more than the AKT pathway. Carcinogenesis 2007; 28: 1379–1386.

    Article  CAS  Google Scholar 

  27. Castro-Rivera E, Ran S, Brekken RA, Minna JD . Semaphorin 3B inhibits the phosphatidylinositol 3-kinase/Akt pathway though neuropilin-1 in lung and breast cancer cells. Cancer Res 2008; 68: 8295–8303.

    Article  CAS  Google Scholar 

  28. Rena G, Woods YL, Prescott AR, Peggie M, Unterman TG, Williams MR et al. Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion. EMBO J 2002; 21: 2263–2271.

    Article  CAS  Google Scholar 

  29. Accili D, Arden KC . FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 2004; 117: 421–426.

    Article  CAS  Google Scholar 

  30. Hu MC, Lee DF, Xia W, Golfman LS, Ou-Yang F, Yang JY et al. IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 2004; 117: 225–237.

    Article  CAS  Google Scholar 

  31. Borkhardt A, Repp R, Hass OA, Leis T, Harbott J, Kreuder J et al. Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t (X; 11)(q13;q23). Oncogene 1997; 14: 195–202.

    Article  CAS  Google Scholar 

  32. Essers MA, de Vries-Smits LM, Barker N, Polderman PE, Burgering BM, Korswagen HC . Functional interaction between beta-catenin and FOXO in oxidative stress signaling. Science 2005; 308: 1181–1184.

    Article  CAS  Google Scholar 

  33. Tang TT, Dowbenko D, Jackson A, Toney L, Lewin DA, Dent AL et al. The forkhead transcription factor AFX activates apoptosis by induction of the BCL-6 transcriptional repressor. J Biol Chem 2002; 277: 14255–14265.

    Article  CAS  Google Scholar 

  34. Lu Y, Lin YZ, LaPushin R, Cuevas B, Fang X, Yu SX et al. The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene 1999; 18: 7034–7045.

    Article  CAS  Google Scholar 

  35. Chakraborty G, Kumar S, Mishra R, Patil TV, Kundu GC . Semaphorin 3A suppresses tumor growth and metastasis in mice melanoma model. PLoS ONE 2012; 7: e33633 pone. 0033633.

    Article  CAS  Google Scholar 

  36. Chakraborty G, Jain S, Kundu GC . Osteopontin promotes vascular endothelial growth factor-dependent breast tumor growth and angiogenesis via autocrine and paracrine mechanisms. Cancer Res 2008; 68: 152–161.

    Article  CAS  Google Scholar 

  37. Miao HQ, Soker S, Feiner L, Alonso JL, Raper JA, Klagsbrun M . Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165. J Cell Biol 1999; 146: 233–242.

    CAS  Google Scholar 

  38. Gabrovska PN, Smith RA, Tiang T, Weinstein SR, Haupt LM, Griffiths LR . Semaphorin-plexin signalling genes associated with human breast tumourigenesis. Gene 2011; 489: 63–69.

    Article  CAS  Google Scholar 

  39. Nho RS, Kahm J . beta1-Integrin-collagen interaction suppresses FoxO3a by the coordination of Akt and PP2A. J Bio Chem 2010; 285: 14195–14209.

    Article  CAS  Google Scholar 

  40. Liu JL, Sheng X, Hortobagyi ZK, Mao Z, Gallick GE, Yung WK . Nuclear PTEN-mediated growth suppression is independent of Akt down-regulation. Mol Cell Bio 2005; 25: 6211–6224.

    Article  CAS  Google Scholar 

  41. Emerling BM, Weinberg F, Liu JL, Mak TW, Chandel NS . PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a). Proc Natl Acad Sci USA 2008; 105: 2622–2627.

    Article  CAS  Google Scholar 

  42. Gil A, Andres-Pons A, Fernandez E, Valiente M, Torres J, Cervera J et al. Nuclear localization of PTEN by a Ran-dependent mechanism enhances apoptosis: Involvement of an N-terimnal nuclear localization domain and multiple nuclear exclusion motifs. Mol Bio Cell 2006; 17: 4002–4013.

    Article  CAS  Google Scholar 

  43. Trotman LC, Wang X, Alimonti A, Chen Z, Teruya-Feldstein J, Yang H et al. Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 2007; 128: 141–156.

    Article  CAS  Google Scholar 

  44. Paik JH, Kollipara R, Chu G, Ji H, Xiao Y, Ding Z et al. FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 2007; 128: 309–323.

    Article  CAS  Google Scholar 

  45. Li P, Lee H, Guo S, Unterman TG, Jenster G, Bai W . AKT-independent protection of prostate cancer cells from apoptosis mediated through complex formation between the androgen receptor and FKHR. Mol Cell Biol 2003; 23: 104–118.

    Article  Google Scholar 

  46. Seoane J, Le HV, Shen L, Anderson SA, Massague J . Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004; 117: 211–223.

    Article  CAS  Google Scholar 

  47. Bouchard C, Marquardt J, Bras A, Medema RH, Eilers M . Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins. EMBO J 2004; 23: 2830–2840.

    Article  CAS  Google Scholar 

  48. Potente M, Urbich C, Sasaki K, Hofmann WK, Heeschen C, Aicher A et al. Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularisation. J Clin Invest 2005; 115: 2382–2392.

    Article  CAS  Google Scholar 

  49. Wang L, Dutta SK, Kojima T, Xu X, Khosravi-Far R, Ekker SC et al. Neuropilin-1 modulates p53/caspases axis to promote endothelial cell survival. PLoS ONE 2007; 2: e1161.

    Article  Google Scholar 

  50. Acevedo LM, Barillas S, Weis SM, Göthert JR, Cheresh DA . Semaphorin 3A suppresses VEGF-mediated angiogenesis yet acts as a vascular permeability factor. Blood 2008; 111: 2674–2680.

    Article  CAS  Google Scholar 

  51. Nasarre C, Roth M, Jacob L, Roth L, Koncina E, Thien A et al. Peptide-based interference of the transmembrane domain of neuropilin-1 inhibits glioma growth in vivo. Oncogene 2010; 29: 2381–2392.

    Article  CAS  Google Scholar 

  52. Yang L, Xie S, Jamaluddin MS, Altuwaijri S, Ni J, Kim E et al. Induction of androgen receptor expression by phosphatidylinositol 3-kinase/Akt downstream substrate, FOXO3a, and their roles in apoptosis of LNCaP prostate cancer cells. J Biol Chem 2005; 280: 33558–33565.

    Article  CAS  Google Scholar 

  53. Sinha S, Malonia SK, Mittal SP, Singh K, Kadreppa S, Kamat R et al. Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MR element. EMBO J 2010; 29: 830–842.

    Article  CAS  Google Scholar 

  54. Rampalli S, Pavithra L, Bhatt A, Kundu TK, Chattopadhyay S . Tumor suppressor SMAR1 mediates cyclin D1 repression by recruitment of the SIN3/histone deacetylase 1 complex. Mol Cell Biol 2005; 25: 8415–8429.

    Article  CAS  Google Scholar 

  55. Jain S, Chakraborty G, Raja R, Kale S, Kundu GC . Prostaglandin E2 regulates tumor angiogenesis in prostate cancer. Cancer Res 2008; 68: 7750–7759.

    Article  CAS  Google Scholar 

  56. Raja R, Kale S, Thorat D, Soundararajan G, Lohite K, Mane A et al. Hypoxia-driven osteopontin contributes to breast tumor growth through modulation of HIF1α-mediated VEGF-dependent angiogenesis. Oncogene 2014; 33: 2053–2064.

    Article  CAS  Google Scholar 

  57. Kumar V, Behera R, Lohite K, Karnik S, Kundu GC . p38 kinase is crucial for osteopontin-induced furin expression that supports cervical cancer progression. Cancer Res 2010; 70: 10381–10391.

    Article  CAS  Google Scholar 

  58. Sharma P, Kumar S, Kundu GC . Transcriptional regulation of human osteopontin promoter by histone deacetylase inhibitor, trichostatin A in cervical cancer cells. Mol Cancer 2010; 9: 178–189.

    Article  Google Scholar 

  59. Vacca A, Scavelli C, Serini G, Di Pietro G, Cirulli T, Merchionne F et al. Loss of inhibitory semaphorin 3A (SEMA3A) autocrine loops in bone marrow endothelial cells of patients with multiple myeloma. Blood 2006; 108: 1661–1667.

    Article  CAS  Google Scholar 

  60. Gyorffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q et al. An online survival analysis tools to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat 2010; 123: 725–731.

    Article  Google Scholar 

Download references

Acknowledgements

We thank Dr B Ramanamurthy, In-charge, Experimental Animal Facility, NCCS for breeding and maintaining NOD–SCID mice. This work was supported by a grant from the Council of Scientific and Industrial Research (CSIR) (to RM and GS) and the Department of Science and Technology (DST) (to DT), Government of India. Financial support: This project was funded by the CSIR and DST, Government of India.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to G C Kundu.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mishra, R., Thorat, D., Soundararajan, G. et al. Semaphorin 3A upregulates FOXO 3a-dependent MelCAM expression leading to attenuation of breast tumor growth and angiogenesis. Oncogene 34, 1584–1595 (2015). https://doi.org/10.1038/onc.2014.79

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2014.79

This article is cited by

Search

Quick links