The Autophagic Process Occurs in Human Bone Metastasis and Implicates Molecular Mechanisms Differently Affected by Rab5a in the Early and Late Stages

Int J Mol Sci. 2016 Mar 25;17(4):443. doi: 10.3390/ijms17040443.

Abstract

Autophagy favours metastatic growth through fuelling energy and nutrients and resistance to anoikis, typical of disseminated-tumour cells. The autophagic process, mediated by a unique organelle, the autophagosome, which fuses with lysosomes, is divided into three steps. Several stages, especially early omegasome formation and isolation-membrane initiation, remain controversial; molecular mechanisms involve the small-GTPase Rab5a, which regulates vesicle traffic for autophagosome formation. We examined Rab5a involvement in the function of key members of ubiquitin-conjugation systems, Atg7 and LC3-lipidated, interacting with the scaffold-protein p62. Immunohistochemistry of Rab5a was performed in human specimens of bone metastasis and pair-matched breast carcinoma; the autophagic-molecular mechanisms affected by Rab5a were evaluated in human 1833 bone metastatic cells, derived from breast-carcinoma MDA-MB231 cells. To clarify the role of Rab5a, 1833 cells were transfected transiently with Rab5a-dominant negative, and/or stably with the short-hairpin RNA Atg7, were exposed to two inhibitors of autolysosome function, and LC3II and p62 expression was measured. We showed basal autophagy in bone-metastatic cells and the pivotal role of Rab5a together with Beclin 1 between the early stages, elongation of isolation membrane/closed autophagosome mediated by Atg7, and the late-degradative stages. This regulatory network might occur in bone-metastasis and in high-grade dysplastic lesions, preceding invasive-breast carcinoma and conferring phenotypic characteristics for dissemination.

Keywords: Rab5a; autophagy; bone metastasis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy
  • Autophagy-Related Protein 7 / antagonists & inhibitors
  • Autophagy-Related Protein 7 / genetics
  • Autophagy-Related Protein 7 / metabolism
  • Beclin-1 / genetics
  • Beclin-1 / metabolism
  • Blotting, Western
  • Bone Neoplasms / metabolism
  • Bone Neoplasms / pathology*
  • Bone Neoplasms / secondary
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology*
  • Cell Line, Tumor
  • Female
  • Humans
  • Immunohistochemistry
  • Microscopy, Fluorescence
  • Microtubule-Associated Proteins / metabolism
  • Neoplasm Staging
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • RNA-Binding Proteins / metabolism
  • rab5 GTP-Binding Proteins / genetics
  • rab5 GTP-Binding Proteins / metabolism*

Substances

  • BECN1 protein, human
  • Beclin-1
  • MAP1LC3A protein, human
  • Microtubule-Associated Proteins
  • P62 protein, human
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • RAB5C protein, human
  • rab5 GTP-Binding Proteins
  • ATG7 protein, human
  • Autophagy-Related Protein 7