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A proteomic approach to identify endosomal cargoes controlling cancer invasiveness
Jesica Diaz-Vera1, Sarah Palmer1, Juan Ramon Hernandez-Fernaud1
1Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK.
Abstract:
We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype.
Insights
Rab17 loss promotes cancer cell invasion by reducing Vamp8 levels, leading to increased cell surface Neuropilin-2 (NRP2). This NRP2 is crucial for breaking down the basement membrane in invasive ductal carcinoma.
Area of Science:
- Cell Biology
- Molecular Oncology
- Cancer Research
Background:
- Rab17, a small GTPase, regulates epithelial polarity and its suppression by ERK2 (MAPK1) signaling promotes cancer invasiveness.
- The precise mechanisms by which Rab17 loss facilitates invasion and the specific endosomal cargoes involved remain largely uncharacterized.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying Rab17-mediated regulation of cancer cell invasiveness.
- To identify key endosomal trafficking pathways and cargoes affected by Rab17 suppression.
Main Methods:
- Quantitative mass spectrometry-based proteomics to identify proteins affected by Rab17 knockdown.
- Immunofluorescence microscopy to localize protein interactions and cellular distribution.
- Proteomic analysis to identify endosomal receptors and their trafficking dynamics.
Main Results:
- Rab17 knockdown selectively reduces cellular levels of the v-SNARE Vamp8, which localizes to late endosomes with Rab17.
- Reduced Vamp8 levels promote the transition from ductal carcinoma in situ (DCIS) to an invasive phenotype.
- Neuropilin-2 (NRP2) was identified as a key pro-invasive cargo regulated by Rab17 and Vamp8 trafficking, with reduced levels of either protein increasing cell surface NRP2 expression.
- NRP2 is essential for the basement membrane degradation observed during the transition to an invasive phenotype.
Conclusions:
- Rab17 and Vamp8 are critical regulators of endosomal trafficking, controlling the cell surface presentation of the pro-invasive receptor NRP2.
- The Rab17-Vamp8-NRP2 axis plays a significant role in promoting cancer cell invasion and basement membrane disruption.
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