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The CXCR4-Dependent LASP1-Ago2 Interaction in Triple-Negative Breast Cancer
Augustus M C Tilley1, Cory M Howard1, Sangita Sridharan1
1Department of Cancer Biology, College of Medicine and Life Sciences, University of Toledo Health Science Campus, Toledo, OH 43614, USA.
Abstract:
The CXCR4-LASP1 axis is an emerging target in the field of breast cancer metastasis. C-X-C chemokine receptor type 4 (CXCR4) mediates directed cell migration when activated by its cognate ligand CXCL12. LIM and SH3 Protein 1 (LASP1) is a critical node in the CXCR4 signaling pathway, as its deficiency blocks CXCR4-dependent Matrigel invasion. The mechanism by which LASP1 facilitates this invasive ability of tumor cells when CXCR4 is activated is unknown. Our previous proteomics work had revealed several components of the RNA interference (RNAi) machinery as being potential LASP1 interacting proteins. Here we report that argonaute 2 (Ago2), a protein with central involvement in RNAi, associates with LASP1 in triple-negative breast cancer (TNBC) cells. We demonstrate that LASP1 co-immunoprecipitates with Ago2 endogenously in a CXCL12-dependent manner, with further confirmation of this interaction by proximity ligation assay. Furthermore, this association is specific to CXCR4 as it can be abrogated by the CXCR4 antagonist, AMD3465. By GST-pulldown approach, we identify that LASP1 directly binds to Ago2 through its LIM and SH3 domains, and that this binding is dictated by the S146 and Y171 phosphorylation sites of LASP1. Additionally, the phosphorylation status of LASP1 affected tumor suppressor microRNA (miRNA) Let-7a-guided Ago2 activity. Levels of several endogenous targets of Let-7a were found to be altered including C-C chemokine receptor type 7 (CCR7), which is another critical chemokine receptor involved in metastasis to lymph nodes. Our results suggest a novel role for the LASP1-Ago2 module in shaping the RNAi landscape, functionally impacting the invasive ability of cancer cells.
Insights
The study reveals that LIM and SH3 Protein 1 (LASP1) interacts with argonaute 2 (Ago2) in breast cancer cells, influencing RNA interference and cell invasion. This LASP1-Ago2 interaction is crucial for CXCR4-mediated metastasis.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- The CXCR4-LASP1 axis is a key player in breast cancer metastasis.
- CXCR4 activation by CXCL12 drives cell migration, with LASP1 being essential for invasion.
- The precise mechanism of LASP1's role in CXCR4-mediated invasion was previously unknown.
Purpose of the Study:
- To elucidate the mechanism by which LASP1 facilitates tumor cell invasion in response to CXCR4 activation.
- To investigate the interaction between LASP1 and components of the RNA interference (RNAi) machinery.
- To determine the functional impact of the LASP1-Ago2 interaction on breast cancer cell invasiveness.
Main Methods:
- Co-immunoprecipitation and proximity ligation assays to confirm LASP1-Ago2 interaction in triple-negative breast cancer (TNBC) cells.
- Use of CXCR4 antagonist AMD3465 to assess specificity of the interaction.
- GST-pulldown assays to identify binding domains and phosphorylation sites (S146, Y171) on LASP1 crucial for Ago2 binding.
- Analysis of microRNA (miRNA) Let-7a activity and its endogenous targets (e.g., CCR7) in relation to LASP1 phosphorylation status.
Main Results:
- LASP1 was found to associate with argonaute 2 (Ago2), a key RNAi protein, in TNBC cells.
- This LASP1-Ago2 interaction is dependent on CXCL12 and specific to CXCR4 signaling.
- LASP1 directly binds Ago2 via its LIM and SH3 domains, modulated by LASP1 phosphorylation at S146 and Y171.
- LASP1 phosphorylation status influences Let-7a miRNA-guided Ago2 activity, altering levels of Let-7a targets like CCR7.
Conclusions:
- A novel LASP1-Ago2 module is identified, playing a significant role in regulating the RNAi landscape.
- This module functionally impacts the invasive capabilities of breast cancer cells.
- The findings provide new insights into the molecular mechanisms driving breast cancer metastasis via the CXCR4 pathway.
