Abstract
Simple SummaryCXCR4 is critically involved in triple-negative breast cancer metastasis. LASP1 was previously found to be necessary for CXCR4-dependent tumor cell invasion. Here we aimed to understand how LASP1 can have such a powerful role this process that is critical to metastasis. We found Ago2, a master regulator protein, to associate with LASP1 in response to CXCR4 activity. Furthermore, we found that this association was responsible for affecting the expression of proteins regulated by Ago2.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.
Highlights
Triple-negative breast cancer (TNBC) has the lowest pathological complete response of all breast cancers
We found that Ago1 associated with LIM and SH3 Protein 1 (LASP1), it only interacted through the SH3 domain (Figure S1)
The present novel study demonstrates that LASP1 is capable of binding to argonaute 2 (Ago2) in a CXCL12-dependent manner, establishing Ago2 as a member of the CXCR4 signaling pathway
Summary
Triple-negative breast cancer (TNBC) has the lowest pathological complete response (pCR) of all breast cancers. Though it initially responds to therapy, it quickly becomes refractory to current neoadjuvant chemotherapies (NACT) involving platinum drugs, taxanes and anthracyclines [1,2,3]. One of the defining features of TNBC is its high incidence of metastasis [7], to the lungs, bone and brain when compared with other types of breast cancer [3]. Primary, localized breast cancer is rarely lethal, but it can metastasize to other critical organs throughout the body. Between metastasis and chemotherapy resistance [8], treating TNBC patients is an extreme challenge with a generally poor clinical outcome. It is imperative to identify novel therapeutic targets critically involved in the metastatic process, in the hopes of developing drugs effective at improving the quality of life and longevity of TNBC patients
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