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The alpha-arrestin ARRDC3 functions as a metastasis suppressor by regulating GPCR trafficking and differential signaling to the Hippo pathway in breast cancer


G protein-coupled receptor (GPCR) signaling regulates cancer cell proliferation, invasion, migration and survival at metastatic sites. However, despite the success and promise of GPCRs as therapeutic targets, there are currently no FDA-approved drugs targeting GPCRs for cancer. Protease-activated receptor-1 (PAR1) is a GPCR that promotes breast cancer progression. PAR1 is overexpressed in breast cancer patient tissue biopsies and in breast carcinoma cell lines, and correlates with increased rates of metastasis and poor prognosis and increased invasion and metastatic potential, respectively. One mechanism that leads to PAR1 aberrant over-expression is defective lysosomal trafficking and degradation of the receptor, leading to persistent G protein signaling. Our lab recently showed that arrestin domain containing protein-3 (ARRDC3), an adaptor protein for E3 ubiquitin ligases, functions in regulating proper lysosomal trafficking and degradation. ARRDC3 has been identified as a tumor suppressor in aggressive breast cancer and I further examined the role of ARRDC3 in PAR1 trafficking in invasive breast carcinoma cells. The Hippo pathway, which converges on the transcriptional co-activators YAP and TAZ, is a well-established mediator of tumorigenesis and cancer progression and is also activated by GPCR stimulation, including through PAR1, in breast carcinoma. However, the mechanisms by which ARRDC3 regulates GPCR-stimulated Hippo signaling to promote breast cancer metastasis remains unknown. In the work described in this thesis, I discovered that ARRDC3 displays a multifunctional role in suppressing breast cancer growth and invasion: 1) by controlling proper PAR1 trafficking and degradation, thus inhibiting persistent G protein signaling and 2) by interacting with TAZ, thus sequestering TAZ in the cytoplasm and blocking downstream Hippo pathway gene transcription to occur. I used a tetracycline-inducible pSLIK lentiviral vector to restore expression of ARRDC3 in highly invasive, basal-like MDA-MB-231 cells, which exhibit high PAR1 and low ARRDC3 expression. Re-expression of ARRDC3 restored agonist-induced PAR1 degradation, attenuated JNK and Hippo-YAP signaling, and further inhibited PAR1-mediated breast carcinoma cell invasion. Thus, the dysregulation of PAR1 trafficking due to loss of ARRDC3 expression leads to persistent JNK signaling and promotes breast cancer invasion. ARRDC3 re-expression in invasive breast carcinoma cells also attenuates GPCR-stimulated Hippo signaling and invasion that is mediated by activation of TAZ but not YAP. Furthermore, siRNA-targeted depletion of TAZ, but not YAP, inhibits GPCR-induced Hippo signaling and invasion. Our studies suggest a crucial role for ARRDC3 and TAZ in GPCR-Hippo pathway signaling in breast carcinoma invasion and metastasis. An understanding of the mechanisms by which the Hippo pathway is regulated by GPCRs may lead to new potential therapeutic targets for the treatment or prevention of metastatic breast cancer.

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