Abstract G protein coupled receptors (GPCRs) are the largest family of cell surface receptors with over 800 members. Aberrant activation of GPCRs has been implicated in cancer initiation and development. However, many of GPCRs are overexpressed in cancer cells, making it difficult to target individual GPCRs for cancer treatment. In this study, we have tested a new approach of targeting GPCRs in HER2+ breast cancer by blocking their common pathways downstream of G proteins. By mammary gland-specific expression of the pertussis toxin (PTx) catalytic subunit, a selective inhibitor that uncouples a subgroup of GPCRs from activating Gi/o proteins, we demonstrated that blocking GPCR downstream signaling delayed the onset of HER2-driven spontaneous mammary tumor formation and suppressed lung metastasis. Moreover, we showed that aberrant HER2 signaling upregulated Gi/o-GPCR expression in breast cancer cells, which in turn activated the ErbB/HER family of protein-tyrosine kinases via Src and PI3K pathways to maintain cancer stem cell tumorigenicity. Targeting Gi/o-GPCR signaling by PTx, Src or PI3K inhibitors sensitizes HER2+ breast cancer to HER2-targeted therapies. Together, our data demonstrate that targeting GPCR downstream signal pathways may represent a new approach to ablate CSCs to block tumor progression and augment HER2-targeted therapeutics. Citation Format: Wei Wang, Dharmendra Bhargava, Yuan Chao Ye, Songhai Chen. Targeting GPCR signaling in HER2+ breast cancer suppresses cancer stem cell tumorigenicity and sensitizes HER2-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4683.