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Mishra and Sahin patent info for Conflict of Interest

This file contains supplementary methods, 7 supplementary figures and 5 supplementary tables.

Purpose: Tamoxifen remains an important hormonal therapy for ER-positive breast cancer; however, development of resistance is a major obstacle in clinics. Here, we aimed to identify novel mechanisms of tamoxifen resistance and provide actionable drug targets overcoming resistance.Experimental Design: Whole-transcriptome sequencing, downstream pathway analysis, and drug repositioning approaches were used to identify novel modulators [here: phosphodiesterase 4D (PDE4D)] of tamoxifen resistance. Clinical data involving tamoxifen-treated patients with ER-positive breast cancer were used to assess the impact of PDE4D in tamoxifen resistance. Tamoxifen sensitization role of PDE4D was tested in vitro and in vivo. Cytobiology, biochemistry, and functional genomics tools were used to elucidate the mechanisms of PDE4D-mediated tamoxifen resistance.Results: PDE4D, which hydrolyzes cyclic AMP (cAMP), was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. Higher PDE4D expression predicted worse survival in tamoxifen-treated patients with breast cancer (n = 469, P = 0.0036 for DMFS; n = 561, P = 0.0229 for RFS) and remained an independent prognostic factor for RFS in multivariate analysis (n = 132, P = 0.049). Inhibition of PDE4D by either siRNAs or pharmacologic inhibitors (dipyridamole and Gebr-7b) restored tamoxifen sensitivity. Sensitization to tamoxifen is achieved via cAMP-mediated induction of unfolded protein response/ER stress pathway leading to activation of p38/JNK signaling and apoptosis. Remarkably, acetylsalicylic acid (aspirin) was predicted to be a tamoxifen sensitizer using a drug repositioning approach and was shown to reverse resistance by targeting PDE4D/cAMP/ER stress axis. Finally, combining PDE4D inhibitors and tamoxifen suppressed tumor growth better than individual groups in vivo.Conclusions: PDE4D plays a pivotal role in acquired tamoxifen resistance via blocking cAMP/ER stress/p38-JNK signaling and apoptosis. Clin Cancer Res; 24(8); 1987–2001. ©2018 AACR.

Flow chart of the reference construction. (PDF 34Â kb)

Length distribution of the sequences after oxidation for medaka and Xiphophorus. (PDF 667Â kb)

Length distribution of the sequences after oxidation for medaka and Xiphophorus. (PDF 667Â kb)

Pie charts of the base distribution of piRNA sequences: medaka at the first position (A) and at the 10th position (B) and of Xiphophorus at sequence position 1 (C) and 10 (D). (PDF 310Â kb)

Pie charts of the base distribution of piRNA sequences: medaka at the first position (A) and at the 10th position (B) and of Xiphophorus at sequence position 1 (C) and 10 (D). (PDF 310Â kb)

Histogram of bases within piRNA cluster 3. Red rectangles indicate regions selected for primer design. (PDF 42Â kb)

Histogram of bases within piRNA cluster 3. Red rectangles indicate regions selected for primer design. (PDF 42Â kb)