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. 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 and Cytobiology, biochemistry, and functional genomics tools were used to elucidate the mechanisms of PDE4D-mediated tamoxifen resistance. 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 ( = 469, = 0.0036 for DMFS; = 561, = 0.0229 for RFS) and remained an independent prognostic factor for RFS in multivariate analysis ( = 132, = 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 PDE4D plays a pivotal role in acquired tamoxifen resistance via blocking cAMP/ER stress/p38-JNK signaling and apoptosis. .

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BackgroundA growing number of studies report an abnormal expression of Piwi-interacting RNAs (piRNAs) and the piRNA processing enzyme Piwi in many cancers. Whether this finding is an epiphenomenon of the chaotic molecular biology of the fast dividing, neoplastically transformed cells or is functionally relevant to tumorigenesisis is difficult to discern at present. To better understand the role of piRNAs in cancer development small laboratory fish models can make a valuable contribution. However, little is known about piRNAs in somatic and neoplastic tissues of fish.ResultsTo identify piRNA clusters that might be involved in melanoma pathogenesis, we use several transgenic lines of medaka, and platyfish/swordtail hybrids, which develop various types of melanoma. In these tumors Piwi, is expressed at different levels, depending on tumor type. To quantify piRNA levels, whole piRNA populations of testes and melanomas of different histotypes were sequenced. Because no reference piRNA cluster set for medaka or Xiphophorus was yet available we developed a software pipeline to detect piRNA clusters in our samples and clusters were selected that were enriched in one or more samples. We found several loci to be overexpressed or down-regulated in different melanoma subtypes as compared to hyperpigmented skin. Furthermore, cluster analysis revealed a clear distinction between testes, low-grade and high-grade malignant melanoma in medaka.ConclusionsOur data imply that dysregulation of piRNA expression may be associated with development of melanoma. Our results also reinforce the importance of fish as a suitable model system to study the role of piRNAs in tumorigenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2697-z) contains supplementary material, which is available to authorized users.

Alternative splicing of mRNAs is known to involve a major regulation of gene expression at RNA level in mammalian cells. The PTEN (Phosphatase and TENsin homologue deleted from the human chromosome 10), TPTE (Transmembrane Phosphatase with TEnsin homology) and TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) belong to a family of dual-specific lipid and protein phosphatases. PTEN is a well characterized tumor suppressor, which plays crucial role in cell survival, cell cycle regulation, cell proliferation as well as adhesion, motility and migration of cells. The C2-domain of PTEN is essential for PTEN-functions. We have isolated a novel 1019 bp human TPIP cDNA (TPIP-C2) from a human testis cDNA library. In silico analysis of the cDNA revealed that it is produced from the TPIP-locus on the human chromosome 13 by alternative RNA-splicing. It has a unique 5′-Alu sequence, a LINE sequence followed by a 582 bp Open Reading Frame (ORF) encoding a 193 aa polypeptide with a partial phosphatase domain and a C2-domain. TPIP-C2 mRNA is expressed in human testis and in mouse tissues. Mouse testis and brain showed higher levels of TPIP-C2 mRNA in comparison to the heart, liver and kidney under normal physiological conditions. TPIP-C2 mRNAs from human and mouse testes show extensive sequence identity. Over-expression of TPIP-C2 cDNA in HeLa cells strongly (up to 85%) inhibited cell growth/proliferation and caused apoptosis in a caspase 3-dependent manner. These findings suggest for the first time that a TPIP splice-variant mRNA with a partial phosphatase domain and a C2-domain is expressed in cells and tissues of human and murine origins under normal physiological conditions. Inhibition of cell growth/proliferation and induction of apoptosis by overexpression of TPIP-C2 mRNA in HeLa cells suggest that it may be involved in negative regulation of cell growth/proliferation.

The human TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) belongs to the PTEN (Phosphatase and TENsin homologue deleted on chromosome 10) family of dual-specific phosphatases and is expressed from the human chromosome 13 as multiple splice-variants, e.g., TPIPα, β, γ mRNAs. PTEN is a well characterized tumor suppressor, which controls survival, adhesion, motility and migration of mammalian cells, its C2-domain plays crucial role in controlling these functions. However, role of isolated C2-domain protein in regulation of cell proliferation and apoptosis is not reported. We report sequence analysis and function of a novel human TPIP (TPIP-C2) cDNA encoding a 193 amino acid C2-domain in cell proliferation and apoptosis regulation. In silico analysis and homology modelling revealed that the C2-domain of TPIP-C2 is similar to that of PTEN but with short disorder sequences overlapping or adjacent to the post-translational modification sites. Overexpression of TPIP-C2 cDNA in human embryonic kidney (HEK-293) cells caused cell cycle arrest, inhibition of cell proliferation and induced apoptosis in an activated caspase 3 and PARP-dependent manner in comparison to overexpression of the full length human PTEN cDNA. TPIP-C2 overexpressed cells also showed S-phase cell cycle arrest. We suggest that C2-domain of TPIP-C2 may act as a dominant negative effector, which may bind to and arrest the cell proliferation signalling complex and isolated TPIP-C2-domain-like proteins expressed in mammalian cells/tissues may play important role in regulation of cell proliferation and apoptosis. The TPIP-C2 cDNA may be exploited for inducing cell cycle-inhibition and apoptosis in human cancer cells and tissues.