Potentiation of the HDAC Pin signaling module by

Potentiation of the HDAC6-Pin1 signaling module by GRK2 in specific breast cancer cells could be envisaged as an bax pathway to enhanced activity of different tumor-promoting cascades that would trigger a self-perpetuating positive feedback cycle promoting cell proliferation and increased survival. Notably, HDAC6 is required for oncogenic Ras- and ErbB2-dependent fibroblast transformation, for sustained ERK1/2 and AKT signaling, for anchorage-independent growth of MCF7 cells and cancer cell survival (Lee et al., 2008; Li et al., 2013). Consistent with a regulatory role of GRK2 on HDAC6 tumoral competences, we find that extra GRK2 levels lessen the cytotoxic effectiveness of HDAC6 inhibitors (West and Johnstone, 2014). Therefore, it is tempting to suggest that GRK2-mediated phosphorylation and activation of HDAC6 makes this molecule less sensitive to its inhibitors, thereby debilitating their therapeutic effects. Such notion would be consistent with ineffective or partial responses shown by pan-HDAC inhibitors in GRK2-overexpressing (MCF7, MDA-MB361) (Tate et al., 2012) and with different efficacies among breast tumor cells with p53 mutant status (Li et al., 2011) and different pS670-GRK2 ratios. Therefore, treatment of certain types of breast cancer may benefit from the combined inhibition of GRK2 and HDAC6. It is worth noting that the HDAC6\'s substrate Pin1, which levels are over-expressed in human breast cancers (Ryo et al., 2003) and transcriptionally enhanced by oncogenic HER2 or Ras signaling, is also critical for the HER2-Ras-triggered transformation of mammary epithelial cells (Ryo et al., 2002), for EGF signaling and HER-2 expression in breast cancer cells (Khanal et al., 2010), for regulation of the activity of estrogen receptors (Lucchetti et al., 2013), and for coordination of cell cycle progression (Liou et al., 2011). By using silencing approaches and (or) HDAC6 or Pin1 mutants unable to undergo GRK2-mediated phosphorylation or de-acetylation, our data support a relevant role of the GRK2-HDAC6-Pin1 signaling axis in reinforcing growth factor signaling, proliferation and anchorage-independent growth. Since the higher growth rates of breast tumor cells rely on Pin1 binding to cell-cycle associated proteins (Khanal et al., 2010; Liou et al., 2011; Lucchetti et al., 2013; Ryo et al., 2002), our data suggest that GRK2 would foster proliferation by triggering HDAC6-mediated Pin1 deacetylation, leading to enhanced interaction with mitotic regulators, particularly which those displaying an allosteric binding mode such as PLK-1 or Cdc25 phosphatase (Innes et al., 2013). Therefore, concurrent Pin1 and GRK2 up-regulation also emerges as a relevant component of the tumorigenesis pathway. On the other hand, the simultaneous GRK2-dependent down-modulation of wild-type p53 protein and extra activation of the pro-survival AKT route might also help to counteract the effects of relevant cytotoxic compounds. We observe that down-regulating GRK2 levels increases sensitivity to apoptotic agents in cultured cells and triggers a strong p53 response in xenograft-derived tumors in vivo. Conversely, increasing GRK2 expression in non-transformed breast cells reduces genotoxic-dependent cell death and the p53 response, as previously noted in genotoxic cell cycle-arrested cells (Penela et al., 2010b). These results are consistent with the occurrence of an inverse GRK2-p53 correlation in the xenograft tumor model and in tumors of patients with breast cancer scored as p53-negative (either by gene-deletion or by protein down-modulation), suggesting that GRK2 might be an important modulator of the intact p53 pathway either through direct modulation of HDAC6 (Tang et al., 2008) or of other upstream regulators, with potential implications in therapy. An important proportion of breast cancer ERα-+ patients treated with estradiol antagonists or aromatase inhibitors become resistant to these compounds, what urges to identify new therapeutic targets (Renoir et al., 2013). We found that tamoxifen-resistant MCF7 cells can build stronger apoptotic responses in the absence of GRK2, suggesting that pharmacological inhibition of this kinase might be effective to inactivate tamoxifen-resistance pathways and to sensitize cells to general chemotherapeutic agents.