Hepatic stellate cells (HSC) undergo transdifferentiation (activation) from lipid-storing pericytes to myofibroblastic cells to participate in liver fibrogenesis. Our recent work demonstrates that depletion of peroxisome proliferator-activated receptor ␥ (PPAR␥) constitutes one of the key molecular events for HSC activation and that ectopic expression of this nuclear receptor achieves the phenotypic reversal of activated HSC to the quiescent cells. The present study extends these findings to test a novel hypothesis that adipogenic transcriptional regulation is required for the maintenance of HSC quiescence. Transdifferentiation of vitamin A-storing hepatic stellate cells (HSC)1 to vitamin A-depleted myofibroblastic cells represents a key cellular event in the genesis of cirrhosis, for which no effective medial treatments are currently available except for liver transplantation. Transdifferentiated (activated) HSC are proliferative, proinflammatory, and fibrogenic with induced ability to synthesize and deposit extracellular matrices (1). Thus, better understanding of the mechanism underlying HSC transdifferentiation is the pivotal step toward identification of molecular targets for new and effective treatments for the disease. The most fundamental prerequisite for the understanding of HSC transdifferentiation is defining the cell type of differentiated HSC. This question relates to the origin of HSC that continues to puzzle the field. HSC are believed to serve as pericytes for hepatic capillaries called sinusoids. They represent 5-8% of total liver cells and 15-23% of nonparenchymal cells in the normal liver (2). HSC are positive for a mesenchymal marker such as vimentin. Rodent HSC express desmin (3) and glial fibrillary acidic protein (4), suggesting smooth muscle cell and glial cell lineage, respectively. Upon activation, both rodent and human HSC lose vitamin A and begin to express ␣-smooth muscle actin (5, 6). Interestingly, undifferentiated HSC in fetal livers that do not yet exhibit vitamin A storage also express ␣-smooth muscle actin (7), supporting a smooth muscle cell lineage. Synaptophysin, which controls exocytosis and the release of neurotransmitters in neurons and neuroendocrine cells, is also expressed in both rodent and human HSC (8). Neurotrophins such as nerve growth factor, brain-derived neurotrophic factor (BDNF), neutrophin NT-3, and NT-4/5 are also expressed (9), and so are their receptors, Trk-A, B, and C (9, 10), further supporting the neural and glial lineage.Peroxisome proliferator-activated receptor ␥ (PPAR␥) has been proposed as a potential molecular target for inhibition of HSC transdifferentiation (11-13). PPAR␥ level and activity are reduced in activated HSC, and the treatment of HSC with synthetic ligands for PPAR␥ such as thiazolidinediones effectively suppresses fibrogenic activity of and in vivo in experimental animals (13). However, these ligands are known to have PPAR␥-independent effects (14), and it was yet to be tested whether PPAR␥ per se had a direct effect to suppress ...

Depletion of peroxisome proliferator-activated receptor ␥ (PPAR␥) accompanies myofibroblastic transdifferentiation of hepatic stellate cells (HSC), the primary cellular event underlying liver fibrogenesis. The treatment of activated HSC in vitro or in vivo with synthetic PPAR␥ ligands suppresses the fibrogenic activity of HSC. However, it is uncertain whether PPAR␥ is indeed a molecular target of this effect, because the ligands are also known to have receptor-independent actions. To test this question, the present study examined the effects of forced expression of PPAR␥ via an adenoviral vector on morphologic and biochemical features of culture-activated HSC. The vector-mediated expression of PPAR␥ itself is sufficient to reverse the morphology of activated HSC to the quiescent phenotype with retracted cytoplasm, prominent dendritic processes, reduced stress fibers, and accumulation of retinyl palmitate. These effects are abrogated by concomitant expression of a dominant negative mutant of PPAR␥ that prevents transactivation of but not binding to the PPAR response element. PPAR␥ expression also inhibits the activation markers such as the expression of ␣-smooth muscle actin, type I collagen, and transforming growth factor ␤1; DNA synthesis; and JunD binding to the activator protein-1 (AP-1) site and AP-1 promoter activity. Inhibited JunD activity by PPAR␥ is not due to reduced JunD expression or JNK activity or to a competition for p300. But it is due to a JunD-PPAR␥ interaction as demonstrated by co-immunoprecipitation and glutathione S-transferase pull-down analysis. Further, the use of deletion constructs reveals that the DNA binding region of PPAR␥ is the JunD interaction domain. In summary, our results demonstrate that the restoration of PPAR␥ reverses the activated HSC to the quiescent phenotype and suppresses AP-1 activity via a physical interaction between PPAR␥ and JunD.

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Activating mutations of the PIK3CA gene occur frequently in breast cancer and inhibitors that are specific for PI3K p110α, such as BYL719, are being investigated in clinical trials. In a search for correlates of sensitivity to p110α inhibition among PIK3CA-mutant breast cancer cell lines, we observed that sensitivity to BYL719 (as assessed by cell proliferation) was associated with full inhibition of signaling through the TORC1 pathway. Conversely, cancer cells that were resistant to BYL719 had persistently active mTORC1 signaling, although Akt phosphorylation was inhibited. Similarly in patients, pS6 (residues 240/4) expression (a marker of mTORC1 signaling) was associated with tumor response to BYL719, and mTORC1 was found to be reactivated in tumors from patients whose disease progressed after treatment. In PIK3CA-mutant cancer cell lines with persistent mTORC1 signaling despite PI3K p110α blockade (that is, resistance), the addition of the allosteric mTORC1 inhibitor RAD001 to the cells along with BYL719 resulted in reversal of resistance in vitro and in vivo. Finally, we found that growth factors such as IGF1 and neuregulin1 can activate mTOR and mediate resistance to BYL719. Our findings suggest that simultaneous administration of mTORC1 inhibitors may enhance the clinical activity of p110α targeted drugs and delay the appearance of resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B106. Citation Format: Moshe Elkabets, Sadhna Vora, Dejan Juric, Natasha Morse, Mari Mino-Kenudson, Taru Muranen, Jessica Tao, Ana Bosch Campos, Jordi Rodon, Yasir H. Ibrahim, Violeta Serra, Vanessa Rodrik-Outmezguine, Saswati Hazra, Sharat Singh, Phillip Kim, Cornelia Quadt, Liu Manway, Alan Huang, Neal Rosen, Jeffrey A. Engelman, Maurizio Scaltriti, José Baselga. mTORC1 inhibition is required for sensitivity to PI3K p110α inhibitors in PIK3CA-mutant breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B106.

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidyl-inositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.