Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors.

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.

Mammalian Scratch participates in neuronal differentiation in P19 embryonal carcinoma cells.

Mammalian Scratch (Scrt) is a Snail family zinc finger transcription factor that is specifically expressed in newly differentiating, post-mitotic central nervous system neurons. While Scrt-related genes appear essential for invertebrate neurogenesis, the role of Scrt in mammalian neural development is unknown. In this study, we found that neural differentiation of multipotent mouse P19 embryonal carcinoma cells by retinoic acid led to the appearance of Scrt together with neuron-specific class III beta-tubulin (Tuj1), following the earlier elaboration of Mash1. Transient co-transfection in P19 cells with either Mash1 or NeuroD2 plus E12 also induced Scrt gene expression. Moreover, overexpression of Scrt alone was sufficient to confer Tuj1 immunoreactivity and neuronal morphology in a subset of P19 cells. Scrt thus appears to function downstream of proneural bHLH proteins in promoting mammalian neural differentiation in this model system.

Mammalian Scratch: a neural-specific Snail family transcriptional repressor.

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. We report the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, we detect hScrt expression in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix--loop--helix (bHLH) transcription factors. We show that hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation.

Management of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is responsible for a significant amount of morbidity and mortality throughout the world. In many countries, including the United States, a definite increase in the incidence of HCC has been reported recently, largely attributable to the increasing incidence of hepatitis C infection. Unfortunately, the current management of HCC is confusing due to the large number of treatment options available. The difficulty of managing a patient with HCC is compounded by the lack of well-designed, randomized clinical trials comparing the various treatment modalities. Nevertheless, many exciting management options are currently available that may prove valuable in the treatment of this disease. Partial hepatic resection or, in some instances, liver transplantation offers the best chance for cure. However, various ablative therapies, including percutaneous ethanol injection, radiofrequency ablation, and cryosurgery, may produce a survival benefit. In the future, systemic chemotherapy and transarterial chemoembolization, employed either alone or as adjuncts to ablation or resection, may play an increasing role in palliating or down-staging a patient with advanced HCC. This overview of the state-of-the-art management of HCC attempts to guide the practicing physician in selecting the best treatment plan for an individual with HCC.

Long-term survival of solid organ allografts by brief anti-lymphocyte function-associated antigen-1 monoclonal antibody monotherapy.

Strategies targeting lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule-1 (ICAM-1) have previously been shown to produce long-term survival of solid organ allografts in animals only when both CD11a and ICAM-1 are targeted for a brief (6-7 days) time or when extended (14 weeks) treatment with anti-CD11a monoclonal antibody (mAb) is administered. We show that recipient pretreatment followed by a brief (13 days) treatment course with high-dose anti-CD11a mAb alone produces long-term survival of cardiac allografts in the rigorous, nonprimarily vascularized heart allograft model in mice. This treatment regimen induces specific unresponsiveness in our model. In recipients bearing long-term beating cardiac grafts after treatment with anti-CD11a mAb, there still exists a high frequency of potentially antigen-reactive T cells in isolated peripheral blood lymphocyte fractions. Therefore, clonal deletion does not appear to explain the induction of specific unresponsiveness by treatment with anti-CD11a mAb in this model. These findings support the further investigation of the use of high-dose anti-LFA-1 mAb monotherapy in the pre- and early postoperative period to promote solid organ allograft survival.