Faculty Bibliography

RAS proteins require membrane association for their biologic activity, making this association a logical target for anti-RAS therapeutics. Lipid modification of RAS proteins by a farnesyl isoprenoid is an obligate step in that association, and is an enzymatic process. Accordingly, farnesyltransferase inhibitors (FTI) were developed as potential anti-RAS drugs. The lack of efficacy of FTIs as anticancer drugs was widely seen as indicating that blocking RAS membrane association was a flawed approach to cancer treatment. However, a deeper understanding of RAS modification and trafficking has revealed that this was an erroneous conclusion. In the presence of FTIs, KRAS and NRAS, which are the RAS isoforms most frequently mutated in cancer, become substrates for alternative modification, can still associate with membranes, and can still function. Thus, FTIs failed not because blocking RAS membrane association is an ineffective approach, but because FTIs failed to accomplish that task. Recent findings regarding RAS isoform trafficking and the regulation of RAS subcellular localization have rekindled interest in efforts to target these processes. In particular, improved understanding of the palmitoylation/depalmitoylation cycle that regulates RAS interaction with the plasma membrane, endomembranes, and cytosol, and of the potential importance of RAS chaperones, have led to new approaches. Efforts to validate and target other enzymatically regulated posttranslational modifications are also ongoing. In this review, we revisit lessons learned, describe the current state of the art, and highlight challenging but promising directions to achieve the goal of disrupting RAS membrane association and subcellular localization for anti-RAS drug development. Clin Cancer Res; 21(8); 1819-27. (c)2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers."

Transforming growth factor-beta (TGF-beta) induces miR-21 expression which contributes to fibrotic events in the left ventricle (LV) under pressure overload. SMAD effectors of TGF-beta signaling interact with DROSHA to promote primary miR-21 processing into precursor miR-21 (pre-miR-21). We hypothesize that p-SMAD-2 and -3 also interact with DICER1 to regulate the processing of pre-miR-21 to mature miR-21 in cardiac fibroblasts under experimental and clinical pressure overload. The subjects of the study were mice undergoing transverse aortic constriction (TAC) and patients with aortic stenosis (AS). In vitro, NIH-3T3 fibroblasts transfected with pre-miR-21 responded to TGF-beta1 stimulation by overexpressing miR-21. Overexpression and silencing of SMAD2/3 resulted in higher and lower production of mature miR-21, respectively. DICER1 co-precipitated along with SMAD2/3 and both proteins were up-regulated in the LV from TAC-mice. Pre-miR-21 was isolated bound to the DICER1 maturation complex. Immunofluorescence analysis revealed co-localization of p-SMAD2/3 and DICER1 in NIH-3T3 and mouse cardiac fibroblasts. DICER1-p-SMAD2/3 protein-protein interaction was confirmed by in situ proximity ligation assay. Myocardial up-regulation of DICER1 constituted a response to pressure overload in TAC-mice. DICER mRNA levels correlated directly with those of TGF-beta1, SMAD2 and SMAD3. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with TGF-beta1, SMAD2, and SMAD3. Our results support that p-SMAD2/3 interacts with DICER1 to promote pre-miR-21 processing to mature miR-21. This new TGFbeta-dependent regulatory mechanism is involved in miR-21 overexpression in cultured fibroblasts, and in the pressure overloaded LV of mice and human patients.

Oxytocin is important for social interactions and maternal behaviour. However, little is known about when, where and how oxytocin modulates neural circuits to improve social cognition. Here we show how oxytocin enables pup retrieval behaviour in female mice by enhancing auditory cortical pup call responses. Retrieval behaviour required the left but not right auditory cortex, was accelerated by oxytocin in the left auditory cortex, and oxytocin receptors were preferentially expressed in the left auditory cortex. Neural responses to pup calls were lateralized, with co-tuned and temporally precise excitatory and inhibitory responses in the left cortex of maternal but not pup-naive adults. Finally, pairing calls with oxytocin enhanced responses by balancing the magnitude and timing of inhibition with excitation. Our results describe fundamental synaptic mechanisms by which oxytocin increases the salience of acoustic social stimuli. Furthermore, oxytocin-induced plasticity provides a biological basis for lateralization of auditory cortical processing.

RATIONALE: Coronary artery disease (CAD), the direct result of atherosclerosis, is the most common cause of death in Western societies. Vascular smooth muscle cell (VSMC) apoptosis occurs during the progression of atherosclerosis and in advanced lesions, promotes plaque necrosis, a common feature of high-risk/vulnerable atherosclerotic plaques. Akt1, a serine-threonine protein kinase, regulates several key endothelial cell (EC) and VSMC functions including cell growth, migration, survival and vascular tone. While global deficiency of Akt1 results in impaired angiogenesis and massive atherosclerosis, the specific contribution of VSMC Akt1 remains poorly characterized. OBJECTIVE: To investigate the contribution of VSMC Akt1 during atherogenesis and in established atherosclerotic plaques. METHODS AND RESULTS: We generated two mouse models in which Akt1 expression can be suppressed specifically in VSCMs before (Apoe-/-Akt1fl/flSm22alphaCRE) and after (Apoe-/-Akt1fl/flSM-MHC-CreERT2E) the formation of atherosclerotic plaques. This approach allows us to interrogate the role of Akt1 during the initial and late steps of atherogenesis. Absence of Akt1 in VSMCs during the progression of atherosclerosis results in larger atherosclerotic plaques characterized by bigger necrotic core areas, enhanced VSMC apoptosis and reduced fibrous cap and collagen content. In contrast, VSMC Akt1 inhibition in established atherosclerotic plaques does not influence lesion size but markedly reduces the relative fibrous cap area in plaques and increases VSMC apoptosis. CONCLUSIONS: Akt1 expression in VSMCs influences early and late stages of atherosclerosis. Absence of Akt1 in VSMCs induces features of plaque vulnerability including fibrous cap thinning and extensive necrotic core areas. These observations suggest that interventions enhancing Akt1 expression specifically in VSMCs may lessen plaque progression.

The integrated stress response (ISR) modulates mRNA translation to regulate the mammalian unfolded protein response (UPR), immunity and memory formation. A chemical ISR inhibitor, ISRIB, enhances cognitive function and modulates the UPR in vivo. To explore mechanisms involved in ISRIB action we screened cultured mammalian cells for somatic mutations that reversed its effect on the ISR. Clustered missense mutations were found at the N-terminal portion of the delta subunit of guanine nucleotide exchange factor (GEF) eIF2B. When reintroduced by CRISPR-Cas9 gene editing of wildtype cells, these mutations reversed both ISRIB-mediated inhibition of the ISR and its stimulatory effect on eIF2B GEF activity toward its substrate, the translation initiation factor eIF2, in vitro. Thus ISRIB targets an interaction between eIF2 and eIF2B that lies at the core of the ISR.

T cells directed to endogenous tumor antigens are powerful mediators of tumor regression. Recent immunotherapy advances have identified effective interventions to unleash tumor-specific T cell activity in patients who naturally develop them. Eliciting T cell responses to a patient's individual tumor remains a major challenge. Radiation therapy can induce immune responses to model antigens expressed by tumors, but it remains unclear if it can effectively prime T cells specific for endogenous antigens expressed by poorly immunogenic tumors. We hypothesized that TGFbeta activity is a major obstacle hindering the ability of radiation to generate an in situ tumor vaccine. Here we show that antibody-mediated TGFbeta neutralization during radiation therapy effectively generates CD8+ T cell responses to multiple endogenous tumor antigens in poorly immunogenic mouse carcinomas. Generated T cells were effective at causing regression of irradiated tumors and non-irradiated lung metastases or synchronous tumors (abscopal effect). Gene signatures associated with IFNgamma and immune-mediated rejection were detected in tumors treated with radiation therapy and TGFbeta blockade in combination but not as single agents. Upregulation of programmed death (PD) ligand-1 and -2 in neoplastic and myeloid cells and PD-1 on intratumoral T cells limited tumor rejection resulting in rapid recurrence. Addition of anti-PD-1 antibodies extended survival achieved with radiation and TGFbeta blockade. Thus, TGFbeta is a fundamental regulator of radiation therapy ability to generate an in situ tumor vaccine. The combination of local radiation therapy with TGFbeta neutralization offers a novel individualized strategy for vaccinating patients against their tumors.

In mouse models of atherosclerosis, normalization of hyperlipidemia promotes macrophage emigration and regression of atherosclerotic plaques in part by the Liver X Receptor (LXR)-mediated induction of the chemokine receptor CCR7. Here we report that LXRalpha serine 198 (S198) phosphorylation modulates CCR7 expression. Low levels of S198 phosphorylation are observed in plaque macrophages in the regression environment where high levels of CCR7 expression are observed. Consistent with these findings, CCR7 gene expression in human and mouse macrophages cell lines is induced when LXRalpha at S198 is non-phosphorylated. In bone marrow derived-macrophages (BMDMs) we also observe induction of CCR7 by ligands that promote non-phosphorylated LXRalpha S198 and this is lost in LXR deficient BMDMs. LXRalpha occupancy at the CCR7 promoter is enhanced and histone modifications associated with gene repression are reduced in RAW264.7 cells expressing non-phosphorylated (RAW-LXRalphaS198A) compared to phosphorylated LXRalpha (RAW-LXRalphaWT). Expression profiling from ligand treated RAW-LXRalphaS198A compared to RAW-LXRalphaWT cells revealed induction of cell migratory and anti-inflammatory genes, and repression of pro-inflammatory genes. Modeling of LXRalpha S198 in non-phosphorylated and phosphorylated states identified phosphorylation-dependent conformational changes in the hinge region commensurate with sites for protein interaction. Therefore, gene transcription is regulated by LXRalpha S198 phosphorylation including anti-atherogenic genes like CCR7.

BACKGROUND AND PURPOSE: To improve oral delivery of AmB by achieving increased oral bioavailability and synergistically enhanced antileishmanial activity using copaiba oil (Cop) through nano-emulsified carrier system (CopNEC). EXPERIMENTAL APPROACH: The AmB encapsulated nano-emulsified carrier (CopNEC-AmB) comprised of Cop, d-alpha Tocopheryl Polyethylene Glycol 1000 Succinate and phosphatidylcholine was prepared by high pressure homogenization method. Stability study of CopNEC-AmB was carried out in simulated gastric fluid and simulated intestinal fluid. The CopNEC-AmB and plain AmB were compared for in vitro antileishmanial activity, their pharmacokinetics, organ distribution and toxicity profiling. RESULTS: The optimized CopNEC-AmB has globule size of 127+/-21 nm, PDI 0.11+/-0.02, and zeta potential (-)38.5+/-2.7 with encapsulation efficiency 91.9+/-1.4 %w/w. The high resolution transmission electron microscopy illustrates spherical particle geometry with homogeny in their sizes. The optimized CopNEC-AmB was found to be stable in gastro-intestinal fluids showing insignificant changes in globule size and encapsulation efficiency. The AUC0-48 value of CopNEC-AmB in rats was significantly improved showing 7.2 folds higher oral bioavailability than free drug. The in vitro antileishmanial activity of CopNEC-AmB was significantly higher (p<0.05) than the free drug because copaiba oil synergistically enhanced chemotherapy of leishmaniasis by causing drastic changes in morphology of Leishmania parasite and rupture of the plasma membrane with loss of their contents. The CopNEC-AmB showed significantly lesser hemolytic toxicity and cell cytotoxicity, and absence of any changes in histopathology of kidney tissues as compared to free drug. CONCLUSION: This prototype CopNEC formulation showed improved bioavailability and nontoxic synergistic antileishmanial chemotherapy of AmB due to copaiba oil against leishmaniasis.

Determination of a protein's N-terminal sequence can be important for the characterization of protein processing. To increase the confidence of protein N-terminal identification, chemical derivatization of the N-terminal amine group by (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) or dimethyl labeling followed by mass spectrometric analysis is commonly performed. Using this approach, proteins can be separated by SDS-PAGE, and the protein N-terminus of interest is labeled by TMPP or dimethyl in-gel before tryptic digestion and LC-MS analysis. The N-terminus of a protein can thus be easily identified because the N-terminal tryptic peptides are preferentially labeled. Peptides with N-terminal derivatization produce a better fragmentation pattern during tandem mass spectrometric analysis, which significantly facilitates sequencing of these peptides.