Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7^Mtu and Rps7^Zma) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.     

Thermodynamic investigation into the mechanisms of proton-coupled electron transfer events in heme protein maquettes

To study the engineering requirements for proton pumping in energy-converting enzymes such as cytochrome c oxidase, the thermodynamics and mechanisms of proton-coupled electron transfer in two designed heme proteins are elucidated. Both heme protein maquettes chosen, heme b-[H10A24]2 and heme b-[delta7-His]2, are four-alpha-helix bundles that display pH-dependent heme midpoint potential modulations, or redox-Bohr effects. Detailed equilibrium binding studies of ferric and ferrous heme b with these maquettes allow the individual contributions of heme-protein association, iron-histidine ligation, and heme-protein electrostatics to be elucidated. These data demonstrate that the larger, less well-structured [H10A24]2 binds heme b in both oxidation states tighter than the smaller and more well-structured [Delta7-His]2 due to a stronger porphyrin-protein hydrophobic interaction. The 66 mV (1.5 kcal/mol) difference in their heme reduction potentials observed at pH 8.0 is due mostly to stabilization of ferrous heme in [H10A24]2 relative to [delta7-His]2. The data indicate that porphyrin-protein hydrophobic interactions and heme iron coordination are responsible for the Kd value of 37 nM for the heme b-[delta7-His]2 scaffold, while the affinity of heme b for [H10A24]2 is 20-fold tighter due to a combination of porphyrin-protein hydrophobic interactions, iron coordination, and electrostatic effects. The data also illustrate that the contribution of bis-His coordination to ferrous heme protein affinity is limited, <3.0 kcal/mol. The 1H+/1e- redox-Bohr effect of heme b-[H10A24]2 is due to the greater absolute stabilization of the ferric heme (4.1 kcal/mol) compared to the ferrous heme (1.4 kcal/mol) binding upon glutamic acid deprotonation, i.e., an electrostatic response mechanism. The 2H+/1e- redox-Bohr effect observed for heme b-[delta7-His]2 is due to histidine protonation and histidine dissociation of ferrous heme b upon reduction, i.e., a ligand loss mechanism. These results indicate that the contribution of porphyrin-protein hydrophobic interactions to heme affinity is critical to maintaining the heme bound in both oxidation states and eliciting an electrostatic response from these designed heme protein scaffolds.     

Inhibition of autophagy enhances the effects of the AKT inhibitor MK‐2206 when combined with paclitaxel and carboplatin in BRAF wild‐type melanoma

This study investigates the mechanism of action behind the long‐term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK‐2206 in combination with paclitaxel and carboplatin. Although single agent MK‐2206 inhibited phospho‐AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK‐2206 with paclitaxel and carboplatin was cytotoxic in long‐term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase‐dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti‐oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.     

p75 Neurotrophin Receptor Cleavage by α- and γ-Secretases Is Required for Neurotrophin-mediated Proliferation of Brain Tumor-initiating Cells

Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target.     

Dietary fibre and blood pressure

Ninety-four volunteers participated in a three-day weighed dietary survey and had their blood pressures measured once. They were classified according to their fibre intake. Subjects with a high-fibre intake were found to have lower mean blood pressures than those with a low-fibre intake. Forty-two of the volunteers further participated in a group of experiments. Those eating a high-fibre diet decreased their dietary fibre, and those eating a low-fibre diet increased their dietary fibre. The first group (11 subjects) showed increased mean blood pressures after four weeks of eating the experimental diet. The last group (31 subjects) showed decreased mean blood pressures after four weeks of eating the experimental diet. The 11 subjects consuming the low-fibre experimental diet showed a decrease in mean blood pressure when some of the saturated fat in their diet was replaced by polyunsaturated fat. A similar substitution carried out by 14 of the subjects consuming the high-fibre experimental diet also resulted in decreased mean blood pressure, but this was not statistically significant. Twelve more volunteers, with hypertension, were all found to have low-fibre diets. They consumed a high-fibre diet for a six-week experimental period, but their mean blood pressures did not decrease significantly; individual recordings varied substantially during this period.It is suggested that differences in the type and quantity of dietary fibre and fat may be responsible for the lower mean blood pressures of groups of vegetarians compared with similar groups of non-vegetarians.     

Evaluating Melanoma Drug Response and Therapeutic Escape with Quantitative Proteomics

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.Despite excitement about the development of targeted therapy strategies for cancer, few cures have been achieved. In patients with BRAF mutant melanoma, treatment with small molecule BRAF inhibitors typically follows a course of response and tumor shrinkage followed by eventual relapse and resistance (mean progression-free survival is ∼5.3 months) (1). Resistance to BRAF inhibitors is typically accompanied by reactivation of the MAPK signaling pathway, an effect mediated through activating mutations in NRAS and MEK1/2, genomic amplification of BRAF, increased expression of CRAF and Cot, and the acquisition of BRAF splice-form mutants (2–5). There is also evidence that increased PI3K/AKT signaling, resulting from the genetic inactivation of PTEN and NF1 and increased receptor tyrosine kinase (RTK)¹ signaling, may be involved in acquired BRAF inhibitor resistance (5–7). Many of the signaling proteins implicated in the escape from BRAF inhibitor therapy are clients of heat shock protein (HSP)-90 (8). Preclinical evidence now indicates that HSP90 inhibitors can overcome acquired and intrinsic BRAF inhibitor resistance, and clinical trials have been initiated to evaluate the BRAF/HSP90 combination in newly diagnosed patients (8, 9).Although targeted therapy strategies have been promising in BRAF mutant melanoma, few options currently exist for the 15–20% of melanoma patients whose tumors harbor activating NRAS mutations (10). Although there is some evidence that MEK inhibitors have activity in NRAS mutant melanoma patients, responses tend to be short-lived (mean progression-free survival ∼3 months) and resistance is nearly inevitable (11). Our emerging experience suggests that oncogene-driven signaling networks are highly robust with the capacity to rapidly adapt (12, 13). The future success of targeted therapy for melanoma and other cancers will depend upon the development of strategies that identify and overcome these adaptive escape mechanisms.The evaluation of targeted therapy responses in patients has proved to be challenging. The clinical development of HSP90 inhibitors has been hampered in part by the lack of a good pharmacodynamic assay for measuring HSP90 inhibition within tumor specimens (14). Additionally, very little is known about the adaptive changes that occur following the inhibition of MEK/ERK signaling in NRAS mutant melanoma. To address these issues, the optimal technique is liquid chromatography-multiple reaction monitoring mass spectrometry, which been shown to be highly reproducible and portable across laboratories (15–18).In addition to these technical developments, LC-MRM has also been shown to have excellent application to the study of biological pathways, including phosphotyrosine signaling, β-catenin signaling in colon cancer, and the evasion of apoptosis following BRAF inhibition in PTEN null melanoma (19–21). This technique can also be readily translated from cell line models to patient specimens. Here, we have developed a novel multiplexed LC-MRM assay to quantify the expression of >80 key signaling proteins in cell line models and fine needle aspirates from accessible melanoma lesions (22). In this study, we present the proof-of-principle for monitoring multiple signaling proteins in melanomas treated with either HSP90 or MEK inhibitors. Through this method, we identify the degradation of key HSP90 client proteins in vivo and elucidate a novel mechanism of adaptation to MEK inhibition through increased RTK signaling.     

A novel immunocytochemistry technique to measure hsp70 induction in L929 cells exposed to cadmium chloride.

The heat shock response has been suggested as a potential biomarker in toxicology. A vast amount of stimuli have been shown to induce heat shock proteins and new techniques to measure the response are constantly being assessed. In this study we use a novel immunocytochemistry technique to measure heat shock protein 70 (hsp70) induction in L929 cells exposed to cadmium chloride. Hsp70 induction was quantifiably measured using a soluble coloured substrate and qualitatively measured using a coloured substrate that precipitated at the location of hsp70. Using the insoluble coloured substrate hsp70 was identified predominantly within the cytoplasm of control cells. At intermediate cadmium concentrations hsp70 was observed to translocate to the nucleus. At these intermediate concentrations a heterogeneous heat shock response was observed. At lethal concentrations a strong heat shock response was observed with a widespread cellular response. This study demonstrates the potential of this immunocytochemistry technique to measure toxicological effects in cells by identifying the response quantitatively and qualitatively.     

Noradrenaline as a putative neurotransmitter mediating hypotension—induced FOs—like immunoreactivity in the supraoptic nucleus of the rat

Hemorrhage or hypotension induces extensive Fos-like immunoreactivity in the magnocellular neurosecretory cells in the supraoptic nucleus of the hypothalamus in rat,especially in the vasopressin neurons.The present study was to explore the neurotransmitter mediating this effect,Microinfusion of the alpha-adrenergic blocker into the supraoptic nucleus reduced the hypotension-induced FOs.whereas beta-antagonist did not affect it significantly.Alaha1-and alpha2-antagonist,prazosin and yohimbine,both reduced the Fos-Positive cell counts.However,the effective dosage of yohimbine was much larger,Alpha1-agonist,methoxamine,induced abundant Fos-like immunoreactivity in the vasopressin cells in this nucleus,while beta-and alpha2-agonist did not elicit such effect.Administration of the noradrenergic re-uptake inhibitor desipramine,to this nucleus to locally accumulate the spontaneously released noradrenaline from the nerve terminals also induced Fos expression,mostly in the vasopressin cells.