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Supplement 3 from Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer

Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)–induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1. [Cancer Res 2008;68(7):2250–8]

Supplement 2 from Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer

Supplement 1 from Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer

Laryngoceles are best treated with surgery. The goal of this study is to compare patient outcomes and complications in patients undergoing removal of laryngoceles with either transoral endoscopic/microlaryngoscopic or robotic approaches. A systematic review of the published literature was conducted using Pubmed, Web of Science, and the Cochrane Clinical Trials databases. A pooled analysis of individual data was used to compare outcomes between robotic and endoscopic approaches. A total of 30 studies were included. Nine studies with 95 patients were included in the final analysis. Eighty-one (85.26%) were treated with microlaryngoscopic surgery and 14 (14.74%) were treated with robotic-assisted surgery. The rates of tracheostomy (RR = 1.44, 95% CI = 0.389–5.332), complications (RR = 0.329, 95% CI = 0.047–2.294) and recurrence (RR = 0.354, 95% CI = 0.021–5.897) were not statistically different between groups. Within the endoscopic subgroup, 66 laryngoceles (78.57%) were completely excised, while 18 (21.43%) laryngoceles were treated with marsupialization. Marsupialization was associated with an increased risk of recurrence (RR = 4.889, 95% CI = 1.202–19.891). In the robotic subgroup, there was an increased risk of nasogastric tube use (RR = 103.867, 95% CI = 6.379–1619.214) and a longer mean length of hospital stay (p = 0.0001). Transoral treatment of laryngoceles has complication and recurrence rates of 18.95% and 7.37%, respectively. Robotic approaches are associated with higher rates of NGT use and increased hospital stay, however much of this is due to one robotic surgeon’s preference for routine NGT placement and higher rates of combined laryngocele removal via robotic approach. Complete excision of combined laryngoceles is possible with transoral approaches. Marsupialization, reported in traditional endoscopic approaches, is associated with a significantly higher rate of recurrence (22.22% vs. 4.76%).

Objectives. Foreign bodies of the external and middle ear are not uncommon; however, foreign bodies in the eustachian tube are rare. Here we describe the presentation, imaging, and endoscopic-assisted surgical management of a case of eustachian tube foreign body. Methods. A 34-year-old male was seen for evaluation of foreign body of the left eustachian tube while working with metal at a machine shop. Imaging and surgical management are highlighted and review of available literature regarding foreign bodies of the eustachian tube is presented. Results. A CT scan revealed a foreign body present approximately 1 cm into the bony eustachian tube. The patient underwent middle ear exploration which required endoscopic assistance to adequately visualize the foreign body. The foreign body was unable to be removed and required the creation of a bony tunnel lateral to the eustachian tube for visualization and access to the foreign body. Conclusions. This report presents a rare case of eustachian tube foreign body. Use of the endoscope during the surgical removal greatly enhanced the ease and safety of removal. This report also highlights the importance of ear protection with any machining and welding work.

Background: Chronic rhinosinusitis (CRS) is a multifaceted disease with a significant genetic component. The importance of taste receptor signaling has recently been highlighted in CRS; single nucleotide polymorphisms (SNPs) of bitter tastant-responsive G-protein-coupled receptors have been linked with CRS and with altered innate immune responses to multiple bacterially derived signals. Objective: To determine in CRS the frequency of six SNPs in genes with known bitter tastant signaling function. Methods: Genomic DNA was isolated from 74 CRS volunteers in West Virginia, and allele frequency was determined and compared with demographically matched data from the 1,000 Genomes database. Results: For two SNPs in a gene recently associated with bitterant signaling regulation, RGS21, there were no associations with CRS (although the frequency of the minor allele of RGS21, rs7528947, was seen to increase with increasing Lund-Mackay CT staging score). Two TAS2R bitter taste receptor gene variants (TAS2R19 rs10772420 and TAS2R38 rs713598), identified in prior CRS genetics studies, were found to have similar associations in this study. Conclusion: Unique to our study is the establishment of an association between CRS in this patient population and GNB3 SNP rs5443, a variation in an established G protein component downstream of bitterant receptor signal transduction.

To evaluate the frequency and types of humoral immunodeficiencies (HID) in pediatric and adult patients with recurrent (RARS). Patients with HID commonly present with upper respiratory tract infections. Their pathophysiology in children is different than adult counterparts. It is unknown how HID affects those two age groups.We performed a retrospective chart review of pediatric (18 years old) and adult (18 years and older) patients who were evaluated in our pediatric and adult rhinology clinic between July 2010 and December 2020 and had the diagnosis of recurrent (3 times/year) rhinosinusitis. Patients with cystic fibrosis, Aspirin Exacerbated Respiratory Disease (AERD), and ciliary dyskinesia were excluded. Demographic data and associated conditions were reviewed. Immunologic evaluation included complete blood cell count (CBC) with differential, serum immunoglobulin G, A, and M levels, and baseline and post-vaccination pneumococcal antibody titers.There were 135 patients who met the inclusion criteria. 86 patients (63.7%) were children, 49 patients (36.3%) were adults. 46.5% of the pediatric patients and 45% of the adult patients were female. 17.4% of children had abnormal immunologic findings: 8 had hypogammaglobulinemia (p 0.0001), 2 had specific antibody deficiency (SAD), and 5 had selective IgA deficiency. 32.7% of adults (p 0.0001) had abnormal immunologic findings: 4 had hypogammaglobulinemia, 11 had SAD (p 0.0001), and 1 patient had both IgA deficiency and SAD.Humoral immunodeficiency, specifically SAD, seems to be more common in adult versus pediatric RARS that is refractory to treatment.

Laryngoceles are best treated with surgery. The goal of this study is to compare patient outcomes and complications in patients undergoing removal of laryngoceles with either transoral endoscopic/microlaryngoscopic or robotic approaches. A systematic review of the published literature was conducted using Pubmed, Web of Science, and the Cochrane Clinical Trials databases. A pooled analysis of individual data was used to compare outcomes between robotic and endoscopic approaches. A total of 30 studies were included. Nine studies with 95 patients were included in the final analysis. Eighty-one (85.26%) were treated with microlaryngoscopic surgery and 14 (14.74%) were treated with robotic-assisted surgery. The rates of tracheostomy (RR = 1.44, 95% CI = 0.389-5.332), complications (RR = 0.329, 95% CI = 0.047-2.294) and recurrence (RR = 0.354, 95% CI = 0.021-5.897) were not statistically different between groups. Within the endoscopic subgroup, 66 laryngoceles (78.57%) were completely excised, while 18 (21.43%) laryngoceles were treated with marsupialization. Marsupialization was associated with an increased risk of recurrence (RR = 4.889, 95% CI = 1.202-19.891). In the robotic subgroup, there was an increased risk of nasogastric tube use (RR = 103.867, 95% CI = 6.379-1619.214) and a longer mean length of hospital stay (p = 0.0001). Transoral treatment of laryngoceles has complication and recurrence rates of 18.95% and 7.37%, respectively. Robotic approaches are associated with higher rates of NGT use and increased hospital stay, however much of this is due to one robotic surgeon's preference for routine NGT placement and higher rates of combined laryngocele removal via robotic approach. Complete excision of combined laryngoceles is possible with transoral approaches. Marsupialization, reported in traditional endoscopic approaches, is associated with a significantly higher rate of recurrence (22.22% vs. 4.76%).

DJ-1 is a multifunctional protein affecting different biological and cellular processes. In addition, DJ-1 has roles in regulating mitochondrial function. Loss-of-function mutations in DJ-1 were found to cause an autosomal recessive form of Parkinson’s disease. One of the main pathological features of PD is loss of dopamine neurons in the nigrostriatal pathway. DJ-1 knockout (KO) rats exhibit progressive nigral neurodegeneration with about 50% dopaminergic cell loss at 8 months of age. In order to assess the effects of DJ-1 deficiency on neuronal mitochondria prior to neuron loss, we performed proteomic analysis of synaptic mitochondria isolated from the striatum, the location of nigrostriatal pathway nerve terminals, of 3-month-old DJ-1 KO rats. In total, 371 mitochondrial proteins were quantified, and of these 76 were differentially expressed in DJ-1 KO rats. Proteins perturbed by the loss of DJ-1 were involved in several mitochondrial functional pathways, including the tricarboxylic acid cycle and electron transport chain. Thus, synaptic mitochondrial respiration was measured and showed a significant change due to DJ-1 deficiency. The dataset generated here highlights the role of synaptic mitochondria in PD associated with DJ-1. This study improves our understanding of DJ-1 effects in a complex tissue environment and the synaptic mitochondrial changes that accompany its loss.

The purpose of our study is to test the feasibility of transoral thyroid chondrolaryngoplasty using a similar approach to transoral thyroidectomy. This approach would allow for gender-affirming surgery while avoiding an external neck scar. We carried out our cadaveric feasibility study in an anatomy laboratory at an academic center. Five fresh cadavers were used for this study. We used a lower oral vestibular incision, along with retractors and an endoscope to dissect and gain access to the laryngeal prominence of the thyroid cartilage. Portions of the laryngeal prominence were then removed using scissors to achieve a satisfactory neck contour. Endoscopic as well as extracorporeal photographs were taken to demonstrate the approach. We were able to remove the laryngeal prominence successfully in all of our cadaveric specimens with this transoral approach.

Sinusitis is commonly seen by both otolaryngologists and primary care physicians who treat pediatric patients. Judicious use of radiation is especially important in this population. When sinusitis is acute and uncomplicated, there is no indication for imaging. There are several clinical situations which may prompt consideration of imaging including persistent or chronic sinusitis and before a planned sinus surgery. In these situations, a non-contrasted CT is appropriate. Complicated sinusitis or concern for malignancy may prompt consideration of other imaging modalities. Caution must be used in interpreting the incidental finding of sinusitis on CT or MRI obtained for other clinical indications, as sinus changes are exceedingly common in asymptomatic patients. Here we discuss the indications for imaging and considerations for choice of imaging modality. We then highlight imaging findings in chronic and complicated sinusitis and discuss specific imaging changes found in cystic fibrosis and allergic fungal sinusitis. This chapter reviews the use of imaging in pediatric patients with sinusitis and the most common imaging findings.

In pediatric patients, viral upper respiratory infection is common. Approximately 10% of these patients will develop acute bacterial rhinosinusitis. The most common bacteria associated with acute rhinosinusitis are Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae. In chronic rhinosinusitis, defined as symptoms for greater than 3 months, bacteria interact with other factors including anatomic abnormalities, allergy, and immunodeficiencies. Polymicrobial infection is more common in chronic rhinosinusitis. Both acute and chronic sinusitis can have intracranial or extracranial complications. In these cases, there is an increasing role of staphylococci and anaerobes. An understanding of the microbiology of sinusitis guides proper antimicrobial selection. This chapter highlights the current concepts in the microbiology of acute, chronic, and complicated pediatric sinusitis.

The purpose of this article is to differentiate pediatric patients with chronic adenoiditis from those with chronic rhinosinusitis (CRS) based on presenting symptoms. A chart review from a tertiary care facility with pediatric patients who presented with suspected CRS from 2006 to 2014 was identified. We compared patient characteristics, clinical symptoms, duration of symptoms, and past medial history using univariate and multivariate logistic regression models. Based on recent literature, utilizing the computed tomography (CT) score, we identified those children with CRS versus those with chronic adenoiditis. Of the 99 pediatric patients included, 22 patients had diagnosis of adenoiditis and 77 had diagnosis of CRS. When purulent rhinorrhea was present with facial pain, CRS was statistically more prevalent than chronic adenoiditis ( P = .017). Symptoms including cough ( P = .022), rhinorrhea ( P = .27), and facial pressure ( P = .98) were not predictive of one diagnosis over the other. Past medical history of asthma or allergy was similar in both groups. Smoke exposure was associated with CT scores >5 (odds ratio 2.4, 95% confidence interval, 0.799-7.182). We conclude that purulent rhinorrhea in the presence of facial pain is more indicative of CRS versus chronic adenoiditis. For all other children, an adenoidectomy without the need for a CT scan can be entertained.

A 6-year-old male was seen for evaluation of middle ear polyp with associated hearing loss and otorrhea. CT scan revealed canal polyp with a stalk extending to the middle ear with malformation of the malleus and incus. The patient underwent exploratory tympanotomy which revealed a fistula from the bony-cartilaginous junction connecting to a middle ear mass which had eroded the incus. Intraoperative pathology suggested columnar epithelium without cholesteatoma or muscle. Final pathologic diagnosis reported dense fibrous tissue as well as ectopic salivary gland tissue, consistent with salivary gland choristoma. After removal of the mass, a partial prosthesis was successfully placed. Middle ear salivary gland choristoma is a rare entity. It can be confused clinically with cholesteatoma and is usually diagnosed on pathology. This diagnosis is often associated with other external, middle, and inner ear abnormalities.

This article reports changes in the striatal non-synaptic mitochondrial proteome of DJ-1, Parkin, and PINK1 knockout (KO) rats at 3 months of age. DJ-1, Parkin, and PINK1 mutations cause autosomal-recessive parkinsonism. It is thought that loss of function of these proteins contributes to the onset and pathogenesis of Parkinson׳s disease (PD). As DJ-1, Parkin, and PINK1 have functions in the regulation of mitochondria, the dataset generated here highlights protein expression changes, which can be helpful for understanding pathological mitochondrial alterations. In total, 1281 proteins were quantified and 25, 37, and 15 proteins were found to exhibit differential expression due to DJ-1, Parkin, and PINK1 deficiency, respectively. All quantification can be found in the supplemental table and can be searched online at http://genome.unmc.edu/mitorat/index.html. Further, mitochondrial respiration was measured to evaluate mitochondrial function in the striatum of DJ-1, Parkin, and PINK1 KO rats, which was significantly changed only in the DJ-1 KOs.

Parkinson׳s disease (PD), the second most common neurodegenerative disorder, affects roughly 7–10 million people worldwide. A wide array of research has suggested that PD has a mitochondrial component and that mitochondrial dysfunction occurs well in advance of the clinical manifestation of the disease. Previous work by our lab has categorized the mitochondrial disorder associated with Parkinson׳s disease in a PINK1 knockout rat model. This model develops Parkinson׳s disease in a spontaneous, predictable manner. Our findings demonstrated PINK1-deficient rats at 4 months of age had mitochondrial proteomic and functional abnormalities before the onset of Parkinsonian symptoms (6 months) such as the movement disorder, loss of midbrain dopaminergic neurons, or the progressive degeneration present at 9 months. With this in mind, our group investigated the PINK1 knockout genetic rat model at postnatal day 10 to determine if the observed alterations at 4 months were present at an earlier time point. Using a proteomic analysis of brain mitochondria, we identified significant mitochondrial proteomic alterations in the absence of mitochondrial functional changes suggesting the observed alterations are part of the mitochondrial pathways leading to PD. Specifically, we identified differentially expressed proteins in the PINK1 knockout rat involved in glycolysis, the tricarboxylic acid cycle, and fatty acid metabolism demonstrating abnormalities occur well in advance of the manifestation of clinical symptoms. Additionally, 13 of the differentially expressed proteins have been previously identified in older PINK1 knockout animals as differentially regulated suggesting these proteins may be viable markers of the PD pathology, and further, the abnormally regulated pathways could be targeted for therapeutic interventions. All raw data can be found in Supplementary Table 1.

With the advent of the combination antiretroviral therapy era (cART), the development of AIDS has been largely limited in the USA. Unfortunately, despite the development of efficacious treatments, HIV-1-associated neurocognitive disorders (HAND) can still develop, and as many HIV-1 positive individuals age, the prevalence of HAND is likely to rise because HAND manifests in the brain with very low levels of virus. However, the mechanism producing this viral disorder is still debated. Interestingly, HIV-1 infection exposes neurons to proteins including Tat, Nef, and Vpr which can drastically alter mitochondrial properties. Mitochondrial dysfunction has been posited to be a cornerstone of the development of numerous neurodegenerative diseases. Therefore, we investigated mitochondria in an animal model of HAND. Using an HIV-1 transgenic rat model expressing seven of the nine HIV-1 viral proteins, mitochondrial functional and proteomic analysis were performed on a subset of mitochondria that are particularly sensitive to cellular changes, the neuronal synaptic mitochondria. Quantitative mass spectroscopic studies followed by statistical analysis revealed extensive proteome alteration in this model paralleling mitochondrial abnormalities identified in HIV-1 animal models and HIV-1-infected humans. Novel mitochondrial protein changes were discovered in the electron transport chain (ETC), the glycolytic pathways, mitochondrial trafficking proteins, and proteins involved in various energy pathways, and these findings correlated well with the function of the mitochondria as assessed by a mitochondrial coupling and flux assay. By targeting these proteins and proteins upstream in the same pathway, we may be able to limit the development of HAND.

Efavirenz (EFV) is a non-nucleoside reverse-transcriptase inhibitor in wide use for the treatment of human immunodeficiency virus infection. Although EFV is generally well tolerated, neuropsychiatric toxicity has been well documented. The toxic effects of EFV in hepatocytes and keratinocytes have been linked to mitochondrial perturbations and changes in autophagy. Here, we studied the effect of EFV on mitochondria and autophagy in neuronal cell lines and primary neurons. In SH-SY5Y cells, EFV induced a drop in ATP production, which coincided with increased autophagy, mitochondrial fragmentation, and mitochondrial depolarization. EFV-induced mitophagy was also detected by colocalization of mitochondria and autophagosomes and use of an outer mitochondrial membrane tandem fluorescent vector. Pharmacologic inhibition of autophagy with 3-methyladenine increased the cytotoxic effect of EFV, suggesting that autophagy promotes cell survival. EFV also reduces ATP production in primary neurons, induces autophagy, and changes mitochondrial morphology. Overall, EFV is able to acutely induce autophagy and mitochondrial changes in neurons. These changes may be involved in the mechanism leading to central nervous system toxicity observed in clinical EFV use.

Synaptic mitochondria are essential for maintaining calcium homeostasis and producing ATP, processes vital for neuronal integrity and synaptic transmission. Synaptic mitochondria exhibit increased oxidative damage during aging and are more vulnerable to calcium insult than nonsynaptic mitochondria. Why synaptic mitochondria are specifically more susceptible to cumulative damage remains to be determined. In this study, the generation of a super-SILAC mix that served as an appropriate internal standard for mouse brain mitochondria mass spectrometry based analysis allowed for the quantification of the proteomic differences between synaptic and nonsynaptic mitochondria isolated from 10-month-old mice. We identified a total of 2260 common proteins between synaptic and nonsynaptic mitochondria of which 1629 were annotated as mitochondrial. Quantitative proteomic analysis of the proteins common between synaptic and nonsynaptic mitochondria revealed significant differential expression of 522 proteins involved in several pathways including oxidative phosphorylation, mitochondrial fission/fusion, calcium transport, and mitochondrial DNA replication and maintenance. In comparison to nonsynaptic mitochondria, synaptic mitochondria exhibited increased age-associated mitochondrial DNA deletions and decreased bioenergetic function. These findings provide insights into synaptic mitochondrial susceptibility to damage.

HIV infection in the central nervous system (CNS) is responsible for a spectrum of neurocognitive and behavioral disorders. The wide use of appropriate combination antiretroviral therapy (cART) has decreased the severity of cognitive dysfunction. Viral loads are reduced and CD4+ T cell counts are increased. In addition, patients are living longer with improved quality of life. However, cART does not completely eliminate CNS dysfunction in HIV infected patients. There is an increase in milder forms of HIV-associated neurocognitive disorders (HAND), which are the result of a complex interplay between many factors including: aging, vascular disease, Alzheimer’s disease, and psychiatric disease. The underlying biochemical and molecular changes that underlie these changes and the underlying mechanisms of HAND pathogenesis continue to be an area of ongoing research. New advances in imaging and biomarker discovery will continue to advance clinical diagnosis and management. Treatments that can perturb the viral reservoirs and decrease chronic inflammatory response, in addition to continuing cART, may give the clinician additional tools to prevent HAND.

Purpose : Mutations in PTEN-induced putative kinase 1 (Pink1), a mitochondrial serine/threonine kinase, cause a recessive inherited form of Parkinson's disease (PD). Pink1 deletion in rats results in a progressive PD-like phenotype, characterized by significant motor deficits starting at 4 months of age. Despite the evidence of mitochondrial dysfunction, the pathogenic mechanism underlying disease due to Pink1-deficiency remains obscure. Experimental design : Striatal synaptic mitochondria from 3-month-old Pink1-deficient rats were characterized using bioenergetic and mass spectroscopy (MS)-based proteomic analyses. Results : Striatal synaptic mitochondria from Pink1-deficient rats exhibit decreased complex I-driven respiration and increased complex II-mediated respiration compared with wild-type rats. MS-based proteomics revealed 69 of the 811 quantified mitochondrial proteins were differentially expressed between Pink1-deficient rats and controls. Down-regulation of several electron carrier proteins, which shuttle electrons to reduce ubiquinone at complex III, in the Pink1-knockouts suggests disruption of the linkage between fatty acid, amino acid, and choline metabolism and the mitochondrial respiratory system. Conclusions and clinical relevance : These results suggest that complex II activity is increased to compensate for loss of electron transfer mechanisms due to reduced complex I activity and loss of electron carriers within striatal nerve terminals early during disease progression. This may contribute to the pathogenesis of PD. This article is protected by copyright. All rights reserved

Background With the emergence of Src inhibitors in clinical trials, improved knowledge of the molecular responses of cancer cells to these agents is warranted. This will facilitate the development of tests to identify patients who may benefit from these agents, allow drug activity to be monitored and rationalize the combination of these agents with other treatment modalities. Methods This study evaluated the molecular and functional effects of Src inhibitor AZD0530 in human lung cancer cells, by Western blotting and reverse transcription-polymerase chain reaction, and by assays for cell viability, migration, and invasion. Results Src was activated in four of five cell lines tested and the level corresponded with the invasive potential and the histologic subtype. Clinically relevant, submicromolar concentrations of AZD0530 blocked Src and focal adhesion kinase, resulting in significant inhibition of cell migration and Matrigel invasion. Reactivation of STAT3 and up-regulation of JAK indicated a potential mechanism of resistance. AZD0530 gave a potent and sustained blockage of AKT and enhanced the sensitivity to irradiation. Conclusions The results indicated that AZD0530, aside from being a potent inhibitor of tumor cell invasion which could translate to inhibition of disease progression in the clinic, may also lower resistance of lung cancer cells to pro-apoptotic signals.

The human aurora family of serine-threonine kinases comprises three members, which act in concert with many other proteins to control chromosome assembly and segregation during mitosis. Aurora dysfunction can cause aneuploidy, mitotic arrest, and cell death. Aurora kinases are strongly expressed in a broad range of cancer types. Aurora A expression in tumors is often associated with gene amplification, genetic instability, poor histologic differentiation, and poor prognosis. Aurora B is frequently expressed at high levels in a variety of tumors, often coincidently with aurora A, and expression level has also been associated with increased genetic instability and clinical outcome. Further, aurora kinase gene polymorphisms are associated with increased risk or early onset of cancer. The expression of aurora C in cancer is less well studied. In recent years, several small-molecule aurora kinase inhibitors have been developed that exhibit preclinical activity against a wide range of solid tumors. Preliminary clinical data from phase I trials have largely been consistent with cytostatic effects, with disease stabilization as the best response achieved in solid tumors. Objective responses have been noted in leukemia patients, although this might conceivably be due to inhibition of the Abl kinase. Current challenges include the optimization of drug administration, the identification of potential biomarkers of tumor sensitivity, and combination studies with cytotoxic drugs. Here, we summarize the most recent preclinical and clinical data and discuss new directions in the development of aurora kinase inhibitors as antineoplastic agents.

PTEN-induced kinase 1 (PINK1) mutations are responsible for an autosomal recessive, familial form of Parkinson’s disease. PINK1 protein is a Ser/Thr kinase localized to the mitochondrial membrane and is involved in many processes including mitochondrial trafficking, mitophagy, and proteasomal function. Using a new PINK1 knockout (PINK1 KO) rat model, we found altered brain metabolomic markers using magnetic resonance spectroscopy, identified changes in mitochondrial pathways with quantitative proteomics using sequential window acquisition of all theoretical spectra (SWATH) mass spectrometry, and demonstrated mitochondrial functional alterations through measurement of oxygen consumption and acidification rates. The observed alterations included reduced creatine, decreased levels of complex I of the electron transport chain, and increased proton leak in the electron transport chain in PINK1 KO rat brains. In conjunction, these results demonstrate metabolomic and mitochondrial alterations occur during the asymptomatic phase of Parkinson’s disease in this model. These results indicate both potential early diagnostic markers and therapeutic pathways that can be used in PD.

Mitochondria are the main cellular source of reactive oxygen species and are recognized as key players in several age-associated disorders and neurodegeneration. Their dysfunction has also been linked to cellular aging. Additionally, mechanisms leading to the preservation of mitochondrial function promote longevity. In this study we investigated the proteomic and functional alterations in brain mitochondria isolated from mature (5 months old), old (12 months old), and aged (24 months old) mice as determinants of normal "healthy" aging. Here the global changes concomitant with aging in the mitochondrial proteome of mouse brain analyzed by quantitative mass-spectrometry based super-SILAC identified differentially expressed proteins involved in several metabolic pathways including glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Despite these changes, the bioenergetic function of these mitochondria was preserved. Overall, this data indicates that proteomic changes during aging may compensate for functional defects aiding in preservation of mitochondrial function. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001370 (http://proteomecentral.proteomexchange.org/dataset/PXD001370).

Aging correlates with a progressive impairment of mitochondrial homeostasis and is an influential factor for several forms of neurodegeneration. However, the mechanisms underlying age-related alterations in synaptosomal mitochondria, a neuronal mitochondria population highly susceptible to insults and critical for brain function, remain incompletely understood. Therefore this study investigates the synaptic mitochondrial proteomic and bioenergetic alterations that occur with age. The utilization of a state of the art quantitative proteomics approach allowed for the comparison of protein expression levels in synaptic mitochondria isolated from 5 (mature), 12 (old), and 24 (aged) month old mice. During the process of aging we find that dynamic proteomic alterations occur in synaptic mitochondria. Despite direct (mitochondrial DNA deletions) and indirect (increased antioxidant protein levels) signs of mitochondrial damage in the aged mice, there was an overall maintenance of mitochondrial function. Therefore the synaptic mitochondrial proteomic changes that occur with aging correlate with preservation of synaptic mitochondrial function.

Background Drp1 is the primary protein responsible for mitochondrial fission. Perturbations of mitochondrial morphology and increased fission are seen in neurodegeneration. While Drp1 degradation induced by Parkin overexpression can be prevented by proteasome inhibition, there are numerous links between proteasomal and autophagic processes in mitochondrial protein degradation. Here we investigated the role of autophagy in Drp1 regulation. Results We demonstrate that autophagy plays a major role in the control of Drp1 levels. In HEK-293T cells, inhibitors of autophagy increase total Drp1 and levels of Drp1 in the mitochondrial cellular fraction. Similarly by silencing ATG7, which is required for initiation of autophagy, there is an increased level of Drp1. Because of the role of increased Drp1 in neurodegeneration, we then examined the ability to modulate Drp1 levels in neurons by inducing autophagy. We are able to decrease Drp1 levels in a time- and dose-dependent manner with the potent neuronal autophagy inducer 10-NCP, as well as structurally related compounds. Further, 10-NCP was able increase average mitochondrial size and length verifying a functional result of Drp1 depletion in these neurons. Conclusions These pharmacological and genetic approaches indicate that autophagy targets Drp1 for lysosomal degradation. Additionally these data suggest a mechanism, through Drp1 downregulation, which may partly explain the ability of autophagy to have a neuroprotective effect.

The interaction between the transmembrane receptor CD44 on epithelial tumor cells and its ligand hyaluronan in the surrounding extracellular matrix is important in tumor progression and metastasis. CD44 is encoded by a single 20-exon gene and expressed in standard form (CD44s), as well as a myriad of CD44 variants (CD44v) generated by alternative splicing of the CD44 mRNA. Previously, we demonstrated that hyaluronan (HA) production is increased at tumor-stroma interface in invasive and metastatic human breast cancers when compared with benign or premalignant lesions. We hypothesize that CD44 expression on breast cancer cells is a major contributing factor to cell adhesion, migration and invasion. To evaluate this hypothesis we examined the effects of 3 distinct anti-CD44s and 2 anti-CD44v6 monoclonal antibodies on breast cancer cell lines that expressed high and low CD44s and CD44v6. Using these antibodies we assessed the role of CD44 in cell adhesion, cell motility, and cell invasion using immobilized HA-coated wells, wound healing assays, and modified Boyden chamber respectively. Our results showed that anti-CD44s could inhibit breast cancer cell adhesion, motility and invasion, while anti-CD44v6 inhibits cell motility. In conclusion, our data suggests that CD44s is involved in breast cancer cell adhesion, motility and invasion through interaction with HA but CD44v6 is involved only in cell motility. Furthermore we concluded that antibodies against different epitopes on CD44 mediate distinct functional effects on breast cancer cells.

Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)–induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1. [Cancer Res 2008;68(7):2250–8]

13108 Background: AZD0530 is a highly selective, orally available, dual specific Src/Abl kinase inhibitor in clinical development. We tested this agent in multiple lung cancer cell lines in vitro. We hypothesized that activity of AZD0530 may depend on the level of activated (pY416-)Src, and that Src inhibition may decrease Bcl-xL protein levels and lower the barrier to apoptosis. Methods: NSCLC (A549, Calu-1, Calu-6) and SCLC (H69, H526) cells were incubated with 0.001–100 μM AZD0530 for 1–72hrs. Proliferation (MTT), DNA-content (flow cytometry), and protein levels of Src, pY416-Src, PARP, and Bcl-xL (Western blotting) were assessed. Results: Basal pY416-Src was detectable in most cell lines except H526. AZD0530 decreased pY416-Src levels at submicromolar concentrations in pY416-Src positive cells. In A549, Calu-1 and Calu-6, AZD0530 blocked cell growth in a time- and dose-dependent way (IC50 = 7–25 μM) by arrest in G1, retaining a cytostatic effect at submicromolar concentrations in A549 and Calu-1. AZD0530 induced apoptosis in 10–22% of these cells at micromolar concentrations, accompanied by a decrease of Bcl-xL protein in A549 and Calu-1. In H69 and H526, growth inhibition by AZD0530 was limited (IC50 >100 μM). Conclusions: 1) AZD0530 induced apoptosis at micromolar concentrations, and inhibited cell growth at micromolar to submicromolar concentrations in some cell lines. 2) pY416-Src is a potential marker for drug responsiveness, but other factors should also be tested. 3) Decrease of Bcl-xL by AZD0530 may render cancer cells more sensitive to chemotherapy. These data suggest that Src kinase inhibitors merit further testing in lung cancer, both alone and in combination with other agents. (Support: Swiss National Science Foundation, Swiss Cancer League, AstraZeneca). [Table: see text]

Mitochondria are dynamic organelles critical for many cellular processes, including energy generation. Thus, mitochondrial dysfunction likely plays a role in the observed alterations in brain glucose metabolism during aging. Despite implications of mitochondrial alterations during brain aging, comprehensive quantitative proteomic studies remain limited. Therefore, to characterize the global age-associated mitochondrial proteomic changes in the brain, we analyzed mitochondria isolated from the brain of 5-, 12-, and 24-month old mice using quantitative mass spectrometry. We identified changes in the expression of proteins important for biological processes involved in the generation of precursor metabolites and energy through the breakdown of carbohydrates, lipids, and proteins. These results are significant because we identified age-associated proteomic changes suggestive of altered mitochondrial catabolic reactions during brain aging. The proteomic data described here can be found in the PRIDE Archive using the reference number PXD001370. A more comprehensive analysis of this data may be obtained from the article “Proteomic analysis and functional characterization of mouse brain mitochondria during aging reveal alterations in energy metabolism” in PROTEOMICS.