A Rare Case of Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disease due to mutations in the thymidine phosphorylase gene, leading to mitochondrial alterations and dysfunctions in oxidative phosphorylation. MNGIE is a multisystem disorder with gastrointestinal symptoms arising in large part from gut dysmotility and neurological manifestations including peripheral neuropathy. We discuss a patient with chronic vomiting, diarrhea, and weight loss with a prior unrevealing extensive workup who was hospitalized for severe protein-calorie malnutrition. The patient was found to have gastrointestinal dysmotility on a gastric emptying scan and persistently elevated lactate levels and was subsequently diagnosed with MNGIE after confirmatory testing.

An Unfortunate Cause of Chronic Nausea and Vomiting: Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE).

We present a unique case of a 24-year-old male who was admitted for intractable nausea, emesis, weight loss, and abdominal discomfort. The patient underwent an extensive workup and was diagnosed with mitochondrial neurogastrointestinal encephalopathy. Early diagnosis is critical to proper management of this disease process. MGNIE is a difficult disorder to diagnose given the complexity of the disease, and this case provides clinicians the proper understanding and management of such a unique and difficult diagnosis.

Sodium Polystyrene Sulfonate-Induced Gastric Pneumatosis: A Rare Side Effect.

Kayexalate has been used in the USA since 1975 for the treatment of hyperkalemia. Prior case reports have shown that sorbitol added to kayexalate has been known to cause rare side effects of colonic necrosis. We present a unique case report of gastric pneumatosis as a complication of kayexalate.

In-Hospital Outcomes of Patients With Pulmonary Hypertension and Cirrhosis: A 6-Year Population Cohort Study of Over One Million Patients.

INTRODUCTION: There is a paucity of data on the influence of sex, race, insurance, pulmonary hypertension-related complications, and cirrhosis-related complications on mortality, hospital length of stay (LOS), and total hospital charges. The aim of this study was to identify risk factors in a national population cohort (in the USA) admitted to hospital between 2012 and 2017. METHODS: All patients aged > 18 years with pulmonary hypertension and cirrhosis, who had been admitted to hospital between 2012 and 2017, were identified from the US Nationwide Inpatient Sample (NIS), a large publicly available all-payer inpatient care database in the USA. Multivariate regression analysis was used to estimate the odds ratios of in-hospital mortality, average length of hospital stay, and hospital charges, after adjusting for age, gender, race, primary insurance payer status, hospital type and size (number of beds), hospital region, hospital teaching status, and other demographic characteristics. RESULTS: Our study identified 1,111,594 patients who had been discharged from hospital from 2012 to 2017. Of these patients, 355,455 were admitted with pulmonary hypertension, with 9.8% having cirrhosis as a complication (n = 34,986). The analysis revealed that patients with both pulmonary hypertension and cirrhosis compared to patients with only pulmonary hypertension experience increased mortality, hospital LOS, total hospital charges, and pulmonary hypertension-related and cirrhosis-related complications. Independent positive predictors of mortality were Asian/Pacific Islander race and "other" insurance status (worker's compensation; other US health benefits plans [CHAMPUS/TRICARE, CHAMPVA, Title V]). Independent positive predictors of increased hospital LOS were black race and "other" patients (more than one race/mixed). Independent positive predictors of increased total hospital charges were male gender, Hispanic ethnicity, Asian/Pacific Islander race, and other insurance status. Pulmonary hypertension-related complications (cor pulmonale, pulmonary embolism, hemoptysis, cardiac arrest, atrial fibrillation, ventricular tachycardia) and cirrhosis-related complications (ascites, hepatorenal syndrome, hepatic encephalopathy, variceal bleeding, portal hypertension) were independent positive predictors of mortality, hospital LOS, and total hospital charges. CONCLUSIONS: Patients with pulmonary hypertension and cirrhosis have increased mortality and hospital utilization compared to patients with only pulmonary hypertension. We identified key drivers for these outcomes. Targeted interventions, such as novel medications, right-to-left shunts, more evaluations for lung transplantation, and reversal of pulmonary vacular remodeling, are needed for the subgroups identified in this study in order to improve outcomes.

Impact of United States Preventive Services Task Force recommendations on prostate biopsy characteristics and disease presentation at a tertiary-care medical center.

BACKGROUND: To evaluate early consequences of 2012 United States Preventive Services Task Force (USPSTF) recommendations for decreased prostate-specific antigen (PSA) screening on prostate biopsy characteristics and prostate cancer presentation. MATERIALS AND METHODS: A single tertiary-care institution, multisurgeon, prospectively maintained database was queried for patients undergoing prostate biopsy from October 2005 to September 2016. Patient demographics, biopsy characteristics, and extent of disease were reported. Patient cohorts before and after USPSTF recommendations were compared using two-sample t test, Chi-square test, and Wilcoxon rank sum test with significance at P < 0.05. RESULTS: A total of 2,000 patients were analyzed, including 1,440 patients before and 560 patients after USPSTF recommendations. Following the recommendations, patients had higher prebiopsy PSA (5.90 vs. 6.70, P < 0.001). Overall, 817 (40.9%) patients had prostate cancer detected at biopsy with an increase from 37.0% before to 50.8% after (P < 0.001). Biopsies detected less low-risk Gleason ≤6 prostate cancer (47.4% vs. 41.1%) and more intermediate-risk Gleason 7 cancer (30.9% vs. 39.7%), with comparable findings of high-risk Gleason ≥8 cancer (21.7% vs. 19.2%), P = 0.042. In addition, greater percentage of core involvement (P < 0.001) was seen. At the time of diagnosis, extraprostatic extension identified by pelvic imaging increased from 12.6% to 18.9%, P = 0.039, with a trend toward lymph node positivity (1.1% vs. 2.2%, P = 0.078). Of those with metastatic disease, bony involvement occurred more often (1.7% vs. 3.2%, P = 0.041). CONCLUSIONS: After 2012 USPSTF guidelines, patients presented with higher PSA with prostate cancer were detected more frequently. More adverse, pathologic prostate cancer features were found on biopsy with the extent of disease implicating locally advanced/metastatic disease. These findings should be considered when counseling patients about prostate cancer screening importance.

Predicted versus observed 30-day perioperative outcomes using the ACS NSQIP surgical risk calculator in patients undergoing partial nephrectomy for renal cell carcinoma.

PURPOSE: The purpose of the study was to evaluate the accuracy of the American College of Surgeons NSQIP Surgical Risk Calculator for predicting risk-adjusted 30-day outcomes for patients undergoing partial nephrectomy (PN) for renal cell carcinoma (RCC). METHODS: A single institution, multi-surgeon, prospectively maintained database was queried for patients undergoing PN for RCC from 1998 to 2015. 21 preoperative factors were analyzed for each patient with predicted risk for 30-day complications, mortality, and length of stay (LOS) calculated. Differences between the mean predicted risk and observed rate of surgical outcomes were determined using two-sided one-sample t test with significance at p < 0.05. Subgroup analyses of outcomes stratified by surgical approach were also performed. RESULTS: 470 patients undergoing PN for RCC were analyzed. Comparing NSQIP predicted to observed outcomes, clinically significant underestimations occurred with rates of overall complications (9.16 vs. 16.81%, p < 0.001), surgical site infections [SSI] (1.65 vs. 2.77%, p < 0.001), urinary tract infection [UTI] (1.41 vs. 3.40%, p < 0.001), and LOS (3.25 vs. 3.73 days, p < 0.001). On subgroup analysis, 209 open PN and 261 minimally invasive PN (MIPN) were performed. The NSQIP calculator consistently underestimated overall complications, SSI, UTI, and LOS (p < 0.001) among both surgical approaches, while overestimating MIPN severe complications (p < 0.001). Clinically important differences persisted when stratifying the MIPN group by laparoscopic (N = 111) and robotic (N = 150) approaches. CONCLUSIONS: The ACS NSQIP Surgical Risk Calculator had significant discrepancies among observed and predicted outcomes. Additional analyses confirmed these differences remained significant irrespective of surgical approach. These findings emphasize the need for urologic oncology-specific calculators to better predict surgical outcomes in this complex patient population.

Longitudinal Rupture of Distal Corpus Cavernosum With Concomitant Urethral Injury: An Uncommon Result of a Common Mechanism.

Fracture of the penis is a well-recognized yet relatively uncommon urologic event. Forceful, blunt trauma with lateral bending of the penis in an erect state typically results in a transverse rupture of the tunica albuginea of the corpus cavernosum. Longitudinal tears of the corpus cavernosum are by themselves considered infrequent. We present a rare case of a patient with longitudinal rupture of the distal corpus cavernosum with concomitant extension to the corpus spongiosum causing partial urethral disruption as a result of trauma during sexual intercourse.

Activating Mutations in PIK3CA Lead to Widespread Modulation of the Tyrosine Phosphoproteome.

The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis.

A phosphoproteomic screen demonstrates differential dependence on HER3 for MAP kinase pathway activation by distinct PIK3CA mutations.

The PIK3CA gene encodes for the p110 alpha isoform of PI3 kinase and is one of the most frequently mutated oncogenes in human cancers. However, the mechanisms by which PIK3CA mutations activate cell signaling are not fully understood. Here we used a phosphoproteomic approach to compare differential phosphorylation patterns between human breast epithelial cells and two isogenic somatic cell knock in derivatives, each harboring a distinct PIK3CA mutation. We demonstrated differential phosphorylation patterns between isogenic cell lines containing a PIK3CA helical domain mutation (E545K) compared to cells with a PIK3CA kinase domain mutation (H1047R). In particular, the receptor tyrosine kinase, HER3, showed increased phosphorylation at tyrosine 1328 in H1047R cells versus E545K cells. Genetic studies using shRNA demonstrated that H1047R cells have a profound decrease in growth factor independent proliferation upon HER3 knock down, but this effect was attenuated in E545K cells. In addition, HER3 knock down led to reductions in both PI3 kinase and MAP kinase pathway activation in H1047R cells, but in E545K cells only PI3 kinase pathway diminution was observed. These studies demonstrate the power of using paired isogenic cell lines for proteomic analysis to gain new insights into oncogenic signal transduction pathways.

MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers.

Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.

Detection of cancer DNA in plasma of patients with early-stage breast cancer.

PURPOSE: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection. EXPERIMENTAL DESIGN: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR. RESULTS: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery. CONCLUSIONS: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients.

Somatic alterations as the basis for resistance to targeted therapies.

Recent advances in genetics and genomics have revealed new genes and pathways that are somatically altered in human malignancies. This wealth of knowledge has translated into molecularly defined targets for therapy over the past two decades, serving as key examples that translation of laboratory findings can have great impact on the ability to treat patients with cancer. However, given the genetic instability and heterogeneity that are characteristic of all human cancers, drug resistance to virtually all therapies has emerged, posing further and future challenges for clinical oncology. Here we review the history of targeted therapies, including examples of genetically defined cancer targets and their approved therapies. We also discuss resistance mechanisms that have been uncovered, with an emphasis on somatic genetic alterations that lead to these phenotypes.

Reduced fluoroscopy protocol for percutaneous nephrostolithotomy: feasibility, outcomes and effects on fluoroscopy time.

PURPOSE: Radiation exposure from fluoroscopy during percutaneous nephrostolithotomy contributes to patient overall exposure, which may be significant. We compared fluoroscopy times and treatment outcomes before and after implementing a reduced fluoroscopy protocol during percutaneous nephrostolithotomy. MATERIALS AND METHODS: We retrospectively reviewed the charts of patients treated with percutaneous nephrostolithotomy at a single academic institution by a single surgeon. We compared 40 patients treated before implementation of a reduced fluoroscopy protocol to 40 post-protocol patients. The reduced protocol included visual and tactile cues, fixed lowered mAs and kVp, a laser guided C-arm and designated fluoroscopy technician, and single pulse per second fluoroscopy. Preoperative characteristics, fluoroscopy and operative time, complications and treatment success were examined using univariate and multivariate analysis. RESULTS: There was no significant difference in body mass index, stone size, success rate, operative time or complications between the groups. After protocol implementation fluoroscopy time decreased from 175.6 to 33.7 seconds (p<0.001). A longer average hospital stay was seen in the pre-protocol group (3.9 vs 3.6 days, p=0.027). Stays greater than 2 days were associated with a body mass index of greater than 30 kg/m2 on multivariate analysis. No complication in either group was attributable to fluoroscopic technique. CONCLUSIONS: Implementing a decreased fluoroscopy protocol during percutaneous nephrostolithotomy resulted in an 80.9% reduction in fluoroscopy time while maintaining success rates, operative times and complications similar to those of the conventional technique. Adopting this reduced fluoroscopy protocol safely decreased radiation exposure to patients, surgeons and operating room staff during percutaneous nephrostolithotomy.

Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation.

The selective pressures leading to cancers with mutations in both KRAS and PIK3CA are unclear. Here, we show that somatic cell knockin of both KRAS G12V and oncogenic PIK3CA mutations in human breast epithelial cells results in cooperative activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in vitro, and leads to tumor formation in immunocompromised mice. Xenografts from double-knockin cells retain single copies of mutant KRAS and PIK3CA, suggesting that tumor formation does not require increased copy number of either oncogene, and these results were also observed in human colorectal cancer specimens. Mechanistically, the cooperativity between mutant KRAS and PIK3CA is mediated in part by Ras/p110α binding, as inactivating point mutations within the Ras-binding domain of PIK3CA significantly abates pathway signaling. In addition, Pdk1 activation of the downstream effector p90RSK is also increased by the combined presence of mutant KRAS and PIK3CA. These results provide new insights into mutant KRAS function and its role in carcinogenesis.

Detection of tumor PIK3CA status in metastatic breast cancer using peripheral blood.

PURPOSE: We sought to evaluate the feasibility of detecting PIK3CA mutations in circulating tumor DNA (ctDNA) from plasma of patients with metastatic breast cancer using a novel technique called BEAMing. EXPERIMENTAL DESIGN: In a retrospective analysis, 49 tumor and temporally matched plasma samples from patients with breast cancer were screened for PIK3CA mutations by BEAMing. We then prospectively screened the ctDNA of 60 patients with metastatic breast cancer for PIK3CA mutations by BEAMing and compared the findings with results obtained by screening corresponding archival tumor tissue DNA using both sequencing and BEAMing. RESULTS: The overall frequency of PIK3CA mutations by BEAMing was similar in both patient cohorts (29% and 28.3%, respectively). In the retrospective cohort, the concordance of PIK3CA mutation status by BEAMing between formalin-fixed, paraffin-embedded (FFPE) samples and ctDNA from temporally matched plasma was 100% (34 of 34). In the prospective cohort, the concordance rate among 51 evaluable cases was 72.5% between BEAMing of ctDNA and sequencing of archival tumor tissue DNA. When the same archival tissue DNA was screened by both sequencing and BEAMing for PIK3CA mutations (n = 41 tissue samples), there was 100% concordance in the obtained results. CONCLUSIONS: Analysis of plasma-derived ctDNA for the detection of PIK3CA mutations in patients with metastatic breast cancer is feasible. Our results suggest that PIK3CA mutational status can change upon disease recurrence, emphasizing the importance of reassessing PIK3CA status on contemporary (not archival) biospecimens. These results have implications for the development of predictive biomarkers of response to targeted therapies.

Left ventricular assist device support as a bridge to recovery in young children.

OBJECTIVE: Left ventricular assist device (LVAD) experience and follow-up data in children are limited. We report the deployment and successful weaning from LVAD in young children with severe heart failure (HF). DESIGN: From 2004--2009, 13 children suffering from HF were placed on LVAD. All presented with a dilated left ventricle (LV) with severely reduced contractility, secondary to myocarditis, atrial arrhythmia or idiopathic HF. This study reports their outcomes and longitudinal follow-up. RESULTS: Of 13 young children with HF (ages 1 month--6 years; mean 19.2 months) placed on LVAD: eight weaned to recovery and successful hospital discharge, one was transplanted and four died. Echo follow-up in the weaned patients (mean age 22.1 months) revealed significant improvements from pre-LVAD measurements: LV end-diastolic dimension (LVED) mean z-score decreased from +4.8 to +0.95 (P < .001); fractional shortening (FS %) improved from a mean of 9.3% to 33% (P < .001); and the degree of mitral regurgitation (MR) significantly improved (P < .05). Time to LVAD deployment from HF diagnosis was more likely to be less than 30 days in the successfully weaned patients (100%) than patients who died or were transplanted (20%); P = .007. CONCLUSIONS: LVAD support can be utilized as a bridge to recovery in young children with HF. Following LVAD weaning, children sustain improvements in LV size, function and degree of MR. LVAD deployment less than 30 days from HF diagnosis improves the likelihood of successful weaning and illustrates that children with acute etiologies of HF are more likely to achieve recovery.

Copper transporter 2 regulates endocytosis and controls tumor growth and sensitivity to cisplatin in vivo.

Copper transporter 2 (CTR2) is one of the four copper transporters in mammalian cells that influence the cellular pharmacology of cisplatin and carboplatin. CTR2 was knocked down using a short hairpin RNA interference. Robust expression of CTR2 was observed in parental tumors grown in vivo, whereas no staining was found in the tumors formed from cells in which CTR2 had been knocked down. Knockdown of CTR2 reduced growth rate by 5.8-fold, increased the frequency of apoptotic cells, and decreased the vascular density, but it did not change copper content. Knockdown of CTR2 increased the tumor accumulation of cis-diamminedichloroplatinum(II) [cisplatin (cDDP)] by 9.1-fold and greatly increased its therapeutic efficacy. Because altered endocytosis has been implicated in cDDP resistance, uptake of dextran was used to quantify the rate of macropinocytosis. Knockdown of CTR2 increased dextran uptake 2.5-fold without reducing exocytosis. Inhibition of macropinocytosis with either amiloride or wortmannin blocked the increase in macropinocytosis mediated by CTR2 knockdown. Stimulation of macropinocytosis by platelet-derived growth factor coordinately increased dextran and cDDP uptake. Knockdown of CTR2 was associated with activation of the Rac1 and cdc42 GTPases that control macropinocytosis but not activation of the phosphoinositide-3 kinase pathway. We conclude that CTR2 is required for optimal tumor growth and that it is an unusually strong regulator of cisplatin accumulation and cytotoxicity. CTR2 regulates the transport of cDDP in part through control of the rate of macropinocytosis via activation of Rac1 and cdc42. Selective knockdown of CTR2 in tumors offers a strategy for enhancing the efficacy of cDDP.

The role of the methionines and histidines in the transmembrane domain of mammalian copper transporter 1 in the cellular accumulation of cisplatin.

Mammalian copper transporter 1 (CTR1) is a high-affinity copper influx transporter that also mediates the uptake of platinum-containing chemotherapeutic agents including cisplatin (cDDP). Methionines 150, 154, and histidine 139 have been proposed to form a series of stacked rings in the pore formed by the CTR1 homotrimer, each of which is required for maximal copper transport. To examine the mechanism by which hCTR1 also transports cDDP, variant forms of hCTR1 in which methionines 150 and 154 were converted to isoleucines or in which histidine 139 was converted to alanine were re-expressed in cells in which both alleles of CTR1 had been knocked out. Each of these conversions disabled copper transport and increased cellular resistance to the cytotoxic effect of copper. In contrast, conversion of the methionines increased the uptake and cytotoxicity of cDDP well above that attained with wild-type hCTR1. Conversion of His139 to alanine did not impair cDDP uptake and actually enhanced cytotoxicity. Thus, although Met150 and Met154 facilitate the movement of copper through the pore, they serve to obstruct the passage of cDDP. None of the modifications altered the ability of cDDP to trigger the degradation of hCTR1, indicating that cDDP must interact with hCTR1 at other sites as well. Although both copper and cDDP may rely on a series of transchelation reactions to pass through the hCTR1 trimeric complex, the details of the molecular interactions must be different, which provides a potential basis for selective pharmacological modulation of copper versus cDDP cytotoxicity.

The role of the N-terminus of mammalian copper transporter 1 in the cellular accumulation of cisplatin.

The mammalian copper transporter 1 (CTR1) is responsible for the uptake of copper (Cu) from the extracellular space, and has been shown to play a major role in the initial accumulation of platinum-based drugs. In this study we re-expressed wild type and structural variants of hCTR1 in mouse embryo fibroblasts in which both alleles of mCTR1 had been knocked out (CTR1(-/-)) to examine the role of the N-terminal extracellular domain of hCTR1 in the accumulation of cisplatin (cDDP). Deletion of either the first 45 amino acids or just the (40)MXXM(45) motif in the N-terminal domain did not alter subcellular distribution or the amount of protein in the plasma membrane but it eliminated the ability of hCTR1 to mediate the uptake of Cu. In contrast it only partially reduced cDDP transport capacity. Neither of these structural changes prevented cDDP from triggering the rapid degradation of hCTR1. However, they did alter the potency of the cDDP that achieved cell entry, possibly reflecting the fact that hCTR1 may mediate the transport of cDDP both through the pore it forms in the plasma membrane and via endocytosis. We conclude that cDDP interacts with hCTR1 both at (40)MXXM(45) and at sites outside the N-terminal domain that produce the conformational changes that trigger degradation.

Regulation of copper transporter 2 expression by copper and cisplatin in human ovarian carcinoma cells.

Down-regulation of copper transporter 1 (CTR1) reduces uptake and sensitivity, whereas down-regulation of CTR2 enhances both. Cisplatin (DDP) triggers the rapid degradation of CTR1 and thus limits its own accumulation. We sought to determine the effect of DDP and copper on the expression of CTR2. Changes in CTR1 and CTR2 mRNA and protein levels in human ovarian carcinoma 2008 cells and ATOX1(+/+) and ATOX1(-/-) mouse embryo fibroblasts in response to exposure to DDP and copper were measured by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and deconvolution microscopy. DDP triggered rapid degradation of CTR1 in 2008 human ovarian cancer cells. However, it increased the expression of CTR2 mRNA and protein levels. Expression of CTR2 was heavily modulated by changes in intracellular copper concentration; copper depletion produced rapid disappearance of CTR2, whereas excess copper increased the level of CTR2 protein. This increase was associated with an increase in CTR2 mRNA and prolongation of the CTR2 half-life. Consistent with prior observations that short hairpin RNA interference-mediated knockdown of CTR2 enhanced DDP uptake and tumor cell kill, reduction of CTR2 by copper starvation also enhanced DDP uptake and cytotoxicity. Comparison of the ability of copper and DDP to modulate the expression of CTR1 in ATOX1(+/+) and ATOX1(-/-) indicated that ATOX1 participates in the regulation of CTR2 expression. Unlike CTR1, the expression of CTR2 is increased rather than decreased by DDP. Therefore, these two copper transporters have opposite effects on DDP sensitivity. CTR2 expression is regulated by copper availability via the copper-dependent regulator ATOX1.